Abstract:
Compositions and methods are provided to identify, characterize, and optimize immunostimulatory compounds, their agonists and antagonists, working through TLR3.

Description:
RELATED APPLICATION  
         [0001]    This application claims benefit of U.S. provisional patent application Serial No. 60/327,520, filed Oct. 5, 2001.  
         FIELD OF THE INVENTION  
         [0002]    The invention pertains to signal transduction by Toll-like receptor 3 (TLR3), which is believed to be involved in innate immunity. More specifically, the invention pertains to screening methods useful for the identification and characterization of TLR3 ligands, TLR3 signaling agonists, and TLR3 signaling antagonists.  
         BACKGROUND OF THE INVENTION  
         [0003]    Toll-like receptors (TLRs) are a family of at least ten highly conserved receptor proteins (TLR1-TLR10) which recognize pathogen-associated molecular patterns (PAMPs) and act as key elements in innate immunity. As members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, TLRs share homologies in their cytoplasmic domains called Toll/IL-1R homology (TIR) domains. PCT published applications PCT/US98/08979 and PCT/USO1/16766. Intracellular signaling mechanisms mediated by TIRs appear generally similar, with MyD88 (Wesche H et al. (1997)  Immunity  7:837-47; Medzhitov R et al. (1998)  Mol Cell  2:253-8; Adachi O et al. (1998)  Immunity  9:143-50; Kawai T et al. (1999)  Immunity  11:115-22) and tumor necrosis factor receptor-associated factor 6 (TRAF6; Cao Z et al. (1996)  Nature  383:443-6; Lomaga M A et al. (1999)  Genes Dev  13:1015-24) believed to have critical roles. Signal transduction between MyD88 and TRAF6 is known to involve members of the serine-threonine kinase IL-1 receptor-associated kinase (IRAK) family, including at least IRAK-1 and IRAK-2. Muzio M et al. (1997)  Science  278:1612-5.  
           [0004]    Ligands for many but not all of the TLRs have been described. For instance, it has been reported that TLR2 signals in response to peptidoglycan and lipopeptides. Yoshimura A et al. (1999)  J Immunol  163:1-5; Brightbill H D et al. (1999)  Science  285:732-6; Aliprantis A O et al. (1999)  Science  285:736-9; Takeuchi O et al. (1999)  Immunity  11:443-51; Underhill D M et al. (1999)  Nature  401:811-5. TLR4 has been reported to signal in response to lipopolysaccharide (LPS). Hoshino K et al. (1999)  J Immunol  162:3749-52; Poltorak A et al. (1998)  Science  282:2085-8; Medzhitov R et al. (1997)  Nature  388:394-7. Bacterial flagellin has been reported to be a natural ligand for TLR5. Hayashi F et al. (2001)  Nature  410:1099-1103. TLR6, in conjunction with with TLR2, has been reported to signal in response to proteoglycan. Ozinsky A et al. (2000)  PNAS USA  97:13766-71; Takeuchi O et al. (2001)  Int Immunol  13:933-40. Recently it was recently reported that TLR9 is a receptor for CpG DNA. Hemmi H et al. (2000)  Nature  408:740-5.  
         SUMMARY OF THE INVENTION  
         [0005]    The invention provides screening methods and compositions useful for the identification and characterization of compounds which themselves signal through Toll-like receptor 3 (TLR3) or which influence signaling through TLR3. Compounds which themselves signal through TLR3 are presumptively immunostimulatory. Compounds which influence signaling through TLR3 include both agonists and antagonists of TLR3 signaling activity. The methods provided by the invention are adaptable to high throughput screening, thus accelerating the identification and characterization of previously unknown inducers, agonists, and antagonists of TLR3 signaling activity.  
           [0006]    The methods of the invention rely at least in part on the ability to assess TLR3 signaling activity. It has surprisingly been discovered according to the present invention that reporter constructs having reporter genes under control of certain promoter response elements sensitive to TLR3 signaling activity are useful in the screening assays of the invention. For example it has been surprisingly discovered according to the present invention that a reporter gene under control of interferon-specific response element (ISRE) is sensitive to TLR3 signaling activity.  
           [0007]    It has also surprisingly been discovered according to the present invention that screening assays for TLR ligands and other assays involving TLR signaling activity can benefit from optimization for at least one of the variables of (a) concentration of test and/or reference compound, (b) kinetics of the assay, and (c) selection of reporter. Interpretation of assay data can be influenced by each of these variables.  
           [0008]    In one aspect the invention provides a screening method for identifying an immunostimulatory compound. The method according to this aspect of the invention involves the steps of (a) contacting a functional TLR3 with a test compound under conditions which, in absence of the test compound, permit a negative control response mediated by a TLR3 signal transduction pathway; (b) detecting a test response mediated by the TLR3 signal transduction pathway; and (c) determining the test compound is an immunostimulatory compound when the test response exceeds the negative control response. In this and in all aspects of the invention, in one embodiment the screening method is performed on a plurality of test compounds. A test compound according to this and all aspects of the invention is in one embodiment a member of a library of compounds, preferably a combinatorial library of compounds. Also in this and in all aspects of the invention, a test compound is preferably a small molecule, a nucleic acid, a polypeptide, an oligopeptide, or a lipid. In more preferred embodiments, the test compound is a small molecule or a nucleic acid. In one embodiment a test compound that is a nucleic acid is a CpG nucleic acid.  
           [0009]    In another aspect the invention provides a screening method for identifying an immunostimulatory compound. The method according to this aspect of the invention involves the steps of (a) contacting a functional TLR3 with a test compound under conditions which, in presence of a reference immunostimulatory compound, permit a reference response mediated by a TLR3 signal transduction pathway; (b) detecting a test response mediated by the TLR3 signal transduction pathway; and (c) determining the test compound is an immunostimulatory compound when the test response equals or exceeds the reference response. In this and other aspects of the invention, a reference immunostimulatory compound is preferably a small molecule, a nucleic acid, a polypeptide, an oligopeptide, or a lipid. In one embodiment the reference immunostimulatory compound is a CpG nucleic acid.  
           [0010]    In a further aspect the invention provides a screening method for identifying a compound that modulates TLR3 signaling activity. The method according to this aspect of the invention involves the steps of (a) contacting a functional TLR3 with a test compound and a reference immunostimulatory compound under conditions which, in presence of the reference immunostimulatory compound alone, permit a reference response mediated by a TLR3 signal transduction pathway; (b) detecting a test-reference response mediated by the TLR3 signal transduction pathway; (c) determining the test compound is an agonist of TLR3 signaling activity when the test-reference response exceeds the reference response; and (d) determining the test compound is an antagonist of TLR3 signaling activity when the reference response exceeds the test-reference response.  
           [0011]    In yet another aspect the invention provides a screening method for identifying species specificity of an immunostimulatory compound. The method according to this aspect of the invention involves the steps of (a) measuring a first species-specific response mediated by a TLR3 signal transduction pathway when a functional TLR3 of a first species is contacted with a test compound; (b) measuring a second species-specific response mediated by the TLR3 signal transduction pathway when a functional TLR3 of a second species is contacted with the test compound; and (c) comparing the first species-specific response with the second species-specific response. In a preferred embodiment the functional TLR3 of the first species is a human TLR3. In one preferred embodiment the functional TLR3 of the first species is a human TLR3 and the functional TLR3 of the second species is a mouse TLR3.  
           [0012]    In preferred embodiments of the foregoing aspects of the invention, the response mediated by the TLR3 signal transduction pathway is measured quantitatively.  
           [0013]    Also in preferred embodiments of the foregoing aspects of the invention, the functional TLR3 is expressed in a cell. For example, in one embodiment the cell is an isolated mammalian cell that naturally expresses the functional TLR3. Alternatively, in another embodiment the cell is an isolated mammalian cell that does not naturally express the functional TLR3, wherein the cell has an expression vector for TLR3. For example, in one preferred embodiment the cell is a human 293 fibroblast. In other embodiments, the functional TLR3 is part of a cell-free system.  
           [0014]    Particularly useful in embodiments of the invention involving cells which express functional TLR3 are cells which include a reporter construct sensitive to TLR3 signaling. In one embodiment the cell includes an expression vector having an isolated nucleic acid which encodes a reporter construct selected from the group of nuclear factor-kappa B-luciferase (NF-κB-luc), IFN-specific response element-luciferase (ISRE-luc), interleukin-6-luciferase (IL-6-luc), interleukin 8-luciferase (IL-8-luc), interleukin 12 p40 subunit-luciferase (IL-12 p40-luc), interleukin 12 p40 subunit-beta galactosidase (IL-12 p40-β-Gal), activator protein 1-luciferase (AP1-luc), interferon alpha-luciferase (IFN-α-luc), interferon beta-luciferase (IFN-β-luc), RANTES-luciferase (RANTES-luc), tumor necrosis factor-luciferase (TNF-luc), IP-10-luciferase (IP-10-luc), and interferon-inducible T cell alpha chemoattractant-luciferase (I-TAC-luc). In a preferred embodiment the reporter construct is ISRE-luc.  
           [0015]    In one embodiment according to each of the foregoing aspects of the invention, the functional TLR3 is part of a complex with a non-TLR protein selected from the group consisting of MyD88, IL-1 receptor associated kinase 1-3 (IRAK1, IRAK2, IRAK3), tumor necrosis factor receptor-associated factor 1-6 (TRAF1-TRAF6), IκB, NF-κB, MyD88-adapter-like (Mal), Toll-interleukin 1 receptor (TIR) domain-containing adapter protein (TIRAP), Tollip, Rac, and functional homologues and derivatives thereof. In a related embodiment functional TLR3 is part of a complex with a non-TLR protein listed above, excluding MyD88.  
           [0016]    Also according to each of the foregoing aspects of the invention, in one embodiment the response mediated by a TLR3 signal transduction pathway is induction of a reporter gene under control of a promoter response element selected from the group consisting of ISRE, IL-6, IL-8, IL-12 p40, IFN-α, IFN-β, IFN-ω, RANTES, TNF, IP-10, and I-TAC. For example, in a preferred embodiment the reporter gene under control of a promoter response element is selected from the group consisting of ISRE-luc, IL-6-luc, IL-8-luc, IL-12 p40-luc, IL-12 p40-β-Gal, IFN-α-luc, IFN-β-luc, RANTES-luc, TNF-luc, IP-10-luc, and I-TAC-luc. In one preferred embodiment the reporter gene under control of a promoter response element is ISRE-luc. In yet another preferred embodiment the reporter gene is selected from the group consisting of IFN-α1-luc and IFN-α4-luc.  
           [0017]    In yet another embodiment according to each of the foregoing aspects of the invention, the response mediated by a TLR3 signal transduction pathway is selected from the group consisting of (a) induction of a reporter gene under control of a minimal promoter responsive to a transcription factor selected from the group consisting of AP1, NF-κB, ATF2, IRF3, and IRF7; (b) secretion of a chemokine; and (c) secretion of a cytokine. For example, in one preferred embodiment the response mediated by a TLR3 signal transduction pathway is induction of a reporter gene selected from the group consisting of AP1-luc and NF-κB-luc. In another preferred embodiment the response mediated by a TLR3 signal transduction pathway is secretion of a type 1 IFN. In yet another preferred embodiment the response mediated by a TLR3 signal transduction pathway is secretion of a chemokine selected from the group consisting of CCL5 (RANTES), CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (1-TAC).  
           [0018]    The sensitivity and interpretation of the screening methods of the present invention can be optimized. Such optimization involves proper selection of any one or combination of (a) concentration of test and/or reference compound, (b) kinetics of the assay, and (c) reporter. Thus, further according to each of the first three aspects of the invention, in one embodiment the contacting a functional TLR3 with a test compound further entails, for each test compound, contacting with the test compound at each of a plurality of concentrations. For example, each test compound may be evaluated at various concentrations which differ by log increments. Also according to each of the foregoing aspects of the invention, in one embodiment the detecting is performed 4-12 hours, preferably 6-8 hours, following the contacting. Similarly, in yet another embodiment according to each of the foregoing aspects of the invention, the detecting is performed 16-24 hours following the contacting. Detecting performed 4-12 hours, preferably 6-8 hours, following the contacting is believed to be more sensitive to affinity of interaction than is detecting at later times. Detecting performed 16-24 hours or later following the contacting is believed to be more sensitive to stability and duration of receptor/ligand interaction. Furthermore, because certain reporter constructs are more sensitive to certain TLRs than others, proper matching of reporter to TLR assay is important to increase signal-to-noise ratio in the readout of a particular assay. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0019]    This application includes examples which refer to figures or other drawings. It is to be understood that the referenced figures are illustrative only and are not essential to the enablement of the claimed invention.  
         [0020]    [0020]FIG. 1 is two paired bar graphs showing (A) the induction of NF-κB and (B) the amount of IL-8 produced by 293 fibroblast cells transfected with human TLR9 in response to exposure to various stimuli, including CpG-ODN, GpC-ODN, LPS, and medium.  
         [0021]    [0021]FIG. 2 is a bar graph showing the induction of NF-κB produced by 293 fibroblast cells transfected with murine TLR9 in response to exposure to various stimuli, including CpG-ODN, methylated CpG-ODN (Me-CpG-ODN), GpC-ODN, LPS, and medium.  
         [0022]    [0022]FIG. 3 is a series of gel images depicting the results of reverse transcriptase-polymerase chain reaction (RT-PCR) assays for murine TLR9 (mTLR9), human TLR9 (hTLR9), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in untransfected control 293 cells, 293 cells transfected with mTLR9 (293-mTLR9), and 293 cells transfected with hTLR9 (293-hTLR9).  
         [0023]    [0023]FIG. 4 is a graph showing the degree of induction of NF-κB-luc by various stimuli in stably transfected 293-hTLR9 cells.  
         [0024]    [0024]FIG. 5 is a graph showing the degree of induction of NF-κB-luc by various stimuli in stably transfected 293-mTLR9 cells.  
         [0025]    [0025]FIG. 6 is a graph showing fold induction of response as a function of concentration for a series of four related immunostimulatory nucleic acids contacted with human 293 fibroblast cells stably transfected with murine TLR9 and NF-κB-luc. Concentrations listed correspond to EC50 for each ligand.  
         [0026]    [0026]FIG. 7 is a graph showing kinetics of EC50 determinations for a series of five immunostimulatory nucleic acids contacted with human 293 fibroblast cells stably transfected with murine TLR9 and NF-κB-luc.  
         [0027]    [0027]FIG. 8 is a graph showing kinetics of EC50 determinations for the same series of five immunostimulatory nucleic acids as in FIG. 7 contacted with human 293 fibroblast cells stably transfected with human TLR9 and NF-κB-luc.  
         [0028]    [0028]FIG. 9 is a graph showing kinetics of maximal activity (fold induction of response) for the same series of five immunostimulatory nucleic acids as in FIG. 7 contacted with human 293 fibroblast cells stably transfected with murine TLR9 and NF-κB-luc.  
         [0029]    [0029]FIG. 10 is a graph showing kinetics of maximal activity (fold induction of response) for the same series of five immunostimulatory nucleic acids as in FIG. 7 contacted with human 293 fibroblast cells stably transfected with human TLR9 and NF-κB-luc.  
         [0030]    [0030]FIG. 11 is a bar graph showing fold induction of response as measured using various luciferase reporter constructs (NF-κB-luc, IP-10-luc, RANTES-luc, ISRE-luc, and IL-8-luc) in combination with TLR7, TLR8, and TLR9, each TLR contacted with a specific reference TLR ligand. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0031]    The invention in certain aspects provides screening methods useful for the identification, characterization, and optimization of immunostimulatory compounds, including but not limited to immunostimulatory nucleic acids and immunostimulatory small molecules, as well as assays for the identification and optimization of agonists and antagonists of TLR3 signaling. The methods according to the invention include both cell-based and cell-free assays. In certain preferred embodiments the screening methods are performed in a high throughput manner. The methods can be used to screen libraries of compounds for their ability to modulate immune activation that involves TLR3 signaling.  
         [0032]    In one aspect the invention provides a screening method for identifying an immunostimulatory compound. The method according to this aspect of the invention involves the steps of (a) contacting a functional TLR3 with a test compound under conditions which, in absence of the test compound, permit a negative control response mediated by a TLR3 signal transduction pathway; (b) detecting a test response mediated by the TLR3 signal transduction pathway; and (c) determining the test compound is an immunostimulatory compound when the test response exceeds the negative control response. In a second aspect the invention provides a screening method for identifying an immunostimulatory compound. The method according to this aspect of the invention involves the steps of (a) contacting a functional TLR3 with a test compound under conditions which, in presence of a reference immunostimulatory compound, permit a reference response mediated by a TLR3 signal transduction pathway; (b) detecting a test response mediated by the TLR3 signal transduction pathway; and (c) determining the test compound is an immunostimulatory compound when the test response equals or exceeds the reference response. It will be appreciated that these two aspects of the invention differ in that one involves comparison of the test compound against a negative control and the other involves comparison of the test compound against a positive control.  
         [0033]    For these and other aspects of the invention, the TLR3 is preferably a mammalian TLR3, such as human TLR3 or mouse TLR3. Nucleotide and amino acid sequences for human TLR3 and murine TLR3 have previously been described. The nucleotide sequence for human TLR3 cDNA can be found as GenBank accession no. NM — 003265 (SEQ ID NO:1), and the deduced amino acid sequence for human TLR3, encompassing 904 amino acids, can be found as GenBank accession nos NP — 003256 (SEQ ID NO:2). The nucleotide sequence for murine TLR3 cDNA can be found as GenBank accession no. AF355152 (SEQ ID NO:3), and the deduced amino acid sequence for murine TLR3, encompassing 905 amino acids, can be found as GenBank accession no. AAK26117 (SEQ ID NO:4).  
         [0034]    As used herein, a “functional TLR3” shall refer to a polypeptide, including a full length naturally occurring TLR3 polypeptide as described above, which specifically binds a TLR3 ligand and signals via a Toll/interleukin-1 receptor (TIR) domain. In addition to full length naturally occurring TLR3, a functional TLR3 thus also refers to allelic variants, fusion proteins, and truncated versions of the same, provided the polypeptide specifically binds a TLR3 ligand and signals via a TIR domain. In a preferred embodiment, the functional TLR3 includes a human TLR3 extracellular domain having an amino acid sequence provided by amino acids 38-707 according to SEQ ID NO:2. In another preferred embodiment, the functional TLR3 includes a murine TLR3 extracellular domain having an amino acid sequence provided by amino acids 39-708 according to SEQ ID NO:4. Preferably, the functional TLR3 signals through a TIR domain of TLR3.  
         [0035]    In certain embodiments of this and other aspects of the invention, the functional TLR3 is expressed, either naturally or artifically, in a cell. In some embodiments, a cell expressing TLR3 for use in the methods of the invention expresses TLR3 and no other TLR. Alternatively, in some embodiments a cell expressing TLR3 for use in the methods of the invention expresses both TLR3 and at least one other TLR, e.g., TLR7, TLR8, or TLR9. In one embodiment the cell is an isolated mammalian cell that naturally expresses functional TLR3. Cells and tissues known to express TLR3 include dendritic cells (DCs), intraepithelial cells, and placenta. Muzio M et al. (2000)  J Immunol  164:5998-6004; Cario E et al. (2000)  Infect Immun  68:7010-7; Rock F L et al. (1998)  Proc Natl Acad Sci USA  95:588-93. The term “isolated” as used herein, with reference to a cell or to a compound, means substantially free of or separated from components with which the cell or compound is normally associated in nature, e.g., other cells, nucleic acids, proteins, lipids, carbohydrates or in vivo systems to an extent practical and appropriate for its intended use.  
         [0036]    In another embodiment the cell can be one that, as it occurs in nature, is not capable of expressing TLR3 but which is rendered capable of expressing TLR3 through the artificial introduction of an expression vector for TLR3. Examples of cell lines lacking TLR3 include, but are not limited to, human 293 fibroblasts (ATCC CRL-1573) and HEp-2 human epithelial cells (ATCC CCL-23). Examples of cell lines lacking TLR9 include, but are not limited to, human 293 fibroblasts (ATCC CRL-1573), MonoMac-6, THP-1, U937, CHO, and any TLR9 knock-out. Typically the cell, whether it is capable of expressing TLR3 naturally or artificially, preferably has all the necessary elements for signal transduction initiated through the the TLR3 receptor. For example, it is believed that TLR9 signaling requires the adapter protein MyD88 in an early step of signal transduction. In contrast, TLR3 appears not to require MyD88 but may require other factors further downstream, e.g., factors that induce mitogen-activated protein kinase (MAPK) and factors downstream of MAPK.  
         [0037]    When indicated, introduction of a particular TLR into a cell or cell line is preferably accomplished by transient or stable transfection of the cell or cell line with a TLR-encoding nucleic acid sequence operatively linked to a gene expression sequence (as described herein). For example, a cell artificially induced to express TLR3 for use in the methods of the invention includes a cell that has been transiently or stably transfected with a TLR3 expression vector. Any suitable method of transient or stable transfection can be employed for this purpose.  
         [0038]    An expression vector for TLR3 will include at least a nucleotide sequence coding for a functional TLR3 polypeptide, operably linked to a gene expression sequence which can direct the expression of the TLR3 nucleic acid within a eukaryotic or prokaryotic cell. A “gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the nucleic acid to which it is operably linked. With respect to TLR3 nucleic acid, the “gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the TLR3 nucleic acid to which it is operably linked. The gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus (RSV), cytomegalovirus (CMV), the long terminal repeats (LTR) of Moloney murine leukemia virus and other retroviruses, and the thymidine kinase (TK) promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein (MT) promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.  
         [0039]    In general, the gene expression sequence shall include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined TLR3 nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.  
         [0040]    Generally a nucleic acid coding sequence and a gene expression sequence are said to be “operably linked” when they are covalently linked in such a way as to place the transcription and/or translation of the nucleic acid coding sequence under the influence or control of the gene expression sequence. Thus the TLR3 nucleic acid sequence and the gene expression sequence are said to be “operably linked” when they are covalently linked in such a way as to place the transcription and/or translation of the TLR3 coding sequence under the influence or control of the gene expression sequence. If it is desired that the TLR3 sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5′ gene expression sequence results in the transcription of the TLR3 sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the TLR3 sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a TLR3 nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that TLR3 nucleic acid sequence such that the resulting transcript might be translated into the desired protein or polypeptide.  
         [0041]    In certain embodiments a TLR expression vector is constructed so as to permit tandem expression of two distinct TLRs, e.g., both TLR3 and a second TLR. Such a tandem expression vector can be used when it is desired to express two TLRs using a single transformation or transfection. Alternatively, a TLR3 expression vector can be used in conjunction with a second expression vector constructed so as to permit expression of a second TLR.  
         [0042]    The screening assays can have any of a number of possible readout systems based upon a TLR/IL-1R signal transduction pathway. In preferred embodiments, the readout for the screening assay is based on the use of native genes or, alternatively, transfected or otherwise artificially introduced reporter gene constructs which are responsive to the TLR/IL-1R signal transduction pathway involving MyD88, TRAF, p38, and/or ERK. Häcker H et al. (1999)  EMBO J.  18:6973-82. These pathways activate kinases including KB kinase complex and c-Jun N-terminal kinases. Thus reporter genes and reporter gene constructs particularly useful for the assays include, e.g., a reporter gene operatively linked to a promoter sensitive to NF-κB. Examples of such promoters include, without limitation, those for NF-κB, IL-1β, IL-6, IL-8, IL-12 p40, CD80, CD86, and TNF-α. The reporter gene operatively linked to the TLR-sensitive promoter can include, without limitation, an enzyme (e.g., luciferase, alkaline phosphatase, β-galactosidase, chloramphenicol acetyltransferase (CAT), etc.), a bioluminescence marker (e.g., green-fluorescent protein (GFP, U.S. Pat. No. 5,491,084), etc.), a surface-expressed molecule (e.g., CD25), and a secreted molecule (e.g., IL-8, IL-12 p40, TNF-α). In certain preferred embodiments the reporter is selected from IL-8, TNF-α, NF-κB-luciferase (NF-κB-luc; Häcker H et al. (1999)  EMBO J.  18:6973-82), IL-12 p40-luc (Murphy T L et al. (1995)  Mol Cell Biol  15:5258-67), and TNF-luc (Häcker H et al. (1999)  EMBO J.  18:6973-82). In assays relying on enzyme activity readout, substrate can be supplied as part of the assay, and detection can involve measurement of chemiluminescence, fluorescence, color development, incorporation of radioactive label, drug resistance, or other marker of enzyme activity. For assays relying on surface expression of a molecule, detection can be accomplished using flow cytometry (FACS) analysis or functional assays. Secreted molecules can be assayed using enzyme-linked immunosorbent assay (ELISA) or bioassays. These and other suitable readout systems are well known in the art and are commercially available.  
         [0043]    Thus a cell expressing a functional TLR3 and useful for the methods of the invention has, in some embodiments, an expression vector comprising an isolated nucleic acid which encodes a reporter construct useful for detecting TLR signaling. The expression vector comprising an isolated nucleic acid which encodes a reporter construct useful for detecting TLR signaling can include a reporter gene under control of a minimal promoter responsive to a transcription factor believed by the applicant to be activated as a consequence of TLR3 signaling. Examples of such minimal promoters include, without limitation, promoters for the following genes: AP1, NF-κB, ATF2, IRF3, and IRF7. In other embodiments the expression vector comprising an isolated nucleic acid which encodes a reporter construct useful for detecting TLR signaling can include a gene under control of a promoter response element selected from IL-6, IL-8, IL-12 p40 subunit, a type 1 IFN, RANTES, TNF, IP-10, I-TAC, and ISRE. The promoter response element generally will be present in multiple copies, e.g., as tandem repeats. For example, an ISRE-luciferase reporter construct useful in the invention is available from Stratagene (catalog no. 219092) and includes a 5×ISRE tandem repeat joined to a TATA box upstream of a luciferase reporter gene. As discussed further elsewhere herein, the reporter itself can be any gene product suitable for detection by methods recognized in the art. Such methods for detection can include, for example, measurement of spontaneous or stimulated light emission, enzyme activity, expression of a soluble molecule, expression of a cell surface molecule, etc.  
         [0044]    As mentioned above, the functional TLR3 is contacted with a test compound in order to identify an immunostimulatory compound. An immunostimulatory compound is a natural or synthetic compound that is capable of inducing an immune response when contacted with an immune cell. In the context of the methods of the invention, an immunostimulatory compound refers to a natural or synthetic compound that is capable of inducing an immune response when contacted with an immune cell expressing a functional TLR3 polypeptide. Preferably the immune response is or involves activation of a TLR3 signal transduction pathway. Thus immunostimulatory compounds identified and characterized using the methods of the invention specifically include TLR3 ligands, i.e., compounds which selectively bind to TLR3 and induce a TLR3 signal transduction pathway. Immunostimulatory compounds in general include but are not limited to nucleic acids, including oligonucleotides and polynucleotides; oligopeptides; polypeptides; lipids, including lipopolysaccharides; carbohydrates, including oligosaccharides and polysaccharides; and small molecules. Accordingly, a “test compound” refers to nucleic acids, including oligonucleotides and polynucleotides; oligopeptides; polypeptides; lipids, including lipopolysaccharides; carbohydrates, including oligosaccharides and polysaccharides; and small molecules. Test compounds include compounds with known biological activity as well as compounds without known biological activity.  
         [0045]    A “reference immunostimulatory compound” refers to an immunostimulatory compound that characteristically induces an immune response when contacted with an immune cell expressing a functional TLR polypeptide. In the screening methods of the invention, the reference immunositmulatory compound is a natural or synthetic compound that that characteristically induces an immune response when contacted with an immune cell expressing a functional TLR3 polypeptide. Preferably the immune response is or involves activation of a TLR3 signal transduction pathway. Thus a reference immunostimulatory compound will characteristically induce a reference response mediated by a TLR3 signal transduction pathway when contacted with a functional TLR3 under suitable conditions. The reference response can be measured according to any of the methods described herein. Importantly, a reference immunostimulatory compound specifically includes a test compound identified as an immunostimulatory compound according to any one of the methods of the invention. Therefore a reference immunostimulatory compound can be a nucleic acid, including oligonucleotides and polynucleotides; an oligopeptide; a polypeptide; a lipid, including lipopolysaccharides; a carbohydrate, including oligosaccharides and polysaccharides; or a small molecule.  
         [0046]    Small molecules include naturally occurring, synthetic, and semisynthetic organic and organometallic compounds with molecular weight less than about 1.5 kDa. Examples of small molecules include most drugs, subunits of polymeric materials, and analogs and derivatives thereof.  
         [0047]    A “nucleic acid” as used herein with respect to test compounds and reference compounds used in the methods of the invention, shall refer to any polymer of two or more individual nucleoside or nucleotide units. Typically individual nucleoside or nucleotide units will include any one or combination of deoxyribonucleosides, ribonucleosides, deoxyribonucleotides, and ribonucleotides. The individual nucleotide or nucleoside units of the nucleic acid can be naturally occurring or not naturally occurring. For example, the individual nucleotide units can include deoxyadenosine, deoxycytidine, deoxyguanosine, thymidine, and uracil. In addition to naturally occurring 2′-deoxy and 2′-hydroxyl forms, individual nucleosides also include synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., as described in Uhlmann E et al. (1990)  Chem Rev  90:543-84. The linkages between individual nucleotide or nucleoside units can be naturally occurring or not naturally occurring. For example, the linkages can be phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, as well as peptide linkages and other covalent linkages, known in the art, suitable for joining adjacent nucleoside or nucleotide units. The nucleic acid test compounds and nucleic acid reference compounds typically range in size from 3-4 units to a few tens of units, e.g., 18-40 units.  
         [0048]    The substituted purines and pyrimidines of the ISNAs include standard purines and pyrimidines such as cytosine as well as base analogs such as C-5 propyne substituted bases. Wagner R W et al. (1996)  Nat Biotechnol  14:840-4. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.  
         [0049]    Libraries of compounds that can be used as test compounds are available from various commercial suppliers, and they can be made to order using techniques well known in the art, including combinatorial chemistry techniques. Especially in combination with high throughput screening methods, such methods including in particular automated multichannel methods of screening, large libraries of test compounds can be screened according to the methods of the invention. Large libraries can include hundreds, thousands, tens of thousands, hundreds of thousands, and even millions of compounds.  
         [0050]    Thus in preferred embodiments, the methods for screening test compounds can be performed on a large scale and with high throughput by incorporating, e.g., an array-based assay system and at least one automated or semi-automated step. For example, the assays can be set up using multiple-well plates in which cells are dispensed in individual wells and reagents are added in a systematic manner using a multiwell delivery device suited to the geometry of the multiwell plate. Manual and robotic multiwell delivery devices suitable for use in a high throughput screening assay are well known by those skilled in the art. Each well or array element can be mapped in a one-to-one manner to a particular test condition, such as the test compound. Readouts can also be performed in this multiwell array, preferably using a multiwell plate reader device or the like. Examples of such devices are well known in the art and are available through commercial sources. Sample and reagent handling can be automated to further enhance the throughput capacity of the screening assay, such that dozens, hundreds, thousands, or even millions of parallel assays can be performed in a day or in a week. Fully robotic systems are known in the art for applications such as generation and analysis of combinatorial libraries of synthetic compounds. See, for example, U.S. Pat. Nos. 5,443,791 and 5,708,158.  
         [0051]    A “CpG nucleic acid” or a “CpG immunostimulatory nucleic acid” as used herein is a nucleic acid containing at least one unmethylated CpG dinucleotide (cytosine-guanine dinucleotide sequence, i.e. “CpG DNA” or DNA containing a 5′ cytosine followed by 3′ guanine and linked by a phosphate bond) and activates a component of the immune system. The entire CpG nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5′ CG 3′ must be unmethylated.  
         [0052]    In one embodiment a CpG nucleic acid is represented by at least the formula:  
         5′-N 1 X 1 CGX 2 N 2 -3′ 
         [0053]    wherein X 1  and X 2  are nucleotides, N is any nucleotide, and N 1  and N 2  are nucleic acid sequences composed of from about 0-25 N&#39;s each. In some embodiments X 1  is adenine, guanine, or thymine and/or X 2  is cytosine, adenine, or thymine. In other embodiments X 1  is cytosine and/or X 2  is guanine.  
         [0054]    Examples of CpG nucleic acids according to the invention include but are not limited to those listed in Table 1.  
                                     TABLE 1                       Exemplary CpG Nucleic Acids                                    AA CG TTCT                   AAG CG AAAATGAAATTGACT   SEQ ID NO:39                       ACCATGGA CG AACTGTTTCCCCTC   SEQ ID NO:40                       ACCATGGA CG ACCTGTTTCCCCTC   SEQ lD NO:41                       ACCATGGA CG AGCTGTTTCCCCTC   SEQ ID NO:42                       ACCATGGA CG ATCTGTTTCCCCTC   SEQ ID NO:43                       ACCATGGA CG GTCTGTTTCCCCTC   SEQ ID NO:44                       ACCATGGA CG TACTGTTTCCCCTC   SEQ ID NO:45                       ACCATGGA CG TTCTGTTTCCCCTC   SEQ ID NO:46                       AG CG GGGG CG AG CG GGGG CG     SEQ lD NO:47                       AGCTATGA CG TTCCAAGG   SEQ ID NO:48                       AT CG ACTCT CG AG CG TTCTC   SEQ ID NO:49                       ATGA CG TTCCTGA CG TT   SEQ ID NO:50                       ATGGAAGGTCCAA CG TTCTC   SEQ ID NO:51                       ATGGAAGGTCCAG CG TTCTC   SEQ ID NO:52                       ATGGACTCTCCAG CG TTCTC   SEQ ID NO:53                       ATGGAGGCTCCAT CG TTCTC   SEQ ID NO:54                       CAA CG TT                       CA CG TTGAGGGGCAT   SEQ ID NO:55                       CAGGCATAA CG GTTC CG TAG   SEQ ID NO:56                       CCAA CG TT                       CTGATTTCCC CG AAATGATG   SEQ ID NO:57                       GAGAA CG ATGGACCTTCCAT   SEQ ID NO:58                       GAGAA CG CTCCAGCACTGAT   SEQ ID NO:59                       GAGAA CG CT CG ACCTTCCAT   SEQ ID NO:60                       GAGAA CG CT CG ACCTT CG AT   SEQ ID NO:61                       GAGAA CG CTGGACCTTCCAT   SEQ ID NO:62                       GATTGCCTGA CG TCAGAGAG   SEQ ID NO:63                       GCATGA CG TTGAGCT   SEQ ID NO:64                       G CG G CG GG CG G CGCGCG CCC   SEQ ID NO:65                       G CG TG CG TTGT CG TTGT CG TT   SEQ ID NO:66                       GCTAGA CG TTAG CG T   SEQ ID NO:67                       GCTAGA CG TTAGTGT   SEQ ID NO:68                       GCTAGATGTTAG CG T   SEQ ID NO:69                       GCTTGATGACTCAGC CG GAA   SEQ ID NO:70                       GGAATGA CG TTCCCTGTG   SEQ ID NO:71                       GGGGTCAA CG TTGA CG GGG   SEQ ID NO:72                       GGGGTCAGTCTTGA CG GGG   SEQ ID NO:73                       GTCCATTTCC CG TAAATCTT   SEQ ID NO:74                       GT CG CT                       GT CG TT                       TAC CGCG TG CG ACCCTCT   SEQ ID NO:75                       TCAA CG TC                       TCAA CG TT                       TCAG CG CT                       TCAG CG TG CG CC   SEQ ID NO:76                       TCAT CG AT                       TCCA CG A CG TTTT CG A CG TT   SEQ ID NO:77                       TCCATAA CG TTCCTGATGCT   SEQ ID NO:78                       TCCATAG CG TTCCTAG CG TT   SEQ ID NO:79                       TCCATCA CG TGCCTGATGCT   SEQ ID NO:80                       TCCATGA CG GTCCTGATGCT   SEQ ID NO:81                       TCCATGA CG TCCCTGATGCT   SEQ ID NO: 82                       TCCATGA CG TGCCTGATGCT   SEQ ID NO:83                       TCCATGA CG TTCCTGA CG TT   SEQ ID NO:84                       TCCATGA CG TTCCTGATGCT   SEQ ID NO:18                       TCCATGC CG GTCCTGATGCT   SEQ ID NO:85                       TCCATG CG TG CG TG CG TTTT   SEQ ID NO:86                       TCCATG CG TTG CG TTG CG TT   SEQ ID NO:87                       TCCATGG CG GTCCTGATGCT   SEQ ID NO:88                       TCCATGT CG ATCCTGATGCT   SEQ ID NO:89                       TCCATGT CG CTCCTGATGCT   SEQ ID NO:90                       TCCATGT CG GTCCTGATGCT   SEQ ID NO:91                       TCCATGT CG GTCCTGCTGAT   SEQ ID NO:92                       TCCATGT CG TCCCTGATGCT   SEQ ID NO:93                       TCCATGT CG TTCCTGATGCT   SEQ ID NO:94                       TCCATGT CG TTCCTGT CG TT   SEQ ID NO:95                       TCCATGT CG TTTTTGT CG TT   SEQ ID NO:96                       TCCTGA CG TTCCTGA CG TT   SEQ ID NO:97                       TCCTGT CG TTCCTGT CG TT   SEQ ID NO:98                       TCCTGT CG TTCCTTGT CG TT   SEQ ID NO:99                       TCCTGT CG TTTTTTGT CG TT   SEQ ID NO:100                       TCCTTGT CG TTCCTGT CG TT   SEQ ID NO:101                       T CG AT CG GGG CG GGG CG AGC   SEQ ID NO:102                       T CG T CG CTGTCTC CG CTTCTT   SEQ ID NO:103                       T CG T CG CTGTCTC CG CTTCTTCTTGCC   SEQ ID NO:104                       T CG T CG CTGTCTGCCCTTCTT   SEQ ID NO:105                       T CG T CG CTGTTGT CG TTTCTT   SEQ ID NO:106                       T CG T CG T CG T CG TT   SEQ ID NO:107                       T CG T CG TTGT CG TTGT CG TT   SEQ ID NO:108                       T CG T CG TTGT CG TTTTGT CG TT   SEQ ID NO:109                       T CG T CG TTTTGT CG TTTTGT CG TT   SEQ ID NO:15                       TCTCCCAG CGCGCG CCAT   SEQ ID NO:110                       TCTCCCAG CG GG CG CAT   SEQ ID NO:111                       TCTCCCAG CG TG CG CCAT   SEQ ID NO:112                       TCTT CG AA                       TGCAGATTG CG CAATCTGCA   SEQ ID NO:113                       TGT CG CT                       TGT CG TT                       TGT CG TTGT CG TT   SEQ ID NO:114                       TGT CG TTGT CG TTGT CG TT   SEQ ID NO: 115                       TGT CG TTGT CG TTGT CG TTGT CG TT   SEQ ID NO:116                       TGT CG TTTGT CG TTTGT CG TT   SEQ ID NO:117                      
 
         [0055]    As used herein the term “response mediated by a TLR signal transduction pathway” refers to a response which is characteristic of an interaction between a TLR and an immunostimulatory compound that induces signaling events through the TLR. Such responses typically involve usual elements of Toll/IL-1R signaling, e.g., MyD88, TRAF, and IRAK molecules, although in the case of TLR3 the role of MyD88 is less clear than for other TLR family members. As demonstrated herein such responses include the induction of a gene under control of a specific promoter such as a NF-κB promoter, increases in particular cytokine levels, increases in particular chemokine levels etc. The gene under the control of the NF-κB promoter may be a gene which naturally includes an NF-κB promoter or it may be a gene in a construct in which an NF-κB promoter has been inserted. Genes which naturally include the NF-κB promoter include but are not limited to IL-8, IL-12 p40, NF-κB-luc, IL-12 p40-luc, and TNF-luc. Increases in cytokine levels may result from increased production or increased stability or increased secretion of the cytokines in response to the TLR-immunostimulatory compound interaction. Th1 cytokines include but are not limited to IL-2, IFN-γ, and IL-12. It has unexpectedly been discovered, according to the instant invention, that the promoter response element ISRE is directly activated as a result of signaling through the TLR3 signal transduction pathway, i.e., independent of IFN-γ production. Th2 cytokines include but are not limited to IL-4, IL-5, and IL-10. Chemokines of particular significance in the invention include but are not limited to CCL5 (RANTES), CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC).  
         [0056]    In another aspect the invention provides a screening method for identifying a compound that modulates TLR3 signaling activity. The method according to this aspect of the invention involves the steps of (a) contacting a functional TLR3 with a test compound and a reference immunostimulatory compound under conditions which, in presence of the reference immunostimulatory compound alone, permit a reference response mediated by a TLR3 signal transduction pathway; (b) detecting a test-reference response mediated by the TLR3 signal transduction pathway; (c) determining the test compound is an agonist of TLR3 signaling activity when the test-reference response exceeds the reference response; and (d) determining the test compound is an antagonist of TLR3 signaling activity when the reference response exceeds the test-reference response. A test-reference response refers to a type of test response as determined when a test compound and a reference immunostimulatory compound are simultaneously contacted with the TLR3. When a test compound is neither an agonist nor an antagonist of TLR3 signaling activity, the test-reference response and the reference response are indistinguishable.  
         [0057]    An agonist as used herein is a compound which causes an enhanced response of a TLR to a reference stimulus. The enhanced response can be additive or synergistic with respect to the response to the reference stimulus by itself. Furthermore, an agonist can work directly or indirectly to cause the enhanced response. Thus an agonist of TLR3 signaling activity as used herein is a compound which causes an enhanced response of a TLR to a reference stimulus.  
         [0058]    An antagonist as used herein is a compound which causes a diminished response of a TLR to a reference stimulus. Furthermore, an antagonist can work directly or indirectly to cause the diminished response. Thus an antagonist of TLR3 signaling activity as used herein is a compound which causes a diminished response of a TLR to a reference stimulus.  
         [0059]    In addition to identification and characterization of immunostimulatory compounds, agonists of TLR3 signaling, and antagonists of TLR3 signaling, the methods of the invention also permit optimization of lead compounds. Optimization of a lead compound involves an iterative application of a screening method of the invention, further including the steps of selecting the best candidate at any given stage or round in the screening and then substituting it as a benchmark or reference in a subsequent round of screening. This latter process can further include selection of parameters to modify in choosing and generating candidate test compounds to screen. For example, a lead compound from a particular round of screening can be used as a basis to develop a focused library of new test compounds for use in a subsequent round of screening.  
         [0060]    In another aspect the invention provides a screening method for identifying species specificity of an immunostimulatory compound. The method according to this aspect of the invention involves the steps of (a) measuring a first species-specific response mediated by a TLR3 signal transduction pathway when a functional TLR3 of a first species is contacted with a test compound; (b) measuring a second species-specific response mediated by the TLR3 signal transduction pathway when a functional TLR3 of a second species is contacted with the test compound; and (c) comparing the first species-specific response with the second species-specific response.  
         [0061]    A species-specific TLR, including TLR3, is not limited to a human TLR, but rather can include a TLR derived from human or non-human sources. Examples of non-human sources include, but are not limited to, murine, rat, bovine, canine, feline, ovine, porcine, and equine. Other species include chicken and fish, e.g., aquaculture species.  
         [0062]    The species-specific TLR, including TLR3, also is not limited to native TLR polypeptides. In certain embodiments the TLR can be, e.g., a chimeric TLR in which the extracellular domain and the cytoplasmic domain are derived from TLR polypeptides from different species. Such chimeric TLR polypeptides, as described above, can include, for example, a human TLR extracellular domain and a murine TLR cytoplasmic domain, each domain derived from the corresponding TLR of each species. In alternative embodiments, such chimeric TLR polypeptides can include chimeras created with different TLR splice variants or allotypes. Other chimeric TLR polypeptides useful for the screening methods of the invention include chimeric polypeptides created with a TLR of a first type, e.g., TLR3, and another TLR, e.g., TLR7, TLR8, or TLR9, of the same or another species as the TLR of the first type. Also contemplated are chimeric polypeptides which incorporate sequences derived from more than two polypeptides, e.g., an extracellular domain, a transmembrane domain, and a cytoplasmic domain all derived from different polypeptide sources, provided at least one such domain derives from a TLR3 polypeptide. As a further example, also contemplated are constructs such as include an extracellular domain of one TLR3, an intracellular domain of another TLR3, and a non-TLR reporter such as luciferase, GFP, etc. Those of skill in the art will recognize how to design and generate DNA sequences coding for such chimeric TLR polypeptides.  
         [0063]    It has also been discovered, according to the instant invention, that TLR-based screening assays, including but not limited to the TLR3-based assays described herein, are sensitive to parameters such as concentration of test compound, stability of test compound, kinetics of detection, and selection of reporter. These parameters can be optimized in order to derive the most information from a given screening assay. Importantly, the kinetics of detection appear to afford separation of types of information such as affinity of interaction and stability or duration of interaction. For example, measurements taken at earlier timepoints, e.g., after 6-8 hours of contact between TLR and test and/or reference compound, appear to reflect more information about affinity of interaction than do measurements obtained at later timepoints, e.g., after 16-24 or more hours of contact. In addition, while NF-κB-driven reporters are generally useful in TLR-based screening assays like those of the instant invention, in some instances a reporter other than an NF-κB-driven reporter will afford greater sensitivity. For example, the IL-8-luc reporter is significantly more sensitive to TLR7 and TLR8 than NF-κB-luc. Selection of reporter thus appears to be TLR-dependent, while parameters relating to kinetics and concentration appear to be more compound-dependent. Thus in performing the screening methods of the instant invention, it is expected that the methods will be enhance by inclusion of measurements obtained using at least two concentrations and two time points for each test compound. Typically at least three concentrations will be employed, spanning a two to three log-fold range of concentrations. Finer ranges of concentration can of course be employed under suitable circumstances, for instance based on results of an earlier screening performed using a wider initial range of concentrations.  
         [0064]    The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate certain embodiments of the invention and are not to be construed to limit the scope of the invention.  
       EXAMPLES  
     Example 1  
     Expression Vectors for Human TLR3 (hTLR3) and Murine TLR3 (mTLR3)  
       [0065]    To create an expression vector for human TLR3, human TLR3 cDNA was amplified by the polymerase chain method (PCR) from a cDNA made from human 293 cells using the primers 5′-GAAACTCGAGCCACCATGAGACAGACTTTGCCTTGTATCTAC-3′ (sense, SEQ ID NO:9) and 5′-GAAAGAATTCTTAATGTACAGAGTTTTTGGATCCAAG-3′ (antisense, SEQ ID NO:10). The primers introduce Xho I and EcoRI restriction endonuclease sites at their 5′ ends for use in subsequent cloning into the expression vector. The resulting amplication product fragment was cloned into pGEM-T Easy vector (Promega), isolated, cut with Xho I and EcoRI restriction endonucleases, ligated into an Xho I/EcoRI-digested pcDNA3.1 expression vector (Invitrogen). The insert was fully sequenced and translated into protein. The cDNA sequence corresponds to the published cDNA sequence for hTLR3, available as GenBank accession no. NM — 003265 (SEQ ID NO:1). The open reading frame codes for a protein 904 amino acids long, having the sequence corresponding to GenBank accession no. NP — 003256 (SEQ ID NO:2).  
                                           TABLE 2                       cDNA Sequence for Human TLR3                                (GenBank Accession No. NM 003265: SEQ ID NO:1)                gcggccgcgt cgacgaaatg tctggatttg gactaaagaa aaaaggaaag gctagcagtc   60                   atccaacaga atcatgagac agactttgcc ttgtatctac ttttgggggg gccttttgcc   120               ctttgggatg ctgtgtgcat cctccaccac caagtgcact gttagccatg aagttgctga   180               ctgcagccac ctgaagttga ctcaggtacc cgatgatcta cccacaaaca taacagtgtt   240               gaaccttacc cataatcaac tcagaagatt accagccgcc aacttcacaa ggtatagcca   300               gctaactagc ttggatgtag gatttaacac catctcaaaa ctggagccag aattgtgcca   360               gaaacttccc atgttaaaag ttttgaacct ccagcacaat gagctatctc aactttctga   420               taaaaccttt gccttctgca cgaatttgac tgaactccat ctcatgtcca actcaatcca   480               gaaaattaaa aataatccct ttgtcaagca gaagaattta atcacattag atctgtctca   540               taatggcttg tcatctacaa aattaggaac tcaggttcag ctggaaaatc tccaagagct   600               tctattatca aacaataaaa ttcaagcgct aaaaagtgaa gaactggata tctttgccaa   660               ttcatcttta aaaaaattag agttgtcatc gaatcaaatt aaagagtttt ctccagggtg   720               ttttcacgca attggaagat tatttggcct ctttctgaac aatgtccagc tgggtcccag   780               ccttacagag aagctatgtt tggaattagc aaacacaagc attcggaatc tgtctctgag   840               taacagccag ctgtccacca ccagcaatac aactttcttg ggactaaagt ggacaaatct   900               cactatgctc gatctttcct acaacaactt aaatgtggtt ggtaacgatt cctttgcttg   960               gcttccacaa ctagaatatt tcttcctaga gtataataat atacagcatt tgttttctca   1020               ctctttgcac gggcttttca atgtgaggta cctgaatttg aaacggtctt ttactaaaca   1080               aagtatttcc cttgcctcac tccccaagat tgatgatttt tcttttcagt ggctaaaatg   1140               tttggagcac cttaacatgg aagataatga tattccaggc ataaaaagca atatgttcac   1200               aggattgata aacctgaaat acttaagtct atccaactcc tttacaagtt tgcgaacttt   1260               gacaaatgaa acatttgtat cacttgctca ttctccctta cacatactca acctaaccaa   1320               gaataaaatc tcaaaaatag agagtgatgc tttctcttgg ttgggccacc tagaagtact   1380               tgacctgggc cttaatgaaa ttgggcaaga actcacaggc caggaatgga gaggtctaga   1440               aaatattttc gaaatctatc tttcctacaa caagtacctg cagctgacta ggaactcctt   1500               tgccttggtc ccaagccttc aacgactgat gctccgaagg gtggccctta aaaatgtgga   1560               tagctctcct tcaccattcc agcctcttcg taacttgacc attctggatc taagcaacaa   1620               caacatagcc aacataaatg atgacatgtt ggagggtctt gagaaactag aaattctcga   1680               tttgcagcat aacaacttag cacggctctg gaaacacgca aaccctggtg gtcccattta   1740               tttcctaaag ggtctgtctc acctccacat ccttaacttg gagtccaacg gctttgacga   1800               gatcccagtt gaggtcttca aggatttatt tgaactaaag atcatcgatt taggattgaa   1860               taatttaaac acacttccag catctgtctt taataatcag gtgtctctaa agtcattgaa   1920               ccttcagaag aatctcataa catccgttga gaagaaggtt ttcgggccag ctttcaggaa   1980               cctgactgag ttagatatgc gctttaatcc ctttgattgc acgtgtgaaa gtattgcctg   2040               gtttgttaat tggattaacg agacccatac caacatccct gagctgtcaa gccactacct   2100               ttgcaacact ccacctcact atcatgggtt cccagtgaga ctttttgata catcatcttg   2160               caaagacagt gccccctttg aactcttttt catgatcaat accagtatcc tgttgatttt   2220               tatctttatt gtacttctca tccactttga gggctggagg atatcttttt attggaatgt   2280               ttcagtacat cgagttcttg gtttcaaaga aatagacaga cagacagaac agtttgaata   2340               tgcagcatat ataattcatg cctataaaga taaggattgg gtctgggaac atttctcttc   2400               aatggaaaag gaagaccaat ctctcaaatt ttgtctggaa gaaagggact ttgaggcggg   2460               tgtttttgaa ctagaagcaa ttgttaacag catcaaaaga agcagaaaaa ttatttttgt   2520               tataacacac catctattaa aagacccatt atgcaaaaga ttcaaggtac atcatgcagt   2580               tcaacaagct attgaacaaa atctggattc cattatattg gttttccttg aggagattcc   2640               agattataaa ctgaaccatg cactctgttt gcgaagagga atgtttaaat ctcactgcat   2700               cttgaactgg ccagttcaga aagaacggat aggtgccttt cgtcataaat tgcaagtagc   2760               acttggatcc aaaaactctg tacattaaat ttatttaaat attcaattag caaaggagaa   2820               actttctcaa tttaaaaagt tctatggcaa atttaagttt tccataaagg tgttataatt   2880               tgtttattca tatttgtaaa tgattatatt ctatcacaat tacatctctt ctaggaaaat   2940               gtgtctcctt atttcaggcc tatttttgac aattgactta attttaccca aaataaaaca   3000               tataagcacg caaaaaaaaa aaaaaaaaa 3029                  
 
         [0066]    [0066]                                           TABLE 3                       Amino Acid Sequence for Human TLR3                                (GenBank Accession No. NP 003256; SEQ ID NO:2)                MRQTLPCIYF WGGLLPFGML CASSTTKCTV SHEVADCSHL KLTQVPDDLP TNITVLNLTH   60                   NQLRRLPAAN FTRYSQLTSL DVGFNTISKL EPELCQKLPM LKVLNLQHNE LSQLSDKTFA   120               FCTNLTELHL MSNSIQKIKN NPFVKQKNLI TLDLSHNGLS STKLGTQVQL ENLQELLLSN   180               NKIQALKSEE LDIFANSSLK KLELSSNQIK EFSPGCFHAI GRLFGLFLNN VQLGPSLTEK   240               LCLELANTSI RNLSLSNSQL STTSNTTFLG LKWTNLTMLD LSYNNLNVVG NDSFAWLPQL   300               EYFFLEYNNI QHLFSHSLHG LFNVRYLNLK RSFTKQSISL ASLPKIDDFS FQWLKCLEHL   360               NMEDNDIPGI KSNMFTGLIN LKYLSLSNSF TSLRTLTNET FVSLAHSPLH ILNLTKNKIS   420               KIESDAFSWL GHLEVLDLGL NEIGQELTGQ EWRGLENIFE IYLSYNKYLQ LTRNSFALVP   480               SLQRLMLRRV ALKNVDSSPS PFQPLRNLTI LDLSNNNIAN INDDMLEGLE KLEILDLQHN   540               NLARLWKHAN PGGPIYFLKG LSHLHILNLE SNGFDEIPVE VFKDLFELKI IDLGLNNLNT   600               LPASVFNNQV SLKSLNLQKN LITSVEKKVF GPAFRNLTEL DMRFNPFDCT CESIAWFVNW   660               INETHTNIPE LSSHYLCNTP PHYHGFPVRL FDTSSCKDSA PFLEFFMINT SILLIFIFIV   720               LLIHFEGWRI SFYWNVSVHR VLGFKEIDRQ TEQFEYAAYI IHAYKDKDWV WEHFSSMEKE   780               DQSLKFCLEE RDFEAGVFEL EAIVNSIKRS RKIIFVITHH LLKDPLCKRF KVHHAVQQAI   840               EQNLDSIILV FLEEIPDYKL NHALCLRRGM FKSHCILNWP VQKERIGAFR HKLQVALGSK   900               NSVH 904                    
         [0067]    Corresponding nucleotide and amino acid sequences for murine TLR3 (mTLR3) are known. The nucleotide sequence of mTLR3 cDNA has been reported as GenBank accession no. AF355152, and the amino acid sequence of mTLR3 has been reported as GenBank accession no. AAK26117.  
                                       TABLE 4                       cDNA Sequence for Murine TLR3                                (GenBank Accession No. AF355152; SEQ ID NO:3)                tagaatatga tacagggatt gcacccataa tctgggctga atcatgaaag ggtgttcctc   60                   ttatctaatg tactcctttg ggggactttt gtccctatgg attcttctgg tgtcttccac   120               aaaccaatgc actgtgagat acaacgtagc tgactgcagc catttgaagc taacacacat   180               acctgatgat cttccctcta acataacagt gttgaatctt actcacaacc aactcagaag   240               attaccacct accaacttta caagatacag ccaacttgct atcttggatg caggatttaa   300               ctccatttca aaactggagc cagaactgtg ccaaatactc cctttgttga aagtattgaa   360               cctgcaacat aatgagctct ctcagatttc tgatcaaacc tttgtcttct gcacgaacct   420               gacagaactc gatctaatgt ctaactcaat acacaaaatt aaaagcaacc ctttcaaaaa   480               ccagaagaat ctaatcaaat tagatttgtc tcataatggt ttatcatcta caaagttggg   540               aacgggggtc caactggaga acctccaaga actgctctta gcaaaaaata aaatccttgc   600               gttgcgaagt gaagaacttg agtttcttgg caattcttct ttacgaaagt tggacttgtc   660               atcaaatcca cttaaagagt tctccccggg gtgtttccag acaattggca agttattcgc   720               cctcctcttg aacaacgccc aactgaaccc ccacctcaca gagaagcttt gctgggaact   780               ttcaaacaca agcatccaga atctctctct ggctaacaac cagctgctgg ccaccagcga   840               gagcactttc tctgggctga agtggacaaa tctcacccag ctcgatcttt cctacaacaa   900               cctccatgat gtcggcaacg gttccttctc ctatctccca agcctgaggt atctgtctct   960               ggagtacaac aatatacagc gtctgtcccc tcgctctttt tatggactct ccaacctgag   1020               gtacctgagt ttgaagcgag catttactaa gcaaagtgtt tcacttgctt cacatcccaa   1080               cattgacgat ttttcctttc aatggttaaa atatttggaa tatctcaaca tggatgacaa   1140               taatattcca agtaccaaaa gcaatacctt cacgggattg gtgagtctga agtacctaag   1200               tctttccaaa actttcacaa gtttgcaaac tttaacaaat gaaacatttg tgtcacttgc   1260               tcattctccc ttgctcactc tcaacttaac gaaaaatcac atctcaaaaa tagcaaatgg   1320               tactttctct tggttaggcc aactcaggat acttgatctc ggccttaatg aaattgaaca   1380               aaaactcagc ggccaggaat ggagaggtct gagaaatata tttgagatct acctatccta   1440               taacaaatac ctccaactgt ctaccagttc ctttgcattg gtccccagcc ttcaaagact   1500               gatgctcagg agggtggccc ttaaaaatgt ggatatctcc ccttcacctt tccgccctct   1560               tcgtaacttg accattctgg acttaagcaa caacaacata gccaacataa atgaggactt   1620               gctggagggt cttgagaatc tagaaatcct ggattttcag cacaataact tagccaggct   1680               ctggaaacgc gcaaaccccg gtggtcccgt taatttcctg aaggggctgt ctcacctcca   1740               catcttgaat ttagagtcca acggcttaga tgaaatccca gtcggggttt tcaagaactt   1800               attcgaacta aagagcatca atctaggact gaataactta aacaaacttg aaccattcat   1860               ttttgatgac cagacatctc taaggtcact gaacctccag aagaacctca taacatctgt   1920               tgagaaggat gttttcgggc cgccttttca aaacctgaac agtttagata tgcgcttcaa   1980               tccgttcgac tgcacgtgtg aaagtatttc ctggtttgtt aactggatca accagaccca   2040               cactaatatc tttgagctgt ccactcacta cctctgtaac actccacatc attattatgg   2100               cttccccctg aagcttttcg atacatcatc ctgtaaagac agcgccccct ttgaactcct   2160               cttcataatc agcaccagta tgctcctggt ttttatactt gtggtactgc tcattcacat   2220               cgagggctgg aggatctctt tttactggaa tgtttcagtg catcggattc ttggtttcaa   2280               ggaaatagac acacaggctg agcagtttga atatacagcc tacataattc atgcccataa   2340               agacagagac tgggtctggg aacatttctc cccaatggaa gaacaagacc attctctcaa   2400               attttgccta gaagaaaggg actttgaagc aggcgtcctt ggacttgaag caattgttaa   2460               tagcatcaaa agaagccgaa aaatcatttt cgttatcaca caccatttat taaaagaccc   2520               tctgtgcaga agattcaagg tacatcacgc agttcagcaa gctattgagc aaaatctgga   2580               ttcaattata ctgatttttc tccagaatat tccagattat aaactaaacc atgcactctg   2640               tttgcgaaga ggaatgttta aatctcattg catcttgaac tggccagttc agaaagaacg   2700               gataaatgcc tttcatcata aattgcaagt agcacttgga tctcggaatt cagcacatta   2760               aactcatttg aagatttgga gtcggtaaag ggatagatcc aatttataaa ggtccatcat   2820               gaatctaagt tttacttgaa agttttgtat atttatttat atgtatagat gatgatatta   2880               catcacaatc caatctcagt tttgaaatat ttcggcttat ttcattgaca tctggtttat   2940               tcactccaaa taaacacatg ggcagttaaa aacatcctct attaatagat tacccattaa   3000               ttcttgaggt gtatcacagc tttaaagggt tttaaatatt tttatataaa taagactgag   3060               agttttataa atgtaatttt ttaaaactcg agtcttactg tgtagctcag aaaggcctgg   3120               aaattaatat attagagagt catgtcttga acttatttat ctctgcctcc ctctgtctcc   3180               agagtgttgc ttttaagggc atgtagcacc acacccagct atgtacgtgt gggattttat   3240               aatgctcatt tttgagacgt ttatagaata aaagataatt gcttttatgg tataaggcta   3300               cttgaggtaa 3310                  
 
         [0068]    [0068]                                           TABLE 5                       Amino Acid Sequence for Murine TLR3                                (GenBank Accession No. AAK26117; SEQ ID NO:4)                MKGCSSYLMY SFGGLLSLWI LLVSSTNQCT VRYNVADCSH LKLTHIPDDL PSNITVLNLT   60                   HNQLRRLPPT NFTRYSQLAI LDAGFNSISK LEPELCQILP LLKVLNLQHN ELSQISDQTF   120               VFCTNLTELD LMSNSIHKIK SNPFKNQKNL IKLDLSHNGL SSTKLGTGVO LENLQELLLA   180               KNKILALRSE ELEFLGNSSL RKLDLSSNPL KEFSPGCFQT IGKLFALLLN NAQLNPHLTE   240               KLCWELSNTS IQNLSLANNQ LLATSESTFS GLKQTNLTQL DLSYNNLHDV GNGSFSYLPS   300               LRYLSLEYNN IQRLSPRSFY GLSNLRYLSL KRAFTKQSVS LASHPNIDDF SFQWLKYLEY   360               LNMDDNNIPS TKSNTFTGLV SLKYLSLSKT FTSLQTLTNE TFVSLAHSPL LTLNLTKNHI   420               SKISNGTFSW LGQLRILDLG LNEIEQKLSG QEWRGLRNIF EIYLSYNKYL QLSTSSFALV   480               PSLQRLMLRR VALKNVDISP SPFRPLRNLT ILDLSNNNIA NINEDLLEGL ENLEILDFQH   540               NNLARLWKRA NPGGPVNFLK GLSHLHILNL ESNGLDEIPV GVFKNLFELF SINLGLNNLN   600               KLEPFIFDDQ TSLRSLNLQK NLITSVEKDV FGPPFQNLNS LDMRFNPFDC TCESISWFVN   660               WINQTHTNIF ELSTHYLCNT PHHYYGFPLK LFDTSSCKDS APFELLFIIS TSMLLVFILV   720               VLLIHIEGWR ISFYWNVSVH RILGFKEIDT QARQFEYTAY IIHAHKDRDW VWEHFSPMEE   780               QDQSLKFCLE ERDFEAGVLG LEAIVNSIKR SRKIIFVITH HLLKDPLCRR FKVHHAVQQA   840               IEQNLDSIIL IFLQNIPDYK LNHALCLRRG MFKSHCILNW PVQKERINAF HHKLQVALGS   900               RNSAH 905                    
       Example 2  
     Method of Making IFN-α4 Reporter Vector  
       [0069]    A number of reporter vectors may be used in the practice of the invention. Some of the reporter vectors are commercially available, e.g., the luciferase reporter vectors pNF-κB-Luc (Stratagene) and pAP1-Luc (Stratagene). These two reporter vectors place the luciferase gene under control of an upstream (5′) promoter region derived from genomic DNA for NF-κB or AP1, respectively. Other reporter vectors can be constructed following standard methods using the desired promoter and a vector containing a suitable reporter, such as luciferase, β-galactosidase (β-gal), chloramphenicol acetyltransferase (CAT), and other reporters known by those skilled in the art. Following are some examples of reporter vectors constructed for use in the present invention.  
         [0070]    IFN-α4 is an immediate-early type 1 IFN. Sequence-specific PCR products for the −620 to +50 promoter region of IFN-α4 were derived from genomic DNA of human 293 cells and cloned into SmaI site of the pGL3-Basic Vector (Promega). The resulting expression vector includes a luciferase gene under control of an upstream (5′) −620 to +50 promoter region of IFN-α4. The sequence of the −620 to +50 promoter region of IFN-α4 is provided as SEQ ID NO:11 in Table 6.  
                                           TABLE 6                       Nucleotide Sequence of the −620 to +50 Promoter Region of           Human IFN-α4                                (SEQ ID NO:11)                agaaaaattt taaaaaatta ttcattcata tttttaggag ttttgaatga ttggatatgt   60                   aattatattc atattattaa tgtgtatcta tatagatttt tattttgcat atgtactttg   120               atacaaaatt tacatgaaca aattacacta aaagttattc cacaaatata cttatcaaat   180               taagttaaat gtcaatagct tttaaactta aattttagtt taacttttct gtcattcttt   240               actttgaata aaaagagcaa actttgtagt ttttatctgt gaagtagagg tatacgtaat   300               atacataaat agatatgcca aatctgtgtt attaaaattt catgaagatt tcaattagaa   360               aaaaatacca taaaaggctt tgagtgcagg tgaaaaatag gcaatgatga aaaaaaatga   420               aaaacttttt aaacacatgt agagagtgcg taaagaaagc aaaaacagag atagaaagta   480               caactaggga atttagaaaa tggaaattag tatgttcact atttaagacc tatgcacaga   540               gcaaagtctt cagaaaacct agaggccgaa gttcaaggtt atccatctca agtagcctag   600               caatatttgc aacatcccaa tggccctgtc cttttcttta ctgatggccg tgctggtgct   660               cagctacaaa 670                  
 
       Example 3  
     Method of Making IFN-α1 Reporter Vector  
       [0071]    IFN-α1 is a late type 1 IFN. Sequence-specific PCR products for the −140 to +9 promoter region of IFN-α1 were derived from genomic DNA of human 293 cells and cloned into SmaI site of the pGL3-Basic Vector (Promega). The resulting expression vector includes a luciferase gene under control of an upstream (5′) −140 to +9 promoter region of IFN-α1.  
       Example 4  
     Method of Making IFN-β Reporter Vector  
       [0072]    IFN-β is an immediate-early type 1 IFN. The −280 to +20 promoter region of IFN-β was derived from the pUCβ26 vector (Algarté M et al. (1999)  J Virol  73(4):2694-702) by restriction at EcoRI and TaqI sites. The 300 bp restriction fragment was filled in by Klenow enzyme and cloned into NheI-digested and filled in pGL3-Basic Vector (Promega). The resulting expression vector includes a luciferase gene under control of an upstream (5′) −280 to +20 promoter region of IFN-β. The sequence of the −280 to +20 promoter region of IFN-β is provided as SEQ ID NO:12 in Table 7.  
                                           TABLE 7                       Nucleotide Sequence of the −280 to +20 Promoter Region of           Human IFn-β                                (SEQ ID NO:12)                ttctcaggtc gtttgctttc ctttgctttc tcccaagtct tgttttacaa tttgctttag   60                   tcattcactg aaactttaaa aaacattaga aaacctcaca gtttgtaaat ctttttccct   120               attatatata tcataagata ggagcttaaa taaagagttt tagaaactac taaaatgtaa   180               atgacatagg aaaactgaaa gggagaagtg aaagtgggaa attcctctga atagagagag   240               gaccatctca tataaatagg ccatacccac ggagaaagga cattctaact gcaacctttc   300                  
 
       Example 5  
     Method of Making RANTES Reporter Vector  
       [0073]    Transcription of the chemokine RANTES is believed to be regulated at least in part by IRF3 and by NF-κB. Lin R et al. (1999)  J Mol Cell Biol  19(2):959-66; Genin P et al. (2000)  J Immunol  164:5352-61. A 483 bp sequence-specific PCR product including the −397 to +5 promoter region of RANTES was derived from genomic DNA of human 293 cells, restricted with PstI and cloned into pCAT-Basic Vector (Promega) using HindIII (filled in with Klenow) and PstI sites (filled in). The −397 to +5 promoter region of RANTES was then isolated from the resulting RANTES/chloramphenicol acetyltransferase (CAT) reporter plasmid by restriction with BglII and SalI, filled in with Klenow enzyme, and cloned into the NheI site (filled in with Klenow) of the pGL3-Basic Vector (Promega). The resulting expression vector includes a luciferase gene under control of an upstream (5′) −397 to +5 promoter region of RANTES. Comparison of the insert sequence −397 to +5 of Genin P et al. (2000)  J Immunol  164:5352-61 and GenBank accession no. AB023652 (SEQ ID NO:13) revealed two point deletions (at positions 105 and 273 of SEQ ID NO:13) which do not create new restriction sites. The sequence of the −397 to +5 promoter region of RANTES is provided as SEQ ID NO:14 in Table 8.  
                                           TABLE 8                       Nucleotide Sequence of the −397 to +5 Promoter Region of           Human RANTES                                SEQ ID NO:14)                gatctgtaat gaataagcag gaactttgaa gactcagtga ctcagtgagt aataaagact   60                   cagtgacttc tgatcctgtc ctaactgcca ctccttgttg tcccaagaaa gcggcttcct   120               gctctctgag gaggacccct tccctggaag gtaaaactaa ggatgtcagc agagaaattt   180               ttccaccatt ggtgcttggt caaagaggaa actgatgagc tcactctaga tgagagagca   240               gtgagggaga gacagagact cgaatttccg gagctatttc agttttcttt tccgttttgt   300               gcaatttcac ttatgatacc ggccaatgct tggttgctat tttggaaact ccccttaggg   360               gatgcccctc aactggccct ataaagggcc agcctgagct g 401                  
 
         [0074]    [0074]                                       TABLE 9                       Nucleotide Sequence of GenBank Accession No. AB023652                                (SEQ ID NO:13)                agaaggcctt acagtgagat gggatcccag tatttattga gtttcctcat tcataaaatg   60                   gggataataa tagtaaatga gttgacacgc gctaagacag tggaatagtg gctggcacag   120               ataagccctc ggtaaatggt agccaataat gatagagtat gctgtaagat atctttctct   180               ccctctgctt ctcaacaagt ctctaatcaa ttattccact ttataaacaa ggaaatagaa   240               ctcaaagaca ttaagcactt ttcccaaagg tcgcttagca agtaaatggg agagacccta   300               tgaccaggat gaaagcaaga aattcccaca agaggactca ttccaactca tatcttgtga   360               aaaggttccc aatgcccagc tcagatcaac tgcctcaatt tacagtgtga gtgtgctcac   420               ctcctttggg gactgtatat ccagaggacc ctcctcaata aaacacttta taaataacat   480               ccttccatgg atgagggaaa ggaggtaaga tctgtaatga ataagcagga actttgaaga   540               ctcagtgact cagtgagtaa taaagactca gtgacttctg atcctgtcct aactgccact   600               ccttgttgtc cccaagaaag cggcttcctg ctctctgagg aggacccctt ccctggaagg   660               taaaactaag gatgtcagca gagaaatttt tccaccattg gtgcttggtc aaagaggaaa   720               ctgatgagct cactctagat gagagagcag tgagggagag acagagactc gaatttccgg   780               aggctatttc agttttcttt tccgttttgt gcaatttcac ttatgatacc ggccaatgct   840               tggttgctat tttggaaact ccccttaggg gatgcccctc aactggccct ataaagggcc   900               agcctgagct gcagaggatt cctgcagagg atcaagacag cacgtggacc tcgcacagcc   960               tctcccacag gtaccatgaa ggtctccgcg gcagccctcg ctgtcatcct cattgctact   1020               gccctctgcg c 1031                    
       Example 6  
     Method of Making Human IL-12 p40 Reporter Vectors  
       [0075]    Reporter constructs have been made using truncated (−250 to +30) and full length (−860 to +30) promoter regions derived from human IL-12 p40 genomic DNA. In one reporter construct the truncated IL-12 p40 promoter was cloned as a KpnI-XhoI insert into pβgal-Basic (Promega). The resulting expression vector includes a β gal gene under control of an upstream (5′) −250 to +30 promoter region of human IL-12 p40. In a second reporter construct the full length IL-12 p40 promoter was cloned as a KpnI-XhoI insert into pβgal-Basic (Promega). The resulting expression vector includes a β gal gene under control of an upstream (5′) −860 to +30 promoter region of human IL-12 p40. In a third reporter construct the truncated −250 to +30 promoter region of human IL-12 p40 was cloned into the pGL3-Basic Vector (Promega). The resulting expression vector includes a luciferase gene under control of an upstream (5′) −250 to +30 promoter region of human IL-12 p40. In a fourth reporter construct the full length IL-12 p40 promoter of human IL-12 p40 was cloned into the pGL3-Basic Vector (Promega). The resulting expression vector includes a luciferase gene under control of an upstream (5′) −860 to +30 promoter region of human IL-12 p40.  
       Example 7  
     Method of Making Human IL-6 Reporter Vectors  
       [0076]    Reporter constructs are made using the −235 to +7 promoter region derived from human IL-6 genomic DNA. In one reporter construct the IL-6 promoter region is cloned as a KpnI-XhoI insert into pGL3-Basic Vector (Promega). The resulting expression vector includes a luciferase gene under control of an upstream (5′) −235 to +7 promoter region derived from human IL-6 genomic DNA.  
       Example 8  
     Method of Making Human IL-8 Reporter Vectors  
       [0077]    Reporter constructs have been made using a −546 to +44 and a truncated −133 to +44 promoter region derived from human IL-8 genomic DNA. Mukaida N et al. (1989)  J Immunol  143:1366-71. In each reporter construct the IL-8 promoter region was cloned as a KpnI-XhoI insert into pGL3-Basic Vector (Promega). One of the resulting expression vectors includes a luciferase gene under control of an upstream (5′) −546 to +44 promoter region derived from human IL-8 genomic DNA. Another of the resulting expression vectors includes a luciferase gene under control of an upstream (5′) −133 to +44 promoter region derived from human IL-8 genomic DNA.  
       Example 9  
     Sequence Comparison of Human TLR3 and Human TLR9  
       [0078]    Human TLR3 and TLR9 are homologous proteins with several structural commonalities. Both appear to be transmembrane proteins with an extracellular domain and an intracellular domain. Common characteristics include a signal sequence and transmembranal domain. Similarities common to most TLRs include a cysteine rich domain and a TIR domain. Most TLRs have leucine rich repeats (LRR) in their extracellular domain. TLR3, TLR7, TLR8, and TLR9 appear to have similar structures. The regularity of the leucine repeats are shown below for TLR3 and TLR9. These four TLRs can be broken into two extracellular subdomains, domain 1 and 2, by virtue of a separation by an unstructured hinge region. TLR7, TLR8, and TLR9 have 14 LRR in domain 1 and 12 LRR in domain 2. TLR9 is a known nucleic acid binder, interacting with CpG-DNA. It has been suspected that TLR7 and TLR8 most likely also interact with nucleic acids. TLR3 has a similar 11 LRR in domain 1 and has 12 LRR in domain 2, lacking the initial 3 repeats common to TLR7, TLR8, and TLR9. Based on structural consideration it is hypothesized that TLR3 interacts with nucleic acids or similar structures.  
         [0079]    The structure of TLR3 differs from TLR7, TLR8, and TLR9 in an interesting character. Referring to Table 13, within the TIR domain it has been shown that a proline (shown in bold) is required for MyD88 interaction. MyD88 is required for TLR9 to transduce signal for the activation of NF-κB. Both TLR7 and TLR8 also have this proline. TLR3 however has an alanine at this position (also shown in bold). It is believed by the applicant that this difference may disallow MyD88 interaction with TLR3 and thus result in an altered signal transduction pattern compared to, e.g., TLR9.  
                                                                                                                                                                                 TABLE 10                       Sequence Alignment of hTLR9 (SEQ ID NO:6)       and hTLR3 (SEQ ID NO:2)                   SIGNAL SEQUENCE            hTLR9   MGFCRSALHPLSLLVQAIMLAMTLALGTLPAFLPCELQPHGLVNCNW   47                   hTLR3   MRQTLPCIYFWGGLLPFGMLCASSTTKCTVSHEVADC   37                    DOMAIN 1 LEUCINE RICH REPEATS            hTLR9   LFLKSVPHFSMAAPRGNVTSLSLSSN   73                   hTLR9   RIHHLHDSDFAHLPSLRHLNLKWN   97               hTLR9   CPPVGLSPMHFPCHMTIEPSTFLAVPTLEELNLSYN   133               hTLR9   NIMTVPALPKSLISLSLSHT   153               hTLR3   SHLKLTQVPDDLPTNITVLNLTHN   61               hTLR9   NILMLDSASLAGLHALRFLFMDGN   177               hTLR3   QLRRLPAANFTRYSQLTSLDVGFN   85               hTLR9   CYYKNPCRQALEVAPGALLGLGNLTHLSLKYN   209               hTLR3   TISKLEPELCQKLPMLKVLNLQHN   109               hTLR9   NLTVVPRNLPSSLEYLLLSYN   230               hTLR3   ELSQLSDKTFAFCTNLTELHLMSN   133               hTLR9   RIVKLAPEDLANLTALRVLDVGGN   254               hTLR3   SIQKIKNNPFVKQKNLITLDLSHN   157               hTLR9     CRRCDHAPNPCMECPRHFPQ LHPDTFSHLSRLEGLVLKDS   294               hTLR3   GLSSTKLGTQVQLENLQELLLSNN   181               hTLR9   SLSWLNASWFRGLGNLRVLDLSEN   318               hTLR3   KIQALKSEELDIFANSSLKKLELSSN   207               hTLR9   FLYKCITKTKAFQGLTQLRKLNLSFN   344               hTLR3   QIKEFSPGCFHAIGRLFGLFLNNV   231               hTLR9   YQKRVSFAHLSLAPSFGSLVALKELDMHGI   374               hTLR3   QLGPSLTEKLCLELANTSIRNLSLSNS   258               hTLR9   FFRSLDETTLRPLARLPMLQTLRLQMN   401               hTLR3   QLSTTSNTTFLGLKWTNLTMLDLSYN   284               hTLR9   FINQAQLGIFRAFPGLRYVDLSDN   425               hTLR3   NLNVVGNDSFAWLPQLEYFFLEYN   308                    HINGE REGION            hTLR9   RISGASELTATMGEADGGEKVWLQPGDLAPAPV   458                   hTLR3   NIQHLFSHSLHGLFNVRYLNLKRSFTKQSISLA   341                    DOMAIN 2 LEUCINE RICH REPEATS            hTLR9   DTPSSEDFRPNCSTLNFTLDLSRN   482                   hTLR3   SLPKIDDFSFQWLKCLEHLNMEDN   365               hTLR9   NLVTVQPEMFAQLSHLQCLRLSHN   506               hTLR3   DIPGIKSNMFTGLINLKYLSLSNS   389               hTLR9   CISQAVNGSQFLPLTGLQVLDLSHN   531               hTLR3   FTSLRTLTNETFVSLAHSPLHILNLTKN   417               hTLR9   KL DLY HEHSFTELPRLEALDLSYN   555               hTLR3   KISKIESDAFSWLGHLEVLDLGLN   441               hTLR9   SQPFGMQGVGHNFSFVAHLRTLRHLSLAHN   585               hTLR3   EIGQELTGQEWRGLENIFEIYLSYN   466               hTLR9   NIHSQVSQQLCSTSLRALDFSGN   608               hTLR3   KYLQLTRNSFALVPSLQRLMLRRV   490               hTLR9   ALGHMWAEGDLYLHFFQGLSGLIWLDLSQN   638               hTLR3   ALKNVDSSPSPFQPLRNLTILDLSNN   516               hTLR9   RLHTLLPQTLRNLPKSLQVLRLRDN   663               hTLR3   NIANINDDMLEGLEKLEILDLQHN   540               hTLR9   YLAFFKWWSLHFLPKLEVLDLAGN   687               hTLR3   NLARLWKHANPGGPIYFLKGLSHLHILNLESN   572               hTLR9   QLKALTNGSLPAGTRLRRLDVSCN   711               hTLR3   GFDEIPVEVFKDLFELKIIDLGLN   596               hTLR9   SISFVAPGFFSKAKELRELNLSAN   735               hTLR3   NLNTLPASVFNNQVSLKSLNLQKN   620               hTLR9   ALKTVDHSWFGPLASALQILDVSAN   760               hTLR3   LITSVEKKVFGPAFRNLTELDMRFN   645                    CYSTEINE RICH DOMAIN            hTLR9   PLHCACG**AAFMDFLLEVQAAVPGLPSRVKCGSPGQLQGLSIFAQD   805                   hTLR3   PFDCTCESIAWFVNWINETHTNIPELSSHYLCNTPPHYHGFPVRLFD   692               hTLR9   LRLCLDEALSWDCFA   820               hTLR3   TSSCKDSAPFELFFM   707                    TRANSMEMBRANAL DOMAIN            hTLR9   LSLLAVALGLGVPMLHHL   838                   hTLR3   INTSILLIFIFIVLLIHF   725                    TIR DOMAIN            hTLR9   CGWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVAD   885                   hTLR3   EGWRISFYWNVSVHRVLGFKEIDRQTEQFE*YAAYIIHAYK***DKD   768               hTLR9   WVYNELRGQLEECRGRWALRLCLEERDWLPGKTLFENLWASVYGSRK   932               hTLR3   WVW***EHFSSMEKEDQSLKFCLEERDFEAGVFELEAIVNSIKRSRK   812               hTLR9   TLFVLAHTD*RVSGLLRASFLLAQQRLLEDRKDVVVLVILSPDGRRS   978               hTLR3   IIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYK   859               hTLR9   ***RYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHHFYN   1022               hTLR3   LNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH   904               hTLR9   RNFCQGPTAE   1032                  
 
       Example 10  
     Reconstitution of TLR9 Signaling in 293 Fibroblasts  
       [0080]    Methods for cloning murine and human TLR9 have been described in pending U.S. patent application Ser. No. 09/954,987 and corresponding published PCT application PCT/US01/29229, both filed Sep. 17, 2001, the contents of which are incorporated by reference. Human TLR9 cDNA and murine TLR9 cDNA in pT-Adv vector (from Clonetech) were individually cloned into the expression vector pcDNA3.1(−) from Invitrogen using the EcoRI site. Utilizing a “gain of function” assay it was possible to reconstitute human TLR9 (hTLR9) and murine TLR9 (mTLR9) signaling in CpG-DNA non-responsive human 293 fibroblasts (ATCC, CRL-1573). The expression vectors mentioned above were transfected into 293 fibroblast cells using the calcium phosphate method.  
                                       TABLE 11                           cDNA Sequence for Human TLR9                (GenBank Accession No. AF245704; SEQ ID NO:5)                    aggctggtat aaaaatctta cttcctctat tctctgagcc gctgctgccc ctgtgggaag   60                   ggacctcgag tgtgaagcat ccttccctgt agctgctgtc cagtctgccc gccagaccct   120               ctggagaagc ccctgccccc cagcatgggt ttctgccgca gcgccctgca cccgctgtct   180               ctcctggtgc aggccatcat gctggccatg accctggccc tgggtacctt gcctgccttc   240               ctaccctgtg agctccagcc ccacggcctg gtgaactgca actggctgtt cctgaagtct   300               gtgccccact tctccatggc agcaccccgt ggcaatgtca ccagcctttc cttgtcctcc   360               aaccgcatcc accacctcca tgattctgac tttgcccacc tgcccagcct gcggcatctc   420               aacctcaagt ggaactgccc gccggttggc ctcagcccca tgcacttccc ctgccacatg   480               accatcgagc ccagcacctt cttggctgtg cccaccctgg aagagctaaa cctgagctac   540               aacaacatca tgactgtgcc tgcgctgccc aaatccctca tatccctgtc cctcagccat   600               accaacatcc tgatgctaga ctctgccagc ctcgccggcc tgcatgccct gcgcttccta   660               ttcatggacg gcaactgtta ttacaagaac ccctgcaggc aggcactgga ggtggccccg   720               ggtgccctcc ttggcctggg caacctcacc cacctgtcac tcaagtacaa caacctcact   780               gtggtgcccc gcaacctgcc ttccagcctg gagtatctgc tgttgtccta caaccgcatc   840               gtcaaactgg cgcctgagga cctggccaat ctgaccgccc tgcgtgtgct cgatgtgggc   900               ggaaattgcc gccgctgcga ccacgctccc aacccctgca tggagtgccc tcgtcacttc   960               ccccagctac atcccgatac cttcagccac ctgagccgtc ttgaaggcct ggtgttgaag   1020               gacagttctc tctcctggct gaatgccagt tggttccgtg ggctgggaaa cctccgagtg   1080               ctggacctga gtgagaactt cctctacaaa tgcatcacta aaaccaaggc cttccagggc   1140               ctaacacagc tgcgcaagct taacctgtcc ttcaattacc aaaagagggt gtcctttgcc   1200               cacctgtctc tggccccttc cttcgggagc ctggtcgccc tgaaggagct ggacatgcac   1260               ggcatcttct tccgctcact cgatgagacc acgctccggc cactggcccg cctgcccatg   1320               ctccagactc tgcgtctgca gatgaacttc atcaaccagg cccagctcgg catcttcagg   1380               gccttccctg gcctgcgcta cgtggacctg tcggacaacc gcatcagcgg agcttcggag   1440               ctgacagcca ccatggggga ggcagatgga ggggagaagg tctggctgca gcctggggac   1500               cttgctccgg ccccagtgga cactcccagc tctgaagact tcaggcccaa ctgcagcacc   1560               ctcaacttca ccttggatct gtcacggaac aacctggtga ccgtgcagcc ggagatgttt   1620               gcccagctct cgcacctgca gtgcctgcgc ctgagccaca actgcatctc gcaggcagtc   1680               aatggctccc agttcctgcc gctgaccggt ctgcaggtgc tagacctgtc ccgcaataag   1740               ctggacctct accacgagca ctcattcacg gagctaccgc gactggaggc cctggacctc   1800               agctacaaca gccagccctt tggcatgcag ggcgtgggcc acaacttcag cttcgtggct   1860               cacctgcgca ccctgcgcca cctcagcctg gcccacaaca acatccacag ccaagtgtcc   1920               cagcagctct gcagtacgtc gctgcgggcc ctggacttca gcggcaatgc actgggccat   1980               atgtgggccg agggagacct ctatctgcac ttcttccaag gcctgagcgg tttgatctgg   2040               ctggacttgt cccagaaccg cctgcacacc ctcctgcccc aaaccctgcg caacctcccc   2100               aagagcctac aggtgctgcg tctccgtgac aattacctgg ccttctttaa gtggtggagc   2160               ctccacttcc tgcccaaact ggaagtcctc gacctggcag gaaaccggct gaaggccctg   2220               accaatggca gcctgcctgc tggcacccgg ctccggaggc tggatgtcag ctgcaacagc   2280               atcagcttcg tggcccccgg cttcttttcc aaggccaagg agctgcgaga gctcaacctt   2340               agcgccaacg ccctcaagac agtggaccac tcctggtttg ggcccctggc gagtgccctg   2400               caaatactag atgtaagcgc caaccctctg cactgcgcct gtggggcggc ctttatggac   2460               ttcctgctgg aggtgcaggc tgccgtgccc ggtctgccca gccgggtgaa gtgtggcagt   2520               ccgggccagc tccagggcct cagcatcttt gcacaggacc tgcgcctctg cctggatgag   2580               gccctctcct gggactgttt cgccctctcg ctgctggctg tggctctggg cctgggtgtg   2640               cccatgctgc atcacctctg tggctgggac ctctggtact gcttccacct gtgcctggcc   2700               tggcttccct ggcgggggcg gcaaagtggg cgagatgagg atgccctgcc ctacgatgcc   2760               ttcgtggtct tcgacaaaac gcagagcgca gtggcagact gggtgtacaa cgagcttcgg   2820               gggcagctgg aggagtgccg tgggcgctgg gcactccgcc tgtgcctgga ggaacgcgac   2880               tggctgcctg gcaaaaccct ctttgagaac ctgtgggcct cggtctatgg cagccgcaag   2940               acgctgtttg tgctggccca cacggaccgg gtcagtggtc tcttgcgcgc cagcttcctg   3000               ctggcccagc aqcgcctgct ggaggaccgc aaggacgtcg tggtgctggt gatcctgagc   3060               cctgacggcc gccgctcccg ctacgtgcgg ctgcgccagc gcctctgccg ccagagtgtc   3120               ctcctctggc cccaccagcc cagtggtcag cgcagcttct gggcccagct gggcatggcc   3180               ctgaccaggg acaaccacca cttctataac cggaacttct gccagggacc cacggccgaa   3240               tagccgtgag ccggaatcct gcacggtgcc acctccacac tcacctcacc tctgcctgcc   3300               tggtctgacc ctcccctgct cgcctccctc accccacacc tgacacagag ca 3352                  
 
         [0081]    [0081]                                       TABLE 12                           Amino Acid Sequence for Human TLR9                (GenBank Accession No. AAF78037, SEQ ID NO:6)+HZ,1/44            MGFCRSALHP LSLLVQAIML AMTLALGTLP AFLPCELQPH GLVNCNWLFL KSVPHFSMAA   60                   PRGNVTSLSL SSNRIHHLHD SDFAHLPSLR HLNLKWNCPP VGLSPMHFPC HMTIEPSTFL   120               AVPTLEELNL SYNNIMTVPA LPKSLISLSL SHTNILMLDS ASLAGLHALR FLFMDGNCYY   180               KNPCRQALEV APGALLGLGN LTHLSLKYNN LTVVPRNLPS SLEYLLLSYN RIVKLAPEDL   240               ANLTALRVLD VGGNCRRCDH APNPCMECPR HFPQLHPDTF SHLSRLEGLV LKDSSLSWLN   300               ASWFRGLGNL RVLDLSENFL YKCITKTKAF QGLTQLRKLM LSFNYQKRVS FAHLSLAPSF   360               GSLVALKELD MHGIFFRSLD ETTLRPLARL PMLQTLRLQM NFINQAQLGI FRAFPGLRYV   420               DLSDNRISGA SELTATMGEA DGGEKVWLQP GDLAPAPVDT PSSEDFRPNC STLNFTLDLS   480               RNNLVTVQPE MFAQLSHLQC LRLSHNCISQ AVNGSQFLPL TGLQVLDLSR NKLDLYHEHS   540               FTELPRLEAL DLSYNSQPFG MQGVGHNFSF VAHLRTLRHL SLAHNNTHSQ VSQQLCSTSL   600               RALDFSGNAL GHMWAEGDLY LHFFQGLSGL IWLDLSQNRL HTLLPQTLRN LPKSLQVLRL   660               RDNYLAFFKW WSLHFLPKLE VLDLAGNRLK ALTNGSLPAG TRLRRLDVSC NSISFVAPGF   720               FSKAKELREL NLSANALKTV DHSWFGPLAS ALQILDVSAN PLHCACGAAF MDFLLEVQAA   780               VPGLPSRVKC GSPGQLQGLS IFAQDLRLCL DEALSWDCFA LSLLAVALCL GVPMLHHLCG   840               WDLWYCFHLC LAWLPWRGRQ SGRDEDALPY DAFVVFDKTQ SAVADWVYNE LRGQLEECRG   900               RWALRLCLEE RDWLPGKTLF ENLWASVYGS RKTLFVLAHT DRVSGLLRAS FLLAQQRLLE   960               DRKDVVVLVI LSPDGRRSRY VRLRQRLCRQ SVLLWPHQPS GQRSFWAQLG MALTRDNHHF   1020               YNRNFCQGPT AE 1032                    
         [0082]    [0082]                                 TABLE 13                           cDNA Sequence for Murine TLR9           (GenBank Accession No. AF348140; SEQ ID NO:7)                    tgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg tcgaaggact   60                   ctgcacccct tgtccctcct ggtacaggct gcagtgctgg ctgagactct ggccctgggt   120               accctgcctg ccttcctacc ctgtgagctg aagcctcatg gcctggtgga ctgcaattgg   180               ctgttcctga agtctgtacc ccgtttctct gcggcagcat cctgctccaa catcacccgc   240               ctctccttga tctccaaccg tatccaccac ctgcacaact ccgacttcgt ccacctgtcc   300               aacctgcggc agctgaacct caagtggaac tgtccaccca ctggccttag ccccctgcac   360               ttctcttgcc acatgaccat tgagcccaga accttcctgg ctatgcgtac actggaggag   420               ctgaacctga gctataatgg tatcaccact gtgccccgac tgcccagctc cctggtgaat   480               ctgagcctga gccacaccaa catcctggtt ctagatgcta acagcctcgc cggcctatac   540               agcctgcgcg ttctcttcat ggacgggaac tgctactaca agaacccctg cacaggagcg   600               gtgaaggtga ccccaggcgc cctcctgggc ctgagcaatc tcacccatct gtctctgaag   660               tataacaacc tcacaaaggt gccccgccaa ctgcccccca gcctggagta cctcctggtg   720               tcctataacc tcattgtcaa gctggggcct gaagacctgg ccaatctgac ctcccttcga   780               gtacttgatg tgggtgggaa ttgccgtcgc tgcgaccatg cccccaatcc ctgtatagaa   840               tgtggccaaa agtccctcca cctgcaccct gagaccttcc atcacctgag ccatctggaa   900               ggcctggtgc tgaaggacag ctctctccat acactgaact cttcctggtt ccaaggtctg   960               gtcaacctct cggtgctgga cctaagcgag aactttctct atgaaagcat caaccacacc   1020               aatgcctttc agaacctaac ccgcctgcgc aagctcaacc tgtccttcaa ttaccgcaag   1080               aaggtatcct ttgcccgcct ccacctggca agttccttca agaacctggt gtcactgcag   1140               gagctgaaca tgaacggcat cttcttccgc tcgctcaaca agtacacgct cagatggctg   1200               gccgatctgc ccaaactcca cactctgcat cttcaaatga acttcatcaa ccaggcacag   1260               ctcagcatct ttggtacctt ccgagccctt cgctttgtgg acttgtcaga caatcgcatc   1320               agtgggcctt caacgctgtc agaagccacc cctgaagagg cagatgatgc agagcaggag   1380               gagctgttgt ctgcggatcc tcacccagct ccactgagca cccctgcttc taagaacttc   1440               atggacaggt gtaagaactt caagttcacc atggacctgt ctcggaacaa cctggtgact   1500               atcaagccag agatgtttgt caatctctca cgcctccagt gtcttagcct gagccacaac   1560               tccattgcac aggctgtcaa tggctctcag ttcctgccgc tgactaatct gcaggtgctg   1620               gacctgtccc ataacaaact ggacttgtac cactggaaat cgttcagtga gctaccacag   1680               ttgcaggccc tggacctgag ctacaacagc cagcccttta gcatgaaggg tataggccac   1740               aatttcagtt ttgtggccca tctgtccatg ctacacagcc ttagcctggc acacaatgac   1800               attcataccc gtgtgtcctc acatctcaac agcaactcag tgaggtttct tgacttcagc   1860               ggcaacggta tgggccgcat gtgggatgag gggggccttt atctccattt cttccaaggc   1920               ctgagtggcc tgctgaagct ggacctgtct caaaataacc tgcatatcct ccggccccag   1980               aaccttgaca acctccccaa gagcctgaag ctgctgagcc tccgagacaa ctacctatct   2040               ttctttaact ggaccagtct gtccttcctg cccaacctgg aagtcctaga cctggcaggc   2100               aaccagctaa aggccctgac caatggcacc ctgcctaatg gcaccctcct ccagaaactg   2160               gatgtcagca gcaacagtat cgtctctgtg gtcccagcct tcttcgctct ggcggtcgag   2220               ctgaaagagg tcaacctcag ccacaacatt ctcaagacgg tggatcgctc ctggtttggg   2280               cccattgtga tgaacctgac agttctagac gtgagaagca accctctgca ctgtgcctgt   2340               ggggcagcct tcgtagactt actgttggag gtgcagacca aggtgcctgg cctggctaat   2400               ggtgtgaagt gtggcagccc cggccagctg cagggccgta gcatcttcgc acaggacctg   2460               cggctgtgcc tggatgaggt cctctcttgg gactgctttg gcctttcact cttggctgtg   2520               gccgtgggca tggtggtgcc tatactgcac catctctgcg gctgggacgt ctggtactgt   2580               tttcatctgt gcctggcatg gctacctttg ctggcccgca gccgacgcag cgcccaagct   2640               ctcccctatg atgccttcgt ggtgttcgat aaggcacaga gcgcagttgc ggactgggtg   2700               tataacgagc tgcgggtgcg gctggaggag cggcgcggtc gccgagccct acgcttgtgt   2760               ctggaggacc gagattggct gcctggccag acgctcttcg agaacctctg ggcttccatc   2820               tatgggagcc gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg   2880               cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga cgtggtggtg   2940               ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg tgcgactgcg ccagcgtctc   3000               tgccgccaga gtgtgctctt ctggccccag cagcccaacg ggcagggggg cttctgggcc   3060               cagctgagta cagccctgac tagggacaac cgccacttct ataaccagaa cttctgccgg   3120               ggacctacag cagaatagct cagagcaaca gctggaaaca gctgcatctt catgcctggt   3180               tcccgagttg ctctgcctgc 3200                    
         [0083]    [0083]                                       TABLE 14                           Amino Acid Sequence for Murine TLR9                (GenBank Accession No. AAK29625; SEQ ID NO:8)                    MVLRRRTLHP LSLLVQAAVL AETLALGTLP AFLPCELKPH GLVDCNWLFL KSVPRFSAAA   60                   SCSNITRLSL ISNRIHHLHN SDFVHLSNLR QLNLKWNCPP TGLSPLHFSC HMTIEPRTFL   120               AMRTLEELNL SYNGITTVPR LPSSLVNLSL SHTNILVLDA NSLAGLYSLR VLFMDGNCYY   180               KNPCTGAVKV TPGALLGLSN LTHLSLKYNN LTKVPRQLPP SLEYLLVSYN LIVKLGPEDL   240               ANLTSLRVLD VGGNCRRCDH APNPCIECGQ KSLHLHPETF HHLSHLEGLV LKDSSLHTLN   300               SSWFQGLVNL SVLDLSENFL YESTNBTNAF QNLTRLRKLN LSFNYRKKVS FARLHLASSF   360               KNLVSLQELN MNGIFFRSLN KYTLRWLADL PKLHTLHLQM NFINQAQLSI FGTFRALRFV   420               DLSDNRISGP STLSEATPEE ADDAEQEELL SADPHPAPLS TPASKNFMDR CKIFKFTMDL   480               SRNNLVTIKP EMFVNLSRLQ CLSLSHNSIA QAVNGSQFLP LTNLQVLDLS HNKLDLYHWK   540               SFSELPQLQA LDLSYNSQPF SMKGIGHNFS FVAHLSMLHS LSLAHNDIHT RVSSHLNSNS   600               VRFLDFSGNG MGRMWDEGGL YLHFFQGLSG LLKLDLSQNN LHILRPQNLD NLPKSLKLLS   660               LRDNYLSFFN WTSLSFLPNL EVLDLAGNQL KALTNGTLPN GTLLQKLDVS SNSIVSVVPA   720               FFALAVELKE VNLSHNTLKT VDRSWFGPTV MNLTVLDVRS NPLHCACGAA FVDLLLEVQT   780               KVPGLANGVK CGSPGQLQGR SIFAQDLRLC LDEVLSWDCF GLSLLAVAVG MVVPILHHLC   840               GWDVWYCFHL CLAWLPLLAR SRRSAQALPY DAFVVFDKAQ SAVADWVYNE LRVRLEERRG   900               RRALRLCLED RDWLPGQTLF ENLWASIYGS RKTLFVLAHT DRVSGLLRTS FLLAQQRLLE   960               DRKDVVVLVI LRPDAHRSRY VRLRQRLCRQ SVLFWPQQPN GQOGFWAQLS TALTRDNRHF   1020               YNQNFCRGPT AE 1032                    
         [0084]    Since NF-κB activation is central to the IL-1/TLR signal transduction pathway (Medzhitov R et al. (1998)  Mol Cell  2:253-258 (1998); Muzio M et al. (1998)  J Exp Med  187:2097-101), cells were transfected with hTLR9 or co-transfected with hTLR9 and an NF-κB-driven luciferase reporter construct. Human 293 fibroblast cells were transiently transfected with (FIG. 1A) hTLR9 and a six-times NF-κB-luciferase reporter plasmid (NF-κB-luc, kindly provided by Patrick Baeuerle, Munich, Germany) or (FIG. 1B) with hTLR9 alone. After stimulus with CpG-ODN (2006, 2 μM, T CG T CG TTTTGT CG TTTTGT CG TT, SEQ ID NO:15), GpC-ODN (2006-GC, 2 μM, TGCTGCTTTTGTGCTTTTGTGCTT, SEQ ID NO:16), LPS (100 ng/ml) or media, NF-κB activation by luciferase readout (8 h, FIG. 1A) or IL-8 production by ELISA (48 h, FIG. 1B) were monitored. Results are representative of three independent experiments. FIG. 1 shows that cells expressing hTLR9 responded to CpG-DNA but not to LPS.  
         [0085]    [0085]FIG. 2 demonstrates the same principle for the transfection of mTLR9. Human 293 fibroblast cells were transiently transfected with mTLR9 and the NF-κB-luc construct (FIG. 2). Similar data was obtained for IL-8 production (not shown). Thus expression of TLR9 (human or mouse) in 293 cells results in a gain of function for CpG-DNA stimulation similar to hTLR4 reconstitution of LPS responses.  
         [0086]    To generate stable clones expressing human TLR9, murine TLR9, or either TLR9 with the NF-κB-luc reporter plasmid, 293 cells were transfected in 10 cm plates (2×10 6  cells/plate) with 16 μg of DNA and selected with 0.7 mg/ml G418 (PAA Laboratories GmbH, Cölbe, Germany). Clones were tested for TLR9 expression by RT-PCR, for example as shown in FIG. 3. The clones were also screened for IL-8 production or NF-κB-luciferase activity after stimulation with ODN. Four different types of clones were generated.  
                                                       293-hTLR9-luc:   expressing human TLR9 and               6-fold NF-κB-luciferase reporter           293-mTLR9-luc:   expressing murine TLR9 and               6-fold NF-κB-luciferase reporter           293-hTLR9:   expressing human TLR9           293-mTLR9:   expressing murine TLR9                      
 
         [0087]    [0087]FIG. 4 demonstrates the responsiveness of a stable 293-hTLR9-luc clone after stimulation with CpG-ODN (2006, 2 μM), GpC-ODN (2006-GC, 2 μM), Me-CpG-ODN (2006 methylated, 2 μM; TZGTZGTTTTGTZGTTTTGTZGTT, Z=5-methylcytidine, SEQ ID NO:17), LPS (100 ng/ml) or media, as measured by monitoring NF-κB activation. Similar results were obtained utilizing IL-8 production with the stable clone 293-hTLR9. 293-mTLR9-luc were also stimulated with CpG-ODN (1668, 2μM; TCCATGA CG TTCCTGATGCT, SEQ ID NO:18), GpC-ODN (1668-GC, 2 μM; TCCATGAGCTTCCTGATGCT, SEQ ID NO:19), Me-CpG-ODN (1668 methylated, 2 μM; TCCATGAZGTTCCTGATGCT, Z=5-methylcytidine, SEQ ID NO:20), LPS (100 ng/ml) or media, as measured by monitoring NF-κB activation (FIG. 5). Similar results were obtained utilizing IL-8 production with the stable clone 293-mTLR9. Results are representative of at least two independent experiments. These results demonstrate that CpG-DNA non-responsive cell lines can be stably genetically complemented with TLR9 to become responsive to CpG-DNA in a motif-specific manner. These cells can be used for screening of optimal ligands for innate immune responses driven by TLR9 in multiple species.  
       Example 11  
     Reconstitution of TLR3 Signaling in 293 Fibroblasts  
       [0088]    Human TLR3 cDNA and murine TLR3 cDNA in pT-Adv vector (from Clonetech) were individually cloned into the expression vector pcDNA3.1 (−) from Invitrogen using the EcoRI site. The resulting expression vectors mentioned above were transfected into CpG-DNA non-responsive human 293 fibroblast cells (ATCC, CRL-1573) using the calcium phosphate method. Utilizing a “gain of function” assay it was possible to reconstitute human TLR3 (hTLR3) and murine TLR3 (mTLR3) signaling in 293 fibroblast cells.  
         [0089]    Since NF-κB activation is central to the IL-1/TLR signal transduction pathway (Medzhitov R et al. (1998)  Mol Cell  2:253-8; Muzio M et al. (1998)  J Exp Med  187:2097-101), in a first set of experiments human 293 fibroblast cells were transfected with hTLR3 alone or co-transfected with hTLR3 and an NF-κB-driven luciferase reporter construct.  
         [0090]    Likewise, in a second set of experiments, 293 fibroblast cells were transfected with hTLR3 alone or co-transfected with hTLR3 and an IFN-α4-driven luciferase reporter construct (described in Example 2 above).  
         [0091]    In a third group of experiments, 293 fibroblast cells were transfected with hTLR3 alone or co-transfected with hTLR3 and a RANTES-driven luciferase reporter construct (described in Example 5 above).  
       Example 12  
     Proline to Histidine Mutation P915H in the TIR Domain of Human and MurineTLR9 Alters TLR9 Signaling  
       [0092]    Toll-like receptors have a cytoplasmic Toll/IL-1 receptor (TIR) homology domain which initiates signaling after binding of the adapter molecule MyD88. Medzhitov R et al. (1998)  Mol Cell  2:253-8; Kopp E B et al. (1999)  Curr Opin Immunol  11:15-8. Reports by others have shown that a single point mutation in the signaling TIR domain in murine TLR4 (Pro712 to His, P712H) or human TLR2 (Pro681 to His, P681H) abolishes host immune response to lipopolysaccharide or gram-positive bacteria, respectively. Poltorak A et al. (1998)  Science  282:2085-8; Underhill D M et al. (1999)  Nature  401:811-5. Through site-specific mutagenesis the equivalent proline (P) at position 915 of human TLR9 and murine TLR9 were mutated to histidine (H; P915H). These mutations were generated by the use of the primers 5′-GCGACTGGCTGCATGGCAAAACCCTCTTTG-3′ (SEQ ID NO:21) and 5′-CAAAGAGGGTTTTGCCATGCAGCCAGTCGC-3′ (SEQ ID NO:22) for human TLR9 and the primers 5′-CGAGATTGGCTGCATGGCCAGACGCTCTTC-3′ (SEQ ID NO:23) and 5′-GAAGAGCGTCTGGCCATGCAGCCAATCTCG-3′ (SEQ ID NO:24) for murine TLR9. Expression vectors for the mutant TLR9s, hTLR9-P915H and mTLR9-P915H, were constructed and verified using standard recombinant DNA techniques.  
         [0093]    For the stimulation of human TLR9 variant, hTLR9-P915H, 293 cells were transiently transfected with expression vector for hTLR9 or hTLR9-P915H and stimulated after 16 hours with ODN 2006 or ODN 1668 at various concentrations. Likewise for the stimulation of murine TLR9 variant, mTLR9-P915H, 293 cells were transiently transfected with expression vector for mTLR9 or mTLR9-P915H and stimulated after 16 hours with ODN 2006 or ODN 1668 at various concentrations. After 48 hours of stimulation, supernatant was harvested and IL-8 production was measured by ELISA. Results demonstrated that TLR9 activity can be destroyed by the P915H mutation in the TIR domain of both human and murine TLR9.  
       Example 13  
     Exchange of the TIR Domain Between Human TLR3 and Human TLR9 (hTLR3-TIR9 and hTLR9-TIR3)  
       [0094]    While TLR3 and TLR9 share many structural features, TLR3, by virtue of its having an alanine rather than proline at a critical position in the TIR domain, may not be able to signal via MyD88 as does TLR9. The chimeric TLRs described here can be used in the screening assays of the invention. To generate molecules consisting of human extracellular TLR3 and the TIR domain of human TLR9 (hTLR3-TIR9), the following approach can be used. Through site-specific mutagenesis a ClaI restriction site is introduced in human TLR3 and human TLR9. For human TLR9 the DNA sequence 5′-GGCCTCAGCATCTTT-3′ (3026-3040, SEQ ID NO:25) is mutated to 5′-GGCCT ATCGAT TTTT-3′ (SEQ ID NO:26), introducing a ClaI site (underlined in the sequence) but leaving the amino acid sequence (GLSIF, aa 798-802) unchanged. For human TLR3 the DNA sequence 5′-GGGTTCCCAGTGAGA-3′ (2112-2126, SEQ ID NO:27) is mutated to 5′-GGGTT ATCGAT TAGA-3′ (SEQ ID NO:28), introducing a ClaI site and creating the amino acid sequence (GLSIR, aa 685-689) which differs in three positions (aa 686, 687, 688) from the wildtype human TLR3 sequence (GFPVR, aa 685-689).  
         [0095]    hTLR3-TIR9. The primers used for human TLR9 are 5′-CAGCTCCAGGGCCTATCGATTTTTGCACAGGACC-3′ (SEQ ID NO:29) and 5′-GGTCCTGTGCAAAAATCGATAGGCCCTGGAGCTG-3′ (SEQ ID NO:30). For creating an expression vector containing the extracellular portion of human TLR3 connected to the TIR domain of human TLR9, the human TLR3 expression vector is cut with ClaI and limiting amounts of EcoRI and the fragment coding for the TIR domain of human TLR9 generated by a ClaI and EcoRI digestion of human TLR9 expression vector is ligated in the vector fragment containing the extracellular portion of hTLR3. Transfection into  E. coli  yields the expression vector hTLR3-TIR9 (human extracellular TLR3-human TLR9 TIR domain). The expressed product of hTLR3-TIR9 can interact with TLR3 ligands and also signal through an MyD88-mediated signal transduction pathway.  
         [0096]    hTLR9-TIR3. A fusion construct with the extracellular domain of hTLR9 and the TIR domain of hTLR3 is prepared using an analogous strategy. For creating an expression vector containing the extracellular portion of human TLR9 connected to the TIR domain of human TLR3, the human TLR9 expression vector is cut with ClaI and limiting amounts of EcoRI and the fragment coding for the TIR domain of human TLR3 generated by a ClaI and EcoRI digestion of human TLR3 expression vector is ligated in the vector fragment containing the extracellular portion of hTLR9. Transfection into  E. coli  yields the expression vector hTLR9-TIR3 (human extracellular TLR9-human TLR3 TIR domain). The expressed product of hTLR9-TIR3 can interact with TLR9 ligands, e.g., CpG DNA, and signal through a signal transduction pathway in a manner like TLR3.  
       Example 14  
     Sensitive in vitro Assay for Detecting Ligand Affinity Differences for a TLR  
       [0097]    Human 293 fibroblast cells stably transfected with murine TLR9 and an NF-κB-luciferase reporter were stimulated for 16 hours with the following fully phosphorothioated oligodeoxynucleotides (ODN):  
                                                                                                                 (SEQ ID NO:31)                5890:   T*C*C*A*T*G*A*C*G*T*T*T*T*T*G*A*T*G*T*T                        (SEQ ID NO:32)                5895:   T*C*C*A*T*G*A*C*G*T*T*T*T*T*G*A*T*G                        (SEQ ID NO:33)                5896:   T*C*C*A*T*G*A*C*G*T*T*T*T*T*G*A                        (SEQ ID NO:34)                5897:   T*C*C*A*T*G*A*C*G*T*T*T*T*T              
 
         [0098]    Concentration of the stimulus was titrated between 10 μM and 2 nM. The data is plotted in FIG. 6 as fold induction of NF-κB luciferase, relative to unstimulated background, versus ODN concentration. The data displays typical first-order binding from which EC50 or maximal activity can be determined. EC50 is defined as the concentration of the ligand stimulus that results in 50% maximal activation. As shown in the figure, the EC50 ranges from 42 nM for ODN 5890 to 1220 nM for ODN 5897. The assay demonstrates sensitive differentiation between subtle changes in ligand.  
       Example 15  
     Influence of Assay Kinetics on TLR Screening Assays  
       [0099]    Curves were prepared as in the previous Example 14 with the following ODN ligands, where * indicates phosphrothioate and _ indicates phosophodiester linkage:  
                                   5890:   T*C*C*A*T*G*A*C*G*T*T*T*T*T*G*A*T*G*T*T   (SEQ ID NO:35)                   5497:   T*C*G*T*C*G*T*T*T*T_G_T_C_G_T*T*T*T*G*T*C*G*T*T   (SEQ ID NO:36)               5746:   T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T*G*T*C_G*T*T   (SEQ ID NO:37)               2006:   T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T   (SEQ ID NO:15)               5902:   T*C*C*A*T*G*A*C_G_T*T*T*T*T*G*A*T_G*T*T   (SEQ ID NO:38)          
 
         [0100]    A family of stimulation curves was determined at various times of assay incubation between 1 and 24 hours. The EC50 was determined for each ligand at each time point. The EC50 was then plotted versus time to yield the resultant curves shown in FIG. 7.  
         [0101]    As evident from FIG. 7, it is demonstrated that the kinetics of activation vary dependent on the ligand tested. Because luciferase has a three-hour half-life, the signal is transient and requires constant promoter-driven activation to be maintained. The maintenance is directly related to the signal delivered by the ligand/receptor complex. Thus analysis of time kinetics in such a fashion allows one to determine both affinity of ligand/receptor interaction and the availability of the ligand to the receptor through time. The principle is demonstrated as follows. The ODN 5890 is of higher affinity compared to the ODN 2006. When the ligand is made more labile to destruction by incorporating less stable diester linkages, the activity curves turn upward with time such as for ODN 5746, 5902 and 5497.  
         [0102]    In the context of a screening assay for TLR/ligand interactions, limiting the assay to one time point would bias the assay. At 24 hours it would appear that only ODN 2006 and 5890 were ligand candidates, however this is clearly not the case. The assay also demonstrates that earlier time points, such as 6 hours in this example, would be the optimal time point for determining the greatest difference between receptor/ligand affinities. Thus optimization of the screening assay can be adjusted depending on the desired information to be obtained from the screen, e.g., higher affinity of interaction versus stability and duration of receptor/ligand interaction.  
         [0103]    [0103]FIG. 8 demonstrates the same principles shown with a murine TLR as in this example can be applied independent of the TLR utilized. For this set of data a 293 cell stably transfected with human TLR9 and NF-κB-luciferase was used.  
       Example 16  
     Influence of Assay Kinetics on Maximal Activities in TLR Screening Assays  
       [0104]    Data was collected as in the previous Example 15, however the maximal activity (maximal fold induction) was plotted versus time in FIGS. 9 and 10. Such data analysis results in a prediction of biological efficacy. As can be seen from these figures, the lower affinity ODN, e.g., ODN 2006 and 5890 as demonstrated by the EC50 curves of Example 15, are clearly less efficient at delivering high activity.  
       Example 17  
     Differential Outcomes of TLR Screening Assays Dependent on Promoter Utilization  
       [0105]    Human 293 fibroblast cells were transiently transfected with expression vector for TLR 7, TLR8, or TLR9 and one of the following reporter constructs bearing the following promoters driving the luciferase gene: NF-κB-luc, IP-10-luc, RANTES-luc, ISRE-luc, and IL-8-luc. The cells were stimulated for 16 h with the maximal activity concentration of specific ligand. TLR9 was stimulated with CpG ODN 2006; TLR8 and TLR7 were stimulated with the imidazolquinalone R848. Results are shown in FIG. 11. As evident from the figure, the promoter used influences the outcome of the screening assay dependent on the TLR in question. For example, NF-κB is a reliable marker for all TLRs tested, whereas in this set of experiments ISRE was only functional to some extent for TLR8. The IL-8 promoter is particularly sensitive for TLR7 or TLR8 screening assays but would be much less efficient in TLR9 assays.  
     
       
       
         1 
         
           
             117  
           
           
             1  
             3029  
             DNA  
             Homo sapiens  
           
            1 

gcggccgcgt cgacgaaatg tctggatttg gactaaagaa aaaaggaaag gctagcagtc     60 

atccaacaga atcatgagac agactttgcc ttgtatctac ttttgggggg gccttttgcc    120 

ctttgggatg ctgtgtgcat cctccaccac caagtgcact gttagccatg aagttgctga    180 

ctgcagccac ctgaagttga ctcaggtacc cgatgatcta cccacaaaca taacagtgtt    240 

gaaccttacc cataatcaac tcagaagatt accagccgcc aacttcacaa ggtatagcca    300 

gctaactagc ttggatgtag gatttaacac catctcaaaa ctggagccag aattgtgcca    360 

gaaacttccc atgttaaaag ttttgaacct ccagcacaat gagctatctc aactttctga    420 

taaaaccttt gccttctgca cgaatttgac tgaactccat ctcatgtcca actcaatcca    480 

gaaaattaaa aataatccct ttgtcaagca gaagaattta atcacattag atctgtctca    540 

taatggcttg tcatctacaa aattaggaac tcaggttcag ctggaaaatc tccaagagct    600 

tctattatca aacaataaaa ttcaagcgct aaaaagtgaa gaactggata tctttgccaa    660 

ttcatcttta aaaaaattag agttgtcatc gaatcaaatt aaagagtttt ctccagggtg    720 

ttttcacgca attggaagat tatttggcct ctttctgaac aatgtccagc tgggtcccag    780 

ccttacagag aagctatgtt tggaattagc aaacacaagc attcggaatc tgtctctgag    840 

taacagccag ctgtccacca ccagcaatac aactttcttg ggactaaagt ggacaaatct    900 

cactatgctc gatctttcct acaacaactt aaatgtggtt ggtaacgatt cctttgcttg    960 

gcttccacaa ctagaatatt tcttcctaga gtataataat atacagcatt tgttttctca   1020 

ctctttgcac gggcttttca atgtgaggta cctgaatttg aaacggtctt ttactaaaca   1080 

aagtatttcc cttgcctcac tccccaagat tgatgatttt tcttttcagt ggctaaaatg   1140 

tttggagcac cttaacatgg aagataatga tattccaggc ataaaaagca atatgttcac   1200 

aggattgata aacctgaaat acttaagtct atccaactcc tttacaagtt tgcgaacttt   1260 

gacaaatgaa acatttgtat cacttgctca ttctccctta cacatactca acctaaccaa   1320 

gaataaaatc tcaaaaatag agagtgatgc tttctcttgg ttgggccacc tagaagtact   1380 

tgacctgggc cttaatgaaa ttgggcaaga actcacaggc caggaatgga gaggtctaga   1440 

aaatattttc gaaatctatc tttcctacaa caagtacctg cagctgacta ggaactcctt   1500 

tgccttggtc ccaagccttc aacgactgat gctccgaagg gtggccctta aaaatgtgga   1560 

tagctctcct tcaccattcc agcctcttcg taacttgacc attctggatc taagcaacaa   1620 

caacatagcc aacataaatg atgacatgtt ggagggtctt gagaaactag aaattctcga   1680 

tttgcagcat aacaacttag cacggctctg gaaacacgca aaccctggtg gtcccattta   1740 

tttcctaaag ggtctgtctc acctccacat ccttaacttg gagtccaacg gctttgacga   1800 

gatcccagtt gaggtcttca aggatttatt tgaactaaag atcatcgatt taggattgaa   1860 

taatttaaac acacttccag catctgtctt taataatcag gtgtctctaa agtcattgaa   1920 

ccttcagaag aatctcataa catccgttga gaagaaggtt ttcgggccag ctttcaggaa   1980 

cctgactgag ttagatatgc gctttaatcc ctttgattgc acgtgtgaaa gtattgcctg   2040 

gtttgttaat tggattaacg agacccatac caacatccct gagctgtcaa gccactacct   2100 

ttgcaacact ccacctcact atcatgggtt cccagtgaga ctttttgata catcatcttg   2160 

caaagacagt gccccctttg aactcttttt catgatcaat accagtatcc tgttgatttt   2220 

tatctttatt gtacttctca tccactttga gggctggagg atatcttttt attggaatgt   2280 

ttcagtacat cgagttcttg gtttcaaaga aatagacaga cagacagaac agtttgaata   2340 

tgcagcatat ataattcatg cctataaaga taaggattgg gtctgggaac atttctcttc   2400 

aatggaaaag gaagaccaat ctctcaaatt ttgtctggaa gaaagggact ttgaggcggg   2460 

tgtttttgaa ctagaagcaa ttgttaacag catcaaaaga agcagaaaaa ttatttttgt   2520 

tataacacac catctattaa aagacccatt atgcaaaaga ttcaaggtac atcatgcagt   2580 

tcaacaagct attgaacaaa atctggattc cattatattg gttttccttg aggagattcc   2640 

agattataaa ctgaaccatg cactctgttt gcgaagagga atgtttaaat ctcactgcat   2700 

cttgaactgg ccagttcaga aagaacggat aggtgccttt cgtcataaat tgcaagtagc   2760 

acttggatcc aaaaactctg tacattaaat ttatttaaat attcaattag caaaggagaa   2820 

actttctcaa tttaaaaagt tctatggcaa atttaagttt tccataaagg tgttataatt   2880 

tgtttattca tatttgtaaa tgattatatt ctatcacaat tacatctctt ctaggaaaat   2940 

gtgtctcctt atttcaggcc tatttttgac aattgactta attttaccca aaataaaaca   3000 

tataagcacg caaaaaaaaa aaaaaaaaa                                     3029 

 
           
             2  
             904  
             PRT  
             Homo sapiens  
           
            2 

Met Arg Gln Thr Leu Pro Cys Ile Tyr Phe Trp Gly Gly Leu Leu Pro 
1               5                   10                  15 

Phe Gly Met Leu Cys Ala Ser Ser Thr Thr Lys Cys Thr Val Ser His 
            20                  25                  30 

Glu Val Ala Asp Cys Ser His Leu Lys Leu Thr Gln Val Pro Asp Asp 
        35                  40                  45 

Leu Pro Thr Asn Ile Thr Val Leu Asn Leu Thr His Asn Gln Leu Arg 
    50                  55                  60 

Arg Leu Pro Ala Ala Asn Phe Thr Arg Tyr Ser Gln Leu Thr Ser Leu 
65                  70                  75                  80 

Asp Val Gly Phe Asn Thr Ile Ser Lys Leu Glu Pro Glu Leu Cys Gln 
                85                  90                  95 

Lys Leu Pro Met Leu Lys Val Leu Asn Leu Gln His Asn Glu Leu Ser 
            100                 105                 110 

Gln Leu Ser Asp Lys Thr Phe Ala Phe Cys Thr Asn Leu Thr Glu Leu 
        115                 120                 125 

His Leu Met Ser Asn Ser Ile Gln Lys Ile Lys Asn Asn Pro Phe Val 
    130                 135                 140 

Lys Gln Lys Asn Leu Ile Thr Leu Asp Leu Ser His Asn Gly Leu Ser 
145                 150                 155                 160 

Ser Thr Lys Leu Gly Thr Gln Val Gln Leu Glu Asn Leu Gln Glu Leu 
                165                 170                 175 

Leu Leu Ser Asn Asn Lys Ile Gln Ala Leu Lys Ser Glu Glu Leu Asp 
            180                 185                 190 

Ile Phe Ala Asn Ser Ser Leu Lys Lys Leu Glu Leu Ser Ser Asn Gln 
        195                 200                 205 

Ile Lys Glu Phe Ser Pro Gly Cys Phe His Ala Ile Gly Arg Leu Phe 
    210                 215                 220 

Gly Leu Phe Leu Asn Asn Val Gln Leu Gly Pro Ser Leu Thr Glu Lys 
225                 230                 235                 240 

Leu Cys Leu Glu Leu Ala Asn Thr Ser Ile Arg Asn Leu Ser Leu Ser 
                245                 250                 255 

Asn Ser Gln Leu Ser Thr Thr Ser Asn Thr Thr Phe Leu Gly Leu Lys 
            260                 265                 270 

Trp Thr Asn Leu Thr Met Leu Asp Leu Ser Tyr Asn Asn Leu Asn Val 
        275                 280                 285 

Val Gly Asn Asp Ser Phe Ala Trp Leu Pro Gln Leu Glu Tyr Phe Phe 
    290                 295                 300 

Leu Glu Tyr Asn Asn Ile Gln His Leu Phe Ser His Ser Leu His Gly 
305                 310                 315                 320 

Leu Phe Asn Val Arg Tyr Leu Asn Leu Lys Arg Ser Phe Thr Lys Gln 
                325                 330                 335 

Ser Ile Ser Leu Ala Ser Leu Pro Lys Ile Asp Asp Phe Ser Phe Gln 
            340                 345                 350 

Trp Leu Lys Cys Leu Glu His Leu Asn Met Glu Asp Asn Asp Ile Pro 
        355                 360                 365 

Gly Ile Lys Ser Asn Met Phe Thr Gly Leu Ile Asn Leu Lys Tyr Leu 
    370                 375                 380 

Ser Leu Ser Asn Ser Phe Thr Ser Leu Arg Thr Leu Thr Asn Glu Thr 
385                 390                 395                 400 

Phe Val Ser Leu Ala His Ser Pro Leu His Ile Leu Asn Leu Thr Lys 
                405                 410                 415 

Asn Lys Ile Ser Lys Ile Glu Ser Asp Ala Phe Ser Trp Leu Gly His 
            420                 425                 430 

Leu Glu Val Leu Asp Leu Gly Leu Asn Glu Ile Gly Gln Glu Leu Thr 
        435                 440                 445 

Gly Gln Glu Trp Arg Gly Leu Glu Asn Ile Phe Glu Ile Tyr Leu Ser 
    450                 455                 460 

Tyr Asn Lys Tyr Leu Gln Leu Thr Arg Asn Ser Phe Ala Leu Val Pro 
465                 470                 475                 480 

Ser Leu Gln Arg Leu Met Leu Arg Arg Val Ala Leu Lys Asn Val Asp 
                485                 490                 495 

Ser Ser Pro Ser Pro Phe Gln Pro Leu Arg Asn Leu Thr Ile Leu Asp 
            500                 505                 510 

Leu Ser Asn Asn Asn Ile Ala Asn Ile Asn Asp Asp Met Leu Glu Gly 
        515                 520                 525 

Leu Glu Lys Leu Glu Ile Leu Asp Leu Gln His Asn Asn Leu Ala Arg 
    530                 535                 540 

Leu Trp Lys His Ala Asn Pro Gly Gly Pro Ile Tyr Phe Leu Lys Gly 
545                 550                 555                 560 

Leu Ser His Leu His Ile Leu Asn Leu Glu Ser Asn Gly Phe Asp Glu 
                565                 570                 575 

Ile Pro Val Glu Val Phe Lys Asp Leu Phe Glu Leu Lys Ile Ile Asp 
            580                 585                 590 

Leu Gly Leu Asn Asn Leu Asn Thr Leu Pro Ala Ser Val Phe Asn Asn 
        595                 600                 605 

Gln Val Ser Leu Lys Ser Leu Asn Leu Gln Lys Asn Leu Ile Thr Ser 
    610                 615                 620 

Val Glu Lys Lys Val Phe Gly Pro Ala Phe Arg Asn Leu Thr Glu Leu 
625                 630                 635                 640 

Asp Met Arg Phe Asn Pro Phe Asp Cys Thr Cys Glu Ser Ile Ala Trp 
                645                 650                 655 

Phe Val Asn Trp Ile Asn Glu Thr His Thr Asn Ile Pro Glu Leu Ser 
            660                 665                 670 

Ser His Tyr Leu Cys Asn Thr Pro Pro His Tyr His Gly Phe Pro Val 
        675                 680                 685 

Arg Leu Phe Asp Thr Ser Ser Cys Lys Asp Ser Ala Pro Phe Glu Leu 
    690                 695                 700 

Phe Phe Met Ile Asn Thr Ser Ile Leu Leu Ile Phe Ile Phe Ile Val 
705                 710                 715                 720 

Leu Leu Ile His Phe Glu Gly Trp Arg Ile Ser Phe Tyr Trp Asn Val 
                725                 730                 735 

Ser Val His Arg Val Leu Gly Phe Lys Glu Ile Asp Arg Gln Thr Glu 
            740                 745                 750 

Gln Phe Glu Tyr Ala Ala Tyr Ile Ile His Ala Tyr Lys Asp Lys Asp 
        755                 760                 765 

Trp Val Trp Glu His Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu 
    770                 775                 780 

Lys Phe Cys Leu Glu Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu 
785                 790                 795                 800 

Glu Ala Ile Val Asn Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val 
                805                 810                 815 

Ile Thr His His Leu Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val 
            820                 825                 830 

His His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile Ile 
        835                 840                 845 

Leu Val Phe Leu Glu Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Leu 
    850                 855                 860 

Cys Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro 
865                 870                 875                 880 

Val Gln Lys Glu Arg Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala 
                885                 890                 895 

Leu Gly Ser Lys Asn Ser Val His 
            900 

 
           
             3  
             3310  
             DNA  
             Mus musculus  
           
            3 

tagaatatga tacagggatt gcacccataa tctgggctga atcatgaaag ggtgttcctc     60 

ttatctaatg tactcctttg ggggactttt gtccctatgg attcttctgg tgtcttccac    120 

aaaccaatgc actgtgagat acaacgtagc tgactgcagc catttgaagc taacacacat    180 

acctgatgat cttccctcta acataacagt gttgaatctt actcacaacc aactcagaag    240 

attaccacct accaacttta caagatacag ccaacttgct atcttggatg caggatttaa    300 

ctccatttca aaactggagc cagaactgtg ccaaatactc cctttgttga aagtattgaa    360 

cctgcaacat aatgagctct ctcagatttc tgatcaaacc tttgtcttct gcacgaacct    420 

gacagaactc gatctaatgt ctaactcaat acacaaaatt aaaagcaacc ctttcaaaaa    480 

ccagaagaat ctaatcaaat tagatttgtc tcataatggt ttatcatcta caaagttggg    540 

aacgggggtc caactggaga acctccaaga actgctctta gcaaaaaata aaatccttgc    600 

gttgcgaagt gaagaacttg agtttcttgg caattcttct ttacgaaagt tggacttgtc    660 

atcaaatcca cttaaagagt tctccccggg gtgtttccag acaattggca agttattcgc    720 

cctcctcttg aacaacgccc aactgaaccc ccacctcaca gagaagcttt gctgggaact    780 

ttcaaacaca agcatccaga atctctctct ggctaacaac cagctgctgg ccaccagcga    840 

gagcactttc tctgggctga agtggacaaa tctcacccag ctcgatcttt cctacaacaa    900 

cctccatgat gtcggcaacg gttccttctc ctatctccca agcctgaggt atctgtctct    960 

ggagtacaac aatatacagc gtctgtcccc tcgctctttt tatggactct ccaacctgag   1020 

gtacctgagt ttgaagcgag catttactaa gcaaagtgtt tcacttgctt cacatcccaa   1080 

cattgacgat ttttcctttc aatggttaaa atatttggaa tatctcaaca tggatgacaa   1140 

taatattcca agtaccaaaa gcaatacctt cacgggattg gtgagtctga agtacctaag   1200 

tctttccaaa actttcacaa gtttgcaaac tttaacaaat gaaacatttg tgtcacttgc   1260 

tcattctccc ttgctcactc tcaacttaac gaaaaatcac atctcaaaaa tagcaaatgg   1320 

tactttctct tggttaggcc aactcaggat acttgatctc ggccttaatg aaattgaaca   1380 

aaaactcagc ggccaggaat ggagaggtct gagaaatata tttgagatct acctatccta   1440 

taacaaatac ctccaactgt ctaccagttc ctttgcattg gtccccagcc ttcaaagact   1500 

gatgctcagg agggtggccc ttaaaaatgt ggatatctcc ccttcacctt tccgccctct   1560 

tcgtaacttg accattctgg acttaagcaa caacaacata gccaacataa atgaggactt   1620 

gctggagggt cttgagaatc tagaaatcct ggattttcag cacaataact tagccaggct   1680 

ctggaaacgc gcaaaccccg gtggtcccgt taatttcctg aaggggctgt ctcacctcca   1740 

catcttgaat ttagagtcca acggcttaga tgaaatccca gtcggggttt tcaagaactt   1800 

attcgaacta aagagcatca atctaggact gaataactta aacaaacttg aaccattcat   1860 

ttttgatgac cagacatctc taaggtcact gaacctccag aagaacctca taacatctgt   1920 

tgagaaggat gttttcgggc cgccttttca aaacctgaac agtttagata tgcgcttcaa   1980 

tccgttcgac tgcacgtgtg aaagtatttc ctggtttgtt aactggatca accagaccca   2040 

cactaatatc tttgagctgt ccactcacta cctctgtaac actccacatc attattatgg   2100 

cttccccctg aagcttttcg atacatcatc ctgtaaagac agcgccccct ttgaactcct   2160 

cttcataatc agcaccagta tgctcctggt ttttatactt gtggtactgc tcattcacat   2220 

cgagggctgg aggatctctt tttactggaa tgtttcagtg catcggattc ttggtttcaa   2280 

ggaaatagac acacaggctg agcagtttga atatacagcc tacataattc atgcccataa   2340 

agacagagac tgggtctggg aacatttctc cccaatggaa gaacaagacc aatctctcaa   2400 

attttgccta gaagaaaggg actttgaagc aggcgtcctt ggacttgaag caattgttaa   2460 

tagcatcaaa agaagccgaa aaatcatttt cgttatcaca caccatttat taaaagaccc   2520 

tctgtgcaga agattcaagg tacatcacgc agttcagcaa gctattgagc aaaatctgga   2580 

ttcaattata ctgatttttc tccagaatat tccagattat aaactaaacc atgcactctg   2640 

tttgcgaaga ggaatgttta aatctcattg catcttgaac tggccagttc agaaagaacg   2700 

gataaatgcc tttcatcata aattgcaagt agcacttgga tctcggaatt cagcacatta   2760 

aactcatttg aagatttgga gtcggtaaag ggatagatcc aatttataaa ggtccatcat   2820 

gaatctaagt tttacttgaa agttttgtat atttatttat atgtatagat gatgatatta   2880 

catcacaatc caatctcagt tttgaaatat ttcggcttat ttcattgaca tctggtttat   2940 

tcactccaaa taaacacatg ggcagttaaa aacatcctct attaatagat tacccattaa   3000 

ttcttgaggt gtatcacagc tttaaagggt tttaaatatt tttatataaa taagactgag   3060 

agttttataa atgtaatttt ttaaaactcg agtcttactg tgtagctcag aaaggcctgg   3120 

aaattaatat attagagagt catgtcttga acttatttat ctctgcctcc ctctgtctcc   3180 

agagtgttgc ttttaagggc atgtagcacc acacccagct atgtacgtgt gggattttat   3240 

aatgctcatt tttgagacgt ttatagaata aaagataatt gcttttatgg tataaggcta   3300 

cttgaggtaa                                                          3310 

 
           
             4  
             905  
             PRT  
             Mus musculus  
           
            4 

Met Lys Gly Cys Ser Ser Tyr Leu Met Tyr Ser Phe Gly Gly Leu Leu 
1               5                   10                  15 

Ser Leu Trp Ile Leu Leu Val Ser Ser Thr Asn Gln Cys Thr Val Arg 
            20                  25                  30 

Tyr Asn Val Ala Asp Cys Ser His Leu Lys Leu Thr His Ile Pro Asp 
        35                  40                  45 

Asp Leu Pro Ser Asn Ile Thr Val Leu Asn Leu Thr His Asn Gln Leu 
    50                  55                  60 

Arg Arg Leu Pro Pro Thr Asn Phe Thr Arg Tyr Ser Gln Leu Ala Ile 
65                  70                  75                  80 

Leu Asp Ala Gly Phe Asn Ser Ile Ser Lys Leu Glu Pro Glu Leu Cys 
                85                  90                  95 

Gln Ile Leu Pro Leu Leu Lys Val Leu Asn Leu Gln His Asn Glu Leu 
            100                 105                 110 

Ser Gln Ile Ser Asp Gln Thr Phe Val Phe Cys Thr Asn Leu Thr Glu 
        115                 120                 125 

Leu Asp Leu Met Ser Asn Ser Ile His Lys Ile Lys Ser Asn Pro Phe 
    130                 135                 140 

Lys Asn Gln Lys Asn Leu Ile Lys Leu Asp Leu Ser His Asn Gly Leu 
145                 150                 155                 160 

Ser Ser Thr Lys Leu Gly Thr Gly Val Gln Leu Glu Asn Leu Gln Glu 
                165                 170                 175 

Leu Leu Leu Ala Lys Asn Lys Ile Leu Ala Leu Arg Ser Glu Glu Leu 
            180                 185                 190 

Glu Phe Leu Gly Asn Ser Ser Leu Arg Lys Leu Asp Leu Ser Ser Asn 
        195                 200                 205 

Pro Leu Lys Glu Phe Ser Pro Gly Cys Phe Gln Thr Ile Gly Lys Leu 
    210                 215                 220 

Phe Ala Leu Leu Leu Asn Asn Ala Gln Leu Asn Pro His Leu Thr Glu 
225                 230                 235                 240 

Lys Leu Cys Trp Glu Leu Ser Asn Thr Ser Ile Gln Asn Leu Ser Leu 
                245                 250                 255 

Ala Asn Asn Gln Leu Leu Ala Thr Ser Glu Ser Thr Phe Ser Gly Leu 
            260                 265                 270 

Lys Trp Thr Asn Leu Thr Gln Leu Asp Leu Ser Tyr Asn Asn Leu His 
        275                 280                 285 

Asp Val Gly Asn Gly Ser Phe Ser Tyr Leu Pro Ser Leu Arg Tyr Leu 
    290                 295                 300 

Ser Leu Glu Tyr Asn Asn Ile Gln Arg Leu Ser Pro Arg Ser Phe Tyr 
305                 310                 315                 320 

Gly Leu Ser Asn Leu Arg Tyr Leu Ser Leu Lys Arg Ala Phe Thr Lys 
                325                 330                 335 

Gln Ser Val Ser Leu Ala Ser His Pro Asn Ile Asp Asp Phe Ser Phe 
            340                 345                 350 

Gln Trp Leu Lys Tyr Leu Glu Tyr Leu Asn Met Asp Asp Asn Asn Ile 
        355                 360                 365 

Pro Ser Thr Lys Ser Asn Thr Phe Thr Gly Leu Val Ser Leu Lys Tyr 
    370                 375                 380 

Leu Ser Leu Ser Lys Thr Phe Thr Ser Leu Gln Thr Leu Thr Asn Glu 
385                 390                 395                 400 

Thr Phe Val Ser Leu Ala His Ser Pro Leu Leu Thr Leu Asn Leu Thr 
                405                 410                 415 

Lys Asn His Ile Ser Lys Ile Ala Asn Gly Thr Phe Ser Trp Leu Gly 
            420                 425                 430 

Gln Leu Arg Ile Leu Asp Leu Gly Leu Asn Glu Ile Glu Gln Lys Leu 
        435                 440                 445 

Ser Gly Gln Glu Trp Arg Gly Leu Arg Asn Ile Phe Glu Ile Tyr Leu 
    450                 455                 460 

Ser Tyr Asn Lys Tyr Leu Gln Leu Ser Thr Ser Ser Phe Ala Leu Val 
465                 470                 475                 480 

Pro Ser Leu Gln Arg Leu Met Leu Arg Arg Val Ala Leu Lys Asn Val 
                485                 490                 495 

Asp Ile Ser Pro Ser Pro Phe Arg Pro Leu Arg Asn Leu Thr Ile Leu 
            500                 505                 510 

Asp Leu Ser Asn Asn Asn Ile Ala Asn Ile Asn Glu Asp Leu Leu Glu 
        515                 520                 525 

Gly Leu Glu Asn Leu Glu Ile Leu Asp Phe Gln His Asn Asn Leu Ala 
    530                 535                 540 

Arg Leu Trp Lys Arg Ala Asn Pro Gly Gly Pro Val Asn Phe Leu Lys 
545                 550                 555                 560 

Gly Leu Ser His Leu His Ile Leu Asn Leu Glu Ser Asn Gly Leu Asp 
                565                 570                 575 

Glu Ile Pro Val Gly Val Phe Lys Asn Leu Phe Glu Leu Lys Ser Ile 
            580                 585                 590 

Asn Leu Gly Leu Asn Asn Leu Asn Lys Leu Glu Pro Phe Ile Phe Asp 
        595                 600                 605 

Asp Gln Thr Ser Leu Arg Ser Leu Asn Leu Gln Lys Asn Leu Ile Thr 
    610                 615                 620 

Ser Val Glu Lys Asp Val Phe Gly Pro Pro Phe Gln Asn Leu Asn Ser 
625                 630                 635                 640 

Leu Asp Met Arg Phe Asn Pro Phe Asp Cys Thr Cys Glu Ser Ile Ser 
                645                 650                 655 

Trp Phe Val Asn Trp Ile Asn Gln Thr His Thr Asn Ile Phe Glu Leu 
            660                 665                 670 

Ser Thr His Tyr Leu Cys Asn Thr Pro His His Tyr Tyr Gly Phe Pro 
        675                 680                 685 

Leu Lys Leu Phe Asp Thr Ser Ser Cys Lys Asp Ser Ala Pro Phe Glu 
    690                 695                 700 

Leu Leu Phe Ile Ile Ser Thr Ser Met Leu Leu Val Phe Ile Leu Val 
705                 710                 715                 720 

Val Leu Leu Ile His Ile Glu Gly Trp Arg Ile Ser Phe Tyr Trp Asn 
                725                 730                 735 

Val Ser Val His Arg Ile Leu Gly Phe Lys Glu Ile Asp Thr Gln Ala 
            740                 745                 750 

Glu Gln Phe Glu Tyr Thr Ala Tyr Ile Ile His Ala His Lys Asp Arg 
        755                 760                 765 

Asp Trp Val Trp Glu His Phe Ser Pro Met Glu Glu Gln Asp Gln Ser 
    770                 775                 780 

Leu Lys Phe Cys Leu Glu Glu Arg Asp Phe Glu Ala Gly Val Leu Gly 
785                 790                 795                 800 

Leu Glu Ala Ile Val Asn Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe 
                805                 810                 815 

Val Ile Thr His His Leu Leu Lys Asp Pro Leu Cys Arg Arg Phe Lys 
            820                 825                 830 

Val His His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile 
        835                 840                 845 

Ile Leu Ile Phe Leu Gln Asn Ile Pro Asp Tyr Lys Leu Asn His Ala 
    850                 855                 860 

Leu Cys Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn Trp 
865                 870                 875                 880 

Pro Val Gln Lys Glu Arg Ile Asn Ala Phe His His Lys Leu Gln Val 
                885                 890                 895 

Ala Leu Gly Ser Arg Asn Ser Ala His 
            900                 905 

 
           
             5  
             3352  
             DNA  
             Homo sapiens  
           
            5 

aggctggtat aaaaatctta cttcctctat tctctgagcc gctgctgccc ctgtgggaag     60 

ggacctcgag tgtgaagcat ccttccctgt agctgctgtc cagtctgccc gccagaccct    120 

ctggagaagc ccctgccccc cagcatgggt ttctgccgca gcgccctgca cccgctgtct    180 

ctcctggtgc aggccatcat gctggccatg accctggccc tgggtacctt gcctgccttc    240 

ctaccctgtg agctccagcc ccacggcctg gtgaactgca actggctgtt cctgaagtct    300 

gtgccccact tctccatggc agcaccccgt ggcaatgtca ccagcctttc cttgtcctcc    360 

aaccgcatcc accacctcca tgattctgac tttgcccacc tgcccagcct gcggcatctc    420 

aacctcaagt ggaactgccc gccggttggc ctcagcccca tgcacttccc ctgccacatg    480 

accatcgagc ccagcacctt cttggctgtg cccaccctgg aagagctaaa cctgagctac    540 

aacaacatca tgactgtgcc tgcgctgccc aaatccctca tatccctgtc cctcagccat    600 

accaacatcc tgatgctaga ctctgccagc ctcgccggcc tgcatgccct gcgcttccta    660 

ttcatggacg gcaactgtta ttacaagaac ccctgcaggc aggcactgga ggtggccccg    720 

ggtgccctcc ttggcctggg caacctcacc cacctgtcac tcaagtacaa caacctcact    780 

gtggtgcccc gcaacctgcc ttccagcctg gagtatctgc tgttgtccta caaccgcatc    840 

gtcaaactgg cgcctgagga cctggccaat ctgaccgccc tgcgtgtgct cgatgtgggc    900 

ggaaattgcc gccgctgcga ccacgctccc aacccctgca tggagtgccc tcgtcacttc    960 

ccccagctac atcccgatac cttcagccac ctgagccgtc ttgaaggcct ggtgttgaag   1020 

gacagttctc tctcctggct gaatgccagt tggttccgtg ggctgggaaa cctccgagtg   1080 

ctggacctga gtgagaactt cctctacaaa tgcatcacta aaaccaaggc cttccagggc   1140 

ctaacacagc tgcgcaagct taacctgtcc ttcaattacc aaaagagggt gtcctttgcc   1200 

cacctgtctc tggccccttc cttcgggagc ctggtcgccc tgaaggagct ggacatgcac   1260 

ggcatcttct tccgctcact cgatgagacc acgctccggc cactggcccg cctgcccatg   1320 

ctccagactc tgcgtctgca gatgaacttc atcaaccagg cccagctcgg catcttcagg   1380 

gccttccctg gcctgcgcta cgtggacctg tcggacaacc gcatcagcgg agcttcggag   1440 

ctgacagcca ccatggggga ggcagatgga ggggagaagg tctggctgca gcctggggac   1500 

cttgctccgg ccccagtgga cactcccagc tctgaagact tcaggcccaa ctgcagcacc   1560 

ctcaacttca ccttggatct gtcacggaac aacctggtga ccgtgcagcc ggagatgttt   1620 

gcccagctct cgcacctgca gtgcctgcgc ctgagccaca actgcatctc gcaggcagtc   1680 

aatggctccc agttcctgcc gctgaccggt ctgcaggtgc tagacctgtc ccgcaataag   1740 

ctggacctct accacgagca ctcattcacg gagctaccgc gactggaggc cctggacctc   1800 

agctacaaca gccagccctt tggcatgcag ggcgtgggcc acaacttcag cttcgtggct   1860 

cacctgcgca ccctgcgcca cctcagcctg gcccacaaca acatccacag ccaagtgtcc   1920 

cagcagctct gcagtacgtc gctgcgggcc ctggacttca gcggcaatgc actgggccat   1980 

atgtgggccg agggagacct ctatctgcac ttcttccaag gcctgagcgg tttgatctgg   2040 

ctggacttgt cccagaaccg cctgcacacc ctcctgcccc aaaccctgcg caacctcccc   2100 

aagagcctac aggtgctgcg tctccgtgac aattacctgg ccttctttaa gtggtggagc   2160 

ctccacttcc tgcccaaact ggaagtcctc gacctggcag gaaaccggct gaaggccctg   2220 

accaatggca gcctgcctgc tggcacccgg ctccggaggc tggatgtcag ctgcaacagc   2280 

atcagcttcg tggcccccgg cttcttttcc aaggccaagg agctgcgaga gctcaacctt   2340 

agcgccaacg ccctcaagac agtggaccac tcctggtttg ggcccctggc gagtgccctg   2400 

caaatactag atgtaagcgc caaccctctg cactgcgcct gtggggcggc ctttatggac   2460 

ttcctgctgg aggtgcaggc tgccgtgccc ggtctgccca gccgggtgaa gtgtggcagt   2520 

ccgggccagc tccagggcct cagcatcttt gcacaggacc tgcgcctctg cctggatgag   2580 

gccctctcct gggactgttt cgccctctcg ctgctggctg tggctctggg cctgggtgtg   2640 

cccatgctgc atcacctctg tggctgggac ctctggtact gcttccacct gtgcctggcc   2700 

tggcttccct ggcgggggcg gcaaagtggg cgagatgagg atgccctgcc ctacgatgcc   2760 

ttcgtggtct tcgacaaaac gcagagcgca gtggcagact gggtgtacaa cgagcttcgg   2820 

gggcagctgg aggagtgccg tgggcgctgg gcactccgcc tgtgcctgga ggaacgcgac   2880 

tggctgcctg gcaaaaccct ctttgagaac ctgtgggcct cggtctatgg cagccgcaag   2940 

acgctgtttg tgctggccca cacggaccgg gtcagtggtc tcttgcgcgc cagcttcctg   3000 

ctggcccagc agcgcctgct ggaggaccgc aaggacgtcg tggtgctggt gatcctgagc   3060 

cctgacggcc gccgctcccg ctacgtgcgg ctgcgccagc gcctctgccg ccagagtgtc   3120 

ctcctctggc cccaccagcc cagtggtcag cgcagcttct gggcccagct gggcatggcc   3180 

ctgaccaggg acaaccacca cttctataac cggaacttct gccagggacc cacggccgaa   3240 

tagccgtgag ccggaatcct gcacggtgcc acctccacac tcacctcacc tctgcctgcc   3300 

tggtctgacc ctcccctgct cgcctccctc accccacacc tgacacagag ca           3352  
           
             6  
             1032  
             PRT  
             Homo sapiens  
           
            6 

Met Gly Phe Cys Arg Ser Ala Leu His Pro Leu Ser Leu Leu Val Gln 
1               5                   10                  15 

Ala Ile Met Leu Ala Met Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe 
            20                  25                  30 

Leu Pro Cys Glu Leu Gln Pro His Gly Leu Val Asn Cys Asn Trp Leu 
        35                  40                  45 

Phe Leu Lys Ser Val Pro His Phe Ser Met Ala Ala Pro Arg Gly Asn 
    50                  55                  60 

Val Thr Ser Leu Ser Leu Ser Ser Asn Arg Ile His His Leu His Asp 
65                  70                  75                  80 

Ser Asp Phe Ala His Leu Pro Ser Leu Arg His Leu Asn Leu Lys Trp 
                85                  90                  95 

Asn Cys Pro Pro Val Gly Leu Ser Pro Met His Phe Pro Cys His Met 
            100                 105                 110 

Thr Ile Glu Pro Ser Thr Phe Leu Ala Val Pro Thr Leu Glu Glu Leu 
        115                 120                 125 

Asn Leu Ser Tyr Asn Asn Ile Met Thr Val Pro Ala Leu Pro Lys Ser 
    130                 135                 140 

Leu Ile Ser Leu Ser Leu Ser His Thr Asn Ile Leu Met Leu Asp Ser 
145                 150                 155                 160 

Ala Ser Leu Ala Gly Leu His Ala Leu Arg Phe Leu Phe Met Asp Gly 
                165                 170                 175 

Asn Cys Tyr Tyr Lys Asn Pro Cys Arg Gln Ala Leu Glu Val Ala Pro 
            180                 185                 190 

Gly Ala Leu Leu Gly Leu Gly Asn Leu Thr His Leu Ser Leu Lys Tyr 
        195                 200                 205 

Asn Asn Leu Thr Val Val Pro Arg Asn Leu Pro Ser Ser Leu Glu Tyr 
    210                 215                 220 

Leu Leu Leu Ser Tyr Asn Arg Ile Val Lys Leu Ala Pro Glu Asp Leu 
225                 230                 235                 240 

Ala Asn Leu Thr Ala Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg 
                245                 250                 255 

Arg Cys Asp His Ala Pro Asn Pro Cys Met Glu Cys Pro Arg His Phe 
            260                 265                 270 

Pro Gln Leu His Pro Asp Thr Phe Ser His Leu Ser Arg Leu Glu Gly 
        275                 280                 285 

Leu Val Leu Lys Asp Ser Ser Leu Ser Trp Leu Asn Ala Ser Trp Phe 
    290                 295                 300 

Arg Gly Leu Gly Asn Leu Arg Val Leu Asp Leu Ser Glu Asn Phe Leu 
305                 310                 315                 320 

Tyr Lys Cys Ile Thr Lys Thr Lys Ala Phe Gln Gly Leu Thr Gln Leu 
                325                 330                 335 

Arg Lys Leu Asn Leu Ser Phe Asn Tyr Gln Lys Arg Val Ser Phe Ala 
            340                 345                 350 

His Leu Ser Leu Ala Pro Ser Phe Gly Ser Leu Val Ala Leu Lys Glu 
        355                 360                 365 

Leu Asp Met His Gly Ile Phe Phe Arg Ser Leu Asp Glu Thr Thr Leu 
    370                 375                 380 

Arg Pro Leu Ala Arg Leu Pro Met Leu Gln Thr Leu Arg Leu Gln Met 
385                 390                 395                 400 

Asn Phe Ile Asn Gln Ala Gln Leu Gly Ile Phe Arg Ala Phe Pro Gly 
                405                 410                 415 

Leu Arg Tyr Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Ala Ser Glu 
            420                 425                 430 

Leu Thr Ala Thr Met Gly Glu Ala Asp Gly Gly Glu Lys Val Trp Leu 
        435                 440                 445 

Gln Pro Gly Asp Leu Ala Pro Ala Pro Val Asp Thr Pro Ser Ser Glu 
    450                 455                 460 

Asp Phe Arg Pro Asn Cys Ser Thr Leu Asn Phe Thr Leu Asp Leu Ser 
465                 470                 475                 480 

Arg Asn Asn Leu Val Thr Val Gln Pro Glu Met Phe Ala Gln Leu Ser 
                485                 490                 495 

His Leu Gln Cys Leu Arg Leu Ser His Asn Cys Ile Ser Gln Ala Val 
            500                 505                 510 

Asn Gly Ser Gln Phe Leu Pro Leu Thr Gly Leu Gln Val Leu Asp Leu 
        515                 520                 525 

Ser Arg Asn Lys Leu Asp Leu Tyr His Glu His Ser Phe Thr Glu Leu 
    530                 535                 540 

Pro Arg Leu Glu Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe Gly 
545                 550                 555                 560 

Met Gln Gly Val Gly His Asn Phe Ser Phe Val Ala His Leu Arg Thr 
                565                 570                 575 

Leu Arg His Leu Ser Leu Ala His Asn Asn Ile His Ser Gln Val Ser 
            580                 585                 590 

Gln Gln Leu Cys Ser Thr Ser Leu Arg Ala Leu Asp Phe Ser Gly Asn 
        595                 600                 605 

Ala Leu Gly His Met Trp Ala Glu Gly Asp Leu Tyr Leu His Phe Phe 
    610                 615                 620 

Gln Gly Leu Ser Gly Leu Ile Trp Leu Asp Leu Ser Gln Asn Arg Leu 
625                 630                 635                 640 

His Thr Leu Leu Pro Gln Thr Leu Arg Asn Leu Pro Lys Ser Leu Gln 
                645                 650                 655 

Val Leu Arg Leu Arg Asp Asn Tyr Leu Ala Phe Phe Lys Trp Trp Ser 
            660                 665                 670 

Leu His Phe Leu Pro Lys Leu Glu Val Leu Asp Leu Ala Gly Asn Arg 
        675                 680                 685 

Leu Lys Ala Leu Thr Asn Gly Ser Leu Pro Ala Gly Thr Arg Leu Arg 
    690                 695                 700 

Arg Leu Asp Val Ser Cys Asn Ser Ile Ser Phe Val Ala Pro Gly Phe 
705                 710                 715                 720 

Phe Ser Lys Ala Lys Glu Leu Arg Glu Leu Asn Leu Ser Ala Asn Ala 
                725                 730                 735 

Leu Lys Thr Val Asp His Ser Trp Phe Gly Pro Leu Ala Ser Ala Leu 
            740                 745                 750 

Gln Ile Leu Asp Val Ser Ala Asn Pro Leu His Cys Ala Cys Gly Ala 
        755                 760                 765 

Ala Phe Met Asp Phe Leu Leu Glu Val Gln Ala Ala Val Pro Gly Leu 
    770                 775                 780 

Pro Ser Arg Val Lys Cys Gly Ser Pro Gly Gln Leu Gln Gly Leu Ser 
785                 790                 795                 800 

Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu Asp Glu Ala Leu Ser Trp 
                805                 810                 815 

Asp Cys Phe Ala Leu Ser Leu Leu Ala Val Ala Leu Gly Leu Gly Val 
            820                 825                 830 

Pro Met Leu His His Leu Cys Gly Trp Asp Leu Trp Tyr Cys Phe His 
        835                 840                 845 

Leu Cys Leu Ala Trp Leu Pro Trp Arg Gly Arg Gln Ser Gly Arg Asp 
    850                 855                 860 

Glu Asp Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Thr Gln 
865                 870                 875                 880 

Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Gly Gln Leu Glu 
                885                 890                 895 

Glu Cys Arg Gly Arg Trp Ala Leu Arg Leu Cys Leu Glu Glu Arg Asp 
            900                 905                 910 

Trp Leu Pro Gly Lys Thr Leu Phe Glu Asn Leu Trp Ala Ser Val Tyr 
        915                 920                 925 

Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser 
    930                 935                 940 

Gly Leu Leu Arg Ala Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu 
945                 950                 955                 960 

Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Ser Pro Asp Gly Arg 
                965                 970                 975 

Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val 
            980                 985                 990 

Leu Leu Trp Pro His Gln Pro Ser  Gly Gln Arg Ser Phe  Trp Ala Gln 
        995                 1000                 1005 

Leu Gly  Met Ala Leu Thr Arg  Asp Asn His His Phe  Tyr Asn Arg 
    1010                 1015                 1020 

Asn Phe  Cys Gln Gly Pro Thr  Ala Glu 
    1025                 1030 

 
           
             7  
             3200  
             DNA  
             Mus musculus  
           
            7 

tgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg tcgaaggact     60 

ctgcacccct tgtccctcct ggtacaggct gcagtgctgg ctgagactct ggccctgggt    120 

accctgcctg ccttcctacc ctgtgagctg aagcctcatg gcctggtgga ctgcaattgg    180 

ctgttcctga agtctgtacc ccgtttctct gcggcagcat cctgctccaa catcacccgc    240 

ctctccttga tctccaaccg tatccaccac ctgcacaact ccgacttcgt ccacctgtcc    300 

aacctgcggc agctgaacct caagtggaac tgtccaccca ctggccttag ccccctgcac    360 

ttctcttgcc acatgaccat tgagcccaga accttcctgg ctatgcgtac actggaggag    420 

ctgaacctga gctataatgg tatcaccact gtgccccgac tgcccagctc cctggtgaat    480 

ctgagcctga gccacaccaa catcctggtt ctagatgcta acagcctcgc cggcctatac    540 

agcctgcgcg ttctcttcat ggacgggaac tgctactaca agaacccctg cacaggagcg    600 

gtgaaggtga ccccaggcgc cctcctgggc ctgagcaatc tcacccatct gtctctgaag    660 

tataacaacc tcacaaaggt gccccgccaa ctgcccccca gcctggagta cctcctggtg    720 

tcctataacc tcattgtcaa gctggggcct gaagacctgg ccaatctgac ctcccttcga    780 

gtacttgatg tgggtgggaa ttgccgtcgc tgcgaccatg cccccaatcc ctgtatagaa    840 

tgtggccaaa agtccctcca cctgcaccct gagaccttcc atcacctgag ccatctggaa    900 

ggcctggtgc tgaaggacag ctctctccat acactgaact cttcctggtt ccaaggtctg    960 

gtcaacctct cggtgctgga cctaagcgag aactttctct atgaaagcat caaccacacc   1020 

aatgcctttc agaacctaac ccgcctgcgc aagctcaacc tgtccttcaa ttaccgcaag   1080 

aaggtatcct ttgcccgcct ccacctggca agttccttca agaacctggt gtcactgcag   1140 

gagctgaaca tgaacggcat cttcttccgc tcgctcaaca agtacacgct cagatggctg   1200 

gccgatctgc ccaaactcca cactctgcat cttcaaatga acttcatcaa ccaggcacag   1260 

ctcagcatct ttggtacctt ccgagccctt cgctttgtgg acttgtcaga caatcgcatc   1320 

agtgggcctt caacgctgtc agaagccacc cctgaagagg cagatgatgc agagcaggag   1380 

gagctgttgt ctgcggatcc tcacccagct ccactgagca cccctgcttc taagaacttc   1440 

atggacaggt gtaagaactt caagttcacc atggacctgt ctcggaacaa cctggtgact   1500 

atcaagccag agatgtttgt caatctctca cgcctccagt gtcttagcct gagccacaac   1560 

tccattgcac aggctgtcaa tggctctcag ttcctgccgc tgactaatct gcaggtgctg   1620 

gacctgtccc ataacaaact ggacttgtac cactggaaat cgttcagtga gctaccacag   1680 

ttgcaggccc tggacctgag ctacaacagc cagcccttta gcatgaaggg tataggccac   1740 

aatttcagtt ttgtggccca tctgtccatg ctacacagcc ttagcctggc acacaatgac   1800 

attcataccc gtgtgtcctc acatctcaac agcaactcag tgaggtttct tgacttcagc   1860 

ggcaacggta tgggccgcat gtgggatgag gggggccttt atctccattt cttccaaggc   1920 

ctgagtggcc tgctgaagct ggacctgtct caaaataacc tgcatatcct ccggccccag   1980 

aaccttgaca acctccccaa gagcctgaag ctgctgagcc tccgagacaa ctacctatct   2040 

ttctttaact ggaccagtct gtccttcctg cccaacctgg aagtcctaga cctggcaggc   2100 

aaccagctaa aggccctgac caatggcacc ctgcctaatg gcaccctcct ccagaaactg   2160 

gatgtcagca gcaacagtat cgtctctgtg gtcccagcct tcttcgctct ggcggtcgag   2220 

ctgaaagagg tcaacctcag ccacaacatt ctcaagacgg tggatcgctc ctggtttggg   2280 

cccattgtga tgaacctgac agttctagac gtgagaagca accctctgca ctgtgcctgt   2340 

ggggcagcct tcgtagactt actgttggag gtgcagacca aggtgcctgg cctggctaat   2400 

ggtgtgaagt gtggcagccc cggccagctg cagggccgta gcatcttcgc acaggacctg   2460 

cggctgtgcc tggatgaggt cctctcttgg gactgctttg gcctttcact cttggctgtg   2520 

gccgtgggca tggtggtgcc tatactgcac catctctgcg gctgggacgt ctggtactgt   2580 

tttcatctgt gcctggcatg gctacctttg ctggcccgca gccgacgcag cgcccaagct   2640 

ctcccctatg atgccttcgt ggtgttcgat aaggcacaga gcgcagttgc ggactgggtg   2700 

tataacgagc tgcgggtgcg gctggaggag cggcgcggtc gccgagccct acgcttgtgt   2760 

ctggaggacc gagattggct gcctggccag acgctcttcg agaacctctg ggcttccatc   2820 

tatgggagcc gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg   2880 

cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga cgtggtggtg   2940 

ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg tgcgactgcg ccagcgtctc   3000 

tgccgccaga gtgtgctctt ctggccccag cagcccaacg ggcagggggg cttctgggcc   3060 

cagctgagta cagccctgac tagggacaac cgccacttct ataaccagaa cttctgccgg   3120 

ggacctacag cagaatagct cagagcaaca gctggaaaca gctgcatctt catgcctggt   3180 

tcccgagttg ctctgcctgc                                               3200 

 
           
             8  
             1032  
             PRT  
             Mus musculus  
           
            8 

Met Val Leu Arg Arg Arg Thr Leu His Pro Leu Ser Leu Leu Val Gln 
1               5                   10                  15 

Ala Ala Val Leu Ala Glu Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe 
            20                  25                  30 

Leu Pro Cys Glu Leu Lys Pro His Gly Leu Val Asp Cys Asn Trp Leu 
        35                  40                  45 

Phe Leu Lys Ser Val Pro Arg Phe Ser Ala Ala Ala Ser Cys Ser Asn 
    50                  55                  60 

Ile Thr Arg Leu Ser Leu Ile Ser Asn Arg Ile His His Leu His Asn 
65                  70                  75                  80 

Ser Asp Phe Val His Leu Ser Asn Leu Arg Gln Leu Asn Leu Lys Trp 
                85                  90                  95 

Asn Cys Pro Pro Thr Gly Leu Ser Pro Leu His Phe Ser Cys His Met 
            100                 105                 110 

Thr Ile Glu Pro Arg Thr Phe Leu Ala Met Arg Thr Leu Glu Glu Leu 
        115                 120                 125 

Asn Leu Ser Tyr Asn Gly Ile Thr Thr Val Pro Arg Leu Pro Ser Ser 
    130                 135                 140 

Leu Val Asn Leu Ser Leu Ser His Thr Asn Ile Leu Val Leu Asp Ala 
145                 150                 155                 160 

Asn Ser Leu Ala Gly Leu Tyr Ser Leu Arg Val Leu Phe Met Asp Gly 
                165                 170                 175 

Asn Cys Tyr Tyr Lys Asn Pro Cys Thr Gly Ala Val Lys Val Thr Pro 
            180                 185                 190 

Gly Ala Leu Leu Gly Leu Ser Asn Leu Thr His Leu Ser Leu Lys Tyr 
        195                 200                 205 

Asn Asn Leu Thr Lys Val Pro Arg Gln Leu Pro Pro Ser Leu Glu Tyr 
    210                 215                 220 

Leu Leu Val Ser Tyr Asn Leu Ile Val Lys Leu Gly Pro Glu Asp Leu 
225                 230                 235                 240 

Ala Asn Leu Thr Ser Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg 
                245                 250                 255 

Arg Cys Asp His Ala Pro Asn Pro Cys Ile Glu Cys Gly Gln Lys Ser 
            260                 265                 270 

Leu His Leu His Pro Glu Thr Phe His His Leu Ser His Leu Glu Gly 
        275                 280                 285 

Leu Val Leu Lys Asp Ser Ser Leu His Thr Leu Asn Ser Ser Trp Phe 
    290                 295                 300 

Gln Gly Leu Val Asn Leu Ser Val Leu Asp Leu Ser Glu Asn Phe Leu 
305                 310                 315                 320 

Tyr Glu Ser Ile Asn His Thr Asn Ala Phe Gln Asn Leu Thr Arg Leu 
                325                 330                 335 

Arg Lys Leu Asn Leu Ser Phe Asn Tyr Arg Lys Lys Val Ser Phe Ala 
            340                 345                 350 

Arg Leu His Leu Ala Ser Ser Phe Lys Asn Leu Val Ser Leu Gln Glu 
        355                 360                 365 

Leu Asn Met Asn Gly Ile Phe Phe Arg Ser Leu Asn Lys Tyr Thr Leu 
    370                 375                 380 

Arg Trp Leu Ala Asp Leu Pro Lys Leu His Thr Leu His Leu Gln Met 
385                 390                 395                 400 

Asn Phe Ile Asn Gln Ala Gln Leu Ser Ile Phe Gly Thr Phe Arg Ala 
                405                 410                 415 

Leu Arg Phe Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Pro Ser Thr 
            420                 425                 430 

Leu Ser Glu Ala Thr Pro Glu Glu Ala Asp Asp Ala Glu Gln Glu Glu 
        435                 440                 445 

Leu Leu Ser Ala Asp Pro His Pro Ala Pro Leu Ser Thr Pro Ala Ser 
    450                 455                 460 

Lys Asn Phe Met Asp Arg Cys Lys Asn Phe Lys Phe Thr Met Asp Leu 
465                 470                 475                 480 

Ser Arg Asn Asn Leu Val Thr Ile Lys Pro Glu Met Phe Val Asn Leu 
                485                 490                 495 

Ser Arg Leu Gln Cys Leu Ser Leu Ser His Asn Ser Ile Ala Gln Ala 
            500                 505                 510 

Val Asn Gly Ser Gln Phe Leu Pro Leu Thr Asn Leu Gln Val Leu Asp 
        515                 520                 525 

Leu Ser His Asn Lys Leu Asp Leu Tyr His Trp Lys Ser Phe Ser Glu 
    530                 535                 540 

Leu Pro Gln Leu Gln Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe 
545                 550                 555                 560 

Ser Met Lys Gly Ile Gly His Asn Phe Ser Phe Val Ala His Leu Ser 
                565                 570                 575 

Met Leu His Ser Leu Ser Leu Ala His Asn Asp Ile His Thr Arg Val 
            580                 585                 590 

Ser Ser His Leu Asn Ser Asn Ser Val Arg Phe Leu Asp Phe Ser Gly 
        595                 600                 605 

Asn Gly Met Gly Arg Met Trp Asp Glu Gly Gly Leu Tyr Leu His Phe 
    610                 615                 620 

Phe Gln Gly Leu Ser Gly Leu Leu Lys Leu Asp Leu Ser Gln Asn Asn 
625                 630                 635                 640 

Leu His Ile Leu Arg Pro Gln Asn Leu Asp Asn Leu Pro Lys Ser Leu 
                645                 650                 655 

Lys Leu Leu Ser Leu Arg Asp Asn Tyr Leu Ser Phe Phe Asn Trp Thr 
            660                 665                 670 

Ser Leu Ser Phe Leu Pro Asn Leu Glu Val Leu Asp Leu Ala Gly Asn 
        675                 680                 685 

Gln Leu Lys Ala Leu Thr Asn Gly Thr Leu Pro Asn Gly Thr Leu Leu 
    690                 695                 700 

Gln Lys Leu Asp Val Ser Ser Asn Ser Ile Val Ser Val Val Pro Ala 
705                 710                 715                 720 

Phe Phe Ala Leu Ala Val Glu Leu Lys Glu Val Asn Leu Ser His Asn 
                725                 730                 735 

Ile Leu Lys Thr Val Asp Arg Ser Trp Phe Gly Pro Ile Val Met Asn 
            740                 745                 750 

Leu Thr Val Leu Asp Val Arg Ser Asn Pro Leu His Cys Ala Cys Gly 
        755                 760                 765 

Ala Ala Phe Val Asp Leu Leu Leu Glu Val Gln Thr Lys Val Pro Gly 
    770                 775                 780 

Leu Ala Asn Gly Val Lys Cys Gly Ser Pro Gly Gln Leu Gln Gly Arg 
785                 790                 795                 800 

Ser Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu Asp Glu Val Leu Ser 
                805                 810                 815 

Trp Asp Cys Phe Gly Leu Ser Leu Leu Ala Val Ala Val Gly Met Val 
            820                 825                 830 

Val Pro Ile Leu His His Leu Cys Gly Trp Asp Val Trp Tyr Cys Phe 
        835                 840                 845 

His Leu Cys Leu Ala Trp Leu Pro Leu Leu Ala Arg Ser Arg Arg Ser 
    850                 855                 860 

Ala Gln Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Ala Gln 
865                 870                 875                 880 

Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Val Arg Leu Glu 
                885                 890                 895 

Glu Arg Arg Gly Arg Arg Ala Leu Arg Leu Cys Leu Glu Asp Arg Asp 
            900                 905                 910 

Trp Leu Pro Gly Gln Thr Leu Phe Glu Asn Leu Trp Ala Ser Ile Tyr 
        915                 920                 925 

Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser 
    930                 935                 940 

Gly Leu Leu Arg Thr Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu 
945                 950                 955                 960 

Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Arg Pro Asp Ala His 
                965                 970                 975 

Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val 
            980                 985                 990 

Leu Phe Trp Pro Gln Gln Pro Asn  Gly Gln Gly Gly Phe  Trp Ala Gln 
        995                 1000                 1005 

Leu Ser  Thr Ala Leu Thr Arg  Asp Asn Arg His Phe  Tyr Asn Gln 
    1010                 1015                 1020 

Asn Phe  Cys Arg Gly Pro Thr  Ala Glu 
    1025                 1030 

 
           
             9  
             42  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            9 

gaaactcgag ccaccatgag acagactttg ccttgtatct ac                        42 

 
           
             10  
             37  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            10 

gaaagaattc ttaatgtaca gagtttttgg atccaag                              37 

 
           
             11  
             670  
             DNA  
             Homo sapiens  
           
            11 

agaaaaattt taaaaaatta ttcattcata tttttaggag ttttgaatga ttggatatgt     60 

aattatattc atattattaa tgtgtatcta tatagatttt tattttgcat atgtactttg    120 

atacaaaatt tacatgaaca aattacacta aaagttattc cacaaatata cttatcaaat    180 

taagttaaat gtcaatagct tttaaactta aattttagtt taacttttct gtcattcttt    240 

actttgaata aaaagagcaa actttgtagt ttttatctgt gaagtagagg tatacgtaat    300 

atacataaat agatatgcca aatctgtgtt attaaaattt catgaagatt tcaattagaa    360 

aaaaatacca taaaaggctt tgagtgcagg tgaaaaatag gcaatgatga aaaaaaatga    420 

aaaacttttt aaacacatgt agagagtgcg taaagaaagc aaaaacagag atagaaagta    480 

caactaggga atttagaaaa tggaaattag tatgttcact atttaagacc tatgcacaga    540 

gcaaagtctt cagaaaacct agaggccgaa gttcaaggtt atccatctca agtagcctag    600 

caatatttgc aacatcccaa tggccctgtc cttttcttta ctgatggccg tgctggtgct    660 

cagctacaaa                                                           670 

 
           
             12  
             300  
             DNA  
             Homo sapiens  
           
            12 

ttctcaggtc gtttgctttc ctttgctttc tcccaagtct tgttttacaa tttgctttag     60 

tcattcactg aaactttaaa aaacattaga aaacctcaca gtttgtaaat ctttttccct    120 

attatatata tcataagata ggagcttaaa taaagagttt tagaaactac taaaatgtaa    180 

atgacatagg aaaactgaaa gggagaagtg aaagtgggaa attcctctga atagagagag    240 

gaccatctca tataaatagg ccatacccac ggagaaagga cattctaact gcaacctttc    300 

 
           
             13  
             1031  
             DNA  
             Homo sapiens  
           
            13 

agaaggcctt acagtgagat gggatcccag tatttattga gtttcctcat tcataaaatg     60 

gggataataa tagtaaatga gttgacacgc gctaagacag tggaatagtg gctggcacag    120 

ataagccctc ggtaaatggt agccaataat gatagagtat gctgtaagat atctttctct    180 

ccctctgctt ctcaacaagt ctctaatcaa ttattccact ttataaacaa ggaaatagaa    240 

ctcaaagaca ttaagcactt ttcccaaagg tcgcttagca agtaaatggg agagacccta    300 

tgaccaggat gaaagcaaga aattcccaca agaggactca ttccaactca tatcttgtga    360 

aaaggttccc aatgcccagc tcagatcaac tgcctcaatt tacagtgtga gtgtgctcac    420 

ctcctttggg gactgtatat ccagaggacc ctcctcaata aaacacttta taaataacat    480 

ccttccatgg atgagggaaa ggaggtaaga tctgtaatga ataagcagga actttgaaga    540 

ctcagtgact cagtgagtaa taaagactca gtgacttctg atcctgtcct aactgccact    600 

ccttgttgtc cccaagaaag cggcttcctg ctctctgagg aggacccctt ccctggaagg    660 

taaaactaag gatgtcagca gagaaatttt tccaccattg gtgcttggtc aaagaggaaa    720 

ctgatgagct cactctagat gagagagcag tgagggagag acagagactc gaatttccgg    780 

aggctatttc agttttcttt tccgttttgt gcaatttcac ttatgatacc ggccaatgct    840 

tggttgctat tttggaaact ccccttaggg gatgcccctc aactggccct ataaagggcc    900 

agcctgagct gcagaggatt cctgcagagg atcaagacag cacgtggacc tcgcacagcc    960 

tctcccacag gtaccatgaa ggtctccgcg gcagccctcg ctgtcatcct cattgctact   1020 

gccctctgcg c                                                        1031 

 
           
             14  
             401  
             DNA  
             Homo sapiens  
           
            14 

gatctgtaat gaataagcag gaactttgaa gactcagtga ctcagtgagt aataaagact     60 

cagtgacttc tgatcctgtc ctaactgcca ctccttgttg tcccaagaaa gcggcttcct    120 

gctctctgag gaggacccct tccctggaag gtaaaactaa ggatgtcagc agagaaattt    180 

ttccaccatt ggtgcttggt caaagaggaa actgatgagc tcactctaga tgagagagca    240 

gtgagggaga gacagagact cgaatttccg gagctatttc agttttcttt tccgttttgt    300 

gcaatttcac ttatgatacc ggccaatgct tggttgctat tttggaaact ccccttaggg    360 

gatgcccctc aactggccct ataaagggcc agcctgagct g                        401 

 
           
             15  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            15 

tcgtcgtttt gtcgttttgt cgtt                                            24 

 
           
             16  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            16 

tgctgctttt gtgcttttgt gctt                                            24 

 
           
             17  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            17 

tngtngtttt gtngttttgt ngtt                                            24 

 
           
             18  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            18 

tccatgacgt tcctgatgct                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            19 

tccatgagct tcctgatgct                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            20 

tccatgangt tcctgatgct                                                 20 

 
           
             21  
             30  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            21 

gcgactggct gcatggcaaa accctctttg                                      30 

 
           
             22  
             30  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            22 

caaagagggt tttgccatgc agccagtcgc                                      30 

 
           
             23  
             30  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            23 

cgagattggc tgcatggcca gacgctcttc                                      30 

 
           
             24  
             30  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            24 

gaagagcgtc tggccatgca gccaatctcg                                      30 

 
           
             25  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            25 

ggcctcagca tcttt                                                      15 

 
           
             26  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            26 

ggcctatcga ttttt                                                      15 

 
           
             27  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            27 

gggttcccag tgaga                                                      15 

 
           
             28  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            28 

gggttatcga ttaga                                                      15 

 
           
             29  
             34  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            29 

cagctccagg gcctatcgat ttttgcacag gacc                                 34 

 
           
             30  
             34  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            30 

ggtcctgtgc aaaaatcgat aggccctgga gctg                                 34 

 
           
             31  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            31 

tccatgacgt ttttgatgtt                                                 20 

 
           
             32  
             18  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            32 

tccatgacgt ttttgatg                                                   18 

 
           
             33  
             16  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            33 

tccatgacgt ttttga                                                     16 

 
           
             34  
             14  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            34 

tccatgacgt tttt                                                       14 

 
           
             35  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            35 

tccatgacgt ttttgatgtt                                                 20 

 
           
             36  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            36 

tcgtcgtttt gtcgttttgt cgtt                                            24 

 
           
             37  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            37 

tcgtcgtttt gtcgttttgt cgtt                                            24 

 
           
             38  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            38 

tccatgacgt ttttgatgtt                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            39 

aagcgaaaat gaaattgact                                                 20 

 
           
             40  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            40 

accatggacg aactgtttcc cctc                                            24 

 
           
             41  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            41 

accatggacg acctgtttcc cctc                                            24 

 
           
             42  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            42 

accatggacg agctgtttcc cctc                                            24 

 
           
             43  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            43 

accatggacg atctgtttcc cctc                                            24 

 
           
             44  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            44 

accatggacg gtctgtttcc cctc                                            24 

 
           
             45  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            45 

accatggacg tactgtttcc cctc                                            24 

 
           
             46  
             24  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            46 

accatggacg ttctgtttcc cctc                                            24 

 
           
             47  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            47 

agcgggggcg agcgggggcg                                                 20 

 
           
             48  
             18  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            48 

agctatgacg ttccaagg                                                   18 

 
           
             49  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            49 

atcgactctc gagcgttctc                                                 20 

 
           
             50  
             17  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            50 

atgacgttcc tgacgtt                                                    17 

 
           
             51  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            51 

atggaaggtc caacgttctc                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            52 

atggaaggtc cagcgttctc                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            53 

atggactctc cagcgttctc                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            54 

atggaggctc catcgttctc                                                 20 

 
           
             55  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            55 

cacgttgagg ggcat                                                      15 

 
           
             56  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            56 

caggcataac ggttccgtag                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            57 

ctgatttccc cgaaatgatg                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            58 

gagaacgatg gaccttccat                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            59 

gagaacgctc cagcactgat                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            60 

gagaacgctc gaccttccat                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            61 

gagaacgctc gaccttcgat                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            62 

gagaacgctg gaccttccat                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            63 

gattgcctga cgtcagagag                                                 20 

 
           
             64  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            64 

gcatgacgtt gagct                                                      15 

 
           
             65  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            65 

gcggcgggcg gcgcgcgccc                                                 20 

 
           
             66  
             21  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            66 

gcgtgcgttg tcgttgtcgt t                                               21 

 
           
             67  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            67 

gctagacgtt agcgt                                                      15 

 
           
             68  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            68 

gctagacgtt agtgt                                                      15 

 
           
             69  
             15  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            69 

gctagatgtt agcgt                                                      15 

 
           
             70  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            70 

gcttgatgac tcagccggaa                                                 20 

 
           
             71  
             18  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            71 

ggaatgacgt tccctgtg                                                   18 

 
           
             72  
             19  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            72 

ggggtcaacg ttgacgggg                                                  19 

 
           
             73  
             19  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            73 

ggggtcagtc ttgacgggg                                                  19 

 
           
             74  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            74 

gtccatttcc cgtaaatctt                                                 20 

 
           
             75  
             18  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            75 

taccgcgtgc gaccctct                                                   18 

 
           
             76  
             12  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            76 

tcagcgtgcg cc                                                         12 

 
           
             77  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            77 

tccacgacgt tttcgacgtt                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            78 

tccataacgt tcctgatgct                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            79 

tccatagcgt tcctagcgtt                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            80 

tccatcacgt gcctgatgct                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            81 

tccatgacgg tcctgatgct                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            82 

tccatgacgt ccctgatgct                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            83 

tccatgacgt gcctgatgct                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            84 

tccatgacgt tcctgacgtt                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            85 

tccatgccgg tcctgatgct                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            86 

tccatgcgtg cgtgcgtttt                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            87 

tccatgcgtt gcgttgcgtt                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            88 

tccatggcgg tcctgatgct                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            89 

tccatgtcga tcctgatgct                                                 20 

 
           
             90  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            90 

tccatgtcgc tcctgatgct                                                 20 

 
           
             91  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            91 

tccatgtcgg tcctgatgct                                                 20 

 
           
             92  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            92 

tccatgtcgg tcctgctgat                                                 20 

 
           
             93  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            93 

tccatgtcgt ccctgatgct                                                 20 

 
           
             94  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            94 

tccatgtcgt tcctgatgct                                                 20 

 
           
             95  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            95 

tccatgtcgt tcctgtcgtt                                                 20 

 
           
             96  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            96 

tccatgtcgt ttttgtcgtt                                                 20 

 
           
             97  
             19  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            97 

tcctgacgtt cctgacgtt                                                  19 

 
           
             98  
             19  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            98 

tcctgtcgtt cctgtcgtt                                                  19 

 
           
             99  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            99 

tcctgtcgtt ccttgtcgtt                                                 20 

 
           
             100  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            100 

tcctgtcgtt ttttgtcgtt                                                 20 

 
           
             101  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            101 

tccttgtcgt tcctgtcgtt                                                 20 

 
           
             102  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            102 

tcgatcgggg cggggcgagc                                                 20 

 
           
             103  
             21  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            103 

tcgtcgctgt ctccgcttct t                                               21 

 
           
             104  
             27  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            104 

tcgtcgctgt ctccgcttct tcttgcc                                         27 

 
           
             105  
             21  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            105 

tcgtcgctgt ctgcccttct t                                               21 

 
           
             106  
             21  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            106 

tcgtcgctgt tgtcgtttct t                                               21 

 
           
             107  
             14  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            107 

tcgtcgtcgt cgtt                                                       14 

 
           
             108  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            108 

tcgtcgttgt cgttgtcgtt                                                 20 

 
           
             109  
             22  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            109 

tcgtcgttgt cgttttgtcg tt                                              22 

 
           
             110  
             18  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            110 

tctcccagcg cgcgccat                                                   18 

 
           
             111  
             17  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            111 

tctcccagcg ggcgcat                                                    17 

 
           
             112  
             18  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            112 

tctcccagcg tgcgccat                                                   18 

 
           
             113  
             20  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            113 

tgcagattgc gcaatctgca                                                 20 

 
           
             114  
             13  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            114 

tgtcgttgtc gtt                                                        13 

 
           
             115  
             19  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            115 

tgtcgttgtc gttgtcgtt                                                  19 

 
           
             116  
             25  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            116 

tgtcgttgtc gttgtcgttg tcgtt                                           25 

 
           
             117  
             21  
             DNA  
             Artificial sequence  
             
               Synthetic oligonucleotide  
             
           
            117 

tgtcgtttgt cgtttgtcgt t                                               21