BACKGROUND OF THE INVENTION Metal binding proteins, including metallothioneins, ferritins, and selenium-binding proteins, function in many aspects of metabolism, ranging from detoxification to storage. Mutation of the genes encoding metal-binding proteins can lead to life-threatening disorders, and, where examined, expression of these genes is often tightly regulated in the cell (Aisen, P. et al. (2001) Int. J. Bioch.

Cell Bio. 33: 940-959).

Metallothioneins (MTs) are a group of small (61 amino acids), cysteine rich proteins that bind heavy metals such as cadmium, zinc, mercury, lead, and copper and are thought to play a role in metal detoxification or the metabolism and homeostasis of metals. They are present in a wide variety of eukaryotes including invertebrates, vertebrates, plants, and fungi. The primary structure of the vertebrate MTs is strongly conserved between species, particularly the positions of the cysteine residues which serve to chelate heavy metal ions via thiolate complexes.

At least two closely related but chromatographically distinct isoforms of MT, MT-I and MT-ll, have been observed in every vertebrate species examined. All isoforms of MT sequenced thus far share 20 cysteine residues found in the same positions and lack aromatic amino acids or leucine (Schmidt, C. J. and D. H. Hamer (1983) Gene 24: 137-146; Schmidt, C. J. et al. (1985) J. Biol Chem.

260: 7731-7737). MT-II isoforms differ from MT-I only in the presence of an aspartate residue at position 10 or 11 while MT-I contains either glycine or valine at these positions. Recently, a third class of MT, MT-0, has been discovered in human liver that is characterized by a negatively charged amino acid at position 8 (glutamate) and a lysine substitution at the highly conserved Glu23 (Soumillion, A. et al. (1992) Eur. J. Biochem. 209: 999-1004). In humans, only a single sequence of MT-0 and MT-II have been found, while at least six isoforms of MT-I have been identified. These various isoforms of MT-I differ only by a few residues distributed throughout the molecule. The localization and identification of 12 functional MT genes on human chromosome 16 indicates that several additional MT isoforms exist.

The reason for multiple human MT genes is unclear. It is possible that different isoforms are

responsible for binding different metals, or that different control sequences for the transcription of these various isoforms respond to different stimuli, such as heavy metals, glucocorticoids, or enterotoxin (Schmidt, et al. supra). A third possibility is that multiple isoforms could play different roles in metal homeostasis during development or in different tissues.

Acute or chronic exposure to heavy metals such as lead, arsenic, mercury or cadmium leads to a variety of diseases and disorders involving neuromuscular, CNS, cardiovascular, and gastrointestinal effects. MTs may play a role in the prevention or alleviation of these conditions. In addition, MTs are transcriptionally regulated by glucocorticoids which suggests that MTs have a direct role in the effects of glucocorticoids to treat inflammatory disease, immune disorders, and cancer.

Metal ions such as iron, zinc, copper, cobalt, manganese, molybdenum, selenium, nickel, and chromium are important as cofactors for a number of enzymes. For example, copper is involved in hemoglobin synthesis, connective tissue metabolism, and bone development, by acting as a cofactor in oxidoreductases such as superoxide dismutase, ferroxidase (ceruloplasmin), and lysyl oxidase.

Copper and other metal ions must be provided in the diet, and are absorbed by transporters in the gastrointestinal tract. Plasma proteins transport the metal ions to the liver and other target organs, where specific transporters move the ions into cells and cellular organelles as needed. Imbalances in metal ion metabolism have been associated with a number of disease states (Danks, D. M. (1986) J.

Med. Genet. 23: 99-106).

Iron plays an essential role in oxygen transport and redox reactions, particularly cell respiration; however, iron is also toxic when present in excess. In humans, unregulated iron absorption leads to cirrhosis, endocrine failure, arthritis and cardiomyopathy, as well as hepatocellular carcinoma (Griffiths, W. J. H. et al. (1999) Mol. Med. Today 5: 431-438). Iron- overloading is also the cause of hereditary hemochromatosis, a disorder that leads to death if left untreated. Iron metabolism requires functioning iron-transporter proteins, such as transferrins, as well as iron storage proteins, such as ferritin.

Transferrins are eukaryotic iron-binding glycoproteins that control the level of free iron in biological fluids (Crichton, R. R. and M. Charloteaux-Wauters (1987) Eur. J. Biochem. 164: 485-506).

Transferrin family members are proteins of 650 to 700 residues which have evolved from the duplication of a domain of around 340 residues. Each of the duplicated domains binds one atom of iron. Each iron atom is bound by four conserved residues: an aspartic acid, two tyrosines, and a histidine (Anderson, B. F. et al. (1989) J. Mol. Biol. 209: 711-734). The importance of the function of the transferrins in maintaining homeostasis of the mechanisms regulating iron absorption and transport is appropriately illustrated in conditions which result from iron overload. Briefly described, iron overload occurs when transferrin fails to bind all of the nonheme iron in the circulation. In this

state, the unbound circulating iron may accumulate in specific organs, including the liver, heart, pancreas and gonads. Excess iron in these tissues catalyzes oxidative damage, resulting in cirrhosis, hepatoma, cardiomyopathy, diabetes, hypogonadism and arthritis (Aisen, (2001), supra).

Atransferrinemia (also called hypotransferrinemia) is a condition caused by a genetic mutation that causes severe deficiency in plasma transferrin, resulting in massive deposition of iron in non- hematopoietic tissues. The consequence of this condition is severe iron deficiency anemia leading to death from liver failure (for a review of genetics of iron storage, see Beutler, E. (2001) Drug Metab.

Dispos. 29: 495-499). Aberrant expression of transferrin which results in the disruption of iron homeostasis is associated with neurodegenerative disorders such as Alzheimer's disease and Restless legs syndrome (Thompson, K. J. et al. (2001) Brain Res. Bull. 55: 155-164).

Ferritin is a ubiquitous iron-binding protein that is involved in iron storage and detoxification in microbes, plants, and animals. Mammalian ferritin consists of 24 subunits of two types, H (for heart, or heavy) and L (for light or liver). These subunits assemble into a spherical structure which can accommodate up to 4,000 iron atoms as ferrihydrite, FeOOH (Aisen, P. et al. (1999) Curr. Opin.

Chem. Biol. 3: 200-206). Ferritin expression is regulated at the post-transcriptional level in response to intracellular iron levels. At low intracellular iron concentrations, iron regulatory proteins (IRPs) bind to iron responsive elements of ferritin mRNA, preventing translation of the protein. At high intracellular iron concentrations, when ferritin is needed to sequester iron in order to prevent oxidative damage, IRPs are prevented from binding to the iron responsive elements (Aisen, (2001), supra).

Selenium is a micronutrient that has been implicated in cancer prevention by reducing the number of DNA adducts by carcinogens (Harrison, P. R. et al. (1997) Biomed. Environ. Sci. 10: 235- 245). In mammals, selenium-containing proteins can be divided into three classes: proteins that incorporate selenium in a non-specific manner, selenocysteine-containing selenoproteins, and specific selenium-binding proteins, such as glutathione peroxidase (Behne, D. and A. Kyriakopoulos (2001) Annu. Rev. Nutr. 21: 453-473). In the genetic code, UGA serves as either a signal for termination or as a codon for selenocysteine (Sec). Sec differs from other amino acids in much of the biosynthetic machinery governing its incorporation into protein. Sec-containing proteins have diverse functions.

The more recently evolved selenoproteins appear to take advantage of unique redox properties of Sec that are superior to those of Cys for specific biological functions (Gladyshev V. N. and Kryukov G. V.

(2001) Biofactors 14: 87-92). Selenium-binding proteins are thought to mediate the inhibitory effects of selenium on growth in mammalian cells and tumorigenesis in rodents. Several selenium-binding proteins have been identified in mouse, rat and human, and are expressed in tissues involved in detoxification, such as the liver (Bansal, M. P. et al. (1989) Carcinogenesis 10: 541-546; Bansal, M. P. et al. (1989) J. Biol. Chem. 264: 13780-13784). In mouse and rat, selenium-binding proteins were

also found in blood, duodenum, kidney, pancreas testis, ovary and in mammary tumors (Morrison, D. G. et al. (1989) In Vivo 3: 167-172).

In humans, a 56 kDa selenium-binding protein is expressed in liver, kidney and lung tissue.

Using a cell-free transport assay, this protein has been shown to participate in late stages of intra- Golgi protein transport (Porat, A. et al. (2000) J. Biol. Chem. 275: 14457-14465). A 56 kDa protein that differs from the 56 kDa selenium-binding protein by only 14 residues has been found to associate with an acetamenophen metabolite correlated with hepatotoxicity (Lanfear, J. et al. (1993) 14: 335- 340; Pumford, N. R. et al. (1992) Biochem. Biophys. Res. Commun. 182: 1348-55). Both the selenium-binding and the acetamenophen-binding proteins may mediate detoxification mechanisms and anti-carcinogenic functions of selenium (Lanfear et al., supra).

Expression profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.

One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants.

When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.

Colon Cancer While soft tissue sarcomas are relatively rare, more than 50% of new patients diagnosed with the disease will die from it. The molecular pathways leading to the development of sarcomas are relatively unknown, due to the rarity of the disease and variation in pathology. Colon cancer evolves through a multi-step process whereby pre-malignant colonocytes undergo a relatively defined sequence of events leading to tumor formation. Several factors participate in the process of tumor progression and malignant transformation including genetic factors, mutations, and selection.

To understand the nature of gene alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. Familial adenomatous polyposis (FAP), is caused by mutations in the adenomatous polyposis coli gene (APC), resulting in truncated or inactive forms of the protein.

This tumor suppressor gene has been mapped to chromosome 5q. Hereditary nonpolyposis colorectal

cancer (HNPCC) is caused by mutations in mis-match repair genes. Although hereditary colon cancer syndromes occur in a small percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generally applied. For instance, somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in all of these genes lead to gene expression changes in colon cancer.

There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflammatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders.

SUMMARY OF THE INVENTION Various embodiments of the invention provide purified polypeptides, metal binding proteins, referred to collectively as'MBP'and individually as'MBP-1,''MBP-2,'and'MBP-3'and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified metal binding proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified metal binding proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.

An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1- 3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-3.

Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3,

and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-3. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID N0 : 4-6.

Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3.

Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.

Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3.

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.

Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3.

Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group

consisting of SEQ ID NO : 4-6, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a) -d). In other embodiments, the polynucleotide can comprise at least about 20,30, 40, 60,80, or 100 contiguous nucleotides.

Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of 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. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20,30, 40,60, 80, or 100 contiguous nucleotides.

Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of 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. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an

amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, and a pharmaceutically acceptable excipient.

In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional MBP, comprising administering to a patient in need of such treatment the composition.

Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional MBP, comprising administering to a patient in need of such treatment the composition.

Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional MBP, comprising administering to a patient in need of such treatment the composition.

Another embodiment provides a method of screening for a compound that specifically binds

to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. 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.

Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. 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.

Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

Another embodiment 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 selected from the group consisting of i) a polynucleotide

comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 4-6, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).

Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide 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 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.

Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog (s) are also shown.

Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.

Table 5 shows representative cDNA libraries for polynucleotide embodiments.

Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.

DESCRIPTION OF THE INVENTION Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, 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 invention.

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 various embodiments of 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 "MBP"refers to the amino acid sequences of substantially purified MBP 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 MBP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of MBP either by directly interacting with MBP or by acting on components of the biological pathway in which MBP participates.

An"allelic variant"is an alternative form of the gene encoding MBP. 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 MBP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as MBP or a polypeptide with at least one functional characteristic of MBP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding MBP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding MBP. 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 MBP.

Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of MBP 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 glutamin ; 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"can refer to an oligopeptide, a peptide, a polypeptide, or a 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.

Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.

The term"antagonist"refers to a molecule which inhibits or attenuates the biological activity of MBP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of MBP either by directly interacting with MBP or by acting on components of the biological pathway in which MBP 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 MBP 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"aptamer"refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e. g. , SELEX (Systematic Evolution of Ligands by EXponential Enrichment), described in U. S. Patent No.

5,270, 163), which selects for target-specific aptamer sequences from large combinatorial libraries.

Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.

The nucleotide components of an aptamer may have modified sugar groups (e. g. , the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH), which may improve a desired property, e. g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e. g. , a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.

Aptamers may be specifically cross-linked to their cognate ligands, e. g. , by photo-activation of a cross-linker (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74: 5-13).

The term"intramer"refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96: 3606-3610).

The term"spiegelmer"refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.

The term"antisense"refers to any composition capable of base-pairing with the"sense" (coding) strand of a polynucleotide having 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"immunogenic" refers to the capability of the natural, recombinant, or synthetic MBP, 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"and a"composition comprising a given polypeptide"can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding MBP or fragments of MBP 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., NaCl), 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 (Applied 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 (Accelrys, Burlington MA) 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, He 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 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.

"Differential expression"refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.

"Exon shuffling"refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the

evolution of new protein functions.

A"fragment"is a unique portion of MBP or a polynucleotide encoding MBP which can be 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 about 5 to about 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 NO : 4-6 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ N NO : 4-6, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO : 4-6 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0 : 4-6 from related polynucleotides. The precise length of a fragment of SEQ ID N0 : 4-6 and the region of SEQ ID N0 : 4-6 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 : 1-3 is encoded by a fragment of SEQ ID NO : 4-6. A fragment of SEQ ID NO : 1-3 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO : 1-3. For example, a fragment of SEQ ID NO : 1-3 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO : 1-3. The precise length of a fragment of SEQ ID NO : 1-3 and the region of SEQ ID NO : 1-3 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.

A"full length"polynucleotide 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, alternatively, 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 identical nucleotide 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 one or more computer algorithms or programs known in the art or described herein. For example, percent identity can 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.

Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used 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 2.0. 12 (April-21-2000) set at default parameters. Such default parameters may be, for example: Matrix : BLOSUM62 Rewardfor match : 1 Penalty mismatch :-2 Open Gap: 5 and Extension Gap : 2 penalties Gap x drop-off. 50 Expect : 10 Word Size : 11 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 identical 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. The phrases"percent similarity"and"% similarity, "as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.

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=l, gap penalty=3, window=5, and"diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table.

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 2.0. 12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example: Matrix : BLOSUM62 Open Gap : 11 and Extension Gap : 1 penalties Gap x drop-of : 50 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 step (s) 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 ug/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 T. and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. and D. W.

Russell (2001; Molecular Cloning : A Laboratory Manual, 3rd ed. , vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 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 jMg/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 acids 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 present in solution and another nucleic acid 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 polynucleotide 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 MBP 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 MBP 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, antibodies, or other chemical compounds on a substrate.

The terms"element"and"array element"refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.

The term"modulate"refers to a change in the activity of MBP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of MBP.

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 MBP 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 MBP.

"Probe"refers to nucleic acids encoding MBP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. 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 polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e. g. , by the polymerase 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, for example, Sambrook, J. and D. W. Russell (2001; Molecular Cloning : A Laboratory Manual, 3rd ed. , vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY), Ausubel, F. M. et al. (1999; Short Protocols in

Molecular Biology, 4 ed., John Wiley & Sons, New York NY), and Innis, M. et al. (1990; PCR Protocols, A Guide to Methods and Applications, 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 nucleic acid 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 and Russell (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 molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule 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 MBP, nucleic acids encoding MBP, or fragments thereof 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 about 60% free, preferably at least about 75% free, and most preferably at least about 90% 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"or"expression profile"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. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295: 868-872). 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 and Russell (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 2.0. 9 (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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 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 alternate splicing during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.

Species variants are polynucleotides that vary from one species to another. The resulting polypeptides will generally 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 or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the"BLAST 2 Sequences"tool Version 2.0. 9 (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 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.

THE INVENTION Various embodiments of the invention include new human metal binding proteins (MBP), the polynucleotides encoding MBP, and the use of these compositions for the diagnosis, treatment, or prevention of heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflamrnatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders.

Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.

Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ

ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog (s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog (s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.

Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows amino acid residues comprising signature sequences, domains, motifs, potential phosphorylation sites, and potential glycosylation sites. Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are metal binding proteins. For example, SEQ ID NO : 3 is 100% identical, from residue E168 to residue P635, to human transferrin precursor (GenBank ID g339453) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2. ) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO : 3 also has homology to proteins that function as small molecule-binding proteins and are transferrins, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO : 3 also contains transferrin domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein families/domains. (See Table 3. ) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO : 3 is a transferrin. SEQ ID NO : 1-2 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO : 1-3 are described in Table 7.

As shown in Table 4, full length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs. Column 2 lists fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO : 4-6 or that distinguish between

SEQ ID NO : 4-6 and related polynucleotides. Column 3 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide embodiments. Columns 4 and 5 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 3 relative to their respective full length sequences.

The identification numbers in Column 3 of Table 4 may refer specifically, for example, to Incyte cDNAs along with their corresponding cDNA libraries. For example, 5081695H1 is the identification number of an Incyte cDNA sequence, and LNODNOT11 is the cDNA library from which it is derived. Incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e. g., 71971850V1). Alternatively, the identification numbers in column 3 may refer to GenBank cDNAs or ESTs (e. g. , g2028330) which contributed to the assembly of the full length polynucleotides. In addition, the identification numbers in column 3 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i. e., those sequences including the designation"ENST"). Alternatively, the identification numbers in column 3 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i. e. , those sequences including the designation"NM"or"NT") or the NCBI RefSeq Protein Sequence Records (i. e., those sequences including the designation"NP"). Alternatively, the identification numbers in column 3 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an"exon stitching"algorithm. For example, FL_XXXXXX_N, _N2_YYYYY_N3_N4 represents a"stitched" sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and NI, 2 3, if present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the identification numbers in column 3 may refer to assemblages of exons brought together by an"exon-stretching"algorithm. For example, FLXXXXXXXgAA. AAA_gBBBBB_l lV is the identification number of a"stretched"sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the"exon-stretching"algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the"exon-stretching"algorithm, a RefSeq identifier (denoted by"NM, ""NP,"or"NT") may be used in place of the GenBank identifier (i. e., gBBBBB).

Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).

Prefix Type of analysis and/or examples of programs GNN, GFG, Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES (Computer Genomics Group, The Sanger Centre, Cambridge, UK). GBI Hand-edited analysis of genomic sequences. FL Stitched or stretched genomic sequences (see Example V). INCY Full length transcript and exon prediction from mapping of EST sequences to the genome. Genomic location and EST composition data are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.

Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.

Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with allele frequencies in different human populations. Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PID) for polynucleotides of the invention. Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID). Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full- length polynucleotide sequence (CB 1 SNP). Column 7 shows the allele found in the EST sequence.

Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST. Columns 11- 14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of allele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.

The invention also encompasses MBP variants. Various embodiments of MBP variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the

MBP amino acid sequence, and can contain at least one functional or structural characteristic of MBP.

Various embodiments also encompass polynucleotides which encode MBP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO : 4-6, which encodes MBP. The polynucleotide sequences of SEQ ID N0 : 4-6, 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 variants of a polynucleotide encoding MBP. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding MBP. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO : 4-6 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO : 4-6. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of MBP.

In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding MBP. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding MBP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding MBP over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding MBP.

For example, a polynucleotide comprising a sequence of SEQ ID NO : 4 and a polynucleotide comprising a sequence of SEQ ID NO : 5 are splice variants of each other. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of MBP.

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 MBP, 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 MBP, and all such variations are to be considered as being specifically disclosed.

Although polynucleotides which encode MBP and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring MBP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding MBP 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 MBP 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 polynucleotides which encode MBP and MBP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide 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 polynucleotide encoding MBP or any fragment thereof.

Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO : 4-6 and fragments thereof, under various conditions of stringency (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 polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA). 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 (Applied Biosystems).

Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which

are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).

The nucleic acids encoding MBP 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 (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 (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 (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 (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, Applied 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, polynucleotides or fragments thereof which encode MBP may be cloned in recombinant DNA molecules that direct expression of MBP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express MBP.

The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter MBP-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 No.

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 MBP, 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, polynucleotides encoding MBP may be synthesized, in whole or in part, using one or more chemical methods well known in the art (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, MBP itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH Freeman, New

York NY, pp. 55-60; Roberge, J. Y. et al. (1995) Science 269: 202-204). Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of MBP, 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 (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 (Creighton, supra, pp. 28-53).

In order to express a biologically active MBP, the polynucleotides encoding MBP 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 polynucleotides encoding MBP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding MBP. Such signals include the ATG initiation codon and adjacent sequences, e. g. the Kozak sequence. In cases where a polynucleotide sequence encoding MBP 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 (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 polynucleotides encoding MBP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and Russell, supra, ch. 1-4, and 8 ; Ausubel et al., supra, ch. 1,3, and 15).

A variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding MBP. 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 (Sambrook and Russell, supra ; Ausubel et al., supra ; Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264: 5503-5509; 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: 307-311 ; 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; 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 polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5: 350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6340-6344; Buller, R. M. et al. (1985) Nature 317: 813-815; McGregor, D. P. et al. (1994) Mol. Immunol. 31: 219-226; Verma, I. M. 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 polynucleotides encoding MBP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding MBP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).

Ligation of polynucleotides encoding MBP 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 (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264: 5503-5509). When large quantities of MBP are needed, e. g. for the production of antibodies, vectors which direct high level expression of MBP may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of MBP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharonzyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra ; Bitter, G. A. et al. (1987) Methods Enzymol. 153: 516-544; Scorer, C. A. et al. (1994) Bio/Technology 12: 181-184).

Plant systems may also be used for expression of MBP. Transcription of polynucleotides encoding MBP may be driven by 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 (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224: 838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (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, polynucleotides encoding MBP 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 MBP in host cells (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 (Harrington, J. J. et al. (1997) Nat. Genet. 15: 345-355).

For long term production of recombinant proteins in mammalian systems, stable expression of MBP in cell lines is preferred. For example, polynucleotides encoding MBP 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 (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 (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 (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), glucuronidase and its substrate p-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 (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 MBP is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding MBP can be identified by the absence of marker gene function.

Alternatively, a marker gene can be placed in tandem with a sequence encoding MBP 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 polynucleotide encoding MBP and that express MBP 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 MBP 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 MBP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art (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; 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 MBP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Alternatively, polynucleotides encoding MBP, 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 Biosciences, 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 polynucleotides encoding MBP 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 MBP may be designed to contain signal sequences which direct secretion of MBP 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 polynucleotides 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 polynucleotides encoding MBP 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 MBP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of MBP 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 MBP encoding sequence and the heterologous protein sequence, so

that MBP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.

In another embodiment, synthesis of radiolabeled MBP 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.

MBP, fragments of MBP, or variants of MBP may be used to screen for compounds that specifically bind to MBP. One or more test compounds may be screened for specific binding to MBP. In various embodiments, 1,2, 3,4, 5,10, 20,50, 100, or 200 test compounds can be screened for specific binding to MBP. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e. g. , ligands or receptors), or small molecules.

In related embodiments, variants of MBP can be used to screen for binding of test compounds, such as antibodies, to MBP, a variant of MBP, or a combination of MBP and/or one or more variants MBP. In an embodiment, a variant of MBP can be used to screen for compounds that bind to a variant of MBP, but not to MBP having the exact sequence of a sequence of SEQ ID NO : 1-3. MBP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to MBP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.

In an embodiment, a compound identified in a screen for specific binding to MBP can be closely related to the natural ligand of MBP, e. g. , a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J. E. et al. (1991) Current Protocols in Immunology 1 (2) : Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor MBP (Howard, A. D. et al. (2001) Trends Pharmacol. Sci. 22: 132- 140; Wise, A. et al. (2002) Drug Discovery Today 7: 235-246).

In other embodiments, a compound identified in a screen for specific binding to MBP can be closely related to the natural receptor to which MBP binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket. For example, the compound may be a receptor for MBP which is capable of propagating a signal, or a decoy receptor for MBP which is not capable of propagating a signal (Ashkenazi, A. and V. M. Divit (1999) Curr. Opin. Cell Biol. 11: 255-260; Mantovani, A. et al. (2001) Trends Immunol. 22: 328-336).

The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; Amgen Inc. , Thousand Oaks

CA), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG 1 (Taylor, P. C. et al. (2001) Curr. Opin. Immunol. 13: 611-616).

In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to MBP, fragments of MBP, or variants of MBP.

The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of MBP. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of MBP. In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of MBP.

In an embodiment, anticalins can be screened for specific binding to MBP, fragments of MBP, or variants of MBP. Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7: R177-R184 ; Skerra, A.

(2001) J. Biotechnol. 74: 257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered itt vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e. g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.

In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit MBP involves producing appropriate cells which express MBP, either as a secreted protein or on the cell membrane. Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing MBP or cell membrane fractions which contain MBP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either MBP 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 MBP, either in solution or affixed to a solid support, and detecting the binding of MBP 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 compound (s) may be free in solution or affixed to a solid support.

An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio-labeling assays such as those described in U. S. Patent No. 5,914, 236 and U. S. Patent No.

6,372, 724. In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1: 25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B. C. and J. A. Wells (1991) Proc. Natl. Acad. Sci. USA 88: 3407-3411; Lowman, H. B. et al. (1991) J. Biol. Chem. 266: 10982- 10988).

MBP, fragments of MBP, or variants of MBP may be used to screen for compounds that modulate the activity of MBP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for MBP activity, wherein MBP is combined with at least one test compound, and the activity of MBP in the presence of a test compound is compared with the activity of MBP in the absence of the test compound. A change in the activity of MBP in the presence of the test compound is indicative of a compound that modulates the activity of MBP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising MBP under conditions suitable for MBP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of MBP 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 MBP 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 MBP 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 MBP 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 MBP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastula 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 MBP, e. g. , by secreting MBP 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 MBP and metal binding proteins. In addition, examples of tissues expressing MBP can be found in Table 6 and can also be found in Example XI. Therefore, MBP appears to play a role in heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflammatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders. In the treatment of disorders associated with increased MBP expression or activity, it is desirable to decrease the expression or activity of MBP. In the treatment of disorders associated with decreased MBP expression or activity, it is desirable to increase the expression or activity of MBP.

Therefore, in one embodiment, MBP 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 MBP.

Examples of such disorders include, but are not limited to, heavy metal toxicity ; a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, 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 cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris,

myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation, congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; 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 including Down syndrome, 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, hemiplegic migraine, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as diabetes, GRACILE syndrome, obesity, and osteoporosis; an immune system disorder, such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a lipid disorder such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy,

adrenoleukodystrophy, GM2 gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin: cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; and a vesicle trafficking disorder, such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS), allergies including hay fever, asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage, and viral, bacterial, fungal, helminthic, and protozoal infections.

In another embodiment, a vector capable of expressing MBP 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 MBP including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantially purified MBP 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 MBP including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity of MBP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of MBP including, but not limited to, those listed above.

In a further embodiment, an antagonist of MBP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of MBP. Examples of such disorders include, but are not limited to, those heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflammatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders described above. In one aspect, an antibody which specifically binds MBP 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 MBP.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding MBP may be administered to a subject to treat or prevent a disorder associated with

increased expression or activity of MBP including, but not limited to, those described above.

In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments 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 MBP may be produced using methods which are generally known in the art.

In particular, purified MBP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind MBP. Antibodies to MBP 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. In an embodiment, neutralizing antibodies (i. e., those which inhibit dimer formation) can be used therapeutically. Single chain antibodies (e. g. , from camels or llamas) may be potent enzyme inhibitors and may have application in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J.

Biotechnol. 74 : 277-302).

For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with MBP 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 MBP 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 substantially identical to a portion of the amino acid sequence of the natural protein. Short stretches of MBP 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 MBP 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 (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; 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 (Morrison, S. L. et al. (1984) Proc. Natl. Acad.

Sci. USA 81: 6851-6855; Neuberger, M. S. et al. (1984) Nature 312: 604-608; 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 MBP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (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 (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 MBP 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 (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 MBP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering MBP 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 MBP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of MBP-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 MBP epitopes, represents the average affinity, or avidity, of the antibodies for

MBP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular MBP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the MBP-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 MBP, 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 MBP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra ; Coligan et al., supra).

In another embodiment of the invention, polynucleotides encoding MBP, 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 MBP. 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 MBP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, 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 (Slater, J. E. et al. (1998) J. Allergy Clin. Immunol. 102: 469-475; Scanlon, K. J. et al. (1995) 9: 1288-1296).

Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A. D. (1990) Blood 76: 271; Ausubel et al., supra ; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63: 323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J. J. (1995) Br. Med. Bull. 51: 217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.

87: 1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res. 25: 2730-2736).

In another embodiment of the invention, polynucleotides encoding MBP 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)-Xl 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 VDI or Factor IX deficiencies (Crystal, R. G. (1995) Science 270: 404-410; Verma, I. M. 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 Plasmodiumfalciparum and Trypanosoma cruzi). In the case where a genetic deficiency in MBP expression or regulation causes disease, the expression of MBP 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 MBP are treated by constructing mammalian expression vectors encoding MBP and introducing these vectors by mechanical means into MBP-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. Récipon (1998) Curr. Opin. Biotechnol.

9: 445-450).

Expression vectors that may be effective for the expression of MBP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA 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). MBP may be expressed using (i) a constitutively active promoter, (e. g. , from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or p-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 FIND ; 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 MBP from a normal individual.

Commercially 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 MBP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding MBP 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, 1. 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 No. 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 an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding MBP to cells which have one or more genetic abnormalities with respect to the expression of MBP. 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 No. 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, I. M. and N. Somia (1997; Nature 18: 389: 239-242).

In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding MBP to target cells which have one or more genetic abnormalities with respect to the expression of MBP. The use of herpes simplex virus (HSV) -based vectors may be especially valuable for introducing MBP 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 No. 5,804, 413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U. S. Patent No. 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). 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 embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding MBP 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 MBP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of MBP- coding RNAs and the synthesis of high levels of MBP 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 MBP 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 (Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, 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 RNA molecules encoding MBP.

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 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 iii vivo transcription of DNA molecules encoding MBP. 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'andlor 3' ends of the molecule, or the use of phosphorothioate or 2'0-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.

In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art. RNAi is a post-transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i. e. , the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene. PTGS can also be accomplished by use of DNA or DNA fragments as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391: 806-811) and Gura, T. (2000; Nature 404: 804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.

RNAi can be induced in mammalian cells by the use of small interfering RNA also known as siRNA. SiRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.

SiRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3'overhangs. The use of siRNA for inducing RNAi in mammalian cells is described by Elbashir, S. M. et al. (2001; Nature 411: 494-498).

SiRNA can either be generated indirectly by introduction of dsRNA into the targeted cell, or directly by mammalian transfection methods and agents described herein or known in the art (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/introductory methods). Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e. g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3'adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred. Regions to be avoided for target siRNA sites include the 5'and 3'untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. The

selected target sites for siRNA can then be compared to the appropriate genome database (e. g., human, etc. ) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration.

The selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commercially available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).

In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e. g. , Brummelkamp, T. R. et al. (2002) Science 296: 550-553; and Paddison, P. J. et al. (2002) Genes Dev. 16: 948-958). In these and related embodiments, shRNAs can be delivered to target cells using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PSILENCER1. 0-U6 (circular) plasmid (Ambion). Once delivered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene- specific silencing.

In various embodiments, the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis. Expression levels of the mRNA of a targeted gene, can be determined by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion) ; by microarray methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein. Expression levels of the protein encoded by the targeted gene can be determined by Western analysis using standard techniques known in the art.

An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding MBP. 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 MBP expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding MBP may be therapeutically useful, and in the treatment of disorders associated with decreased MBP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding MBP may be therapeutically useful.

In various embodiments, one or more 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 MBP 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 MBP 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 MBP. 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 Schizosaccharonzyces poiizbe gene expression system (Atkins, D. et al. (1999) U. S. Patent No. 5,932, 435; Arndt, 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 (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 MBP, antibodies to MBP, and mimetics, agonists, antagonists, or inhibitors of MBP.

In various embodiments, the compositions described herein, such as pharmaceutical compositions, 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 allows 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 MBP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, MBP 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 MBP or fragments thereof, antibodies of MBP, and agonists, antagonists or inhibitors of MBP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by

calculating the ED50 (the dose therapeutically effective in 50% of the population) or LD50 (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD5O/ED50 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 ED50 with little or no toxicity.

The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination (s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to94 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0. 1 ug to 100, 000, ug, 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 MBP may be used for the diagnosis of disorders characterized by expression of MBP, or in assays to monitor patients being treated with MBP or agonists, antagonists, or inhibitors of MBP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for MBP include methods which utilize the antibody and a label to detect MBP 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 MBP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of MBP expression. Normal or standard values for MBP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to MBP under conditions suitable for complex formation. The amount of standard complex formation may be

quantitated by various methods, such as photometric means. Quantities of MBP 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, polynucleotides encoding MBP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, 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 MBP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of MBP, and to monitor regulation of MBP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding MBP or closely related molecules may be used to identify nucleic acid sequences which encode MBP. 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 MBP, 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 MBP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO : 4-6 or from genomic sequences including promoters, enhancers, and introns of the MBP gene.

Means for producing specific hybridization probes for polynucleotides encoding MBP include the cloning of polynucleotides encoding MBP or MBP 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 32p or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotides encoding MBP may be used for the diagnosis of disorders associated with expression of MBP. Examples of such disorders include, but are not limited to, heavy metal toxicity; a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, 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 cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular

replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation, congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; 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 including Down syndrome, 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, hemiplegic migraine, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as diabetes, GRACILE syndrome, obesity, and osteoporosis; an immune system disorder, such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a lipid disorder such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's

disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2 gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin: cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; and a vesicle trafficking disorder, such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS), allergies including hay fever, asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage, and viral, bacterial, fungal, helminthic, and protozoal infections.

Polynucleotides encoding MBP 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 MBP expression.

Such qualitative or quantitative methods are well known in the art.

In a particular embodiment, polynucleotides encoding MBP may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding MBP 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 polynucleotides encoding MBP 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 MBP, 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 MBP, 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 MBP 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 MBP, or a fragment of a polynucleotide complementary to the polynucleotide encoding MBP, 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 polynucleotides encoding MBP 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 polynucleotides encoding MBP 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).

SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity. For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations (Taylor, J. G. et al. (2001) Trends Mol. Med. 7: 507-512; Kwok, P. -Y. and Z. Gu (1999) Mol. Med. Today 5: 538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11: 637-641).

Methods which may also be used to quantify the expression of MBP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P. C. et al. (1993) 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 polynucleotides 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 below. 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, MBP, fragments of MBP, or antibodies specific for MBP 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 (Seilhamer et al. ,"Comparative Gene Transcript Analysis, "U. S. Patent No. 5,840, 484; hereby 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-occurring 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. 112-113: 467-471). 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 an embodiment, the toxicity of a test compound can be 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 embodiment relates to the use of the polypeptides disclosed herein 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 interest. In some cases, further sequence data may be obtained for definitive protein identification.

A proteomic profile may also be generated using antibodies specific for MBP to quantify the levels of MBP 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- 111; 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 (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 W095/25116 ; Shalon, D. et al. (1995) PCT application W095/35505 ; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155; Heller, M. J. et al. (1997) U. S. Patent No. 5,605, 662). Various types of microarrays are well known and thoroughly described in Schena, M. , ed. (1999; DNA Microarrays : A Practical Approach, Oxford University Press, London).

In another embodiment of the invention, nucleic acid sequences encoding MBP 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 PI constructions, or single chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat. Genet.

15: 345-355; Price, C. M. (1993) Blood Rev. 7: 127-134; Trask, B. J. (1991) Trends Genet. 7: 149-154).

Once mapped, the nucleic acid sequences 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) (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 (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 MBP 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 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (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, MBP, 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 MBP 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 (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 MBP, or fragments thereof, and washed. Bound MBP is then detected by methods well known in the art. Purified MBP 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 MBP specifically compete with a test compound for binding MBP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with MBP.

In additional embodiments, the nucleotide sequences which encode MBP 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 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, including U. S. Ser. No. 60/457, 538 are hereby expressly incorporated by reference.

EXAMPLES I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 3. 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 (Invitrogen), 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 (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). 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 S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) 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 (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2- TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.

Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XL1- BlueMRF, or SOLR from Stratagene or DH5a, DH10B, or ElectroMAX DH10B from Invitrogen.

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 (Applied 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 Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).

Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Applied 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 (Ausubel et al., supra, ch.

7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIM.

The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly (A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof 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; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic Acids Res. 29: 41-43) ; and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30: 242-244). (HMM is a probabilistic approach which analyzes consensus primary structures of gene families ; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct. Biol.

6: 361-365. ) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages 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 polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein

databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using 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.

Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 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 or the lower the probability value, the greater the identity between two sequences).

The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID NO : 4-6. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.

IV. Identification and Editing of Coding Sequences from Genomic DNA Putative metal binding proteins were initially identified by running the Genscan gene identification program against public genomic sequence databases (e. g. , gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268: 78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8: 346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode metal binding proteins, the encoded polypeptides were analyzed by querying against PFAM models for metal binding proteins. Potential metal binding proteins were also identified by homology to Incyte cDNA sequences that had been annotated as metal binding proteins.

These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by

Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.

When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example m. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched"Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example m were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified were then"stitched"together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.

Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.

"Stretched"Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs

(HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore"stretched"or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.

VI. Chromosomal Mapping of MBP Encoding Polynucleotides The sequences which were used to assemble SEQ ID NO : 4-6 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 NO : 4-6 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). 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 Généthon 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, of 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 Généthon 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.

VII. 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 (Sambrook and Russell, supra, ch. 7; Ausubel et al. , supra, ch. 4).

Analogous computer techniques applying BLAST were used to search for identical or related molecules in 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.

Alternatively, polynucleotides encoding MBP are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example Vit). Each cDNA sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.

The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue-and disease-specific expression of cDNA encoding MBP. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).

VIII. Extension of MBP Encoding Polynucleotides Full length polynucleotides are 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 was synthesized 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) 2SO4, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (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 rnin ; 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 Al PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE and 0.5 Itl 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, ul to 10, ul aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose 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 Biosciences). 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 Biosciences), treated with Pfu DNA polymerase (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 polymerase (Amersham Biosciences) and Pfu DNA polymerase (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 Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).

In like manner, full length polynucleotides are verified using the above procedure or are used to obtain 5'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in MBP Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ID NO : 4-6 using the LIFESEQ database (Incyte Genomics). Sequences from the same gene were clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants. An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.

Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc. ) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown

of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.

X. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID NO : 4-6 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, uCi of [7_32p] adenosine triphosphate (Amersham Biosciences), 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 Biosciences). 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 It, 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.

XI. 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 et al., 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, M. , ed.

(1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). 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 (Schena, M. et al. (1995) Science 270: 467-470; Shalon, 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/jul oligo- (dT) primer (21mer), 1X first strand buffer, 0.03 units/, al RNase inhibitor, 500 uM dATP, 500, uM dGTP, 500, uM dTTP, 40 , uM dCTP, 40 yM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly (A) + RNA with GEMBRIGHT kits (Incyte Genomics). 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 0. 5M 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, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 mi 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 ttl 5X 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 g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).

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-Aldrich, St. Louis MO) in 95% ethanol. Coated slides are cured in a 110°C oven.

Array elements are applied to the coated glass substrate using a procedure described in U. S.

Patent No. 5,807, 522, incorporated herein by reference. 1 Itl of the array element DNA, at an average concentration of 100 ng/jMl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER W-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 Al of sample mixture consisting of 0. 2 ag each of Cy3 and Cy5 labeled cDNA synthesis products in 5X 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 ftl of 5X 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. 1X 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 Cy5. 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 R1477, 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 Cy5.

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 Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to- background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentially expressed.

Expression For example, expression of SEQ ID NO : 4-5 was downregulated in cancerous colon tissue as compared to normal colon tissue as determined by microarray analysis. Matched normal colon tissue and colon tumor tissue samples from an 81-year-old male diagnosed with colon cancer (Huntsman Cancer Institute, Salt Lake City, UT) were compared by competitive hybridization. Expression of SEQ ID NO : 4-5 was decreased at least two-fold in colon adenocarcinoma tumor tissue, compared to normal colon tissue. Therefore, in various embodiments, SEQ ID Nove4-5 can be used for one or more of the following: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and iii) developing therapeutics and/or other treatments for colon cancer.

XII. Complementary Polynucleotides

Sequences complementary to the MBP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring MBP. 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 MBP. 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 MBP-encoding transcript.

XIII. Expression of MBP Expression and purification of MBP is achieved using bacterial or virus-based expression systems. For expression of MBP 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 MBP upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG). Expression of MBP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding MBP 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 Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.

Infection of the latter requires additional genetic modifications to baculovirus (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, MBP 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 Schistosonza japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). Following purification, the GST moiety can be proteolytically cleaved from MBP 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 et al. (supra, ch. 10 and 16). Purified MBP obtained by these methods can be used directly in the assays shown in Examples XVI and XVIII, where applicable.

XIV. Functional Assays MBP function is assessed by expressing the sequences encoding MBP 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 (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 Ig 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, ug 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 YorkNY).

The influence of MBP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding MBP 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 MBP and other genes of interest can be analyzed by northern analysis or microarray techniques.

XV. Production of MBP Specific Antibodies MBP 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 animals (e. g. , rabbits, mice, etc. ) and to produce antibodies using standard protocols.

Alternatively, the MBP 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 (Ausubel et al., supra, ch. 11).

Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Applied 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 (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-MBP activity by, for example, binding the peptide or MBP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

XVI. Purification of Naturally Occurring MBP Using Specific Antibodies Naturally occurring or recombinant MBP is substantially purified by immunoaffinity chromatography using antibodies specific for MBP. An immunoaffinity column is constructed by covalently coupling anti-MBP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing MBP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of MBP (e. g. , high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/MBP 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 MBP is collected.

XVII. Identification of Molecules Which Interact with MBP MBP, or biologically active fragments thereof, are labeled with zsI Bolton-Hunter reagent (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 MBP, washed, and any wells with labeled MBP complex are assayed. Data obtained using different concentrations of MBP are used to calculate values for the number, affinity, and association of MBP with the candidate molecules.

Alternatively, molecules interacting with MBP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340: 245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).

MBP 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).

XVIII. Demonstration of MBP Activity MBP activity can be measured using a modified double antibody radioimmunoassay for metallothionein (Hogstrand, C. and C. Haux (1990) Toxicol. Appl. Pharmacol. 103: 56-65, with modifications as described in Hogstrand, C. et al. ( (1994) J. Exp. Biol. 186: 55-73). Alternatively, a radioimmunoassay for ferritin can be carried out (Spectria, Orion Diagnostics; Punnonen, K. et al.

(1997) Blood 89: 1052-1057). MBP activity can also be assayed for binding of radioactive metal isotopes, such as 75Se (Bansal, M. P. et al. (1989) Carcinogenesis 10: 541-546; Bansal, M. P. et al.

(1990) Carcinogenesis 11: 2071-2073). Metal content of cells or tissues can be determined using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) in a Thermo Jarrel Ash, Polyscan 61E to assess metal-to-protein ratios (Bongers, J. et al. (1988) Anal. Chem. 60: 2683-2686; Valls, M. et al. (2001) J. Biol. Chem. 276: 32835-32843).

MBP activity can be measured using a cell-free intra-Golgi transport assay (Porat, A. et al.

(2000) J. Biol. Chem. 275: 14457-14465). MBP activity can also be measured by its inclusion in coated vesicles. MBP can be expressed by transforming a mammalian cell line such as COS7, HeLa, or CHO with an eukaryotic expression vector encoding MBP. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A small amount of a second plasmid, which expresses any one of a number of marker genes, such as p-galactosidase, is co-transformed into the cells in order to allow rapid identification of those cells which have taken up and expressed the foreign DNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of MBP and ß-galactosidase.

Transformed cells are collected and cell lysates are assayed for vesicle formation. A non- hydrolyzable form of GTP, GTPyS, and an ATP regenerating system are added to the lysate and the mixture is incubated at 37° C for 10 minutes. Under these conditions, over 90% of the vesicles remain coated (Orci, L. et al (1989) Cell 56: 357-368). Transport vesicles are salt-released from the Golgi membranes, loaded under a sucrose gradient, centrifuged, and fractions are collected and analyzed by SDS-PAGE. Co-localization of MBP with clathrin or COP coatamer is indicative of MBP activity in vesicle formation. The contribution of MBP to vesicle formation can be confirmed by incubating lysates with antibodies specific for MBP prior to GTPyS addition. The antibody will bind to MBP and interfere with its activity, thus preventing vesicle formation.

In the alternative, MBP activity is measured by its ability to alter vesicle trafficking

pathways. Vesicle trafficking in cells transformed with MBP is examined using fluorescence microscopy. Antibodies specific for vesicle coat proteins or typical vesicle trafficking substrates such as transferrin or the mannose-6-phosphate receptor are commercially available. Various cellular components such as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and the plasmalemma are examined. Alterations in the numbers and locations of vesicles in cells transformed with MBP as compared to control cells are characteristic of MBP activity.

Various modifications and variations of the described compositions, 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. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions.

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.

Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Table 1 Incyte Project ID Polypeptide Incyte Polynucleotide Incyte SEQ ID NO : Polypeptide ID SEQ ID NO : Polynucleotide ID 7513406 1 7513406CD1 4 7513406CB1 7513407 2 7513407CD1 5 7513407CB1 7512697 3 7512697CD1 6 7512697CB1 Table 2 Polypeptide SEQ Incyte GenBank ID NO: Probability Annotation IN NO: Polypeptide ID or PROTEOME Score ID NO: 1 7513406CD1 g1353859 4.2E-13 [Saccharomyces cerevisiae] Atx2p Lin, S. J. et al., Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which cncodes a manganese-trafficking protein that localizes to Golgi-like vesicles. Mol. Cell. Biol. 16, 6303-6312 (1996) 598972#FLJ1127 6.7E-145 [Homo sapiens] Protein with weak similarity to S. cerevisiae Atx2p, which is a manganese- 4 trafficking protein 249420#T01D3.5 2.8E-52 [Caenorhabditis elegans][Secretory vesicles] Protein with weak similarity to S. cerevisiae Atx2p (a manganese-trafficking protein), has weak similarity to C. elegans T28F3.3 Bateman, A. et al., Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins., Nucleic Acids Res 27, 260-2 (1999). 2 7513407CD1 g1353859 5.7E-14 [Saccharomyces cerevisiae] Atx2p Lin, S. J. et al. (supra) 598972#FLJ1127 2.0E-86 [Homo sapiens] Protein with weak similarity to. S. cerevisiae Atx2p, which is a manganese- 4 trafficking protein 249420#T01D3.5 9.6E-56 [Caenorhabditis elegans][Secretory vesicles] Protein with weak similarity to S. cerevisiae Atx2p 9a manganese-trafficking protein), has weak similarity to C. elegans T28F3.3 Jiang, M. et al., Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans., Proc Natl Acad Sci U S A 98, 218-223 (2001). 3 7512697CD1 g339453 0.0 [Homo sapiens] transferrin precursor Yang, F. et al., Human transferrin: cDNA characterization and chromosomal localization. Proc. Natl. Acad. Sci. U.S.A. 81, 2752-2756 (1984) 339610#TF 0.0 [Homo sapiens][Ligand; Small molecule-binding protein] [Endosome/Endosomal vesicles; Cytoplasmic; Extracellular (excluding cell wall)] Transferrin, an iron-binding protein involved in iron transport, has a role in cell proliferation, may have roles in the immune response and phagocytosis Table 2 Polypeptide SEQ Incyte GenBank ID NO: Probability Annotation ID NO: Polypeptide ID or PROTEOME Score ID NO: Kawabata, H. et al., Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo., J Biol Chem 275, 16618-25 (2000). 591483#Tf 1.0E-242 [Rattus norvegicus][Small molecule-binding protein][Extracellular (excluding cell wall)] Transferrin, an iron-binding protein involved in iron transport, has a role in cell proliferation Stallard, B. J. et al., A transferrinlike (hemiferrin) mRNA is expressed in the germ cells of rat testis., Mol Cell Biol 11, 1448-53 (1991).

Table 3 SEQ Incyte Amino Acid Signature Sequences, Domains and Motifs Analytical Methods ID Polypeptide Residues and Databases NO : ID 1 Peptide : M1-G22 ZIP Zinc transporter : G109-S279HMMERPFAM Cytosolic domains : M1-F4, H60-Q103, H176-H187, L240-R258 TMHMMER Transmembrane domains : I5-A27, L37-V59, L104-G126, L153-M175, L188-S207, V217-V239, G259-G281 Non-cytosolic domains : V28-K36, N127-Q152, K208-E216, H282-H284 Potential Phosphorylation Sites : S83 S209 S249 S261 T133 Potential Sites : N29 N218 MOTIFS i 2 7513407CD1 Signal Peptide : M1-G22 HMMER ZIP Zinc transporter : R74-S236 Cytosolic domains L197-R215 TMHMMER Transmembrane domains : L10-F30, L37-V59, L110-M132, L145-S164, V174-V196, G216-G238 Non-cytosolic domains : Ml-L9, H60-Q109, K165-E173, H239-H241 PROTEIN GUFA TRANSMEMBRANE MEMBRANE INTERGENIC REGION INNER BLAST CONSERVED SIMILARITY MYXOCOCCUS PD004603 : E68-V200 Potential Phosphorylation Sites : S166 S206 S218 Potential Sites : N29 N175 i 3 : Ml-A19 SPSCAN Signal Peptide : Ml-C17 Signal Peptide : MU-ARMER Signal Peptide : M1-P21MER Signal Peptide : M1-D22HMMER Signal Peptide : M1-V25MER Signal Peptide : M1-C28HMMER Signal Peptide : M1-T24HMMER HMAMR Transferrin : V298-S624, V25-P297 Transferrin : V25-E284, V298-K620HMMERPFAM Transferrin : V25-C38, T112-G152, L461-T498 Transferrins : N95-L141, G356-V410, C451-N502, N497-L563, R162-H229 75134Table 3 SEQ Incyte Amino Acid Signature Sequences, Domains and Motifs Analytical Methods ID Polypeptide Residue and Databases NO : ID Transferrin signature PR00422 : C58-A76, A76-Y90, TI 124, L131-L154, E284 PRECURSOR SIGNAL GLYCOPROTEIN IRON TRANSPORT METALBINDING REPEAT SEROTRANSFERRIN SIDEROPHILIN BETA1METAL PD000899 : V25-L154, E434-K620, V298 E463, L158-E284 TRANSFERRINS DM00330 : K296-K620BLASTDOMO TRANSFERRINS DM003301P02787120-349 : V20-N172 TRANSFERRINS DM00330|P02787|351-680 : K23-Y155 TRANSFERRINS DM00330 : P288-L618 TRANSFERRINS DM00330lP19134|350-676 : K23-Y155 E168-L282BLASTDOMO TRANSFERRINS 350-676 : P288-L618 TRANSFERRINS DM00330|P27425|353-688 : K23-L158 TRANSFERRINS DM00330|P274251353-688 : P288-N617 Potential Phosphorylation Sites : S31 S40 S63 S S304 S391 S444 S448 T24 MOTIFS T187 T286 T329 T523 T545 T631 Y515 Y603 Potential Glycosylation Sites : N369 N567 Transferrins signature 1 : Y114-D123, Transferrins signature 2 : Y473-F488 Transferrins signature 3 : Q178-V208, D514-V544 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 4/7513406CB 5360 5098628H2 (EPIMNON05) 1 422566H1 (CARCTXT01) 1 423640H1 (CARCTXT01) 1 7513406CT1 3458893H1 (293TF1T01) 6 7280508H1 (BMARTXE01) 7 2905274H1 (THYMNOT05) 14 2508921H1 (CONUTUT01) 15 2507695H1 (CONUTUT01) 15 5044759H1 (PLACFER01) 15 3391868H1 (LUNGNOT28) 15 5044759F6 (PLACFER01)-15 2508921F6 (CONUTUT01) 15 7191004H1 (BRATDIC01) 15 7277410H1 (BMARTXE01) 18 5764760H1 (PROSBPT02) 37 8194753H1 (PROSUNRO1) 41 71971850V1 71972608V1 7950249H1 (BRABNOE02) 133 7951018H1 (BRABNOE02) 134 71984863V1 8109734H1 (PITUDIR02) 208 8109734J2 (PITUDIR02) 208 71969850V1 71971695V 71970884V 71975288V13211044 71972551V1 71971489V1 90008423J1 90008315H1 90007938J1 72675469V1 6467129H1 (PLACFEB01) 477 72445006D1 '562 71975251V1 594 71971614V1 71986602V1 71974944V1 5640842H1 (UTRSTMR01) 779 71973108V1 71972667V1 FragmentsTable 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/Fragments Incyte ID/ Sequence Length 7l969611Vl 71970688V1 71974752V1 71969863V1 71973584V 72001754V1 71975454V19051355 5044759R6 (PLACFER01) 974 71973111V1 71984824V1 71058952Vl 72050261V1 9468577UI 949859OUl 9468578U1 71057436V1 71072583V1 9481062U1 72666270V111111854 9493772U1 9475972U1 71072616V1 9493767U1 9475967U1 7344833H1 (SYNODIN02) 1671905H1 (BLADNOT05) 1173 1671905F6 (BLADNOT05) 11751532 71247230V1 9475973U1 72052332V1 7761045J1 (THYMNOE02) 1215 9498589U1 9481089U1 7761045H1 (THYMNOE02) 1225 9498561U112362132 3125952H1 (LUNGTUT12) 3125952F6 (LUNGTUT12) 1270 7038373R6 (UTRSTMR02) 1279 6348809H1 (LUNGDIS03) 1326 71112244V1 9475791U1 9493773U1 71070824V1 9481061U1 72444250D1 72665318V1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 60148921D2 1538 2119 6481382H1 (PROSTMC01) 1561 9475966U1 6800092H1 (COLENOR03) 1670 72673986V1 60148921B2 90008235J1 90011785H1 90007703H1 90011777H1 72910538V1 3204062H1 (PENCNOT02) 1836 3204062F6 (PENCNOT02) 1847 90007719J1 90008415H1 8266760H1 (TLYJTXF02) 1860 90007819H1 90008423H1 72443660D1 90008243J1 90007803J1 90008307J1 90011877J1 90007711H1 90008323H1 90008038H1 1385618H1 (CARGDIT02) 1965 7007393H1 (COLNFEC01) 1971 90008407J1 60148929D2 60148929B2 g2028330 60148931B2 60148931D1 60148931D2 60148635D2 60148936B2 60148936D2 60148953D1 60148930B2 60148930D2 3454141H1 (KIDNNOT26) 2358 60148934B1 g4284128 g5445992 3615912H1 (EPIPNOT01) 2430 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/Fragments Incyte ID/ Sequence Length 6467129R6 (PLACFEBO1) 3943862H1 (SCORNOT04) 2525 3944057H1 (SCORNOT04) 2525 71878706V1 3944057F6 (SCORNOT04) 2525 3255492H1 (OVARTUN01) 2530 g2000720 gl277490 4406857H1 (PROSDIT01) 2555 2641087H1 (LUNGTUT08) 2571 2641087F6 (LUNGTUT08) 2571 7220014H1 (SPLNDIC01) 2586 7440525H1 (ADRETUE02) 2588 3125952T6 (LUNGTUT12) 2599 8759178H1 (MYEPUNN01) 2621 g8601040 7370670H1 (ADREFEC01) 2692 60148635B2 60148633B2 1387213H1 (CARGDIT02) 2857 1387104H1 (CARGDIT02) 2857 60148636B2 60148637B2 60148637B1 60148682B1 7644938H1 (UTRSTUE01) 2987 g2179217 g5707000 60148679B 1911014H1 (CONNTUT01) 3043 7697493J1 (KIDPTDE01) 3044 gl237886 g669809 g574251 g900572 7439534H1 (ADRETUE02) 3076 4066325H1 (SEMVNOT05) 3088 4139536H1 (BRSTTMT01) 3201 5681630H1 (BRAENOT02) 3207 4647714H1 (PROSTUT20) 3207 6073905H1 (UTREDIT09) 3220 2433672H1 (BRAVUNT02) 3234 2285289H1 (BRAINON01) 3234 70557881V1 70557989V1 70557816V1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 70558195V1 3234 70558408V1 7038373R6 (UTRSTMR02) 3242 7644938J1 (UTRSTUE01) 3265 2693292H1 (LUNGNOT23) 3276 2665567H1 (ADRENOT08) 3297 929163H1 (BRAINOT04) 3338 70558710V1 4980914H1 (HELATXT04) 3349 6389668H1 (LUNGNON07) 3353 1950293H1 (PITUNOT01) 3370 1644485H1 (HEARFET01) 3374 1644485F6 (HEARFET01) 3374 1969778H1 (UCMCL5T01) 3390 2626705H1 (PROSTUT12) 3406 2628419H1 (PROSTUT12) 3406 g2013240 gl919817 70558367V1 2958203H1 (ADRENOT09) 3538 3497833H1 (PROSTUT13) 3544 70557946V1 70558358V1 2402634H1 (BRAINON01) 3619 70558090V1 7311948J1 (LUNLTUE01) 3680 2369484H1 (ADRENOT07) 3698 2369484F6 (ADRENOT07) 3698 2855535H1 (CONNNOT02) 3709 g2023040 70558079V1 56055944J1 2109137H1 (BRAITUT03) 3719 70558343V1 3752522H1 (BRSTTUT17) 3735 6746579H1 (BRAFNOT02) 3742 6584986H1 (KIDNNOC01) 3750 gl463552 gl779404 4139977H1 (BRSTTMT01) 3783 70558715V1 5691045H1 (BRAUNOT02) 3804 2493566H1 (ADRETUT05) 3826 4551013H1 (HELAUNT01) 3835 70557955V1 3852 5384092H1 (COLNNOT38) 3856 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO : Fragments Incyte ID/ Sequence Length 6134449H1 (BMARTXT02) 4129 70557961V1 g900488 70558261V1 5924941H1 (BRAIFET02) 3941 7761529H1 (THYMNOE02) 3947 4525326H1 (HNT2TXT01) 3955 2047058H1 (THP1T7T01) 3964 6526736H1 (CONFNOT07) 3967 7358466H1 (BRAIFEE05) 3974 894361H1 (BRSTNOT05) 3983 894385H1 (BRSTNOT05) 3985 894361R1 (BRSTNOT05) 3985 3495656H1 (ADRETUT07) 3999 2409294H1 (BSTMNON02) 4002 70558550V1 70557839V1 4513410H1 (EPIMNOT01) g566529 g645789 4729260H1 (GBLADIT01) 4056 4729251H1 (GBLADIT01) 4056 2439738H1 (EOSITXT01) 4083 2115564H1 (BRSTTUT02) 4083 2191269H1 (THYRTUT03) 4329080H1 (KIDNNOT32) 7311948H1 (LUNLTUE01) 4097 4512708H1 (EPIMNOT01) 4102 6517315H1 (THYMDIT01) 4108 6729995H1 (COLITUT02) 4119 3242505H1 (BRAINOT19) 4125 1355009H1 (LUNGNOT09) 4141 1355009F1 (LUNGNOT09) 4141 7318324H2 (BRABDIK02) 4142 7682513H1 (BRABDIK02) 4142 7681669H1 (BRABDIK02) 4142 7686173H1 (BRABDIK02) 4142 039161H1 (HUVENOB01) 4147 5591016H1 (ENDINOT02) 4181 g746905 3393724H1 (LUNGNOT28) 4190 1687080H1 (PROSNOT15) 4198 1647338H1 (PROSTUT09) 4198 1686979H1 (PROSNOT15) 4198 1687080F6 (PROSNOT15) 4198 043461H1 (TBLYNOT01) 4199 Table 4 Polynucleotide SEQ ID NO :/ Incyte ID/ Sequence Length 70558467V1 4201 70557872V1 5682409H1 (BRAENOT02) 4215 1257176H1 (MENITUT03) 4222 1257176F1 (MENITUT03) 4222 6016439H1 (HNT2UNN03) 4236 4704738H1 3327293H1 4903811H2 (TLYMNOT08) 4253 2230829H1 (PROSNOT16) 4275 70558555V1 688809H1 (LUNGTUT02) 4291 1879742H1 (LEUKNOT03) 4291 70558747V1 glO18475 059627H1 (LUNGNOT01) 4305 6631089H1 (LIVRNOT21) 4305 4086944H1 (LIVRNOT06) 73145576V3 73144568D2 g718442 71874831V1 70558433V1 4539421H1 (THYRTMT01) 4318 2275279H1 (PROSNON01) 4350 gl240870 3944371H1 (SCORNOT04) 4365 417371H1 (BRSTNOT01) 4392 417728H1 (BRSTNOT01) 4392 417566H1 (BRSTNOT01) 4392 419811H1 (BRSTNOT01) 4392 413724H1 (BRSTNOT01) 4392 414278H1 (BRSTNOT01) 4392 417077H1 (BRSTNOT01) 4392 416887H1 (BRSTNOT01) 4392 413724R1 (BRSTNOT01) 4394 7697493H2 (KIDPTDE01) 4398 414329H1 (BRSTNOT01) 4401 2646935H1 (CONNNOT02) 4418 4291360F8 (BRABDIR01) 4418 2846558H1 (DRGLNOT01) 4419 4853219H1 (TESTNOT10) 5741806H1 (LUNGNON03) 4423 2640854H1 (LUNGTUT08) 4425 3205960H1 (PENCNOT03) 4432 3469642H1 (BRAIDIT01) 4445 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte Sequence Length 4254043H1 (BSCNNOT03) 4445 4715 3254350H1 (OVARTUN01) 4447 70558176V1 698779H1 (SYNORAT03) 6296203H1 (BRARTUT04) 4484 2815437H1 (OVARNOT10) 4485 71869756V1 4024079H1 (BRAXNOT02) 4489 70558281VI 6550910H1 (BRAFNON02) 4503 6550810H1 (BRAFNON02) 4503 71873390V1 60148932B2 1371517H1 (BSTMNON02) 4510 g2058956 5099587H1 (PROSTUS20) 4514 2183391F6 (SININOT01) 4514 2183391H1 (SININOTO1) 4514 3222358H1 (COLNNON03 1233392H1 (LUNGFET03) 4521 1252114H1 (LUNGFET03) 4521 1233392F1 (LUNGFET03) 4521 71874025V1 71875287V1 1578882H1 (DUODNOT01) 4548 60148923B2 1420740H1 (KIDNNOT09) 4575 60148924B2 g4684262 3806991H1 (CONTTUT01) 4600 3806891H1 (CONTTUT01) 4600 1347869H1 (PROSNOT11) 3468496H1 (BRAIDIT01) 4604 gl063233 4531661H1 (PROSTUT18) 4608 71876695V1 4610 g850366 g851287 71873369V1 3881350H1 (SPLNNOT11) 4615 1456869H1 (COLNFET02) 4616 gl321252 gl312616 60148935B2 g6698282 1893833H1 (THP1TXT04) 4625 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte ID/ Sequence Length gll684037 2997982H1 (OVARTUT07) 4628 1644485T6 (HEARFET01) 70558302V1 7438759H1 (ADRETUE02) 4641 395222R1 (TMLR2DT01) 4648 413724F1 (BRSTNOT01) 4662 71112244V1 72050261V1 71072583V1 71072616V1 72052332V1 71070824V1 71251667V1 71870068V1 71870522V1 70558122V1 60148639B2 71069185V1 2370844T6 (ADRENOT07) 4676 1307686H1 (COLNFET02) 4678 71252088V1 71069238V1 6014897OB1 71071514V1 70557865V1 71072178V1 71070879V1 71873347V1 gl523211 563254H1 (NEUTLPT01) 4719 1687177H1 (PROSTUT10) 4721 gl735618 gl 1394087H1 (THYRNOT03) 4730 71252515V1 71069118V1 2704235H1 (PONSAZT01) 4744 2959396H1 (ADRENOT09) 4748 71252555V1 1687080T6 (PROSNOT15) 4760 g2058862 71251185V1 2701589H1 (OVARTUT10) 4780 71252334V1 glS23212 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 60148631B1 4789 5305 71070874V1 407831H1 (EOSIHET02) 4797 5026 4274795H1 (PROSTMT01) 4798 1879742T6 (LEUKNOT03) 4806 4183672H1 (HEAADIT01) 4820 1732046H1 (BRSTTUT08) 4821 1732046F6 (BRSTTUT08) 4821 1732046T6 (BRSTTUT08) g1137818 72335326V1 g2327877 016702H1 (HUVELPB01) 4832 016160H1 (HUVELPBO1) 4832 018721H1 (HUVELPBO1) 4832 015755H1 (HUVELPB01) 4832 016072H1 (HUVELPB01) 4832 018205H1 (HUVELPBO1) 4832 017260H1 (HUVELPB01) 4832 017196H1 (HUVELPB01) 4832 015290H1 (HUVELPBO1) 4832 015293H1 (HUVELPB01) 4832 015463H1 (HUVELPB01) 4832 016440H1 (HUVELPB01) 4832 018574H1 (HUVELPBO1) 4832 g3400110 glO18752 4588356H1 (MASTTXT01) 71875619V1 g7703609 gl 71070350V1 g2703443 g3214458 g6709266 gl2750200 g4194341 g4373507 g958959 g5768390 g3254761 g3756962 g7154754 g6658873 g2703116 g3693923 FragmentsTable 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length g3924211 5322 g8365916 gl203506 gl289933 g9704507 5640842R6 (UTRSTMR01) 4896 g6660391 gl289792 g6974624 g746952 g6713492 g6656514 g6656516 g6656515 g4900231 gl785037 gl784912 gl784972 g3644747 g7281402 g5594015 g4650710 g7701145 g5594864 g2557440 gl785038 g3884092 g9704110 g4621971 gl463553 4899640H1 (OVARDIT01) 4952 7014965H1 (KIDNNOC01) 4971 gl735619 g3888403 g7044817 g6577048 g4333042 g3961346 g4987844 3843647H1 (DENDNOT01) 4985 g5547054 g6086239 g2882648 g2255481 1677694H1 (STOMFET01) 4999 4206121H1 (BRONNOT02) 4999 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 901088H1 (BRSTTUT03) 901088T2 (BRSTTUT03) 5015 901088T1 (BRSTTUT03) 5015 901088R1 (BRSTTUT03) 5015 g3277900 676328H1 (CRBLNOT01) 5036 2824251H1 (ADRETUT06) 5041 1367924H1 (SCORNON02) 5044 1368141T1 (SCORNON02) 5044 1367924R1 (SCORNON02) 5044 g3740454 g6640747 g5446301 2325919H1 (OVARNOT02) 5087 g6833827 g10034164 g6837758 6517322H1 (THYMDIT01) 5099 g2355312 71263630V1 g1219249 6705103H1 (DRGCNOT02) 5163 g2000719 72062732V1 g5444930 3750936H1 (UTRSNOT18) 5182 g4486607 g2753218 5244 5327 5/7513407CB1/1-872, 5200 5098628H2 (EPIMNON05) 1 422566H1 (CARCTXT01) 1 423640H1 (CARCTXT01) 1 7513407CT1 3458893H1 (293TF1T01) 6 7280508H1 (BMARTXE01) 7 2905274H1 (THYMNOT05) 14 2508921H1 (CONUTUT01) 15 2507695H1 (CONUTUT01) 15 5044759H1 3391868H1 (LUNGNOT28) 15 5044759F6 (PLACFER01) 15 2508921F6 (CONUTUT01) 15 7191004H1 (BRATDIC01) 15 7277410H1 (BMARTXE01) 18 5764760H1 (PROSBPT02) 37 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 8194753H1 (PROSUNR01) 41 841 71971850V1 71972608V1 7950249H1 (BRABNOE02) 133 7951018H1 (BRABNOE02) 71984863V1 8109734J2 (PITUDIR02) 8109734H1 (PITUDIR02) 208 71969850V1283767 71970884V1-321 90011877H1 90011885H1 72675469V1 71986602V1 5584679H1 (FIBAUNT01) 772 72599987D 72444126D 56041554J1 71066522V19101274 71252162V1 71058952VI 9468577U1 9498590U1 71252266V 72050261V1 71984824V1 9468578U1 71057436V1 71072583V1 9481062U1 72666270V1 9493772U1 9475972U1 71072616V1 9493767U1 9475967U1 7344833H1 (SYNODIN02) 1035 1671905H1 (BLADNOT05) 1044 1671905F6 (BLADNOT05) 1046 71247230V1 9475973U1 72052332V1 7761045J1 (THYMNOE02) 1086 9498589U1 9481089U1 7761045H1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/Fragments Incyte ID/ Sequence Length 9498561U1 2003 3125952H1 (LUNGTUT12) 1138 3125952F6 (LUNGTUT12) 1141 7038373R6 (UTRSTMR02) 1144 6348809H1 (LUNGDIS03) 1197 71112244V1 9475791U1 9493773U1 71070824V1 9481061U1 72444250D1 72665318V1 60148921D2 6481382H1 (PROSTMC01) 1432 9475966U1 6800092H1 (COLENOR03) 1541 72673986V1 60148921B2 90008235J1 90011785H1 90007703H1 90011777H116642513 72910538V1 3204062H1 (PENCNOT02) 1707 3204062F6 (PENCNOT02) 1718 l 90007719J1 90008415H1 8266760H1 (TLYJTXF02) 1731 90007819H1 90008423H1 72443660D1 90008243J1 90007803J1 90008307J1 90011877J1 1814 90007711H1 90008323H1 90008038H1 1385618H1 (CARGDIT02) 1836 7007393H1 (COLNFEC01) 1842 90008407J1 60148929D2 60148929B2 g2028330 60148931B2 60148931D1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 60148931D2 1996 2519 60148635D2 60148936B2 60148936D2 60148953DI 60148930B2 60148930D2 3454141H1 (KIDNNOT26) 2229 2448 60148934B1 g4284128 g5445992 3615912H1 (EPIPNOT01) 2301 6467129R6 (PLACFEB01) 2382 3943862H1 (SCORNOT04) 2396 3944057H1 (SCORNOT04) 2396 71878706V1 3944057F6 (SCORNOT04) 2396 3255492H1 (OVARTUN01) 2401 g2000720 gl277490 4406857H1 (PROSDIT0l) 2426 2641087H1 (LUNGTUT08) 2442 2641087F6 (LUNGTUT08) 2442 7220014H1 (SPLNDIC01) 2457 7440525H1 (ADRETUE02) 2459 3125952T6 (LUNGTUT12)'2470 8759178H1 (MYEPUNN01) 2492 g8601040 7370670H1 (ADREFEC01) 2563 60148635B2 60148633B2 1387213H1 (CARGDIT02) 2728 1387104H1 (CARGDIT02) 60148636B2 60148637B2 60148637B1 60148682B1 7644938H1 (UTRSTUE01) 2858 g2179217 g5707000 60148679B1 1911014H1 (CONNTUT01) 2914 7697493J1 (KIDPTDE01) 2915 gl237886 g669809 g57425129323243 .

Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/Fragments Incyte ID/ Sequence Length g900572 3486 7439534H1 (ADRETUE02) 2947 4066325H1 (SEMVNOT05) 2959 4139536H1 (BRSTTMT01) 3072 5681630H1 (BRAENOT02) 3078 4647714H1 (PROSTUT20) 3078 6073905H1 (UTREDIT09) 3091 2433672H1 (BRAVUNT02) 3105 2285289H1 (BRAINON01) 3105 70557881V1 70557989Vl 70557816V1 70558195V1 70558408V1 7038373R6 (UTRSTMR02) 3113 7644938J1 (UTRSTUE01) 3136 2693292H1 (LUNGNOT23) 3147 2665567H1 (ADRENOT08) 3168 929163H1 (BRAINOT04) 3209 70558710V1 4980914H1 (HELATXT04) 3220 6389668H1 (LUNGNON07) 3224 1950293H1 (PITUNOT01) 3241 1644485H1 (HEARFET01) 3245 1644485F6 (HEARFET01) 3245 1969778H1 (UCMCL5T01) 3261 2626705H1 (PROSTUT12) 3277 2628419H1 (PROSTUT12) 3277 g2013240 gl919817 70558367V1 2958203H1 (ADRENOT09) 3409 3497833H1 (PROSTUT13) 3415 70557946V1 70558358V1 2402634H1 (BRAINON01) 70558090V1 7311948J1 (LUNLTUE01) 3551 2369484H1 (ADRENOT07) 3569 2369484F6 (ADRENOT07) 3569 2855535H1 (CONNNOT02) 3580 g2023040 70558079V1 56055944J1 2109137H1 (BRAITUT03) 3590 70558343V1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/Fragments Incyte ID/ Sequence Length 3752522H1 (BRSTTUT17) 3919 6746579H1 (BRAFNOT02) 3613 6584986H1 (KIDNNOC01) 3621 gl463552 gl779404 4139977H1 (BRSTTMT01) 3654 70558715V1 3669 5691045H1 (BRAUNOT02) 3675 2493566H1 (ADRETUT05) 3697 4551013H1 (HELAUNT01) 3706 70557955V1 5384092H1 (COLNNOT38) 3727 6134449H1 (BMARTXT02) 3731 70557961V1 g900488 70558261V1 5924941H1 (BRAIFET02) 3812 7761529H1 (THYMNOE02) 3818 4525326H1 (HNT2TXT01) 3826 2047058H1 (THP1T7T01) 3835 6526736H1 (CONFNOT07) 3838 7358466H1 (BRAIFEE05) 3845 894361H1 (BRSTNOT05) 3854 894385H1 (BRSTNOT05) 3856 894361R1 (BRSTNOT05) 3856 3495656H1 (ADRETUT07) 2409294H1 (BSTMNON02) 3873 70558550V1 70557839V1 4513410H1 (EPIMNOT01) 3910 g566529 g645789 4729260H1 (GBLADIT01) 3927 4729251H1 (GBLADIT01) 3927 2439738H1 (EOSITXT01) 3954 2115564H1 (BRSTTUT02) 3954 2191269H1 (THYRTUT03) 3960 4329080H1 (KIDNNOT32) 3964 7311948H1 (LUNLTUE01) 3968 4512708H1 (EPIMNOT01) 3973 6517315H1 (THYMDIT01) 3979 6729995H1 (COLITUT02) 3990 3242505H1 (BRAINOT19) 3996 1355009H1 (LUNGNOT09) 4012 1355009F1 (LUNGNOT09) 4012 7318324H2 (BRABDIK02) 4013 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 7682513H1 (BRABDIK02) 4013 4585 7681669H1 (BRABDIK02) 4013 7686173H1 (BRABDIK02) 4013 039161H1 (HUVENOB01) 4018 5591016H1 (ENDINOT02) 4052 g746905 3393724H1 (LUNGNOT28) 4061 1687080H1 (PROSNOT15) 4069 1647338H1 (PROSTUT09) 4069 1686979H1 (PROSNOT15) 4069 1687080F6 (PROSNOT15) 4069 043461H1 (TBLYNOT01) 4070 70558467V1 70557872V1 5682409H1 (BRAENOT02) 4086 1257176H1 (MENITUT03) 4093 1257176F1 (MENITUT03) 4093 6016439H1 (HNT2UNN03) 4107 4704738H1 (SMCRTXT01) 4109 3327293H1 (HEAONOT04) 4115 4903811H2 2230829H1 (PROSNOT16) 4146 70558555V1 688809H1 (LUNGTUT02) 4162 1879742H1 (LEUKNOT03) 4162 70558747V1 glO18475 059627H1 (LUNGNOT01) 4176 6631089H1 (LIVRNOT21) 4176 4086944H1 (LIVRNOT06) 4181 73145576V3 73144568D2 g718442 71874831V1 70558433V1 4539421H1 (THYRTMT01) 4189 2275279H1 (PROSNON01) 4221 gl240870 3944371H1 (SCORNOT04) 4236 417371H1 (BRSTNOT01) 4263 417728H1 (BRSTNOT01) 4263 417566H1 (BRSTNOT01) 4263 419811H1 (BRSTNOT01) 4263 413724H1 (BRSTNOT01) 4263 414278H1 (BRSTNOT01) 4263 417077H1 (BRSTNOT01) 4263 FragmentsTable 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 416887H1 (BRSTNOT01) 4263 4500 413724R1 (BRSTNOT01) 4265 7697493H2 (KIDPTDE01) 4269 414329H1 (BRSTNOT01) 4272 2646935H1 (CONNNOT02) 4289 4291360F8 (BRABDIR01) 4289 2846558H1 (DRGLNOT01) 4290 4853219H1 (TESTNOT10) 4294 5741806H1 (LUNGNON03) 4294 2640854H1 (LUNGTUT08) 4296 3205960H1 (PENCNOT03) 4303 3469642H1 (BRAIDIT01) 4316 4254043H1 (BSCNNOT03) 4316 3254350H1 (OVARTUN01) 4318 70558176V1 698779H1 (SYNORAT03) 4343 6296203H1 (BRARTUT04) 4355 2815437H1 (OVARNOT10) 4356 71869756V1 4024079H1 (BRAXNOT02) 4360 70558281V1 6550910H1 (BRAFNON02) 4374 6550810H1 (BRAFNON02) 4374 71873390V1 60148932B2 1371517H1 (BSTMNON02) 4381 g2058956 5099587H1 (PROSTUS20) 4385 2183391F6 (SININOT01) 4385 2183391H1 (SININOT01) 4385 3222358H1 (COLNNON03) 4386 1233392H1 (LUNGFET03) 4392 1252114H1 (LUNGFET03) 4392 1233392F1 (LUNGFET03) 4392 71874025V1 4394 71875287V1 1578882H1 (DUODNOT01) 4419 60148923B2 1420740H1 (KIDNNOT09) 4446 60148924B2 g4684262 3806991H1 (CONTTUT01) 4471 3806891H1 (CONTTUT01) 4471 1347869H1 (PROSNOT11) 4472 3468496H1 (BRAIDIT01) 4475 g1063233 Table 4 Polynucleotide Sequence Fragments 5'Position 3'Position SEQ Incyte ID/ Sequence Length 4531661H1 (PROSTUT18) 4479 4750 71876695V1 g850366 g851287 71873369V1 3881350H1 (SPLNNOT11) 1456869H1 (COLNFET02) 4487 gl321252 4488 g1312616 60148935B2 g6698282 1893833H1 (THP1TXT04) 4496 g11684037 2997982H1 (OVARTUT07) 4499 1644485T6 (HEARFET01) 4499 70558302V1 7438759H1 (ADRETUE02) 4512 395222R1 (TMLR2DT01) 4519 413724F1 (BRSTNOT01) 4533 71252266V1 71112244V1 72050261V1 71072583V1 71072616V1 72052332V1 71070824V1 71251667V1 71870068V1 71870522V1 70558122V1 60148639B2 71069185V1 2370844T6 (ADRENOT07) 4547 1307686H1 (COLNFET02) 4549 71252088V1 71069238V1 60148970B1 71071514V1 70557865V1 71072178V1 71070879V1 71873347V1 4584 gl523211 563254H1 (NEUTLPT01) 4590 1687177H1 (PROSTUT10) 4592 1735618 | Table 4 Polynucleotide SEQ ID NO :/Fragments Incyte ID/ Sequence Length "gl128759"4598 1394087H1 (THYRNOT03) 4601 71252515V1 71069118V1 2704235H1 (PONSAZT01) 4615 2959396H1 (ADRENOT09) 4619 71252555V1 1687080T6 (PROSNOT15) 4631 g2058862 71251185V1 4642 2701589H1 (OVARTUT10) 4651 71252334V1 gl523212 60148631B1 71070874V1 407831H1 (EOSIHET02) 4668 4274795H1 (PROSTMT01) 4669 1879742T6 (LEUKNOT03) 4677 4183672H1 (HEAADIT01) 4691 1732046H1 (BRSTTUT08) 4692 1732046F6 (BRSTTUT08) 4692 1732046T6 (BRSTTUT08) 4692 gl 72335326V1 g2327877 016702H1 (HUVELPB01) 4703 016160H1 (HUVELPB01) 4703 018721H1 (HUVELPBO1) 4703 015755H1 (HUVELPB01) 4703 016072H1 (HUVELPB01) 4703 018205H1 (HUVELPB01) 4703 017260H1 (HUVELPB01) 4703 015290H1 (HUVELPB01) 4703 017196H1 (HUVELPB01) 4703 015293H1 (HUVELPB01) 4703 015463H1 (HUVELPB01) 4703 016440H1 (HUVELPBO1) 4703 018574H1 (HUVELPB01) 4703 g3400110 glO18752 4588356H1 (MASTTXT01) 4715 71875619V1 g7703609 gl 71070350V1 g2703443 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length g3214458 5183 g6709266 gl2750200 g4194341 g4373507 g958959 g5768390 g3254761 g3756962 g7154754 g6658873 g2703116 g3693923 g3924211 g8365916 gl203506 gl289933 g9704507 5640842R6 (UTRSTMR01) 4767 g6660391 gl289792 g6974624 g746952 g6713492 g6656514 g6656516 g6656515 g4900231 gl785037 gl784912 gl784972 g3644747 g7281402 g5594015 g4650710 g7701145 g5594864 g2557440 gl785038 g9704110 g3884092 g4621971 gl463553 4899640H1 (OVARDIT01) 4823 7014965H1 (KIDNNOC01) 4842 gl735619 Table 4 Polynucleotide SEQ Incyte ID/ Sequence Length g3888403 5189 g7044817 g6577048 g4333042 g3961346 g4987844 3843647H1 (DENDNOT01) 4856 _ 4862 g2255481 1677694H1 (STOMFETO1) 4870 4206121H1 (BRONNOT02) 4870 901088H1 (BRSTTUT03) 4886 901088T2 (BRSTTUT03) 4886 901088T1 (BRSTTUT03) 4886 901088R1 (BRSTTUT03) 4886 g3277900 676328H1 (CRBLNOT01) 4907 2824251H1 (ADRETUT06) 4912 1367924H1 (SCORNON02) 4915 1368141T1 (SCORNON02) 4915 1367924R1 (SCORNON02) 4915 g3740454 g6640747 g5446301 2325919H1 (OVARNOT02) 4958 g6833827 gl0034164 g6837758 6517322H1 (THYMDIT01) 4970 g2355312 71263630V1 gl21924950315200 6705103H1 (DRGCNOT02) 5034 g2000719 72062732V1 g5444930 3750936H1 (UTRSNOT18) 5053 g4486607 g2753218 5115 5198 6/7512697CB (BRATNOT03) 98 2487 2487 4894228H1 153 088671H1 (LIVRNOT01) 1 4661925H1 (LIVRTUT09) 1 Table 4 Polynucleotide SEQ ID NO :/ Incyte ID/ Sequence Length 4987182H1 (LIVRTUT10) 1 165 3685651H1 (HEAANOT01) 1 56022619J1 56022511J1 3684318H1 (HEAANOT01) 1 56022511H1 3268669H1 (BRAINOT20) 1 1992081H1 (CORPNOT02) 1 4990250H1 (LIVRTUT11) 1 4992737H1 (LIVRTUT11) 2512694H1 (LIVRTUT04) 1 1481626H1 (CORPNOT02) 1 5808553H2 (BRAHNOT02) 1 4992158H1 (LIVRTUT11) 1 2512758H1 (LIVRTUT04) 1 2782385H1 (BRSTNOT13) 1 3245068H1 (BRAINOT19) 1 2485255H1 (LIVRTUT04) 1 4984780H1 (LIVRTUT10) 1 4988942H1 (LIVRTUT10) 1 4988758H1 (LIVRTUT10) 1 1482657H1 (CORPNOT02) 1 4227071H1 (BRAMDIT01) 1 4989714H1 (LIVRTUT11) 1 4984592H1 (LIVRTUT10) 1 3350434H1 (BRAITUT24) 1 2513108H1 (LIVRTUT04) 1 4994669H1 (LIVRTUT11) 1 4984691H1 (LIVRTUT10) 1 4797623H1 (LIVRTUT09) 1 4794835H1 (LIVRTUT09) 1 4989911H1 (LIVRTUT 4028241H1 (BRAINOT23) 1 4984970H1 (LIVRTUT10) 1 5399986H1 (LIVRTUT13) 1 4984573H1 (LIVRTUT10) 1 3347186H1 (BRAITUT24) 1 383518H1 (HYPONOB01) 1 4794269H1 (LIVRTUT09) 1 1484343H1 (CORPNOT02) 1 4896307H1 (LIVRTUT12) 1 3044382H1 (HEAANOT01) 1 4796272H1 (LIVRTUT09) 1 2517195H2 (LIVRTUT04) 4893666H1 (LIVRTUT12) 1 4895882H1 (LIVRTUT12) 1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 1909854H1 (CONNTUT01) 262 4795418H1 (LIVRTUT09) 1 4793988H1 (LIVRTUT09) 1 4177251H1 (BRAINOT22) 1 4985714H1 (LIVRTUT10) 1 4028005H1 (BRAINOT23) 1 1483470H1 (CORPNOT02) 1 4094669H1 (BSCNSZT01) 1 1485914H1 (CORPNOT02) 1 4796235H1 (LIVRTUT09) 1 4892776H1 (LIVRTUT12) 1 4994581H1 (LIVRTUT11) 1 4994326H1 (LIVRTUT11) 4994405H1 (LIVRTUT11) 1 1481535H1 (CORPNOT02) 1 4797516H1 (LIVRTUT09) 1 3393363H1 (LUNGNOT28) 1 4892855H2 (LIVRTUT12) 1 1484574H1 (CORPNOT02) 1 1484575H1 (CORPNOT02) 1 5842311H1 (BRAENOT04) 1 4794505H1 (LIVRTUT09) 1 4991206H1 (LIVRTUT11) 1 3788757H1 (BRAHNOT05) 1 4990739H1 (LIVRTUT11) 1 3760177H1 (BRAHDIT03) 1 2816717H1 (BRSTNOT14) 1 4993514H1 (LIVRTUT11) 1 2708505H1 (PONSAZT01) 1 2515218H1 (LIVRTUT04) 1 2516703H1 (LIVRTUT04) 1 2512572H1 (LIVRTUT04) 1 gl976931 6898839H1 (LIVRTMR01) 1 56044777H1 6896849H1 (LIVRTMR01) 1 56077554H1 g68944. 6894473H1 (BRAITDR03) 1 6900037H1 (LIVRTMR01) 1 7177971H1 (BRAXDIC01) 1 6770649H1 (BRAUNOR01) 1 7189712H2 (BRATDIC01) 1 56073527H1 7259724H1 (BRAWNOC01) 1 6450062H1 (BRAINOC01) 1 Table 4 Polynucleotide SEQ ID NO :/Fragments Incyte ID/ Sequence Length 7193835H2 (BRATDICOI) 6897090H1 (LIVRTMR01) 1 7142646H1 (LIVRDIT07) 1 1481535F6 (CORPNOT02) 6897130H1 (LIVRTMR01) 1 6450729H1 (BRAINOC01) 1 7141867H1 (LIVRDIT07) 1 7096839H1 (BRACDIR02) 1 7185352H1 (BONRFEC01) 1 6149114H1 (BRANDIT03) 1 7267041H2 (NOSEDIC01) 1 5775051H1 (BRAINOT20) 1 7512697CT1 4985275H1 (LIVRTUT10) 2 gl995915 4987328H1 (LIVRTUT10) 2 4795478H1 (LIVRTUT09) 2 4794082H1 (LIVRTUT09) 2 4991651H1 (LIVRTUT11) 2 3349643H1 (BRAITUT24) 2 4795812H1 (LIVRTUT09) 2 5975185H1 (BRAZNOT01) 2 6979607H1 (BRAHTDR04) 2 5966558H1 (BRATNOT05) 2 5975275H1 (BRAZNOT01) 2 4990461H1 (LIVRTUT11) 3 7142987H1 (LIVRDIT07) 4 4990182H1 (LIVRTUT11) 4 1989462H1 (CORPNOT02) 4 4893331H1 (LIVRTUT12) 4 4990456H1 (LIVRTUT 2516902H1 (LIVRTUT04) 4 4988750H1 (LIVRTUT10) 5 6451018H1 (BRAINOC01) 7 4794294H1 (LIVRTUT09) 8 087746H1 (LIVRNOT01) 9 089029H1 (LIVRNOT01) 9 3467033H1 (BRAIDIT01) 11 7142746H1 (LIVRDIT07) 22 4794521H1 (LIVRTUT09) 24 4984309H1 (LIVRTUT10) 30 g678975 g663416 5657366H1 (BSCNNOT03) 41 4897605H1 (LIVRTUT12) 48 70305659D1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 5372278H1 (BRAINOT22) 343 4992274H1 (LIVRTUT11) 4342713H1 (BRAUNOT02) 82 4795243H1 (LIVRTUT09) 95 5424238H1 (PROSTMT07) 101 4990178H1 (LIVRTUT11) 108 4989526H1 (LIVRTUT11) 3346950H1 (BRAITUT24) 116 4989155H1 (LIVRTUT10) 153 4986451H1 (LIVRTUT10) 154 g680514 167916H1 (LIVRNOT01) 156 1479323H1 (CORPNOT02) 161 4178646H1 (BRAINOT22) 2964826H1 (SCORNOT04) 192 2965815H1 (SCORNOT04) 192 4993494H1 (LIVRTUT11) 203 4991916H1 (LIVRTUT11) 211 1438181H1 (PANCNOT08) 213 4989681H1 (LIVRTUT11) 4989879H1 (LIVRTUT11) 214 3471041H1 (BRAIDIT01) 221 70306691D1 167870H1 (LIVRNOT01) 243 4985789H1 (LIVRTUT10) 267 4894554H1 (LIVRTUT12) 268 4016402H1 (BRAXNOT01) 269 5524490H1 (LIVRDIR01) 269 5524590H1 (LIVRDIR01) 269 5860492H1 (BRAYDIT01) 288 139731H1 (LIVRNOT01) 314 3760251H1 (BRAHDIT03) 331 4987844H1 (LIVRTUT10) 340 g663312 3783604H1 (BRAHDIT04) 386 4992361H1 (LIVRTUT11) 4985889H1 (LIVRTUT10) 401 4985582H1 (LIVRTUT10) 424 4892513H2 (LIVRTUT12) 424 025652H1 (SPLNFET01) 424 g678923 6900125H1 (LIVRTMR01) 424 4896903H1 (LIVRTUT12) 424 4798120H1 (LIVRTUT09) 424 5351145H1 (BRAIDIT05) 424 4993864H1 (LIVRTUT11) Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 4775431H1 (BRAQNOT01) 424 4991784H1 (LIVRTUT11) 4797695H1 (LIVRTUT09) 424 3953263H1 6861754F6 (BRAGNON02) 424 6861754F8 (BRAGNON02) 424 90001875H1 gl301549 4793726H1 (LIVRTUT09) 424 70308391D1 4161906H1 (BRSTNOT32) 424 4895978H1 (LIVRTUT12) 424 027781H1 (SPLNFET01) 424 4988346H1 (LIVRTUT10) 424 4418272H1 (LIVRDIT02) 424 5964825H1 (BRATNOT05) 424 5399702H1 (LIVRTUT13) 424 4892584H2 (LIVRTUT12) 424 7052422H1 (BRACNOK02) 424 5601658911 4230490H1 (BRAMDIT01) 424 gl970266 271694H1 (LIVRNOT02) 424 4896362H1 (LIVRTUT12) 424 73245215DI 4895691H1 (LIVRTUT12) 424 6772378F6 (BRAUNOR01) 4985466H1 (LIVRTUT10) 424 70308689D1 4989014H1 (LIVRTUT10) 424 5970837H1 (BRAZNOT01) 424 6733367H1 (LIVRTUT13) 424 5997426H1 (BRAZDIT04) 424 4892940H1 (LIVRTUT12) 424 1840893H1 (COLNNOT07) 424 6733414H1 (LIVRTUT13) 424 73246633D 6736038H1 (LIVRTUT13) 424 7512697CT1 7252122H1 (PROSTMY01) 424 4991756H1 (LIVRTUT11) 3093681H1 (BRSTNOT19) 424 6890763H1 (BRAITDR03) 424 56047235H1 6899846H1 (LIVRTMR01) 424 70306231D Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 5400279H1 (LIVRTUT13) 424 4794220H1 (LIVRTUT09) 55089649J1 (PROTDNV03) 424 4897029H1 (LIVRTUT12) 424 5689257H1 (BRAUNOT02) 424 70307718D 55089680J1 (PROTDNV03) 424 6733503H1 (LIVRTUT13) 424 4990110H1 1484431H1 (CORPNOT02) 424 70308405D1 3944996H1 (SCORNOT04) 424 6124294H1 (BRAHNON05) 424 4794941H1 (LIVRTUT09) 424 4986382H1 (LIVRTUT10) 424 3955420H1 (PONSAZT01) 424 5997317H1 (BRAZDIT04) 424 70308340D1 58061824J1 4992834H1 (LIVRTUT11) 424 7027659H1 (LIVRNOT21) 424 7025579H1 (LIVRDIT06) 424 70306756D1 166252H1 (LIVRNOT01) 424 4837251H1 (BRAWNOT01) 424 667948H1 (SCORNOT01) 424 4987449F6 (LIVRTUT10) 424 4795014H1 (LIVRTUT09) 424 6509586H1 (BRAHNOT02) 424 1907936H1 (CONNTUT01) 424 6443884H1 (BRAENOT02) 424 70306965D1 g654989 4837283H1 (BRAWNOT01) 424 4420478H1 (LIVRDIT02) 424 4798157H1 (LIVRTUT09) 424 g680784 gl484798 73246633V1 6896064H1 (LIVRTMR01) 424 4989185H1 (LIVRTUT10) 424 8598971H2 (LIVRFEF03) 424 7028432H1 (LIVRNOT21) 424 6892231J1 (BRAITDR03) 424 70305921D1 3664421H1 (PANCNOT16) 424 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte IDl Sequence Length gl970644 484 4895944H1 (LIVRTUT12) 424 1824640H1 (LSUBNOT03) 424 3469134H1 (BRAIDIT01) 424 4897203H1 (LIVRTUT12) 424 8684539H1 (BRAIUNF01) 424 088616H1 (LIVRNOT01) 424 70305742D 70306890D 4990651H1 90001875J1 4985406H1 (LIVRTUT10) 424 4286603H1 (LIVRDIR01) 424 gl754209 6890763J1 (BRAITDR03) 424 4797590H1 (LIVRTUT09) 424 5035934H1 (LIVRTUT13) 424 3944406H1 (SCORNOT04) 424 6506838H1 (BRAHNOT02) 424 1481993H1 (CORPNOT02) 424 5397848H1 (LIVRTUT13) 424 g985042 4338872H1 (BRAUNOT02) 424 4897579H1 (LIVRTUT12) 424 1486062H1 (CORPNOT02) 424 7023242H1 (LIVRDIT06) 424 6620013H1 (BRAUDIR01) 424 4988472H1 (LIVRTUT10) 424 70306454D 4893554H1 (LIVRTUT12) 424 7025961H1 (LIVRDIT06) 424 g689855 4987343H1 (LIVRTUT10) 424 55089606J1 (PROTDNV03) 424 6896077H1 (LIVRTMR01) 424 4993795H1 (LIVRTUT11) 424 530511H1 (BRAINOT03) 424 4698918H1 (BRALNOT01) 424 6836243H1 (BRSTNON02) 424 4892727H1 (LIVRTUT12) 424 7389029H2 (LIVRFEE02) 424 6897313H1 (LIVRTMR01) 424 5204551H1 (BRAFNOT02) 424 4892754H1 (LIVRTUT12) 424 1412846H1 (BRAINOT12) 424 6769066J1 (BRAUNOR01) 424 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 4284113H1 484 4993563H1 (LIVRTUT11) 424 2572094H1 (HIPOAZT01) 425 4893486H1 (LIVRTUT12) 70305993D 2410744H1 (BSTMNON02) 427 4984357H1 (LIVRTUT10) 427 4897226H1 (LIVRTUT12) 427 4985527H1 (LIVRTUT10) 427 3758794H1 (BRAHDIT03) 428 55089649H1 (PROTDNV03) 429 6731641H1 (LIVRTUT13) 429 4314214H1 (BRAFNOT01) 429 4796329H1 (LIVRTUT09) 430 1907835H1 (CONNTUT01) 433 2761563H1 (BRAINOS12) g690197 4895344H1 (LIVRTUT12) 452 4988720H1 (LIVRTUT10) 452 4992322H1 (LIVRTUT11) 530 5408855H1 (BRAMNOT01) 530 4987377H1 (LIVRTUT10) 530 4985655H1 (LIVRTUT10) 530 4990440H1 (LIVRTUT 4893402H1 (LIVRTUT12) 530 70306669D1 70307284D1 g691153 g680831 6444080H1 (BRAENOT02) 530 8598825H2 (LIVRFEF03) 530 4760646H1 (BRAMNOT01) 537 4988649H1 (LIVRTUT10) 538 5519616H1 (LIVRDIR01) 538 4793535H1 (LIVRTUT09) 538 4286254H1 (LIVRDIROI) g653027 4993230H1 (LIVRTUT11) 544 027508H1 (SPLNFET01) 547 6866822H1 (BRAGNON02) 547 70307646D1 6764210J1 (BRAUNOR01) 551 087795H1 (LIVRNOT01) 553 166450H1 (LIVRNOT01) 553 4793661H1 (LIVRTUT09) 554 531614H1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte ID/ Sequence Length 4423039H1 (BRAPDIT01) 555 811 531614R6 (BRAINOT03) 555 4986552H1 (LIVRTUT10) 557 168962H1 (LIVRNOT01) 559 3347996H1 (BRAITUT24) 559 139157H1 (LIVRNOT01) 559 6764942H1 (BRAUNOR01) 559 4284911H1 (LIVRDIR01) 560 2706088H1 (PONSAZT01) 562 4990590H1 (LIVRTUT 4284512H1 (LIVRDIR01) 566 166792H1 (LIVRNOT01) 567 70307753D 4985561H1 (LIVRTUT10) 4177688H1 (BRAINOT22) 576 6507403H1 (BRAHNOT02) 576 g685677 280144H1 (LIVRNOT02) 582 7141559H1 (LIVRDIT07) 583 1460576H1 (COLNFET02) 588 4986357H1 (LIVRTUT10) 589 6765778H1 (BRAUNOR01) 589 3954381H1 (PONSAZT01) 590 2965624H1 (SCORNOT04) 590 1991716H1 (CORPNOT02) 6772436H1 (BRAUNOR01) 594 4895579H1 (LIVRTUT12) 598 4225595H1 (BRAMDIT01) 599 6896649H1 (LIVRTMR01) 602 4987986H1 (LIVRTUT10) 603 6893220J1 (BRAITDR03) 608 088732H1 (LIVRNOT01) 613 5451059H1 (BSCNDIT02) 617 g664277 4990190H1 6893024J1 (BRAITDR03) 621 3687988H1 (HEAANOT01) 622 4991129H1 (LIVRTUT11) 90001875H1 4984877H1 (LIVRTUT10) 635 6896427H1 (LIVRTMR01) 635 73246633D1 7023422H1 (LIVRDIT06) 639 8684285H1 (BRAIUNF01) 644 5515462H1 (LIVRDIR01) 645 55089680H1 (PROTDNV03) 646 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 55089649H1 (PROTDNV03) 1310 2966284H1 (SCORNOT04) 647 7072439H1 (BRAUTDR02) 649 70306097D1 70305426D 70305075D1 8683251H1 (BRAIUNF01) 655 7091870H1 (BRAUTDR03) 655 70306070D1 g680832 167403H1 (LIVRNOT01) 658 6883473J1 (BRAHTDR03) 658 2708594H1 (PONSAZT01) 659 55087934H1 (PROTDNV04) 660 4895142H1 (LIVRTUT12) 666 6509002H1 (BRAHNOT02) 666 73246633V1 g679837 4797479H1 (LIVRTUT09) 668 3130793H1 (PONSAZT01) 673 4992159H1 (LIVRTUT 4793424H1 (LIVRTUT09) 673 56047235H1 4985310H1 (LIVRTUT10) 675 4986670H1 (LIVRTUT10) 676 6898638H1 663953H1 (SCORNOT01) 686 1907408H1 (CONNTUT01) 687 6899523H1 (LIVRTMR01) 689 1482448H1 (CORPNOT02) 695 55089649J1 (PROTDNV03) 696 55089606J1 (PROTDNV03) 704 55089680J1 (PROTDNV03) 704 4059105H1 (BRAINOT21) 707 4662053H1 (LIVRTUT09) 707 5524977H1 (LIVRDIR01) 709 4991202H1 2514002H1 (LIVRTUT04) 711 4987987H1 (LIVRTUT10) 714 4689407H1 (LIVRTUT11) 4760360H1 (BRAMNOT01) 719 086971H1 (LIVRNOT01) 725 4987927H1 (LIVRTUT10) 725 7023911H1 (LIVRDIT06) 725 70306965D 7252122H1 (PROSTMY01) 733 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Fragments Incyte ID/ Sequence Length 5355870H1 (BRAIDIT05) 995 gl982459 g2035539 g679809 4985350H1 (LIVRTUT10) 748 4084815H1 (LIVRNOT06) 748 2516431H2 (LIVRTUT04) 749 4084884H1 (LIVRNOT06) 749 4895790H1 (LIVRTUT12) 751 4994310H1 (LIVRTUT11) 751 70308391D1 4989828H1 (LIVRTUT11) 4993796H1 (LIVRTUT11) 762 4288220H1 (LIVRDIR01) 764 4991052H1 (LIVRTUT11) 2967636H1 (SCORNOT04) 764 4893030H2 (LIVRTUT12) 765 5406505H1 (BRAMNOT01) 769 4892661H1 (LIVRTUT12) 769 2537527H1 (BRAINOT18) 771 4987449H1 (LIVRTUT10) 771 4987449F6 (LIVRTUT10) 771 3349843H1 (BRAITUT24) 772 4793416H1 (LIVRTUT09) 773 6861754F8 (BRAGNON02) 773 6861754F6 (BRAGNON02) 773 4793941H1 (LIVRTUT09) 774 5104964H1 (PROSTUS19) 775 5105172H1 (PROSTUS19) 775 4987725H1 (LIVRTUT10) 775 4987825H1 (LIVRTUT10) 775 70306231D 4986690H1 (LIVRTUT10) 778 4183244H1 (LIVRDIT02) 778 5997317H1 (BRAZDIT04) 779 5997426H1 (BRAZDIT04) 779 4260119H1 (BSCNDIT02) 784 56016589J1 3471006H1 (BRAIDIT01) 787 4990839H1 (LIVRTUT11) 787 4986579H1 (LIVRTUT10) 789 4059832H1 (BRAINOT21) 794 4989759H1 (LIVRTUT11) 4794705H1 (LIVRTUT09) 805 6443884H1 (BRAENOT02) 805 086321H1 (LIVRNOT01) 813 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 6736038H1 (LIVRTUT13) 820 90001875Jl 4699852H1 (BRALNOT01) 822 087902H1 (LIVRNOT01) 823 4988992H1 (LIVRTUT10) 826 6896064H1 (LIVRTMR01) 827 g673776 2293567H1 (BRAINON01) 837 4796621H1 (LIVRTUT09) 837 70305522D 70305628D1 6772378F6 (BRAUNOR01) 845 g691300 4987973H1 (LIVRTUT10) 858 4796870H1 (LIVRTUT09) 858 4896538H1 (LIVRTUT12) 858 70305146D1 4798261H1 (LIVRTUT09) 865 g689707 g663583 gl969783 4992286H1 (LIVRTUT11) 2967829H1 (SCORNOT04) 873 167785H1 (LIVRNOT01) 874 087673H1 (LIVRNOT01) 875 4986173H1 (LIVRTUT10) 875 4984613H1 (LIVRTUT10) 875 4989529H1 (LIVRTUT11) 168085H1 (LIVRNOT01) 875 166712H1 138047H1 (LIVRNOT01) 877 6900125H1 (LIVRTMR01) 877 4419180H1 (LIVRDIT02) 878 4418261H1 (LIVRDIT02) 878 g678923 168664H1 (LIVRNOT01) 883 3346745H1 (BRAITUT24) 886 1486515H1 (CORPNOT02) 886 1486510H1 (CORPNOT02) 886 4985818H1 (LIVRTUT10) 889 4894072H1 (LIVRTUT12) 891 139144H1 (LIVRNOT01) 891 5966813H1 (BRAZNOT01) 892 2708630H1 (PONSAZT01) 892 70305921D1 5355771H1 (BRAIDIT05) 896 Table 4 Polynucleotide Selected Sequence SEQ Incyte ID/ Sequence Length 5691167H1 (BRAUNOT02) 905 4793419H1 (LIVRTUT09) 905 g650039 4793624H1 (LIVRTUT09) 911 7023242H1 (LIVRDIT06) 915 4989242H1 (LIVRTUT10) 916 138289H1 (LIVRNOT01) 917 70305742D1 70308689D1 5386405H1 (BRAINOT19) 919 5386345H1 (BRAINOT19) 919 4089878H1 (LIVRNOT06) 928 1478266H1 (CORPNOT02) 930 7025579H1 (LIVRDIT06) 930 4797414H1 (LIVRTUT09) 932 gl754209 6897313H1 (LIVRTMR01) 932 2049444H1 (LIVRFET02) 933 4897553H1 (LIVRTUT12) 933 4896747H1 (LIVRTUT12) 934 4991708H1 (LIVRTUT1 4986233H1 (LIVRTUT10) 936 70308405D1 7389029H2 (LIVRFEE02) 940 70306454D 3130847H1 (PONSAZT01) 946 4138382H1 (BRAITUT29) 947 4797280H1 (LIVRTUT09) 948 1483523H1 (CORPNOT02) 955 1485633H1 (CORPNOT02) 955 6892231J1 (BRAITDR03) 959 6174705H1 (BRAGNOT03) 967 3629480H1 (COLNNOT38) 967 3945513H1 (SCORNOT04) 974 4689281H1 g655825 6509586H1 (BRAHNOT02) 982 70306890D1 4892513H2 (LIVRTUT12) 986 8684539H1 (BRAIUNF01) 986 4794746H1 (LIVRTUT09) 987 4797619H1 (LIVRTUT09) 987 4893486H1 (LIVRTUT12) 987 4286603H1 (LIVRDIR01) 987 7025961H1 (LIVRDIT06) 987 4897661H1 (LIVRTUT12) 9881246 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte ID/ Sequence Length 4985846H1 (LIVRTUT10) 1250 4286347H1 (LIVRDIR01) 990 4896903H1 (LIVRTUT12) 992 2302315H1 (BRSTNOT05) 994 4794831H1 (LIVRTUT09) 994 4895978H1 (LIVRTUT12) 994 4984719H1 (LIVRTUT10) 996 gl970644 1618458H1 (BRAITUT12) 1003 5282058H1 (TESTNON04) 1003 4798120H1 (LIVRTUT09) 1003 6836243H1 (BRSTNON02) 1003 5351145H1 (BRAIDIT05) 1005 4775431H1 (BRAQNOT01) 1005 4993864H1 (LIVRTUT11) 1006 4991784H1 (LIVRTUT11) 1006 4989788H1 (LIVRTUT11) 1007 4895944H1 (LIVRTUT12) 1007 4897203H1 (LIVRTUT12) 1007 6733503H1 (LIVRTUT13) 1007 5964825H1 (BRATNOT05) 1007 g680784 4992250H1 (LIVRTUT11) 4797695H1 (LIVRTUT09) 1017 4988346H1 (LIVRTUT10) 1017 4793726H1 (LIVRTUT09) 1017 4985582H1 (LIVRTUT10) 1021 4892727H1 (LIVRTUT12) 1021 4892940H1 (LIVRTUT12) 1021 4418272H1 (LIVRDIT02) 1031 4892584H2 (LIVRTUT12) 1039 4338872H1 (BRAUNOT02) 1042 4419879H1 (LIVRDIT02) 1046 026881H1 (SPLNFET01) 1046 4993795H1 (LIVRTUT11) 1048 4230490H1 (BRAMDIT01) 1049 70305993D1 70306756D 70308340D1 4989185H1 (LIVRTUT10) 1055 5397848H1 (LIVRTUT13) 1055 6620013H1 1907936H1 (CONNTUT01) 1061 4420478H1 (LIVRDIT02) 1061 4798157H1 (LIVRTUT09) 1066 3955420H1 (PONSAZT01) 1066 Table 4 Polynucleotide Selected Sequence 5'Position 3'Position SEQ Incyte ID/ Sequence Length 70307718D1 1068 1634 4897579H1 (LIVRTUT12) 1072 530511H1 (BRAINOT03) 1076 027781H1 (SPLNFET01) 1077 7027659H1 (LIVRNOT21) 1077 5204551H1 (BRAFNOT02) 1077 7028432H1 (LIVRNOT21) 1078 6506838H1 (BRAHNOT02) 1078 4797590H1 (LIVRTUT09) 1082 7052422H1 (BRACNOK02) 1088 4992834H1 (LIVRTUT11) 1091 4895691H1 (LIVRTUT12) 1091 4837283H1 (BRAWNOT01) 1092 5400279H1 (LIVRTUT13) 1093 027504H1 (SPLNFET01) 1095 3953263H1 (PONSAZT01) 1099 1840893H1 (COLNNOT07) 1110 4314214H1 (BRAFNOT01) 1111 58061824J1 025652H1 (SPLNFET01) 1112 4991756H1 (LIVRTUT11) 4893554H1 (LIVRTUT12) 1116 1481993H1 (CORPNOT02) 1119 6769066J1 (BRAUNOR01) 1119 gl301549 73245215D1 4837251H1 (BRAWNOT01) 1125 6890763H1 (BRAITDR03) 1131 gl970266 6890763J1 (BRAITDR03) 1134 3664421H1 (PANCNOT16) 1139 4795014H1 (LIVRTUT09) 1142 4990651H1 (LIVRTUT11) 1142 4284113H1 (LIVRDIR01) 1142 667948H1 (SCORNOT01) 1144 4896362H1 (LIVRTUT12) 1150 g714835 3469134H1 (BRAIDIT01) 1152 8598971H2 (LIVRFEF03) 1152 4985406H1 (LIVRTUT10) 1153 4990110H1 (LIVRTUT11) 6733367H1 (LIVRTUT13) 1163 5689257H1 (BRAUNOT02) 1165 6896077H1 (LIVRTMR01) 1171 4985466H1 (LIVRTUT10) 1173 3944996H1 (SCORNOT04) 1175 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte ID/ Sequence Length 8737339J1 2112 1824640H1 (LSUBNOT03) 1178 4987343H1 (LIVRTUT10) 1178 4892754H1 (LIVRTUT12) 1178 6899846H1 (LIVRTMR01) 1181 4993563H1 (LIVRTUT11) 1184 4989014H1 (LIVRTUT10) 1190 4794220H1 (LIVRTUT09) 1190 g985042 088616H1 (LIVRNOT01) 1193 4988472H1 (LIVRTUT10) 1193 5399702H1 (LIVRTUT13) 1194 4698918H1 (BRALNOT01) 1194 4161906H1 (BRSTNOT32) 1195 3944406H1 (SCORNOT04) 1195 166252H1 (LIVRNOT01) 1196 g654989 271694H1 (LIVRNOT02) 1204 4794941H1 (LIVRTUT09) 5035934H1 (LIVRTUT13) 1211 g689855 4985527H1 (LIVRTUT10) 1212 4897029H1 (LIVRTUT12) 1215 1484431H1 (CORPNOT02) 1216 3093681H1 (BRSTNOT19) 1216 6124294H1 (BRAHNON05) 1216 gl484798 8729535J1 (BRAJNON03) 1219 1412846H1 (BRAINOT12) 1222 1486062H1 (CORPNOT02) 1226 4986382H1 6733414H1 (LIVRTUT13) 1226 5970837H1 (BRAZNOT01) 1226 5524490H1 (LIVRDIR01) 1231 7189712H2 (BRATDIC01) 1231 1438181H1 (PANCNOT08) 1231 3471041H1 (BRAIDIT01) 1231 7096839H1 (BRACDIR02) 1231 56073527H1 1481535F6 (CORPNOT02) 1231 3783604H1 (BRAHDIT04) 1231 4989681H1 (LIVRTUT11) 1231 g680514 5966558H1 (BRATNOT05) 1231 7142746H1 (LIVRDIT07) 1231 5975185H1 (BRAZNOT01) 1231 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte ID/ Sequence Length 4992361H1 (LIVRTUT11) 1231 1291 7185352H1 (BONRFEC01) 1231 6897130H1 (LIVRTMR01) 1231 2964826H1 (SCORNOT04) 1231 5524590H1 7193835H2 (BRATDIC01) 1231 2965815H1 (SCORNOT04) 1231 3760251H1 (BRAHDIT03) 1231 6149114H1 (BRANDIT03) 1231 167870H1 (LIVRNOT01) 1231 6897090H1 (LIVRTMR01) 1231 6450729H1 (BRAINOC01) 1231 g663312 7259724H1 (BRAWNOC01) 1231 5975275H1 (BRAZNOT01) 1231 4894554H1 (LIVRTUT12) 1231 139731H1 (LIVRNOT01) 1231 7267041H2 (NOSEDIC01) 1231 6450062H1 (BRAINOC01) 1231 5775051H1 (BRAINOT20) 1231 4991916H1 (LIVRTUT11) 7512697CTI 7141867H1 (LIVRDIT07) 1231 7142646H1 (LIVRDIT07) 1231 2572094H1 (HIPOAZT01) 1232 4987844H1 (LIVRTUT10) 1233 4984357H1 (LIVRTUT10) 1234 2410744H1 (BSTMNON02) 1234 4897226H1 (LIVRTUT12) 1234 3758794H1 (BRAHDIT03) 1235 4989879H1 (LIVRTUT11) 6731641H1 (LIVRTUT13) 1236 4796329H1 (LIVRTUT09) 1237 1907835H1 (CONNTUT01) 1240 2761563H1 (BRAINOS12) 1240 4988720H1 (LIVRTUT10) 1244 4895344H1 (LIVRTUT12) 1252 g690197 6732840H1 (LIVRTUT13) 1268 6893024H1 (BRAITDR03) 1269 4894516H1 (LIVRTUT12) 1273 4896504H1 (LIVRTUT12) 1273 5575154H1 (BRAPNOT04) 1275 8590428T1 (SCOMDIC01) 1278 4896025H1 (LIVRTUT12) 1280 70306713D1 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte Sequence Length 6446952H1 (BRAINOC01) 1285 7104606H1 (BRAWTDR02) 1286 70308693D1 7099229H2 (BRAWTDR02) 1289 7024515H1 (LIVRDIT06) 1292 6449152H1 (BRAINOC01) 1292 g3203668 5517147H1 (LIVRDIR01) 1295 7103347H1 (BRAWTDR02) 1298 5956655H1 (BRATNOT05) 1302 4893567H1 (LIVRTUT12) 1312 4987478H1 (LIVRTUT10) 1316 4993151H1 (LIVRTUT11) 1316 5575541H1 (BRAPNOT04) 1317 4991819H1 (LIVRTUT11) 7088948H1 (BRAUTDR03) 1317 4987808H1 (LIVRTUT10) 1320 4987708H1 (LIVRTUT10) 1320 087739H1 (LIVRNOT01) 1321 4893995H1 (LIVRTUT12) 1327 5575378H1 (BRAPNOT04) 1328 4796890H1 (LIVRTUT09) 1328 4794586H1 (LIVRTUT09) 1328 5971513H1 (BRAZNOT01) 1328 g1024346 g1004581 58061824H1 2965827H1 (SCORNOT04) 1330 087833H1 (LIVRNOT01) 1331 70308161D1 70305008D1 5331426H1 (BRAIDIT05) 1338 7188704H2 (BRATDIC01) 1341 4416736H1 (LIVRDIT02) 1343 4343041H1 (BRAUNOT02) 1343 843421H1 (PROSTUT05) 1344 3636093H1 (LIVRNOT03) 1344 2957589H1 (KIDNFET01) 1344 843421R6 (PROSTUT05) 1344 4894405H1 (LIVRTUT12) 1345 4062252H1 (BRAINOT21) 1347 2051051H1 (LIVRFET02) 1349 6620013J1 (BRAUDIR01) 1349 6437712H1 (BRAENOT02) 1351 1485222H1 (CORPNOT02) 1352 4420392H1 (LIVRDIT02) 13521617 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte Sequence Length 6732586H1 (LIVRTUT13) 1896 8588183T1 (SCOMDIC01) 1355 4339617H1 (BRAUNOT02) 1356 _ gl039737 gl039502 4798240H1 (LIVRTUT09) 1361 g1242414 g703655 481478H1 (LIVRBCT01) 1364 3466541H1 (BRAIDIT01) 1364 138207H1 (LIVRNOT01) 1368 4794334H1 (LIVRTUT09) 1369 4794366F8 (LIVRTUT09) 1369 8739217J1 (BRAJNON03) 1377 4418985H1 (LIVRDIT02) 1383 4416312H1 (LIVRDIT02) 1383 168618H1 (LIVRNOT01) 1383 168604R6 (LIVRNOT01) 1385 1908240H1 (CONNTUT01) 1388 4255806H1 (BSCNNOT03) 1394 4785484T9 (BRATNOT03) 1394 087859H1 (LIVRNOT01) 1396 2706212H1 (PONSAZT01) 1396 138279H1 (LIVRNOT01) 1402 4992759H1 (LIVRTUT11) 4992863H1 (LIVRTUT11) 1403 70306103D1 58033886H1 58033786H1 58033878H1 58033870H1 58033794H1 58033794J1 58033878J1 4658593H1 (NOSEDIT01) 1412 6733739H1 (LIVRTUT13) 1413 7066548H1 (BRATNOR01) 1414 56030707H1 56030723J1 088614H1 (LIVRNOT01) g678976 1615949H1 (BRAITUT12) 1420 805065H1 (BSTMNOT01) 14201615 1309840H1 (COLNFET02) 1420 4420830H1 (LIVRDIT02) 1420 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 4894680H1 (LIVRTUT12) 1691 4987323H1 (LIVRTUT10) 1423 58033886J1 1458964H1 (COLNFET02) 1426 1459024H1 (COLNFET02) 1426 1909630H1 (CONNTUT01) 1426 4794691H1 (LIVRTUT09) 1430 g681659 087893H1 (LIVRNOT01) 1434 8739227J1 (BRAJNON03) 1437 7191450T9 (BRATDIC01) 1440 4992140H1 (LIVRTUT11) 4657719H1 (NOSEDIT01) 1441 4002962H1 (HNT2AZS07) 1441 4989120T8 (LIVRTUT10) 1442 g660643 gl970005 70307708D1 70308564D1 70305638D1 70306970D1 4893555H1 (LIVRTUT12) 1445 4892569H2 (LIVRTUT12) 1445 70305410D1 4421042H1 (LIVRDIT02) 1447 g2221613 g2242304 1954427H1 (CONNNOT01) 1451 6998896H1 (BRAXTDR17) 1451 55075503H1 4689507H1 (LIVRTUT12) 1465 g649999 2753525H1 (THP1AZS08) 1468 4989143H1 (LIVRTUT10) 1477 480442H1 (LIVRBCT01) 1479 g686836 027346H1 (SPLNFET01) 1480 139630H1 (LIVRNOT01) 1481 4770684H1 (BRATNOT02) 1484 5974378H1 (BRAZNOT01) 1484 5396915T1 (LIVRTUT13) 1488 gl484741 6972755H1 (BRAHTDR04) 1493 4794366T9 (LIVRTUT09) 1493 70306049D1 4086928H1 (LIVRNOT06) 1494 Table 4 Polynucleotide SEQ ID NO :/ Incyte ID/ Sequence Length 4985501H1 (LIVRTUT10) 1495 1751 4992279H1 (LIVRTUT11) 1495 4892667H1 (LIVRTUT12) 1501 g647748 g668052 g647727 g680290 4838578H1 (BRAWNOT01) 1508 gl754210 4793464H1 (LIVRTUT09) 1513 4421025H1 (LIVRDIT02) 1514 3788049H1 (BRAHNOT05) 1516 g984995 7141505T8 (LIVRDIT07) 1521 g679756 4059605H1 (BRAINOT21) 1527 g3250257 4260119T1 (BSCNDIT02) 1528 4717532H1 (BRAIHCT02) 1529 g668043 g665298 4416770H1 (LIVRDIT02) 1531 5974587H1 (BRAZNOT01) 1534 168604T6 (LIVRNOT01) 1534 4987449T6 (LIVRTUT10) 1537 7143890T8 (LIVRDIT07) 1537 1479986H1 (CORPNOT02) 1541 1481418T6 (CORPNOT02) 1542 g678572 4419642H1 (LIVRDIT02) 1546 70305233D1 70306649D1 70308544D1 g678847 4418169H1 (LIVRDIT02) 2506918H1 (CONUTUT01) 1550 806470H1 (BSTMNOT01) 1550 g668163 g689575 g680757 1481535T6 (CORPNOT02) 1555 4417753H1 (LIVRDIT02) 1556 7024908H1 (LIVRDIT06) 1558 2963778H1 (SCORNOT04) 1560 4689692H1 (LIVRTUT12) 1562 4793686H1 (LIVRTUT09) 1562 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO : Fragments Incyte ID/ Sequence Length g680606 1563 2103 g673633 g691279 4256911H1 (BSCNNOT03) 1566 4447623H1 (SINDNOT01) 1567 1484343T6 (CORPNOT02) 1569 4894092H1 (LIVRTUT12) 1570 gl758005 1624141H1 (BRAITUT13) 1575 280648H1 (LIVRNOT02) 1576 481284H1 (LIVRBCT01) 1580 g668282 g680186 4793525H1 (LIVRTUT09) 1582 4984996H1 (LIVRTUT10) 1584 4893071H2 (LIVRTUT12) 1585 087840H1 (LIVRNOT01) 1586 g691461 3784014H1 (BRAHDIT04) 1592 4796821H1 (LIVRTUT09) 1593 6897879H1 (LIVRTMR01) 1593 8597996H1 (OVARDIF03) 1597 5664218H1 (BRAUNOT01) 1598 4987753H1 (LIVRTUT10) 1599 4986439H1 (LIVRTUT10) 1599 g686775 g678869 4987646H1 (LIVRTUT10) 1602 4796420H1 (LIVRTUT09) 1604 243516H1 (HIPONOT01) 1613 g680758 g689757 240533H1 (HIPONOT01) 1616 g664168 g689528 g1191833 3468181H1 (BRAIDIT01) 1619 4893091H2 (LIVRTUT12) 1624 1910169H1 (CONNTUT01) 1625 480498H1 (LIVRBCT01) 1625 g691586 g690779 gl0372718 gl039597 7025926H1 (LIVRDIT06) 1634 __ Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ Incyte ID/ Sequence Length 280137H1 (LIVRNOT02) 1641 1975 g686781 480472H1 (LIVRBCT01) 1642 843421T6 (PROSTUT05) 1642 g648869 4985019H1 (LIVRTUT10) 1643 gl0032474 g2706358 g4649746 g3331295 gl024347 4416027H1 (LIVRDIT02) 1652 g691225 g4736869 5036983H1 (LIVRTUT13) 1664 gl209964 g3433957 g5803596 1673 4420525H1 4897251H1 (LIVRTUT12) 1675 g3280351 g663510 1483979H1 (CORPNOT02) 1678 4793134H1 (LIVRTUT09) 1678 6147650H1 (BRANDIT03) 1680 8459520J1 (NERLNOL02) 1683 4893604H1 (LIVRTUT12) 1690 g7700599 g2714524 531614T6 (BRAINOT03) 1694 gl757781 4342290H1 (BRAUNOT02) 1695 4793645H1 (LIVRTUT09) 1696 4796239H1 (LIVRTUT09) 1696 g655692 g654990 2302332H1 (BRSTNOT05) 1708 1481424H1 (CORPNOT02) 1708 4793579H1 (LIVRTUT09) 1708 4797816H1 (LIVRTUT09) 1708 g1224738 g680045 2051065H1 (LIVRFET02) 1714 gl994732 1381847H1 (BRAITUT08) 1717 6055619H1 (BRAENOT04) 1717 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length g648569 2119 g4265903 g4089355 g3246431 g4224019 g665055 g680716 g665049 3633074F6 (LIVRNOT03) 1724 g2555745 gl994787 g5545265 4895286H1 (LIVRTUT12) 1734 4896637H1 (LIVRTUT12) 1734 g9706311 g7152436 5405638H1 (BRAMNOT01) 1739 g4111288 55102564H1 (NUHRDRV02) 1746 g2837928 g3306697 g2355171 5973410H1 (BRAZNOT01) 1751 g3246021 4986465H1 (LIVRTUT10) 1764 2706788H1 (PONSAZT01) 1767 4772470H1 (BRAQNOT01) 1774 g518188 7022975H1 (LIVRDIT06) 1777 g3595435 4419548H1 (LIVRDIT02) 1792 4797130H1 (LIVRTUT09) 1802 2535669H1 (BRAINOT18) 1811 2011912H1 g660576 2634826H1 (BONTNOT01) 1813 4029535H1 (BRAINOT23) 1823 2050662H1 (LIVRFET02) 1825 gl997463 g6087048 4085951H1 (LIVRNOT06) 1836 2049244H1 (LIVRFET02) 1838 g1210594 5289839H1 (LIVRTUS02) 1858 2050248H1 (LIVRFET02) 1862 5947278H1 (LIVRTUN04) 1864 Table 4 Polynucleotide Selected Sequence Fragments 5'Position 3'Position SEQ ID NO :/ Incyte ID/ Sequence Length 4772525H1 (BRAQNOT01) 2139 2292866H1 (BRAINON01) 1868 70308478D1 70305816D1 7023217H1 (LIVRDIT06) 1881 551493H1 (SCORNOT01) 1882 g4892525 1890 479924H1 (LIVRBCT01) 1891 1833141H1 (BRAINON01) 1899 gl gl273065 70305482D1 g3736152 1969325T6 (BRSTNOT04) 1934 1969325R6 (BRSTNOT04) 1934 1969325H1 (BRSTNOT04) 1934 5947652H1 (LIVRTUN04) 1935 5974184H1 (BRAZNOT01) 1938 1286994H1 (BRAINOT11) 1942 4191561H1 (BRAPDIT01) 1942 4191522H1 (BRAPDIT01) 1942 g3742411 g665120 g6030283 g3744552 6549924H1 (BRAFNON02) 1963 g663439 2289436H1 (BRAINON01) 1977 5946033H1 (LIVRTUN04) 1978 gll93472 1997 4795838H1 (LIVRTUT09) 5290161F6 (LIVRTUS02) 2012 5290161F8 (LIVRTUS02) 2012 5290158H1 (LIVRTUS02) 2012 272608H1 (LIVRNOT02) 2012 272761H1 (LIVRNOT02) 2013 272607H1 (LIVRNOT02) 2013 4895822H1 (LIVRTUT12) 2021 Table 5 Polynucleotide SEQ Incyte Project ID : Representative Library ID NO : 4 5 6 75134Table 6 Librar HUVELPB01 Library was constructed using RNA isolated from HUV-EC-C (ATCC CRL 1730) cells that were stimulated with cytokine/LPS. RNA was isolated from two pools of HUV-EC-C cells that had been treated with either gamma IFN and TNF- alpha or IL-1 beta and LPS. In the first instance, HUV-EC-C cells were treated with 4 units/ml TNF and 2 units/ml IFNg for 96 hours. In the second instance, cells were treated with 1 units/ml IL-1 and 100 ng/ml LPS for 5 hours. LIVRTUT10 pINCY Library was constructed using RNA isolated from a treated C3A hepatocyte cell line, which is a derivative of Hep G2, a cell line derived from a hepatoblastoma removed from a 15-year-old Caucasian male. The cells were treated with acetaminophen, 1 mM hours.

VectoTable 7 Program Threshold ABI A program that removes vector sequences and masks Applied Biosystems, Foster City, CA. ambiguous bases in nucleic acid sequences. ABI/PARACEL in comparing and Applied Biosystems, Foster City, CA ; Mismatch <50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA. r pro em's ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA. BLAST A Basic Local Alignment Search Tool useful in Altschul, S. F. et al. (1990) J. Mol. Biol. ESTs sequence similarity search for amino acid and nucleic 215 : 403-410 ; Altschul, S. F. et al. (1997) 8 or less ; Full Length sequences : acid sequences. BLAST includes five functions : Nucleic Acids Res. 25 : 3389-3402. Probability value = 1. OE-10 or blastp, blastn, blastx, tblastn, and tblastx. less A ear n and an al h that a he a FASTA A Pearson and Lipman algorithm that searches for Pearson, W. R. and D. J. Lipman (1988) Proc. ESTs : fasta E value = 1. 06E-6 ; similarity between a query sequence and a group of Natl. Acad Sci. USA 85 : 2444-2448 ; Pearson, Assembled ESTs : fasta Identity sequences of the same type. FASTA comprises as W. R. (1990) Methods Enzymol. 183 : 63-98 ; = 95% or greater and Match least five functions : fasta, tfasta, fastx, tfastx, and and Smith, T. F. and M. S. Waterman (1981) length = 200 bases or greater ; ssearch. Adv. Appl. Math. 2 : 482-489. fastx E value = 1. OE-8 or less ; Full Length sequences : fastx score = 100 or greater BLIMPS A that matches a Henikoff, S. and J. G. Henikoff (1991) Probability value = or sequence against those in BLOCKS, PRINTS, Nucleic Acids Res. 19 : 6565-6572 ; Henikoff, less DOMO, PRODOM, and PFAM databases to search J. G. and S. Henikoff (1996) Methods for gene families, sequence homology, and structural Enzymol. 266 : 88-105 ; and Attwood, T. K. et fingerprint al. (1997) J. Chem. Inf. Comput. Sci. 37 : 417- 424.

_ _ |Table 7 Program Threshold HMMER An algorithm for searching a query sequence against Krogh, A. et al. (1994) J. Mol. Biol. hidden Markov model (HMM)-based databases of 235 : 1501-1531 ; Sonnhammer, E. L. L. et al. TIGRFAM hits : Probability protein family consensus sequences, such as PFAM, (1988) Nucleic Acids Res. 26 : 320-322 ; value = 1. OE-3 or less ; Signal INCY, SMART and TIGRFAM. Durbin, R. et al. (1998) Our World View, in peptide hits : Score = 0 or greater a Nutshell, Cambridge Univ. Press, pp. 1- 350. ProfileScan An algorithm that searches for structural and Gribskov, M. et al. (1988) CABIOS 4 : 61-66 ; Normalized quality score > GCG sequence motifs in protein sequences that match Gribskov, M. et al. (1989) Methods specified"HIGH"value for that sequence patterns defined in Prosite. Enzymol. 183 : 146-159 ; Bairoch, A. et al. particular Prosite motif. (1997) Nucleic Acids Res. 25 : 217-221. Generally, score = 1. 4-2. 1. Phred A base-calling algorithm that examines automated B. et al. (1998) Genome Res. 8 : 175- sequencer traces with high sensitivity and probability. 185 ; Ewing, B. and P. Green (1998) Genome Res. 8 : 186-194. Phrap A Phils Revised Assembly Program including Smith, T. F. and M. S. Waterman (1981) Adv. Score = or greater Match SWAT and CrossMatch, programs based on efficient Appl. Math. 2 : 482-489 ; Smith, T. F. and length = or greater implementation of the Smith-Waterman algorithm, M. S. Waterman (1981) J. Mol. Biol. 147 : 195- useful in searching sequence homology and 197 ; and Green, P., University of assembling DNA sequences. Washington, Seattle, WA. Consed A graphical tool for viewing and editing Phrap D. et al. (1998) Genome Res. 8 : 195- assemblies. SPScan A weight matrix analysis program that scans protein Nielson, H. et al. (1997) Protein Engineering 3. 5 or greater sequences for the presence of secretory signal 10 : 1-6 ; Claverie, J. M. and S. Audic (1997) peptides. CABIOS 12 : 431-439. TMAP program that uses weight matrices to delineate Persson, B. and P. Argos (1994) J. Mol. Biol. transmembrane segments on protein sequences and 237 : 182-192 ; Persson, B. and P. Argos determine orientation. (1996) Protein Sci. 5 : 363-371.

ReferTable 7 Program Description Reference Threshold TMHMMER A program that uses a hidden Markov model (HMM) Sonnhammer, E. L. et al. (1998) Proc. Sixth to delineate transmembrane segments on protein Intl. Conf. On Intelligent Systems for Mol. sequences and determine orientation. Biol., Glasgow et al., eds., The Am. for Artificial Intelligence (AAAI) Press, Menlo Park, CA, and MIT Press, Cambridge, MA, pp. 175-182. Motifs A program that searches amino acid sequences for Bairoch, A. et al. (1997) Nucleic Acids Res. patterns that matched those defined in Prosite. 25 : 217-221 ; Wisconsin Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI.

ParamTable 8 SEQ PID EST EST CB1 EST Allele Allele Amino Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele NO : frequency frequency frequency frequency 4 7513406 19 4159 C C T noncoding n/a n/a n/a 4 4 SNP00004462 113 5156 AAGnoncoding 4 1644485F6 SNP00025253 32 4 1644485T6 SNP00004462 110 5157 A 4 SNP00004462 94 4 1732046T6 SNP00004462 93 4 SNP00124317 150 3192 A 4 SNP00124317 112 3202 A 4 SNP00025253 539 3404 A 5 SNP00025254 19 5 SNP00025255 95 5 SNP00004462 113 5027 A 5 SNP00025253 32 5 1644485T6 SNP00004462 110 5028 A 5 SNP00004462 94 5 1732046T6 SNP00004462 93 5 SNP00124317 150 3063 A 5 SNP00124317 112 3073 A 5 SNP00025253 539 3275 A 6 SNP00128003 103 649 C 6 SNP00011452 91 6 6 SNP00042263 157 979 C 6 SNP00042265 211213 6 SNP00011453 185 1604 C 0. 86 n/a 6 SNP00131332 95 6 SNP00042262 156 726 A ID SNTalbe 8 SEQ PID Caucasian African m ID SNP Allele 1 2 NO : frequency 6 7512697 1478266H1 G G 6 1481418F6 SNP00011450 6 1481418F6 SNP00042260 141 G 6 1481535T6 SNP00011453 6 6 168604T6 SNP00011453 395 1667 6 SNP00053100 541293 6 SNP00124859 51