SIGNAL PEPTIDE-CONTAINING PROTEINS
The invention provides cDNAs which encodes a signal peptide-containing proteins. It also provides for the use of a cDNA, protein, and antibody in the diagnosis, prognosis, treatment and evaluation of therapies for cancer. The invention further provides vectors and host cells for the production of the protein and transgenic model systems.
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This application is a Continuation of U.S. patent application Ser. No. 11/979,577, filed Nov. 6, 2007, which is a Divisional of U.S. patent application Ser. No. 09/968,433, filed Oct. 1, 2001, now U.S. Pat. No. 7,321,023, which is a Continuation-in-Part of U.S. patent application Ser. No. 09/271,110, filed Mar. 19, 1999, now abandoned, which is a Continuation-in-Part of U.S. patent application Ser. No. 08/966,316, filed Nov. 7, 1997, now U.S. Pat. No. 5,932,445. The entire contents of these applications are incorporated herein by reference in their entirety.
FIELD OF THE INVENTIONThis invention relates to signal peptide-containing proteins, their encoding cDNAs, and antibodies which binds the proteins, which can be used in the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
BACKGROUND OF THE INVENTIONProtein transport is a quintessential process for both prokaryotic and eukaryotic cells. Transport of an individual protein usually occurs via an amino-terminal signal sequence which directs, or targets, the protein from its ribosomal assembly site to a particular cellular or extracellular location. Transport may involve any combination of several of the following steps: contact with a chaperone, unfolding, interaction with a receptor and/or a pore complex, addition of energy, and refolding. Moreover, an extracellular protein may be produced as an inactive precursor. Once the precursor has been exported, removal of the signal sequence by a signal peptidase activates the protein.
Although amino-terminal signal sequences vary substantially, many patterns and overall properties are shared. Recently, hidden Markov models (HMM5), statistical alternatives to FASTA and Smith Waterman algorithms, have been used to find shared patterns, specifically consensus sequences (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; Smith and Waterman (1981) J. Mol. Biol. 147:195-197). Although they were initially developed to examine speech recognition patterns, HMMs have been used in biology to analyze protein and DNA sequences and to model protein structure (Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; Collin et al. (1993) Protein Sci. 2:305-314). HMMs have a formal probabilistic basis and use position-specific scores for amino acids or nucleotides and for opening and extending an insertion or deletion. The algorithms are quite flexible in that they incorporate information from newly identified sequences to build even more successful patterns. To find signal sequences, multiple unaligned sequences are compared to identify those which encode a peptide of 20 to 50 amino acids with an N-terminal methionine.
Some examples of the protein families which are known to have signal sequences are receptors (nuclear, 4 transmembrane, G protein coupled, and tyrosine kinase), cytokines (chemokines), hormones (growth and differentiation factors), neuropeptides and vasomediators, protein kinases, phosphatases, phospholipases, phosphodiesterases, nucleotide cyclases, matrix molecules (adhesion, cadherin, extracellular matrix molecules, integrin, and selectin), G proteins, ion channels (calcium, chloride, potassium, and sodium), proteases, transporter/pumps (amino acid, protein, sugar, metal and vitamin; calcium, phosphate, potassium, and sodium) and regulatory proteins. Receptors, kinases, and matrix proteins and diseases associated with their dysfunction are described below.
G-protein coupled receptors, GPCRs are a large group of receptors which transduce extracellular signals. GPCRs include receptors for biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin; for lipid mediators of inflammation such as prostaglandins, platelet activating factor, and leukotrienes; for peptide hormones such as calcitonin, C5a anaphylatoxin, follicle stimulating hormone, gonadotropin releasing hormone, neurokinin, oxytocin, and thrombin; and for sensory signal mediators such as retinal photopigments and olfactory stimulatory molecules. The structure of these highly-conserved receptors consists of seven hydrophobic transmembrane regions, an extracellular N-terminus, and a cytoplasmic C-terminus. The N-terminus interacts with ligands, and the C-terminus interacts with intracellular G proteins to activate second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or ion channel proteins. Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. The most conserved parts of these proteins are the transmembrane regions and the first two cytoplasmic loops. A conserved, acidic-Arg-aromatic triplet present in the second cytoplasmic loop may interact with the G proteins. The consensus pattern, [GSTALIVMYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYWCSH]-x(2)-[LIVM] is characteristic of most proteins belonging to this group (Bolander (1994) Molecular Endocrinology, Academic Press. San Diego Calif.; Strosberg (1991) Eur J Biochem 196: 1-10).
The kinases comprise the largest known group of proteins, a superfamily of enzymes with widely varied functions and specificities. Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Receptor mediated extracellular events trigger the transfer of these high energy phosphate groups and activate intracellular signaling cascades. Activation is roughly analogous to the turning on a molecular switch, and in cases where signaling is uncontrolled. may be associated with or produce inflammation and cancer.
Kinases are usually named after their substrate, their regulatory molecule, or after some aspect of a mutant phenotype. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VIA-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.
The kinases may be categorized into families by the different amino acid sequences (between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domain is conserved and contains specific residues and identifiable motifs or patterns of amino acids. The serine threonine kinases represent one family which preferentially phosphorylates serine or threonine residues. Many serine threonine kinases, including those from human, rabbit, rat, mouse, and chicken cells and tissues, have been described (Hardie and Hanks (1995) The Protein Kinase Facts Books, Vol 1:7-20 Academic Press, San Diego, Calif.).
The matrix proteins (MPs) provide structural support. cell and tissue identity. and autocrine, paracrine and juxtacrine properties for most eukaryotic cells (McGowan (1992) FASEB J 6:2895-2904). MPs include adhesion molecules, integrins and selectins, cadherins, lectins, lipocalins, and extracellular matrix proteins (ECMs). MPs possess many different domains which interact with soluble, extracellular molecules. These domains include collagen-like domains. EGF-like domains. immunoglobulin-like domains, fibronectin-like domains, type A domain of von Willebrand factor (vWFA)-like modules. ankyrin repeat modules. RDG or RDG-like sequences. carbohydrate-binding domains, and calcium ion-binding domains.
For example. multidomain or mosaic proteins play an important role in the diverse functions of the ECMs (Engel et al. (1994) Development S35-42). ECM proteins (ECMPs) are frequently characterized by the presence of one or more domains which may contain a number of potential intracellular disulphide bridge motifs. For example, domains which match the epidermal growth factor tandem repeat consensus are present within several known extracellular proteins that promote cell growth, development. and cell signaling. Other domains share internal homology and a regular distribution of single cysteines and cysteine doublets. In the serum albumin family, cysteine arrangement generates the characteristic “double-loop” structure (Soltysik-Espanola et al. (1994) Dev Biol 165:73-85) important for ligand-binding (Kragh-Hansen (1990) Danish Med Bull 37:57-84). Other ECMPs are members of the vWFA-like module superfamily, a diverse group of proteins with a module sharing high sequence similarity. The vWFA-like module is found not only in plasma proteins, but also in plasma membrane and ECMPs (Colombatti and Bonaldo (1991) Blood 77:2305-2315). Crystal structure analysis of an integrin vWFA-like module shows a classic “Rossmann” fold and suggests a metal ion-dependent adhesion site for binding protein ligands (Lee et al. (1995) Cell 80:631-638).
The diversity, distribution and biochemistry of MPs is indicative of their many, overlapping roles in cell proliferation and cell signaling. MPs function in the formation, growth, remodeling, and maintenance of bone, and in the mediation and regulation of inflammation. Biochemical changes that result from congenital, epigenetic, or infectious diseases affect the expression and balance of MPs. This balance, in turn, affects the activation, proliferation, differentiation, and migration of leukocytes and determines whether the immune response is appropriate or self-destructive (Roman (1996) Immunol. Res. 15:163-178).
Adenylyl cyclases (AC) are a group of second messenger molecules which actively participate in cell signaling processes. There are at least eight types of mammalian ACs which show regions of conserved sequence and are responsive to different stimuli. For example, the neural-specific type I AC is a Ca++-stimulated enzyme whereas the human type VII is unresponsive to CA++ and responds to prostaglandin E1 and isoproterenol. Characterization of these ACs, their tissue distribution, and the activators and inhibitors of the different types of ACs is the subject of various investigations (Nielsen et al. (1996) J. Biol. Chem. 271:33308-16; Hellevuo et al. (1995) J. Biol. Chem. 270:11581-9). AC interactions with kinases and G proteins in the intracellular signaling pathways of all tissues make them interesting candidate molecules for pharmaceutical research.
ATP diphosphohydrolase (ATPDase) is an enzyme expressed and secreted by quiescent endothelial cells and involved in vasomediation. The physiological role of ATPDase is to convert ATP and ADP to AMP. When this conversion occurs in the blood vessels during inflammatory response, it prevents extracellular ATP from causing vascular injury by inhibiting platelet activation and modulating vascular thrombosis (Robson et al. (1997) J. Exp. Med. 185:153-63).
The discovery of new signal peptide-containing proteins, their encoding cDNAs, and antibodies which bind the proteins satisfies a long standing need in the art by providing molecules and compositions which can be used in the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
SUMMARY OF THE INVENTIONThe present invention is based on the discovery of signal peptide-containing proteins, their encoding cDNAs and antibodies which specifically binds the proteins that are useful in the diagnosis, prognosis. treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
The invention provides an isolated cDNA comprising a nucleic acid molecule selected from SEQ ID NOs: 1-15 and 17-78. SEQ ID NO: 17 encodes a protein having an amino acid sequence of SEQ ID NO: 16. The invention also provides isolated cDNAs comprising SEQ ID NOs: 18-78 which have from about 80% to about 100% sequence identity with NOs: 1-15 and 17. The invention additionally encompasses a complement of the cDNAs selected from SEQ ID NOs: 1-15 and 17-78. In one aspect, the cDNA of SEQ ID NO: 17 is a fragment or an oligonucleotide comprising a nucleic acid molecule selected from A24 to G44, G159 to C182, G561 to A596, or A1011 and T1046.
The invention provides compositions comprising the cDNAs or their complements and a heterologous nucleotide sequence or a labeling moiety which may be used in methods of the invention, on a substrate, or in solution. The invention further provides a vector containing the cDNA. a host cell containing the vector, and a method for using the cDNA to make the human protein. The invention still further provides a transgenic cell line or organism comprising the vector containing a cDNA selected from SEQ ID NOs: 1-15 and 17-78. In a second aspect. the invention provides a cDNA or the complement thereof which can be used in methods of detection. screening, and purification. In a further aspect, the cDNA is a single-stranded RNA or DNA molecule, a peptide nucleic acid, a branched nucleic acid, and the like.
The invention provides a method for using a cDNA to detect differential expression of a nucleic acid in a sample comprising hybridizing a cDNA to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with at least one standard, wherein the comparison indicates differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose a cancer.
The invention additionally provides a method for using a composition of the invention to screen a plurality of molecules or compounds to identify or purify at least one ligand which specifically binds the cDNA, the method comprising combining the composition with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the composition, thereby identifying or purifying a ligand which binds the composition. In one aspect. the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions. peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO: 16, a variant of SEQ ID NO: 16, an antigenic epitope of SEQ ID NO: 16, and a biologically active portion of SEQ ID NO: 16. The invention also provides a composition comprising the purified protein and a labeling moiety or a pharmaceutical carrier. The invention further provides a method of using the protein to treat a subject with cancer comprising administering to a patient in need of such treatment a composition containing the purified protein and a pharmaceutical carrier. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify or purify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying or purifying a ligand which specifically binds the protein. In one aspect. the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins. inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with a cancer.
The invention provides a method of using a protein having the amino acid sequence of SEQ ID NO: 16 to screen a plurality of antibodies to identify an antibody which specifically binds the protein comprising contacting isolated antibodies with the protein under conditions to form an antibody:protein complex, and dissociating the antibody from the protein, thereby obtaining antibody which specifically binds the protein.
The invention also provides methods for using a protein having the amino acid sequence of SEQ ID NO: 16 to prepare and purify polyclonal and monoclonal antibodies which specifically bind the protein. The method for preparing a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies. The method for preparing and purifying monoclonal antibodies comprises immunizing a animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating from culture monoclonal antibodies which specifically bind the protein.
The invention provides purified polyclonal and monoclonal antibodies which bind specifically to a protein. The invention also provides a method for using an antibody to detect expression of a protein in a sample, the method comprising combining the antibody with a sample under conditions which allow the formation of antibody:protein complexes; and detecting complex formation, wherein complex formation indicates expression of the protein in the sample. In one aspect, the amount of complex formation when compared to standards is diagnostic of cancer.
The invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method•for using a cDNA to produce a model system, the method comprising constructing a vector containing a DNA selected from SEQ ID NOs: 1-15 and 17-78 transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, model system.
Table I shows the sequence identification numbers, reference, Incyte Clone number, cDNA library, NCB1 sequence identifier and GenBank description for each of the signal peptide-containing proteins encoded by the cDNAs.
DESCRIPTION OF THE INVENTIONIt is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. For example, a reference to “a host cell” includes a plurality of such host cells known to those skilled in the art.
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. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS“Array” refers to an ordered arrangement of at least two cDNAs or antibodies on a substrate. At least one of the cDNAs or antibodies represents a control or standard, and the other, a cDNA or antibody of diagnostic or therapeutic interest. The arrangement of two to about 40,000 cDNAs or of two to about 40,000 monoclonal or polyclonal antibodies on the substrate assures that the size and signal intensity of each labeled hybridization complex, formed between each cDNA and at least one nucleic acid, or antibody:protein complex, formed between each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.
The “complement” of a cDNA of the Sequence Listing refers to a nucleic acid molecule which is completely complementary to the cDNA over its full length and which will hybridize to the cDNA or an mRNA under conditions of maximal stringency.
“cDNA” refers to an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, may be double-stranded or single-stranded, represents coding and noncoding 3′ or 5′ sequence, and generally lacks introns.
A “composition” refers to the polynucleotide and a labeling moiety, a purified protein and a pharmaceutical carrier or a labeling moiety. an antibody and a labeling moiety, and the like.
“Derivative” refers to a cDNA or a protein that has been subjected to a chemical modification. Derivatization of a cDNA can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. Derivatization of a protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer advantages such as longer lifespan or enhanced activity.
“Differential expression” refers to an increased or up-regulated or a decreased or down-regulated expression as detected by presence. absence or at least two-fold change in the amount or abundance of a transcribed messenger RNA or translated protein in a sample.
Disorders associated with cell proliferation and cell signaling include cancers, genetic. and immune conditions. Each disorder is associated with expression of a signal peptide-containing protein or its specific encoding cDNA. These disorders include, but are not limited to, adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular, and adenocarcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma.
“Fragment” refers to a chain of consecutive nucleotides from about 50 to about 4000 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Such ligands are useful as therapeutics to regulate replication, transcription or translation.
A “hybridization complex” is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′. Hybridization conditions, degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
“Labeling moiety” refers to any visible or radioactive label than can be attached to or incorporated into a cDNA or protein. Visible labels include but are not limited to anthocyanins, green fluorescent protein (OFP), 6 glucuronidase. luciferase, Cy3 and Cy5, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
“Ligand” refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats. and lipids.
“Oligonucleotide” refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplimer, primer, and oligomer.
An “oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
“Portion” refers to any part of a protein used for any purpose; but especially, to an epitope for the screening of ligands or for the production of antibodies.
“Post-translational modification” of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
“Probe” refers to a cDNA that hybridizes to at least one nucleic acid in a sample. Where targets are single-stranded, probes are complementary single strands. Probes can be labeled with reporter molecules for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.
“Protein” refers to a polypeptide or any portion thereof. A “portion” of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic epitope of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR).
“Purified” refers to any molecule or compound that is separated from its natural environment and is from about 60% free to about 90% free from other components with which it is naturally associated.
“Sample” is used in its broadest sense as containing nucleic acids, proteins. antibodies. and the like. A sample may comprise a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a biopsy, a cell; a tissue; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like.
“Similarity” refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standard algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Bioi 147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402). BLAST2 may be used in a reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. Particularly in proteins, similarity is greater than identity in that conservative substitutions (for example, valine for leucine or isoleucine) are counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art.
“Specific binding” refers to a special and precise interaction between two molecules which is dependent upon their structure. particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
“Substrate” refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers. magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
A “transcript image” is a profile of gene transcription activity in a particular tissue at a particular time.
“Variant” refers to molecules that are recognized variations of a cDNA or a protein encoded by the cDNA. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure.
The InventionThe invention is based on the discovery of signal peptide-containing proteins, individually SP-1 through SP-16, and their encoding or regulating cDNAs, SEQ ID NOs: 1-15 and 17 which are characterized in TABLE I. U.S. Ser. No. 09/271,110, filed 17 Mar. 1999, is incorporated by reference herein in its entirety. The cDNAs and fragments thereof, the proteins and portions thereof, and an antibody which specifically binds each protein can be used directly or as compositions for the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
SP-1 was identified in Incyte Clone 1221102 from the NEUTGMTOI cDNA library using a computer search for amino acid sequence alignments. A cDNA comprising the nucleic acid shown in SEQ ID NO: 1 and derived using Incyte Clone 1221102, which encompasses nucleotides 300-514 also found in Incyte clone 5269342F6 (SEQ ID NO:18) which was used on HumanGenomeGEM1 microarray, encodes a GPCR with homology to g1575512, the GPR19 gene. Electronic northern analysis showed the expression of this sequence in neuronal tissues and in stimulated granulocytes. The transcript image found in EXAMPLE VIII supported the northern analysis and showed four-fold, up-regulated expression of the cDNA encoding SP-1 in the brain from a subject diagnosed with Huntington's disease.
SP-2 was identified in Incyte Clone 1457779 from the COLNFET02 cDNA library using a computer search for amino acid sequence alignments. A cDNA comprising the nucleic acid shown in SEQ ID NO: 2 and derived from Incyte Clone 1457779, which encompasses nucleotides 1-466 also found in Incyte clone 1457779F6 (SEQ ID NO: 22) which was used on LifeGEM1 microarray, encodes an ATP diphosphohydrolase with homology to g1842120. Electronic northern analysis showed the expression of this sequence in fetal colon, and transcript imaging showed that differential expression of SP-2 is diagnostic of stomach tumor.
SP-3 was identified in Incyte Clone 1682433 from the PROSNOT15 cDNA library using a computer search for amino acid sequence alignments. A cDNA comprising the nucleic acid shown in SEQ ID NO: 3 and derived from Incyte Clone 1682433, which encompasses nucleotides 1-481 also found in Incyte clone 2444114F6 (SEQ ID NO: 26) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g1010391, a transmembrane protein. Electronic northern analysis showed the expression of this sequence in fetal, cancerous or inflamed cells and tissues. Transcript imaging showed that differential expression of SP-3 is diagnostic of ductal carcinoma of the breast.
SP-4 was identified in Incyte Clone 1899132 from the BLADTUT06 cDNA library using a 35 (;omputer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 4. derived from Incyte Clone 1899132, which encompasses nucleotides 272-625 also found in Incyte clone 1899132F6 (SEQ ID NO: 31) which was used on LifeGEM1 microarray encodes a signal peptide containing protein with homology to g887602, a Saccharomyces cerevisiae protein. Electronic northern analysis showed the expression of this sequence in cancerous and inflamed cells and tissues; transcript imaging showed that differential expression of SP-4 is diagnostic of uterine adenosquamous carcinoma.
SP-5 was identified in Incyte Clone 1907344 from the CONNTUT01 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 5, derived from Incyte Clone 1907344, which encompasses nucleotides 17-450 also found in Incyte clone 2487075F6 (SEQ ID NO: 35) which was used on HumanGenomeGEM1 microarray, encodes a signal peptide containing protein with homology to g33715, immunoglobulin light chain. Electronic northern analysis showed the expression of this sequence in cancerous and fetal or infant cells and tissues; transcript imaging showed that differential expression of SP-5 is diagnostic for adenocarcinoma of the breast.
SP-6 was identified in Incyte Clone 1963651 from the BRSTNOT04 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 6, derived from Incyte Clone 1963651, which encompasses nucleotides 651-1090 also found in Incyte clone 1414964F6 (SEQ ID NO: 41) which was used on LifeGEM1 microarray, encodes a GPCR with homology to g1657623, orphan receptor RDC1. Although electronic northern analysis showed expression in ductal carcinoma; transcript imaging showed that differential expression of SP6 in ovary is diagnostic for mucinous cystadenoma.
SP-7 was identified in Incyte Clone 1976095 from the PANCTUT02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 7, derived from Incyte Clone 1976095, which encompasses nucleotides 74-525 also found in Incyte clone 1976095F6 (SEQ ID NO: 44) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g2117185, a Mycobacterium tuberculosis protein. Electronic northern analysis showed the expression of this sequence in cancerous and inflamed tissues; transcript imaging showed that' differential expression of SP-7 in synovium or cartilage is diagnostic for arthritis.
SP-8 was identified in Incyte Clone 2417676 from the HNT3AZT01 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 8, derived from Incyte Clone 2417676, which encompasses nucleotides 2-363 also found in Incyte clone 2890678F6 (SEQ ID NO: 49) which was used on HumanGenomeGEM1 microarray, encodes a signal peptide-containing protein with homology to g2150012, a human transmembrane protein. Electronic northern analysis showed this sequence to be expressed in proliferating. cancerous or inflamed tissues; transcript imaging shows that differential expression of SP-8 is diagnostic for testicular seminoma.
SP-9 was identified in Incyte Clone 1805538 from the SINTNOT13 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 9, derived from Incyte Clone 1805538, which encompasses nucleotides 15-419 also found in Incyte clone 2183094F6 (SEQ ID NO: 53) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g294502, an extracellular matrix protein. Electronic northern analysis showed this sequence to be expressed in inflamed tissues; transcript imaging showed that differential expression of SP-9 is diagnostic of adenofibromatous hyperplasia and prognostic for prostate cancer.
SP-10 was identified in Incyte Clone 1869688 from the SKINBIT01 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 10, derived from Incyte Clone 1869688, which encompasses nucleotides 1124-1380 also found in Incyte clone 2182042F6 (SEQ ID NO: 57) which was used on HumanGenomeGEM1 microarray, encodes a signal peptide-containing protein with homology to g1562, a G3 serine/threonine kinase. Electronic northern analysis showed this sequence to be expressed in proliferating tissues; transcript imaging showed that differential expression of SP-10 is diagnostic of transitional cell carcinoma of the bladder.
SP-11 was identified in Incyte Clone 1880692 from the LEUKNOT03 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 11, derived from Incyte Clone 1880692, which encompasses nucleotides 12-309 also found in Incyte clone 1880692F6 (SEQ ID NO: 60) which was used on LifeGEMI microarray, encodes a signal peptide-containing protein with homology to g1487910, a C. elegans protein. Electronic northern analysis showed this sequence to be expressed in cancer and blood cells; transcript imaging showed that differential expression of SP-11 is diagnostic for renal cell cancer.
SP-12 was identified in Incyte Clone 318060 from the EOSIHET02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 12, derived from Incyte Clone 318060, which encompasses nucleotides 193-1244 also found in Incyte clone 1266985F6 (SEQ ID NO: 64) which was used on HumanGenomeGEM1 microarray, encodes a receptor with homology to g606788, an opioid GPCR. Although electronic northern analysis showed this sequence to be expressed in nerve and blood cells; transcript imaging showed that differential expression of SP-12 is diagnostic for adenocarcinoma of the breast.
SP-13 was identified in Incyte Clone 396450 from the PITUNOT02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 13, derived from Incyte Clone 396450, which encompasses nucleotides 1-277 also found in Incyte clone 396450R6 (SEQ ID NO: 65) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g342279, opiomelanocortin. Electronic northern analysis showed this sequence to be expressed in hormone producing cells and tissues and inflamed cells and tissues; transcript imaging showed that differential expression of SP-13 is diagnostic for schizophrenia.
SP-14 was identified in Incyte Clone 506333 from the TMLR3DT02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence. SEQ ID NO: 14, derived from Incyte Clone 506333, which encompasses nucleotides 1-514 also found in Incyte clone 506333T6 (SEQ ID NO: 68) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g2204110, adenylyl cyclase. Electronic northern analysis showed this sequence to be expressed in cancerous and inflamed cells and tissues; transcript imaging showed that differential expression of SP-14 is diagnostic of breast cancer, in particular lobular carcinoma of the breast.
SP-15 was identified in Incyte Clone 764465 from the LUNGNOT04 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 15, derived from Incyte Clone 764465, which encompasses nucleotides 49-528 also found in Incyte clone 764465R6 (SEQ ID NO: 69) which was used on LifeGEM1 microarray, encodes a receptor with homology to GI 1902984, lectin-like oxidized LDL receptor. Electronic northern analysis showed this sequence to be expressed in lung and in fetal liver; transcript imaging confirms the northern analysis and shows that differential expression of SP-15 when used with lung samples is diagnostic for asthma.
SP-16 (SEQ ID NO: 16) was identified in Incyte Clone 2547002 from the UTRSNOT11 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO: 17, was derived from the extension and assembly of the overlapping nucleic acid sequences of Incyte Clones 2741185T6, 2741185T6F6.comp, and 2741185H1 (BRSTIUT14) and 2547002F6 and 2547002H1 (UTRSNOT11), SEQ ID NOs: 72-76, respectively.
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, as shown in
cDNA fragments encoding or regulating signal peptide-containing proteins were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics, Palo Alto Calif.). These cDNAs have from about 80% to about 95% sequence identity to the human cDNA as shown in the table below. The first column shows the SEQ IDH for the human cDNA; the second column, the SEQ IDFR for fragment cDNAs; the third column, the sequence numbers for the fragments; the fourth column, the species; the fifth column, percent identity to the human cDNA; and the sixth column, the nucleotide, alignment (NtH) of the human and fragment cDNAs.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of cDNAs encoding signal peptide-containing proteins, some bearing minimal similarity to the cDNAs of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of cDNA 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 encoding naturally occurring signal peptide-containing proteins, and all such variations are to be considered as being specifically disclosed.
The cDNAs of SEQ ID NOs: 1-15 and 17-78 may be used in hybridization, amplification, and screening technologies to identify and distinguish among the identical and related molecules in a sample. The cDNAs may also be used to produce transgenic cell lines or organisms which are model systems for cancers and upon which the toxicity and efficacy of potential therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the cDNAs, proteins, antibodies and molecules and compounds identified using the cDNAs and proteins of the present invention.
Characterization and Use of the InventioncDNA Libraries
In a particular embodiment disclosed herein, mRNA is isolated from cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries. The Incyte cDNAs were isolated from cDNA libraries prepared as described in the EXAMPLES. The consensus sequences are chemically and/or electronically assembled from fragments including Incyte cDNAs and extension and/or shotgun sequences using computer programs such as PHRAP (P Green, University of Washington, Seattle Wash.), and the AUTOASSEMBLER application (ABI). After verification of the 5′ and 3′ sequence, at least one of the representative cDNAs which encode a signal peptide-containing protein is designated a reagent. These reagent cDNAs are also used in the construction of human microarrays and are represented among the sequences on the Human Genome Gem Arrays (Incyte Genomics).
SequencingMethods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Pharmacia Biotech (APB), Piscataway N.J.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg Md.). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton. Reno Nev.) and the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.). Machines commonly used for sequencing include the ABI PRISM 3700, 377 or 373 DNA sequencing systems (ABI), the MEGABACE 1000 DNA sequencing system (APB), and the like. The sequences may be analyzed using a variety of algorithms well known in the art and described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).
Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the cDNAs of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8; 195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, or deleted sequences can be removed or restored, respectively, organizing the incomplete assembled sequences into finished sequences.
Extension of a Nuclic Acid MoleculeThe sequences of the invention may be extended using various PCR-based methods known in the art. For example, the XL-PCR kit (ABI), nested primers, and commercially available cDNA or genomic DNA libraries may be used to extend the . . . For all PCR-based methods, primers may be designed using commercially available software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55 C to about 68 C: When extending a sequence to recover regulatory elements, it is preferable to use genomic, rather than cDNA libraries.
HybridizationThe cDNA and fragments thereof can be used in hybridization technologies for various purposes. A probe may be designed or derived from unique regions such as the 5′ regulatory region or from a nonconserved region (i.e., 5′ or 3′ of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding a signal peptide-containing protein, allelic variants, or related molecules. The probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to a nucleic acid molecule selected from SEQ ID NOs: 1-15 and 17-78. Hybridization probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of a reporter molecule. A vector containing the cDNA or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using commercially available kits.
The stringency of hybridization is determined by G+C content of the probe. salt concentration. and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. Hybridization can be performed at low stringency with buffers, such as 5×SSC with 1% sodium dodecyl sulfate (SDS) at 60 C, which permits the formation of a hybridization complex between s that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2×SSC with 0.1% SDS at either 45 C (medium stringency) or 68 C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals can be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.
Arrays incorporating cDNAs or antibodies may be prepared and analyzed using methods well known in the art. Oligonucleotides or cDNAs may be used as hybridization probes or targets to monitor the expression level of large numbers of genes simultaneously or to identify genetic variants, mutations, and single nucleotide polymorphisms. Monoclonal or polyclonal antibodies may be used to detect or quantify expression of a protein in a sample. Such arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., Brennan et al. (1995) U.S. Pat. No. 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; Heller et al. (1997) U.S. Pat. No. 5,605,662; and deWildt et al. (2000) Nature Biotechnol 18:989-994.)
Hybridization probes are also useful in mapping the naturally occurring genomic sequence. The probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction. Such constructions include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacterial PI constructions, or the cDNAs of libraries made from single chromosomes.
ExpressionAnyone of a multitude of cDNAs encoding a signal peptide-containing protein may be cloned into a vector and used to express the protein, or portions thereof, in host cells. The can be engineered by such methods as DNA shuffling, as described in U.S. Pat. No. 5,830,721, and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life. and the like. The expression vector may contain transcriptional and translational control elements (promoters, enhancers. specific initiation signals. and polyadenylated 3′ sequence) from various sources which have been selected for their efficiency in a particular host. The vector, cDNA, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).
A variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors; plant cell systems transformed with expression vectors containing viral and/or bacterial elements, or animal cell systems (Ausubel supra, unit 16). For example. an adenovirus transcription/translation complex may be utilized in mammalian cells. After sequences are ligated into the E1 or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells. The Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.
Routine cloning, subcloning, and propagation of s can be achieved using the multifunctional PBLUESCRIPT vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Introduction of a into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. 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.
For long term production of recombinant proteins. the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media-and-then are-transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired cDNA is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification techniques.
The host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a “prepro” form may also be used to specify protein targeting, folding, and/or activity. Different host cells available from the ATCC (Manassas Va.) which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein.
Recovery of Proteins from Cell Culture
Heterologous moieties engineered into a vector for ease of purification include glutathione Stransferase (GST), 6×His, FLAG, MYC, and the like. GST and 6×His are purified using commercially available affinity matrices such as immobilized glutathione and metal-chelate resins, respectively. FLAG and MYC are purified using commercially available monoclonal and polyclonal antibodies. For ease of separation following purification, a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16) and are commercially available.
Chemical Synthesis of PeptidesProteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds α-amino-and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylarnine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N-α-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative. and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N,N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. These processes are described in the Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook (San Diego Calif., pp. S1-S20). Automated synthesis may also be carried out on machines such as the ABI 431 A peptide synthesizer (ABI). A protein or portion thereof may be purified by preparative high performance liquid chromatography and its composition confirmed by amino acid analysis or by sequencing (Creighton (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York N.Y.).
Preparation and Screening of AntibodiesVarious hosts including, but not limited to, goats, rabbits, rats, mice, and human cell lines may be immunized by injection with a signal peptide-containing protein or any immunogenic portion thereof. Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol may be used to increase immunological response. The oligopeptide, peptide, or portion of protein used to induce antibodies should consist of at least about five amino acids, more preferably ten amino acids, which are identical to a portion of the natural protein. Oligopeptides may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule.
Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human β-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al (1984) Mol Cell Biol 62:109-120.)
Alternatively, techniques described for antibody production may be adapted, using methods known in the art, to produce epitope-specific, single chain antibodies. Antibody fragments which contain specific binding sites for epitopes of the protein 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 Flib fragments with the desired specificity. (See, e.g., Huse et al. (1989) Science 246:1275-1281.)
A signal peptide-containing protein, or a portion thereof, may be used in screening assays of phagemid or β-lymphocyte immunoglobulin libraries to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoassays 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 the protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).
Labeling of Molecules for AssayA wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using commercially available kits (Promega, Madison Wis.) for incorporation of a labeled nucleotide such as 32P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Operon Technologies, Alameda Calif.), or amino acid such as 35S-methionine (APB). Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
Diagnostics Nucleic Acid AssaysThe cDNAs, fragments, oligonucleotides, complementary RNA and DNA molecules, and PNAs may be used to detect and quantify differential gene expression for diagnostic purposes. Disorders associated with expression of SP-1 through SP-16 include, but are not limited to, adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular. and adeno-carcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma. The diagnostic assay may use hybridization or quantitative PCR to compare gene expression in a biological or biopsied subject sample to standard samples in order to detect differential gene expression. Qualitative and quantitative methods for this comparison are commercially available and well known in the art.
For example, the cDNA or probe may be labeled by standard methods and added to a biological sample from a subject under conditions for the formation of hybridization complexes. After an incubation period. the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. If complex formation in the subject sample is significantly altered (higher or lower) in comparison to either, a normal or disease standard, then differential expression indicates the presence of a disorder.
In order to provide standards for establishing differential expression, normal and disease expression profiles are established. This is accomplished by combining a sample taken from normal subjects, either animal or human, with a cDNA under conditions for hybridization to occur. Standard hybridization complexes may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose that disorder.
Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic 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 a 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 years.
Protein AssaysDetection and quantification of a protein using either labeled amino acids or specific polyclonal or monoclonal antibodies which specifically bind the protein are known in the art. Examples of such techniques include two-dimensional polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). These assays and their quantitation against purified. labeled standards are well known in the art (Ausubel, Supra, unit 10.1-10.6). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed. (See. e.g. Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience, New York N.Y.; and Pound. supra.)
TherapeuticsChemical and structural similarity, in particular annotation and motifs that suggest function. are described for SEQ ID NO:16 in THE INVENTION section and transcript images that suggest function for the proteins encoded or regulated by SEQ ID NO:1-15 are described in EXAMPLE VIII and EXAMPLE IX. In addition. the differential expression of each of the cDNAs was shown to be tissue-specific and associated with a particular disorder in EXAMPLE VIII. Thus, each protein clearly plays a role in at least one of the described disorders (adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular, and adena-carcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma) and SP-1 through SP-16 may be used either directly as a therapeutic or as a target for drug discovery.
In one embodiment, increased expression of the protein may be treated by the delivery of an inhibitor, antagonist, antibody and the like or a pharmaceutical composition containing one or more of these molecules. Such delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the diseased tissue.
In another embodiment, decreased expression of the protein late in the disease process may be treated by the delivery of the protein, an agonist, enhancer and the like or a pharmaceutical composition containing one or more of these molecules. Such delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the diseased tissue.
Any of these compositions may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular cancer at a lower dosage of each agent alone.
Modification of Gene Expression Using Nucleic AcidsGene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5′, 3′, or other regulatory regions of the gene encoding a signal peptidecontaining protein. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA. In one alternative. a library or plurality of cDNAs may be screened to identify those which specifically bind a regulatory, nontranslated sequence.
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 at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate wgets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary nucleic acids and ribozymes of the invention may be prepared via recombinant expression, in vitro or in vivo, or using solid phase phosphoramidite chemical synthesis. In addition, RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5′ and/or 3′ ends of the molecule or by the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules. Either the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio-groups renders the molecule less available to endogenous endonucleases.
Screening and Purification AssaysA cDNA encoding a signal peptide-containing protein may be used to screen a library or a plurality of molecules or compounds for specific binding affinity. The libraries may be aptamers, DNA molecules, RNA molecules, PNAs, peptides, proteins such as transcription factors, enhancers, or repressors, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene. The assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the single-stranded or double-stranded molecule.
In one embodiment, the cDNA of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gelretardation assay (U.S. Pat. No. 6,010,849) or a commercially available reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay.
In another embodiment, the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.
In a further embodiment, the protein or a portion thereof may be used to purify a ligand from a sample. A method for using a protein or a portion thereof to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.
In a preferred embodiment, a signal peptide-containing protein may be used to screen a plurality of molecules or compounds in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. For example, in one method, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured. Depending on the particular kind of molecules or compounds being screened, the assay may be used to identify DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs or any other ligand, which specifically binds the protein.
In one aspect, this invention comtemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in U.S. Pat. No. 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding. In another aspect, this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.
PharmacologyPharmaceutical compositions contain active ingredients in an effective amount to achieve a desired and intended purpose and a pharmaceutical carrier. The determination of an effective dose is well within the capability of those skilled in the art. For any compound. the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models. The animal model is also used to achieve a desirable concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of protein or inhibitor which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such agents may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indexes are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
Model SystemsAnimal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models. and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.
ToxicologyToxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess potential consequences on human health following exposure to the agent.
Genetic toxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations. Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny. Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy. or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.
Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.
Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals.
Chronic toxicity tests, with a duration of a year or more, are used to demonstrate either the absence of toxicity or the carcinogenic potential of an agent. When studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment.
Transgenic Animal ModelsTransgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
Embryonic Stem CellsEmbryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gen, the latter serves to identify the presence of the introduced disease gene. The vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BU6 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.
ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes.
Knockout AnalysisIn gene knockout analysis, a region of a gene is enzymatically modified to include a nonmammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene. Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene. In one example, the mammalian gene is a human gene.
Knockin AnalysisES cells can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases. With knockin technology. a region of a human gene is injected into animal ES cells. and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastulae and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases.
Non-Human Primate ModelThe field of animal testing deals with data and methodology from basic sciences such as physiology, genetics, chemistry, pharmacology and statistics. These data are paramount in evaluating the effects of therapeutic agents on non-human primates as they can be related to human health. Monkeys are used as human surrogates in vaccine and drug evaluations, and their responses are relevant to human exposures under similar conditions. Cynomolgus and Rhesus monkeys (Macaca fascicularis and Macaca mulatta, respectively) and Common Marmosets (Callithrix jacchus) are the most common non-human primates (NHPs) used in these investigations. Since great cost is associated with developing and maintaining a colony of NHPs. early research and toxicological studies are usually carried out in rodent models. In studies using behavioral measures such as drug addiction, NHPs are the first choice test animal. In addition, NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from “extensive metabolizers” to “poor metabolizers” of these agents.
In additional embodiments, the cDNAs which encode the protein may be used in any molecular biology techniques that have yet to be developed. provided the new techniques rely on properties of cDNAs that are currently known. including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
EXAMPLES I cDNA Library ConstructionThe UTRSNOT11 cDNA library was constructed from microscopically normal uterine tissue obtained from a 43-year-old female during a vaginal hysterectomy following diagnosis of uterine leiomyoma. Pathology indicated that the myometrium contained an intramural leiomyoma and a submucosal leiomyoma. The endometrium was proliferative, however, the cervix and fallopian tubes were unremarkable. The right and left ovaries contained corpus lutea. The patient presented with metrorrhagia and deficiency anemia. Patient history included benign hypertension and atherosclerosis. Medications included PROVERA tablets (Pharmacia, Peapack N.J.), iron, and vitamins. Family history included benign hypertension, atherosclerosis, and malignant colon neoplasms.
The frozen tissue was homogenized and lysed in TRIZOL reagent (1 gm tissue/10 ml reagent; Life Technologies) using a POLYTRON homogenizer (PT-3000; Brinkmann Instruments, Westbury N.Y.). After a brief incubation on ice, chloroform was added (1:5 v/v), and the lysate was centrifuged. The upper chloroform layer was removed to a fresh tube, and the RNA was extracted with isopropanol, resuspended in DEPC-treated water, and treated with DNAse for 25 min at 37 C. The RNA was re-extracted three times with acid phenol-chloroform, pH 4.7, and precipitated with 0.3M sodium acetate and 2.5 volumes ethanol. The mRNA was isolated with the OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used to construct the cDNA library.
The mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were fractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY1 plasmid. The plasmid was subsequently transformed into DH5a competent cells (Life Technologies).
II Isolation of cDNA ClonesPlasmid DNA was released from the cells and purified using the REAL PREP 96 plasmid kit (Qiagen). This kit enabled the simultaneous purification of 96 samples in a 96-well block using multichannel reagent dispensers. The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, San Jose Calif.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after incubation for 19 hours, the cultures were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4 C.
III SequencingThe cDNAs were prepared for sequencing using the MICROLAB 2200 system (Hamilton) in combination with the DNA ENGINE thermal cyclers (MJ Research). The cDNAs were sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM 373 or 377 sequencing system (ABI). Most of the isolates were sequenced according to standard ABI protocols and kits with solution volumes of 0.25×-1.0× concentrations or using standard solutions and dyes from APB.
IV Extension of cDNA SequencesThe cDNA sequence may be extended to full length using the Incyte clone, for example, SEQ ID NO:17, 2547002H1. A set of nested deletion sequencing templates was prepared from overnight liquid culture of clone 496071 using the ERASE-A-BASE system (Promega).
Sequencing reactions were performed with the ABI PRISM Dye Terminator cycle sequencing kit with AMPLITAQ FS DNA polymerase (ABI). PCR was performed on a DNA ENGINE thermal cycler (MI Research). Reactions were analyzed on an ABI PRISM 310 genetic analyzer (ABI). Individual sequences were assembled and edited using ABI AutoAssembler software (ABI).
In the alternative, extension is accomplished using oligonucleotide primers synthesized to initiate 5′ and 3′ extension of the known fragment. These primers are designed using commercially available primer analysis software 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 that would result in hairpin structures and primer-primer dimerizations is avoided.
Selected cDNA libraries are used as templates to extend the sequence. If more than one extension is necessary, additional or nested sets of primers are designed. Preferred libraries have been size-selected to include larger cDNAs and random primed to contain more sequences with 5′ or upstream regions of genes. Genomic libraries are used to obtain regulatory elements, especially extension into the 5′ promoter binding region.
High fidelity amplification is obtained by PCR using methods such as that taught in U.S. Pat. No. 5,932,451. PCR is performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4,)2SO4, and β-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): Step 1: 94 C, three min; Step 2: 94 C, 15 sec; Step 3: 60 C, one min; Step 4: 68 C, two min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 C, five min; Step 7: storage at 4 C. In the alternative, the parameters for primer pair T7 and SK+ (Stratagene) are as follows: Step 1: 94 C, three min; Step 2: 94 C, 15 sec; Step 3: 57 C, one min; Step 4: 68 C, two min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 C, five min; Step 7: storage at 4 C.
The concentration of DNA in each well is determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% reagent in 1×TE, v/v; Molecular Probes) and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning, Acton Mass.) and allowing the DNA to bind to the reagent. The plate is scanned in a Fluoroskan II (Labsystems Oy, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reactions are successful in extending the sequence.
The extended clones are desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC18 vector (APB). For shotgun sequences, the digested nucleotide sequences are separated on low concentration (0.6 to 0.8%) agarose gels, fragments are excised, and the agar is digested with AGARACE enzyme (Promega). Extended clones are religated using T4 DNA ligase (New England Biolabs) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into E. coli competent cells. Transformed cells are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37 C in 384-well plates in LB/2× carbenicillin liquid media.
The cells are lysed, and DNA is amplified using primers, Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94 C, three min; Step 2: 94 C, 15 sec; Step 3: 60 C, one min; Step 4: 72 C, two min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72 C, five min; Step 7: storage at 4 C. DNA is quantified using PICOGREEN quantitation reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamplified using the conditions described above. Samples are diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the PRISM BIGDYE terminator cycle sequencing kit (ABI).
V Homology Searching of cDNA Clones and their Deduced ProteinsThe cDNAs of the Sequence Listing or their deduced amino acid sequences were used to query databases such as GenBank, SwissProt, BLOCKS, and the like. These databases that contain previously identified and annotated sequences or domains were searched using BLAST or BLAST2 to produce alignments and to determine which sequences were exact matches or homologs. The alignments were to sequences of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively, algorithms such as the one described in Smith and Smith (1992, Protein Engineering 5:35-51) could have been used to deal with primary sequence patterns and secondary structure gap penalties. All of the sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
As detailed in Karlin (supra), BLAST matches between a query sequence and a database sequence were evaluated statistically and only reported when they satisfied the threshold of 10−25 for nucleotides and 10−14 for peptides. Homology was also evaluated by product score calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. In comparison with hybridization procedures used in the laboratory, the stringency for an exact match was set from a lower limit of about 40 (with 1-2% error due to uncalled bases) to a 100% match of about 70.
The BLAST software suite (available on the National Center for Biotechnology Information (“NCBI”) website, Bethesda Md., includes various sequence analysis programs including “blastn” that is used to align nucleotide sequences and BLAST2 that is used for direct pairwise comparison of either nucleotide or amino acid sequences. BLAST programs are commonly used with gap and other parameters set to default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: −2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; and Filter: on. Identity is measured over the entire length of a sequence. Brenner et al., (1998; Proc Natl Acad Sci 95:6073-6078, incorporated herein by reference) analyzed BLAST for its ability to identify structural homologs by sequence identity and found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40%, for alignments of at least 70 residues.
The cDNAs of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database (Incyte Genomics). Component sequences from cDNA, extension, full length, and shotgun sequencing projects were subjected to PHRED analysis and assigned a quality score. All sequences with an acceptable quality score were subjected to various pre-processing and editing pathways to remove low quality 3′ ends, vector and linker sequences, polyA tails, Alu repeats, mitochondria) and ribosomal sequences, and bacterial contamination sequences. Edited sequences had to be at least 50 bp in length, and low-information sequences and repetitive elements such as dinucleotide repeats, Alu repeats, and the like, were replaced by “Ns” or masked.
Edited sequences were subjected to assembly procedures in which the sequences were assigned to gene bins. Each sequence could only belong to one bin, and sequences in each bin were assembled to produce a template. Newly sequenced components were added to existing bins using BLAST and CROSSMATCH. To be added to a bin, the component sequences had to have a BLAST quality score greater than or equal to 150 and an alignment of at least 82% local identity. The sequences in each bin were assembled using PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation of each template was determined based on the number and orientation of its component sequences.
Bins were compared to one another, and those having local similarity of at least 82% were combined and reassembled. Bins having templates with less than 95% local identity were split. Templates were subjected to analysis by STITCHER/EXON MAPPER algorithms that determine the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, and the like. Assembly procedures were repeated periodically, and templates were annotated using BLAST against GenBank databases such as GBpri. An exact match was defined as having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs and a homolog match as having an E-value (or probability score) of <1×10−8. The templates were also subjected to frameshift FASTx against GENPEPT, and homolog match was defined as having an E-value of <1×10−8. Template analysis and assembly was described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999.
Following assembly, templates were subjected to BLAST, motif, and other functional analyses and categorized in protein hierarchies using methods described in U.S. Ser. No. 08/812,290 and U.S. Ser. No. 08/811,758, both filed Mar. 6, 1997; in U.S. Ser. No. 08/947,845, filed Oct. 9, 1997; and in U.S. Ser. No. 09/034,807, filed Mar. 4, 1998. Then templates were analyzed by translating each template in all three forward reading frames and searching each translation against the PFAM database of hidden Markov model-based protein families and domains using the HMMER software package (Washington University School of Medicine, St. Louis Mo.; http://pfam.wustl.edu/). The cDNA was further analyzed using MACDNASIS PRO software (Hitachi Software Engineering), and LASERGENE software (DNASTAR) and queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
VI Chromosome MappingRadiation 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 are used to determine if any of the cDNAs presented in the Sequence Listing have been mapped. Any fragment of a cDNA encoding a signal peptide-containing protein that has been mapped result in the assignment of all related fragments and regulatory sequences to the same location. The genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cM (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the terminus of the chromosomal p-arm.
VII Hybridization Technologies and AnalysesImmobilization of cDNAs on a Substrate
The cDNAs are applied to a substrate by one of the following methods. A mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library. The cDNAs are then arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37 C for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2×SSC for 10 min each. The membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene).
In the second method, cDNAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above. Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in U.S. Pat. No. 5,807,522. Polymer-coated slides are prepared by cleaning glass microscope slides (Corning, Acton Mass.) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a 110 C oven. The slides are washed extensively with distilled water between and after treatments. The nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATALINKER UV-crosslinker (Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford Mass.) for 30 min at 60 C; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before.
Probe Preparation for Membrane HybridizationHybridization probes derived from the cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 μl TE buffer, denaturing by heating to 100 C for five min, and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five μl of [32P]dCTP is added to the tube, and the contents are incubated at 37 C for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 10° C. for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below.
Probe Preparation for Polymer Coated Slide HybridizationHybridization probes derived from mRNA isolated from samples are employed for screening cDNAs of the Sequence Listing in array-based hybridizations. Probe is prepared using the GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 μl TE buffer and adding 5 μl 5× buffer, 1 μl 0.1 M DTT, 3 μl Cy3 or Cy5 labeling mix, 1 μl RNase inhibitor, 1 μl reverse transcriptase, and 5 μl 1× yeast control mRNAs. Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng. 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine mRNA differential expression patterns, a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w). The reaction mixture is mixed and incubated at 37 C for two hr. The reaction mixture is then incubated for 20 min at 85 C, and probes are purified using two successive CHROMA SPIN+TE 30 columns (Clontech, Palo Alto Calif.). Purified probe is ethanol precipitated by diluting probe to 90 μl in DEPC-treated water, adding 2 μl 1 mg/ml glycogen, 60 μl 5 M sodium acetate, and 300 μl 100% ethanol. The probe is centrifuged for 20 min at 20,800×g, and the pellet is resuspended in 12 μl resuspension buffer, heated to 65 C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below.
Membrane-Based HybridizationMembranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and 1× high phosphate buffer (0.5 M NaCl, 0.1 M Na2HPO4, 5 mM EDTA, pH 7) at 55 C for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55 C for 16 hr. Following hybridization, the membrane is washed for 15 min at 25 C in 1 mM Tris (pH 80), 1% Sarkosyl, and four times for 15 min each at 25 C in 1 mM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed to the membrane overnight at −70 C, developed, and examined visually.
Polymer Coated Slide-Based HybridizationProbe is heated to 65 C for five min, centrifuged five min at 9400 rpm in a 5415C microcentrifuge (Eppendorf Scientific, Westbury N.Y.), and then 18 μl is aliquoted onto the array surface and covered with a 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 μl of 5×SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hr at 60 C. The arrays are washed for 10 min at 45 C in 1×SSC, 0.1% SDS, and three times for 10 min each at 45 C in 0.1×SSC, and dried.
Hybridization reactions are performed in absolute or differential hybridization formats. In the absolute hybridization format, probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample. In the differential hybridization format, differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).
Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) 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 20× microscope objective (Nikon, Melville N.Y.). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers. In the differential hybridization format, the two fluorophores are sequentially excited by the laser. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tubes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. 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.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood Mass.) 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 the emission spectrum for each fluorophore. 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 program (Incyte Genomics).
VIII Transcript ImagingA transcript image was performed using the LIFESEQ GOLD database (June 1 release, Incyte Genomics). This process allowed assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries and reconfirmed the data submitted in U.S. Ser. No. 08/966,316, filed 7 Nov. 1997. Criteria for transcript imaging can be selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like.
All sequences and cDNA libraries in the LIFESEQ database have been categorized by system, organ/tissue and cell type. For each category, the number of libraries in which the sequence was expressed were counted and shown over the total number of libraries in that category. In some transcript images, all normalized or pooled libraries, which have high copy number sequences removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject's tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be disregarded or removed where clinical relevance is emphasized. Conversely, fetal tissue may be emphasized wherever elucidation of inherited disorders or differentiation of particular cells or organs from stem cells (such as nerves, heart or kidney) would be furthered by removing clinical samples from the analysis. Transcript imaging can also be used to support data from other methodologies such as microarray analysis.
The transcript images for SEQ ID NOs:1-15 and 17 are shown below. The first column shows library name; the second column, the number of cDNAs sequenced in that library; the third column, the description of the library; the fourth column, absolute abundance of the transcript in the library; and the fifth column, percentage abundance of the transcript in the library.
In clinically-relevant brain samples, SEQ ID NO:1 is expressed four-fold higher in Huntington's disease with its associated dementia than in normal brain. Even though this GPCR is very sparsely expressed in human tissues; when SEQ ID NO:1 is used in a brain tissue-specific assay, it is diagnostic for Huntington's disease.
SEQ ID NO:2 was greater than two-fold differentially expressed in biopsied sample from the stomach of a subject diagnosed with adenocarcinoma over cytologically normal tissue from the same subject. Expression was not found in any other cytologically normal stomach tissue which included STOMNOT01, STOMNOT08, and STOMTMR02. SEQ ID NO:2, when used in a stomach-specific assay, is diagnostic for adenocarcinoma.
SEQ ID NO:3 is differentially expressed in ductal carcinoma of the breast as compared with its matched cytologically normal BRSTTMTOI. In addition, SEQ ID NO:3 was not expressed in BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries, and was not as highly expressed in tissues diagnosed with any other disease states or their cytologically normal matched tissues. SEQ ID NO:3, when used in a breast-specific assay including, but not limited to, ductal lavage, is diagnostic for ductal carcinoma.
SEQ ID NO:4 is more than five-fold differentially expressed in adenosquamous carcinoma of the uterus. It was not differentially expressed in tissues from subjects diagnosed with cervicitis (UTRCNOP01, UTRCDIE01), endometriosis (UTREDIT07, UTREDIT14), cervical tumor (UTRCTUP01), endometrial adenocarcinoma (UTRSTUP03, UTRSTUP04, UTRSTUP07), or leiomyoma (UTRSTUE01, UTRSTUT04, UTRSTUT05, UTRSTUT07) or in cytologically normal tissues (UTRCNOP01, UTREDME05, UTREDME06, UTREDMF01, UTREDMF02, UTREDMT07, UTRENON03, UTRENOT10, UTRETMC01, UTRETUP01, UTRMTMR02, UTRMTMT01, UTRPNOM01, UTRSNON03, UTRSNOP01, UTRSNOR01, UTRSNOT01, UTRSNOT02, UTRSNOT06, UTRSNOT08, UTRSNOT10, UTRSNOT11; UTRSNOTI2, UTRSNOT16, UTRSNOT18, UTRSTDT01, UTRSTMC01, UTRSTME01, UTRSTMR01, and UTRSTMR02). SEQ ID NO:4, when used in a uterus-specific assay, is diagnostic for adenosquamous carcinoma.
SEQ ID NO:5 is differentially expressed more than two-fold in adenocarcinoma of the breast when compared to expression in cytologically normal BRSTNOT31, BRSTNOT32 and matched BRSTNOT33. SEQ ID NO:5 was not differentially expressed in BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries, and was not as highly expressed in tissues diagnosed with any other disease states or their cytologically normal matched tissues. SEQ ID when used in a breast-specific assay including, but not limited to, ductal lavage, is diagnostic for adenocarcinoma.
SEQ ID NO:6 is differentially expressed more than two-fold in mucinous cystadenoma of the ovary when compared to expression in cytologically normal OVARNOT07, OVARNOT13, and OVARNOTO2 and in ovary tissue from a subject diagnosed with adenocarcinoma. SEQ ID NO:6 when used in a ovary-specific assay, is diagnostic for mucinous cystadenoma.
SEQ ID NO:7 is preferentially expressed in IL-1 treated chrondrocytes cultured from knee cartilage, in cartilage and synovia from subjects with rheumatoid and osteoarthritis. It was not expressed in normal control CARGNOT01. SEQ ID NO:7, when used in a tissue-specific assay, is diagnostic for arthritis.
SEQ ID NO:8 was significantly expressed in testicular seminoma; it was not expressed in normal tissue from TESTNOC01, TESTNOF01, TESTNOM01, TESTNON04, TESTNOP01, TESTNOT01, TESTNOT03, TESTNOT04, TESTNOT07, TESTNOT10, and TESTNOT11, or in embryonal carcinomas from TESTTUE02 and TESTTUT02. SEQ ID NO:7, when used in a clinically relevant, testicle-specific assay, is a diagnostic for testicular seminoma.
SEQ ID NO:9 was specifically expressed in prostate tissue cytologically showing adenofibromatous hyperplasia and matched with adenocarcinoma of the prostate (see PROSTUT matches above). It was not expressed in tissues from subjects diagnosed with benign prostatic hyperplasia (PROSBPS05, PROSBPT03, PROSDIP01, PROSDIP02, and PROSDIP03), or prostatic IN (PROETMP06, PROETMP07). SEQ ID NO:9, when used in a prostate-specific assay, is diagnostic for AH and may serve as an early diagnostic marker for prostatic adenocarcinoma.
SEQ ID NO:10 showed five-fold differential expression in a cytologically normal bladder library which was matched with transitional cell carcinoma of the bladder. Expression of SEQ ID NO:10 was clearly distinct from that seen in tissue affected by chronic cystitis and was not seen in normal tissues, BLADNOR01, BLADNOT01, BLADNOT03, BLADNOT04, BLADNOT06, and BLADNOT08 or in the tumor libraries, BLADTUE01, BLADTUT02, BLADTUT03, BLADTUT04, BLADTUT05, BLADTUT06, BLADTUT07 and BLADTUT08, SEQ ID NO:10, when used in a bladder-specific assay, serves as an early diagnostic marker for transitional cell carcinoma of the bladder.
SEQ ID NO:11 is expressed in renal cell cancers and not expressed in cytologically normal kidney libraries (KIDNNOT01, KIDNNOT02, KIDNNOT20, KIDNNOT25, KIDNNOT26, KIDNNOT31, KIDNNOT32) or in KIDPTDE01 from a subject diagnosed with interstitial nephritis. SEQ ID NO:10, when used in a kidney-specific assay, serves as a diagnostic for renal cell cancer.
SEQ ID NO:12 is expressed in adenocarcinoma of the breast and not expressed in cytologically normal matched tissue. SEQ ID NO:12, when used in a breast-specific assay including, but not limited to, ductal lavage, serves as a diagnostic for adenocarcinoma of the breast.
SEQ ID NO:13 is highly overexpressed in the pituitary gland removed from a schizophenic subject with chronic pulmonary pulmonary disease. Such high expression levels were not seen in pooled normal tissue or in the pituitaries of subjects with cancers and Alzheimer's disease (AD). SEQ ID NO:13, when used in a tissue-specific assay, serves as a diagnostic for schizophrenia.
SEQ ID NO:14 is differentially expressed in breast cancer, in particular, in lobular carcinoma. When used in a breast-specific assay including, but not limited to, ductal lavage, SEQ ID NO:14 serves as a diagnostic for breast cancer.
SEQ ID NO:15 is 4-fold differentially expressed in peripheral blood, particularly eosinophils of asthmatics. When used in an assay of a lung sample, SEQ ID NO:15 is a diagnostic for asthma.
The transcript image confirms the information obtained in the original northern analysis (7 Nov. 1997). SEQ ID NO:17 is expressed in adenocarcinoma of the breast and not expressed in cytologically normal matched tissue, BRSTNOT14. Expression was absent from BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries. When used in a breastspecific assay, including, but not limited to, ductal lavage, and compared with cancerous and normal standards, expression of SEQ ID NO:17 is diagnostic for breast adenocarcinoma.
In assays using normal and cancerous standards and patient samples, the cDNA, an mRNA, or an antibody specifically binding the protein can serve a clinically relevant diagnostic marker for disorders associated with cell proliferation and cell signaling.
IX Northern AnalysesSEQ ID NOs:1-15 and 17 were compared with all the other sequences in the LIFESEQ database (Incyte Genomics, Palo Alto Calif.) using BLAST analysis (Altschul (1993) supra); Altschul(1990) Supra). The results of the BLAST analyses were reported in THE INVENTION section above.
Each of the Incyte clones is also used to screen northern blots. A probe is generated by EcoR1 digestion of the plasmid containing the cDNA. The restriction digest is fractionated on a 1% agarose gel, a restriction fragment from about 400 to about 1400 nt in length is excised from the gel and purified on a QIAQUICK column (Qiagen). The fragment is comprised of the 5′ most region of the insert. The probe is prepared by random priming using the REDIPRIME labeling kit (APB) with REDIVUE [−32P]d-CTP (3000 Ci/mmol; APB). Unincorporated radioactivity is removed by column chromatography using a SEPHADEX G-50 NICK column (APB).
Each commercial MTN blot (Clontech) contained approximately 2 ug of poly A+ per lane from various tissues. Otherwise, RNA was electrophoresed on a denaturing formaldehyde, 1.2% agarose gel, blotted on a nylon membrane, and fixed by UV irradiation.
Blots are pre-hybridized in RAPID-HYB hybridization buffer (APB) for 1 hour at 65 C. Hybridizations are performed at 65 C using 0.5×10 cpm/ml probe for 1 hour. Blots are washed for 2×10 minutes in 1×SSC, 0.1% SDS at room temperature followed by 2 stringent washes at 65 C in 0.2×SSC, 0.1% SDS for 10 minutes each. Blots are wrapped in SARAN WRAP plastic film (Dow Chemical, Midland Mich.) and autoradiographed at −70 C using 2 intensifying screens and HYPERFILM-MP (APB).
The northern analysis for SEQ ID NO:17, Incyte clone 2547002, performed Wednesday, 5 Nov. 1997 showed expression in the following libraries of the LIFESEQ database (Incyte Genomics).
When used in a breast sample specific assay and compared with cancerous and normal standards, SEQ ID NO:17 is diagnostic for breast adenocarcinoma (bold above)
X Complementary MoleculesMolecules complementary to the cDNA, from about 5 (PNA) to about 5000 bp (complement of a cDNA insert), are used to detect or inhibit gene expression. Detection is described in Example VII. To inhibit transcription by preventing promoter binding, the complementary molecule is designed to bind to the most unique 5′ sequence and includes nucleotides of the 5′ UTR upstream of the initiation codon of the open reading frame. Complementary molecules include genomic sequences (such as enhancers or introns) and are used in “triple helix” base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. To inhibit translation, a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein.
Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy. Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system.
Stable transformation of dividing cells with a vector encoding the complementary molecule produces a transgenic cell line, tissue, or organism (U.S. Pat. No. 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough complementary molecules to compromise or entirely eliminate activity of the cDNA encoding the protein.
XI Protein ExpressionExpression and purification of the protein are achieved using either a mammalian or an insect cell expression system. The pUB61V5-His vector system (Invitrogen, Carlsbad Calif.) is used to express signal peptide-containing proteins in CHO cells. The vector contains the selectable bsd gene, multiple cloning sites, the promoter/enhancer sequence from the human ubiquitin C gene, a C-terminal V5 epitope for antibody detection with anti-V5 antibodies, and a C-terminal polyhistidine (6×His) sequence for rapid purification on PROBOND resin (Invitrogen). Transformed cells are selected on media containing blasticidin.
Spodoptera frueiperda (Sf9) insect cells are infected with recombinant Autographica californica nuclear polyhedrosis virus (baculovirus). The polyhedrin gene is replaced with the cDNA by homologous recombination and the polyhedrin promoter drives cDNA transcription. The protein is synthesized as a fusion protein with 6× his which enables purification as described above. Purified protein is used in the following activity and to make antibodies.
XII Production of AntibodiesA signal peptide-containing protein is purified using polyacrylamide gel electrophoresis and used to immunize mice or rabbits. Antibodies are produced using the protocols well known in the art and summarized below. Alternatively, the amino acid sequence of signal peptide-containing proteins is analyzed using LASERGENE software (DNASTAR) to determine regions of high antigenicity. An antigenic epitope, usually found near the C-terminus or in a hydrophilic region is selected, synthesized, and used to raise antibodies. Typically, epitopes of about 15 residues in length are produced using an 431A peptide synthesizer (ABI) using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase antigenicity.
Rabbits are immunized with the epitope-KLH complex in complete Freund's adjuvant. Immunizations are repeated at intervals thereafter in incomplete Freund's adjuvant. After a minimum of seven weeks for mouse or twelve weeks for rabbit, antisera are drawn and tested for antipeptide activity.
Testing involves binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. Methods well known in the art are used to determine antibody titer and the amount of complex formation.
XIII Purification of Naturally Occurring Protein Using Specific AntibodiesNaturally occurring or recombinant protein is purified by immunoaffinity chromatography using antibodies which specifically bind the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected.
XIV Screening Molecules for Specific Binding with the cDNA or ProteinThe cDNA, or fragments thereof, or the protein, or portions thereof, are labeled with 3 P-dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or with BIODIPY or FITC (Molecular Probes, Eugene Oreg.), respectively. Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled cDNA or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.
XV Two-Hybrid ScreenA yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories, Palo Alto Calif.), is used to screen for peptides that bind the protein of the invention. A cDNA encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coli cDNA, prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli construct a cDNA library. The pLexA plasmid and pB42AD-cDNA library constructs are isolated from E. coli and used in a 2:1 ratio to co-transform competent yeast EGY48[p8oplacZ] cells using a polyethylene glycol/lithium acetate protocol. Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (His), tryptophan (Trp), and uracil (Ura), and incubated at 30 C until the colonies have grown up and are counted. The colonies are pooled in a minimal volume of 1×TE (pH 7.5), replaced on SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies. Interaction between expressed protein and cDNA fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu). Interaction also activates expression of β-galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal.
Positive interactions between expressed protein and cDNA fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trpf-Ura liquid medium for 1 to 2 days at 30 C. A sample of the culture is plated on SD/-Trp/-Ura media and incubated at 30 C until colonies appear. The sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SD containing histidine but not on media lacking histidine have lost the pLexA plasmid. Histidinerequiring colonies are grown on SD/GallRaf/X-Gal/-Trp/-Ura, and white colonies are isolated and propagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding a protein that physically interacts with the protein, is isolated from the yeast cells and characterized.
XVI Demonstration of Protein ActivityCell Proliferation
SP can be expressed in a mammalian cell line such as DLD-1 or HCT116 (ATCC; Manassas Va.) by transforming the cells with a eukaryotic expression vector encoding SF. Other eukaryotic expression vectors, such as those mentioned in EXAMPLE XI above, are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. The effect of SP on cell morphology can be visualized by microscopy; the effect on cell growth can be determined by measuring cell doubling-time; and the effect on tumorigenicity can be assessed by the ability of transformed cells to grow in a soft agar growth assay (Groden et al. (1995) Cancer Res. 55:1531-1539).
Receptor SPs such as those encoded by SEQ ID NOs: 17, 15, 12, 6, and I can be expressed in heterologous expression systems and their biological activity tested utilizing the purinergic receptor system (P2u) as published by Erb et al. (1993; Proc Natl Acad Sci 90:10449-53). Because cultured K562 human leukemia cells lack P2U receptors, they can be transfected with expression vectors containing either normal or chimeric P2u and loaded with fura-a, fluorescent probe for Ca++. Activation of properly assembled and functional extracellular SP-transmembrane/intracellular P2U receptors with extracellular UTP or ATP mobilizes intracellular Ca++ which reacts with fura-a and is measured spectrofluorometrically. Bathing the transfected K562 cells in microwells containing appropriate ligands will trigger binding and fluorescent activity identifying effectors of SP. The P2u system is also useful for identifying antagonists or inhibitors which block binding and prevent such fluorescent reactions.
All patents and publications mentioned in the specification are incorporated by reference herein. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.
Claims
1. A purified protein comprising an amino acid sequence selected from:
- a) a protein having an amino acid sequence of SEQ ID NO:16,
- b) a naturally occurring protein having an amino acid sequence at least 90% identical to an amino acid sequence of SEQ ID NO:16,
- c) a biologically active fragment of a protein having an amino acid sequence of SEQ ID NO: 16, and
- d) an epitope of a protein having an amino acid sequence of SEQ ID NO:16.
2. A purified protein of claim 1, consisting of an amino acid sequence of SEQ ID NO:16.
3. A composition comprising a protein of claim 1, and a labeling moiety or a pharmaceutical carrier.
4. A method for using a protein to screen a plurality of molecules or compounds to identify at least one ligand, the method comprising:
- a) combining a protein of claim 1, with the molecules or compounds under conditions to allow specific binding; and
- b) detecting specific binding, thereby identifying a ligand which specifically binds the protein.
5. The method of claim 4, wherein the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs.
6. An isolated antibody which specifically binds to a protein of claim 1.
7. A method of using a protein to prepare and purify a polyclonal antibody comprising:
- a) immunizing a animal with a protein of claim 1, under conditions to elicit an antibody response;
- b) isolating animal antibodies;
- c) attaching the protein to a substrate;
- d) contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein;
- e) dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies.
8. A method of using a protein to prepare a monoclonal antibody comprising:
- a) immunizing a animal with a protein of claim 1, under conditions to elicit an antibody response;
- b) isolating antibody producing cells from the animal;
- c) fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells;
- d) culturing the hybridoma cells; and
- e) isolating from culture monoclonal antibodies which specifically bind the protein.
9. A polyclonal antibody produced by the method of claim 7.
10. A monoclonal antibody produced by the method of claim 8.
11. A method for using an antibody to detect expression of a protein in a sample, the method comprising:
- a) combining the antibody of claim 6, with a sample under conditions which allow the formation of antibody:protein complexes; and
- b) detecting complex formation, wherein complex formation indicates expression of the protein in the sample.
12. The method of claim 11, wherein complex formation is compared with standards and is diagnostic of cancer.
13. A composition comprising an antibody of claim 6, and a therapeutic agent.
14. A method for testing a compound for effectiveness as an agonist, the method comprising:
- a) exposing a sample comprising a protein of claim 1, to a compound, and
- b) detecting agonist activity in the sample.
15. A composition comprising an agonist identified by the method of claim 13, and a pharmaceutical carrier.
16. A method for testing a compound for effectiveness as an antagonist, the method comprising:
- a) exposing a sample comprising a protein of claim 1, to a compound, and
- b) detecting antagonist activity in the sample.
17. A composition comprising an antagonist identified by the method of claim 16, and a pharmaceutical carrier.
18. A method for treating a disorder associated with decreased expression of a signal peptide-containing protein comprising administering to a subject in need of such treatment the composition of claim 15.
19. A method for treating a disorder associated with increased expression of a signal peptide-containing protein comprising administering to a subject in need of such treatment the composition of claim 17.
20. An array containing the antibody of claim 6.
Type: Application
Filed: Feb 8, 2013
Publication Date: Sep 19, 2013
Applicant: Incyte Corporation (Wilmington, DE)
Inventors: Preeti G. LAL (Santa Clara, CA), Janice Au-Young (Brisbane, CA), Roopa Reddy (Sunnyvale, CA), Lynn E. Murry (Fayetteville, AR), Preete Mathur (Fremont, CA)
Application Number: 13/763,102
International Classification: C07K 14/435 (20060101);
