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WO2002028999A2 - Profils d'expression genique dans les cellules granulocytaires - Google Patents

Profils d'expression genique dans les cellules granulocytaires Download PDF

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Publication number
WO2002028999A2
WO2002028999A2 PCT/US2001/030821 US0130821W WO0228999A2 WO 2002028999 A2 WO2002028999 A2 WO 2002028999A2 US 0130821 W US0130821 W US 0130821W WO 0228999 A2 WO0228999 A2 WO 0228999A2
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mrna
human
complete cds
gene
protein
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WO2002028999A3 (fr
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Yasmin Beazer-Barclay
Sherman M. Weissman
Shigeru Yamaga
Joseph Vockley
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Ore Pharmaceuticals Inc
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Ore Pharmaceuticals Inc
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Publication of WO2002028999A3 publication Critical patent/WO2002028999A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Granulocytes i.e., neutrophils, eosinophils and basophils
  • Inflammation is a localized protective response elicited by injury or destruction of tissues which serves to destroy, dilute or wall off both the injurious agent and the injured tissue. It is characterized by fenestration of the microvasculature, leakages of the elements of blood into the interstitial spaces, and migration of leukocytes into the inflamed tissue. On a macroscopic level, this is usually accompanied by the familiar clinical signs of erythema, edema, tenderness (hyperalgesia), and pain.
  • Inflammation is initiated by, among other things, trauma, tissue necrosis, infection or immune reactions.
  • the immediate response is temporary vasoconstriction.
  • vascularconstriction is followed within seconds by the acute vascular response resulting in increased blood flow (hyperemia) and edema.
  • the acute phase is also characterized by the margination of polymorphonuclear white blood cells (neutrophils) next to endothelial cells, followed by emigration of neutrophils into the adjacent tissue. Margination is recognized by the lining up of neutrophils along the endothelium of vessels. Emigration occurs by passage of the inflammatory cells between endothelial cells.
  • Neutrophils are the first wave of cellular attack on invading organisms and are the characteristic cells of acute inflammation.
  • the appearance of neutrophils in areas of iriflarnmation may be caused by chemicals released from bacteria, factors produced nonspecifically from necrotic tissue or antibody reacting with antigen.
  • Neutrophils use an actin-rich cytoskeleton to move in a directed manner along a chemotactic gradient from the bloodstream to an inflammatory site where they ingest particles (e.g. bacteria) and immune complexes bearing IgG (via FcR) and/or breakdown products of the complement component C3.
  • Neutrophils belong to a category of white blood cells known as polymorphonuclear white blood cells.
  • the blood cells with single nuclei form the white blood cell population that includes macrophages, T and B cells.
  • White blood cells that contain segmented nuclei are broadly classified as polymorphonuclear.
  • Polymo ⁇ honuclear white blood cells are further subdivided into three major populations on the basis of the staining properties of their cytoplasmic granules in standard hematologic smears or tissue preparations: neutrophils staining pink, eosinophils staining red and basophils staining blue.
  • Neutrophils also referred to as polymorphonuclear neutrophils-PMNs
  • WBCs white blood cells
  • neutrophils are produced from precursor cells in the bone marrow and released into the blood when mature. After entering the circulation, neutrophils are thought to last only 1 or 2 days.
  • Neutrophils are characterized by numerous cytoplasmic granules that contain highly destructive enzymes that must be kept isolated from the cytoplasm. These granules contain a number of oxygen-independent enzymes as well as oxygen-dependent mechanisms of killing.
  • neutrophils Upon attraction to sites of inflammation, neutrophils attempt to engulf and digest bacteria coated with antibody and complement. Phagocytosis by neutrophils is also usually accompanied by release of the lysosomal enzymes into the tissue spaces, particularly if the organism is difficult for the neutrophil to digest.
  • At least three cytoplasmic granules are identifiable in neutrophils: specific granules containing lactoferrin, B cytochrome, the complement receptor CR3 and ⁇ 2 -integrin; azurophilic granules containing acid hydrolases and other enzymes; and a third granule containing gelatinase.
  • neutrophils and other granulocytic cells play in immune response to pathogens, including bacterial infection
  • neutrophils and other granulocytic cells play an unwanted role in many chronic inflammatory diseases.
  • diseases are characterized by massive neutrophil infiltration, such as psoriasis, inflammatory bowel disease, Crohn's disease, asthma, cardiac and renal reperfusion injury, adult respiratory distress syndrome, rheumatoid arthritis, thrombosis and glomerulonephritis. All of these diseases are associated with increased IL-8 production which may be responsible for the chemotaxis of neutrophils into the inflammatory site.
  • neutrophils synthesize de novo important macromolecules including, but not limited to interleukin (TL) 1, 11-6, 11-8, tumor necrosis factor (TNF ), granulocyte and macrophage colony-stimulating factors, interferon (IFN ), intercellular adhesion molecule (ICAM-1) and membrane and cystoskeletal molecules, such as major histocompatibility class I antigens and actin (Beaulieu et al (1992) J. Biolog. Chem. 267(l):426-432; Arnold et al. (1993) Infect. Immun. 61(6):2545-2552; and Eisner et al. (1995) Immunobiol 193:456-464).
  • TNF tumor necrosis factor
  • IFN interferon
  • IAM-1 intercellular adhesion molecule
  • membrane and cystoskeletal molecules such as major histocompatibility class I antigens and actin (Beaulieu et al (1992) J. Biolog. Chem. 267(l
  • Eosinophils are another granulocytic or polymorphonuclear white blood cell that are involved in the inflammatory response. Eosinophils are found predominately in two types of inflammation: allergy and parasite infections.
  • Eosinophils The role of eosinophils in the host response to parasites is thought to be mediated through the components of the eosinophilic granules. Eosinophils are cytotoxic to schistosome larvae through an antibody-dependent cell-mediated mechanism. Eosinopliil cationic proteins are highly toxic for schistosomes and may be responsible for binding of eosinophils to parasitic worms as well as fragmentation of the parasite.
  • eosinophils The role of eosinophils in acute inflammation is not fully understood. On one hand, there is evidence that enzymes in eosinophils may serve to limit the extent of inflammation by neutralizing mediators of anaphylaxis, such as LTC4 , histamine and platelet-activating factor. On the other hand, there is increasing evidence that cationic proteins in eosinophilic granules are mediators of acute inflammation. Eosinopliil activation is associated with acute tissue injury and cause an intense vasoconstriction in lung microvasculature, followed by increased pulmonary vascular permeability and pulmonary edema.
  • Basophils or mast cells are the other major cell type characterized as a granulocytic or polymorphonuclear white blood cell.
  • Mast cells contain granules with a variety of biologically active agents which, when released extracellularly (degranulation), cause dilation of the smooth muscle of arterioles (vasodilation), increased blood flow, and contraction of endothelial cells, thereby opening up vessel walls to permit egress of antibodies, complement or inflammatory cells into tissue spaces.
  • the present invention identifies the global changes in gene expression associated with the activation of granulocytic cells.
  • the present invention also identifies expression profiles which serve as useful diagnostic markers as well as markers that can be used to monitor disease states, disease progression, drug toxicity, drug efficacy and drug metabolism.
  • the present inventors have systematically assessed the transcriptional response from granulocytic cells activated through contact with a pathogen or from granulocytic cells isolated from a subject with a sterile inflammatory disease.
  • the present invention provides a method of detecting granulocyte activation comprising detecting the level of expression in a sample of one or more genes from Tables 2-8 and comparing the expression level to an expression level in an un- activated granulocyte, wherein differential expression of the genes in Tables 2-8 is indicative of granulocyte activation.
  • the present invention also provides a method of modulating granulocyte activation comprising contacting a granulocyte with an agent, wherein the agent alters the expression of at least one gene in Tables 2-8 thereby modulating granulocyte activation.
  • the present invention provides a method of screening for an agent capable of modulating granulocyte activation comprising preparing a first gene expression profile of a cell population comprising granulocytes, wherein the expression profile determines the expression level of one or more genes from Tables 2-8, exposing the cell population to the agent, preparing a second gene expression profile of the agent-exposed cell population and comparing the first and second gene expression profiles.
  • the present invention provides a method of detecting inflamation in a tissue comprising detecting the level of expression in a sample of the tissue of one or more genes from Tables 2-8; wherein the level of expression of the genes in Tables 2-8 is indicative of inflammation.
  • the present invention also provides a method of treating inflammation in a tissue comprising contacting a tissue undergoing n inflammatory response with an agent, wherein the agent alters the expression in the tissue of at least one gene in Tables 2-8 thereby treating the inflammation.
  • the present invention provides a method of screening for an agent capable of modulating inflammation in a tissue comprising preparing a first gene expression profile of a sample of the tissue, wherein the expression profile determines the expression level of one or more genes from Tables 2-8, exposing the tissue to the agent, preparing second gene expression profile of the agent-exposed tissue and comparing the first and second gene expression profiles.
  • the present invention provides a method of detecting a chronic inflamation in a tissue comprising detecting the level of expression in a sample of the tissue of one or more genes from Tables 2-8, wherein the level of expression of the genes in Tables 2-8 is indicative of a chronic inflammation.
  • the present invention also provides a method of treating a chronic inflammation in a tissue comprising contacting a tissue having a chronic inflammation with an agent, wherein the agent alters the expression in the tissue of at least one gene in Tables 2-8 thereby treating the chronic inflammation.
  • the present invention provides a method of screening for an agent capable of modulating a chronic inflammation in a tissue comprising preparing a first gene expression profile of a sample of the tissue, wherein the expression profile determines the expression level of one or more genes from Tables 2-8, exposing the tissue to the agent, preparing second gene expression profile of the agent-exposed tissue and comparing the first and second gene expression profiles.
  • Some embodiments of the present invention provide a method of detecting an allergic response in a subject comprising obtaining a sample from the subject, the sample comprising granulocytes, preparing a gene expression profile of the sample, wherein the expression profile determines the expression level of one or more genes from Tables 2-8, comparing the expression level to an expression level in a sample from a normal individual, wherein differential expression of the genes in Tables 2-8 is indicative of an allergic response.
  • the invention also provides a method of treating an allergic response in a subject comprising administering to the subject an agent, wherein the agent alters the expression in the tissue of at least one gene in Tables 2-8 thereby treating the allergic response, h a related embodiment, the present invention provides a method of screening for an agent capable of modulating an allergic response in a subject comprising preparing a first gene expression profile of a sample from the subject wherein the expression profile determines the expression level of one or more genes from Tables 2-8, administering to the subject an agent, preparing a second gene expression profile of a sample from the agent- exposed subject and comparing the first and second gene expression profiles.
  • the present invention is a method of detecting exposure of a subject to a pathogen comprising preparing a first gene expression profile of a granulocyte population from the subject wherein the expression profile determines the expression level of one or more genes from Tables 2-8, comparing the first gene expression profile to a second gene expression profile from a granulocyte population exposed to the pathogen and to a third gene expression profile from a granulocyte population not exposed to the pathogen, and determining whether the subject was exposed to the pathogen.
  • the invention provides a method of treating a subject exposed to a pathogen comprising administering to the subject an agent, wherein the agent affects the expression of at least one gene in Tables 2-8 thereby treating the subject
  • the invention provides a method of screening for an agent that modulates a response of a granulocyte population to a pathogen comprising preparing a first gene expression profile of a first sample from the granulocyte population wherein the expression profile determines the expression level of one or more genes from Tables 2-8, exposing a second sample of the granulocyte population to a pathogen and preparing a second gene expression profile from the second sample, contacting the pathogen-exposed granulocyte population with an agent and preparing a third gene expression profile from the agent- contacted pathogen-exposed population, comparing the first, second and third gene expression profiles and identifying agents that modulate the response of a granulocyte population to the pathogen.
  • the present invention provides a method of detecting a sterile inflammatory disease in a subject comprising detecting the level of expression in a sample from the subject of one or more genes from Tables 2-8 wherein the level of expression of the genes in Tables 2-8 is indicative of a sterile inflammatory disease.
  • the present invention provides a method of treating a sterile inflammatory disease in a subject comprising contacting the subject with an agent wherein the agent alters the expression in the tissue of at least one gene in Tables 2-8 thereby treating the sterile inflammatory disease.
  • the present invention is a method of screening for an agent capable of modulating a sterile inflammatory disease in a subject comprising preparing a first gene expression profile of a sample from the subject wherein the expression profile determines the expression level of one or more genes from Tables 2- 8, exposing the subject to the agent, preparing a second gene expression profile of a sample obtained from the agent-exposed subject and comparing the first and second gene expression profiles.
  • the present invention provides a composition comprising at least two ohgonucleotides wherein each of the ohgonucleotides comprises a sequence that specifically hybridizes to a gene in Tables 2-8. In some preferred embodiments, the invention provides compositions comprising at least 3, 4, 5, 6, 7, 8, 9 or 10 or more ohgonucleotides wherein each of the ohgonucleotides comprises a sequence that specifically hybridizes to a gene in Tables 2-8.
  • At least one oligonucleotide is attached to a solid support which may be a membrane, a glass support, a filter, a tissue culture dish, a polymeric material, a bead, a silica support or any other solid support known to those skilled in the art.
  • a solid support which may be a membrane, a glass support, a filter, a tissue culture dish, a polymeric material, a bead, a silica support or any other solid support known to those skilled in the art.
  • the present invention provides a solid support comprising at least two ohgonucleotides wherein each of the ohgonucleotides comprises a sequence that specifically hybridizes to a gene in Tables 2-8.
  • the ohgonucleotides maybe attached covalently or non-covalently to the solid support and a given support may comprise both covalently attached and non-covalently attached ohgonucleotides.
  • the solid supports of the present invention may comprise ohgonucleotides attached at varying densities, for example, at least 10 different ohgonucleotides may be attached in discrete locations per square centimeter, at least 100 different ohgonucleotides maybe attached in discrete locations per square centimeter, at least 1,000 different ohgonucleotides maybe attached in discrete locations per square centimeter, at least 10,000 different ohgonucleotides may be attached in discrete locations per square centimeter.
  • the present invention also provides a computer system comprising a database containing information identifying an expression level in a cell population comprising granulocytes of a set of genes comprising at least two genes in Tables 2-8 and a user interface to view the information.
  • the computer system of the present invention may further comprise sequence information for the genes and/or information identifying the expression level for the set of genes in a cell population comprising non-activated granulocytes and/or information identifying the expression level of the set of genes in a cell population comprising activated granulocytes.
  • the computer system of the present invention may comprise records including descriptive information from an external database (for example, GenBank), which information correlates said genes to records in the external database.
  • the present invention also includes methods of using a computer system to present information identifying the expression level in a tissue or cell of at least one gene in Tables 2-8 comprising comparing the expression level of at least one gene in Tables 2-8 in the tissue or cell to the level of expression of the gene in the database.
  • the methods may include comparison of the expression levels of 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genes in Tables 2-8.
  • the methods may comprise displaying the level of expression of at least one gene in the tissue or cell sample compared to the expression level in a cell population comprising activated granulocytes.
  • the present invention also includes a method of identifying virulence factor genes in a pathogen by preparing a first gene expression profile of a quiescent granulocyte population, preparing a second gene expression profile of a granulocyte population exposed to a virulent or avirulent bacterial strain, preparing a third gene expression profile from a granulocyte population exposed to a bacterial strain with a mutation in a putative bacterial virulence factor gene, comparing the first, second and third gene expression profiles and identifying a bacterial virulence factor gene.
  • RNA processing e.g., through control of initiation, provision of RNA precursors, RNA processing, etc.
  • translational control e.g., through control of initiation, provision of RNA precursors, RNA processing, etc.
  • fundamental biological processes such as cell cycle, cell differentiation and cell death, are often characterized by the variations in the expression levels of groups of genes.
  • Changes in gene expression also are associated with pathogenesis.
  • changes in the expression levels of particular genes e.g., oncogenes, tumor suppressors, cytokines and the like
  • Monitoring changes in gene expression may also provide certain advantages during drug screening development. Often drugs are screened and prescreened for the ability to interact with a major target without regard to other effects the drugs have on cells. Often such other effects cause toxicity in the whole animal, which prevent the development and use of the potential drug.
  • the present inventors have examined two sets of cell populations comprising quiescent and activated granulocytes to identify the global changes in gene expression associated with granulocyte, and in particular neutrophil, activation. These global changes in gene expression, also referred to as expression profiles, provide useful markers for diagnostic uses as well as markers that can be used to monitor disease states, disease progression, drug toxicity, drug efficacy and drug metabolism.
  • Expression profiles of genes in particular tissues, disease states or disease progression stages provide molecular tools for evaluating toxicity, drug efficacy, drug metabolism, development, and disease monitoring. Changes in the expression profile from a baseline profile can be used as an indication of such effects.
  • Those skilled in the art can use any of a variety of known techniques to evaluate the expression of one or more of the genes and/or ESTs identified in the instant application in order to observe changes in the expression profile.
  • the response of neutrophils to pathogens, including bacterial pathogens is a subject of primary importance in view of the need to find ways to modulate the immune response to infection.
  • neutrophils Similarly, the response of neutrophils to agonists (pro-inflammatory molecules) is a subject of primary importance in view of the need to find better ways of controlling inflammation in various disease states.
  • One means of assessing the response of neutrophils to pathogens and agonists is to measure the ability of neutrophils to synthesize specific RNA de novo upon contact with the pathogen or agonist.
  • Granulocytic cells also known as polymorphonuclear white blood cells
  • neutrophils also known as polymorphonuclear neutrophils or peripheral blood neutrophils, eosinophils, and basophils, also referred to a mast cells.
  • pathogen refers to any infectious organism including bacteria, viruses, parasites, mycoplasma, protozoans, and fungi (including molds and yeast).
  • Pathogenic bacteria include, but are not limited to Staphylococci (e.g. aureus), Streptococci (e.g. pneurnoniae), Clostridia (e.g. perfringens), Neisseria (e.g. gonorrhoeae), Enterobacteriaceae (e.g. coli as well as Klebsiella, Salmonella, Shigella, Yersinia and Proteus), Helicobacter (e.g. pylori), Vibrio (e.g.
  • cholerae Campylobacter (e.g. jejuni), Pseudomonas (e.g. aeruginosa), Haemophilus (e.g. influenzae), Bordetella (e.g. pertussis), Mycoplasma (e.g. pneurnoniae), Ureaplasma (e.g. urealyticum), Legionella (e.g. pneumophila), Spirochetes (e.g. Treponema, Leptospira and Borrelia), Mycobacteria (e.g. tuberculosis, smegmatis), Actinomyces (e.g. (israelii), Nocardia (e.g. asteroides),
  • Chlamydia e.g. trachomatis
  • Rickettsia Coxiella
  • Ehrilichia e.g. trachomatis
  • Rochalimaea e.g. Brucella
  • Brucella e.g. Brucella
  • Yersinia e.g. Brucella
  • Fracisella e.g. Yersinia
  • Pasteurella e.g. trachomatis
  • sterile inflammatory disease refers to any inflammatory disease caused by immune or nonimmune mechanisms not directly linked to infection (see Stewart et al).
  • sterile inflammatory diseases include, but are not limited to psoriasis, rheumatoid arthritis, glomerulonephritis, asthma, cardiac and renal reperfusion injury, thrombosis, adult respiratory distress syndrome, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis and periodontal disease.
  • solid support refers to any support to which nucleic acids can be bound or immobilized.
  • Preferred solid supports include, but are not limited to, nitrocellulose, nylon, glass, polymeric material, other solid supports which are positively charged and nanochannel glass arrays disclosed by Beattie (WO 95/1175).
  • Solid supports may be in any convenient form including, but not limited to, a membrane, a filter, a tissue culture dish, a strip, a bead and the like.
  • gene expression profile also referred to as a "differential expression profile” or “expression profile” refers to any representation of the expression of at least one mRNA species in a cell sample or population. A gene expression profile may be used to detect the level of expression of one or more genes of interest.
  • the present invention provides compositions and methods to detect the level of expression of genes that may be differentially expressed dependent upon the state of the cell, i. e., quiescent versus activated.
  • the phrase "detecting the level of expression” is seen to include determining whether a gene of interest is expressed at all.
  • an assay which provides a yes or no result without necessarily providing quantification of an amount of expression is seen to be an assay that requires "detecting the level of expression” as that phrase is used herein.
  • a gene expression profile can refer to an autoradiograph of labeled cDNA fragments produced from total cellular mRNA separated on the basis of size by known procedures. Such procedures include slab gel electrophoresis, capillary gene electrophoresis, high performance liquid chromatography, and the like. Digitized representations of scanned electrophoresis gels are also included as are two and three dimensional representations of the digitized data.
  • a gene expression profile also can be prepared using "DNA chip” technology as described below.
  • oligonucleotide sequences that are complementary to one or more of the genes described herein refers to ohgonucleotides that are capable of hybridizing under stringent conditions to at least part of the nucleotide sequence of said genes. Such hybridizable ohgonucleotides will typically exhibit at least about 75% sequence identity at the nucleotide level to said genes, preferably about 80% or 85% sequence identity or more preferably about 90% or 95% or more sequence identity to said genes.
  • Bind(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
  • background or “background signal intensity” refer to hybridization signals resulting from non-specific binding, or other interactions, between the labeled target nucleic acids and components of the oligonucleotide array (e.g., the oligonucleotide probes, control probes, the array substrate, etc.). Background signals may also be produced by intrinsic fluorescence of the array components themselves.
  • a single background signal can be calculated for the entire array, or a different background signal may be calculated for each target nucleic acid.
  • background is calculated as the average hybridization signal intensity for the lowest 5% to 10% of the probes in the array, or, where a different background signal is calculated for each target gene, for the lowest 5% to 10% of the probes for each gene.
  • the probes to a particular gene hybridize well and thus appear to be specifically binding to a target sequence, they should not be used in a background signal calculation.
  • background may be calculated, as the average hybridization signal intensity produced by hybridization to probes that are not complementary to any sequence found in the sample (e.g., probes directed to nucleic acids of the opposite sense or to genes not found in the sample such as bacterial genes where the sample is mammalian nucleic acids). Background can also be calculated as the average signal intensity produced by regions of the array that lack any probes at all.
  • hybridizing specifically to refers to the binding, duplexing or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • mismatch control or "mismatch probe” refer to a probe whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence.
  • MM mismatch
  • PM perfect match
  • the mismatch may comprise one or more bases.
  • mismatch(s) may be located anywhere in the mismatch probe, terminal mismatches are less desirable as a terminal mismatch is less likely to prevent hybridization of the target sequence, hi a particularly preferred embodiment, the mismatch is located at or near the center of the probe such that the mismatch is most likely to destabilize the duplex with the target sequence under the test hybridization conditions.
  • perfect match probe refers to a probe that has a sequence that is perfectly complementary to a particular target sequence.
  • the test probe is typically perfectly complementary to a portion (subsequence) of the target sequence.
  • the perfect match (PM) probe can be a "test probe”, a "normalization control” probe, an expression level control probe and the like.
  • a perfect match control or perfect match probe is, however, distinguished from a “mismatch control” or “mismatch probe.”
  • a "probe” is defined as a nucleic acid, capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, U, C or T) or modified bases (7- deazaguanosine, inosine, etc.).
  • the bases in probes may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • stringent conditions refers to conditions under which a probe will hybridize to its target subsequence, but with only insubstantial hybridization to other sequences or to other sequences such that the difference may be identified. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • the "percentage of sequence identity” or “sequence identity” is determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the portion of the polynucleotide sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical submit (e.g., nucleic acid base or amino acid residue) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Percentage sequence identity when calculated using the programs GAP or BESTFIT (see below) is calculated using default gap weights.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blastx, tblastn and tblastx Karlin et al, (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268 and Altschul, (1993) J. Mol. Evol. 36, 290-300, fully incorporated by reference
  • the approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance.
  • granulocyte-containing sample from a subject may be assayed by any of the methods described herein, and the expression levels from a gene or genes from the Tables, in particular the genes in Tables 2-8, may be compared to the expression levels found in activated and/or quiescent granulocytes.
  • the samples obtained from subjects with a disease affecting granulocyte activation may be compared to similar samples from normal subjects. Differences and/or similarities of the expression profiles maybe used to diagnose diseases. Comparison of the expression data, as well as available sequence or other information may be done by researcher or diagnostician or may be done with the aid of a computer and databases as described herein.
  • the genes and gene expression information provided in Tables 2-8 may also be used as markers for the monitoring of disease progression, for instance, the progress of an infection or a sterile inflammatory disease.
  • a granulocyte- containing sample from a subject may be assayed by any of the methods described herein, and the expression levels in the sample from a gene or genes from Tables 2-8 may be compared to the expression levels found in activated and/or quiescent granulocytes.
  • Expression profiles generated from a granulocyte-containing sample from normal or diseased subjects may be used, for instance, to monitor disease progression. Comparison of the expression data, as well as available sequence or other information may be done by researcher or diagnostician or may be done with the aid of a computer and databases as described herein.
  • the genes identified in Tables 2-8 may be used as markers to evaluate the effects of a candidate drug or agent on a cell, particularly a cell undergoing an inflammatory response.
  • a candidate drug or agent can be screened for the ability to simulate the transcription or expression of a given marker or markers or to down- regulate or counteract the transcription or expression of a marker or markers.
  • Agents that are assayed in the methods described herein can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the a protein of the invention alone or with its associated substrates, binding partners, etc.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.
  • the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Dominant negative proteins, DNA encoding these proteins, antibodies to these proteins, peptide fragments of these protems or mimics of these proteins may be introduced into cells to affect function. "Mimic” as used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide (see Grant, (1995) in Molecular Biology and Biotechnology Meyers (editor) VCH Publishers). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • the genes identified as being differentially expressed in quiescent versus activated granulocytes may be used in a variety of nucleic acid detection assays to detect or quantititate the expression level of a gene or multiple genes in a given sample. For example, traditional Northern blotting, nuclease protection, RT-PCR and differential display methods may be used for detecting gene expression levels. Those methods are useful for some embodiments of the invention.
  • Gene expression profiles can be produced by any means known in the art, including, but not limited to the methods disclosed by: Liang et al. (1992) Science 257:967-971; Ivanova et al. (1995) Nucleic Acids Res. 23:2954-2958; Guilfoyl et al.
  • gene expression profiles are produced by the methods of Prashar et al (WO 97/05286) and Prashar et al. (1996) Proc. Natl. Acad. Sci. USA 93:659-663.
  • gene expression profiles as described herein are made to identify one or more genes whose expression levels are modulated in an activated granulocytic cell population such as one exposed to a pathogen or isolated from a subject having a sterile inflammatory disease.
  • the assaying of the modulation of gene expression via the production of a gene expression profile may involve the production of cDNA from polyA RNA (mRNA) isolated from granulocytes as described below .
  • mRNAs are isolated from a granulocytic cell source.
  • the cells may be obtained from an in vivo source, such as a peripheral blood.
  • any granulocytic cell type may be used, however, neutrophils are preferred.
  • peripheral blood cells that are initially obtained may be subjected to various separation techniques (e.g., flow cytometry, density gradients).
  • mRNAs are isolated from cells by any one of a variety of techniques. Numerous techniques are well known (see e.g., Sambrook et al, Molecular Cloning: A Laboratory Approach, Cold Spring harbor Press, NY, 1987; Ausubel et., Current Protocols in Molecular Biology, Greene Publishing Co. NY, 1995). In general, these techniques first lyse the cells and then enrich for or purify RNA. In one such protocol, cells are lysed in a Tris-buffered solution containing SDS. The lysate is extracted with phenol/chloroform, and nucleic acids are precipitated.
  • RNAs may, however, be purified from crude preparations of nucleic acids or from total RNA by chromatography, such as binding and elution from oligo(dT)- cellulose or poly(U)-Sepharose®. As stated above, other protocols and methods for isolation of RNAs may be substituted.
  • RNA-directed DNA polymerase such as reverse transcriptase isolated from AMV, MoMuLV or recombinantly produced.
  • RNA-directed DNA polymerase such as reverse transcriptase isolated from AMV, MoMuLV or recombinantly produced.
  • Many commercial sources of enzyme are available (e.g., Pharmacia, New England Biolabs, Stratagene Cloning Systems). Suitable buffers., cofactors, and conditions are well known and supplied by manufacturers (see also, Sambrook et al, supra; Ausubel et al, supra).
  • Various ohgonucleotides are used in the production of cDNA. h particular, the methods utilize oligonucleotide primers for cDNA synthesis, adapters, and primers for amplification.
  • Ohgonucleotides are generally synthesized as single strands by standard chemistry techniques, including automated synthesis. Ohgonucleotides are subsequently de-protected and may be purified by precipitation with ethanol, chromatographed using a sized or reversed-phase column, denaturing polyacrylamide gel electrophoresis, high- pressure liquid chromatography (HPLC), or other suitable method.
  • a functional group such as biotin
  • a biotin moiety may be inco ⁇ orated at any position in the oligonucleotide, for example, at the 5'- or 3'- terminal nucleotide or at internal nucleotide positions.
  • biotinylated oligonucleotide may be synthesized using pre-coupled nucleotides, or alternatively, biotin may be conjugated to the oligonucleotide using standard chemical reactions.
  • Other functional groups such as florescent dyes, radioactive molecules, digoxigenin, and the like, may also be inco ⁇ orated.
  • Partially-double stranded adaptors are formed from single stranded ohgonucleotides by annealing complementary single-stranded ohgonucleotides that are chemically synthesized or by enzymatic synthesis. Following synthesis of each strand, the two oligonucleotide strands are mixed together in a buffered salt solution (e.g., 1 M NaCl, 100 mM Tris-HCl pH.8.0, 10 mM EDTA) or in a buffered solution containing Mg 2+ (e.g., 10 mM MgCl 2 ) and annealed by heating to high temperature and slow cooling to room temperature.
  • a buffered salt solution e.g., 1 M NaCl, 100 mM Tris-HCl pH.8.0, 10 mM EDTA
  • Mg 2+ e.g., 10 mM MgCl 2
  • the oligonucleotide primer that primes first strand DNA synthesis comprises a 5' sequence incapable of hybridizing to a polyA tail of the mRNAs, and a 3' sequence that hybridizes to a portion of the polyA tail of the mRNAs and at least one non-polyA nucleotide immediately upstream of the polyA tail.
  • the 5' sequence is preferably a sufficient length that can serve as a primer for amplification.
  • the 5' sequence also preferably has an average G+C content and does not contain large palindromic sequence; some palindromes, such as a recognition sequence for a restriction enzyme, may be acceptable. Examples of suitable 5' sequences are CTCTCAAGGATCTACCGCT (SEQ ID No. 1370), CAGGGTAGACGACGCTACGC (SEQ ID No. 1371), and TAATACCGCGCCACATAGCA (SEQ ID No. 1372).
  • the 5' sequence is joined to a 3' sequence comprising sequence that hybridizes to a portion of the polyA tail of mRNAs and at least one non-polyA nucleotide immediately upstream.
  • the polyA-hybridizing sequence is typically a homopolymer of dT or dU, it need only contain a sufficient number of dT or dU bases to hybridize to polyA under the conditions employed. Both oligo-dT and oligo-dU primers have been used and give comparable results. Thus, other bases may be interspersed or concentrated, as long as hybridization is not impeded. Typically, 12 to 18 bases or 12 to 30 bases of dT or dU will be used.
  • the non-polyA nucleotide is A, C, or G, or a nucleotide derivative, such as inosinate. If one non-polyA nucleotide is used, then three oligonucleotide primers are needed to hybridize to all mRNAs. If two non-polyA nucleotides are used, then 12 primers are needed to hybridize to all mRNAs. The 12 primers would have 3 '-terminal sequences capable of hybridizing to the two nucleotides immediately preceding the polyA tail of the mRNA, i.
  • mRNAs are either subdivided into three (if one non- polyA nucleotide is used) or 12 (if two non-polyA nucleotides are used) fractions, each containing a single oligonucleotide primer, or the primers may be pooled and contacted with a mRNA preparation. Other subdivisions may alternatively be used.
  • first strand cDNA is initiated from the oligonucleotide primer by reverse transcriptase (RTase).
  • RTase reverse transcriptase
  • RTase may be obtained from numerous sources and protocols are well known.
  • Second strand synthesis may be performed by RTase (Gubler and Hoffman, Gene 25: 263, 1983), which also has a DNA-directed DNA polymerase activity, with or without a specific primer, by DNA polymerase 1 in conjunction with RNaseH and DNA ligase, or other equivalent methods.
  • RTase Gubler and Hoffman, Gene 25: 263, 1983
  • DNA polymerase 1 in conjunction with RNaseH and DNA ligase, or other equivalent methods.
  • the double-stranded cDNA is generally treated by phenol: chloroform extraction and ethanol precipitation to remove protein and free nucleotides.
  • Double-stranded cDNA is subsequently digested with an agent that cleaves in a sequence-specific manner.
  • cleaving agents include restriction enzymes. Restriction enzyme digestion is preferred; enzymes that are relatively infrequent cutters (e.g., 5 bp recognition site) are preferred and those that leave overhanging ends are especially preferred.
  • a restriction enzyme with a six base pair recognition site cuts approximately 8% of cDNAs, so that approximately 12 such restriction enzymes should be needed to digest every cDNA at least once. By using 30 restriction enzymes, digestion of every cDNA is assured.
  • the adapters for use in the present invention are designed such that the two strands are only partially complementary and only one of the nucleic acid strands that the adapter is ligated to can be amplified.
  • the adapter is partially double-stranded (i.e., comprising two partially hybridized nucleic acid strands), wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other.
  • the adapter is "Y-shaped" or "bubble- shaped.” When the 5' region is non-paired, the 3' end of other strand cannot be extended by a polymerase to make a complementary copy.
  • the ligated adapter can also be blocked at the 3' end to eliminate extension during subsequent amplifications.
  • Blocking groups include dideoxynucleotides or any other agent capable of blocking the 3'-OH.
  • the non-complementary portion of the upper strand of the adapters is preferably a length that can serve as a primer for amplification.
  • the non-complementary portion of the lower strand need only be one base, however, a longer sequence is preferable (e.g., 3 to 20 bases; 3 to 15 bases; 5 to 15 bases; or 14 to 24 bases).
  • the complementary portion of the adapter should be long enough to form a duplex under conditions of litigation.
  • the non-complementary portion of the upper strands is preferably a length that can serve as a primer for amplification.
  • this portion is preferably 15 to 30 bases.
  • the adapter can have a structure similar to the Y- shaped adapter, but has a 3' end that contains a moiety that a DNA polymerase cannot extend from.
  • Amplification primers are also used in the present invention. Two different amplification steps are performed in the preferred aspect, hi the first, the 3' end
  • the sequence of the single primer comprises at least a portion of the 5' sequence of the oligonucleotide primer used for first strand cDNA synthesis.
  • the portion need only be long enough to serve as an amplification primer
  • the primer pair consists of a first primer whose sequence comprises at least a portion of the 5' sequence of the oligonucleotide primer as described herein; and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion.
  • the primer will generally contain all the sequence of the non-complementary potion, but may contain less of the sequence, especially when the non-complementary portion is very long, or more of the sequence, especially when the non-complementary portion is very short.
  • the primer will contain sequence of the complementary portion, as long as that sequence does not appreciably hybridize to the other strand of the adapter under the amplification conditions employed, for example, in one embodiment, the primer sequence comprises four bases of the complementary region to yield a 19 base primer, and amplification cycles are performed at 56 °C (annealing temperature), 72 °C (extension temperature), and 94 °C (denaturation temperature).
  • the primer is 25 bases long and has 10 bases of sequence in the complementary portion. Amplification cycles for this primer are performed at 68 °C (annealing and extension temperature) and 94 °C (denaturation temperature). By using these longer primers, the specificity of priming is increased.
  • the design of the amplification primers will generally follow well-known guidelines, such as average G-C content, absence of hanpin structures, inability to form primer-dimers and the like. At times, however, it will be recognized that deviations from such guidelines may be appropriate or desirable.
  • the lengths of the amplified fragments are determined. Any procedure that separate nucleic acids on the basis of size and allows detection or identification of the nucleic acids is acceptable. Such procedures include slap get electrophoresis, capillary gel electrophoresis, high performance liquid chromatography, and the like.
  • Electrophoresis is technique based on the mobility of DNA in an electric flied. Negatively charged DNA migrates towards a positive electrode at a rate dependent on their total charge, size, and shape. Most often, DNA is electrophoresed in agarose or polyacrylamide gels. For maximal resolution, polyacrylamide is preferred and for maximal linearity, a denaturant, such as urea is present.
  • a typical get setup uses a 19:1 mixture of acrylamide:bisacrylamide and a Tris-borate buffer. DNA samples are denatured and applied to the gel, which is usually sandwiched between glass plates. A typical procedure can be found in Sambrook et al (Molecular Cloning: A Laboratory
  • Capillary electrophoresis in its various manifestations (free solution, isotachophoresis, isoelectric focusing, polyacrylamide get. micellar electrokinetic "chromatography") allows high resolution separation of very small sample volumes.
  • a neutral coated capillary such as a 50 ⁇ m X 37 cm column (eCAP neutral, Beckman Instruments, CA)
  • a linear polyacrylamide e.g. 0.2% polyacrylamide
  • a sample is introduced by high-pressure injection followed by an injection of running buffer (e.g., IX TBE).
  • running buffer e.g., IX TBE
  • Capillaries may be used in parallel for increased throughput (Smith et al. (1990) Nuc. Acids. Res. 18:4417; Mathies and Huang (1992) Nature 359:167). Because of the small sample volume that can be loaded onto a capillary, a sample may be concentrated to increase level of detection.
  • concentration is sample stacking (Chien and Burgi (1992) Anal. Chem 64:489A). In sample stacking, a large volume of sample in a low concentration buffer is introduced to the capillary column. The capillary is then filled with a buffer of the same composition, but at higher concentration, such that when the sample ions reach the capillary buffer with a lower electric field, they stack into a concentrated zone.
  • HPLC High-performance liquid chromatography
  • HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting an aliquot of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase.
  • IP-RO-HPLC on non-porous PS/DNB particles with chemically bonded alkyl chains can also be used to analyze nucleic acid molecules on the basis of size (Huber et al (1993) Anal Biochem. 121:351; Huber et al. (1993) Nuc. Acids Res. 21:1061; Huber et al (1993) Biotechniques 16:898). In each of these analysis techniques, the amplified fragments are detected.
  • a variety of labels can be used to assist in detection. Such labels include, but are not limited to, radioactive molecules (e.g., S, P, P) fluorescent molecules, and mass spectrometric tags.
  • the labels may be attached to the oligonucleotide primers or to nucleotides that are inco ⁇ orated during D ⁇ A synthesis, including amplification.
  • Radioactive nucleotides may be obtained from commercial sources; radioactive primers may be readily generated by transfer of label from ⁇ - P-ATP to a 5'-OH group by a kinase (e.g., T4 polynucleotide kinase).
  • Detection systems include autoradiograph, phosphor image analysis and the like.
  • Fluorescent nucleotides maybe obtained from commercial sources (e.g., ABI, Foster city, CA) or generated by chemical reaction using appropriately derivatized dyes. Oligonucleotide primers can be labeled, for example, using succinimidyl esters to conjugate to amine-modified ohgonucleotides. A variety of florescent dyes maybe used, including 6 carboxyfluorescein, other carboxyfluorescein derivatives, carboxyrhodamine derivatives, Texas red derivatives, and the like. Detection systems include photomultiplier tubes with appropriate wave-length filters for the dyes used. DNA sequence analysis systems, such as produced by ABI (Foster City, CA), may be used.
  • cDNA fragments which correspond to differentially expressed mRNA species are isolated, reamplified and sequenced according to standard procedures. For instance, bands conesponding the cDNA fragments can be cut from the electrophoresis gel, reamplified and subcloned into any available vector, including pCRscript using the PCR script cloning kit (Stratagene). The insert is then sequenced using standard procedures, such as cycle sequencing on an ABI sequencer.
  • gene expression profiles may be prepared using a hybridization assay format. Any hybridization assay format may be used, including solution-based and solid support-based assay formats.
  • Oligonucleotide probe anays for expression monitoring can be made and used according to any techniques known in the art (see for example, Lockhart et al, (1996) Nat. Biotechnol. 14, 1675-1680; McGall et al, (1996) Proc. Nat. Acad. Sci. USA 93, 13555- 13460).
  • Such probe anays may contain at least two or more ohgonucleotides that are complementary to or hybridize to two or more of the genes described herein.
  • Such anays may also contain ohgonucleotides that are complementary or hybridize to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 70 or more the genes described herein.
  • Assays and methods of the invention may utilize available formats to simultaneously screen at least about 100, preferably about 1000, more preferably about 10,000 and most preferably about 1,000,000 different nucleic acid hybridizations.
  • the genes which are assayed according to the present invention are typically in the form of mRNA or reverse transcribed mRNA.
  • the genes may be cloned or not and the genes may be amplified or not. The cloning itself does not appear to bias the representation of genes within a population. However, it may be preferable to use polyA+ RNA as a source, as it can be used with less processing steps.
  • sequences of the expression marker genes are in the public databases, i. e., GenBank.
  • Tables 2-8 provide the GenBank Accession numbers and name for each of the sequences.
  • the sequences of the genes in GenBank have been submitted on an electronic medium in computer readable form in compliance with Al ⁇ 801(a) of the PCT and are expressly inco ⁇ orated by reference as are identical or related sequences with difference GenBank numbers.
  • Assays to monitor the expression of a marker or markers as defined in Tables 2-8 may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention.
  • an agent is said to modulate the expression of a nucleic acid of the invention if it is capable of up- or down-regulating expression of the nucleic acid in a cell.
  • gene chips containing probes to at least two genes from Tables 2-8 may be used to directly monitor or detect changes in gene expression in the treated or exposed cell as described in more detail above.
  • cell lines that contain reporter gene fusions between the open reading frame of a gene in Tables 2-8 and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al, (1990) Anal. Biochem. 188, 245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of the nucleic acid.
  • Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a gene identified in Tables 2-8.
  • mRNA expression may be monitored directly by hybridization of probes to the nucleic acids of the invention.
  • Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al, (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press).
  • cells or cell lines are first identified which express the gene products of the invention physiologically.
  • Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface fransduction mechanisms and/or the cytosolic cascades.
  • such cells or cell lines may be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5'- promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag.
  • an expression vehicle e.g., a plasmid or viral vector
  • the agent comprises a pharmaceutically acceptable excipient and is contacted with cells comprised in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and serum incubated at 37°C.
  • PBS phosphate buffered saline
  • BSS Eagles balanced salt solution
  • Said conditions may be modulated as deemed necessary by one of skill in the art.
  • the cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot).
  • immunological assay e.g., ELISA, immunoprecipitation or Western blot.
  • the pool of proteins isolated from the agent- contacted sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent-contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.
  • Another embodiment of the present invention provides methods for identifying agents that modulate at least one activity of a protein(s) encoded by the genes in Tables 2- 8. Such methods or assays may utilize any means of monitoring or detecting the desired activity.
  • the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed.
  • probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations.
  • Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
  • Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe, such as a specific antibody.
  • the high density array will typically include a number of probes that specifically hybridize to the sequences of interest. See WO 99/32660 for methods of producing probes for a given gene or genes.
  • the array will include one or more control probes.
  • Test probes may be ohgonucleotides that range from about 5 to about 45 or 5 to about 500 nucleotides, more preferably from about 10 to about 40 nucleotides and most preferably from about 15 to about 40 nucleotides in length. In other particularly prefened embodiments the probes are 20 or 25 nucleotides in length, hi another prefened embodiment, test probes are double or single strand DNA sequences. DNA sequences are isolated or cloned from natural sources or amplified from natural sources using natural nucleic acid as templates. These probes have sequences complementary to particular subsequences of the genes whose expression they are designed to detect. Thus, the test probes are capable of specifically hybridizing to the target nucleic acid they are to detect.
  • Probes based on the sequences of the genes described herein may be prepared by any commonly available method. Oligonucleotide probes for assaying the tissue or cell sample are preferably of sufficient length to specifically hybridize only to appropriate, complementary genes or transcripts. Typically the oligonucleotide probes will be at least 10, 12, 14, 16, 18, 20 or 25 nucleotides in length, h some cases longer probes of at least 30, 40, or 50 nucleotides will be desirable.
  • the high density array can contain a number of control probes.
  • the control probes fall into three categories refened to herein as (1) normalization controls; (2) expression level controls; and (3) mismatch controls.
  • Normalization controls are oligonucleotide or other nucleic acid probes that are complementary to labeled reference ohgonucleotides or other nucleic acid sequences that are added to the nucleic acid sample.
  • the signals obtained from the normalization controls after hybridization provide a control for variations in hybridization conditions, label intensity, "reading" efficiency and other factors that may cause the signal of a perfect hybridization to vary between arrays, h a prefened embodiment, signals (e.g. , fluorescence intensity) read from all other probes in the anay are divided by the signal (e.g., fluorescence intensity) from the control probes thereby normalizing the measurements.
  • any probe may serve as a normalization control.
  • Prefened normalization probes are selected to reflect the average length of the other probes present in the array, however, they can be selected to cover a range of lengths.
  • the normalization control(s) can also be selected to reflect the (average) base composition of the other probes in the array, however in a prefened embodiment, only one or a few probes are used and they are selected such that they hybridize well (i.e., no secondary structure) and do not match any target-specific probes.
  • Expression level controls are probes that hybridize specifically with constitutively expressed genes in the biological sample. Virtually any constitutively expressed gene provides a suitable target for expression level controls. Typical expression level control probes have sequences complementary to subsequences of constitutively expressed "housekeeping genes" including, but not limited to the ⁇ -actin gene, the transferrin receptor gene, the GAPDH gene, and the like.
  • Mismatch controls may also be provided for the probes to the target genes, for expression level controls or for normalization controls.
  • Mismatch controls are oligonucleotide probes or other nucleic acid probes identical to their conesponding test or control probes except for the presence of one or more mismatched bases.
  • a mismatched base is a base selected so that it is not complementary to the corresponding base in the target sequence to which the probe would otherwise specifically hybridize.
  • One or more mismatches are selected such that under appropriate hybridization conditions (e.g., stringent conditions) the test or control probe would be expected to hybridize with its target sequence, but the mismatch probe would not hybridize (or would hybridize to a significantly lesser extent).
  • Prefened mismatch probes contain a central mismatch.
  • a conesponding mismatch probe will have the identical sequence except for a single base mismatch (e.g., substituting a G, a C or a T for an A) at any of positions 6 through 14 (the central mismatch).
  • Mismatch probes thus provide a control for non-specific binding or cross hybridization to a nucleic acid in the sample other than the target to which the probe is directed.
  • Mismatch probes also indicate whether a hybridization is specific or not. For example, if the target is present the perfect match probes should be consistently brighter than the mismatch probes. In addition, if all central mismatches are present, the mismatch probes can be used to detect a mutation. The difference in intensity between the perfect match and the mismatch probe (I(PM) - I(MM>) provides a good measure of the concentration of the hybridized material.
  • nucleic acid samples used in the methods and assays of the invention may be prepared by any available method or process. Methods of isolating total mRNA are also well known to those of skill in the art. For example, methods of isolation and purification of nucleic acids are described in detail in Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I Theory and Nucleic Acid Preparation, Tijssen, (1993) (editor) Elsevier Press. Such samples include RNA samples, but also include cDNA synthesized from a mRNA sample isolated from a cell or tissue of interest. Such samples also include DNA amplified from the cDNA, and an RNA transcribed from the amplified DNA. One of skill in the art would appreciate that it is desirable to inhibit or destroy RNase present in homogenates before homogenates can be used.
  • Biological samples may be of any biological tissue or fluid or cells from any organism as well as cells raised in vitro, such as cell lines and tissue culture cells. Frequently the sample will be a "clinical sample" which is a sample derived from a subject. In some prefened embodiments, subjects may be mammalian, preferably human. Typical clinical samples include, but are not limited to, sputum, blood, blood-cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
  • Biological samples may also include sections of tissues, such as frozen sections or formalin fixed sections taken for histological pu ⁇ oses.
  • Solid supports containing oligonucleotide probes for differentially expressed genes of the invention can be filters, polyvinyl chloride dishes, silicon or glass based chips, etc.
  • An solid or semi-solid material conventionally used to immobilize nucleic acids may be used.
  • Solid supports containing oligonucleotide probes for differentially expressed genes of the invention can be filters, polyvinyl chloride dishes, sihcon or glass based chips, etc.
  • Such wafers and hybridization methods are widely available, for example, those disclosed by Beattie (WO 95/11755). Any solid surface to which ohgonucleotides can be bound, either directly or indirectly, either covalently or non-covalently, can be used.
  • a prefened solid support is a high density array or DNA chip. These contain a particular oligonucleotide probe in a predetermined location on the array. Each predetermined location may contain more than one molecule of the probe, but each molecule within the predetermined location has an identical sequence. Such predetermined locations are termed features. There maybe, for example, from 2, 10, 100, 1000 to 10,000; 100,000 or 400,000 of such features on a single solid support. The solid support, or the area within which the probes are attached may be on the order of a square centimeter.
  • oligonucleotide analogue array can be synthesized on a solid substrate by a variety of methods, including, but not limited to, light-directed chemical coupling, and mechanically directed coupling (see Pirrung et al, (1992) U.S. Patent No. 5J43, 854; Fodor et al, (1998) U.S. Patent No. 5,800,992; Chee et al, (1998) 5,837,832 h brief, the light-directed combinatorial synthesis of oligonucleotide arrays on a glass surface proceeds using automated phosphoramidite chemistry and chip masking tecliniques.
  • a glass surface is derivatized with a silane reagent containing a functional group, e.g., a hydroxyl or amine group blocked by a photolabile protecting group.
  • a functional group e.g., a hydroxyl or amine group blocked by a photolabile protecting group.
  • Photolysis through a photolithogaphic mask is used selectively to expose functional groups which are then ready to react with incoming 5' photoprotected nucleoside phosphoramidites.
  • the phosphoramidites react only with those sites which are illuminated (and thus exposed by removal of the photolabile blocking group).
  • the phosphoramidites only add to those areas selectively exposed from the preceding step. These steps are repeated until the desired array of sequences have been synthesized on the solid surface.
  • Combinatorial synthesis of different oligonucleotide analogues at different locations on the anay is determined by the pattern of illumination during synthesis and the order of addition of coupling reagents
  • High density nucleic acid arrays can also be fabricated by depositing premade or natural nucleic acids in predetermined positions. Synthesized or natural nucleic acids are deposited on specific locations of a substrate by light directed targeting and oligonucleotide directed targeting. Another embodiment uses a dispenser that moves from region to region to deposit nucleic acids in specific spots.
  • Nucleic acid hybridization simply involves contacting a probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing (see Lockhart et al, (1999) WO 99/32660). The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label.
  • nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids.
  • low stringency conditions e.g., low temperature and/or high salt
  • hybrid duplexes e.g., DNA-DNA, RNA-RNA or RNA-DNA
  • specificity of hybridization is reduced at lower stringency.
  • higher stringency e.g., higher temperature or lower salt
  • successful hybridization requires fewer mismatches.
  • hybridization conditions may be selected to provide any degree of stringency, i a prefened embodiment, hybridization is performed at low stringency, in this case in 6x SSPE-T at 37°C (0.005% Triton x-100) to ensure hybridization and then subsequent washes are performed at higher stringency (e.g., lx SSPE-T at 37°C) to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency (e.g., down to as low as 0.25x SSPET at 37°C to 50°C until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide.
  • Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present (e.g., expression level control, normalization control, mismatch controls, etc.).
  • controls e.g., expression level control, normalization control, mismatch controls, etc.
  • the wash is performed at the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity.
  • the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular oligonucleotide probes of interest.
  • the hybridized nucleic acids are typically detected by detecting one or more labels attached to the sample nucleic acids.
  • the labels may be inco ⁇ orated by any of a number of means well known to those of skill in the art (see Lockhart et al, (1999) WO 99/32660).
  • the present invention includes relational databases containing sequence information, for instance for the genes of Tables 2-8, as well as gene expression information in various granulocyte-containing samples.
  • Databases may also contain information associated with a given sequence or tissue sample such as descriptive information about the gene associated with the sequence information, or descriptive information concerning the clinical status of the tissue sample, or the subject from which the sample was derived.
  • the database may be designed to include different parts, for instance a sequences database and a gene expression database. Methods for the configuration and construction of such databases are widely available, for instance, see Akerblom et al, (1999) U.S. Patent 5,953,727, which is herein inco ⁇ orated by reference in its entirety.
  • the databases of the invention may be linked to an outside or external database.
  • the external database is GenBank and the associated databases maintained by the National Center for Biotechnology Information (NCBI).
  • Any appropriate computer platform may be used to perform the necessary comparisons between sequence information, gene expression information and any other information in the database or provided as an input.
  • a large number of computer workstations are available from a variety of manufacturers, such has those available from Silicon Graphics.
  • Client-server environments, database servers and networks are also widely available and appropriate platforms for the databases of the invention.
  • the databases of the invention maybe used to produce, among other things, electronic Northerns to allow the user to determine the cell type or tissue in which a given gene is expressed and to allow determination of the abundance or expression level of a given gene in a particular tissue or cell.
  • the databases of the invention may also be used to present information identifying the expression level in a tissue or cell of a set of genes comprising at least one gene in Tables 2-8 comprising the step of comparing the expression level of at least one gene in Tables 2-8 in the tissue to the level of expression of the gene in the database.
  • Such methods may be used to predict the physiological state of a given tissue by comparing the level of expression of a gene or genes in Tables 2-8 from a sample to the expression levels found in tissue from normal liver, malignant liver or hepatocellular carcinoma. Such methods may also be used in the drug or agent screening assays as described below.
  • RNA expression levels from neutrophils exposed to various pathogens offer a powerful means of identifying genes that are specifically regulated in response to infection.
  • the production of expression profiles from neutrophils exposed to E. coli and Y. pestis allow the identification of neutrophil genes that are specifically regulated in response to bacterial infection.
  • Neutrophils may be isolated from normal donor peripheral blood following any protocol known to those skilled in the art. The LPS-free method of isolation is described below. Peripheral blood is isolated using a butterfly needle and a syringe containing 5 cc ACD, 5 cc of 6% Dextran (in normal saline).
  • HBSS without Ca " * ""1” or Mg
  • the plasma was centrifuged (1500 ⁇ m, for 15 m at 4°C), the supernatant decanted and cold HBSS added to resuspend the cells.
  • the cell suspension was then layered onto a cold Ficoll Hypaq, centrifuged at 500xg for 30m at 4°C.
  • the pellet contains polymo ⁇ honuclear neutrophils. Neutrophils can also be isolated by other commonly used methods such as those disclosed in Current Protocols of Immunology (John Wiley & Sons, Inc.), Babior et al. (1981) ⁇ n:Leokocyte Function, Cline, MJ. ⁇ d., p.1-38 (Church Livingstone, NY), and Haslett et al. (1985) Am. J. Pathol. 119:101-110.
  • neutrophils were incubated with E. coli or one of three strains of 7. pestis ypoH, KTM5 or KIM6 for 30 minutes or two hours and then total RNA was isolated using a standard guanidine «HCl method.
  • bacteria are harvested and washed in phosphate buffered saline and opsonized with either autologous human serum or complement factor C7 deficient human serum (SIGMA).
  • SIGMA complement factor C7 deficient human serum
  • Incubation was at a ratio of approximately a PMN:bacteria ratio of 1 :20 in RPMI 1640 (H ⁇ P ⁇ S buffered) with heat inactivated Fetal Bovine Serum at 37°C with gentle mixing in a rotary shaker bath.
  • LPS bacterial lipopolysaccharide
  • latex beads bacterial lipopolysaccharide
  • LPS was added to approximately 3.38 x 10 8 cells in 100 ml of RPMI containing 6% autologous serum to a final concentration of 1 ng/ml to 1 ⁇ g/1. Incubation proceeded for two hours with gentle rotation in disposable polycarbonate ⁇ rlenmeyer flasks at 37°C. After incubation, the cells were spun down and washed once with HBSS and frozen until RNA isolation.
  • the neutrophils extracted from blood were examined for purity by flow microfluorometry. Preparations with >0.5% monocytes contamination were rejected. Samples of mRNA were later examined for specific expression markers for induced monocytes to bacterial exposure.
  • the neutrophils were cultured with the non-pathogenic bacteria, E. coli, or three pathogenic strains of Yersinia pestis, KTM5, KIM6, and yopH (Perry et al.(1991) Clin. Microbiology Reviewsl0(l):35-66), respectively, and after 2 hours total RNA was extracted by the standard guanidine «HCl method.
  • RNA was processed for the Affymetrix oligonucleotide GeneChip microarrays following Affmetrix's protocol.
  • the final product, cRNA was hybridized on the 42K array set (a combination of the full-length genes and EST's) and the HuGU95A array, containing ⁇ 12,000 full length known genes.
  • the data was analyzed to determine present/absent calls, gene expression levels, and expression differences.
  • a gene identified as present or absent has been calculated by an algorithm in the Affymetrix analysis software. Gene expression levels have been measured as average differences. Gene expression changes have been calculated as the ratios of the expressed genes in uninduced/induced neutrophils. Expression differences with a ratio of ⁇ > 3 fold have been analyzed.
  • RNA yield for each sample was 200-500 ⁇ g.
  • mRNA was isolated using the Oligotex mRNA Midi kit (Qiagen). Since the mRNA was eluted in a final volume of 400 ⁇ l, an ethanol precipitation step was required to bring the concentration to 1 ⁇ g/ ⁇ l. Using 1-5 ⁇ g of mRNA, double stranded cDNA was created using the Superscript Choice system (Gibco- BRL).
  • First strand cDNA synthesis was primed with a T7-(dT 4 ) oligonucleotide.
  • the cDNA was then phenol-chloroform extracted and ethanol precipitated to a final concentration of 1 ⁇ g/ ⁇ l.
  • cRNA was synthesized using Ambion' s T7 MegaScript in vitro Transcription Kit.
  • nucleotides Bio-11-CTP and Bio- 16- UTP Enzo Diagnostics
  • the labeled cRNA was cleaned up according to the Rneasy Mini kit protocol (Qiagen).
  • the cRNA was then fragmented (5x fragmentation buffer: 200 mM Tris- Acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc) for thirty-five minutes at 94°C.
  • Hybridization to the probe arrays was detected by fluorometric scanning (Hewlett Packard Gene Array Scanner). Following hybridization and scanning, the microanay images were analyzed for quality confrol, looking for major chip defects or abnormalities in hybridization signal. After all chips passed QC, the data was analyzed using Affymetrix GeneChip software (v3.0), and Experimental Data Mining Tool (EDMT) software (vl.O).
  • EDMT Experimental Data Mining Tool
  • Each chip contains 16-20 oligonucleotide probe pairs per gene or cDNA clone. These probe pairs include perfectly matched sets and mismatched sets, both of which are necessary for the calculation of the average difference.
  • the average difference is a measure of the intensity difference for each probe pair, calculated by subtracting the intensity of the mismatch from the intensity of the perfect match. This takes into consideration variability in hybridization among probe pairs and other hybridization artifacts that could affect the fluorescence intensities. Using the average difference value that has been calculated, the GeneChip software then makes an absolute call for each gene or EST.
  • 1182 genes have been identified to be present in the uninduced neutrophils. In neutrophils exposed to bacteria, the number of genes present generally decreased, hi neutrophils exposed to E. coli 819 genes were called present. In neutrophils exposed to 7. pestis strain yopH 698 genes were identified and those exposed to strain KIM5 expressed 696 genes, hi contrast, neutrophils exposed to KIM6 expresssed 1258 genes (Table 1).
  • a comparison of the genes called present in the three 7. pestis exposed neutrophil populations identified 526 genes as present in all three. 192 genes were switched on or off, with 121 of those with a ratios > 3.
  • a comparison of all four bacteria-exposed neutrophil populations identified 428 genes that were called present in both E. coli and the three 7. pestis induced neutrophils.
  • a number of genes were identified by the comparison of the different induction conditions. Fourteen genes were called absent in uninduced neutrophils and present in all bacteria-exposed neutrophils (Table 2). Twelve genes were called absent in uninduced neutrophils and E. coli exposed neutrophils, and present in the three Y. pestis strains exposed neutrophils (Table 3) and thus were specifically induced by contact with 7. pestis. 135 genes were called absent in uninduced neutrophils, present in E. coli exposed neutrophils, and showed variable expression in the three different 7. pestis exposed neutrophils (Table 4).
  • TRAF3 TRAF3 receptor-associated factor 3
  • PAC1 Dual specificity phosphatase 2
  • MAP phosphorylation and subsequent activation are important for signal fransduction of growth factors.
  • DUSP2 down regulates intracellular signal fransduction through the dephosphorylation of MAP kinases.
  • Solute carrier family & cationic amino acid transporter, y+ system
  • member 5 SLC7A5
  • GRO2 gene encodes a cytokine involved with inflammatory response and growth regulation (Haskill et al. (1990) Proc. Natl Acad. Sci. 87 '-.1132-1136).
  • Cyclin-dependent kinase inhibitor lA(p21, Cipl) (CDK ⁇ 1 A), is an inhibitor of Gl cyclin-dependent kinases ( ⁇ l-Deiry et al. (1993) Cell 75:817-825).
  • CD44 antigen (CD44) is up regulated in induced lymphoblastoid cell line, KCA ( ⁇ l-Deiry et al (1993) Cell 75:817-825).
  • Colony stimulating factor 3 (granulocyte) (CSF3) has been identified in haematopoietic cell proliferation and differentation (Dougherty et al. (1991) J. Exp. Med 174: 1-5).
  • Pentaxin-related gene rapidly induced by IL-1 beta (PTX3) is an inflammatory cytokine identified in stimulated fibroblast cell lines (Souza et al. (1986) Science 232:61- 65).
  • Nuclear factor erythroid-derived 2), 45kD (NFE2) has been identified in hematopoietic cell lines (Lee et al. (1992) J. Cell Biol. 116:545-557).
  • beta 2 (antigenCDl ⁇ (p95), lymphocyte function-associated antigen 1; macrophage antigen 1 (mac-1) beta subunit) (ITGB2) has been identified with cell surface signaling (Pischedda et al. (1995) Proc. Natl Acad. Sci. 92:3511-3515).
  • Table 7 A complete list of all genes identified in bacteria-exposed neutrophils is presented in Table 7. The table also provides the ratio of the expression observed in the bacteria- exposed neutrophils to the expression level in quiescent neutrophils.
  • Genes differentially expressed in quiescent neutrophils as compared to neutrophils exposed to bacteria are genes that are responsive to an induction from various sources.
  • genes discussed are genes that are specific to cellular induction. Genes not expressed in E. coli exposed neutrophils but expressed in 7 pestis exposed neutrophils are genes which may make the cell susceptible to infection.
  • the 7. pestis bacterium is pathogenic triggering gene expression of genes that inhibit the phagocytic response in neutrophils. Genes expressed in E. coli but not in 7 pestis exposed neutrophils provide another set of genes that are affected by the pathogenic capacity of 7 petis.
  • the genes that were down regulated when neutrophils were exposed to bacteria are genes involved in progression of cell development. One of the many neutrophilic responses to bacteria is the suppression of genes involved in normal cell cycle, this allows the cell to respond to the infection.
  • genes in Tables 2-8 allow one skilled in the art to select an appropriate set of genes in order to assay for exposure to a specific bacterium or strain, hi addition those skilled in the art can select an appropriate gene set from the list of affected genes to conduct assays for agents that modulate the activation response of bacteria- exposed neutrophils.
  • Table 1 shows that a large number of genes are affected in a short amount of time (two hours or less). This quick and complex response is consistent to the nature of neutrophils and the expected response in vivo.
  • the present invention has identified numerous genes that were not previously known to be involved in the neutrophil response to bacterial contact. The present invention also allows the selection of gene sets specific to different strains of bacteria.
  • RNA Ten micrograms of total RNA, the amount obtainable from about 3xl0 6 neutrophils, is sufficient for a complete set of cDNA expression profiles.
  • cDNA was synthesized according to the protocol described in the GIBCO/BRL kit for cDNA synthesis.
  • the reaction mixture for first-strand synthesis included 6 ⁇ g of total RNA, and 200 ng of a mixture of 1-base anchored oligo(dT) primers with all three possible anchored bases.
  • the reaction mixture may include lO ⁇ g of total RNA, and 2 p ol of 1 of the 2-base anchored oligo(dT) primers such as RP5.0 (CTCTCAAGGATCTTACCGCT(T) 18 AT, SEQ ID NO: 1374), or RP6.0 (TAATACCGCGCCACATAGCA(T) 18 CG, SEQ ID NO: 1375), or RP9.2 (CAGGGTAGACGACGCTACGC(T) 18 GA, SEQ ID NO: 1376) along with other components for first-strand synthesis reaction except reverse transcriptase.
  • This mixture was then layered with mineral oil and incubated at 65 °C for 7 min followed by 50 °C for another 7 min.
  • the adapter oligonucleotide sequences were Al (TAGCGTCCGGCGCAGCGACGGCCAG, SEQ ID NO: 1377) and A2 (GATCCTGGCCGTCGGCTGTCTGTCGGCGC, SEQ ID NO: 1378).
  • One microgram of oligonucleotide A2 was first phosphorylated at the 5' end using T4 polynucleotide kinase (PNK).
  • PNK was heated denatured, and 1 ⁇ g of the oligonucleotide Al was added along with 10X annealing buffer (1 M NaCl/100 mM Tris-HCl, ⁇ H8.0/10 mM EDTA, pH8.0) in a final vol of 20 ⁇ l. This mixture was then heated at 65 °C for 10 min followed by slow cooling to room temperature for 30 min, resulting in formation of the Y adapter at a final concentration of 100 ng/ ⁇ l. About 20 ng of the cDNA was digested with 4 units of Bgl II in a final vol of 10 ⁇ l for 30 min at 37 °C.
  • 24 pmol of oligonucleotide Al or Al.l was 5 '-end-labeled using 15 ⁇ l of [ ⁇ - 32 P]ATP (Amersham; 3000 Ci/mmol) and PNK in a final volume of 20 ⁇ l for 30 min at 37 C.
  • the labeled oligonucleotide was diluted to a final concentration of 2 ⁇ M in 80 ⁇ l with unlabeled oligonucleotide AIJ.
  • the PCR mixture (20 ⁇ l) consisted of 2 ⁇ l ( 100 pg) of the template, 2 ⁇ l of 10X PCR buffer (100 mM Tris-HCl, pH 8.3/500 mM KCl), 2 ⁇ l of 15 mM MgCl 2 to yield 1.5 mM final Mg 2+ concentration optimum in the reaction mixture, 200 M dNTPs, 200 nM each 5' and 3' PCR primers, and 1 unit of Amplitaq Gold.
  • PCR was done to avoid amplification artifacts arising out of arbitrary annealing of PCR primers at lower temperature during transition from room temperature to 94 °C in the first PCR cycle.
  • PCR consisted of 5 cycles of 94 °C for 30 sec, 55 °C for 2 min, and 72 °C for 60 sec followed by 25 cycles of 94 °C for 30 sec, 60 °C for 2 min, and 72 °C for 60 sec. A higher number of cycles resulted in smeary gel patterns.
  • PCR products (2.5 ⁇ l) were analyzed on 6% polyacrylamide sequencing gel.
  • Z50194 1358 family A member 1 PHLDA1 A 27.9 24.0 16.4 8.5
  • ABO blood group (transferase A. alph 1-3-N- acetylgalactosaminyltransferase; transferase
  • Integrin, beta 2 (antigenCD18 (p95), lymphocyte function-associated antigen 1;
  • 32335_r_at AB009010 24 Homo sapiens mRNA for polyubiquitin UbC, complete cds. -1.5 2.7 -6.1 2.8
  • IPL Homo sapiens IPL (IPL) mRNA, complete cds. 2.6 46.2 34.0 13.1
  • MIR-7 monocyte/macrophage Ig-related receptor MIR-7 (MIR cl-7) mRNA
  • LIR-4 Homo sapiens leucocyte immunoglobulin-like receptor-4
  • NAPOR-1 neuroblastoma apoptosis-related RNA binding protein
  • RNA binding motif protein 5 (RBM5) mRNA, complete cds. -12.4 -21.5 -9.7 -215
  • IMAGE.645184 3 similar to gb:D00763 PROTEASOME COMPONENT C9 (HUMAN);
  • Human protein phosphatase 2A regulatory subunit alpha-isotype (alpha-PR65) mRNA Human protein phosphatase 2A regulatory subunit alpha-isotype (alpha-PR65) mRNA
  • Human protein phosphatase 2A regulatory subunit alpha-isotype (alpha-PR65) mRNA Human protein phosphatase 2A regulatory subunit alpha-isotype (alpha-PR65) mRNA
  • CGM1 Human carcinoembryonic antigen
  • CRE-BP1 Homo sapiens cAMP response element-binding protein
  • CAMK calcium/calmodulin-dependent protein kinase
  • IL8RB interleukin 8 receptor beta
  • JNK1 Human protein kinase
  • JNK1 B2 Human JNK1 beta2 protein kinase
  • IQGAP1 Homo sapiens ras GTPase-activating-like protein
  • HBB Homo sapiens beta-globin
  • 36690_at M10901 485 Human mRNA for alpha-glucocorticoid receptor (clone 0B7). -5.7 -6.5 -4.8 -6.5
  • HCK Human hemopoietic cell protein-tyrosine kinase
  • IL 1 alpha 1076 at M28983 552 Homo sapiens interleukin 1 alpha (IL 1) mRNA, complete cds. -1.9 -5.1 -5.1 -5.1
  • HBP high density lipoprotein binding protein

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Abstract

L'invention concerne l'identification de modifications globales survenant dans l'expression génique associée à l'activation des granulocytes, ainsi que l'identification de profils d'expression utiles en tant que marqueurs de diagnostic et marqueurs susceptibles d'être utilisés pour contrôler l'état et la progression de maladies, ou la toxicité, l'efficacité et le métabolisme de médicaments
PCT/US2001/030821 2000-10-03 2001-10-03 Profils d'expression genique dans les cellules granulocytaires Ceased WO2002028999A2 (fr)

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