INDUCTION OF TUMOR IMMUNITY BY VARIANTS OF FOLATE BINDING PROTEIN

Abstract

The present invention is directed to variants of antigens comprising folate binding protein epitopes as a composition associated with providing immunity against a tumor in an individual. The variant is effective in inducing cytotoxic T-lymphocytes but preferably not to the extent that they become sensitive to silencing by elimination, such as by apoptosis, or by anergy, as in unresponsiveness.

Description

[0001] The present invention claims priority to U.S. Provisional Patent Application Ser. No. 60/274,676 filed Mar. 9, 2001, incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0003] The present invention is directed to the fields of cancer and immunology. Specifically, the present invention is directed to compositions and methods for tumor vaccines directed to tumor antigens and is directed to specific epitopes on these antigens that are recognized by cytotoxic T-lymphocytes (CTL). More specifically, the present invention regards compositions and methods for variants of folate binding protein (FBP).

BACKGROUND OF THE INVENTION

[0004] Tumor reactive T-cells have been reported to mediate therapeutic responses against human cancers (Rosenberg et al., 1988). In certain instances, in human immunotherapy trials with tumor infiltrating lymphocytes (TIL) or tumor vaccines, these responses correlated either with in vitro cytotoxicity levels against autologous tumors (Aebersold et al., 1991) or with expression of certain HLA-A,B,C gene products (Marincola et al., 1992). Recent studies (Ioannides et al., 1992) have proposed that in addition to virally encoded and mutated oncogenes, overexpressed self-proteins may elicit some degree of tumor-reactive cytotoxic T-lymphocytes (CTLs) in patients with various malignancies (Ioannides et al., 1992; Ioannides et al., 1993; Brichard et al., 1993; Jerome et al., 1991). Autologous tumor reactive CTLs can be generated from lymphocytes infiltrating ovarian malignant ascites (Ioannides et al., 1991), and overexpressed proteins, such as HER-2, may be targets for CTL recognition (Ioannides et al., 1992).

[0005] T-cells play an important role in tumor regression in most murine tumor models. Tumor infiltrating lymphocytes (TIL) that recognize unique cancer antigens can be isolated from many murine tumors. The adoptive transfer of these TIL in addition to interleukin-2 can mediate the regression of established lung and liver metastases (Rosenberg et al., 1986). In addition, the secretion of IFN-.gamma. by injected TIL significantly correlates with in vivo regression of murine tumors suggesting activation of T-cells by the tumor antigens (Barth et al., 1991). The known ability of TIL to mediate the regression of metastatic cancer in 35 to 40% of melanoma patients when adoptively transferred into patients with metastatic melanoma attests to the clinical importance of the antigens recognized (Rosenberg et al., 1988; Rosenberg, 1992).

[0006] Strong evidence that an immune response to cancer exists in humans is provided by the existence of tumor reactive lymphocytes within melanoma deposits. These lymphocytes, when isolated, are capable of recognizing specific tumor antigens on autologous and allogeneic melanomas in an MHC restricted fashion. (Itoh et al., 1986; Muul et al., 1987; Topalian et al., 1989: Darrow et al., 1989; Hom et al., 1991; Kawakami et al., 1992; Hom et al., 1993; O'Neil et al., 1993). TIL from patients with metastatic melanoma recognize shared antigens including melanocyte-melanoma lineage specific tissue antigens in vitro (Kawakami et al., 1993: Anichini et al. 1993). Anti-melanoma T-cells appear to be enriched in TIL, probably as a consequence of clonal expansion and accumulation at the tumor site in vivo (Sensi et al., 1993). The transduction of T-cells with a variety of genes, such as cytokines, has been demonstrated. T-cells have been shown to express foreign gene products. (Blaese, 1993; Hwu et al., 1993; Culver et al., 1991) The fact that individuals mount cellular and humoral responses against tumor associated antigens suggests that identification and characterization of additional tumor antigens is important for immunotherapy of patients with cancer.

[0007] T-cell receptors on CD8.sup.+ T-cells recognize a complex consisting of an antigenic peptide (9-10 amino acids for HLA-A2), .beta.2 microglobulin and class I major histocompatibility complex (MHC) heavy chain (HLA-A, B, C, in humans). Peptides generated by digestion of endogenously synthesized proteins are transported into the endoplastic reticulum, bound to class I MHC heavy chain and .beta.2 microglobulin, and finally expressed in the cell surface in the groove of the class I MHC molecule.

[0008] Information on epitopes of self-proteins recognized in the context of MHC Class I molecules remain limited, despite a few attempts to identify epitopes capable of in vitro priming and Ag-specific expansion of human CTLs. For example, peptide epitopes have been proposed which are likely candidates for binding on particular MHC Class I Ag (Falk et al., 1991), and some studies have attempted to define peptide epitopes which bind MHC Class I antigens.

[0009] Synthetic peptides have been shown to be a useful tool for T-cell epitope mapping. However in vivo and in vitro priming of specific CTLs has encountered difficulties (Alexander et al., 1991; Schild et al., 1991; Carbone et al., 1988). It is generally considered that in vitro CTL priming cannot necessarily be achieved with peptide alone, and in fact, a high antigen density is thought to be required for peptide priming (Alexander et al., 1991). Even in the limited instances when specific priming was achieved, APC or stimulators were also required at high densities (Alexander et al., 1991).

[0010] Short synthetic peptides have been used either as target antigens for epitope mapping or for induction of in vitro primary and secondary CTL responses to viral and parasitic Ags (Bednarek et al., 1991; Gammon et al., 1992; Schmidt et al., 1992; Kos and Mullbacher, 1992; Hill et al., 1992). Unfortunately, these studies failed to show the ability of proto-oncogene peptide analogs to stimulate in vitro human CTLs to lyse tumors endogenously expressing these antigens.

[0011] Identification of tumor antigens (Ag) and of specific epitopes on these Ag recognized by cytotoxic T-lymphocytes enables the development of tumor vaccines (for review of tumor antigens, see Rosenberg (2000), incorporated by reference herein). Tumor Ag are weak or partial agonists for activation of low-avidity (low-affinity) CTL. Attempts to activate CTL by increasing the affinity of peptide for MHC (by modifications in the anchor residues) has produced mixed successes even with powerful APC (dendritic cells, DC) and added B7 costimulation. Some of the resulting cross-reactive CTL recognized tumors with lower affinity than CTL induced by wild type Ag.

[0012] The limited ability of anchor-fixed immunogens to induce and expand high-affinity CTL raises the need for alternative approaches for CTL induction. One approach to this question is to design immunogens which activate "high-affinity" CTL from the existent pool of responders. In human tumor immunology, this approach has been successful in some instances. However, high-affinity CTL are expected to be more sensitive to silencing by elimination (e.g apoptosis) or by anergy (unresponsiveness or diminished reactivity to a specific antigen).

[0013] These processes occur as a consequence of recurrent stimulations with Ag (tumor Ag) and are amplified by a number of cytokines. The general mechanism of activation induced cell death (AICD) is that repeated stimulations with an Ag in the presence of cytokines such as IL-2 activates cell death pathways. This is because stimulation with Ag and IL-2 transduces a signal which is too strong to induce proliferation and instead leads to premature senescence. An alternative death pathway, passive cell death (PCD) occurs when cytokines involved in survival (IL-2, IL-4, IL-7, etc.) are withdrawn. Since tumor Ag are self-Ag, the corresponding responding cells should be even more sensitive to deletion than CTL responding to foreign Ag, because the body's defense mechanisms are programmed to avoid autoimmunity. There is little known as to how the survival of responders to tumor Ag can be induced, and how they can be protected from AICD or PCD.

[0014] Preclinical and clinical trials are underway for the utilization of tumor-specific peptide epitopes for melanoma (Rivoltini et al., 1999; Parkhurst et al., 1998; Kawakami et al., 1998; Lustgarten et al., 1997; Zeng et al., 1997; Reynolds et al., 1998; Nestle et al., 1998; Chakraborty et al., 1998; Rosenberg et al., 1998); breast cancer, such as with MUC1 (Gendler et al., 1998; Xing et al., 1989; Xing et al., 1990; Jerome et al., 1993; Apostolopoulos et al., 1994; Ding et al., 1993; Zhang et al., 1996; Acres et al., 1993; Henderson et al., 1998; Henderson et al., 1996; Samuel et al., 1998; Gong et al., 1997; Apostolopoulos et al., 1995; Pietersz et al., 1998; Lofthouse et al., 1997; Rowse et al., 1998; Gong et al., 1998; Acres et al., 1999; Apostolopoulos et al., 1998; Lees et al., 1999; Xing et al., 1995; Goydos et al., 1996; Reddish et al., 1998; Karanikas et al., 1997), p53 (DeLeo, 1998; McCarty et al., 1998; Hurpin et al., 1998; Gabrilovich et al., 1996), and Her-2/neu (Disis and Cheever, 1998; Ioannides et al., 1993; Fisk et al., 1995; Peoples et al., 1995; Kawashima et al., 1999; Disi et al., 1996); and colon cancer (Kantor et al., 1992; Kantor et al., 1992; Tsang et al., 1995; Hodge et al., 1997; Conry et al., 1998; Kass et al., 1999; Zaremba et al., 1997; Nukaya et al., 1999).

[0015] Recently, peptides of folate binding protein (FBP) were recognized by tumor-associated lymphocytes (Peoples et al., 1998; Peoples et al., 1999; Kim et al., 1999). FBP is a membrane-associated glycoprotein originally found as a mAb-defined Ag in placenta and trophoblastic cells but rarely in other normal tissues (Retrig et al., 1985; Elwood, 1989; Weitman et al., 1992; Garin-Chesa et al., 1993). Of interest, this protein has been found in greater than 90% of ovarian and endometrial carcinomas; in 20-50% of breast, colorectal, lung, and renal cell carcinomas; and in multiple other tumor types. When present in cancerous tissue, the level of expression is usually greater than 20-fold normal tissue expression and has been reported to be as high as 80-90-fold in ovarian carcinomas (Li et al., 1996).

[0016] U.S. Pat. No. 5,846,538 is directed to immune reactivity to peptides of HER-2/neu protein for treatment of malignancies.

[0017] Folate binding protein provides an ideal target for and satisfies a long-felt need in the art for compositions and methods of utilizing the compositions directed to tumor immunity.

SUMMARY OF THE INVENTION

[0018] It is an object of the present invention to provide as a composition of matter an antigen comprising a folate binding protein epitope of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0019] It is another object of the present invention to provide a composition comprising an antigen which includes a folate binding protein epitope of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof in a pharmaceutically acceptable excipient.

[0020] It is another object of the present invention to provide a method for stimulating cytotoxic T-lymphocytes, comprising the step of contacting the cytotoxic T-lymphocytes with an amount of an antigen comprising a folate binding protein epitope selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and a combination thereof, wherein the amount is effective to stimulate the cytotoxic T-lymphocytes. In a specific embodiment of the present invention, the cytotoxic T-lymphocytes are located within a human. In another specific embodiment, the method further comprises the step of administering to the human an antigen comprising a folate binding protein epitope selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3. SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and a combination thereof. In another specific embodiment of the present invention, the epitope is formulated for administration parenterally, topically, or as an inhalant, aerosol or spray.

[0021] It is an additional object of the present invention to provide a method of generating an immune response, comprising the step of administering to a human a pharmaceutical composition comprising an immunologically effective amount of a composition comprising an antigen comprising a folate binding epitope of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof.

[0022] It is another object of the present invention to provide a method of inducing immunity against a tumor in an individual, comprising the steps of administering to the individual an antigen comprising a folate binding protein epitope of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof; and administering to the individual a cancer vaccine. In a specific embodiment of the present invention, the an antigen comprising a folate binding protein epitope is administered prior to the administration of the cancer vaccine. In a specific embodiment of the present invention, an antigen comprising a folate binding protein epitope is administered subsequent to the administration of the cancer vaccine. In another specific embodiment of the present invention, the antigen comprising a folate binding protein epitope is administered both prior to and subsequent to the administration of the cancer vaccine. In a further specific embodiment, the cancer vaccine comprises a polypeptide selected from the group consisting of SEQ ID NO:268 (E39) and SEQ ID NO:269 (E41).

[0023] It is another object of the present invention to provide a method of inducing memory cytotoxic T-lymphocytes in an individual comprising the step of administering an antigen comprising a folate binding epitope of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7. SEQ ID NO:8, or a combination thereof. In a specific embodiment, the individual is substantially susceptible to recurrence of cancer.

[0024] It is another object of the present invention to provide a method of providing immunity against a tumor comprising the step of administering an antigen comprising a folate binding epitope vaccine of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof.

[0025] It is another object of the present invention to provide a method of treating an individual for cancer comprising the steps of administering to the individual a first cancer vaccine; and administering to the individual a second cancer vaccine comprising a peptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof. In a specific embodiment, the first cancer vaccine administration step precedes the second cancer vaccine administration step. In another specific embodiment, the first cancer vaccine administration step is subsequent to the second cancer vaccine administration step.

[0026] It is an additional object of the present invention to provide a pharmaceutical composition comprising an antigen comprising a folate binding protein epitope selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof in a pharmaceutically acceptable excipient.

[0027] It is another object of the present invention to provide a method of treating a proliferative cell disorder in a human, comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising an antigen comprising a folate binding protein epitope selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2. SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof in a pharmaceutically acceptable excipient. In a specific embodiment, the proliferative cell disorder is cancer. In an additional specific embodiment, the cancer is breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, brain cancer, sarcomas, or a combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIG. 1 demonstrates HLA-A2 stabilization by FBP epitope E39 variants.

[0030] FIG. 2A illustrates IFN-.gamma. induction in peripheral blood mononuclear cells (PBMC) with multiple stimulations with J65 or E39.

[0031] FIG. 2B illustrates CTL activity in PBMC with multiple stimulations with J65 or E39.

[0032] FIG. 3 illustrates specific interleukin 2 (IL-2) induction in PBMCs by priming with E39 variants.

[0033] FIG. 4 illustrates expansion of PBMCs stimulated with FBP peptide E39 and its variants.

[0034] FIG. 5 demonstrates expansion of PBMC stimulated with variants of the FBP peptide E39.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[0035] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

[0036] The term "antigen" as used herein is defined as an entity which elicits an immune system response. The term herein may be abbreviated to "Ag."

[0037] The term "cancer" as used herein is defined as a tissue of uncontrolled growth or proliferation of cells, such as a tumor. In a specific embodiment, the cancer is an epithelial cancer. In specific embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, brain cancer, sarcomas, or a combination thereof. In specific embodiments, such cancers in mammals are caused by chromosomal abnormalities, degenerative growth and/or developmental disorders, mitogenic agents, ultraviolet radiation (uv), viral infections, inappropriate tissue expression of a gene, alterations in expression of a gene, carcinogenic agents, or a combination thereof. The term melanoma includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome. The aforementioned cancers can be treated by methods described in the present application.

[0038] The term "epitope" as used herein is defined as a short peptide derived from a protein antigen which binds to an MHC molecule and is recognized by a particular T cell.

[0039] The term "folate binding protein variant" as used herein is defined as a folate binding protein and peptides thereof which are preferably recognized by helper T cells or cytotoxic T cells and may be naturally derived, synthetically produced, genetically engineered, or a functional equivalent thereof, e.g where one or more amino acids may be replaced by other amino acid(s) or non-amino acid(s) which do not substantially affect function. In specific embodiments, the peptides are epitopes which contain alterations, modifications, or changes in comparison to SEQ ID NO:268 (E39) or SEQ ID NO:269 (E41). In further specific embodiments, the variants are of SEQ ID NO:1 through SEQ ID NO:8.

[0040] The term "immune response" as used herein refers to a cellular immune response, including eliciting stimulation of T lymphocytes, macrophages, and/or natural killer cells.

[0041] The term "immunity" as used herein is defined as the ability to provide resistance to a tumor resulting from exposure to an antigen that is a folate binding protein epitope, such as the folate binding protein variants described herein.

[0042] The term "vaccine" as used herein is defined as a composition for generating immunity to a cancer. In specific embodiments, the cancer vaccine is a wild-type epitope of folate binding protein, such as E39 (FBP amino acid residues 191-199) (SEQ ID NO:268) or E41 (FBP amino acid residues 245-253) (SEQ ID NO:269). In other specific embodiments, the cancer vaccine comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or a combination thereof. In a preferred embodiment, administration of the vaccine alternates the signaling through the T cell receptor, thereby reducing the possibility of apoptosis.

[0043] The term "variant" as used herein is defined as a modified or altered form of a wildtype sequence, such as the folate binding protein E39 epitope (SEQ ID NO:268). The variant may contain replacement of at least one amino acid residue or may contain an altered side chain for at least one amino acid residue.

II. The Present Invention

A. Specific Embodiments

[0044] The present invention is directed to folate binding protein tumor Ag modified to attenuate the signaling through T cell receptors, compared with a wild-type folate binding protein tumor Ag, particularly for reducing the possibility of apoptosis that results following repeated exposure to strong antigens. Thus, variants of folate binding protein epitopes such as E39 (SEQ ID NO:268) and E41 (SEQ ID NO:269), which are "strong" antigens, are modified to act as a "weak" antigen. Thus, the present invention utilizes compositions and methods to attenuate signaling through the T cell receptors.

[0045] The invention works as (1) prestimulation prevaccine, to be administered before the tumor Ag; (2) as post vaccine to be given after the tumor Ag; and/or (3) in certain individuals will work as a priming vaccine. The situations (1) and (2) are more related to a protective role for SEQ ID NO:6 (365) and its analogs for tumor reactive CTL. The situation (3) can be encountered in certain individuals where mutations in the histocompatibility Ag binding pocket may transform an attenuator into a strong immunogen.

[0046] The invention allows protection before and after vaccination of either precursors (stand-in) or activated effectors. In specific embodiments, administration of the variants of folate binding protein provide targeted induction of memory CTL.

[0047] The variants described herein, in a particular embodiment SEQ ID NO:6, are intended to attenuate the signaling at recurrent stimulation, thus inducing protection of CTL precursors as of activated T-cells from apoptosis, thereby enabling the immune response to expand, and, in preferred embodiments, have important implications in induction of memory CTL.

[0048] It is well known that the two major arms of the immune system are: (1) cell-mediated immunity with immune T cells; and (2) humoral immunity with antibodies. Further, the immune system normally functions to recognize and destroy any foreign or aberrant cells in the body. Since FBP is expressed by some normal cells, tolerance and/or anergy is expected.

[0049] Development of molecular therapies for cancer have historically focused on specific recognition of Ags by cellular immune effectors. The present invention discloses novel strategies aimed at identification of peptide targets for CTLs, and generation of T-cell immunity against specific epitopes (for a review of T-cell specific immunity, see, e.g, Ioannides et al., 1992; Houbiers et al., 1993).

[0050] To achieve this, the present invention provides novel naturally- and synthetically-derived peptides which bind human leucocyte antigen-(HLA) class I heavy chains. Appropriate criteria for epitope selection in vitro have been defined, and synthetic peptides based on immunogenic epitopes of FBP have also been produced.

[0051] Although the dominant anchors for peptide binding to HLA-A2 are Leu (P2) and Val (P9), a number of residues with similar charge and side chains, such as Ile and/or Met, were identified in CTL epitopes from viral proteins (Falk et al., 1991; Bednarek et al., 1991).

B. General Embodiments

[0052] 1. CTL Epitopes

[0053] CTL epitopes reported to date are mainly derived from foreign (viral) proteins with little or no homology to self-proteins. With respect to CTL responses to self-proteins, it is expected that T-cells expressing TCR with high affinity for self-peptide-MHC class I complexes are eliminated in the thymus during development. Self-peptides eluted from HLA-A2.1 molecules of various cell lines show residues at P3-P5 and P7-P8 which are different from the sequences of viral epitopes recognized by human CTLs. Since these residues are likely to contact and interact with TCR, they may reflect peptides for which autologous T-cells are already tolerant/anergic.

[0054] For T-cell recognizing self-epitopes to be eliminated or anergized, a precondition exists that the peptide-MHC complex is stable enough to engage a sufficient number of TCRs, or at least more stable than other HLA-A2 peptide complexes, where one peptide can be easily displaced by other peptides. Consequently, this would suggest that for self-proteins with extension to FBP, the ones that can bind TCR with high affinity during development will be less likely to be recognized later when expressed on a tumor other target, than peptides that bind HLA-A2 with low affinity, which under appropriate conditions (e.g, high protein concentration) may occupy a higher number of HLA-A2 molecules. For low-affinity peptides, modification of the anchors resulting in stabilization of peptide--HLA-A2 interaction by replacing weak with dominant anchor residues (e.g, (P9) MV, should facilitate the reactivity of CTL with targets expressing such antigens, because TCR interacts mainly with the sequence P4-P8.

[0055] Tumor progression and metastasis are often associated with overexpression of specific cellular proteins. Epitopes of non-mutated overexpressed proteins can be targets of a specific cellular immune response against tumor mediated by T-cells. Moreover, when T-cell epitopes are present, distinction between tumor immunity/autoimmunity and unresponsiveness can be predicated on the protein concentration as a limiting factor of epitope supply.

[0056] 2. Epitopic Core Sequences

[0057] The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically recognized by a CTL.

[0058] As used herein, the term "incorporating an epitope(s) that is immunologically recognized by a CTL" is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a FBP polypeptide. The level of similarity will generally be to such a degree that the same population of CTLs will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen.

[0059] The identification of CTL-stimulating immunodominant epitopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.

[0060] Preferred peptides for use in accordance with the present invention will generally be on the order of 8 to 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that shorter antigenic CTL-stimulating peptides will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

[0061] It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a "universal" epitopic peptide directed to FBP sequences. These epitopic core sequences are identified herein in particular aspects as hydrophilic regions of the FBP polypeptide antigen. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation, and, hence, elicit specific antibody production.

[0062] An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, receptors on CTLs. It will be understood that in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other.

[0063] In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 8 amino acids in length, with sequences on the order of 9 or 10 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.

[0064] A skilled artisan recognizes that numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g, Jameson & Wolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysis programs (e.g, DNAStar Software, DNAStar, Inc., Madison, Wis.) may also be useful in designing synthetic peptides in accordance with the present disclosure.

[0065] Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g, through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

[0066] In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g, up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4.quadrature.C, or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g, in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.

[0067] 3. T lymphocytes

[0068] T lymphocytes recognize antigen in the form of peptide fragments that are bound to class I and class H molecules of the major histocompatibility complex (MHC) locus. Major Histocompatibility Complex (MHC) is a generic designation meant to encompass the histocompatibility antigen systems described in different species including the human leucocyte antigens (HLA). The T-cell receptor for antigen (TCR) is a complex of at least 8 polypeptide chains. ("Basic and Clinical Immunology" (1994) Stites, Terr and Parslow(eds) Appleton and Lange, Nenmack Conn.) Two of these chains (the alpha and beta chains) form a disulfide-linked dimer that recognizes antigenic peptides bound to MHC molecules and therefore is the actual ligand-binding structure within the TCR. The TCR alpha and beta chains are similar in many respects to immunoglobulin proteins. The amino-terminal regions of the alpha and beta chains are highly polymorphic, so that within the entire T-cell population there are a large number of different TCR alpha/beta dimers, each capable of recognizing or binding a particular combination of antigenic peptide and MHC.

[0069] In general, CD4.sup.+ T cell populations are considered to function as helpers/inducers through the release of lymphokines when stimulated by a specific antigen; however, a subset of CD4.sup.+ cells can act as cytotoxic T lymphocytes (CTL). Similarly, CD8.sup.+ T cells are considered to function by directly lysing antigenic targets; however, under a variety of circumstances they can secrete lymphokines to provide helper or DTH function. Despite the potential of overlapping function, the phenotypic CD4 and CD8 markers are linked to the recognition of peptides bound to class H or class I MHC antigens. The recognition of antigen in the context of class II or class I MHC mandates that CD4.sup.+ and CD8.sup.+ T cells respond to different antigens or the same antigen presented under different circumstances. The binding of immunogenic peptides to class II MHC antigens most commonly occurs for antigens ingested by antigen presenting cells. Therefore, CD4.sup.+ T cells generally recognize antigens that have been external to the tumor cells. By contrast, under normal circumstances, binding of peptides to class I MHC occurs only for proteins present in the cytosol and synthesized by the target itself, proteins in the external environment are excluded. An exception to this is the binding of exogenous peptides with a precise class I binding motif which are present outside the cell in high concentration. Thus, CD4.sup.+ and CD8.sup.+ T cells have broadly different functions and tend to recognize different antigens as a reflection of where the antigens normally reside.

[0070] As disclosed within the present invention, the protein product expressed by FBP is recognized by T cells. Such a protein expression product "turns over" within cells, i.e., undergoes a cycle wherein a synthesized protein functions and then eventually is degraded and replaced by a newly synthesized molecule. During the protein life cycle, peptide fragments from the protein bind to major histocompatibility complex (MHC) antigens. By display of a peptide bound to MHC antigen on the cell surface and recognition by host T cells of the combination of peptide plus self MHC antigen, a malignant cell will be immunogenic to T cells. The exquisite specificity of the T cell receptor enables individual T cells to discriminate between protein fragments which differ by a single amino acid residue.

[0071] During the immune response to a peptide, T cells expressing a T cell receptor with high affinity binding of the peptide-MHC complex will bind to the peptide-MHC complex and thereby become activated and induced to proliferate. In the first encounter with a peptide, small numbers of immune T cells will secrete lymphokines, proliferate and differentiate into effector and memory T cells. Subsequent encounters with the same antigen by the memory T cell will lead to a faster and more intense immune response.

[0072] Intact folate binding protein or peptides thereof which are recognized by cytotoxic T cells may be used within the present invention. The peptides may be naturally derived or produced based upon an identified sequence. The peptides for CD8.sup.+ T cell responses (elicited by peptides presented by folate binding protein class I MHC molecules) are generally about 8-10 amino acids in length. Peptides for CD8.sup.+ T cell responses vary according to each individual's class I MHC molecules. Examples of peptides suitable within the present invention for CD8.sup.+ T cell responses include peptides comprising or consisting of SEQ ID NO:1 through SEQ ID NO:8.

[0073] It will be evident to those of ordinary skill in the art that other peptides may be produced for use within the present invention, both for class I MHC molecules as well as for class II molecules. A variety of techniques are well known for isolating or constructing peptides. Suitable peptides are readily identified based upon the disclosure provided herein. Additional suitable peptides include those which are longer in length. Such peptides may be extended (e.g, by the addition of one or more amino acid residues and/or truncated (e.g, by the deletion of one or more amino acid residues from the carboxyl terminus). Alternatively, suitable peptides may be variations on other preferred peptides disclosed herein. Although this particular peptide variation may result in a peptide with the same number of total amino acids (such as nine), a peptide variation on a preferred peptide need not be identical in length. Variations in amino acid sequence that yield peptides having substantially the same desired biological activity are within the scope of the present invention.

[0074] Immunization of an individual with a FBP peptide (i.e., as a vaccine) can induce continued expansion in the number of T cells necessary for therapeutic attack against a tumor in which FBP is associated. Typically, about 0.01 .mu.g/kg to about 100 mg/kg body weight will be administered by the intradermal, subcutaneous or intravenous route. A preferred dosage is about 1 .mu.g/kg to about 1 mg/kg, with about 5 .mu.g/kg to about 200 .mu.g/kg particularly preferred. It will be evident to those skilled in the art that the number and frequency of administrations will be dependent upon the response of the patient. It may be desirable to administer the FBP peptide repetitively. It will be evident to those skilled in this art that more than one FBP peptide may be administered, either simultaneously or sequentially. For example, a combination of about 8-15 peptides may be used for immunization. Preferred peptides for immunization are those that include all or a portion of at least one FBP amino acid SEQ ID NO:1 through SEQ ID NO:68, or variants thereof. One or more peptides from other portions of the amino acid sequence shown in SEQ ID NO:1 through SEQ ID NO:68 may be added to one or more of the preferred peptides.

[0075] In addition to the FBP peptide (which functions as an antigen), it may be desirable to include other components in the vaccine, such as a vehicle for antigen delivery and immunostimulatory substances designed to enhance the protein's immunogenicity. Examples of vehicles for antigen delivery include aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of immunostimulatory substances (adjuvants) include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon and IL-15. It will be evident to those skilled in this art that a FBP peptide may be prepared synthetically or that a portion of the protein (naturally-derived or synthetic) may be used. When a peptide is used without additional sequences, it may be desirable to couple the peptide hapten to a carrier substance, such as keyhole limpet hemocyanin.

[0076] The methods and compositions of the present invention are particularly well-suited for inducing an immune response in a patient who has developed resistance to conventional cancer treatments or who has a high probability of developing a recurrence following treatment. A skilled artisan recognizes that cancer cells are able to evade the immune system or evade an effective immune response because they look like self, they actively anergize the immune system to any antigens which may potentially differentiate between self and tumor, and they may create an immunosuppressive environment by secreting immunosuppressive factors and/or by expressing factors which can induce apoptosis of an offensive tumor antigen-specific killer cell.

[0077] A skilled artisan is aware of multiple reviews concerning cancer vaccines and the generation of cellular immune responses to antigenic tumor peptides (Pietersz et al., 2000; Pardoll, 2000; Rosenberg, 2000; Dalgleish, 2000, each of which are incorporated by reference herein).

[0078] A skilled artisan recognizes that the antigen can be produced in large amounts by recombinant technology, either as soluble molecules in eukaryotic systems or as fusion proteins in bacterial systems. In a specific embodiment, synthetic peptides are made from the tumor antigen. Furthermore, monoclonal antibodies to the tumor antigens are useful in their identification and purification.

[0079] In a peptide approach to tumor immunotherapy, peptides (such as about 8-9mers) are presented by MHC class I molecules, leading to the generation of CD8.sup.+-mediated cellular responses comprising CTLs and cytokine secretion, mostly in the form of IFN-.gamma. and TNF-.alpha..

[0080] A skilled artisan recognizes that the dendritic cell is important in generating CD8.sup.+ CTLs following class I presentation. Esche et al. (1999) demonstrated techniques whereby dendritic cells are obtained from patients, isolated, expanded in vitro, exposed to the peptides and reintroduced into the patient. Others utilize similarly treated dendritic cells for generation of specifically activated T cells in vitro before transfer.

[0081] A crucial initial step in CD8.sup.+ T cell generation is the uptake and presentation of peptides by MHC molecules by antigen-presenting cells. MHC class 1 proteins consist of three subunits, all of which are important for the formation of a stable complex. X-ray crystallography of MHC class I molecules has demonstrated that interaction of peptides with the MHC class 1 groove is determined by the peptide sequence, with discrete amino acids interacting with pockets in the MHC groove (which have a fixed spacing from each other) and also have specificity for anchoring amino acid side chains. Although there are exceptions, the amino and carboxy termini of the peptides are anchored at either end of the groove, often in positions 2 or 3, 5 or 7 (Apostolopoulos et al., 1997a; Apostolopoulos et al., 1997b). The peptides also interact with the T cell receptor, yet only a small amount of the peptide is exposed (Apostolopoulos et al., 1998).

[0082] Given that multiple peptide tumor antigens, such as folate binding protein, have been identified in addition to characterization of T cell epitopes, in a specific embodiment of the present invention peptide antigens are generated synthetically for immunization. The immunogenicity of small peptides can be improved upon by increasing the peptide size, by binding to carriers and also by using adjuvants to activate macrophages and other immune system factors. A skilled artisan is cognizant of recombinant cytokines being used to increase immunogenicity of a synthetic peptide (Tao and Levy, 1993) and furthermore that cytokines can also be directly fused to peptides (Nakao et al., 1994; Disis et al., 1996; Chen et al., 1994).

[0083] In specific embodiments of the present invention, mixtures of separate peptides are administered as a vaccine. Alternatively, multiple epitopes may be incorporated into the same molecule by recombinant technology well known in the art (Mateo et al., 1999; Astori and Krachenbuhl, 1996). In another embodiment, a combinatorial peptide library is used to increase binding peptides by utilizing different amino acids at least one anchor location.

[0084] In another embodiment of the present invention, natural amino acids of a peptide are replaced with unnatural D-amino acids; alternatively, the peptide residues are assembled in reverse order, which renders the peptides resistant to proteases (Briand et al., 1997; Herve et al., 1997; Bartnes et al., 1997; Guichard et al., 1996). In another embodiment, unnatural modified amino acids are incorporated into a peptide, such as .alpha.-aminoisobutyric acid or N-methylserine.

[0085] A skilled artisan recognizes that the binding strength of the 8- or 9-mer to the MHC complex and the subsequent recognition by the T cell receptor determines the immunogenicity of CTL peptides. Van Der Burg et al. (1993) determined that the longer the peptide remains bound to the MHC complex, the better the chance it will induce a T cell response. A skilled artisan also recognizes that there are methods for introducing extraneous peptides directly into the cytoplasm of a cell to allow generation of class I-restricted cellular immune responses. One example includes microbial toxins, which can carry peptides in their cytoplasm for delivery because they enter cells by receptor-mediated endocytosis and thereby deposit cellular toxins into the cytoplasm. Specific examples include shiga toxin (Lee et al., 1998), anthrax toxin (Goletz et al., 1997), diphtheria toxin (Stenmark et al., 1991), Pseudomonas exotoxin (Donnelly et al., 1993), and Bordetella pertussis toxin (Fayolle et al., 1996).

[0086] In alternative embodiments, peptides enter cells through membrane fusion and are beneficial for delivering tumor or other peptides into a cell cytoplasm, including Antennapedia (Derossi et al., 1994; Derossi et al., 1996; Schutze-Redelmeier et al., 1996), Tat protein (Kim et al., 1997), and Measles virus fusion peptide (Partidos et al., 1997).

[0087] In other embodiments, peptides are introduced into a cytoplasm through lipopeptides, which comprise both a lipid and a peptide, by direct insertion into the lipophilic cell membrane (BenMohamed et al., 1997; Obert et al., 1998; Deprez et al., 1996; Beekman et al., 1997). In alternative embodiments, the peptides are delivered in liposomes (for examples, see Nakanishi et al., 1997; Noguchi et al., 1991; Fukasawa et al., 1998; Guan et al., 1998), whereby the immunogenicity is dependent on the size, charge, lipid composition of the liposome itself, and whether or not the antigen is present on the surface of the liposome or within its interior.

[0088] A skilled artisan also recognizes that immune-stimulating complexes (ISCOMs), which comprise Quill A (a mixture of saponins), cholesterol, phospholipid, and proteins, are useful for delivering naturally hydrophobic antigens or antigens made hydrophobic by the addition of myristic or palmitic acid tails (for examples, see Hsu et al., 1996; Sjolander et al., 1997; Villacres-Eriksson, 1995; Tarpey et al., 1996; Rimmelzwaan et al., 1997). ISCOMs facilitate penetration into cells by fusion with their membranes, by endocytosis, or by phagocytosis.

[0089] Antigens may also be directed to particular subcellular compartments through incorporation of sorting signals to the antigen by recombinant technology, including Class 11 LAMP-I (Rowell et al., 1995; Wu et al., 1995), ER targeting peptide (Minev et al., 1994); CLIP (Malcherik et al., 1998), and heat shock proteins (Udono and Srivastava, 1993; Heike et al., 1996; Zhu et al., 1996; Suzue et al., 1997; Ciupitu et al., 1998).

[0090] A skilled artisan recognizes that the present invention provides anti-cancer therapeutic compositions comprising a variety of peptides designated for CD8.sup.+ T cell responses comprising SEQ ID NO:1 through SEQ ID NO:8, or a combination thereof. A skilled artisan also recognizes that the present invention provides anti-cancer therapeutic compositions comprising a variety of peptides designated for CD8.sup.+ T cell responses consisting essentially of SEQ ID NO:1 through SEQ ID NO:8, or a combination thereof.

[0091] A skilled artisan recognizes that references such as Abrams and Schlom (2000) summarize the current views on rational Ag modification. Two types of peptides are described: (1) agonistic peptides which upregulate Ag-specific responses; (2) antagonistic/partial agonistic peptides which downregulate the same responses. However, it is an object of the present invention to provide therapy which stimulate Ag-specific immune responses while at the same time does not elicit activation induced-cell death or death by neglect.

[0092] A skilled artisan recognizes that sequences that encode folate binding protein epitopes for induction of tumor immunity can be obtained from databases such as the National Center for Biotechnology Informations's GenBank database or commercially available databases, such as that of Celera Genomics, Inc. (Rockville, Md.). Examples of folate binding protein sequences which may comprise an epitope or which can be altered to comprise an epitope include the following, denoted by GenBank Accession numbers: P14207 (SEQ ID NO:9); P15328 (SEQ ID NO:10); P13255 (SEQ ID NO:11); NP_000793 (SEQ ID NO:12); AAB05827 (SEQ ID NO:13); AAG36877 (SEQ ID NO:14); S42627 (SEQ ID NO:15); S00112 (SEQ ID NO:16); BFBO (SEQ ID NO:17); S62670 (SEQ ID NO:18); S62669 (SEQ ID NO:19); A55968 (SEQ ID NO:20); A45753 (SEQ ID NO:21); A33417 (SEQ ID NO:22); B40969 (SEQ ID NO:23); A40969 (SEQ ID NO:24); NP_057943 (SEQ ID NO:25); NP_057942 (SEQ ID NO:26); NP_057941 (SEQ ID NO:27); NP_057937 (SEQ ID NO:28); NP_057936 (SEQ ID NO:29); NP_037439 (SEQ ID NO:30); NP_032061 (SEQ ID NO:31); NP_032060 (SEQ ID NO:32); NP_000795 (SEQ ID NO:33); NP_000794 (SEQ ID NO:34); AAF66225 (SEQ ID NO:35); BAA37125 (SEQ ID NO:36); P02752 (SEQ ID NO:37); Q05685 (SEQ ID NO:38); P35846 (SEQ ID NO:39); P02702 (SEQ ID NO:40); AAD53001 (SEQ ID NO:41); AAD33741 (SEQ ID NO:42); AAD33740 (SEQ ID NO:43); AAD19354 (SEQ ID NO:44); AAD19353 (SEQ ID NO:45); AAC98303 (SEQ ID NO:46); AAB81938 (SEQ ID NO:47); AAB81937 (SEQ ID NO:48); AAB49703 (SEQ ID NO:49); AAB35932 (SEQ ID NO:50); 1011184A (SEQ ID NO:51); 0908212A (SEQ ID NO:52); CAA44610 (SEQ ID NO:53); CAA83553 (SEQ ID NO:54); AAA74896 (SEQ ID NO:55); AAA49056 (SEQ ID NO:56); AAA37599 (SEQ ID NO:57); AAA37598 (SEQ ID NO:58); AAA37597 (SEQ ID NO:59); AAA37594 (SEQ ID NO:60); AAA37596 (SEQ ID NO:61); AAA37595 (SEQ ID NO:62); AAA35824 (SEQ ID NO:63); AAA35823 (SEQ ID NO:64); AAA35822 (SEQ ID NO:65); AAA35821 (SEQ ID NO:66); AAA18382 (SEQ ID NO:67); and AAA17370 (SEQ ID NO:68).

[0093] A skilled artisan also recognizes that epitopes of folate binding protein, nucleic acid sequences are encoded by, or altered to encode a variant of, for example, one of the following: U02715 (SEQ ID NO:69); BE518506 (SEQ ID NO:70); BG058247 (SEQ ID NO:71); BG017460 (SEQ ID NO:72); NM_000802 (SEQ ID NO:73); U20391 (SEQ ID NO:74); NM_016731 (SEQ ID NO:75); NM_016730 (SEQ ID NO:76); NM_016729 (SEQ ID NO:77); NM_016725 (SEQ ID NO:78); NM_016724 (SEQ ID NO:79); NM_013307 (SEQ ID NO:80); NM_008035 (SEQ ID NO:81); NM_008034 (SEQ ID NO:82); BF153292 (SEQ ID NO:83); BF114518 (SEQ ID NO:84); BE940806 (SEQ ID NO:85); BE858996 (SEQ ID NO:86); AF219906 (SEQ ID NO:87); AF219905 (SEQ ID NO:88); AF219904 (SEQ ID NO:89); BE687177 (SEQ ID NO:90); BE636622 (SEQ ID NO:91); BE627230 (SEQ ID NO:92); BE506561 (SEQ ID NO:93); BE505048 (SEQ ID NO:94); BE496754 (SEQ ID NO:95); BB114010 (SEQ ID NO:96); BB109527 (SEQ ID NO:97); BB107219 (SEQ ID NO:98); BE206324 (SEQ ID NO:99); BE448392 (SEQ ID NO:100); BE207596 (SEQ ID NO:101); BE206635 (SEQ ID NO:102); BE240998 (SEQ ID NO:103); BE228221 (SEQ ID NO:104); BE225416 (SEQ ID NO:105); BE225404 (SEQ ID NO:106); BB214040 (SEQ ID NO:107); BE199619 (SEQ ID NO:108); BE199597 (SEQ ID NO:109); BE198610 (SEQ ID NO:110); BE198571 (SEQ ID NO:111); BE188055 (SEQ ID NO:112); BE187804 (SEQ ID NO:113); BB032646 (SEQ ID NO:114); BE037278 (SEQ ID NO:115); BE037125 (SEQ ID NO:116); BE037110 (SEQ ID NO:117); BE037009 (SEQ ID NO:118); BE036024 (SEQ ID NO:119); BE035828 (SEQ ID NO:120); BE035751 (SEQ ID NO:121); BE019724 (SEQ ID NO:122); AW913291 (SEQ ID NO:123); AW912445 (SEQ ID NO:124); AW823912 (SEQ ID NO:125); AW823418 (SEQ ID NO:126); AB023803 (SEQ ID NO:127); AB022344 (SEQ ID NO:128); AW475385 (SEQ ID NO:129); AW323586 (SEQ ID NO:130); AW319308 (SEQ ID NO:131); AW239668 (SEQ ID NO:132); AV253136 (SEQ ID NO:133); AW013716 (SEQ ID NO:134); AW013704 (SEQ ID NO:135); AW013702 (SEQ ID NO:136); AW013696 (SEQ ID NO:137); AW013669 (SEQ ID NO:138); AW013647 (SEQ ID NO:139); AW013501 (SEQ ID NO:140); AW013484 (SEQ ID NO:141); AW013428 (SEQ ID NO:142); AW013404 (SEQ ID NO:143); AW013386 (SEQ ID NO:144); AW013284 (SEQ ID NO:145); AW013183 (SEQ ID NO:146); AF061256 (SEQ ID NO:147); AI956572 (SEQ ID NO:148); AI882550 (SEQ ID NO:149); AI822932 (SEQ ID NO:150); AI785988 (SEQ ID NO:151); AI744273 (SEQ ID NO:152); AI727302 (SEQ ID NO:153); AI725714 (SEQ ID NO:154); AF137375 (SEQ ID NO:155); AF137374 (SEQ ID NO:156); AF137373 (SEQ ID NO:157); AF096320 (SEQ ID NO: 158); AF096319 (SEQ ID NO:159); AI663857 (SEQ ID NO:160); AI647841 (SEQ ID NO:161); AI646950 (SEQ ID NO:162); AI607910 (SEQ ID NO:163); AI529173 (SEQ ID NO:164); AI509734 (SEQ ID NO:165); AI506267 (SEQ ID NO:166); AI498269 (SEQ ID NO:167); AI000444 (SEQ ID NO:168); AA956337 (SEQ ID NO:169); AA955042 (SEQ ID NO:170); AA899838 (SEQ ID NO:171); AA899718 (SEQ ID NO:172); AA858756 (SEQ ID NO:173); AI311561 (SEQ ID NO:174); AI385951 (SEQ ID NO:175); AI352406 (SEQ ID NO:176); AF100161 (SEQ ID NO:177); AI326503 (SEQ ID NO:178); AI325517 (SEQ ID NO:179); AI325453 (SEQ ID NO:180); AI325382 (SEQ ID NO:181); AI323700 (SEQ ID NO:182); AI323374 (SEQ ID NO:183); AI313973 (SEQ ID NO:184); AI196928 (SEQ ID NO:185); AF091041 (SEQ ID NO:186); AI156212 (SEQ ID NO:187); AI120374 (SEQ ID NO:188); AI119000 (SEQ ID NO:189); AA408670 (SEQ ID NO:190); AA408072 (SEQ ID NO:191); AA407615 (SEQ ID NO:192); AA995272 (SEQ ID NO:193); C78593 (SEQ ID NO:194); AA999910 (SEQ ID NO:195); AA991491 (SEQ ID NO:196); X99994 (SEQ ID NO:197); X99993 (SEQ ID NO:198); X99992 (SEQ ID NO:199); X99991 (SEQ ID NO:200); X99990 (SEQ ID NO:201); AA958985 (SEQ ID NO:202); AA873222 (SEQ ID NO:203); AA930051 (SEQ ID NO:204); AA895334 (SEQ ID NO:205); AA796142 (SEQ ID NO:206); AA798223 (SEQ ID NO:207); AA734325 (SEQ ID NO:208); AA690871 (SEQ ID NO:209); AA674988 (SEQ ID NO:210); AA674863 (SEQ ID NO:211); AA674821 (SEQ ID NO:212); AA674744 (SEQ ID NO:213); AA671558 (SEQ ID NO:214); AF000381 (SEQ ID NO:215); AF000380 (SEQ ID NO:216); AA637071 (SEQ ID NO:217); AA616314 (SEQ ID NO:218); AA109687 (SEQ ID NO:219); AA608235 (SEQ ID NO:220); AA589050 (SEQ ID NO:221); AA544782 (SEQ ID NO:222); AA522095 (SEQ ID NO:223); AA386821 (SEQ ID NO:224); AA386818 (SEQ ID NO:225); AA386495 (SEQ ID NO:226); AA289278 (SEQ ID NO:227); AA286342 (SEQ ID NO:228); AA276302 (SEQ ID NO:229); AA276123 (SEQ ID NO:230); AA277280 (SEQ ID NO:231); AA273543 (SEQ ID NO:232); U89949 (SEQ ID NO:233); AA208306 (SEQ ID NO:234); AA208089 (SEQ ID NO:235); AA242285 (SEQ ID NO:236); AA139715 (SEQ ID NO:237); AA139709 (SEQ ID NO:238); AA139675 (SEQ ID NO:239); AA139593 (SEQ ID NO:240); AA124010 (SEQ ID NO:241); AA108790 (SEQ ID NO:242); AA108350 (SEQ ID NO:243); AA028831 (SEQ ID NO:244); AA061275 (SEQ ID NO:245); W82933 (SEQ ID NO: 246); AA015571 (SEQ ID NO:247); W71715 (SEQ ID NO:248); W59165 (SEQ ID NO:249); X62753 (SEQ ID NO:250); Z32564 (SEQ ID NO:251); T29279 (SEQ ID NO:252); M25317 (SEQ ID NO:253); M86438 (SEQ ID NO:254); J03922 (SEQ ID NO:255); M64817 (SEQ ID NO:256); L25338 (SEQ ID NO:257); M97701 (SEQ ID NO:258); M97700 (SEQ ID NO:259); M64782 (SEQ ID NO:260); M35069 (SEQ ID NO:261); J05013 (SEQ ID NO:262); M28099 (SEQ ID NO:263); J02876 (SEQ ID NO:264); U08471 (SEQ ID NO:265); U02714 (SEQ ID NO:266); and U02716 (SEQ ID NO:267).

[0094] A skilled artisan also recognizes that the scope of the invention is not limited to the specific nonapeptides described in SEQ ID NO:1 through SEQ ID NO:8. The antigens comprising a FBP epitope may be at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or up to about 30. It is contemplated that any amino acid may be used for additions or filling in for the remainder of sequences in addition to the specific variant sequence provided herein. However, it is preferred that they will be those that will maintain the underlying sequence of FBP.

III. Rational Vaccine Design

[0095] The goal of rational vaccine design is to produce structural analogs of biologically active compounds. By creating such analogs, it is possible to fashion vaccines which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, a skilled artisan generates a three-dimensional structure for the folate binding protein variant of the invention or a fragment thereof. This could be accomplished by X-ray crystallography, computer modeling, or by a combination of both approaches. An alternative approach involves the random replacement of functional groups throughout the folate binding protein variant, and the resulting affect on function is determined.

[0096] It also is possible to isolate a folate binding protein variant specific antibody, selected by a functional assay, and then solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent vaccine design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the vaccine.

[0097] Thus, one may design vaccines which have enhanced and improved biological activity, for example, anti-tumor activity, relative to a starting folate binding protein variant of the invention. By virtue of standard chemical isolation procedures and other descriptions herein, sufficient amounts of the folate binding protein variants of the invention can be produced to perform crystallographic studies. In addition, knowledge of the chemical characteristics of these compounds permits computer-employed predictions of structure-function relationships.

IV. Immunological Reagents

[0098] It is well known in the art that the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, chemokines, cofactors, toxins, plasmodia, synthetic compositions or LEEs or CEEs encoding such adjuvants.

[0099] Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, .gamma.-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated. MHC antigens may even be used. Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

[0100] In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokines such as g-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

[0101] A variety of routes can be used to administer the vaccines including but not limited to subcutaneous, intramuscular, intradermal, intraepidermal, intravenous and intraperitoneal.

[0102] An individual, such as a patient, is injected with vaccine generally as described above. The antigen may be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster administrations with the same vaccine or DNA encoding the same may occur at approximately two-week intervals.

V. Immunotherapeutic Agents

[0103] An immunotherapeutic agent generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, a folate binding protein variant which is or is similar to a tumor cell antigen. The variant alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The variant also may be conjugated to a drug or toxin (e.g. a chemotherapeutic, a radionuclide, a ricin A chain, a cholera toxin, a pertussis toxin, etc.) and serve merely as a targeting agent. Such antibody conjugates are called immunotoxins, and are well known in the art (see U.S. Pat. No. 5,686,072, U.S. Pat. No. 5,578,706, U.S. Pat. No. 4,792,447, U.S. Pat. No. 5,045,451, U.S. Pat. No. 4,664,911, and U.S. Pat. No. 5,767,072, each incorporated herein by reference). Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0104] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist in addition to folate binding protein described herein, and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.

[0105] The disclosures presented herein have significant relevance to immunotherapy of human diseases and disorders, including cancer. In using the immunotherapeutic compositions derived from the antigen of the present invention in treatment methods, other standard treatments also may be employed, such as radiotherapy or chemotherapy. However, it is preferred that the immunotherapy be used alone initially as its effectiveness can be readily assessed. Immunotherapies of cancer can broadly be classified as adoptive, passive and active, as described in the following sections, and may be used or produced with the folate binding protein variant antigen of the present invention.

[0106] A. Immune Stimulators

[0107] A specific aspect of immunotherapy is to use an immune stimulating molecule as an agent, or more preferably in conjunction with another agent, such as, for example, a cytokine such as IL-2, IL-4, IL-12, GM-CSF, tumor necrosis factor; interferons alpha, beta, and gamma; F42K and other cytokine analogs; a chemokine such as, for example, MIP-1, MIP-lbeta, MCP-1, RANTES, IL-8; or a growth factor such as, for example, FLT3 ligand.

[0108] One particular cytokine contemplated for use in the present invention is tumor necrosis factor. Tumor necrosis factor (TNF; Cachectin) is a glycoprotein that kills some kinds of cancer cells, activates cytokine production, activates macrophages and endothelial cells, promotes the production of collagen and collagenases, is an inflammatory mediator and also a mediator of septic shock, and promotes catabolism, fever and sleep. Some infectious agents cause tumor regression through the stimulation of TNF production. TNF can be quite toxic when used alone in effective doses, so that the optimal regimens probably will use it in lower doses in combination with other drugs. Its immunosuppressive actions are potentiated by gamma-interferon, so that the combination potentially is dangerous. A hybrid of TNF and interferon-a also has been found to possess anti-cancer activity.

[0109] Another cytokine specifically contemplate is interferon alpha. Interferon alpha has been used in treatment of hairy cell leukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cell cancer, ovary cancer, bladder cancer, non-Hodgkin's lymphomas, mycosis fungoides, multiple myeloma, and chronic granulocytic leukemia.

[0110] B. Passive Immunotherapy

[0111] A number of different approaches for passive immunotherapy of cancer exist. They may be broadly categorized into the following: injection of vaccine alone; injection of vaccine coupled to toxins or chemotherapeutic agents; injection of vaccine coupled to radioactive isotopes; injection of anti-idiotype vaccine; and finally, purging of tumor cells in bone marrow.

[0112] It may be favorable to administer more than one vaccine associated with two different antigens or even vaccine with multiple antigen specificity. Treatment protocols also may include administration of lymphokines or other immune enhancers (Bajorin et al. 1988).

[0113] C. Active Immunotherapy

[0114] In some embodiments of the invention, active immunotherapy may be employed. In active immunotherapy, a folate binding protein variant (e.g, a peptide or polypeptide), a nucleic acid encoding a folate binding protein variant, and/or additional vaccine components, such as for example, a cell expressing the folate binding protein variant (e.g a dendritic cell fused with a tumor cell, or an autologous or allogeneic tumor cell composition expressing the antigen), an adjuvant, a recombinant protein, an immunomodulator, and the like is administered (Ravindranath and Morton, 1991; Morton and Ravindranath, 1996; Morton et al., 1992; Okamoto et al., 1997; Kugler et al., 2000; Trefzer et al., 2000; Mitchell et al., 1990; Mitchell et al., 1993).

[0115] An antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine" is administered generally with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton and Ravindranath, 1996; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993). In melanoma immunotherapy, those patients who elicit high IgM response often survive better than those who elicit no or low IgM antibodies (Morton et al., 1992). IgM antibodies are often transient antibodies and the exception to the rule appears to be anti-ganglioside or anti-carbohydrate antibodies.

[0116] D. Adoptive Immunotherapy

[0117] In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al., 1988; 1989). To achieve this, one would administer to an animal, or human patient, an immunologically effective amount of activated lymphocytes in combination with an adjuvant-incorporated antigenic peptide composition as described herein. The activated lymphocytes will most preferably be the patient's own cells that were earlier isolated from a blood or tumor sample and activated (or "expanded") in vitro. In certain embodiments, the patient's lymphocytes are cultured or expanded in number or selected for activity, such as immunoreactivity to the antigen. This form of immunotherapy has produced several cases of regression of melanoma and renal carcinoma.

VI. Vaccines

[0118] The present invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared most readily directly from immunogenic CTL-stimulating peptides prepared in a manner disclosed herein. Preferably the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.

[0119] The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables. Either as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.

[0120] Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10 to about 95% of active ingredient, preferably about 25 to about 70%.

[0121] The peptides of the present invention may be formulated into the vaccine as neutral or salt forms. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0122] The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g, the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.

[0123] The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.

[0124] Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol.RTM.) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 700 to about 101.degree. C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA.RTM.) used as a block substitute may also be employed.

[0125] In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.

[0126] For an antigenic composition to be useful as a vaccine, an antigenic composition must induce an immune response to the antigen in a cell, tissue or animal (e.g, a human). As used herein, an "antigenic composition" may comprise an antigen (e.g, a peptide or polypeptide), a nucleic acid encoding an antigen (e.g, an antigen expression vector), or a cell expressing or presenting an antigen. In particular embodiments, the antigenic composition comprises or encodes a folate binding protein variant, or an immunologically functional equivalent thereof. In other embodiments, the antigenic composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination. In certain embodiments, the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.

[0127] In certain embodiments, an antigenic composition or immunologically functional equivalent, may be used as an effective vaccine in inducing an anti-folate binding protein variant humoral and/or cell-mediated immune response in an animal. The present invention contemplates one or more antigenic compositions or vaccines for use in both active and passive immunization embodiments.

[0128] A vaccine of the present invention may vary in its composition of proteinaceous, nucleic acid and/or cellular components. In a non-limiting example, a nucleic acid encoding an antigen might also be formulated with a proteinaceous adjuvant. Of course, it will be understood that various compositions described herein may further comprise additional components. For example, one or more vaccine components may be comprised in a lipid or liposome. In another non-limiting example, a vaccine may comprise one or more adjuvants. A vaccine of the present invention, and its various components, may be prepared and/or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

[0129] A. Proteinaceous Antigens

[0130] It is understood that an antigenic composition of the present invention may be made by a method that is well known in the art, including but not limited to chemical synthesis by solid phase synthesis and purification away from the other products of the chemical reactions by HPLC, or production by the expression of a nucleic acid sequence (e.g, a DNA sequence) encoding a peptide or polypeptide comprising an antigen of the present invention in an in vitro translation system or in a living cell. Preferably the antigenic composition is isolated and extensively dialyzed to remove one or more undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle. It is further understood that additional amino acids, mutations, chemical modification and the like, if any, that are made in a vaccine component will preferably not substantially interfere with the antibody recognition of the epitopic sequence.

[0131] A peptide or polypeptide corresponding to one or more antigenic determinants of the folate binding protein variant of the present invention should generally be at least five or six amino acid residues in length, and may contain up to about 10, about 15, about 20, or more. A peptide sequence may be synthesized by methods known to those of ordinary skill in the art, for example, peptide synthesis using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, Calif.).

[0132] Longer peptides or polypeptides also may be prepared, e.g, by recombinant means. In certain embodiments, a nucleic acid encoding an antigenic composition and/or a component described herein may be used, for example, to produce an antigenic composition in vitro or in vivo for the various compositions and methods of the present invention. For example, in certain embodiments, a nucleic acid encoding an antigen is comprised in, for example, a vector in a recombinant cell. The nucleic acid may be expressed to produce a peptide or polypeptide comprising an antigenic sequence. The peptide or polypeptide may be secreted from the cell, or comprised as part of or within the cell.

[0133] B. Genetic Vaccine Antigens

[0134] In certain embodiments, an immune response may be promoted by transfecting or inoculating an animal with a nucleic acid encoding an antigen. One or more cells comprised within a target animal then expresses the sequences encoded by the nucleic acid after administration of the nucleic acid to the animal. Thus, the vaccine may comprise "genetic vaccine" useful for immunization protocols. A vaccine may also be in the form, for example, of a nucleic acid (e.g, a cDNA or an RNA) encoding all or part of the peptide or polypeptide sequence of an antigen. Expression in vivo by the nucleic acid may be, for example, by a plasmid type vector, a viral vector, or a viral/plasmid construct vector.

[0135] In preferred aspects, the nucleic acid comprises a coding region that encodes all or part of the sequences disclosed as SEQ ID NO:1 through SEQ ID NO:9, or an immunologically functional equivalent thereof. Of course, the nucleic acid may comprise and/or encode additional sequences, including but not limited to those comprising one or more immunomodulators or adjuvants. The nucleotide and protein, polypeptide and peptide encoding sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/). The coding regions for these known genes may be amplified, combined with the nucleic acid sequences encoding the folate binding protein variant disclosed herein (e.g, ligated) and/or expressed using the techniques disclosed herein or by any technique that would be know to those of ordinary skill in the art (e.g, Sambrook et al., 1987). Though a nucleic acid may be expressed in an in vitro expression system, in preferred embodiments the nucleic acid comprises a vector for in vivo replication and/or expression.

[0136] C. Cellular Vaccine Antigens

[0137] In another embodiment, a cell expressing the antigen may comprise the vaccine. The cell may be isolated from a culture, tissue, organ or organism and administered to an animal as a cellular vaccine. Thus, the present invention contemplates a "cellular vaccine." The cell may be transfected with a nucleic acid encoding an antigen to enhance its expression of the antigen. Of course, the cell may also express one or more additional vaccine components, such as immunomodulators or adjuvants. A vaccine may comprise all or part of the cell.

[0138] D. Immunologically Functional Equivalents

[0139] Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:

TABLE-US-00001 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGO AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0140] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode the peptides without appreciable loss of their biological utility or activity. Amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art.

[0141] Numerous scientific publications have also been devoted to the prediction of secondary structure, and to the identification of an epitope, from analyses of an amino acid sequence (Chou and Fasman, 1974a,b; 1978a,b, 1979). Any of these may be used, if desired, to supplement the teachings of U.S. Pat. No. 4,554,101.

[0142] Moreover, computer programs are currently available to assist with predicting an antigenic portion and an epitopic core region of one or more proteins, polypeptides or peptides. Examples include those programs based upon the Jameson-Wolf analysis (Jameson & Wolf, 1988; Wolf et al., 1988), the program PepPlot.RTM. (Brutlag et al., 1990; Weinberger et al., 1985), and other new programs for protein tertiary structure prediction (Fetrow & Bryant, 1993). Another commercially available software program capable of carrying out such analyses is MacVector (IBI, New Haven, Conn.).

[0143] As modifications and changes may be made in the structure of an antigenic composition (e.g, a folate binding protein variant) of the present invention, and still obtain molecules having like or otherwise desirable characteristics, such immunologically functional equivalents are also encompassed within the present invention.

[0144] For example, certain amino acids may be substituted for other amino acids in a peptide, polypeptide or protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules or receptors, DNA binding sites, or such like. Since it is the interactive capacity and nature of a peptide, polypeptide or protein that defines its biological (e.g, immunological) functional activity, certain amino acid sequence substitutions can be made in a amino acid sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a peptide or polypeptide with like (agonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of an antigenic composition such as, for example a folate binding protein variant peptide or polypeptide, or underlying DNA, without appreciable loss of biological utility or activity.

[0145] Accordingly, antigenic composition, particularly an immunologically functional equivalent of the sequences disclosed herein, may encompass an amino molecule sequence comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unnatural amino acid, including but not limited to those shown on Table 2 below.

TABLE-US-00002 TABLE 2 Modified, Unnatural or Rare Amino Acids Abbr. Amino Add Abbr. Amino Add Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3- Aminoadipic acid Hyl Hydroxylysine Ba1a .beta.-alanine, b-Amino-propionic acid Ahyl Allo-Hydroxylysine Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid Aile Allo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine, sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2'-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGly N-Ethylglycine

[0146] In terms of immunologically functional equivalent, it is well understood by the skilled artisan that, inherent in the definition is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent immunological activity. An immunologically functional equivalent peptide or polypeptide are thus defined herein as those peptide(s) or polypeptide(s) in which certain, not most or all, of the amino acid(s) may be substituted.

[0147] In particular, where a shorter length peptide is concerned, it is contemplated that fewer amino acid substitutions should be made within the given peptide. A longer polypeptide may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. Of course, a plurality of distinct polypeptides/peptides with different substitutions may easily be made and used in accordance with the invention.

[0148] It also is well understood that where certain residues are shown to be particularly important to the immunological or structural properties of a protein or peptide, e.g, residues in binding regions or active sites, such residues may not generally be exchanged. This is an important consideration in the present invention, where changes in the folate binding protein variant antigenic site should be carefully considered and subsequently tested to ensure maintenance of immunological function (e.g, antigenicity), where maintenance of immunological function is desired. In this manner, functional equivalents are defined herein as those peptides or polypeptides which maintain a substantial amount of their native immunological activity.

[0149] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Careful selection of a particular amino acid substitution for a peptide, as opposed to a protein, must be considered given the differences in size between peptides and proteins.

[0150] In further embodiments, major antigenic determinants of a peptide or polypeptide may be identified by an empirical approach in which portions of a nucleic acid encoding a peptide or polypeptide are expressed in a recombinant host, and the resulting peptide(s) or polypeptide(s) tested for their ability to elicit an immune response. For example, PCR.TM. can be used to prepare a range of peptides or polypeptides lacking successively longer fragments of the C-terminus of the amino acid sequence. The immunoactivity of each of these peptides or polypeptides is determined to identify those fragments or domains that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinant(s) of the peptide or polypeptide to be more precisely determined.

[0151] Another method for determining a major antigenic determinant of a peptide or polypeptide is the SPOTs.TM. system (Genosys Biotechnologies, Inc., The Woodlands, Tex.). In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody. An antigenic determinant of the peptides or polypeptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive sequence.

[0152] Once one or more such analyses are completed, an antigenic composition, such as for example a peptide or a polypeptide is prepared that contain at least the essential features of one or more antigenic determinants. An antigenic composition is then employed in the generation of antisera against the composition, and preferably the antigenic determinant(s).

[0153] While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. Nucleic acids encoding these antigenic compositions also can be constructed and inserted into one or more expression vectors by standard methods (Sambrook et al., 1987), for example, using PCR.TM. cloning methodology.

[0154] In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide or polypeptide structure or to interact specifically with, for example, an antibody. Such compounds, which may be termed peptidomimetics, may be used in the same manner as a peptide or polypeptide of the invention and hence are also immunologically functional equivalents.

[0155] Certain mimetics that mimic elements of protein secondary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orientate amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.

[0156] E. Antigen Mutagenesis

[0157] In particular embodiments, an antigenic composition is mutated for purposes such as, for example, enhancing its immunogenicity or producing or identifying an immunologically functional equivalent sequence. Methods of mutagenesis are well known to those of skill in the art (Sambrook et al., 1987).

[0158] As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No. 4,237,224, specifically incorporated herein by reference in its entirety.

[0159] In a preferred embodiment, site directed mutagenesis is used. Site-specific mutagenesis is a technique useful in the preparation of an antigenic composition (e.g. a folate binding protein variant-comprising peptide or polypeptide, or immunologically functional equivalent protein, polypeptide or peptide), through specific mutagenesis of the underlying DNA. In general, the technique of site-specific mutagenesis is well known in the art. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of a mutant through the use of specific oligonucleotide sequence(s) which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the position being mutated. Typically, a primer of about 17 to about 75 nucleotides in length is preferred, with about 10 to about 25 or more residues on both sides of the position being altered, while primers of about 17 to about 25 nucleotides in length being more preferred, with about 5 to 10 residues on both sides of the position being altered.

[0160] In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. As will be appreciated by one of ordinary skill in the art, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

[0161] This mutagenic primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as, for example, E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

[0162] Alternatively, a pair of primers may be annealed to two separate strands of a double stranded vector to simultaneously synthesize both corresponding complementary strands with the desired mutation(s) in a PCR.TM. reaction. A genetic selection scheme to enrich for clones incorporating the mutagenic oligonucleotide has been devised (Kunkel et al., 1987). Alternatively, the use of PCR.TM. with commercially available thermostable enzymes such as Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector (Tomic et al., 1990; Upender et al., 1995). A PCR.TM. employing a thermostable ligase in addition to a thermostable polymerase also may be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector (Michael 1994).

[0163] The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

[0164] Additionally, one particularly useful mutagenesis technique is alanine scanning mutagenesis in which a number of residues are substituted individually with the amino acid alanine so that the effects of losing side-chain interactions can be determined, while minimizing the risk of large-scale perturbations in protein conformation (Cunningham et al., 1989).

[0165] F. Vectors

[0166] In order to effect replication, expression or mutagenesis of a nucleic acid, the nucleic acid may be delivered ("transfected") into a cell. The transfection of cells may be used, in certain embodiments, to recombinately produce one or more vaccine components for subsequent purification and preparation into a pharmaceutical vaccine. In other embodiments, the nucleic acid may be comprised as a genetic vaccine that is administered to an animal. In other embodiments, the nucleic acid is transfected into a cell and the cell administered to an animal as a cellular vaccine component. The nucleic acid may consist only of naked recombinant DNA, or may comprise, for example, additional materials to protect the nucleic acid and/or aid its targeting to specific cell types.

[0167] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g, YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0168] The term "expression vector" refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell.

[0169] The nucleic acid encoding the antigenic composition or other vaccine component may be stably integrated into the genome of the cell, or may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. Vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

[0170] 1. Promoters and Enhancers

[0171] A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.

[0172] A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control of" a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0173] The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0174] A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the .beta.-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR.TM., in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0175] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 1989, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[0176] Additionally any promoter/enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0177] Table 3 lists non-limiting examples of elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a RNA. Table 4 provides non-limiting examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.

TABLE-US-00003 TABLE 3 Promoter and/or Enhancer Promoter/Enhancer References Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984: Kiledjian et al., 1988; Porton et al.; 1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984 T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.; 1990 HLA DQ a and/or DQ .beta. Sullivan et al., 1987 .beta.-Interferon Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Sherman et al., 1989 .beta.-Actin Kawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine Kinase (MCK) Jaynes et al., 1988: Horlick et al., 1989; Johnson et al. 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Omitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culotta et al., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin Pinkert et al., 1987; Tronche et al., 1989, 1990 .alpha.-Fetoprotein Godbout et al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987; Perez-Stable et al., 1990 .beta.-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM) .alpha..sub.1-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al., 1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981: Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988: Campbell and/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie, 1983: Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987; Glue et al., 1988 Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989

TABLE-US-00004 TABLE 4 Inducible Elements Element Inducer References MT II Phorbol Ester (TFA) Palmiter et al., 1982; Haslinger et Heavy metals al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammary Glucocorticoids Huang et al., 1981; Lee et al., tumor virus) 1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988 .beta.-Interferon poly(rI)x Tavernier et al., 1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 Gene A23187 Resendez et al., 1988 .alpha.-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2.kappa.b Interferon Blanar et al., 1989 HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a, 1990b Antigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 Tumor Necrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating Thyroid Hormone Chatterjee et al., 1989 Hormone .alpha. Gene

[0178] The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Nonlimiting examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), and human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).

[0179] 2. Initiation Signals and Internal Ribosome Binding Sites

[0180] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0181] In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

[0182] 3. Multiple Cloning Sites

[0183] Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see, for example, Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference.) "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

[0184] 4. Splicing Sites

[0185] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler et al., 1997, herein incorporated by reference.)

[0186] 5. Termination Signals

[0187] The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

[0188] In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0189] Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

[0190] 6. Polyadenylation Signals

[0191] In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

[0192] 7. Origins of Replication

[0193] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

[0194] 8. Selectable and Screenable Markers

[0195] In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0196] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

[0197] 9. Plasmid Vectors

[0198] In certain embodiments, a plasmid vector is contemplated for use to transform a host cell. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.

[0199] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEMTM.lamda.11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.

[0200] Further useful plasmid vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with .beta.-galactosidase, ubiquitin, and the like.

[0201] Bacterial host cells, for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g, by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.

[0202] 10. Viral Vectors

[0203] The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g, mammalian cells). Vaccine components of the present invention may be a viral vector that encode one or more folate binding protein variant antigenic compositions or other components such as, for example, a folate binding protein variant immunomodulator or adjuvant. Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of the present invention are described below.

[0204] a. Adenoviral Vectors

[0205] A particular method for delivery of the nucleic acid involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

[0206] b. AAV Vectors

[0207] The nucleic acid may be introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for use in the folate binding protein variant vaccines of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by reference.

[0208] c. Retroviral Vectors

[0209] Retroviruses have promise as folate binding protein variant antigen delivery vectors in vaccines due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines (Miller, 1992).

[0210] In order to construct a folate binding protein variant vaccine retroviral vector, a nucleic acid (e.g, one encoding an folate binding protein variant antigen of interest) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g, by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0211] Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.

[0212] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.

[0213] d. Other Viral Vectors

[0214] Other viral vectors may be employed as vaccine constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0215] e. Vaccine Delivery Using Modified Viruses

[0216] A nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

[0217] Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989). Thus, it is contemplated that antibodies, specific binding ligands and/or other targeting moieties may be used to specifically transfect APC types.

[0218] 11. Vector Delivery and Cell Transformation

[0219] Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g, DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

[0220] a. Injection

[0221] In certain embodiments, a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection). Methods of injection of nucleic acids are described herein, and are well known to those of ordinary skill in the art. Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection to a cell. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985). The amount of folate binding protein variant used may vary upon the nature of the antigen as well as the organelle, cell, tissue or organism used

[0222] b. Electroporation

[0223] In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384,253, incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.

[0224] Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.

[0225] To effect transformation by electroporation in cells such as, for example, plant cells, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Examples of some species which have been transformed by electroporation of intact cells include maize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987) and tobacco (Lee et al., 1989).

[0226] One also may employ protoplasts for electroporation transformation of plant cells (Bates, 1994; Lazzeri, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application No. WO 9217598, incorporated herein by reference. Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato (Tsukada, 1989).

[0227] c. Calcium Phosphate

[0228] In other embodiments of the present invention, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).

[0229] d. DEAE-Dextran

[0230] In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

[0231] e. Liposome-Mediated Transfection

[0232] In a further embodiment of the invention, one or more vaccine components or nucleic acids may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).

[0233] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).

[0234] In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.

[0235] f. Receptor Mediated Transfection

[0236] One or more vaccine components or nucleic acids, may be employed to delivered using a receptor-mediated delivery vehicle. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in the target cells. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993, incorporated herein by reference).

[0237] Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated herein by reference). In certain aspects of the present invention, a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.

[0238] In other embodiments, a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.

[0239] In still further embodiments, the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner.

[0240] g. Microprojectile Bombardment

[0241] Microprojectile bombardment techniques can be used to introduce a nucleic acid into at least one, organelle, cell, tissue or organism (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and PCT Application WO 94/09699; each of which is incorporated herein by reference). This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). There are a wide variety of microprojectile bombardment techniques known in the art, many of which are applicable to the invention.

[0242] Microprojectile bombardment may be used to transform various cell(s), tissue(s) or organism(s), such as for example any plant species. Examples of species which have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95/06128), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055, incorporated herein by reference), rice (Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum (Casas et al., 1993; Hagio et al., 1991); as well as a number of dicots including tobacco (Tomes et al., 1990; Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783, incorporated herein by reference), sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumes in general (U.S. Pat. No. 5,563,055, incorporated herein by reference).

[0243] In this microprojectile bombardment, one or more particles may be coated with at least one nucleic acid and delivered into cells by a propelling force. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold particles or beads. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.

[0244] For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

[0245] 12. Host Cells

[0246] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced nucleic acid.

[0247] In certain embodiments, it is contemplated that RNAs or proteinaceous sequences may be co-expressed with other selected RNAs or proteinaceous sequences in the same host cell. Co-expression may be achieved by co-transfecting the host cell with two or more distinct recombinant vectors. Alternatively, a single recombinant vector may be constructed to include multiple distinct coding regions for RNAs, which could then be expressed in host cells transfected with the single vector.

[0248] A tissue may comprise a host cell or cells to be transformed with a folate binding protein variant. The tissue may be part or separated from an organism. In certain embodiments, a tissue may comprise, but is not limited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood (e.g, lymphocytes), blood vessel, bone, bone marrow, brain, breast, cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial, esophagus, facia, fibroblast, follicular, ganglion cells, glial cells, goblet cells, kidney, liver, lung, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stem cells, stomach, testes, anthers, ascite tissue, cobs, ears, flowers, husks, kernels, leaves, meristematic cells, pollen, root tips, roots, silk, stalks, and all cancers thereof.

[0249] In certain embodiments, the host cell or tissue may be comprised in at least one organism. In certain embodiments, the organism may be, but is not limited to, a prokaryote (e.g, a eubacteria, an archaea) or an eukaryote, as would be understood by one of ordinary skill in the art (see, for example, webpage http://phylogeny.arizona.edu/tree/phylogeny.html).

[0250] Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Cell types available for vector replication and/or expression include, but are not limited to, bacteria, such as E. coli (e.g, E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F', lambda, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas specie, DH5a, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE.RTM. Competent Cells and SOLOPACKa Gold Cells (STRATAGENE.RTM., La Jolla). In certain embodiments, bacterial cells such as E. coli LE392 are particularly contemplated as host cells for phage viruses.

[0251] Examples of eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

[0252] Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0253] 13. Expression Systems

[0254] Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

[0255] The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC.RTM. 2.0 from INVITROGEN.RTM. and BACPACK.TM. BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH.RTM..

[0256] Other examples of expression systems include STRATAGENE.RTM.'s COMPLETE CONTROLa Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN.RTM., which carries the T-REX.TM. (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN.RTM. also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0257] It is contemplated that the proteins, polypeptides or peptides produced by the methods of the invention may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in cells. Such overexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein, polypeptide or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific protein, polypeptides or peptides in relation to the other proteins produced by the host cell and, e.g, visible on a gel.

[0258] In some embodiments, the expressed proteinaceous sequence forms an inclusion body in the host cell, the host cells are lysed, for example, by disruption in a cell homogenizer, washed and/or centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars, such as sucrose, into the buffer and centrifugation at a selective speed. Inclusion bodies may be solubilized in solutions containing high concentrations of urea (e.g 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents, such as .beta.-mercaptoethanol or DTT (dithiothreitol), and refolded into a more desirable conformation, as would be known to one of ordinary skill in the art.

[0259] G. Vaccine Component Purification

[0260] In any case, a vaccine component (e.g. an antigenic peptide or polypeptide or nucleic acid encoding a proteinaceous composition) may be isolated and/or purified from the chemical synthesis reagents, cell or cellular components. In a method of producing the vaccine component, purification is accomplished by any appropriate technique that is described herein or well known to those of skill in the art (e.g, Sambrook et al., 1987). Although preferred for use in certain embodiments, there is no general requirement that an antigenic composition of the present invention or other vaccine component always be provided in their most purified state. Indeed, it is contemplated that a less substantially purified vaccine component, which is nonetheless enriched in the desired compound, relative to the natural state, will have utility in certain embodiments, such as, for example, total recovery of protein product, or in maintaining the activity of an expressed protein. However, it is contemplate that inactive products also have utility in certain embodiments, such as, e.g. in determining antigenicity via antibody generation.

[0261] The present invention also provides purified, and in preferred embodiments, substantially purified vaccines or vaccine components. The term "purified vaccine component" as used herein, is intended to refer to at least one vaccine component (e.g, a proteinaceous composition, isolatable from cells), wherein the component is purified to any degree relative to its naturally-obtainable state, e.g, relative to its purity within a cellular extract or reagents of chemical synthesis. In certain aspects wherein the vaccine component is a proteinaceous composition, a purified vaccine component also refers to a wild-type or mutant protein, polypeptide, or peptide free from the environment in which it naturally occurs.

[0262] Where the term "substantially purified" is used, this will refer to a composition in which the specific compound (e.g, a protein, polypeptide, or peptide) forms the major component of the composition, such as constituting about 50% of the compounds in the composition or more. In preferred embodiments, a substantially purified vaccine component will constitute more than about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or even more of the compounds in the composition.

[0263] In certain embodiments, a vaccine component may be purified to homogeneity. As applied to the present invention, "purified to homogeneity," means that the vaccine component has a level of purity where the compound is substantially free from other chemicals, biomolecules or cells. For example, a purified peptide, polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully. Various methods for quantifying the degree of purification of a vaccine component will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific protein activity of a fraction (e.g, antigenicity), or assessing the number of polypeptides within a fraction by gel electrophoresis.

[0264] Various techniques suitable for use in chemical, biomolecule or biological purification, well known to those of skill in the art, may be applicable to preparation of a vaccine component of the present invention. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; fractionation, chromatographic procedures, including but not limited to, partition chromatograph (e.g, paper chromatograph, thin-layer chromatograph (TLC), gas-liquid chromatography and gel chromatography) gas chromatography, high performance liquid chromatography, affinity chromatography, supercritical flow chromatography ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity; isoelectric focusing and gel electrophoresis (see for example, Sambrook et al. 1989; and Freifelder, Physical Biochemistry, Second Edition, pages 238-246, incorporated herein by reference).

[0265] Given many DNA and proteins are known (see for example, the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/)), or may be identified and amplified using the methods described herein, any purification method for recombinately expressed nucleic acid or proteinaceous sequences known to those of skill in the art can now be employed. In certain aspects, a nucleic acid may be purified on polyacrylamide gels, and/or cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al. 1989, incorporated herein by reference). In further aspects, a purification of a proteinaceous sequence may be conducted by recombinately expressing the sequence as a fusion protein. Such purification methods are routine in the art. This is exemplified by the generation of an specific protein-glutathione S-transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N- or C-terminus of the protein, and subsequent purification using Ni-affinity chromatography. In particular aspects, cells or other components of the vaccine may be purified by flow cytometry. Flow cytometry involves the separation of cells or other particles in a liquid sample, and is well known in the art (see, for example, U.S. Pat. Nos. 3,826,364, 4,284,412, 4,989,977, 4,498,766, 5,478,722, 4,857,451, 4,774,189, 4,767,206, 4,714,682, 5,160,974 and 4,661,913). Any of these techniques described herein, and combinations of these and any other techniques known to skilled artisans, may be used to purify and/or assay the purity of the various chemicals, proteinaceous compounds, nucleic acids, cellular materials and/or cells that may comprise a vaccine of the present invention. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified antigen or other vaccine component.

[0266] H. Additional Vaccine Components

[0267] It is contemplated that an antigenic composition of the invention may be combined with one or more additional components to form a more effective vaccine. Non-limiting examples of additional components include, for example, one or more additional antigens, immunomodulators or adjuvants to stimulate an immune response to an antigenic composition of the present invention and/or the additional component(s).

[0268] 1. Immunomodulators

[0269] For example, it is contemplated that immunomodulators can be included in the vaccine to augment a cell's or a patient's (e.g, an animal's) response. Immunomodulators can be included as purified proteins, nucleic acids encoding immunomodulators, and/or cells that express immunomodulators in the vaccine composition. The following sections list non-limiting examples of immunomodulators that are of interest, and it is contemplated that various combinations of immunomodulators may be used in certain embodiments (e.g, a cytokine and a chemokine).

[0270] In another aspects of the invention, it is contemplated that the folate binding protein variant composition may further comprise a therapeutically effective composition of an immunomodulator. It is envisioned that an immunomodulator would constitute a cytokine, hematapoietin, colony stimulating factor, interleukin, interferon, growth factor or combination thereof. As used herein certain embodiments, the terms "cytokine" are the same as described in U.S. Pat. No. 5,851,984, incorporated herein by reference in its entirety, which reads in relevant part:

[0271] "The term `cytokine` is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, growth factors and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor, prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-.alpha. and -.beta.; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor, integrin; thrombopoietin (TPO); nerve growth factors such as NGF-.beta.; platelet-growth factor, transforming growth factors (TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -.b, and -g; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0272] a. .beta.-Interferon

[0273] .beta.-interferon (IFN-b) is low molecular weight protein that is produced by many cell types, including epithelial cells, fibroblasts and macrophages. Cells that express endogenous IFN-b are resistant to viral infection and replication. The b-interferon genes from mouse (GenBank accession numbers X14455, X14029) and human (GenBank accession numbers J00218, K00616 and M11029) have been isolated and sequenced. IFN-b is a multifunctional glycoprotein that can inhibit tumor growth both directly, by suppressing cell replication and inducing differentiation or apoptosis and indirectly by activating tumoricidal properties of macrophages and NK cells, by suppressing tumor angiogenesis and by stimulating specific immune response.

[0274] b. Interleukin-2

[0275] Interleukin-2 (IL-2), originally designated T-cell growth factor I, is a highly proficient inducer of T-cell proliferation and is a growth factor for all subpopulations of T-lymphocytes. IL-2 is an antigen independent proliferation factor that induces cell cycle progression in resting cells and thus allows clonal expansion of activated T-lymphocytes. Since freshly Isolated leukemic cells also secrete IL2 and respond to it IL2 may function as an autocrine growth modulator for these cells capable of worsening ATL. IL2 also promotes the proliferation of activated B-cells although this requires the presence of additional factors, for example, IL4. In vitro IL2 also stimulates the growth of oligodendroglial cells. Due to its effects on T-cells and B-cells IL2 is a central regulator of immune responses. It also plays a role in anti-inflammatory reactions, in hematopoiesis and in tumor surveillance. IL-2 stimulates the synthesis of IFN-g in peripheral leukocytes and also induces the secretion of IL-1, TNF-a and TNF-b. The induction of the secretion of tumoricidal cytokines, apart from the activity in the expansion of LAK cells, (lymphokine-activated killer cells) are probably the main factors responsible for the antitumor activity of IL2.

[0276] c. GM-CSF

[0277] GM-CSF stimulates the proliferation and differentiation of neutrophilic, eosinophilic, and monocytic lineages. It also functionally activates the corresponding mature forms, enhancing, for example, to the expression of certain cell surface adhesion proteins (CD-11A, CD-11C). The overexpression of these proteins could be one explanation for the observed local accumulation of granulocytes at sites of inflammation. In addition, GM-CSF also enhances expression of receptors for fMLP (Formyl-Met-Leu-Phe) which is a stimulator of neutrophil activity.

[0278] d. Cytokines

[0279] Interleukins, cytokines, nucleic acids encoding interleukins or cytokines, and/or cells expressing such compounds are contemplated as possible vaccine components. Interleukins and cytokines, include but are not limited to interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18, .beta.-interferon, .alpha.-interferon, .gamma.-interferon, angiostatin, thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH-1, METH-2, tumor necrosis factor, TGFb, LT and combinations thereof.

[0280] e. Chemokines

[0281] Chemokines, nucleic acids that encode for chemokines, and/or cells that express such also may be used as vaccine components. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine coding sequence in combination with, for example, a cytokine coding sequence, to enhance the recruitment of other immune system components to the site of treatment. Such chemokines include, for example, RANTES, MCAF, MIP1-alpha, MIP1-Beta, IP-10 and combinations thereof. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.

[0282] f. Immunogenic Carrier Proteins

[0283] In certain embodiments, an antigenic composition's may be chemically coupled to a carrier or recombinantly expressed with a immunogenic carrier peptide or polypetide (e.g, a antigen-carrier fusion peptide or polypeptide) to enhance an immune reaction. Exemplary and preferred immunogenic carrier amino acid sequences include hepatitis B surface antigen, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as immunogenic carrier proteins. Means for conjugating a polypeptide or peptide to a immunogenic carrier protein are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

[0284] g. Biological Response Modifiers

[0285] It may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), or a gene encoding a protein involved in one or more immune helper functions, such as B-7.

[0286] 2. Adjuvants

[0287] Immunization protocols have used adjuvants to stimulate responses for many years, and as such adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation.

[0288] In one aspect, an adjuvant effect is achieved by use of an agent such as alum used in about 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively, the antigen is made as an admixture with synthetic polymers of sugars (Carbopol.RTM.) used as an about 0.25% solution. Adjuvant effect may also be made my aggregation of the antigen in the vaccine by heat treatment with temperatures ranging between about 70.degree. to about 101.degree. C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cell(s) such as C. parvum or an endotoxin or a lipopolysaccharide components of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA.RTM.) used as a block substitute also may be employed.

[0289] Some adjuvants, for example, are certain organic molecules obtained from bacteria, act on the host rather than on the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. The effects of MDP, as with most adjuvants, are not fully understood. MDP stimulates macrophages but also appears to stimulate B cells directly. The effects of adjuvants, therefore, are not antigen-specific. If they are administered together with a purified antigen, however, they can be used to selectively promote the response to the antigen.

[0290] Adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g, U.S. Pat. No. 4,877,611). This has been attempted particularly in the treatment of cancer. For many cancers, there is compelling evidence that the immune system participates in host defense against the tumor cells, but only a fraction of the likely total number of tumor-specific antigens are believed to have been identified to date. However, using the present invention, the inclusion of a suitable adjuvant into the membrane of an irradiated tumor cell will likely increase the anti-tumor response irrespective of the molecular identification of the prominent antigens. This is a particularly important and time-saving feature of the invention.

[0291] In certain embodiments, hemocyanins and hemoerythrins may also be used in the invention. The use of hemocyanin from keyhole limpet (KLH) is preferred in certain embodiments, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed.

[0292] Various polysaccharide adjuvants may also be used. For example, the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et al., 1989). The doses that produce optimal responses, or that otherwise do not produce suppression, should be employed as indicated (Yin et al., 1989). Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.

[0293] Another group of adjuvants are the muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterial peptidoglycans. Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated.

[0294] U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide which is described for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. It is the to be effective in activating human monocytes and destroying tumor cells, but is non-toxic in generally high doses. The compounds of U.S. Pat. No. 4,950,645 and PCT Patent Application WO 91/16347, are contemplated for use with cellular carriers and other embodiments of the present invention.

[0295] Another adjuvant contemplated for use in the present invention is BCG. BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) and BCG-cell wall skeleton (CWS) may also be used as adjuvants in the invention, with or without trehalose dimycolate. Trehalose dimycolate may be used itself. Trehalose dimycolate administration has been shown to correlate with augmented resistance to influenza virus infection in mice (Azuma et al., 1988). Trehalose dimycolate may be prepared as described in U.S. Pat. No. 4,579,945.

[0296] BCG is an important clinical tool because of its immunostimulatory properties. BCG acts to stimulate the reticulo-endothelial system, activates natural killer cells and increases proliferation of hematopoietic stem cells. Cell wall extracts of BCG have proven to have excellent immune adjuvant activity. Molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990; Martin et al., 1990).

[0297] Live BCG is an effective and safe vaccine used worldwide to prevent tuberculosis. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly 2 billion immunizations, BCG has a long record of safe use in man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination. An exemplary BCG vaccine is sold as TICE.TM. BCG (Organon Inc., West Orange, N.J.).

[0298] In a typical practice of the present invention, cells of Mycobacterium bovis-BCG are grown and harvested by methods known in the art. For example, they may be grown as a surface pellicle on a Sauton medium or in a fermentation vessel containing the dispersed culture in a Dubos medium (Dubos et al., 1947; Rosenthal, 1937). All the cultures are harvested after 14 days incubation at about 37.degree. C. Cells grown as a pellicle are harvested by using a platinum loop whereas those from the fermenter are harvested by centrifugation or tangential-flow filtration. The harvested cells are resuspended in an aqueous sterile buffer medium. A typical suspension contains from about 2.times.10.sup.10 cells/ml to about 2.times.10.sup.12 cells/ml. To this bacterial suspension, a sterile solution containing a selected enzyme which will degrade the BCG cell covering material is added. The resultant suspension is agitated such as by stirring to ensure maximal dispersal of the BCG organisms. Thereafter, a more concentrated cell suspension is prepared and the enzyme in the concentrate removed, typically by washing with an aqueous buffer, employing known techniques such as tangential-flow filtration. The enzyme-free cells are adjusted to an optimal immunological concentration with a cryoprotectant solution, after which they are filled into vials, ampoules, etc., and lyophilized, yielding BCG vaccine, which upon reconstitution with water is ready for immunization.

[0299] Amphipathic and surface active agents, e.g, saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of adjuvants for use with the immunogens of the present invention. Nonionic block copolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) may also be employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et al., 1988). Quil A and lentinen are other adjuvants that may be used in certain embodiments of the present invention.

[0300] One group of adjuvants preferred for use in the invention are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals. Of course, the detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant-incorporated cells. For example, combination of detoxified endotoxins with trehalose dimycolate is particularly contemplated, as described in U.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Pat. No. 4,520,019.

[0301] In other embodiments, the present invention contemplates that a variety of adjuvants may be employed in the membranes of cells, resulting in an improved immunogenic composition. The only requirement is, generally, that the adjuvant be capable of incorporation into, physical association with, or conjugation to, the cell membrane of the cell in question. Those of skill in the art will know the different kinds of adjuvants that can be conjugated to cellular vaccines in accordance with this invention and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others. Certain adjuvants particularly contemplated for use are the teichoic acids from Gram positive cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the invention (Takada et al., 1995a).

[0302] Various adjuvants, even those that are not commonly used in humans, may still be employed in animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result from either the adjuvant or the cells, e.g, as may occur using non-irradiated tumor cells, is irrelevant in such circumstances.

[0303] One group of adjuvants preferred for use in some embodiments of the present invention are those that can be encoded by a nucleic acid (e.g. DNA or RNA). It is contemplated that such adjuvants may be encoded in a nucleic acid (e.g, an expression vector) encoding the antigen, or in a separate vector or other construct. These nucleic acids encoding the adjuvants can be delivered directly, such as for example with lipids or liposomes.

[0304] 3. Excipients, Salts and Auxiliary Substances

[0305] An antigenic composition of the present invention may be mixed with one or more additional components (e.g, excipients, salts, etc.) which are pharmaceutically acceptable and compatible with at least one active ingredient (e.g, antigen). Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and combinations thereof.

[0306] An antigenic composition of the present invention may be formulated into the vaccine as a neutral or salt form. A pharmaceutically-acceptable salt, includes the acid addition salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A salt formed with a free carboxyl group also may be derived from an inorganic base such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and combinations thereof.

[0307] In addition, if desired, an antigentic composition may comprise minor amounts of one or more auxiliary substances such as for example wetting or emulsifying agents, pH buffering agents, etc. which enhance the effectiveness of the antigenic composition or vaccine.

[0308] 1. Vaccine Preparations

[0309] Once produced, synthesized and/or purified, an antigen or other vaccine component may be prepared as a vaccine for administration to a patient. The preparation of a vaccine is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251, 4,601,903, 4,599,231, 4,599,230, and 4,596,792, all incorporated herein by reference. Such methods may be used to prepare a vaccine comprising an antigenic composition comprising folate binding protein epitopes and/or variants as active ingredient(s), in light of the present disclosure. In preferred embodiments, the compositions of the present invention are prepared to be pharmacologically acceptable vaccines.

[0310] Pharmaceutical vaccine compositions of the present invention comprise an effective amount of one or more folate binding protein epitopes and/or variants or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one folate binding protein epitope or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g, human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0311] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). The folate binding protein variant may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0312] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g, methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0313] The folate binding protein variant may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g, those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

[0314] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g, glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g, triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0315] In other embodiments, one may use nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

[0316] In certain embodiments the folate binding protein variant is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g, hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

[0317] In certain preferred embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.

[0318] Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

[0319] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[0320] The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

[0321] In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

[0322] J. Vaccine Administration

[0323] The manner of administration of a vaccine may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. For example, a vaccine may be conventionally administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, rectally, nasally, topically, in eye drops, locally, using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g, liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0324] A vaccination schedule and dosages may be varied on a patient by patient basis, taking into account, for example, factors such as the weight and age of the patient, the type of disease being treated, the severity of the disease condition, previous or concurrent therapeutic interventions, the manner of administration and the like, which can be readily determined by one of ordinary skill in the art.

[0325] A vaccine is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. For example, the intramuscular route may be preferred in the case of toxins with short half lives in vivo. The quantity to be administered depends on the subject to be treated, including, e.g, the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host. Precise amounts of an active ingredient required to be administered depend on the judgment of the practitioner. In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein However, a suitable dosage range may be, for example, of the order of several hundred micrograms active ingredient per vaccination. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per vaccination, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above. A suitable regime for initial administration and booster administrations (e.g, innoculations) are also variable, but are typified by an initial administration followed by subsequent inoculation(s) or other administration(s).

[0326] In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies.

[0327] The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays. Other immune assays can be performed and assays of protection from challenge with the folate binding protein variant can be performed, following immunization.

[0328] K. Enhancement of an Immune Response

[0329] The present invention includes a method of enhancing the immune response in a subject comprising the steps of contacting one or more lymphocytes with a folate binding protein variant antigenic composition, wherein the antigen comprises as part of its sequence a sequence in accordance with SEQ ID NO:1 through SEQ ID NO:8, or a immunologically functional equivalent thereof. In certain embodiments the one or more lymphocytes is comprised in an animal, such as a human. In other embodiments, the lymphocyte(s) may be isolated from an animal or from a tissue (e.g, blood) of the animal. In certain preferred embodiments, the lymphocyte(s) are peripheral blood lymphocyte(s). In certain embodiments, the one or more lymphocytes comprise a T-lymphocyte or a B-lymphocyte. In a particularly preferred facet, the T-lymphocyte is a cytotoxic T-lymphocyte.

[0330] The enhanced immune response may be an active or a passive immune response. Alternatively, the response may be part of an adoptive immunotherapy approach in which lymphocyte(s) are obtained with from an animal (e.g. a patient), then pulsed with composition comprising an antigenic composition. In a preferred embodiment, the lymphocyte(s) may be administered to the same or different animal (e.g, same or different donors).

[0331] 1. Cytotoxic T Lymphocytes

[0332] In certain embodiments, T-lymphocytes are specifically activated by contact with an antigenic composition of the present invention. In certain embodiments, T-lymphocytes are activated by contact with an antigen presenting cell that is or has been in contact with an antigenic composition of the invention.

[0333] T cells express a unique antigen binding receptor on their membrane (T-cell receptor), which can only recognize antigen in association with major histocompatibility complex (MHC) molecules on the surface of other cells. There are several populations of T cells, such as T helper cells and T cytotoxic cells. T helper cells and T cytotoxic cells are primarily distinguished by their display of the membrane bound glycoproteins CD4 and CD8, respectively. T helper cells secret various lymphokines, that are crucial for the activation of B cells, T cytotoxic cells, macrophages and other cells of the immune system. In contrast, a T cytotoxic cell that recognizes an antigen-MHC complex proliferates and differentiates into an effector cell called a cytotoxic T lymphocyte (CTL). CTLs eliminate cells of the body displaying antigen by producing substances that result in cell lysis.

[0334] CTL activity can be assessed by methods described herein or as would be known to one of skill in the art. For example, CTLs may be assessed in freshly isolated peripheral blood mononuclear cells (PBMC), in a phytohaemaglutinin-stimulated IL-2 expanded cell line established from PBMC (Bernard et al., 1998) or by T cells isolated from a previously immunized subject and restimulated for 6 days with DC infected with an adenovirus vector containing antigen using standard 4 h 51.sup.Cr release microtoxicity assays. In another fluorometric assay developed for detecting cell-mediated cytotoxicity, the fluorophore used is the non-toxic molecule alamarBlue (Nociari et al., 1998). The alamarBlue is fluorescently quenched (i.e., low quantum yield) until mitochondrial reduction occurs, which then results in a dramatic increase in the alamarBlue fluorescence intensity (i.e., increase in the quantum yield). This assay is reported to be extremely sensitive, specific and requires a significantly lower number of effector cells than the standard 51Cr release assay.

[0335] In certain aspects, T helper cell responses can be measured by in vitro or in vivo assay with peptides, polypeptides or proteins. In vitro assays include measurement of a specific cytokine release by enzyme, radioisotope, chromaphore or fluorescent assays. In vivo assays include delayed type hypersensitivity responses called skin tests, as would be known to one of ordinary skill in the art.

[0336] 2. Antigen Presenting Cells

[0337] In general, the term "antigen presenting cell" can be any cell that accomplishes the goal of the invention by aiding the enhancement of an immune response (i.e., from the T-cell or -B-cell arms of the immune system) against an antigen (e.g, a folate binding protein variant or a immunologically functional equivalent) or antigenic composition of the present invention. Such cells can be defined by those of skill in the art, using methods disclosed herein and in the art. As is understood by one of ordinary skill in the art (see for example Kuby, 1993, incorporated herein by reference), and used herein certain embodiments, a cell that displays or presents an antigen normally or preferentially with a class II major histocompatability molecule or complex to an immune cell is an "antigen presenting cell." In certain aspects, a cell (e.g, an APC cell) may be fused with another cell, such as a recombinant cell or a tumor cell that expresses the desired antigen. Methods for preparing a fusion of two or more cells is well known in the art, such as for example, the methods disclosed in Goding, pp. 65-66, 71-74 1986; Campbell, pp. 75-83, 1984; Kohler and Milstein, 1975; Kohler and Milstein, 1976, Gefter et al., 1977, each incorporated herein by reference. In some cases, the immune cell to which an antigen presenting cell displays or presents an antigen to is a CD4.sup.+ TH cell. Additional molecules expressed on the APC or other immune cells may aid or improve the enhancement of an immune response. Secreted or soluble molecules, such as for example, immunomodulators and adjuvants, may also aid or enhance the immune response against an antigen. Such molecules are well known to one of skill in the art, and various examples are described herein.

VII. Peptide Formulations

[0338] Peptides containing the epitope motifs described herein are contemplated for use in therapeutics to provide universal FBP targets and antigens for CTLs in the HLA-A2 system. The development of therapeutics based on these novel sequences provides induction of tumor reactive immune cells in vivo through the formulation of synthetic cancer vaccines, as well as induction of tumor-reactive T-cells in vitro through either peptide-mediated (e.g. lipopeptide) or cell-mediated (e.g, EBV-B lines using either autologous or HLA-A2 transfectants where the gene for the peptide of interest is introduced, and the peptide is expressed associated with HLA-A2 on the surface). The use of these novel peptides as components of vaccines to prevent, or lessen the chance of cancer progression is also contemplated.

[0339] The peptides contemplated for use, being smaller than other compositions, such as envelope proteins, will have improved bioavailability and half lives. If desired, stability examinations may be performed on the peptides, including, e.g, pre-incubation in human serum and plasma; treatment with various proteases; and also temperature- and pH-stability analyses. If found to be necessary, the stability of the synthetic peptides may be enhanced by any one of a variety of methods such as, for example, employing D-amino acids in place of L-amino acids for peptide synthesis; using blocking groups like t-boc and the like; or encapsulating the peptides within liposomes. The bio-availability of select mixtures of peptides may also be determined by injecting radio-labeled peptides into experimental animals, such as mice and/or Rhesus monkeys, and subsequently analyzing their tissue distribution.

[0340] If stability enhancement was desired, it is contemplated that the use of dextrorotary amino acids (D-amino acids) would be advantageous as this would result in even longer bioavailability due to the inability of proteases to attack these types of structures. The peptides of the present invention may also be further stabilized, for example, by the addition of groups to the N- or C-termini, such as by acylation or amination. If desired, the peptides could even be in the form of lipid-tailed peptides, formulated into surfactant-like micelles, or other peptide multimers. The preparation of peptide multimers and surfactant-like micelles is described in detail in U.S. Ser. No. 07/945,865, incorporated herein by reference. The compositions of the present invention are contemplated to be particularly advantageous for use in economical and safe anti-tumor/anti-cancer therapeutics, and specific therapeutic formulations may be tested in experimental animal models, such as mice, rats, rabbits, guinea pigs, cats, goats, Rhesus monkeys, chimpanzees, and the like, in order to determine more precisely the dosage forms required.

[0341] In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention. This may be achieved by the techniques of modelling and chemical design known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the terminus of a peptide to mimic a particular terminal motif structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.

[0342] Therapeutic or pharmacological compositions of the present invention will generally comprise an effective amount of a CTL-stimulating peptide or peptides, dissolved or dispersed in a pharmaceutically acceptable medium. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic, toxic, or otherwise adverse reaction when administered to a human. Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

[0343] Supplementary active ingredients can also be incorporated into the therapeutic compositions of the present invention. For example, the stimulatory peptides may also be combined with peptides including cytotoxic T-cell- or T-helper-cell-inducing epitopes (as disclosed in U.S. Ser. No. 07/945,865; incorporated herein by reference) to create peptide cocktails for immunization and treatment.

[0344] The preparation of pharmaceutical or pharmacological compositions containing a CTL-stimulating peptide or peptides, including dextrorotatory peptides, as active ingredients will be known to those of skill in the art in light of the present disclosure. Typically, such compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules; or in any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.

[0345] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0346] Sterile solutions suitable for intravenous administration are preferred in certain embodiments and are contemplated to be particularly effective in stimulating CTLs and/or producing an immune response in an animal. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0347] A peptide or peptides can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, e.g, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0348] The carrier can also be a solvent or dispersion medium containing, e.g, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained by inter alia the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought inter alia by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, e.g, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0349] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0350] The preparation of more- or highly-concentrated solutions for intramuscular injection is also contemplated. This is envisioned to have particular utility in facilitating the treatment of needle stick injuries to animals or even humans. In this regard, the use of DMSO as solvent is preferred as this will result in extremely rapid penetration, delivering high concentrations of the active peptide, peptides or agents to a small area.

[0351] The use of sterile formulations, such as saline-based washes, by veterinarians, technicians, surgeons, physicians or health care workers to cleanse a particular area in the operating field may also be particularly useful. Therapeutic formulations in accordance with the present invention may also be reconstituted in the form of mouthwashes, including the peptides alone, or in conjunction with antifungal reagents. Inhalant forms are also envisioned, which again, may contain active peptides or agents alone, or in conjunction with other agents, such as, e.g, pentamidine. The therapeutic formulations of the invention may also be prepared in forms suitable for topical administration, such as in cremes and lotions.

[0352] Suitable preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like. Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such that the sodium chloride equivalent of the ophthalmic solution is in the range 0.9.+-.0.2%. Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea and the like. Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethyl-propylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the like.

[0353] Upon formulation, therapeutics will be administered in a manner compatible with the dosage formulation, and in such amount as is pharmacologically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. As used herein, "pharmacologically effective amount" means an amount of composition is used that contains an amount of a peptide or peptides sufficient to significantly stimulate a CTL or generate an immune response in an animal.

[0354] In this context, the quantity of peptide(s) and volume of composition to be administered depends on the host animal to be treated, such as, the capacity of the host animal's immune system to produce an immune response. Precise amounts of active peptide required to be administered depend on the judgment of the practitioner and are peculiar to each individual.

[0355] A minimal volume of a composition required to disperse the peptide is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals. For example, for parenteral administration, a suitably buffered, and if necessary, isotonic aqueous solution would be prepared and used for intravenous, intramuscular, subcutaneous or even intraperitoneal administration. One dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).

[0356] In certain embodiments, active compounds may be administered orally. This is contemplated for agents that are generally resistant, or have been rendered resistant, to proteolysis by digestive enzymes. Such compounds are contemplated to include chemically designed or modified agents; dextrorotatory peptides; and peptide and liposomal formulations in timed-release capsules to avoid peptidase, protease and/or lipase degradation.

[0357] Oral formulations may include compounds in combination with an inert diluent or an edible carrier which may be assimilated; those enclosed in hard- or soft-shell gelatin capsules; those compressed into tablets; or those incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should generally contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[0358] Tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, corn starch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparaben as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0359] The peptides may be used in their immunizing capacity by administering an amount effective to generate an immune response in an animal. In this sense, such an "amount effective to generate an immune response" means an amount of composition that contains a peptide or peptide mixture sufficient to significantly produce an antigenic response in the animal.

VIII. Examples

[0360] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Rationale for Variant Design

[0361] Studies in experimental models regarding lymphocyte development in the thymus show that interaction of thymocytes with weak or null (no apparent effect) agonists lead to positive selection (i.e. survival) of responders for a specific Ag, while stimulation with strong agonists leads to negative selection (deletion of reactive CTL). Similarly, recent studies on CD8.sup.+ cell responses from peripheral blood show that Ag variants with null or weak agonistic activity induced expansion of precursors of CTL responding to a model Ag, but not effector function. These results were obtained with transgenic animals, and the recipients for the CTL were heavily irradiated. There is little information concerning how the responders to tumor, and/or their precursors, can be maintained and avoid elimination in healthy individuals, or patients without evidence of disease. However, the presence of such precursors, or of activated CTL recognizing tumor Ag, (Peoples et al., 1998; Hudson et al., 1998; Peoples et al, 1998; Kim et al., 1999; Lee et al., 2000) is proof that such responders exist in the peripheral blood. Approaches to promote their survival, expansion and induction of lytic formation is beneficial for the patients. If the responders targeted for survival are low-affinity CTL, the weak affinity is expected to be compensated by a significant increase in effector numbers. If the responders are of high affinity, protection from AICD will also allow their expansion.

[0362] To design "survival inducing" Ag, the present invention focuses on the FBP epitope E39: EIWTHSYKV. This epitope is recognized, although with low affinity, by ovarian and breast tumor reactive CTL. It was predicted that improved immunogenicity in terms of net gain in cell numbers reacting with the wild-type Ag is achieved by reducing the positive charge at the amino acid in position 5 (histidine) and replacement of histidine with phenylalanine (Phe). Phe is not charged, but its benzene aromatic ring is a close substitution for the imidazole ring of histidine. To ensure a better flexibility of the residues in the peptide, the phenolic structure of tyrosine was replaced with the aliphatic core chain of Threonine (Thr). Both Tyr and Thr contain an OH (hydroxyl) side chain group. Thus, the positive charge in position 5 and the rigid structure of Tyr were eliminated. In a specific embodiment, this increases the flexibility of the residues 5-9 (SYKV) in the peptide and allows for a better fitting of the TCR with the peptide MHC complex. The variant: E I W T F S T K V was designated J65. Additional variants of J65 were created with changes in position 7 (Tyr).fwdarw.Thr only=designated J77, in position 5 only Phe.fwdarw.His=designated J78, and in positions 1 and 6. These analogs/variants are listed in Table 5.

TABLE-US-00005 TABLE 5 Variants of Folate Binding Protein VARIANT SEQUENCE CHANGE E39 EIWTHSYKV (SEQ wild type ID NO: 268) J77 EIWTHSTKV (SEQ Y7.fwdarw.T ID NO: 1) J78 EIWTFSYKV (SEQ ID H5.fwdarw.F NO: 2) J68 FIWTFATKV (SEQ ID E1.fwdarw.F, H5.fwdarw.F, Y7.fwdarw.T NO: 3) J67 EIWTHATKV (SEQ S6.fwdarw.A, Y7.fwdarw.T ID NO: 4) J66 EIWTFSTKV (SEQ ID E1.fwdarw.F, H5.fwdarw.F, Y7.fwdarw.T NO: 5) J65 EIWTFSYKV (SEQ ID H5.fwdarw.F, Y7.fwdarw.T NO: 6) J64 GIWTHSTKV (SEQ E1.fwdarw.G, Y7.fwdarw.T ID NO: 7) J63 FIWTHSTKV (SEQ ID E1.fwdarw.F, Y7.fwdarw.T NO: 8)

[0363] Selection of these Ag variants was made on the principle of Ag alteration aiming to alternate signaling. In addition to substitutions H.fwdarw.F (Pos. 5) and Y.fwdarw.T (pos. 7), substitutions were introduced in the other positions: S.fwdarw.A (Pos. 6 and Glu (B).fwdarw.F and E.fwdarw.Gly (G) (in Pos. 1). The purpose of these substitutions was to remove potential reacting groups with the TCR. In the substitution S.fwdarw.A (Pos. A), this change removes a side chain OH group. In position 1, the substitution E (glutamic acid).fwdarw.glycine, removes the entire aliphatic side chain plus the charged COO group. Also in position 1, the substitution E.fwdarw.F (removes the charged group COO, but introduces an aromatic ring). These substitutions aim to diminish the reactivity of the peptide with the TCR.

Example 2

IFN-.gamma. Induction and CTL Activity

[0364] The HLA-A2 stabilizing ability of the variant peptides has also been determined (FIG. 1). The results show that the stabilizing ability of J65 is almost half of the stabilizing ability of E39. In contrast, substitutions at position 1 increase the binding affinity of the peptide. The results in FIG. 2 show the cytolytic activity of J65-induced CTL compared with E39-induced CTL. The results indicate that J65 was a weaker inducer of IFN-y from 3.times.J65 stimulated cultures than J77 and E39, suggesting that the changes in the sequence had cumulative effects in decreasing IFN-.gamma. induction.

[0365] To address the effects of FBP variants on induction of CTL, activity, PBMC cultures from the healthy donor stimulated three times with J65 were split in three and restimulated with either E39 or J65 or J77. A control culture was made of the same PBMC stimulated three times with E39 and restimulated with E39 for the fourth time. PBMC stimulated three times with E39 (3.times.E39) followed by E39 showed moderate weak recognition of E39. In contrast, 3.times.J65 stimulated CTL showed significantly higher recognition of E39 after stimulation with E39. A similar picture was observed with 3.times.J65 cells restimulated with J65, while 3.times.J65 restimulated with J77 showed significantly lower CTL activity than 3.times.J65 stimulated with the other peptides. It was recently reported that memory CTL reacting with the tumor Ag such as FBP are present in the blood of healthy individuals (Lee et al., 2000). These cells can be easily activated by stimulation with the corresponding peptide presented on dendritic cells (Kim et al., 1999). To evaluate the stimulating ability of the analogs J65 and J77, PBMC from a responding donor were stimulated with E39, J65 and J77. These results show that the potentiating role of J65 in responder proliferation and cytotoxicity does not reflect enhanced IL-2 and/or IFN-.gamma. secretion compared with the wild-type Ag, but its weaker cytokine-inducing activity appears to protect CTL of higher affinity from apoptosis by avoiding overstimulation.

Example 3

Specific IL-2 Induction by Priming with FBP Variants

[0366] In J65-primed CTL, higher CTL activity and IFN-.gamma. secretion can be elicited by the wild-type epitope E39, suggesting a protective effect of the previous stimulations. The results in FIG. 3 show that J65 and J77 induced lower levels of IL-2 in the PBMC of this donor compared with the wild-type peptide E39. To identify which of E39 variants induced higher cell expansion, PBMC from the same donor were stimulated three times with the corresponding peptide, and the resulting live cells were counted a week after each stimulation. The results in FIG. 4 show that cultures stimulated with E39 initially expanded faster than other cultures; however, after the third stimulation, cultures stimulated with J65 increased faster in numbers. In contrast, cultures stimulated with J78 (H.fwdarw.F) and J77 (Y.fwdarw.T) proliferated slower than control cultures which were not stimulated with peptide. Similar results were obtained with J65 in another donor (FIG. 5). In this donor, cells stimulated with E39 died after the third stimulation while cells stimulated by J65 expanded faster. Cells stimulated with J77 and J78 also expanded, but at a slower rate.

REFERENCES

[0367] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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PUBLICATIONS

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[0516] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as described herein.

Sequence CWU 1

1

27119PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 1Glu Ile Trp Thr His Ser Thr Lys Val 1 529PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 2Glu Ile Trp Thr Phe Ser Tyr Lys Val 1 539PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 3Phe Ile Trp Thr Phe Ala Thr Lys Val 1 549PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 4Glu Ile Trp Thr His Ala Thr Lys Val 1 559PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 5Glu Ile Trp Thr Phe Ser Thr Lys Val 1 569PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 6Glu Ile Trp Thr Phe Ser Tyr Lys Val 1 579PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 7Gly Ile Trp Thr His Ser Thr Lys Val 1 589PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 8Phe Ile Trp Thr His Ser Thr Lys Val 1 59255PRTHomo sapiens 9Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu Leu Val Cys Val Ala 1 5 10 15Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val Cys Met 20 25 30Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His 35 40 45Asp Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 50 55 60Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp 65 70 75 80Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 85 90 95Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln 100 105 110Gln Val Asn Gln Thr Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu 115 120 125Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His Thr Ser His 130 135 140Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val145 150 155 160Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe 165 170 175Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 180 185 190Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe 195 200 205Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 210 215 220Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly225 230 235 240Leu Leu Leu Ser Leu Ala Leu Met Leu Gln Leu Trp Leu Leu Gly 245 250 25510257PRTHomo sapiens 10Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser11293PRTRAT 11Met Val Asp Ser Val Tyr Arg Thr Arg Ser Leu Gly Val Ala Ala Glu 1 5 10 15Gly Ile Pro Asp Gln Tyr Ala Asp Gly Glu Ala Ala Arg Val Trp Gln 20 25 30Leu Tyr Ile Gly Asp Thr Arg Ser Arg Thr Ala Glu Tyr Lys Ala Trp 35 40 45Leu Leu Gly Leu Leu Arg Gln His Gly Cys His Arg Val Leu Asp Val 50 55 60Ala Cys Gly Thr Gly Val Asp Ser Ile Met Leu Val Glu Glu Gly Phe 65 70 75 80Ser Val Thr Ser Val Asp Ala Ser Asp Lys Met Leu Lys Tyr Ala Leu 85 90 95Lys Glu Arg Trp Asn Arg Arg Lys Glu Pro Ala Phe Asp Lys Trp Val 100 105 110Ile Glu Glu Ala Asn Trp Leu Thr Leu Asp Lys Asp Val Pro Ala Gly 115 120 125Asp Gly Phe Asp Ala Val Ile Cys Leu Gly Asn Ser Phe Ala His Leu 130 135 140Pro Asp Ser Lys Gly Asp Gln Ser Glu His Arg Leu Ala Leu Lys Asn145 150 155 160Ile Ala Ser Met Val Arg Pro Gly Gly Leu Leu Val Ile Asp His Arg 165 170 175Asn Tyr Asp Tyr Ile Leu Ser Thr Gly Cys Ala Pro Pro Gly Lys Asn 180 185 190Ile Tyr Tyr Lys Ser Asp Leu Thr Lys Asp Ile Thr Thr Ser Val Leu 195 200 205Thr Val Asn Asn Lys Ala His Met Val Thr Leu Asp Tyr Thr Val Gln 210 215 220Val Pro Gly Ala Gly Arg Asp Gly Ala Pro Gly Phe Ser Lys Phe Arg225 230 235 240Leu Ser Tyr Tyr Pro His Cys Leu Ala Ser Phe Thr Glu Leu Val Gln 245 250 255Glu Ala Phe Gly Gly Arg Cys Gln His Ser Val Leu Gly Asp Phe Lys 260 265 270Pro Tyr Arg Pro Gly Gln Ala Tyr Val Pro Cys Tyr Phe Ile His Val 275 280 285Leu Lys Lys Thr Gly 29012257PRTHomo sapiens 12Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser13257PRTHomo sapiens 13Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser14244PRTMus musculus 14Met Ala Gln Trp Trp Gln Ile Leu Leu Gly Leu Trp Ala Val Leu Pro 1 5 10 15Thr Leu Ala Gly Asp Lys Leu Leu Ser Val Cys Met Asn Ser Lys Arg 20 25 30His Lys Gln Glu Pro Gly Pro Glu Asp Glu Leu Tyr Gln Glu Cys Arg 35 40 45Pro Trp Glu Asp Asn Ala Cys Cys Thr Arg Ser Thr Ser Trp Glu Ala 50 55 60His Leu Glu Glu Pro Leu Leu Phe Asn Phe Ser Met Met His Cys Gly 65 70 75 80Leu Leu Thr Pro Ala Cys Arg Lys His Phe Ile Gln Ala Ile Cys Phe 85 90 95His Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Pro Val Val Pro 100 105 110Asn Gly Gln Glu Glu Gln Arg Val Trp Gly Val Pro Leu Cys Gln Glu 115 120 125Asp Cys Glu Asp Trp Trp Arg Ala Cys His Ser Ser Leu Thr Cys Lys 130 135 140Ser Asn Trp Leu His Gly Trp Asp Trp Ser Glu Glu Lys Lys His Cys145 150 155 160Pro Ala His Glu Pro Cys Leu Pro Phe Ser Tyr His Phe Pro Thr Pro 165 170 175Asp Asp Leu Cys Glu Lys Ile Trp Asn Asn Thr Phe Lys Ala Ser Pro 180 185 190Glu Arg Arg Asn Ser Gly Arg Cys Leu Gln Lys Trp Phe Glu Pro Thr 195 200 205Leu Ser Asn Pro Asn Val Glu Val Ala Leu His Phe Ala Gly Ser Ala 210 215 220Leu Ala Pro Gln Leu Ser Tyr Thr Leu Pro Ala Phe Ser Leu Cys Leu225 230 235 240Leu Phe His Pro15295PRTHomo sapiens 15Met Val Asp Ser Val Tyr Arg Thr Arg Ser Leu Gly Val Ala Ala Glu 1 5 10 15Gly Leu Pro Asp Gln Tyr Ala Glu Gly Glu Ala Ala Arg Val Trp Gln 20 25 30Leu Tyr Ile Gly Asp Thr Arg Ser Arg Thr Ala Glu Tyr Lys Ala Trp 35 40 45Leu Leu Gly Leu Leu Arg Gln His Gly Cys Gln Arg Val Leu Asp Val 50 55 60Ala Cys Gly Thr Gly Val Asp Ser Ile Met Leu Val Glu Glu Gly Phe 65 70 75 80Ser Val Thr Ser Val Asp Ala Ser Asp Lys Met Leu Lys Tyr Ala Leu 85 90 95Lys Glu Arg Trp Asn Arg Arg His Glu Pro Ala Phe Asp Lys Trp Val 100 105 110Ile Glu Glu Ala Asn Trp Met Thr Leu Asp Lys Asp Val Pro Gln Ser 115 120 125Ala Glu Gly Gly Phe Asp Ala Val Ile Cys Leu Gly Asn Ser Phe Ala 130 135 140His Leu Pro Asp Cys Lys Gly Asp Gln Ser Glu His Arg Leu Ala Leu145 150 155 160Lys Asn Ile Ala Ser Met Val Arg Ala Gly Gly Leu Leu Val Ile Asp 165 170 175His Arg Asn Tyr Asp His Ile Leu Ser Thr Gly Cys Ala Pro Pro Gly 180 185 190Lys Asn Ile Tyr Tyr Lys Ser Asp Leu Thr Lys Asp Val Thr Thr Ser 195 200 205Val Leu Ile Val Asn Asn Lys Ala His Met Val Thr Leu Asp Tyr Thr 210 215 220Val Gln Val Pro Gly Ala Gly Gln Asp Gly Ser Pro Gly Leu Ser Lys225 230 235 240Phe Arg Leu Ser Tyr Tyr Pro His Cys Leu Ala Ser Phe Thr Glu Leu 245 250 255Leu Gln Ala Ala Phe Gly Gly Lys Cys Gln His Ser Val Leu Gly Asp 260 265 270Phe Lys Pro Tyr Lys Pro Gly Gln Thr Tyr Ile Pro Cys Tyr Phe Ile 275 280 285His Val Leu Lys Arg Thr Asp 290 29516293PRTRAT 16Met Val Asp Ser Val Tyr Arg Thr Arg Ser Leu Gly Val Ala Ala Glu 1 5 10 15Gly Ile Pro Asp Gln Tyr Ala Asp Gly Glu Ala Ala Arg Val Trp Gln 20 25 30Leu Tyr Ile Gly Asp Thr Arg Ser Arg Thr Ala Glu Tyr Lys Ala Trp 35 40 45Leu Leu Gly Leu Leu Arg Gln His Gly Cys His Arg Val Leu Asp Val 50 55 60Ala Cys Gly Thr Gly Val Asp Ser Ile Met Leu Val Glu Glu Gly Phe 65 70 75 80Ser Val Thr Ser Val Asp Ala Ser Asp Lys Met Leu Lys Tyr Ala Leu 85 90 95Lys Glu Arg Trp Asn Arg Arg Lys Glu Pro Ala Phe Asp Lys Trp Val 100 105 110Ile Glu Glu Ala Asn Trp Leu Thr Leu Asp Lys Asp Val Pro Ala Gly 115 120 125Asp Gly Phe Asp Ala Val Ile Cys Leu Gly Asn Ser Phe Ala His Leu 130 135 140Pro Asp Ser Lys Gly Asp Gln Ser Glu His Arg Leu Ala Leu Lys Asn145 150 155 160Ile Ala Ser Met Val Arg Pro Gly Gly Leu Leu Val Ile Asp His Arg 165 170 175Asn Tyr Asp Tyr Ile Leu Ser Thr Gly Cys Ala Pro Pro Gly Lys Asn 180 185 190Ile Tyr Tyr Lys Ser Asp Leu Thr Lys Asp Ile Thr Thr Ser Val Leu 195 200 205Thr Val Asn Asn Lys Ala His Met Val Thr Leu Asp Tyr Thr Val Gln 210 215 220Val Pro Gly Ala Gly Arg Asp Gly Ala Pro Gly Phe Ser Lys Phe Arg225 230 235 240Leu Ser Tyr Tyr Pro His Cys Leu Ala Ser Phe Thr Glu Leu Val Gln 245 250 255Glu Ala Phe Gly Gly Arg Cys Gln His Ser Val Leu Gly Asp Phe Lys 260 265 270Pro Tyr Arg Pro Gly Gln Ala Tyr Val Pro Cys Tyr Phe Ile His Val 275 280 285Leu Lys Lys Thr Gly 29017222PRTbovidaeMOD_RES(55)Xaa = anything 17Ala Gln Ala Pro Arg Thr Pro Arg Ala Arg Thr Asp Leu Leu Asn Val 1 5 10 15Cys Met Asp Ala Lys His His Lys Ala Glu Pro Gly Pro Glu Asp Ser 20 25 30Leu His Glu Gln Cys Ser Pro Trp Arg Lys Asn Ala Cys Cys Ser Val 35 40 45Asn Thr Ser Ile Glu Ala Xaa Lys Asp Ile Ser Tyr Leu Tyr Arg Phe 50 55 60Asn Trp Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe 65 70 75 80Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp 85 90 95Ile Arg Glu Val Asn Gln Arg Trp Arg Lys Glu Arg Val Leu Gly Val 100 105 110Pro Leu Cys Lys Glu Asp Cys Gln Ser Trp Trp Glu Asp Cys Arg Thr 115 120 125Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser 130 135 140Gly Tyr Asn Gln Cys Pro Val Lys Ala Ala His

Cys Arg Phe Asp Phe145 150 155 160Tyr Phe Pro Thr Pro Ala Ala Leu Cys Asn Glu Ile Trp Ser His Ser 165 170 175Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met 180 185 190Trp Phe Asp Pro Phe Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe 195 200 205Tyr Ala Glu Asn Pro Thr Ser Gly Ser Thr Pro Gln Gly Ile 210 215 2201822PRTRAT 18Gln Ala Thr Arg Ala Arg Thr Glu Leu Leu Asn Val Phe Ala Asp Ala 1 5 10 15Lys Arg Glu Lys Pro Lys 201922PRTRAT 19Gln Ala Thr Arg Ala Glu Thr Glu Asn Leu Asn Val Asp Met Asp Ala 1 5 10 15Lys His His Lys Glu Lys 2020589PRTHomo sapiens 20Met Val Pro Ser Ser Pro Ala Val Glu Lys Gln Val Pro Val Glu Pro 1 5 10 15Gly Pro Asp Pro Glu Leu Arg Ser Trp Arg His Leu Val Cys Tyr Leu 20 25 30Cys Phe Tyr Gly Phe Met Ala Gln Ile Arg Pro Gly Glu Ser Phe Ile 35 40 45Thr Pro Tyr Leu Leu Gly Pro Asp Lys Asn Phe Thr Arg Glu Gln Val 50 55 60Thr Asn Glu Ile Thr Pro Val Leu Ser Tyr Ser Tyr Leu Ala Val Leu 65 70 75 80Val Pro Val Phe Leu Leu Thr Asp Tyr Leu Arg Tyr Thr Pro Val Leu 85 90 95Leu Leu Gln Gly Leu Ser Phe Val Ser Val Trp Leu Leu Leu Leu Leu 100 105 110Gly His Ser Val Ala His Met Gln Leu Met Glu Leu Phe Tyr Ser Val 115 120 125Thr Met Ala Ala Arg Ile Ala Tyr Ser Ser Tyr Ile Phe Ser Leu Val 130 135 140Arg Pro Ala Arg Tyr Gln Arg Val Ala Gly Tyr Ser Arg Ala Ala Val145 150 155 160Leu Leu Gly Val Phe Thr Ser Ser Val Leu Gly Gln Leu Leu Val Thr 165 170 175Val Gly Arg Val Ser Phe Ser Thr Leu Asn Tyr Ile Ser Leu Ala Phe 180 185 190Leu Thr Phe Ser Val Val Leu Ala Leu Phe Leu Lys Arg Pro Lys Arg 195 200 205Ser Leu Phe Phe Asn Arg Asp Asp Arg Gly Arg Cys Glu Thr Ser Ala 210 215 220Ser Glu Leu Glu Arg Met Asn Pro Gly Pro Gly Gly Lys Leu Gly His225 230 235 240Ala Leu Arg Val Ala Cys Gly Asp Ser Val Leu Ala Arg Met Leu Arg 245 250 255Glu Leu Gly Asp Ser Leu Arg Arg Pro Gln Leu Arg Leu Trp Ser Leu 260 265 270Trp Trp Val Phe Asn Ser Ala Gly Tyr Tyr Leu Val Val Tyr Tyr Val 275 280 285His Ile Leu Trp Asn Glu Val Asp Pro Thr Thr Asn Ser Ala Arg Val 290 295 300Tyr Asn Gly Ala Ala Asp Ala Ala Ser Thr Leu Leu Gly Ala Ile Thr305 310 315 320Ser Phe Ala Ala Gly Phe Val Lys Ile Arg Trp Ala Arg Trp Ser Lys 325 330 335Leu Leu Ile Ala Gly Val Thr Ala Thr Gln Ala Gly Leu Val Phe Leu 340 345 350Leu Ala His Thr Arg His Pro Ser Ser Ile Trp Leu Cys Tyr Ala Ala 355 360 365Phe Val Leu Phe Arg Gly Ser Tyr Gln Phe Leu Val Pro Ile Ala Thr 370 375 380Phe Gln Ile Ala Ser Ser Leu Ser Lys Glu Leu Cys Ala Leu Val Phe385 390 395 400Gly Val Asn Thr Phe Phe Ala Thr Ile Val Lys Thr Ile Ile Thr Phe 405 410 415Ile Val Ser Asp Val Arg Gly Leu Gly Leu Pro Val Arg Lys Pro Val 420 425 430Ile Leu Arg Val Leu Pro Asp Pro Val His His Leu Leu Leu Gly Gly 435 440 445His Ala Gly Trp Pro Ala Ala Leu Pro Ala Gly Pro Pro Pro Ala Ala 450 455 460Ala Pro Gly Pro Gly Pro Glu Glu Cys Arg Gly Gly Glu Gly Ser Thr465 470 475 480Gly Thr Glu Arg Ala Gly Gln Gly Pro Arg Arg Leu Gln Pro Ala Gln 485 490 495Ser Pro Pro Leu Ser Pro Glu Asp Ser Leu Gly Ala Val Gly Pro Ala 500 505 510Ser Leu Glu Gln Arg Gln Ser Asp Pro Tyr Leu Ala Gln Ala Pro Ala 515 520 525Pro Gln Ala Ala Glu Phe Leu Ser Pro Val Thr Thr Pro Ser Pro Cys 530 535 540Thr Leu Ser Ser Ala Gln Ala Ser Gly Pro Glu Ala Ala Asp Glu Thr545 550 555 560Cys Pro Gln Leu Ala Val His Pro Pro Gly Val Ser Lys Leu Gly Leu 565 570 575Gln Cys Leu Pro Ser Asp Gly Val Gln Asn Val Asn Gln 580 58521257PRTHomo sapiens 21Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser22255PRTHomo sapiens 22Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu Leu Val Cys Val Ala 1 5 10 15Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val Cys Met 20 25 30Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His 35 40 45Asp Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 50 55 60Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp 65 70 75 80Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 85 90 95Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln 100 105 110Gln Val Asn Gln Thr Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu 115 120 125Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His Thr Ser His 130 135 140Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val145 150 155 160Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe 165 170 175Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 180 185 190Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe 195 200 205Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 210 215 220Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly225 230 235 240Leu Leu Leu Ser Leu Ala Leu Met Leu Gln Leu Trp Leu Leu Gly 245 250 25523251PRTMus musculus 23Met Ala Trp Lys Gln Thr Pro Leu Leu Leu Leu Val Tyr Met Val Thr 1 5 10 15Thr Gly Ser Gly Arg Asp Arg Thr Asp Leu Leu Asn Val Cys Met Asp 20 25 30Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His Asp 35 40 45Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Ser Val Asn Thr Ser 50 55 60Gln Glu Leu His Lys Ala Asp Ser Arg Leu Tyr Phe Asn Trp Asp His 65 70 75 80Cys Gly Lys Met Glu Pro Ala Cys Lys Ser His Phe Ile Gln Asp Ser 85 90 95Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln Val 100 105 110Asp Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu Cys Lys 115 120 125Glu Asp Cys His Gln Trp Trp Glu Ala Cys Arg Thr Ser Phe Thr Cys 130 135 140Lys Arg Asp Trp His Lys Gly Trp Asp Trp Ser Ser Gly Ile Asn Lys145 150 155 160Cys Pro Asn Thr Ala Pro Cys His Thr Phe Glu Tyr Tyr Phe Pro Thr 165 170 175Pro Ala Ser Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys Val Ser 180 185 190Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Ser 195 200 205Thr Gln Gly Asn Pro Asn Glu Asp Val Val Lys Phe Tyr Ala Ser Phe 210 215 220Met Thr Ser Gly Thr Val Pro His Ala Ala Val Leu Leu Val Pro Ser225 230 235 240Leu Ala Pro Val Leu Ser Leu Trp Leu Pro Gly 245 25024255PRTMus musculus 24Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Asn Leu His Asp Gln Cys Ser Pro Trp Lys Thr Asn Ser Cys Cys 50 55 60Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Ile Ser Tyr Leu Tyr 65 70 75 80Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Ser Glu Cys Lys Arg 85 90 95His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Gln Trp Trp Glu Asp Cys 130 135 140Gln Ser Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Ser Ser Gly His Asn Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe 165 170 175Thr Phe Tyr Phe Pro Thr Ser Ala Ala Leu Cys Glu Glu Ile Trp Ser 180 185 190His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Arg Phe Tyr Ala Glu Ala Met Ser Gly Ala Gly Leu His Gly Thr Trp225 230 235 240Pro Leu Leu Cys Ser Leu Ser Leu Val Leu Leu Trp Val Ile Ser 245 250 25525257PRTHomo sapiens 25Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser26257PRTHomo sapiens 26Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser27257PRTHomo sapiens 27Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala

Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser28257PRTHomo sapiens 28Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser29257PRTHomo sapiens 29Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser30254PRTHomo sapiens 30Met Ala Ser Val Pro Lys Thr Asn Lys Ile Glu Pro Arg Ser Tyr Ser 1 5 10 15Ile Ile Pro Ser Cys Ser Ile Arg Arg Leu Gly Pro Ala Leu Asn Thr 20 25 30Pro Ile Phe Gln Ser Lys Arg Asn Gly Pro Arg Gly His Ser Ala Tyr 35 40 45Ser Ile Glu Gly Arg Gln Arg Gln Gly Ala Gly Arg Ala Val Val Pro 50 55 60Arg Ala Asp Arg Pro Pro Ala Pro Lys Ile Gln Leu Arg Ala Phe Tyr 65 70 75 80Leu Gln Gln Leu Tyr Tyr Thr Leu Leu Glu Leu Glu Leu Pro Arg Leu 85 90 95Leu Ala Pro Asp Leu Pro Ser Asn Gly Ser Ser Leu Lys Asp Leu Lys 100 105 110Trp Thr His Ser Asn Tyr Arg Ala Ser Lys Glu Ser Cys Ile Val Ile 115 120 125Phe Val Thr Thr Ser Pro Gly Arg Glu Trp Val Ile Cys Ala Pro Ala 130 135 140Ala Phe Leu Gly Cys Gly Ser Leu Gln Ala Pro Ser Pro Glu Ser Glu145 150 155 160Pro Ser Phe Pro Val Thr Arg Gly His His Gly Arg His Gly Asp Tyr 165 170 175His Arg Lys Leu Ile Gly Gln Thr Phe Glu Trp Val Val Val Arg Arg 180 185 190His Gly Gly Arg Ala Ile Gly Pro Arg Leu Ser Arg Val Thr Lys Ala 195 200 205Ala Gly Ala Arg Pro Pro Ala Gly Ala Gly Glu Gly Leu Arg Val Gly 210 215 220Phe Asp Leu Ile Asn Ala Pro Ile Pro Pro Ala Lys Gly Val Ser Ala225 230 235 240Arg Arg His Val Leu Ala Leu Glu Leu Pro Gln Leu Ser Lys 245 25031251PRTMus musculus 31Met Ala Trp Lys Gln Thr Pro Leu Leu Leu Leu Val Tyr Met Val Thr 1 5 10 15Thr Gly Ser Gly Arg Asp Arg Thr Asp Leu Leu Asn Val Cys Met Asp 20 25 30Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His Asp 35 40 45Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Ser Val Asn Thr Ser 50 55 60Gln Glu Leu His Lys Ala Asp Ser Arg Leu Tyr Phe Asn Trp Asp His 65 70 75 80Cys Gly Lys Met Glu Pro Ala Cys Lys Ser His Phe Ile Gln Asp Ser 85 90 95Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln Val 100 105 110Asp Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu Cys Lys 115 120 125Glu Asp Cys His Gln Trp Trp Glu Ala Cys Arg Thr Ser Phe Thr Cys 130 135 140Lys Arg Asp Trp His Lys Gly Trp Asp Trp Ser Ser Gly Ile Asn Lys145 150 155 160Cys Pro Asn Thr Ala Pro Cys His Thr Phe Glu Tyr Tyr Phe Pro Thr 165 170 175Pro Ala Ser Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys Val Ser 180 185 190Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Ser 195 200 205Thr Gln Gly Asn Pro Asn Glu Asp Val Val Lys Phe Tyr Ala Ser Phe 210 215 220Met Thr Ser Gly Thr Val Pro His Ala Ala Val Leu Leu Val Pro Ser225 230 235 240Leu Ala Pro Val Leu Ser Leu Trp Leu Pro Gly 245 25032255PRTMus musculus 32Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Asn Leu His Asp Gln Cys Ser Pro Trp Lys Thr Asn Ser Cys Cys 50 55 60Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Ile Ser Tyr Leu Tyr 65 70 75 80Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Ser Glu Cys Lys Arg 85 90 95His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Gln Trp Trp Glu Asp Cys 130 135 140Gln Ser Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Ser Ser Gly His Asn Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe 165 170 175Thr Phe Tyr Phe Pro Thr Ser Ala Ala Leu Cys Glu Glu Ile Trp Ser 180 185 190His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Arg Phe Tyr Ala Glu Ala Met Ser Gly Ala Gly Leu His Gly Thr Trp225 230 235 240Pro Leu Leu Cys Ser Leu Ser Leu Val Leu Leu Trp Val Ile Ser 245 250 25533243PRTHomo sapiens 33Met Ala Trp Gln Met Met Gln Leu Leu Leu Leu Ala Leu Val Thr Ala 1 5 10 15Ala Gly Ser Ala Gln Pro Arg Ser Ala Arg Ala Arg Thr Asp Leu Leu 20 25 30Asn Val Cys Met Asn Ala Lys His His Lys Thr Gln Pro Ser Pro Glu 35 40 45Asp Glu Leu Tyr Gly Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys 50 55 60Thr Ala Ser Thr Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr 65 70 75 80Asn Phe Asn Trp Asp His Cys Gly Lys Met Glu Pro Thr Cys Lys Arg 85 90 95His Phe Ile Gln Asp Ser Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Arg Gln Val Asn Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Arg Trp Trp Glu Asp Cys 130 135 140Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Thr Ser Gly Ile Asn Glu Cys Pro Ala Gly Ala Leu Cys Ser Thr Phe 165 170 175Glu Ser Tyr Phe Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser 180 185 190His Ser Phe Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Lys Phe Tyr Ala Ala Ala Met Asn Ala Gly Ala Pro Ser Arg Gly Ile225 230 235 240Ile Asp Ser34255PRTHomo sapiens 34Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu Leu Val Cys Val Ala 1 5 10 15Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val Cys Met 20 25 30Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His 35 40 45Asp Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 50 55 60Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp 65 70 75 80Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 85 90 95Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln 100 105 110Gln Val Asn Gln Thr Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu 115 120 125Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His Thr Ser His 130 135 140Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val145 150 155 160Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe 165 170 175Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 180 185 190Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe 195 200 205Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 210 215 220Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly225 230 235 240Leu Leu Leu Ser Leu Ala Leu Met Leu Gln Leu Trp Leu Leu Gly 245 250 25535255PRTRAT 35Met Ala His Leu Met Ala Gly Gln Trp Leu Leu Leu Leu Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Lys Leu His Asp Gln Cys Ser Pro Trp Lys Thr Asn Ala Cys Cys 50 55 60Ser Thr Asn Thr Ser Gln Glu Asp Thr Lys Asp Ile Ser Tyr Leu Tyr 65 70 75 80Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Pro Glu Cys Lys Arg 85 90 95His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asp Val Pro Leu Cys Lys Glu Asp Cys Val Leu Trp Trp Glu Asp Cys 130 135 140Lys Ser Ser Phe Thr Cys Lys Ser Asn Trp Leu Lys Gly Trp Asn Trp145 150 155 160Thr Ser Gly His Asn Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe 165 170 175Thr Phe Tyr Phe Pro Thr Pro Ala Val Leu Cys Glu Lys Ile Trp Ser 180 185 190His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Arg Phe Tyr Ala Glu Val Met Ser Gly Ala Gly Leu Arg Glu Ala Trp225 230 235 240Leu Leu Val Cys Ser Leu Ser Leu Val Leu Phe Cys Val Val Ser 245 250 2553646PRTchicken 36Met Leu Arg Phe Ala Ile Thr Leu Phe Ala Val Ile Thr Ser Ser Thr 1 5 10 15Cys Gln Gln Tyr Gly Cys Leu Glu Gly Asp Thr His Lys Ala Asn Pro 20 25 30Ser Pro Glu Pro Asn Met His Glu Cys Thr Leu Tyr Ser Glu 35 40 4537238PRTchicken 37Met Leu Arg Phe Ala Ile Thr Leu Phe Ala Val Ile Thr Ser Ser Thr 1 5 10 15Cys Gln Gln Tyr Gly Cys Leu Glu Gly Asp Thr His Lys Ala Asn Pro 20 25 30Ser Pro Glu Pro Asn Met His Glu Cys Thr Leu Tyr Ser Glu Ser Ser 35 40 45Cys Cys Tyr Ala Asn Phe Thr Glu Gln Leu Ala His Ser Pro Ile Ile 50 55 60Lys Val Ser Asn Ser Tyr Trp Asn Arg Cys Gly Gln Leu Ser Lys Ser 65 70 75 80Cys Glu Asp Phe Thr Lys Lys Ile Glu Cys Phe Tyr Arg Cys Ser Pro 85 90 95His Ala Ala Arg Trp Ile Asp Pro Arg Tyr Thr Ala Ala Ile Gln Ser 100 105 110Val Pro Leu Cys Gln Ser Phe Cys Asp Asp Trp Tyr Glu Ala Cys Lys 115 120 125Asp Asp Ser Ile Cys Ala His Asn Trp Leu Thr Asp Trp Glu Arg Asp 130 135 140Glu Ser Gly Glu Asn His Cys Lys Ser Lys Cys Val Pro Tyr Ser Glu145 150 155 160Met Tyr Ala Asn Gly Thr Asp Met Cys Gln Ser Met Trp Gly Glu Ser 165 170 175Phe Lys Val Ser Glu Ser Ser Cys Leu Cys Leu Gln Met Asn Lys Lys 180 185 190Asp Met Val Ala Ile Lys His Leu Leu Ser Glu Ser Ser Glu Glu Ser 195 200 205Ser Ser Met Ser Ser Ser Glu Glu His Ala Cys Gln Lys Lys Leu Leu 210 215 220Lys Phe Glu Ala Leu Gln Gln Glu Glu Gly Glu Glu Arg Arg225 230 23538251PRTMus musculus 38Met Ala Trp Lys Gln Thr Pro Leu Leu Leu Leu Val Tyr Met Val Thr 1 5 10 15Thr Gly Ser Gly Arg Asp Arg Thr Asp Leu Leu Asn Val Cys Met Asp 20 25 30Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His Asp 35 40 45Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Ser Val Asn Thr Ser 50 55 60Gln Glu Leu His Lys Ala Asp Ser Arg Leu Tyr Phe Asn Trp Asp His 65 70 75 80Cys Gly Lys Met Glu Pro Ala Cys Lys Ser His Phe Ile Gln Asp Ser 85

90 95Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln Val 100 105 110Asp Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu Cys Lys 115 120 125Glu Asp Cys His Gln Trp Trp Glu Ala Cys Arg Thr Ser Phe Thr Cys 130 135 140Lys Arg Asp Trp His Lys Gly Trp Asp Trp Ser Ser Gly Ile Asn Lys145 150 155 160Cys Pro Asn Thr Ala Pro Cys His Thr Phe Glu Tyr Tyr Phe Pro Thr 165 170 175Pro Ala Ser Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys Val Ser 180 185 190Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Ser 195 200 205Thr Gln Gly Asn Pro Asn Glu Asp Val Val Lys Phe Tyr Ala Ser Phe 210 215 220Met Thr Ser Gly Thr Val Pro His Ala Ala Val Leu Leu Val Pro Ser225 230 235 240Leu Ala Pro Val Leu Ser Leu Trp Leu Pro Gly 245 25039255PRTMus musculus 39Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Asn Leu His Asp Gln Cys Ser Pro Trp Lys Thr Asn Ser Cys Cys 50 55 60Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Ile Ser Tyr Leu Tyr 65 70 75 80Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Ser Glu Cys Lys Arg 85 90 95His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Gln Trp Trp Glu Asp Cys 130 135 140Gln Ser Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Ser Ser Gly His Asn Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe 165 170 175Thr Phe Tyr Phe Pro Thr Ser Ala Ala Leu Cys Glu Glu Ile Trp Ser 180 185 190His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Arg Phe Tyr Ala Glu Ala Met Ser Gly Ala Gly Leu His Gly Thr Trp225 230 235 240Pro Leu Leu Cys Ser Leu Ser Leu Val Leu Leu Trp Val Ile Ser 245 250 25540222PRTCowMOD_RES(55)Xaa = anything 40Ala Gln Ala Pro Arg Thr Pro Arg Ala Arg Thr Asp Leu Leu Asn Val 1 5 10 15Cys Met Asp Ala Lys His His Lys Ala Glu Pro Gly Pro Glu Asp Ser 20 25 30Leu His Glu Gln Cys Ser Pro Trp Arg Lys Asn Ala Cys Cys Ser Val 35 40 45Asn Thr Ser Ile Glu Ala Xaa Lys Asp Ile Ser Tyr Leu Tyr Arg Phe 50 55 60Asn Trp Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe 65 70 75 80Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp 85 90 95Ile Arg Glu Val Asn Gln Arg Trp Arg Lys Glu Arg Val Leu Gly Val 100 105 110Pro Leu Cys Lys Glu Asp Cys Gln Ser Trp Trp Glu Asp Cys Arg Thr 115 120 125Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser 130 135 140Gly Tyr Asn Gln Cys Pro Val Lys Ala Ala His Cys Arg Phe Asp Phe145 150 155 160Tyr Phe Pro Thr Pro Ala Ala Leu Cys Asn Glu Ile Trp Ser His Ser 165 170 175Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met 180 185 190Trp Phe Asp Pro Phe Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe 195 200 205Tyr Ala Glu Asn Pro Thr Ser Gly Ser Thr Pro Gln Gly Ile 210 215 22041261PRTHamster sp. 41Met Ala His Leu Met Thr Met Gln Leu Leu Leu Leu Leu Ile Trp Val 1 5 10 15Ser Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Asn Leu His Asn Gln Cys Ser Pro Trp Lys Lys Asn Ser Cys Cys 50 55 60Ser Thr Asn Thr Ser Gln Glu Ala His Glu Asp Ile Ser Tyr Leu Tyr 65 70 75 80Arg Phe Asn Trp Asp His Cys Gly Lys Met Thr Leu Glu Cys Lys Arg 85 90 95His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys 130 135 140Arg Thr Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Thr Ser Gly Tyr Asn Gln Cys Pro Val Gly Ala Ser Cys Arg His Phe 165 170 175Asp Phe Tyr Phe Pro Thr Pro Ala Ala Leu Cys Glu Glu Ile Trp Ser 180 185 190His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Arg Phe Tyr Ala Glu Ala Met Ser Gly Ala Gly Leu His Gly Ala Trp225 230 235 240Pro Leu Met Cys Ser Leu Ser Leu Val Leu Leu Trp Val Phe Ser Arg 245 250 255Val Pro Leu Thr Phe 26042249PRTPig 42Met Ala Leu Gly Arg Ala Arg Leu Leu Leu Leu Leu Val Cys Val Ala 1 5 10 15Val Thr Trp Ala Ala Arg Pro Asp Leu Leu Asn Ile Cys Met Asp Ala 20 25 30Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Gly Leu His Glu Gln 35 40 45Cys Ser Pro Trp Glu Met Asn Ala Cys Cys Ser Val Asn Thr Ser Gln 50 55 60Glu Ala His Asn Asp Ile Ser Tyr Leu Tyr Lys Phe Asn Trp Glu His 65 70 75 80Cys Gly Lys Met Lys Pro Ala Cys Lys Arg His Phe Ile Gln Asp Thr 85 90 95Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Glu Val 100 105 110Asn Gln Lys Trp Arg Arg Glu Arg Ile Leu Asn Val Pro Leu Cys Lys 115 120 125Glu Asp Cys Gln Asn Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr Cys 130 135 140Lys Ser Asn Trp His Glu Gly Trp Asn Trp Ser Ser Gly Tyr Asn Arg145 150 155 160Cys Pro Ala Asn Ala Ala Cys His Pro Phe Asp Phe Tyr Phe Pro Thr 165 170 175Pro Ala Ala Leu Cys Ser Gln Ile Trp Ser Asn Ser Tyr Lys Gln Ser 180 185 190Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Pro 195 200 205Glu Gln Gly Asn Pro Asn Glu Val Val Ala Arg Tyr Tyr Ala Gln Ile 210 215 220Met Ser Gly Ala Gly Leu Ser Glu Ala Trp Pro Leu Gln Phe Gly Leu225 230 235 240Ala Leu Thr Leu Leu Trp Leu Leu Ser 24543252PRTPig 43Met Ala Trp Arg Leu Thr Leu Phe Val Leu Leu Gly Leu Val Ala Ala 1 5 10 15Val Gly Gly Ala Arg Ala Lys Ser Asp Met Leu Asn Val Cys Met Asp 20 25 30Ala Lys His His Lys Pro Lys Pro Ser Pro Glu Asp Lys Leu His Asp 35 40 45Gln Cys Ser Pro Trp Arg Lys Asn Ser Cys Cys Ser Val Asn Thr Ser 50 55 60Leu Glu Ala His Lys Asp Ile Ser Tyr Leu Tyr Arg Phe Asn Trp Asp 65 70 75 80His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln Asp 85 90 95Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Glu 100 105 110Val Asn Gln Lys Trp Arg Arg Glu Arg Ile Leu Asn Val Pro Leu Cys 115 120 125Lys Glu Asp Cys Gln Ile Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr 130 135 140Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser Gly Tyr Asn145 150 155 160Gln Cys Pro Val Ser Ala Ala Cys His Arg Phe Asp Phe Tyr Phe Pro 165 170 175Thr Pro Ala Ala Leu Cys Asn Glu Ile Trp Ser His Ser Phe Glu Val 180 185 190Ser Ser Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp 195 200 205Pro Ala Gln Gly Asn Pro Asn Glu Ala Val Ala Arg Tyr Tyr Ala Glu 210 215 220Asn Gly Asp Ala Gly Ala Val Ala Gln Gly Ile Gly Pro Leu Leu Thr225 230 235 240Asn Leu Thr Glu Met Val Lys His Trp Val Thr Gly 245 2504442PRTMus musculus 44Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys 35 4045255PRTMus musculus 45Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Asn Leu His Asp Gln Cys Ser Pro Trp Lys Thr Asn Ser Cys Cys 50 55 60Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Ile Ser Tyr Leu Tyr 65 70 75 80Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Ser Glu Cys Lys Arg 85 90 95His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Gln Trp Trp Glu Asp Cys 130 135 140Gln Ser Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Ser Ser Gly His Asn Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe 165 170 175Thr Phe Tyr Phe Pro Thr Ser Ala Ala Leu Cys Glu Glu Ile Trp Ser 180 185 190His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Arg Phe Tyr Ala Glu Ala Met Ser Gly Ala Gly Phe His Gly Thr Trp225 230 235 240Pro Leu Leu Cys Ser Leu Ser Leu Val Leu Leu Trp Val Ile Ser 245 250 2554654PRTHomo sapiens 46Met Ala Trp Gln Met Met Gln Leu Leu Leu Leu Ala Leu Val Thr Ala 1 5 10 15Ala Gly Ser Ala Gln Pro Arg Ser Ala Arg Ala Arg Thr Asp Leu Leu 20 25 30Asn Val Cys Met Asn Ala Lys His His Lys Thr Gln Pro Ser Pro Glu 35 40 45Asp Glu Leu Tyr Gly Gln 504785PRTHomo sapiens 47Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln 1 5 10 15Val Asp Gln Ser Trp Arg Lys Glu Arg Val Leu Asn Val Pro Leu Cys 20 25 30Lys Glu Asp Cys Glu Gln Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr 35 40 45Cys Lys Ser Asn Trp His Lys Gly Cys Asn Trp Thr Ser Gly Phe Asn 50 55 60Lys Cys Ala Val Gly Ala Ala Cys Gln Pro Phe His Phe Tyr Phe Pro 65 70 75 80Thr Pro Ile Ala Arg 8548245PRTHomo sapiens 48Met Leu Pro Ala Ala Thr Glu Val Gln His Arg Leu Gln Gly Gln Lys 1 5 10 15Asp Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu Leu Val Cys Val 20 25 30Ala Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val Cys 35 40 45Met Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu 50 55 60His Asp Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser 65 70 75 80Thr Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn 85 90 95Trp Asp His Cys Gly Lys Met Glu Pro Ala Cys Ser Ala Thr Ser Ser 100 105 110Arg Thr Pro Val Ser Met Ser Ala His Gln Pro Gly Ala Leu Asp Pro 115 120 125Ala Gly Glu Ser Glu Leu Ala Ala Lys Asn Ala Ser Trp Met Cys Pro 130 135 140Tyr Ala Lys Ser Thr Val Ser Ala Gly Gly Arg Ile Val Thr Pro Pro145 150 155 160Thr Arg Ala Arg Ala Thr Gly Thr Glu Asp Gly Thr Gly Pro Gln Glu 165 170 175Leu Thr Ser Ala Gln Leu Gly Leu Ser Ala Ala Pro Leu Ser Pro Thr 180 185 190Ser Pro Leu Gln Leu Pro Phe Val Lys Ala Ser Gly Val Thr His Thr 195 200 205Arg Ser Ala Thr Thr Ala Glu Gly Ala Ala Ala Ala Ser Arg Cys Gly 210 215 220Leu Leu Gln Pro Arg Ala Thr Pro Thr Arg Lys Trp Arg Gly Ser Met225 230 235 240Leu Gln Pro Cys Met 24549253PRTPig 49Met Pro Trp Lys Leu Thr Ala Leu Leu Leu Phe Leu Ala Gly Val Val 1 5 10 15Ser Val Cys Arg Ala Arg Ala Arg Thr Asp Leu Leu Asn Val Cys Met 20 25 30Asp Ala Lys His His Lys Val Glu Pro Gly Pro Glu Asp Glu Leu His 35 40 45Asp Gln Cys Val Pro Trp Lys Lys Asn Ala Cys Cys Ser Ala Arg Val 50 55 60Ser His Glu Leu His Arg Asp Lys Ser Ser Leu Tyr Asn Phe Ser Trp 65 70 75 80Glu His Cys Gly Arg Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 85 90 95Asn Asn Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Phe Gln 100 105 110Glu Val Asn Gln Lys Trp Arg Lys Glu Arg Phe Leu Asn Val Pro Leu 115 120 125Cys Lys Glu Asp Cys Leu Asp Trp Trp Glu Asp Cys Arg Thr Ser Tyr 130 135 140Thr Cys Lys Ser Ser Trp His Lys Gly Trp Asn Trp Ser Ser Gly Ser145 150 155 160Asn Gln Cys Pro Thr Gly Thr Thr Cys Asp Thr Phe Glu Ser Phe Phe 165 170 175Pro Thr Pro Ala Ala Leu Cys Glu Gly Ile Trp Asn His Asp Tyr Lys 180 185 190Phe Thr Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe 195 200 205Asp Ala Ala Glu Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 210 215 220Leu Ala Leu Ser Ala Gly Thr Met Ser Leu Gly Thr Gly Pro Leu Leu225 230 235 240Leu Ser Ala Ala Leu Met Leu Pro Leu Gly Leu Leu Asp 245 2505022PRTRAT 50Gln Ala Thr Arg Ala Glu Thr Glu Asn Leu Asn Val Asp Met Asp Ala 1 5 10 15Lys His His Lys Glu Lys 2051222PRTCowMOD_RES(55)Xaa = anything 51Ala Gln Ala Pro Arg Thr Pro Arg Ala Arg Thr Asp Leu Leu Asn Val 1 5 10 15Cys Met Asp Ala Lys His His Lys Ala Glu Pro Gly Pro Glu Asp Ser 20 25 30Leu His Glu Gln Cys Ser Pro Trp Arg Lys Asn Ala Cys Cys Ser Val 35 40 45Asn Thr Ser Ile Glu Ala Xaa Lys Asp Ile Ser Tyr Leu Tyr Arg Phe 50 55 60Asn Trp Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe 65 70 75 80Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp

85 90 95Ile Arg Glu Val Asn Gln Arg Trp Arg Lys Glu Arg Val Leu Gly Val 100 105 110Pro Leu Cys Lys Glu Asp Cys Gln Ser Trp Trp Glu Asp Cys Arg Thr 115 120 125Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser 130 135 140Gly Tyr Asn Gln Cys Pro Val Lys Ala Ala His Cys Arg Phe Asp Phe145 150 155 160Tyr Phe Pro Thr Pro Ala Ala Leu Cys Asn Glu Ile Trp Ser His Ser 165 170 175Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met 180 185 190Trp Phe Asp Pro Phe Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe 195 200 205Tyr Ala Glu Asn Pro Thr Ser Gly Ser Thr Pro Gln Gly Ile 210 215 22052114PRTHomo SapiensMOD_RES(12)..(92)X = anything 52Ile Ala Trp Ala Arg Thr Glu Leu Leu Asn Val Xaa Met Asn Ala Lys 1 5 10 15His His Lys Glu Lys Pro Gly Pro Glu Asp Lys Leu His Glu Gln Xaa 20 25 30Xaa Pro Trp Arg Lys Asn Ala Xaa Xaa Ser Thr Xaa Thr Xaa Gln Glu 35 40 45Ala Xaa Lys Asp Val Ser Tyr Leu Tyr Arg Phe Asn Ala Pro Ala Cys 50 55 60Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Xaa Ser Pro Asn 65 70 75 80Leu Gly Pro Xaa Ile Gln Gln Val Asp Gln Ser Xaa Arg Lys Glu Arg 85 90 95Val Leu Asn Val Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Gln 100 105 110Val Ala53257PRTHomo Sapiens 53Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser54243PRTHomo Sapiens 54Met Ala Trp Gln Met Met Gln Leu Leu Leu Leu Ala Leu Val Thr Ala 1 5 10 15Ala Gly Ser Ala Gln Pro Arg Ser Ala Arg Ala Arg Thr Asp Leu Leu 20 25 30Asn Val Cys Met Asn Ala Lys His His Lys Thr Gln Pro Ser Pro Glu 35 40 45Asp Glu Leu Tyr Gly Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys 50 55 60Thr Ala Ser Thr Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr 65 70 75 80Asn Phe Asn Trp Asp His Cys Gly Lys Met Glu Pro Thr Cys Lys Arg 85 90 95His Phe Ile Gln Asp Ser Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Arg Gln Val Asn Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Arg Trp Trp Glu Asp Cys 130 135 140Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Thr Ser Gly Ile Asn Glu Cys Pro Ala Gly Ala Leu Cys Ser Thr Phe 165 170 175Glu Ser Tyr Phe Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser 180 185 190His Ser Phe Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Lys Phe Tyr Ala Ala Ala Met Asn Ala Gly Ala Pro Ser Arg Gly Ile225 230 235 240Ile Asp Ser55226PRTHomo Sapiens 55Thr Arg Ile Ala Trp Ala Arg Thr Glu Leu Leu Asn Val Cys Met Asn 1 5 10 15Ala Lys His His Lys Glu Lys Pro Gly Pro Glu Asp Lys Leu His Glu 20 25 30Gln Cys Arg Pro Trp Arg Lys Asn Ala Cys Cys Ser Thr Asn Thr Ser 35 40 45Gln Glu Ala His Lys Asp Val Ser Tyr Leu Tyr Arg Phe Asn Trp Asn 50 55 60His Cys Gly Glu Met Ala Pro Ala Cys Lys Arg His Phe Ile Gln Asp 65 70 75 80Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln 85 90 95Val Asp Gln Ser Trp Arg Lys Glu Arg Val Leu Asn Val Pro Leu Cys 100 105 110Lys Glu Asp Cys Glu Gln Trp Trp Glu Asp Cys Arg Thr Ser Tyr Thr 115 120 125Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser Gly Phe Asn 130 135 140Lys Cys Ala Val Gly Ala Ala Cys Gln Pro Phe His Phe Tyr Phe Pro145 150 155 160Ser Pro Thr Val Leu Cys Asn Glu Ile Trp Thr His Ser Tyr Lys Val 165 170 175Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp 180 185 190Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala Ala 195 200 205Ala Met Ser Gly Ala Gly Pro Trp Ala Ala Trp Pro Phe Leu Leu Ser 210 215 220Leu Ala22556238PRTChicken 56Met Leu Arg Phe Ala Ile Thr Leu Phe Ala Val Ile Thr Ser Ser Thr 1 5 10 15Cys Gln Gln Tyr Gly Cys Leu Glu Gly Asp Thr His Lys Ala Lys Pro 20 25 30Ser Pro Glu Pro Asn Met His Glu Cys Thr Leu Tyr Ser Glu Ser Ser 35 40 45Cys Cys Tyr Ala Asn Phe Thr Glu Gln Leu Ala His Ser Pro Ile Ile 50 55 60Lys Val Ser Asn Ser Tyr Trp Asn Arg Cys Gly Gln Leu Ser Lys Ser 65 70 75 80Cys Glu Asp Phe Thr Lys Lys Ile Glu Cys Phe Tyr Arg Cys Ser Pro 85 90 95His Ala Ala Arg Trp Ile Asp Pro Arg Tyr Thr Ala Ala Ile Gln Ser 100 105 110Val Pro Leu Cys Gln Ser Phe Cys Asp Asp Trp Tyr Glu Ala Cys Lys 115 120 125Asp Asp Ser Ile Cys Ala His Asn Trp Leu Thr Asp Trp Glu Arg Asp 130 135 140Glu Ser Gly Glu Asn His Cys Lys Ser Lys Cys Val Pro Tyr Ser Glu145 150 155 160Met Tyr Ala Asn Gly Thr Asp Met Cys Gln Ser Met Trp Gly Glu Ser 165 170 175Phe Lys Val Ser Glu Ser Ser Cys Leu Cys Leu Gln Met Asn Lys Lys 180 185 190Asp Met Val Ala Ile Lys His Leu Leu Ser Glu Ser Ser Glu Glu Ser 195 200 205Ser Ser Met Ser Ser Ser Glu Glu His Ala Cys Gln Lys Lys Leu Leu 210 215 220Lys Phe Glu Ala Leu Gln Gln Glu Glu Gly Glu Glu Arg Arg225 230 23557251PRTMus musculus 57Met Ala Trp Lys Gln Thr Pro Leu Leu Leu Leu Val Tyr Met Val Thr 1 5 10 15Thr Gly Ser Gly Arg Asp Arg Thr Asp Leu Leu Asn Val Cys Met Asp 20 25 30Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His Asp 35 40 45Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Ser Val Asn Thr Ser 50 55 60Gln Glu Leu His Lys Ala Asp Ser Arg Leu Tyr Phe Asn Trp Asp His 65 70 75 80Cys Gly Lys Met Glu Pro Ala Cys Lys Ser His Phe Ile Gln Asp Ser 85 90 95Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln Val 100 105 110Asp Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu Cys Lys 115 120 125Glu Asp Cys His Gln Trp Trp Glu Ala Cys Arg Thr Ser Phe Thr Cys 130 135 140Lys Arg Asp Trp His Lys Gly Trp Asp Trp Ser Ser Gly Ile Asn Lys145 150 155 160Cys Pro Asn Thr Ala Pro Cys His Thr Phe Glu Tyr Tyr Phe Pro Thr 165 170 175Pro Ala Ser Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys Val Ser 180 185 190Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Ser 195 200 205Thr Gln Gly Asn Pro Asn Glu Asp Val Val Lys Phe Tyr Ala Ser Phe 210 215 220Met Thr Ser Gly Thr Val Pro His Ala Ala Val Leu Leu Val Pro Ser225 230 235 240Leu Ala Pro Val Leu Ser Leu Trp Leu Pro Gly 245 2505830PRTMus musculus 58Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu 20 25 305930PRTMus musculus 59Met Phe Gly Leu Lys Phe Phe Leu Val Leu Glu Ala Leu Leu Phe Leu 1 5 10 15Phe Thr Cys Tyr Ile Val Leu Lys Ile Gly Leu Lys Ile Leu 20 25 306049PRTMus musculus 60Met Ala Trp Lys Gln Thr Pro Leu Leu Leu Leu Val Tyr Met Val Thr 1 5 10 15Thr Gly Ser Gly Arg Asp Arg Thr Asp Leu Leu Asn Val Cys Met Asp 20 25 30Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His Asp 35 40 45Gln6154PRTMus musculus 61Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Asn Leu His Asp Gln 5062255PRTMus musculus 62Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met 1 5 10 15Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu 20 25 30Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu 35 40 45Asp Asn Leu His Asp Gln Cys Ser Pro Trp Lys Thr Asn Ser Cys Cys 50 55 60Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Ile Ser Tyr Leu Tyr 65 70 75 80Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Ser Glu Cys Lys Arg 85 90 95His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Gln Trp Trp Glu Asp Cys 130 135 140Gln Ser Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Ser Ser Gly His Asn Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe 165 170 175Thr Phe Tyr Phe Pro Thr Ser Ala Ala Leu Cys Glu Glu Ile Trp Ser 180 185 190His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Arg Phe Tyr Ala Glu Ala Met Ser Gly Ala Gly Leu His Gly Thr Trp225 230 235 240Pro Leu Leu Cys Ser Leu Ser Leu Val Leu Leu Trp Val Ile Ser 245 250 2556322PRTHomo Sapiens 63Thr Arg Ile Ala Trp Ala Arg Thr Glu Leu Leu Asn Val Cys Met Asn 1 5 10 15Ala Lys His His Lys Glu 2064257PRTHomo Sapiens 64Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser65257PRTHomo sapiens 65Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp145 150 155 160Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala225 230 235 240Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255Ser66255PRTHomo sapiens 66Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu Leu Val Cys Val Ala 1 5 10 15Thr Met Cys Ser Ala Gln Asp Arg Thr Asp

Leu Leu Asn Val Cys Met 20 25 30Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His 35 40 45Asp Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 50 55 60Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp 65 70 75 80Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 85 90 95Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln 100 105 110Gln Val Asn Gln Thr Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu 115 120 125Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His Thr Ser His 130 135 140Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val145 150 155 160Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe 165 170 175Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 180 185 190Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe 195 200 205Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 210 215 220Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly225 230 235 240Leu Leu Leu Ser Leu Ala Leu Met Leu Gln Leu Trp Leu Leu Gly 245 250 25567243PRTHomo sapiens 67Met Ala Trp Gln Met Met Gln Leu Leu Leu Leu Ala Leu Val Thr Ala 1 5 10 15Ala Gly Ser Ala Gln Pro Arg Ser Ala Arg Ala Arg Thr Asp Leu Leu 20 25 30Asn Val Cys Met Asn Ala Lys His His Lys Thr Gln Pro Ser Pro Glu 35 40 45Asp Glu Leu Tyr Gly Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys 50 55 60Thr Ala Ser Thr Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr 65 70 75 80Asn Phe Asn Trp Asp His Cys Gly Lys Met Glu Pro Thr Cys Lys Arg 85 90 95His Phe Ile Gln Asp Ser Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly 100 105 110Pro Trp Ile Arg Gln Val Asn Gln Ser Trp Arg Lys Glu Arg Ile Leu 115 120 125Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Arg Trp Trp Glu Asp Cys 130 135 140Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp145 150 155 160Thr Ser Gly Ile Asn Glu Cys Pro Ala Gly Ala Leu Cys Ser Thr Phe 165 170 175Glu Ser Tyr Phe Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser 180 185 190His Ser Phe Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile 195 200 205Gln Met Trp Phe Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala 210 215 220Lys Phe Tyr Ala Ala Ala Met Asn Ala Gly Ala Pro Ser Arg Gly Ile225 230 235 240Ile Asp Ser68255PRTHomo sapiens 68Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu Leu Val Cys Val Ala 1 5 10 15Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val Cys Met 20 25 30Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His 35 40 45Asp Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr 50 55 60Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp 65 70 75 80Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln 85 90 95Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln 100 105 110Gln Val Asn Gln Thr Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu 115 120 125Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys Leu Thr Ser His 130 135 140Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val145 150 155 160Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe 165 170 175Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys 180 185 190Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe 195 200 205Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala 210 215 220Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly225 230 235 240Leu Leu Leu Arg Leu Ala Leu Met Leu Gln Leu Trp Leu Leu Gly 245 250 25569478DNAHomo sapiens 69tctcattggg tcccattggc ctgaccctaa agcctgggtt cttttccacc agacctaatc 60tccatcgagc tggccttatc ctaagaacca cttggggtat ctataaaatc cagatgcccc 120ctggtgatga gcaattctct agattttgat gaaagttgaa tgtgtggatg ctggaatgag 180taaattaaca agtaaggaga tgaatgcaag caggaatgac taaatggaca gactcaggga 240gccttgaaga gggtggggtc tggaagggaa ggaagagagg aaggagaata gctaagtagg 300gagatttcac tcagtgctta ccagagcgcg ttgtctaccc tgtaccgaag acagaggctg 360tggggacagc ctaggggcct ggatctattg cctacttaga gagaggccaa ctcagacaca 420gccgtgtatg ctcccagcag caacggaggt tcaggcaaga tgcccgaaga gggaaggg 47870701DNASalmon 70gggggctggg acaggcggta gctcgcctcg cggcggaccg ccagctcgat cccgagatcc 60aactacgagc tttttaactg cagcaacttt aagatacgct attggagctg gaattaccgc 120ggctgctggc accagacttg ccctccaatg gatcctcgtt aaaggattta aagtgtactc 180attccaatta cagggcctcg aaagagtcct gtattgttat ttttcgtcac tacctccccg 240agtcgggagt gggtaatttg cgcgcctgct gccttccttg gatgtggtag ccgtttctca 300ggctccctct ccggaatcga accctgattc cccgttaccc gtggtcacca tggtaggcac 360agaaagtacc atcgaaagtt gatagggcag acattcgaat gagacgtcac cgccacaaag 420ggcgcgcgat cggctcgagg ttatctagag tcaccaaagc ggccggggca accgagattg 480gcccgcatgg gttttgggtc tgataaatgc acgcatcccc ggaggtcagc gctcgtctgc 540atgtattagc tctagaattg ccacagttat ccaagtaacg ttggagcgat caaaggaacc 600ataactgatt taatgagcca ttcgcagttt cactgtaccg gccgtgtgta cttagacttg 660catggcttaa tctttgagac aagcatatgc tactggcagg a 70171356DNAHomo sapiens 71gcggccgcct actactacta ctactgctcg aattcaagct tctaacgatg tacggggaca 60tgccgacggg cgctgacccc cttcgcgggg gggatgcgtg catttatcag atcaaaacca 120acccggtcag cccctctccg gccccggccg gggggcgggc gccggcggct ttggtgactc 180tagataacct cgggccgatc gcacgccccc cgtggcggcg acgacccatt cgaacgtctg 240ccctctccct taccaggacc acagctctgt tccttcggcc tctggtcctc tctggtcccc 300tcctgggttt cttacgtagt tgatttttcc tctttagtct cccccgacct gcgccc 35672155DNAFrog 72tttttttttt tttcaaagta aacgcttcgg gcccccggga cactcagtca agagcatcgg 60ggaggcgccg agaggcaggg gctgggacag gcggtagctc gcctcgcggc ggaccgccag 120ctcgatccca agatccaact acgagctttt taact 15573944DNAHomo sapiens 73tcaagattaa acgacaagga cagacatggc tcagcggatg acaacacagc tgctgctcct 60tctagtgtgg gtggctgtag taggggaggc tcagacaagg attgcatggg ccaggactga 120gcttctcaat gtctgcatga acgccaagca ccacaaggaa aagccaggcc ccgaggacaa 180gttgcatgag cagtgtcgac cctggaggaa gaatgcctgc tgttctacca acaccagcca 240ggaagcccat aaggatgttt cctacctata tagattcaac tggaaccact gtggagagat 300ggcacctgcc tgcaaacggc atttcatcca ggacacctgc ctctacgagt gctcccccaa 360cttggggccc tggatccagc aggtggatca gagctggcgc aaagagcggg tactgaacgt 420gcccctgtgc aaagaggact gtgagcaatg gtgggaagat tgtcgcacct cctacacctg 480caagagcaac tggcacaagg gctggaactg gacttcaggg tttaacaagt gcgcagtggg 540agctgcctgc caacctttcc atttctactt ccccacaccc actgttctgt gcaatgaaat 600ctggactcac tcctacaagg tcagcaacta cagccgaggg agtggccgct gcatccagat 660gtggttcgac ccagcccagg gcaaccccaa tgaggaggtg gcgaggttct atgctgcagc 720catgagtggg gctgggccct gggcagcctg gcctttcctg cttagcctgg ccctaatgct 780gctgtggctg ctcagctgac ctccttttac cttctgatac ctggaaatcc ctgccctgtt 840cagccccaca gctcccaact atttggttcc tgctccatgg tcgggcctct gacagccact 900ttgaataaac cagacaccgc acatgtgtct tgagaattat ttgg 944747720DNAHomo sapiens 74taagttgaca cttctcaggt tgtcacaaga ttcaggtatg gctcactgtt gcaggacata 60agctgggatc tcctgggaat tggtctgctt gcaggcccta gagagccttc cttcttggtt 120gattttcctc tagagatcca actgtcttct caggctcccc tgcctgcctc ctccttgggt 180cctttcttgt ggcattgcca gattactggg cccccatttt ccctacactt actgccactc 240atagtctgat ggttcccaca tctgcatcca acctggactc ttcccctgag ctttcccctc 300tacaaccacc ttccccgggc caagggcaca caggcacctc gacaaaacag tgttctatgt 360ttcttcctgc ccaaacctgc ccctccctct cccttttccc atctgtggta ccaccatggg 420ctcagagaat aaaaaaaatg aaggcttctg tcattgactg gggtggagat ggagggaaga 480gttagcccag aatcacaggt gctgtagaaa ggatacctga gttgccggga gagggggtcc 540atgagttggg gatggaagga gagcttggcc cttcaaacaa ttgaagatct gatcaaaaga 600ttcagaacat ctgtgatttt gtggctggtg atgggtgaca cctgggctaa tggggttggg 660ggagttggtg gctctacaat ttatggcctt gggagatcct tgctctctat agctgactgg 720gaggttggaa gcctgggctc tagcccttgc cttgatcctc cggatctcat tttcctcatc 780tgcctaacag gacagagggg ttggaaactg atgagattag ctcaaaggat cctggcagct 840caggctgcaa gatttttttc agacctcagt gtttgggaaa aaattgggta ggtggagctt 900agggactggc cttaggcctg cactgttaat tcaccccctc ccactacccc atggaggcct 960ggctggtgct cacatacaat aattaactgc tgagtggcct tcgcccaatc ccaggctcca 1020ctcctgggct ccattcccac tccctgcctg tctcctaggc cactaaacca cagctgtccc 1080ctggaataag gcaaggggga gtgtagagca gagcagaagc ctgagccaga cggagagcca 1140cctcctctcc caggtatgtg acactcccca tcccccttca gaggccacac accctatggc 1200attcccacca tgtgttaagg attttctgaa ctggaagggc cctctgtttg cctgaaggcc 1260agagaatctt gaagtggaga ctgaggccca gaccagagtg tggcctgctc aagattaaac 1320gacaagttag tgttcatccc cctgaactag tacctgggct ctagcccttc agtccagagc 1380tgagttctca gctcttctag tctggggccc caaggttggg tgtgggggtc atgattgttg 1440gtggggaggg gtcacagctg gactaagacc tgaaggtgag actaggcagg tgggaaagga 1500gcttgcagag tgatgctgct caaaaggaca ggaagagagc ctggcttcag aagcagccac 1560agcaagagag actactgact gaacaggtgg gctccactgg gggctccgga aaggattttc 1620tcagccccca tccccagcac tgtgtgttgg ccgcacccat gagagcctca gcactctgaa 1680ggtgcagggg gcaaaggcca aaagagctct ggcctgaact tgggtggtcc ctactgtgtg 1740acttggggca tggccctcat ctgtgctgaa atgattccac aaagattaaa ctggctatca 1800tttgttgatt tcccccttct tacatttaat ccttgcagga gaaagctaag cctcaagata 1860gtttgcttct ctttccccca aggccaagga gaaggtggag tgagggctgg ggtcgggaca 1920ggttgaacgg gaaccctgtg ctctaaacag ttagggtttg ttcccgcagg aactgaaccc 1980aaaggatcac ctggtattcc ctgagagtac agatttctcc ggcgtggccc tcaaggttag 2040tgagtgagca ggtccacagg ggcatgattg gatcctggaa tgaatgaatc aaccatgaga 2100gagtgaatga acactggaat caatagagta gcagagtaat ggattgtgga gcaggaaaga 2160gagctgctgg gtgggaattc aattccaggc ttatatgagc cctgctgtgc agtcggcctg 2220gagacagccc agctcaggcc ctgcctagac ccctgtcaag gaggccctgt caagaggaga 2280ggaggggcag cacgggggca aggcaagctt gtgagcggga aaggcatgtc cactttagcg 2340actggtatgt ggaagatgag ttagaggaga cagatggaga gaagtcatag gaaataaatt 2400ctgagcattt taggagggcc cagacacctg gtgtccagtg gagtgaagga aacagtcgcc 2460tcccaaaatt cagtgtctga ggtcaaagga ttgaagttct gtgatgacca aggagaagcc 2520agctctgtgg tagggggcac aggagctccc caaggcccca gggctgtcca gctggctgtc 2580ccctgccagc acccatgtcc tgtgacccca ccccaccaag atcccatggt ttccgggaag 2640ggcctactaa actagcttga gtgatgaggc tagaaagggg ctgggaccaa ggtttaaaaa 2700gcaaaacaaa ctaacaaaaa ccacactgca gcccccccaa ctaaaacatt tttataaact 2760tttttttttt ttttgagatg gagtctcgct ctgtcaccca ggctagagtg caatggcaca 2820atcttggctc actgtaacct ccacctcctg gattcaagtg attctcctgc ctcagcctcc 2880cacgtagctg ggactacagg cacacgacac cgcacccagc tcattttgta tttttagtag 2940agacagggtt tcactatgtt ggccaggctg gtctcaaact tctgacctca ggtgatccac 3000ccacctcagc cttccaaagt gctgggatta caggcatgag ccaccgcgcc cagcccattt 3060ttgtaaactt ttacaatgaa gtaatttggt gtcaaaatct gacctgaaaa ttaatgtgag 3120tttatgtata gttttaattt atcccactag tgtaactgtt tcaccccaga atatacactt 3180gattattggg tatatgaaaa aaatattttc tttgaatcac ctttgatgaa atcctaaaaa 3240attttaaccc tgaaacattt gaataaggca ttgtggacct atggcaaact cctggctatt 3300tctgcatttt gcccaaatcc atccttgaat tatatcacct gaacctcgtg accacctgga 3360gaaggcaatg aggctcaagc cagggagggg tggtgtctaa tcctaccttt cattggatct 3420gggaaaactg agggagatgg gggcagggct ctatctgccc caggcttccg tccaggcccc 3480accctcctgg agccctgcac acaacttaag gccccacctc cgcattcctt ggtgccactg 3540accacagctc tttcttcagg gacagacatg gctcagcgga tgacaacaca gctgctgctc 3600cttctagtgt gggtggctgt agtaggggag gctcagacaa ggattgcatg ggccaggact 3660gagcttctca atgtctgcat gaacgccaag caccacaagg aaaagccagg ccccgaggac 3720aagttgcatg agcaggtggg ccagggggtg atctggggtg gtgagggact ggctcaggaa 3780gaggaaacga ggacatggaa atgccaaacc ccattggcac tggtgaactg aagtggagga 3840gcccttcagt ttgcattaat atgggtgact tatttcagag acactgtgcc aaatgtcggt 3900acaatgccaa cagttcacct tcttggttgt tgagtttccg cattacagaa ataaggaagc 3960aggcccaaag gagagcctgg gaaatgaagt tggagtgacc catcctgggg ttgcttgatt 4020tagggattta gactgggaat gactcctcca aagatctgag ggaagaaact gcacactgtg 4080catagtggcc tcttttctgc cagccctaaa cagctcaaga agggagagtc tctcacatta 4140tgaggctgtg tgcaaagcat tctttttttt ttttcctgag acaaagtctc catatgttgc 4200ccaggctggt ctcaaattcc tggactcaag tgatcctccc acctcagccc tcccaaagtg 4260tgggattaca gaaatgagcc gtacgccctc ctgaagcatc ttggttcatg catctcgcaa 4320aactttgggc tgtgtctctc gaccacattg gacctgaggt ctccctataa catttatttt 4380gctaccaccc ctttaatatc ctgaacatga tgatataact aaagaaaaag cagaggaaaa 4440gtaatttgta ggccaggtgt tacggctcac gcctgtaatc ccaacactgt gggatgtcga 4500gatgggcaga tcacttgagc tcaggagttc gagaccagcc tgggcaagat ggcaaaaccc 4560catctctact aaaaaataaa aaaaattagt caggtgtggt ggcacatgcc tgcagtccca 4620gctactcagg aggctgaggt gggcaggtca gttgagccca ggaggcagag attgtagatc 4680gtgccactgc actccagcct gggcaacaga gtgagacctt gtcaaaagaa agaaagaacg 4740aaaaaaagaa agaaaggaag gaaggaaggg gaggaaggaa agggagggag gaaagggagg 4800gaggaaaggg agggaggcaa gggagagaaa cttgtaatac gcatttcttt ttttttttct 4860tgagatagag ttttgctctt gttgcccagg gtggatggca gtggcacaat ctcagctcac 4920tgcaacctcc acctcccagg ttcaagtgat tctcctgcct cagcctcctg agtaggcaca 4980cgccaccaca cccagctaat tttttgtttg tttgtttgtt ttgtttgttg gtatttttag 5040tagagatggg ggtttcacca tgttggccag gctggtctcg aactcctcac ctcataatcc 5100gcccctcttg gcctcccaaa gtgctgagat tacaggtgtg agccactgcg cccggcctta 5160agtgcacatt ttatttattt atttatttat ttatttattg agatggagtc ttgctctgtt 5220gcccaggctg gagtgcagtg gcacaatctc agctcactgc aacctccacc tcccaggttc 5280aagcaattct tctgccttgg cctccagagt agctgggact ataggcacct gccaccatgc 5340ctagctaatt tttgtatttt tagtagaaat ggggttttgc catgttggcc aggctggtct 5400ccattcttga ccttaagtga tctgtccacc tccacctccc aaagtgctgg gattacaggc 5460actatgtgag ccactgtgcc ggcccacatt ttaatattta gcttgtcagc cttaagtaat 5520gagattcagg aagcttgagg ataggcacac aggagcatag tttcaagttg tcctgaattt 5580tgcagccatc acaagttagt ttttaaggaa aaagattagt tcctaagttg tttctcaata 5640acttataata aaataacatc cacaattgat tggctataca ttgttttttt gtatcacaaa 5700ttccacaaac agataatggg tgaggcagct agtcagggac aaaacacttc ccaagtagct 5760gggattacag gtgtccgcca ccacacttgg ctagtttttt gtttgtttat tttttgagat 5820ggagtcttgc tctgtcgccc aggctggagt gcagtggcat gatctcggct cactgcaagc 5880tccacctgcc gggttcacac cattctcctg cctcagcctc ccaagtagct gggactacag 5940gtgccagcca ccacgcccgg ctaatttttt gtatttttag tagagacggg gtttcaccat 6000gttggccagg atggtcttga tctcttagcc tcgtgatcca cccgcctcgg cctcccaaaa 6060tgctgggatt acaggcgtga gccaccgcac ccggcctaat ttttatattt ttagtagaga 6120cggggtttca ccatgttggc caggctggtc tcaaactctt gatctcaggt gatccacctg 6180ccttggcctc ccaaagtgct gggattacac aagtaagcca ctgcacccag cctggggtta 6240caatttaaat tgctttttta ccttcaaatc tttgacacct cagtgaggct taatctgacc 6300gcactattac actacaagtc cccatccgtc tctgcttaat ttttgtccaa agcaaaaatc 6360aggtgatgtg ttcattgttg taaccccagt ttctacaaaa gtacctgggt gagagtaagt 6420aggatctcaa taaaggttga attaacaaat tttgtaatga ctgcaactcc agcaggagct 6480cccttttggg ctcccactgt ctctgacggc cctctcccct aaagaggtcc caatagcaag 6540tattttcctg ggtgacttcc agtgggctgg ggaatcaagg actaagaggg gagacactgc 6600atgtggaata ttctggctgt gctggctgtg ctggctgtgg actgagtcct ctgtcttccc 6660ccatccagtg tcgaccctgg aggaagaatg cctgctgttc taccaacacc agccaggaag 6720cccataagga tgtttcctac ctatatagat tcaactggaa ccactgtgga gagatggcac 6780ctgcctgcaa acggcatttc atccaggaca cctgcctcta cgagtgctcc cccaacttgg 6840ggccctggat ccagcaggta tgcatggctt cctgcaggta caagacctag cggagcagct 6900gagctttcca ggcatctctg caggctgcaa ccccagctcc agttctattc ggggctgagt 6960tgctgggatt cttgaacctg agcccttctt ttgtatcaaa atcacccagg tggatcagag 7020ctggcgcaaa gagcgggtac tgaacgtgcc cctgtgcaaa gaggactgtg agcaatggtg 7080ggaagattgt cgcacctcct acacctgcaa gagcaactgg cacaagggct ggaactggac 7140ttcaggtgag ggctggggtg ggcaggaatg gagggatttg gaagtggagg tgtgtgggtg 7200tggaacaggt atgtgacaat ttggagttgt agggctggca gacctcaaga tagttccggg 7260cccagtggct aaaggtcttc cctcctctct acagggttta acaagtgcgc agtgggagct 7320gcctgccaac ctttccattt ctacttcccc acacccactg ttctgtgcaa tgaaatctgg 7380actcactcct acaaggtcag caactacagc cgagggagtg gccgctgcat ccagatgtgg 7440ttcgacccag cccagggcaa ccccaatgag gaggtggcga ggttctatgc tgcagccatg 7500agtggggctg ggccctgggc agcctggcct ttcctgctta gcctggccct aatgctgctg 7560tggctgctca gctgacctcc ttttaccttc tgatacctgg aaatccctgc cctgttcagc 7620cccacagctc ccaactattt ggttcctgct ccatggtcgg gcctctgaca gccactttga 7680ataaaccaga caccgcacat gtgtcttgag aattatttgg 772075929DNAHomo sapiens

75agggacagac atggctcagc ggatgacaac acagctgctg ctccttctag tgtgggtggc 60tgtagtaggg gaggctcaga caaggattgc atgggccagg actgagcttc tcaatgtctg 120catgaacgcc aagcaccaca aggaaaagcc aggccccgag gacaagttgc atgagcagtg 180tcgaccctgg aggaagaatg cctgctgttc taccaacacc agccaggaag cccataagga 240tgtttcctac ctatatagat tcaactggaa ccactgtgga gagatggcac ctgcctgcaa 300acggcatttc atccaggaca cctgcctcta cgagtgctcc cccaacttgg ggccctggat 360ccagcaggtg gatcagagct ggcgcaaaga gcgggtactg aacgtgcccc tgtgcaaaga 420ggactgtgag caatggtggg aagattgtcg cacctcctac acctgcaaga gcaactggca 480caagggctgg aactggactt cagggtttaa caagtgcgca gtgggagctg cctgccaacc 540tttccatttc tacttcccca cacccactgt tctgtgcaat gaaatctgga ctcactccta 600caaggtcagc aactacagcc gagggagtgg ccgctgcatc cagatgtggt tcgacccagc 660ccagggcaac cccaatgagg aggtggcgag gttctatgct gcagccatga gtggggctgg 720gccctgggca gcctggcctt tcctgcttag cctggcccta atgctgctgt ggctgctcag 780ctgacctcct tttaccttct gatacctgga aatccctgcc ctgttcagcc ccacagctcc 840caactatttg gttcctgctc catggtcggg cctctgacag ccactttgaa taaaccagac 900accgcacatg tgtcttgaga attatttgg 929761331DNAHomo sapiens 76ggaaaggatt ttctcagccc ccatccccag cactgtgtgt tggccgcacc catgagagcc 60tcagcactct gaaggtgcag ggggcaaagg ccaaaagagc tctggcctga acttgggtgg 120tccctactgt gtgacttggg gcatggccct catctgtgct gaaatgattc cacaaagatt 180aaactggcta tcatttgttg atttccccct tcttacattt aatccttgca ggagaaagct 240aagcctcaag atagtttgct tctctttccc ccaaggccaa ggagaaggtg gagtgagggc 300tggggtcggg acaggttgaa cgggaaccct gtgctctaaa cagttagggt ttgttcccgc 360aggaactgaa cccaaaggat cacctggtat tccctgagag tacagatttc tccggcgtgg 420ccctcaaggg acagacatgg ctcagcggat gacaacacag ctgctgctcc ttctagtgtg 480ggtggctgta gtaggggagg ctcagacaag gattgcatgg gccaggactg agcttctcaa 540tgtctgcatg aacgccaagc accacaagga aaagccaggc cccgaggaca agttgcatga 600gcagtgtcga ccctggagga agaatgcctg ctgttctacc aacaccagcc aggaagccca 660taaggatgtt tcctacctat atagattcaa ctggaaccac tgtggagaga tggcacctgc 720ctgcaaacgg catttcatcc aggacacctg cctctacgag tgctccccca acttggggcc 780ctggatccag caggtggatc agagctggcg caaagagcgg gtactgaacg tgcccctgtg 840caaagaggac tgtgagcaat ggtgggaaga ttgtcgcacc tcctacacct gcaagagcaa 900ctggcacaag ggctggaact ggacttcagg gtttaacaag tgcgcagtgg gagctgcctg 960ccaacctttc catttctact tccccacacc cactgttctg tgcaatgaaa tctggactca 1020ctcctacaag gtcagcaact acagccgagg gagtggccgc tgcatccaga tgtggttcga 1080cccagcccag ggcaacccca atgaggaggt ggcgaggttc tatgctgcag ccatgagtgg 1140ggctgggccc tgggcagcct ggcctttcct gcttagcctg gccctaatgc tgctgtggct 1200gctcagctga cctcctttta ccttctgata cctggaaatc cctgccctgt tcagccccac 1260agctcccaac tatttggttc ctgctccatg gtcgggcctc tgacagccac tttgaataaa 1320ccagacaccg c 133177939DNAHomo sapiens 77cattccttgg tgccactgac cacagctctt tcttcaggga cagacatggc tcagcggatg 60acaacacagc tgctgctcct tctagtgtgg gtggctgtag taggggaggc tcagacaagg 120attgcatggg ccaggactga gcttctcaat gtctgcatga acgccaagca ccacaaggaa 180aagccaggcc ccgaggacaa gttgcatgag cagtgtcgac cctggaggaa gaatgcctgc 240tgttctacca acaccagcca ggaagcccat aaggatgttt cctacctata tagattcaac 300tggaaccact gtggagagat ggcacctgcc tgcaaacggc atttcatcca ggacacctgc 360ctctacgagt gctcccccaa cttggggccc tggatccagc aggtggatca gagctggcgc 420aaagagcggg tactgaacgt gcccctgtgc aaagaggact gtgagcaatg gtgggaagat 480tgtcgcacct cctacacctg caagagcaac tggcacaagg gctggaactg gacttcaggg 540tttaacaagt gcgcagtggg agctgcctgc caacctttcc atttctactt ccccacaccc 600actgttctgt gcaatgaaat ctggactcac tcctacaagg tcagcaacta cagccgaggg 660agtggccgct gcatccagat gtggttcgac ccagcccagg gcaaccccaa tgaggaggtg 720gcgaggttct atgctgcagc catgagtggg gctgggccct gggcagcctg gcctttcctg 780cttagcctgg ccctaatgct gctgtggctg ctcagctgac ctccttttac cttctgatac 840ctggaaatcc ctgccctgtt cagccccaca gctcccaact atttggttcc tgctccatgg 900tcgggcctct gacagccact ttgaataaac cagacaccg 939781104DNAHomo sapiens 78tggaggcctg gctggtgctc acatacaata attaactgct gagtggcctt cgcccaatcc 60caggctccac tcctgggctc cattcccact ccctgcctgt ctcctaggcc actaaaccac 120agctgtcccc tggaataagg caagggggag tgtagagcag agcagaagcc tgagccagac 180ggagagccac ctcctctccc agggacagac atggctcagc ggatgacaac acagctgctg 240ctccttctag tgtgggtggc tgtagtaggg gaggctcaga caaggattgc atgggccagg 300actgagcttc tcaatgtctg catgaacgcc aagcaccaca aggaaaagcc aggccccgag 360gacaagttgc atgagcagtg tcgaccctgg aggaagaatg cctgctgttc taccaacacc 420agccaggaag cccataagga tgtttcctac ctatatagat tcaactggaa ccactgtgga 480gagatggcac ctgcctgcaa acggcatttc atccaggaca cctgcctcta cgagtgctcc 540cccaacttgg ggccctggat ccagcaggtg gatcagagct ggcgcaaaga gcgggtactg 600aacgtgcccc tgtgcaaaga ggactgtgag caatggtggg aagattgtcg cacctcctac 660acctgcaaga gcaactggca caagggctgg aactggactt cagggtttaa caagtgcgca 720gtgggagctg cctgccaacc tttccatttc tacttcccca cacccactgt tctgtgcaat 780gaaatctgga ctcactccta caaggtcagc aactacagcc gagggagtgg ccgctgcatc 840cagatgtggt tcgacccagc ccagggcaac cccaatgagg aggtggcgag gttctatgct 900gcagccatga gtggggctgg gccctgggca gcctggcctt tcctgcttag cctggcccta 960atgctgctgt ggctgctcag ctgacctcct tttaccttct gatacctgga aatccctgcc 1020ctgttcagcc ccacagctcc caactatttg gttcctgctc catggtcggg cctctgacag 1080ccactttgaa taaaccagac accg 1104791032DNAHomo sapiens 79ggcaaggggg agtgtagagc agagcagaag cctgagccag acggagagcc acctcctctc 60ccaggaactg aacccaaagg atcacctggt attccctgag agtacagatt tctccggcgt 120ggccctcaag ggacagacat ggctcagcgg atgacaacac agctgctgct ccttctagtg 180tgggtggctg tagtagggga ggctcagaca aggattgcat gggccaggac tgagcttctc 240aatgtctgca tgaacgccaa gcaccacaag gaaaagccag gccccgagga caagttgcat 300gagcagtgtc gaccctggag gaagaatgcc tgctgttcta ccaacaccag ccaggaagcc 360cataaggatg tttcctacct atatagattc aactggaacc actgtggaga gatggcacct 420gcctgcaaac ggcatttcat ccaggacacc tgcctctacg agtgctcccc caacttgggg 480ccctggatcc agcaggtgga tcagagctgg cgcaaagagc gggtactgaa cgtgcccctg 540tgcaaagagg actgtgagca atggtgggaa gattgtcgca cctcctacac ctgcaagagc 600aactggcaca agggctggaa ctggacttca gggtttaaca agtgcgcagt gggagctgcc 660tgccaacctt tccatttcta cttccccaca cccactgttc tgtgcaatga aatctggact 720cactcctaca aggtcagcaa ctacagccga gggagtggcc gctgcatcca gatgtggttc 780gacccagccc agggcaaccc caatgaggag gtggcgaggt tctatgctgc agccatgagt 840ggggctgggc cctgggcagc ctggcctttc ctgcttagcc tggccctaat gctgctgtgg 900ctgctcagct gacctccttt taccttctga tacctggaaa tccctgccct gttcagcccc 960acagctccca actatttggt tcctgctcca tggtcgggcc tctgacagcc actttgaata 1020aaccagacac cg 103280765DNAHomo sapiens 80atggcctcag ttccgaaaac caacaaaata gaaccgcggt cctattccat tattcctagc 60tgcagtatca ggcggctcgg gcctgctttg aacactccaa tttttcaaag taaacgcaac 120gggccccgcg gacactcagc ttacagcatc gaggggcgcc agaggcaagg ggcgggacgg 180gcggtggtcc ctcgcgcgga ccgcccgccc gctcccaaga tccaactacg agctttttac 240ctgcagcaac tttactatac gctattggag ctggaattac cgcggctgct ggcaccagac 300ttgccctcca atggctcctc gttaaaggat ttaaagtgga ctcattccaa ttacagggcc 360tcgaaagagt cctgtattgt tattttcgtc actacctccc cgggtcggga gtgggtaatt 420tgcgcgcctg ctgccttcct tggatgtggt agcctccagg ctccctctcc ggaatctgaa 480ccctcattcc ccgtcacccg tggtcaccat ggtcggcacg gcgactacca tcgaaagttg 540atagggcaga cgttcgaatg ggtcgtcgtc cgccgccacg gggggcgtgc gatcggcccg 600aggttatcta gagtcaccaa agccgccggc gcccgccccc cggccggggc cggagagggg 660ctgagggttg gttttgatct gataaatgca ccgatccccc ccgcgaaggg ggtcagcgcc 720cgtcggcatg tattagctct agaattacca cagttatcca agtag 765811223DNAMus musculus 81gctttagagg cagatcaggg tgtagttttc agctagcgcc gtgccttccc caccatgttc 60cttgccatga tgataatgta ctagacctct gaaactgtag cttctttgtt acagagtctc 120cgtgaatctg gaattcacca attcggcgag tctgaaagcc tcagtgatct ctcaggctcc 180atctgtctcc actccccagt ggaaggcttg cagctgtgtc accgctccag acttcacaca 240ggtgctggaa gactgaacta agacagaaag acatggcctg gaaacagaca ccactcttgc 300ttttggtcta catggtcaca acaggcagtg gccgggacag aacagaccta ctcaacgttt 360gcatggatgc caaacaccat aagacaaagc cgggccccga ggacaagctg catgaccagt 420gtagtccatg gaagaaaaat gcctgttgct cagtcaacac cagccaggag ctacacaagg 480ctgactcccg tctgtacttc aactgggatc actgtggcaa gatggagcct gcctgtaaga 540gtcacttcat ccaagactcc tgcctgtatg agtgctcccc caaccttggg ccttggatcc 600agcaagtgga ccagagttgg cgtaaagagc gtttcctgga tgtgccctta tgcaaagagg 660actgtcacca gtggtgggaa gcctgtcgta cctcctttac ctgcaagaga gactggcata 720aaggctggga ctggtcctca ggcattaaca agtgcccaaa cacagcaccc tgtcacacgt 780ttgagtacta cttcccgaca ccagccagcc tttgcgaggg tctctggagt cactcctaca 840aggtcagcaa ctacagcaga gggagtggcc gctgcatcca gatgtggttt gactcaaccc 900agggcaatcc caatgaggac gtggtgaagt tttatgcttc ctttatgaca tctgggactg 960tgccccatgc agcagtactt cttgtgccca gcctggcccc agtgctgtca ttatggctcc 1020ctggctgaga ggtcagtctt cctctctaga tttctcctct atctaccctt ggtctggttc 1080aactcttcaa agaataagga agtcttgagc ctgcttccac ccctctcctc tgtcatccag 1140ttcctgatcc atgttggggg ttggggtttc tacaatcatt ttcaataaat ctatgacaca 1200tctgggccta atgaaaaaaa aaa 122382979DNAMus musculus 82tggagctgag cacacacttg gaggttccac ttaccttagc tctgccttca gggtctgaca 60tggctcacct gatgactgtg cagttgttgc tcctggtgat gtggatggcc gaatgtgctc 120agtccagagc tactcgggcc aggactgaac ttctcaatgt ctgcatggat gccaagcacc 180acaaagaaaa accgggccct gaggacaatt tacacgacca gtgcagcccc tggaagacga 240attcctgctg ttccacgaac acaagccagg aagcacataa ggacatttcc tacctgtacc 300ggttcaactg gaaccactgc ggaactatga catcggaatg caaacggcac tttatccaag 360acacctgcct ctatgagtgt tccccgaact tgggaccctg gatccagcag gtggaccaga 420gctggcgcaa agagcggatc cttgatgttc ccctgtgcaa agaggactgt cagcagtggt 480gggaggactg ccagagctct tttacctgca agagcaattg gcacaaggga tggaactggt 540cctctgggca taacgagtgt cctgtgggag cctcctgcca tcccttcacc ttctacttcc 600ccacatctgc tgctctgtgt gaggaaatct ggagtcactc ctacaagctc agcaactaca 660gccgagggag cggccgctgc attcagatgt ggtttgaccc agcccagggc aaccccaacg 720aggaagtggc gaggttctat gccgaggcca tgagtggagc tgggcttcat gggacctggc 780cactcttgtg cagcctgtcc ttagtgctgc tctgggtgat cagctgagtt cctgttttac 840cttcagttgt ctggagcgcc accctgcttg gctcagcctc ccagctccca gcctcctttg 900tggtggggct ctgacagcct ctttaataaa ccagacattc cacatgtgcc ttatgaatta 960aaaaaaaaaa aaaaaaaaa 97983647DNASolanum tuberosum 83ctccgatccc gaaggccaac gtaataggac cgaaatccta taatgttatc ccatgctaat 60gtatacagag cgtaggcttg ctttgagcac tctaatttct tcaaagtaac agcgccggag 120gcacgacccg gccaattaag gccaggagcg catcgccgac agaagggacg agacgaccgg 180tgcacaccta gggcggaccg gccggcccat cccaaagtcc aactacgagc tttttaactg 240caacaactta aatatacgct attggagctg gaattaccgc ggctgctggc accagacttg 300ccctccaatg gatcctcgtt aagggattta gattgtactc attccaatta ccagactcat 360agagcccggt attgttattt attgtcacta cctccccgtg tcaggattgg gtaatttgcg 420cgcctgctgc cttccttgga tgtggtagcc gtttctcagg ctccctctcc ggaatcgaac 480cctaattctc cgtcacccgt caccaccatg gtaggccact atcctaccat cgaaagttga 540tagggcagaa atttgaatga tgcgtcgccg gcacgatggc cgtgcgatcc gtcgagttat 600catgaatcat cgcagcaacg ggcagagccc gcgtcgacct tttatct 64784280DNASuaeda maritima supsb. salsa 84cgacgcatca ttcaaatttc tgccctatca actttcgatg gtaggatagt ggcctaccat 60ggtggtgacg ggtgacggag aattagggtt cgattccgga gagggagcct gagaaacggc 120taccacatcc aaggaaggca gtaggcgcgc aaattaccca atcctgacac ggggaggtag 180tgacaataaa taacaatacc gggctcttcg agtctggtaa ttggaatgag tacaatctaa 240atcccttaac gaggatccat tggagggcaa gtctggtgcc 28085300DNAAvicennia marina 85gcacggccct cgtgccggcg acgcatcatt caaatttctg ccctatcaac tttcgatggt 60aggatagtgg cctactatgg tggtgacggg tgacggagaa ttagggttcg attccggaga 120gggagcctga gaaacggcta ccacatccaa ggaaggcagc aggcgcgcaa attacccaat 180cctgatacgg ggaggtagtg acaataaata acaataccgg gctctcagag tctggtaatt 240ggatgagtac aatctaatcc ttaacgagga tccattggag ggcaagtctg gtgcacgagc 30086453DNAHomo sapiens 86gaagacacac gtttagtatt ttattatgaa tcattatttc aaagtcccat actgcatatt 60catataaggc aacacggcac aatttcaggc ttcatcacaa aggatgaaaa agactgtttc 120taactccctc ctaatttgca gacatgcttg aacacttaat ggaaggtgaa gtttattttg 180tggcccctca gttctctttc aagtcctcta gtagaaagtc tccatggtgt gatcttctga 240ctgggtagaa cccgcaattc tctgctgttt ttagtctttg ttccagatga ctaattacat 300gacttggctg catttgtgag gggccgacac caacacaatt aaatcagtgc accattcagg 360gccatagggt aggaggcacc agtggtcacc atggtaggca cggcgactac catcgaaagt 420tgatagggca gacgttcgaa tgggtcgtcg ccg 4538770DNARAT 87gttgaagagt cacctggtgc ttcaacggga ctgatttcct gggcctggag ttggagatca 60gaggtctgac 7088381DNARAT 88cgctgatctg gaagcataaa caagaactga agctgaaggc tctaggggtt cccaacctgt 60gatctccagc agacactcct ggtgtgtcac cggattcagg ctcctgggat aaagaaagca 120aaggaagtct ggagtggaga cgaagaaacc ccaggcactc tgagagctgc taccttttcc 180atgtgtgctg ccagacactt ctcgtcaggg accaaatacc ccaagggagt ggagagaggc 240ctgggctggg ccagacttcc tgggctttaa cctgtgctcc aagtaggtgg gtcacatttt 300ccccagcggg agttgaagag tcacctggtg cttcaacggg actgatttcc tgggcctgga 360gttggagatc agaggtctga c 38189916DNARAT 89ggggctggag ttggagatca gaggtctgac atggctcacc tgatggctgg gcagtggttg 60ctcctgctga tgtggatggc cgaatgtgcc cagtccagag ctactcgggc caggaccgaa 120cttctcaatg tctgcatgga tgccaagcac cacaaagaaa agccaggccc agaggacaag 180ttacacgacc agtgcagccc ctggaagacg aatgcctgct gctccaccaa cacaagccag 240gaagacacta aggacatttc ctacctgtac cgattcaact ggaatcactg tggaactatg 300accccggagt gcaaacgtca ctttatccaa gacacctgcc tctatgagtg ttccccgaac 360ttgggaccct ggatccagca ggtggaccag agctggcgca aagagcggat ccttgatgtt 420cccctgtgca aagaagactg tgtgctgtgg tgggaggact gcaagagctc ttttacctgc 480aagagcaact ggctcaaggg atggaactgg acctcggggc ataatgagtg ccctgtggga 540gcctcctgcc atcccttcac tttctacttc cctacacctg ctgtgctgtg tgagaaaatc 600tggagtcact cctacaagct cagcaactac agccgaggga gcggccgctg catccagatg 660tggttcgacc cagcccaagg caaccccaac gaggaagtgg cgaggttcta tgccgaggtc 720atgagtggag ctgggcttcg cgaggcctgg ctgctggtgt gcagcctgtc cttagtgctg 780ttctgcgtcg tcagctgagt tcctgttact ccttgtctgg agctccaccc tgcccggctt 840agcctcccag ctccagcctc ctttgtggtg gggctctgac agcctgttta gtaaaccaga 900cattctaaaa aaaaaa 91690478DNAMus musculus 90acccggtgag ctccctcccg gctccggccg ggggtcgggc gccggcggct ttggtgactc 60tagataacct cgggccgatc gcacgccccc aggtcaagtt tgtttatgaa ggtattttgg 120tattgttttc ctttgcttaa ttgcctcaca ttttgttctg aaaaacatgg gtccactgtt 180aaaaccgaat gtatgtgtag ctttattctg tttcacaggc gcatgtgatt ggaaaactca 240ttgtctcctc cagcctcagg agacttctaa aaagttttgc gtagctcaag ttgtgcatga 300attaccgaat atattatttt tcagcttttc ttcatgaacg atatttgaca tgtgctttgg 360tacccttctc tgaaagttga aaacctacct acttagtccc ttctgtgcct tttttatttt 420gccaaccatg ttttatggaa aagacattag caattacatt ttgcaaatgg aattatgt 47891821DNAMastigamoeba balamuthimodified_base(560)N = A, C, G, or T/U 91ggcaccagag tagtcatatg cttgtgttaa agattaagcc atgcatgcct aagtacaaac 60tattcttatg gtaaaactgc ggacggctcc atagatcagt aatagttcgt tcagtgattt 120gaaaaagtac ttggataacc ctgttaattg tagagctaat acatgcaccg acggcctgat 180cgggtgaccg agagggtcgc acttgtctta attcacagtg ccccggaact gaggctgttc 240gacgtggtag gggaggacgc tgaatggggc tggtagaaac aactgggggt ataaaaccaa 300ggaggaagca aaaaagccat aacccggcga tggccttggt ggaaacctct gggctcaagg 360ttgttattat gttcattgtg gcctctcggg gttattttga atgtggtaat aaaccgaaag 420caactctatc agtttggttt ggatgtccgt taatcctgcg tggccagcgg ctttggggac 480tccaggggac agggcgaaac gaggcaattc aaagctgatc gctttctaac gagggcgaca 540cactgttcga attcctgacn tatcaactcg atggtaggat agtggcctac catggttata 600acgggtaacg gggaatcagg gctcgattcc ggagagggag cctgagaaac ggctaccact 660tccaaggaag gcagcaggcg cgtaaattac tccctgccga cacggcgagg tagtgacgac 720aaataccaag gaaaaccgcc tttggtggtt ttccattgga atgagcagaa ttcaaacccc 780tctgcaagta acaattggag ggcaagtctg gtgccagcag c 82192110DNAMus musculus 92tccctcgact gtagttgctg agcttgtagg agtgactcca gatttcctca cacagagcag 60cagatgtggg gaagtagaag gtgaagggat ggcaggaggc tcccacagga 11093334DNAFrog 93ctatcgatat ccgatggtac ttgttgtgcc taccatggtg accccagttc atagcgaatg 60agggtgcgat ggcagagagg gaggatgtga tgcagctatc gcatgcggtg gatgctggag 120gcgcgcatgt tgcaccctcc cgacggcgag aggtggtgac tacccatatc gtgcaggact 180ctttcgacgc gctgtagtct gaatgagtac actttaagtc cgtgagcgcg gatctatcgg 240ttggcgagtt tagtgccagc agcgcgaggc tttacagcct caatgtcgtg tatgacagtt 300gcgtgtcctt atggagcgtg agttggatca tggg 33494220DNAHomo sapiensmodified_base(54) (77)N = A, C, G, or T/U 94gcggccgcct actactacta aattcgcggc cgcgtcgacc gacgacccat tcgntcgtct 60gccctatcaa ctttcgntgg ttgtcgccgt gcctaccatg gtgaccacgg gtgacgggga 120ttctgggttc gtttccggtg agggtgcctg tggggcggtt gcctcttctc tggttggctg 180caggcgcgct tttttcctcc tcccggcccg gggtggttgt 22095630DNAHaemonchus contortus 95ctggctgcag gaattcgcac gaggctatat gctcagttta aagattaagc catgcatgtc 60gagttcatct ttgaagagaa actgcgaacg gctcattaga gcagatgtca tttattcgga 120acgtcctttt ggataactgc ggtaattctg gagctaatac atgcaaataa accctgactt 180ttgaaagggt gcaattatta gagcaaatca atcactttcg ggtgcagttt gctgactctg 240aataacgcag catatcggcg gcttgttcgc cgatattccg aaaaagtgtc tgccctatca 300acctgatggt agtctattag tctaccatgg ttattacggg taacggagaa taagggttcg 360actccggaga gggagcctta gaaacggcta ccacatccaa ggaaggcagc aggcgcgaaa 420cttatccaat cttgaacaga tgagatagtg actaaaaata aaaagaccat tcctatggaa 480cggtcatttc aatgagttga tcataaacct tttttcgagg atcaagtgga gggcaagtct 540ggtgccagca gccgcggtaa ttccagctcc actagtgtaa atcgtcattg ctgcggttaa 600aaagctcgta gttggatctg agttacatgc 63096340DNAMus musculus 96atcatccaga

tttcgtttga tttcaccccg ggccttccgg aggaggacct cctgaaattt 60tctccttcct atatgacatt agggactgtg ccccaagcag cagtactttt tgtccccagc 120ctgcccccag tgccgtcatt atggctcccc gctgagaggt cagttttcct ctctagattt 180ttcctctatt tacccttggt ctggttcaac ttttcaaaga ataaggaagt cttgaccctg 240cttccacccc tttcctctgt catccagttc ctgatccatg tggggggttg gggtttctac 300aatcattttc aataaattta tgacacatct gggcctaatg 34097284DNAMus musculus 97aggacgtttg atgtcttatg cttcctttat gaaatccggg attgtgcccc atccagcagt 60attcttgtgc ccagcctggc cccactgcag tcattatgcc tccctggctg agaggtcatt 120cttcctcttt agatttctcc tcaatctacc cttgttctgg ttcaactctt caaagaataa 180ggaagtcttg accctgcttc cacccctttc ctctttcatc cagttcctga tccatgttgg 240gggttggggt ttctacattc attttcaata aatctatgac acac 28498263DNAMus musculus 98tccgggcctt tccccccaca caccaaaaac ttttctgcct actctggccc cagcgctttc 60cttatgcctc cctggctgag aggtcatttt cttctataga tttctcctct atttaccctc 120gctctggttc aactcttcaa agaataagga acttttgagc ctgcttccac ccttttcctc 180tgtcatccag ttcctgatcc atgttggggg ttggggtttc tacaatcatt ttcaataaat 240ctatgacaca tctgggccta atg 26399489DNAHomo sapiens 99tttttgtgcg gtgtctggtt tattcaaagt ggctgtcaga ggcccgacca tggagcagga 60accaaatagt tgggagctgt ggggctgaac agggcaggga tttccaggta tcagaaggta 120aaaggaggtc agctgagcag ccacagcagc attagggcca ggctaagcag gaaaggccag 180gctgcccagg gcccagcccc actcatggct gcagcataga acctcgccac ctcctcattg 240gggttgccct gggctgggtc gaaccacatc tggatgcagc ggccactccc tcggctgtag 300ttgctgacct tgtaggagtg agtccagatt tcattgcaca gaacagtggg tgtggggaag 360tagaaatgga aaggttggca ggcagctccc actgcgcact tgttaaaccc tgaagtccag 420tttcagccct tgtgccagtt gctcttgcag gtgtaggagg tgcgacaatc ttcccaccat 480tgctcacag 489100102DNAMus musculus 100ggatggaact ggtcctcggg gcataacgag tgtcctgtgg gagcctcctg ccatcccttc 60accttctact tccccacatc tgctgctctg tgtgaggaaa tc 102101583DNAHomo sapiens 101tagtgtgggt ggctgtagta ggggaggctc agacaaggat tgcatgggcc aggactgagc 60ttctcaatgt ctgcatgaac gccaagcacc acaaggaaaa gccaggcccc gaggacaagt 120tgcttgtagc agtgtcgacc ctggaggaag aatgcctggt gttctaccaa caccagccag 180gaagcccata aggatgtttc ctacctatat agattcaact ggaaccactg tggagagatg 240gcacctgcct gcaaacggca tttcatccag gacacctgcc tctacgagtg ctcccccaac 300ttggggccct ggatccagca ggtggatcag agctggcgca aagagcgggt actgaacgtg 360cccctgtgca aagaggactg tgagcaatgg tgggaagatt gtcgcacctc ctacacctgc 420aagagcaact ggcacaaggg ctggaactgg acttcagggt ttaacaagtg cgcagtggga 480gctgcctgcc aacctttcca tttctacttc cccacaccca ctgttctgtg caatgaaatc 540tggactcact cctacaggtc agcaactaca gccgagggag tgg 583102514DNAHomo sapiensmodified_base(122) (131)N = A, C, G, or T/U 102tgcggtgtct ggtttattca aagtggctgt cagaggcccg accatggagc aggaaccaaa 60tagttgggag ctgtggggct gaacagggca ttttatttcc aggtatcata ttgtttgttg 120tnggagctga ncagccacag cagcattagg gccaggctaa gcaggaaagg ccaggctgcc 180cagggcccag ccccactcat ggctgcagca tagaacctcg ccacctcctc attggggttg 240ccctgggctg ggtcgaacca catctggatg cagcggccac tccctcggct gtagttgctg 300accttgtagg agtgagtcca gatttcattg cacagaacag tgggtgtggg gaagtagaaa 360tggaaaggtt ggcaggcagc tcccactgcg cacttgttaa accctgaaga ccagttccag 420cccttgtgcc agttgctctt ggaggtgtag gaggtgccac aatcttccca ccattgctca 480cagtccttct tgcacagggg cacgttcaga accc 514103321DNASuaeda maritima supsb. salsa 103cgacgcatca ttcaaatttc tgccctatca actttcgatg gtaggatagt ggcctaccat 60ggtggtgacg ggtgacggag aattagggtt cgattccgga gagggagcct gagaaacggc 120taccacatcc aaggaaggca gtaggcgcgc aaattaccca atcctgacac ggggaggtag 180tgacaataaa taacaatacc gggctcttcg agtctggtaa ttggaatgag tacaatctaa 240atcccttaac gaggatccat tggagggcaa gtctggtgcc agcagccgcg gtaattccag 300ctccaatagc gtatatttaa g 321104504DNAHaemonchus contortus 104tatatgctca gtttaaagat taagccatgc atgtcgagtt catctttgaa gagaaactgc 60gaacggctca ttagagcaga tgtcatttat tcggaacgtc cttttggata actgcggtca 120ttctggagct aatacatgca aataaaccct gacttttgaa agggtgcaat tattagagca 180aatcaatcac tttcgggtgc agtttgctga ctctgaataa cgcagcatat cggcggcttg 240ttcgccgata ttccgaaaaa gtgtctgccc tatcaacctg atggtagtct attagtctac 300catggttatt acgggtaacg gagaataagg gttcgactcc ggagagggag ccttagaaac 360ggctaccaca tccaaggaag gcagcaggcg cgaaacttat ccaatcttga acagatgaga 420tagtgactaa aaataaaaag accattccta tggaacggtc atttcaatga gttgatcata 480aacctttttt ccagttaatt ctac 504105600DNAMeloidogyne incognita 105tggccgggat tagaacaaaa ccacgcggct tcggctgctt cttgttgact cagaataact 60aagctgaccg catggccttg tgccggcggc gtgtctttca agcgtccact ttatcaactt 120gacgggagca taatcgactc ccgtggtggt gacggataac ggaggatcag ggttcgactc 180cggagaaggg gcctgagaaa tggccactac gtctaaggat ggcagcaggc gcgcaaatta 240cccactctcg gctcgaggag gtagtgacga gaaataacga gatcgttctc tttgaggccg 300gtcatcggaa tgagtacaat ttaaaccctt taacgagtat caagcagagg gcaagtctgg 360tgccagcagc cgcggtaatt ccagctctgc taatacatag aattattgct gcggttaaaa 420agctcgtagt tggattcgta tcggtaccct ggaaccctcc gggtgtttct gggtgttatc 480gatttatcgt aatgttcggt tttgagtcct taacaggatt cttaacaggc attgcaagtt 540tactttgaac aaatcagagt gcttcaaaca ggcgtttgcg ctgaatgatc gtgcatggat 600106634DNAMeloidogyne incognita 106tgcctaatgt gccaccgctg agtgtgatga tattgacaat cggtagcatt atggccgggt 60gtgtctattt caaagattaa gccatgcatg tataagttta aatcgttttg acgagaaacc 120gcgaacggct cattacaatg gccatgattt acttgatctt gattatctaa atggattaac 180tgtggaaaag ctagagctaa tacatgcacc aaaacttgtt cctctcggaa aagcgcattt 240attagaacaa aaccacgcgg cttcggctgc ttcttgtgac tcagaataac taagctgacc 300gcatggcctt gtgccggcgg cgtgtctttc aagcgtccac tttatcaact tgacgggagc 360ataatcgact cccgtggtgg tgacggataa cggaggatca gggttcgact ccggagaagg 420ggcctgagaa atggccacta cgtctaagga tggcagcagg cgcgcaaatt acccactctc 480ggctcgagga ggtagtgacg agaaataacg agatcgttct ctttgaggcc ggtcatcgga 540atgggtacaa tttaaaccct ttaacgagta tcaagcagag ggcaagtctg gtgccagcag 600ccgggtattc cagctctgct aatacataga atta 634107222DNAMus musculusmodified_base(40)N = A, C, G, or T/U 107tccccaccct gcccccagtg ctgtcattat ggatccctgn ctgagaggtc aatcttcctt 60tctagatttt tcctctatct acccttggtc tggttcaaat tttcaaagaa taaggaagtc 120ttgagcctgc ttccacccct ctcctctttc atccagttcc taatccatgt tgggggttgg 180ggtttctaca atcattttca ataaatttat gacacatctg gg 222108311DNAMus musculus 108gtagttgctg agcttgtagg agtgactcca gatttcctca cacagagcag cagatgtggg 60gaagtagaag gtgaagggat ggcaggaggc tcccacagga cactcgttat gccccgagga 120ccagttccat cccttgtgcc aattgctctt gcaggtaaaa gagctctggc agtcctccca 180ccactgctga cagtcctctt tgcacagggg aacatcaagg atccgctctt tgcgccagct 240ctggtccacc tgctggatcc agggtcccaa gttcggggaa cactcataga ggcaggtgtc 300ttggataaag t 311109272DNAMus musculus 109acacagagca gcagatgtgg ggaagtagaa ggtgaaggga tggcaggagg ctcccacagg 60acactcgtta tgccccgagg accagttcca tcccttgtgc caattgctct tgcaggtaaa 120agagctctgg cagtcctccc accactgctg acagtcctct ttgcacaggg gaacatcaag 180gatccgctct ttgcgccagc tctggtccac ctgctggatc cagggtccca agttcgggga 240acactcatag aggcaggtgt cttggataaa gt 272110315DNAMus musculusmodified_base(196)N = A, C, G, or T/U 110actgcggaac tatgacatcg gaatgcaaac ggcactttat ccaagacacc tgcctctatg 60agtgttcccc gaacttggga ccctggatcc agcaggtgga ccagagctgg cgcaaagagc 120ggatccttga tgttcccctg tgcaaagagg actgtcagca gtggtgggag gactgccaga 180gctcttttac ctgcangagc aattggcaca agggatggaa ctggtcctcg gggcataacg 240agtgtcctgt gggagcctcc tgccatccct tcaccttcta cttccccaca tctgctgctc 300tgtgtgagga aatct 315111315DNAMus musculus 111actgcggaac tatgacatcg gaatgcaaac ggcactttat ccaagacacc tgcctctatg 60agtgttcccc gaacttggga ccctggatcc agcaggtgga ccagagctgg cgcaaagagc 120ggatccttga tgttcccctg tgcaaagagg actgtcagca gtggtgggag gactgccaga 180gctcttttac ctgcaagagc aattggcaca agggatggaa ctggtcctcg gggcataacg 240agtgtcctgt gggagcctcc tgccatccct tcaccttcta cttccccaca tctgctgctc 300tgtgtgagga aatct 315112685DNACladosporium fulvummodified_base(197)..(683)N = A, C, G, or T/U 112gaggccagta gtcatatgct tgtctcaaag attaagccat gcatgtctaa gtataagcaa 60ctatacggtg aaactgcgaa tggctcatta aatcagttat cgtttatttg atagtacctt 120actacatgga taaccgtggt aattctagag ctaatacatg ctaaaaaccc cgacttcgga 180aggggtgtat ttattanata aaaaaccaac gcccctcggg gctccttggt gaatcataat 240aacttcacga atcgcatggc cttgcgccgg cgatggttca ttcaaatttc tgccctatca 300actttcgatg gtaggataga ggcctaccat ggtttcaacg ggtaacgggg aattagggtt 360cgactccgga gagggagcct gagaaacggc taccacatcc aaggaaggca gcaggcgcgc 420aaattaccca atcccgaccg gggagggagn gacaataaat actgatncng gctntttggg 480gtcttgnaat tggaatgagt ncaattaaat cccttaccag gaacaattgg aggcaanttg 540gngcccccan cncggnattc cactccatag cgttntaaag tttgcaatta aaagttgaat 600taacttggcc tggtggcggc ccctacgggt ctggccggcg gcnttntttg gggccgnncc 660tttatgnggg gggaacngct ttntt 685113440DNACladosporium fulvummodified_base(72)..(433)N = A, C, G, or T/U 113tgacaattga atacggatgc ccccgactat ccctattaat cattacgggg gtcctagaaa 60ccaacaaaat anaaccacnc gtcctattct attattccat gctaatgtat tcgagcaaag 120gcctgctttg aacactntaa ttttttcaaa gnaaaagtcc tggttccccg acncncccag 180ngaagggcat gcggctcccc aaaaggaaag gcccggccgg accagtacac gcggngaggn 240ggaccggcca gccaggccca aggttcaact acgagctttt taactgcaac aactttaata 300tacgctattg gagctggaat taccgnggnt gctggcacca aacttgccct ccaattgttc 360ctcgttaagg ggatttaaat tgtactcatt ccaattacaa gacccaaaag agccctgtat 420cagtatttat tgncactact 440114246DNAMus musculus 114tgtgccccat gcaacagtaa tttttgagcc caccctggcc ccagtgctgt cattatggct 60ccctggctga gaggtcagtt ttcctatcta gatttttcct gtatctaccc ttggtctggt 120tcaaattttc aaagaataag gaagtcttga gcctgcttcc acccctttcc tctgtcatcc 180agttcctgat ccatgttggg ggttggggtt tctacaatca ttttcaataa atctatgaca 240catctg 246115694DNAMesembryanthemum crystallinummodified_base(23) (609)N = A, C, G, or T/U 115ccccctagat gctagtagca gtngncacga ggtcatatgc ttgtctcaaa gattaagcca 60tgcatgtgta agtatgaact aattcagact gtgaaactgc gaatggctca ttaaatcagt 120tatagtttgt ttgatggtac ctgctactcg gataaccgta gtaattctag agctaatacg 180tgcaacaaac cccgacttct ggaagggatg catttattag ataaaaggtc gacgcgggct 240ttgcccgttg ctctgatgat tcatgataac tcgacggatc gcacggnctt tgcgccggcg 300acgcatcatt caaatttctg ccctatcaac tttcgatggt aggatagtgg cctaccatgg 360tggtgacggg tgacggagaa ttagggttcg attccggaga gggagcctga gaaacggcta 420ccacatccaa ggaaggcagc aggcgcgcaa attacccaat cctgcacggg gaggtaggga 480caataaataa caataccggg ctcttcgagt ctggtaattg gaatgagtac aatctaaatc 540ccttaacgag gatacattgg agggccaagt ctgttgccag cagccgcggt atattccagc 600ttcaatagnc gtatatttaa agttgttggc agttaaaaag cttgtatttg gactctgggg 660tgggcgaccc ggtcgtctag cggtgtgcac cggc 6941161266DNAMesembryanthemum crystallinummodified_base(16)..(1230)N = A, C, G or T/U 116gactactcat cagtgncagg ctagctgcac gaggtcatat gctcgtctca tagattaagc 60catgcatgtg taagtatgaa ctaattcaga ctgtgaaact gcgaatggct cattaaatca 120gttatagttt gtttgatggt acctgctact aggataaccg tagtaattct agagctaata 180cgtgcaacaa accccgactt ctggaaggga tgcatttatt agataaaagg tcgacgcggg 240ctttgcccgt tgctctgatg attcatgata actcgacgga tcgcacggcc tttgcgccgg 300cgacgcatca ttcaaatttc tgccctatca actttcgatg gtaggatagt ggcctaccat 360ggtggtgacg ggtgacggag aattagggtt cgattccgga gagggagcct gagaaacggc 420taccacatcc aaggaaggca tcaggcgcgc atattaccca atcctgacac ggcgaggtag 480tgacaataaa taacaatacc gggctcttcg agtctcggta atcggaatga gttcaatcta 540tatcccttta cgaggatcca ttggagggca agtcctgctg ccagcagcct gctgtccttt 600cagctccaat agcgtatatt taagttgttg cagtttaaca agctcttatt cgaccttgtc 660gtgcgaccgt tctcattacg ctatatgcct catcatatgt ccatatctat tctcgacttc 720tcgctcccct cgtcttctct agtacttctg cctcttctat tatattcact atgatctatt 780ctctacgcct cttcctctgc actcttatat tcatcgcact cttcactcta ctctctctta 840tcgtctgcta gtctttcgct tcttcctctt tctactttct catgtctctc atcttatctt 900accctctctc actctttctg ttcgtctcct ctcactctgc gatttctcca ctgtatcacg 960cttcgttctc tctactcttc tacttgttct ctctctatct cgtcctcatc tcctccgtct 1020cgtctctatc gtcgtctacc gatactcttt ccttctctgt catcttcctc tctcttcctc 1080tcttgcttac ttctcgtctc tcttcacgat tatcntctag cacgtcatct ctttactctc 1140tctatcttca tgtctactca ctctctcctg tgcgtactac tcttggctat catcatctcc 1200tagagtggct cgatgaggcg aatgtgcncn tctatctctc tacgttctct tactgatact 1260tctttg 12661171162DNAMesembryanthemum crystallinummodified_base(34)..(960)N = A, C, G or T/U 117gtcgacgcac tagtgctata gtagcgttca tgcnagcngc acgaggagag agagagagag 60agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 120agagagagag agagagcggc acgagcttgt ctcaaagatt aagccatgca tgtgtaagta 180tgaactaatt cagactgtga aactgcgaat ggctcattaa atcagttata gtttgtttga 240tggtacctgc tactaggata accgtagtaa ttctagagct aatacgtgca acaaaccccg 300acttctggaa gggatgcatt tattagataa aaggtcgacg cgggctttgc ccgttgctct 360gatgattcat gataactcga cggatcgcac ggcctttgcg ccggcgacgc atcattcaaa 420tttctgccct atcaactttc gatggtagga tagtggccta ccatggtggt gacgggtgac 480agagaattag ggttcgattc cggagaggga gcctgagaaa cggctaccac atccaaggaa 540ggcagcatgc gcgcaaatta cccaatcctg acacggagag gtagtgacaa tatataacaa 600taccgcgctc ttcgagtctg gtaattggaa tgagtacaat ctatatccct taacgaggat 660ccattgtagg gcatgtctgg tgccagcagt cgcggtaatt tcagttccaa ttagcgatat 720ttaattcgtt gcagtaaaaa gctcgtattt gaactttgcg tgggcccacc taccgtctag 780cggtgtgcac tgtcttctct gcttttttcg gcatagcctc tgccttaaag cttgtctcgc 840actgctctta cttcgatatt tgatcttcat gcgctctctt ggatctcatc atggatacct 900aatgatctgc ctttctttgc ttggattcgc atcatcattg tacctggtct ttcgttctan 960ttagtatttc tcgattttat catcctgcta ccctactcga tttattttaa actatttgtc 1020ttaacctatt tctttctctt cttacttcac tcttcctcgt aatctgtctt attatcactc 1080ttcctcattt ctttattact gttcatttac ttatttactt tatttccttc tacatctttt 1140ctctcatctt ctactcacgt cg 1162118537DNAMesembryanthemum crystallinummodified_base(460)N = A, C, G, or T/U 118cccacactag ttctagagga ttcggcacga ggtctcaaag attaagccat gcatgtgtaa 60gtatgaacta attcagactg tgaaactgcg aatggctcat taaatcagtt atagtttgtt 120tgatggtacc tgctactagg ataaccgtag taattctaga gctaatacgt gcaacaaacc 180ccgacttctg gaagggatgc atttattaga taaaaggtcg acgcgggctt tgcccgttgc 240tctgatgatt catgataact cgacggatcg cacggccttt gcgccggcga cgcatcattc 300aaatttctgc cctatcaact ttcgatggta ggatagtggc ctaccatggt ggtgacgggt 360gacggagaat tagggttcga ttccggagag ggagcctgag aaacggctac cacatccaag 420gaaggcagca ggcgcgcaaa ttacccaatc ctgacacggn gaggtagtga acaataataa 480caataccggg ctcttcgagt ctggtaatgg gaatgagtac aatctaaatt ccttaac 537119968DNAMesembryanthemum crystallinummodified_base(635) (658)N = A, C, G, or T/U 119gcacgagcga cgcgggcttt gcccgttgct ctgatgattc atgataactc gacggatcgc 60acggcctttg cgccggcgac gcatcattca aatttctgcc ctatcaactt tcgatggtag 120gatagtggcc taccatggtg gtgacgggtg acggagaatt agggttcgat tccggagagg 180gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgctaat tacccaatcc 240tgacacgggg aggtagtgac aataaataac aataccgggc tcttcgagtc tggtaattgg 300aatgagtaca atctaaatcc cttaacgagg atccattgga gggcaagtct ggtgccagca 360gccgcggtaa ttccagctcc aatagcgtat atttaagttg ttgcagttaa aaagctcgta 420gttggacctt ggggtgggcc gaccggtccg cctagcggtg tgcaccggtc gtcctgcctc 480ttctgccggc gatgcgctcc tggccttaac tgggccggtc gtgccaccgg gcgctgtact 540ttgaagaaat agagtgctca agcaggccta cgctctggat acattagcat gggataacat 600cataggaatt ccgtcctatt ctgttgccct tcggnattcg agtaattgat aacaggnnac 660agcgggggca ttcgtatttc atagtcagag gtgaaaatct tggattattg aagaccaaca 720actgccaaag catttggcca ggatgttttc attattcaag accgaaagtt ggggcttcga 780agaccaacag attcccgtct aatcttaaac cttaaacata tcccaccagg ggatcgggga 840tgtaactttt aggaccccgc cggcccctta tgagaaatta aagttttggg gtcccggggg 900gagtttggtg ccaaggcttt aacttaaggg aattgcgcgg aggggccccc cccgggaatg 960ggccctgt 968120893DNAMesembryanthemum crystallinummodified_base(660)N = A, C, G, or T/U 120gcacgaggtc tcaaagatta agccatgcat gtgtaagtat gaactaattc agactgtgaa 60actgcgaatg gctcattaaa tcagttatag tttgtttgat ggtacctgct actaggataa 120ccgtagtaat tctagagcta atacgtgcaa caaaccccga cttctggaag ggatgcattt 180attagataaa aggtcgacgc gggctttgcc cgttgctctg atgattcatg ataactcgac 240ggatcgcacg gcctttgcgc cggcgacgca tcattcaaat ttctgcccta tcaactttcg 300atggtaggat agtggcctac catggtggtg acgggtgacg gagaattagg gttcgattcc 360ggagagggag cctgagaaac ggctaccaca tccaaggaag gcagcaggcg cgcaaattac 420ccaatcctga cacggggagg tagtgacaat aaataacaat accgggctct tcgagtctgg 480taattggaat gagtacaatc taaatccctt aacgaggatc cattggaggg caagtctggt 540gccagcagcc gcggtaattc cagctccaat agcgtatatt taagttgttg cagttaaaaa 600gctcgtagtt ggaccttggg gtgggccgac cggtccgcct agcggtgtgc accggtcggn 660cttgcctctt ttgtcggcga tgcgctcctg gcctttaact ggccgggttg tgccaccggc 720gctgttactt ttgaagaaat aagagtgctc aaagcaagcc ctacgctctg gttacattag 780catgggataa caatatagga tttccggtcc tattttgttg gcctttggga tcggagttat 840gaataacagg gaccgtccgg gggcatttct tttttaatat tcaaaggtga aat 893121996DNAMesembryanthemum crystallinummodified_base(676) (854)N = A, C, G, or T/U 121agctggtacg cctgcggtac cggtccggaa ttcccgggtc gacccacgcg tccgcggacg 60cgtgggcgga cgcgtggggc taatacatgc aactcggtct ctaccggaaa

tggtagggac 120gcttttatta gaccaaaacc aatcgggcgt tctcgtccgt tttgccttgg tgactctgaa 180taaattgtgt gcagatcgca cggtcctcgt accggcgacg catctttcaa atgtctgcct 240tatcaacttt cgatggtagg tcctgcgcct accatggttg taacgggtaa cggggaatca 300gggttcgatt ccggagaggg agcctgagaa acggctgcta catccaagga aggcagcagg 360cgcgcaaatt acccactccc ggcacgggga ggtagtgacg acaaataacg atacgggact 420catccgaggc cccgtaatcg gaatgaacac actttaaatc ctttaatgag tatccattgg 480agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta tattaaagtt 540gttgcggtta aaaagctcgt agtcggactt gtgtcacacg ctgccggttc accgcccgtc 600ggtgctaact ggcatgcacg tgttgacgtc ctgctggtgg ccgtagccgg tccgggtgtt 660ctgggatccc ttcggngttt cccggacccc ggtgcttggt gaaggcctac ttgacctacc 720cgtcgcggtg ctcttaaccg agtgtctcga tgggccggca cttttacttt gaacaattag 780agtgcttaaa gcaggcagta tcagccctga tactgagtgc atggaataat ggaataggaa 840cctcggtcta tttntgtggt tttcggaatg ccctagatcg cgagcggccg ctctagaaga 900tccaagctta cgtacgcctg cattgccaag tataagcttt tttatatggg gaaccctaaa 960ttcaatcaac tggcgcgcgg tttaacacac gcggag 996122607DNAMus musculusmodified_base(106)N = A, C, G, or T/U 122tgctgctcct tctagtgtgg gtggctgtag taggggaggc tcagacaagg attgcatggg 60ccaggactga gcttctcaat gtctgcatga acgccaagca ccacanagga aaattctttc 120cccgaggaca agttgcatgt tctgtggggg ccctggagga agaatgcctg ctgttctacc 180aacaccagcc aggaagccca taaggatgtt tcctacctat atagattcaa ctggaaccac 240tgtggagaga tggcacctgc ctgcaaacgg catttcatcc aggacacctg cctctacgag 300tgctccccca acttggggcc ctggatccag caggtggatc agagctggcg caaagagcgg 360gtactgaacg tgcccctgtg caaagaggac tgtgagcaat ggtgggaaga ttgtcgcacc 420tcctacacct gcaagagcaa ctggcacaag ggcctggaac ctggacttca gggttttaac 480aaggtgcgca ggtgggaggc tgccctgccc accttttcca ttttctactt ctctcacacc 540cactgttgct gttgcattgc aaatcttgtc ctcacttctt acaaggtaca gcaactacca 600agaaaaa 607123351DNAMus musculus 123aggacatttc ctacctgtac cggttcaact ggaaccactg cggaactatg acatcggaat 60gcaaacggca ctttatccaa gacacctgcc tctatgagtg ttccccgaac ttgggaccct 120ggatccagca ggtggaccag agctggcgca aagagcggat ccttgatgtt cccctgtgca 180aagaggactg tcagcagtgg tgggaggact gccagagctc ttttacctgc aagagcaatt 240ggcacaaggg atggaactgg tcctcggggc ataacgagtg tcctgtggga gcctcctgcc 300atcccttcac cttctacttc cccacatctg ctgctctgtg tgaggaaatc t 351124365DNAMus musculus 124gcggccgctc cctcgactgt agttgctgag cttgtaggag tgactccaga tttcctcaca 60cagagcagca gatgtgggga agtagaaggt gaagggatgg caggaggctc ccacaggaca 120ctcgttatgc cccgaggacc agttccatcc cttgtgccaa ttgctcttgc aggtaaaaga 180gctctggcag tcctcccacc actgctgaca gtcctctttg cacaggggaa catcaaggat 240ccgctctttg cgccagctct ggtccacctg ctggatccag ggtcccaagt tcggggaaca 300ctcatagagg caggtgtctt ggataaagtg ccgtttgcat tccgatgtca tagttccgca 360gtggt 365125653DNAMus musculusmodified_base(532)N = A, C, G, or T/U 125gggctgtgga cgaagactgt agagactacc cagagtctga cctagggaga ggccaactcg 60gataccccta tgtgcgctcc cagaagctaa ggacattgag acagaaagac atggcctgga 120aacagacacc actcttgctt ttggtctaca tggtcacaac aggcagtggc cgggacagaa 180cagacctact caacgtttgc atggatgcca aacaccataa gacaaagccg ggccccgagg 240acaagctgca tgaccagtgt agtccatgga agaaaaatgc ctgttgctca gtcaacacca 300gccaggagct acacaaggct gactcccgtc tgtacttcaa ctgggatcac tgtggcaaga 360tggagcctgc ctgtaagagt cacttcatcc aagactcctg cctgtatgag tgctccccca 420accttgggcc ttggatccag caagtggacc agagttggcg taaagagcgt gtcctggatg 480tgcccttatg caaagaggac tgtcaccagt ggtgggaagc ctgtcgtacc tnctttacct 540gcaagagaga ctggcataaa ggctgggact ggtcctcagg catttacaag tgcccaaaca 600cagcaccctg tcacacgttt gagtactact tcccgacacc agccagccct tgc 653126649DNAMus musculus 126tttttttttt ttcccaaatg tgtcatagat ttattgaaaa tgattgtaga aaccccaacc 60cccaacatgg atcaggaact ggatgacaga ggaaaggggt ggaagcaggc tcaagacttc 120cttattcttt gaagagttga accaaaccaa gggtagatag aggagaaatc tagagaggaa 180gactgacctc tcagccaggg agccataatg acagcactgg ggccaggctg ggcacaaaaa 240gtactgctgc atggggcaca gtcccagatg tcataaagga agcataaaac ttcaccacgt 300cctcattcgg attgccctgg gttgagtcaa accacatttg gatgcagcgg ccactccctc 360tgctgtagtt gctgaccttg taggagtgac tccagagacc ctcgcaaagg ctggctggtg 420tcgggaagta gtactcaaac gtgtgacagg gtgctgtgtt tgggcacttg ttaatgcctg 480aggaccagtc ccagccttta tgccagtctc tcttgcaggt aaaggaggta cgacaggctt 540cccaccactg gtgacagtcc tttttgcata agggcaccat ccagaaaacg ctctttacgc 600caactcttgt tccacttgct gatccaaagg ccaaagttgg gggagcact 649127668DNAchicken 127cagcctcttg cacacagctt tactctgtca gccccagggt ggaaacaaag ggctggctgt 60tcatcacact gcactttgtg taatcactcg ctctcacaac tggcaaatct cttttgccag 120tggtgggact gaataacatt ttaaagggat gaagtacagc acagagctgt acaagatagt 180ggatgactgc agactttttc ataattttgt accatttcta aaaaagtgat gtttctcaaa 240ttactacaag ttgattttaa ctccattctt tttaaaatgt gattgatgtg tgtttctcat 300tttacacaca gatgtatgca aatgggaccg acatgtgcca gagtatgtgg ggggaatcct 360ttaaggtgag cgaatcctcc tgcctctgct tgcaaatgaa caagaaggac atggtggcaa 420tcaagcacct cctctccgaa agctcagagg aaagctccag tatgagcagc agtgaggagc 480acgcctgcca aaagaaactc ctgaagtttg aggcactgca gcaagaggaa ggggaagaga 540gaagatgaat tttggtggat gaatatcagg aggagaggaa tcattgtgga ggttgtgctc 600ggggcatcac agcagcctgt cttatccctc acttctgaga acacaataaa tcaatggttg 660gctatatt 6681283632DNAchicken 128acaagcagat taatttcatt agcacgcatc accatatata ataaagctgt aataggccaa 60atgctccaat ttacacttgt gaaactccgt ctcactccag ccacactgtt gttacacttt 120catgatgcca aggagggaaa cagatctggc agctgtcaca agttggaagt acaaacaatt 180tttcccttca ccactacagc tttgcagagt taacaaaaat ataaaaccag aaaagcttac 240ttcagtcatt agagagatct gcctcactaa aaagggatca ctgtgttgag ttaggagatg 300tcagtttgac atagatacta actcaatggc cagaagctgt gaagttagca actagctgga 360gttcttgtat ctcttttgca tttttttccc tcattaccca atggtagctc ttgcagaagg 420aattcatgca ggcaggtagc ggctcctgag agctcaaata gctgcgtctg tgatttcgga 480ataaatacat ccttctgcta acatcgctgg ccattatcag atagtcagat gataatgtaa 540taataataat gtacccgtgc cagaattact gtctgtggca atatctgtaa catcatgcat 600gctttaacgc tgtataaaaa ctttgagaag atgaatataa gttcataggg caatgatatt 660aatgttaaaa ataaatgata acaggagttt tatcagtaca aaaatatgag cgagtacttg 720caaataaatt cagcattaac aaatgaggtt aacaacccat tcaagtattg aaagcaataa 780gaaacattct ttaataaatt tctcaatata agacttacgg tcttatactg agacttttct 840tactcagaat aagaaaaaga agactcaaga tgatgaagat gtgtggctga aatctctaga 900agctcctgtg cttgagcctg cctacatcta ttgttaacca aagccaagtc tgagaaatca 960caaacatatg acaattttcc ttcctgctgt tagaaattct gcctaatctc ccagcaagtg 1020gtcccatttg gctcatattc aaagcttgaa aaagatccca gtctcctata gcttaatata 1080atttgtatgt caattccata aacaaaggca ttacatgaaa cctcctggct cctaacacct 1140ttacaagagt gaatacattt catacaaacc aagcagtaag gaacagaaca cgtgcttttt 1200caccaggctg gctagcacag ccactcatcc tcagattgaa aggggatgtt tatgtggcac 1260agtggtcttt actttgtatg aatacactga tcttagtacc aagcaatatg cacaagtcct 1320ttacactaca aatcagcaag aagctccatt aatttcagcc agcacaaaat caagccacat 1380gaagtgaagg cacaggcaat aaggtcttac atttacttca gtttctccta tacttatatt 1440atgtctcttt gtatttgttt taattaaatt cactctggaa agcagaaaac actagggttt 1500caaatgatct gaaaatggtc ttgtaaaggc agcagcactt ttgcctcaag gaaggcttca 1560gccagagcag gaattggtgc ttacagctca gcagagatca ttatcatact gtgagtttgc 1620tcagtgagat tcattccaca cttccactgt gccagtgttt gttttattca agcaaaaaag 1680ttttgtaaat actgacccac agttactatt tgacaaacca ctgttgtgtt ttaaaataga 1740aacaagagat gctattttcc atttgcatct gaataattgc aaagtagtca gtggcgtgtt 1800gctagttagg gagctcactg ggatttgacc tatggaagta agtgcaccta tttgtaatga 1860ccacgtctgc tttctgtgat ggtccatgtt cagatgtgga atcccctctg cagaaagcac 1920acctggtaag gaaatccagt cagcaactgc tgtcagtggt actcgcaaca gtttctccta 1980gtgtttgtga cacccttgga aagcacaaac atggcaggta gagaaagaag gacaaacatc 2040agcaggttaa aaaaagaatc ttctgggcaa agagcaaagg cctgagaatc aggagaccaa 2100ttctccttca ggggctgcag taaaattact gagtaaccca aagcaaactg atatgtttac 2160ggtcaaaatt aagccagaca ggttgaaata tgaagagttg tttgaaatgt ctataattca 2220gtgaagttgg tgataagaac ccaattaagc tgttgtagaa atgaatctaa taattataac 2280aaaaggaatc attgcaaaat caggcagggg gtgggaggta gttgtattgg gtacactgga 2340gagctgttgt ttttctaatt ctagtctatg tttgtacttt cctgtttatt atgtccacat 2400ttgcaagcaa taaaagggca ttatgtgctg gtcattccat ctgcttttga gataaatcta 2460tgttagcatt tcaaagggtc aaggaactct ccagggcaaa caaattctgg agcgctgctg 2520ccagatggcg cgtatataaa gtggaaagcg agaaaagcaa tttgctgtgt ttctgttcca 2580gggagaagtc tcacccagaa ggacagcaaa agaggtgaga aactaccgag aaattgtaca 2640ggcggttttc ttctgtaaca tgttgctttc tttgcatctg aaaagtttag gtacggagag 2700aagctcagtt cttgttcagg caaagctctt ccaaaaaggt atcaggaata tttaaccaaa 2760gaattgaagg ttaagttaat aacacctata aagaattatg cacttcttta tgtgggaggt 2820tctagattta tctgtataac tcactaatat gtagtctgta cttacagaaa ctctatgctc 2880gcagaccaaa tggtggttat cttgcatatt tgactgaact ctacaaaagc agacacaaaa 2940ccattgatca gattattagg ttcaaataag cgtgacctca acaaaggcaa gttatctgca 3000taatttatcc agctcaattg ccaccttatg ctctgctatt agcttgtcaa ttctgtaaac 3060agaagcactg caattaaatg ggtaatttcc cagcacacaa aagaactctg taagtttcgg 3120agctgatcaa tcttgccttc aaatctagtg tagcagtggg atgggaaatc catatctgca 3180tgagaaattt aaaaaccttt tgttaaatac tgaaaaccat aacatatagc cttcattctt 3240catatagcct gtattcttca taggtcacca gaaactgaaa atatgtagca gaagcattaa 3300gtgtttggac atgagcaaag gaaagggaga atgagtgacc caatatttat atgcgtacct 3360ctcttgagca tatttaattg tatatatatg tagctttttt acagcagccc ttctttttac 3420tatcaggact tttcctacaa ataaaggata tcagtaaaga cttctctccg cacaggaaaa 3480gaagggaaca acaatgctga ggtttgccat caccctcttt gctgtcatca catcatctac 3540ctgccagcag tatggatgtc tggaagggga cacccacaaa gcgaatccaa gtcctgagcc 3600aaacatgcat gaatgcactc tgtattctga at 3632129713DNAMus musculus 129tttccccagt cagctggctg atctggaagt ataaacaaga aaggaggctg acggctctag 60aagtccccaa cctgttgtga tcttcagtag acaaacactc ctggtgtgtc acaggattca 120ggccactaaa cctcggccgg ctgtctcctg gaatgaagaa agcaaaggaa gcctagagtg 180gagacaaaga aacccgaggc actctgagag ctgccatctt atccttgttt gccgcctgac 240acttctcagc aggatccaca taccctaagg agtggaagac tccttggcgc ttggtgcttc 300aaccggactg acttcctggg cctggagttg gcgattagag gtctgacatg gctcacctga 360tgactgtgca gttgttgctc ctggtgatgt ggatggccga atgtgctcag tccagagcta 420ctcgggccag gactgaactt ctcaatgtct gcatggatgc catacaccac agagaaaaac 480cgggccctga tgacaattta cacgaccagt gcagcctctg gaaacgaatt cctgctgttc 540cacgaacact agccatgaag cacataagga catgtcctac ctgttccaga tcaactggaa 600ccactgcggg actatgacat cggaatgcag actgcactgt atgcaagaca cctgcctcta 660tgagtgtaca cagaacttgg gacgctggat tcatctagtg aaccaaagct ggc 713130444DNAMus musculus 130cacctgatga ctgtgcagtt gttgctcctg gtgatgtgga tggccgaatg tgctcagtcc 60agagctactc gggccaggac tgaacttctc aatgtctgca tggatgccaa acaccacaaa 120gaaaaaccgg gccctgagga caatttacac gaccagtgca gcccctggaa gacgaattcc 180tgctgttcca cgaacacaag ccaggaagca cataaggaca tttcctacct gtaccggttc 240aactggaacc actgcggaac tatgacatcg gaatgcaaac ggcactttat ccaagacacc 300tgcctctatg agtgttcccc gaacttggga ccctggatcc agcaggtgga ccagagctgg 360cgcaaagagc ggatccttga tgttcccctg tgcaaagagg actgtcagca gtggtgggag 420gactgccaga gctcttttac ctgc 444131286DNAMus musculusmodified_base(263)N = A, C, G, or T/U 131caacaaccca ttcaaacatc taccctatca actttcaata atagtcacca tacctaccat 60aataaccacg aataacaaaa aatcataatt caattccaaa taagaatcct aagaaactac 120taccacatcc aaataataca gcatacactc aaattaccca ctcccgaccc aagaaaattt 180aacgaaaaat aacaatacaa tactctttcg aagccctata attaaaataa atccacttta 240aatcctttaa cgaagatcca ttngagaaca attctgctga tatcac 286132590DNAHomo sapiensmodified_base(18)..(577)N = A, C, G, or T/U 132aagattatgc ctcccccnaa ttcggcacga ngcggggagc gagcggnccc cctccctgtc 60cgtctcctgg tcggggtcct tttttaataa cgcgtaaacc tatccaangg tacacaacga 120agaagcttgg acaaaaggcg gaaaagcgtc ttgccaaaag ggggactgga ngtnaactgg 180aaaaaaacta attttccaag agaagaactt ggnagaangg ggaattgngt ttcnggggtg 240nccttctcgn tctccggggn cgnanttctg natncgcaac aagcaaggac caatccaatc 300ccgggnacgc gggcggnccc anccgcgaag nttttcannc ccganaatcc aaacaatcct 360ggccnaagaa atatgccctt gngtaacaaa ccntcccaat ttttttaata tatcccaaan 420tnttattatt aaaacaaatg ctnaaanccc tccactcccn nanggttaaa naaatggggt 480ccnnttggca ccaactttaa tgggangttt gggnttanaa anaaacaccc cttccntttt 540cccggggngc gttatttggg gnngcacccc ccccgcnctt taattttgtt 590133247DNAMus musculus 133atgggaatat cccccataca atagtacttc ttgtgcccaa tctggcccca gtgccgtcat 60tatgggtccc tgcgtgagag gtcattttct tctttagatt tttcctctat ttacccttgg 120tctggttcaa ttcttcaaag aataaggaag ttttgagcct gcttccaccc ctttcttctt 180tcatccagtt cctgatccat gttgggggtt ggggtttcta caatcatttt caataaatct 240atgacac 247134665DNAPseudopleuronectes americanusmodified_base(40) (596)N = A, C, G, or T/U 134gaattcggca cgagccagta gcatatgctt gtctcaaagn ttaagccatg caagtctaag 60tacacacggc cggtacagtg aaactgcgaa tggctcatta aatcagttat ggttcctttg 120atcgctctca cgttacttgg ataactgtgg caattccaga gctaatacat gccaacgggc 180gctgacctcc ggggacgcgt gcatttatca gacccaaaac ccatgcgggg tgctcctcac 240ggggtgcccc ggccgctttg gtgactctag ataacctcga gctgatcgct ggccctcgtg 300gcggcgacgt ctcattcgaa tgtctgccct atcaactttc gatggtactt tttgtgccta 360ccatggtgac cacgggtaac ggggaatcag ggttcgattc cggagaggga gcctgagaaa 420cggctaccac atccaaggaa ggcagcaggc gcgcaaatta cccactcccg actcggggag 480gtagtgacga aaaataacaa tacaggactc tttcgaggcc ctgtaattgg aatgagtaca 540ctttaaatcc tttaacgaag atccattgga gggcaagtct ggtgccagca gccgcnggta 600attcagctcc aatagcgtat cttaaagttg ctgcaattaa aaagctccgt attggacctc 660ggatc 665135666DNAPseudopleuronectes americanus 135gaattcggca cgagcagtag catatgcttg tctcaaagat taagccatgc aagtctaagt 60acacacggcc ggtacagtga aactgcgaat ggctcattaa atcagttatg gttcctttga 120tcgctctcac gttacttgga taactgtggc aattccagag ctaatacatg ccaacgggcg 180ctgacctccg gggacgcgtg catttatcag acccaaaacc catgcggggt gctcctcacg 240gggtgccccg gccgctttgg tgactctaga taacctcgag ctgatcgctg gccctcgtgg 300cggcgacgtc tcattcgaat gtctgcccta tcaactttcg atggtacttt ttgtgcctac 360catggtgacc acgggtaacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 420ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga ctcggggagg 480tagtgacgaa aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtacac 540tttaaatcct ttaacgagga tccattggaa ggcaagtctg gtgccagcag ccgcggtaat 600tccagctcca atagcgtatc ttaaagttgc tgcagttcaa caagcctcgt attggacctc 660ggattc 666136645DNAPseudopleuronectes americanusmodified_base(508)..(569)N = A, C, G or T/U 136gaattcggca cgaggcggta ttcaggcgac cgggcctgct ttgaacactc taattttttc 60aaagtaaacg cttcggaccc cgcgggacac tcagctaaga gcatcgaggg ggcgccgaga 120ggcaggggct gggacagacg gtagctcgcc tcgcggcgga ccgtcagctc gatcccgagg 180tccaactacg agctttttaa ctgcagcaac tttaagatac gctattggag ctggaattac 240cgcggctgct ggcaccagac ttgccctcca atggatcctc gttaaaggat ttaaagtgta 300ctcattccaa ttacagggcc tcgaaagagt cctgtattgt tatttttcgt cactacctcc 360ccgagtcggg agtgggtaat ttgcgcgcct gctgccttcc ttggatgtgg tagccgtttc 420tcaggctccc tctccggaat cgaaccctga ttccccgtta cccgtggtca ccatggtagg 480cacaaaaagt accatcgaaa gttgatangg cagacattcg aatgagacgt cccgccacga 540aggccagcga tcagctcgag gttatctana gtcaccacag cggccggggc cacccgttga 600ggaaccaccg ccgcattggg ggttttgggt ctgaataaat tgcac 645137542DNAPseudopleuronectes americanusmodified_base(18)..(504)N = A, C, G, or T/U 137gaattcggca cgaggctnga cctccgggga cgcgtgcatt tatcagaccc aaaacccatg 60cggggtgctc ctcacggggt gccccggccg ctttggtgac tctagataac ctcgagctga 120tcgctggccc tcgtggcggc gacgtctcat tcgaatgtct gccctatcaa ctttcgatgg 180tactttttgt gcctaccatg gtgaccacgg gtaacgggga atcagggttc gattccggag 240agggagcctg agaaacggct accacatcca aggaaggcag caggcgcgca aattacccac 300tcccgactcg gggaggtagt gacgaaaaat aacaatacag gactctttcg aggccctgta 360attggaatga gtacacttta aatcctttaa cnaggatcca ttggagggca agtctggtgc 420catcagccgc ggtaattcca gctccaatan cgtatcttaa agttggctgc acttaaaaag 480ctcntanttg gacctcggga tccnagctga cggtccgccg ctaagcgaac ttaccgtctg 540tc 542138650DNAPseudopleuronectes americanusmodified_base(626)N = A, C, G or T/U 138gaattcggca cgagcagtag catatgcttg tctcaaagat taagccatgc aagtctaagt 60acacacggcc ggtacagtga aactgcgaat ggctcattaa atcagttatg gttcctttga 120tcgctctcac gttacttgga taactgtggc aattccagag ctaatacatg ccaacgggcg 180ctgacctccg gggacgcgtg catttatcag acccaaaacc catgcggggt gctcctcacg 240gggtgccccg gccgctttgg tgactctaga taacctcgag ctgatcgctg gccctcgtgg 300cggcgacgtc tcattcgaat gtctgcccta tcaactttcg atggtacttt ttgtgcctac 360catggtgacc acgggtaacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 420ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga ctcggggagg 480tagtgacgaa aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtacac 540tttaaatcct ttaacgagga tccattggag ggcaagtctg gtgccagcag ccgccggtaa 600ttccagctcc atagcgtatc ttaaanttgc ctgccagtta aataagcctc 650139709DNAPseudopleuronectes americanus 139gaattcggca cgagtggccg tccctcttaa tcatggcccc agttcagaga agaaaaccca 60caaaatagaa ccggagtcct attccattat tcctagctgc ggtattcagg cgaccgggcc 120tgctttgaac actctaattt tttcaaagta aacgcttcgg accccgcggg acactcagct 180aagagcatcg agggggcgcc gagaggcagg ggctgggaca gacggtagct cgcctcgcgg 240cggaccgtca gctcgatccc gaggtccaac tacgagcttt ttaactgcag caactttaag 300atacgctatt ggagctggaa ttaccgcggc tgctggcacc agacttgccc tccaatggat 360cctcgttaaa ggatttaaag tgtactcatt ccaattacag ggcctcgaaa gagtcctgta 420ttgttatttt tcgtcactac

ctccccgagt cgggagtggg taatttgcgc gcctgctgcc 480ttccttggat gtggtagccg tttctcaggc tccctctccg gaatcgaacc ctgattcccc 540gttacccgtg gtcaccatgg taggcacaaa aagtaccatc gaaagttgat agggcagaca 600ttccgaatga gacgtcgccg ccaccgaggg ccagcggatc tagctcgagg ttatctagag 660tcaccaaaag ccggccgggg caccccgtga ggaacacccc gccattggg 709140679DNAPseudopleuronectes americanus 140gaattcggca cgaggtcagc tcgatcccga ggtccaacta cgagcttttt aactgcagca 60actttaagat acgctattgg agctggaatt accgcggctg ctggcaccag acttgccctc 120caatggatcc tcgttaaagg atttaaagtg tactcattcc aattacaggg cctcgaaaga 180gtcctgtatt gttatttttc gtcactacct ccccgagtcg ggagtgggta atttgcgcgc 240ctgctgcctt ccttggatgt ggtagccgtt tctcaggctc cctctccgga atcgaaccct 300gattccccgt tacccgtggt caccatggta ggcacaaaaa gtaccatcga aagttgatag 360ggcagacatt cgaatgagac gtcgccgcca cgagggccag cgatcagctc gaggttatct 420agagtcacca aagcggccgg ggcaccccgt gaggagcacc ccgcatgggt tttgggtctg 480ataaatgcac gcgtccccgg aggtcagcgc ccgttggcat gtattagctc tggaattgcc 540acagttatcc aagtaacgtg agagcgatca aaggaaccat aactgattta atgagccatt 600cgcagtttca ctgtaccggc cgtgtgtatt agacttgcat ggcttaatct ttgagacaag 660catatctcgt gccgaattc 679141611DNAPseudopleuronectes americanus 141gaattcggca cgaggcccta tcaactttcg atggtacttt ttgtgcctac catggtgacc 60acgggtaacg gggaatcagg gttcgattcc ggagagggag cctgagaaac ggctaccaca 120tccaaggaag gcagcaggcg cgcaaattac ccactcccga ctcggggagg tagtgacgaa 180aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtacac tttaaatcct 240ttaacgagga tccattggag ggcaagtctg gtgccagcag ccgcggtaat tccagctcca 300atagcgtatc ttaaagttgc tgcagttaaa aagctcgtag ttggacctcg ggatcgagct 360gacggtccgc cgcgaggcga gctaccgtct gtcccagccc ctgcctctcg gcgccccctc 420gatgctctta gctgagtgtc ccgcggggtc cgaaacgttt actttgaaaa aattagagtg 480ttcaaagcag gcccggtcgc ctgaataccg catctaggaa taatggaata ggactccggt 540tctattttgt gggttttctt ctctgaactg gggccatgat taagaaggac ggccgggctc 600gtgccgaatt c 611142476DNAPseudopleuronectes americanusmodified_base(335)N = A, C, G, or T/U 142gaattcggca cgaggtgccc ttccgtcaat tcctttaagt ttcagctttg caaccatact 60ccccccggaa cccaaagact ttggtttccc ggacgctgcc cggcgggtca tgggaataac 120gccgccggat cgctagttgg catcgtttac ggtcggaact acgacggtat ctgatcgtct 180tcgaacctcc gactttcgtt cttgattaat gaaaacattc ttggcaaatg ctttcgcttt 240cgtccgtctt gcgccggtcc aagaatttca cctctagcgg cacaatacga atgcccccgg 300ccgtccctct taatcatggc cccagttcag agaanaaaac ccacaaaata gaaccggagt 360cctattccat tattcctagc tgcggtattc aggcgaccgg gcctgctttg aacactctaa 420ttttttcaaa gtaaacgctt cggaccccgc gggacactca gcctcgtgcc gaattc 476143740DNAPseudopleuronectes americanusmodified_base(526)N = A, C, G or T/U 143gaattcggca cgaggctgcg gtattcaggc gaccgggcct gctttgaaca ctctaatttt 60ttcaaagtaa acgcttcgga ccccgcggga cactcagcta agagcatcga gggggcggaa 120ttcggcacga gctgggacag acggtagctc gcctcgcggc ggaccgtcag ctcgatcccg 180aggtccaact acgagctttt taactgcagc aactttaaga tacgctattg gagctggaat 240taccgcggct gctggcacca gacttgccct ccaatggatc ctcgttaaag gatttaaagt 300gtactcattc caattacagg gcctcgaaag agtcctgtat tgttattttt cgtcactacc 360tccccgagtc gggagtgggt aatttgcgcg cctgctgcct tccttggatg tggtagccgt 420ttctcaggct ccctctccgg aatcgaaccc tgattccccg ttacccgtgg tcaccatggt 480aggcacaaaa agtaccatcg aaagttgata gggcagacat tcgaangaga cgtcgccgcc 540acgagggcca gcgatcagct cgaggttatc tagagtcacc aaagcggccg gggcaccccg 600tgaggagcac cccgcatggg ttttgggtct gataaatgca cgcgctctct ctctctctct 660ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct 720ctctccctcg tgccgaattc 740144334DNAPseudopleuronectes americanus 144gaattcggca cgaggctgcg gtattcaggc gaccgggcct gctttgaaca ctctaatttt 60ttcaaagtaa acgcttcgga ccccgcggga cactcagcta agagcatcga gggggcgccg 120agaggcaggg gctgggacag acggtagctc gcctcgcggc ggaccgtcag ctcgatcccg 180aggtccaact acgagctttt taactgcagc aactttaaga tacgctattg gagctggaat 240taccgcggct gctggcacca gacttgccct ccaatggatc ctcgttaaag gatttaaagt 300gtactcattc caattacagg gcctcgaaag agtc 334145542DNAPseudopleuronectes americanusmodified_base(516) (521)N = A, C, G, or T/U 145gaattcggca cgagtacttg gataactgtg gcaattccag agctaataca tgccaacggg 60cgctgacctc cggggacgcg tgcatttatc agacccaaaa cccatgcggg gtgctcctca 120cggggtgccc cggccgcttt ggtgactcta gataacctcg agctgatcgc tggccctcgt 180ggcggcgacg tctcattcga atgtctgccc tatcaacttt cgatggtact ttttgtgcct 240accatggtga ccacgggtaa cggggaatca gggttcgatt ccggagaggg agcctgagaa 300acggctacca catccaagga aggcagcagg cgcgcaaatt acccactccc gactcgggga 360ggtagtgacg aaaaataaca atacaggact ctttcgaggc cctgtaattg gaatgagtac 420actttaaatc ctttaacgag gatccattgg agggcaagtc tggtgccagc agccgcggta 480attccagctc caatagcgta tcttaaagtt gcctcntgcc naatcctgca gccgggggat 540cc 542146532DNAPseudopleuronectes americanus 146cgtccgtctt gggccggtcc aagaatttca cctctagcgg cacaatacga atgcccccgg 60ccgtccctct taatcatggc cccagttcag agaagaaaac ccacaaaata gaaccggagt 120cctattccat tattcctagc tgcggtattc aggcgaccgg gcctgctttg aacactctaa 180ttttttcaaa gtaaacgctt cggaccccgc gggacactca gctaagagca tcgagggggc 240gccgagaggc aggggctggg acagacggta gctcgcctcg cggcggaccg tcagctcgat 300cccgaggtcc aactacgagc tttttaactg cagcaacttt aagatacgct attggagctg 360gaattaccgc ggctgctggc accagacttg ccctccaatg gatcctcgtt aaaggattta 420aagtgtactc attccaatta cagggcctcg aaagagtcct gtattgttat ttttcgtcac 480tacctccccg agtcgggagt gggtaatttg cgcgcctgct gccttccttg ga 5321471135DNACricetulus griseus 147gggcggcgac ggtttcctgg tggccgcgcg ctgctctgtg agcggcgggt ggcagacgga 60cctgggccct caccccagac gcaccgcgga tctggcatgg ctcacctgat gacaatgcag 120ttgctgctcc tgctgatatg ggtatctgag tgtgcccaat caagagctac tcgggccaga 180actgaactgc tcaatgtttg catggatgca aagcaccaca aagaaaagcc aggccctgag 240gacaatttac acaaccagtg cagtccctgg aagaagaatt cctgctgttc caccaacaca 300agccaggaag cccacgagga catttcctac ctgtaccgat tcaactggga ccactgtgga 360aagatgacat tggaatgcaa gcgacacttt atccaggata cctgtctcta tgagtgttct 420cctaacttgg gaccctggat tcagcaggtg gaccagagct ggcgaaaaga gcgaatcctt 480gatgttcctc tgtgcaaaga ggactgtcag cgatggtggg aggactgccg cacctctttc 540acctgcaaga gcaactggca caaggggtgg aactggacct cggggtataa ccagtgccct 600gtgggagcct cctgtcgcca cttcgacttc tatttcccta cacctgctgc tctgtgtgag 660gaaatctgga gtcactccta caaactcagt aactacagcc gagggagtgg ccgctgtatc 720cagatgtggt tcgacccagc ccaaggcaac cccaacgagg aagtggcaag gttctatgct 780gaggccatga gtggagctgg gcttcacggg gcctggccac taatgtgcag cctgtcttta 840gtgctgctct gggtgttcag ccgagttcct ttaaccttct gatccccagg aactccctgc 900cgggcttaga ctcccagctc ccaacctcct ttgtggtggg gcctctgaca ggcattcaat 960atctctctta tgaattattt gggtgtgaat gggaatataa ttattttgca tcctacttac 1020cactgattga agttgtttaa acttggttag ttccctgctc taacacttac tgtgggcaag 1080ttaaataaac ttaattttcc tgtgctgttc cacaaaaaaa aaaaaaaaaa aaaaa 113514870DNAMus musculus 148atcggacgcc ccccgtgtcg gtgacgaccc attcgaacgt ctgccctatc aactttcgat 60ggtagtcgct 70149470DNAMus musculus 149gtggacgaag actgtagaga ctacccagag tctgacctag ggagaggcca actcggatac 60ccctatgtgc gctcccagaa gctaaggaca ttgagacaga aagacatggc ctggaaacag 120acaccactct tgcttttggt ctacatggtc acaacaggca gtggcgggac agaacagacc 180tactcaacgt ttgcatggat gccaaacacc ataagacaaa gccgggcccc gaggacaagc 240tgcatgacca gtgtagtcca tggaagaaaa atgcctgttg ctcagtcaac accagccagg 300agctacacaa ggctgactcc cgtctgtact tcaactggga tcactgtggc aagatggagc 360ctgcctgtaa gagtcacttc atccaagact cctgcctgta tgagtgctcc ccaaccttgg 420ccttggatca gcaagtggac agagttggcg taagagcgtt ctggatgtgc 470150387DNAMus musculus 150gagaattagg gttcgattcc ggagagggag cctgagaaac ggctaccaca tccaaggaag 60gcagcaggcg cgcaaattac ccaatcctga cacggggagg tagtgacaat aaataacaat 120accgggctct tcgagtctgg taattggaat gagtacaatc taaatccctt aacgaggatc 180cattggaggg caagtctggt gccagcagcc gcggtaattc cagctccaat agcgtatatt 240taagttgttg cagttaaaaa gctcgtagtt gctgtcttta ggggactctc actctcctgc 300ttgtcgttgt gttcttaagg tcttgtcttt attgccggtt gatgtactgc tagtcgtaat 360tgctctcatt tgccctgtcg tttccgt 38715122DNAMus musculus 151cgccgtgcct accatggtga cc 2215246DNAHomo sapiens 152gggccccgcg ggacactcag ctaaaagcat cgagggggcg ccgaga 46153635DNAGossypium hirsutummodified_base(565)N = A, C, G, or T/U 153ctaaatccct taacgaggat ccattggagg gcaagtctgg tgccagcagc cgcggtaatt 60ccagctccaa tagcgtatat ttaagttgtt gcagttaaaa agctcgtagt tggacttagg 120ggtgggtcgg ccggtccgcc tcacggtgag caccggtctg ctcgtcccta ctgccggcga 180tgcgctcctg gccttaattg gccgggtcgt tcctccggcg ctgttacttt gaagaaatta 240gagtgctcaa agcaggccta cgcttgtata cattagcatg ggataacatc ataggatttc 300gatcctattg tgttggcctt cgggatcgga gtaatgatta acagggacag tcgggggcat 360tcgtatttca tagtcagagg tgaaattctt ggatttatga aagacgaaca actgcgatag 420catttgccaa ggatgttttc attaatcaag aacgaaagtt gggggctcga aaacgatcag 480ataccgtcct agtctcaacc ataaatctcc tccagttccg gaaccacatc ctccgccagt 540tccagtctat aagaaaacac atccnactcc agttccagta tacaagatac catgtcctcc 600ccagttccag tctataaatc tcctccggtt ccatt 635154584DNAGossypium hirsutummodified_base(389) (513)N = A, C, G, or T/U 154ggataaccgt agtaattcta gagctaatac gtgcaacaaa ccccgacttc tggaagggat 60gcatttatta aataaaaggt cgacgcgggc tttgcccgtt gctctgatga ttcatgataa 120ctcgacggat cgcacggcct ttgtgccggc gacgcatcat tcaaatttct gccctatcaa 180ctttcgatgg taggatagtg gcctactatg gtggtgacgg gtgacggaga attagggttc 240gattccggag agggagcctg agaaacggct accacatcca aggaaggcag caggcgcgca 300aattacccaa tcctgacacg gggaggtatt gacaataaat aacaataccg ggctctatga 360gtctggtaat tggaatgagt acaatctana tcccttaacg aagatccatt ggagggcaat 420tctggtgcca ncanccgcgg taattccact cccatancgt atatttaagt gtttgcagtc 480aaaaagctcg taattggact taggggtggg tcngccggtc cccctcacgg tgagcacggg 540tctgctcttc cctactgcgg gcgatgccct cctggcctta attg 58415565DNAPig 155ggattcctgc tgcttttgac cacagttctt tctgcaggac aagcatggcc cttgggagag 60cacgg 65156959DNAPig 156gatgagggag tccaggagtt ccagcaagct cgacctgctt aacactccca gacggtcaca 60ggattcagga caagcatggc ccttgggaga gcacggctgc tgctgctctt ggtgtgtgtg 120gctgtcacat gggcggcccg gcctgatctc ctcaacatct gcatggacgc caagcaccac 180aagaccaagc ccggcccgga agatggcctg catgagcagt gcagcccctg ggagatgaac 240gcctgctgct ccgtcaacac cagccaagaa gcccataacg acatctccta cctgtacaaa 300ttcaactggg agcactgcgg caagatgaag ccggcctgca agcgccactt cattcaagac 360acctgtctct atgagtgctc gcccaacctg gggccctgga tccaggaggt gaaccagaag 420tggcgcagag agcggatcct gaacgtgccc ctctgcaaag aggactgtca gaactggtgg 480gaagactgcc gcacctccta cacctgcaag agcaactggc acgagggctg gaactggagc 540tcagggtata accggtgccc cgcgaacgcc gcctgccacc ccttcgactt ctacttcccc 600acgcctgctg ccctgtgcag ccagatctgg agcaactcct acaaacaaag caactacagc 660cggggcagcg gccgctgcat ccagatgtgg ttcgacccgg aacagggcaa ccccaacgag 720gtggtggcga gatactacgc ccagatcatg agtggcgctg ggctctccga ggcctggcct 780ctccagttcg gcctggccct gacgctgctc tggctgctga gctgagcttc tgtcttcgga 840gagctggaca gccctcccct gttcggcccc acagcaccca gctcgtcagt gcctcagtgg 900tggtggtagt ggtggtggtg gtggcggcgg ggggactctg aataaaccag tcaccccac 9591571218DNAPig 157gacactgctt ccgggtgggc ctccaggagg gccgaggcag aggagcctct gcctgtgggt 60gaagcactgg ctggcgaact ccggaagggg aggtccggag aggtggtgcc tccccccgca 120gcaaagctca gactgcactg tcctcaggtg gcagtggtgt cctaccactt ggcacagacc 180tccacgggcc cttcatcgct tggctccact gtgctgtggg gtaagcggcg cggggaggga 240cgacgatctg ggcttggaag ggaaacagga aatctggcca agaagcttac ggcagctttc 300tggcagaagt ggatcaacat ggcctggcgg ctgacgctct tcgtgctcct gggtttggtg 360gctgctgtgg ggggcgcccg ggccaagtcg gacatgctca atgtctgcat ggatgccaag 420caccacaagc caaagccaag cccggaggac aagctgcacg accagtgcag cccctggagg 480aagaactcct gctgctcagt caacaccagc ctagaagccc ataaagacat ctcctacctg 540tacagattca actgggacca ctgcggcaag atggagccgg cctgcaagcg ccacttcatt 600caagacacct gtctctatga gtgctcgccc aacctggggc cctggatcca ggaggtgaac 660cagaagtggc gcagagagcg gatcctgaac gtgcccctct gcaaagagga ctgtcagatc 720tggtgggaag actgccgtac ctcctacacc tgcaagagca actggcacaa gggctggaac 780tggacctcag ggtataacca gtgcccagtg agcgccgcct gccaccgctt cgacttctac 840ttccccacgc ccgctgccct gtgcaacgag atctggagcc actcctttga agtcagcagc 900tacagccggg gcagcggccg ctgcatccag atgtggttcg acccggccca gggcaacccc 960aacgaggcgg tggcgagata ctatgcagag aatggggatg ctggggccgt ggcccagggg 1020atcgggcctc tcctgaccaa cttgacggag atggtgaaac actgggtcac cggctaagct 1080gttcccccgc cgacccctgc tttccgccca caccccctgg gttactctcc gggtggcctc 1140agcaccccgg tcattggctc ctgatctaag atccgatggg gagcctctga tggcctcttc 1200caatacaata tccacgtg 1218158406DNAMus musculus 158ctcagtcgca catagataaa attggccttt atttggagac gggtttgttc ttctatgttt 60aatcctcggg tgaaatgacc tgaagatatt tgtgtctgtt ttccgcatgg tcaagcaggg 120agtggagaga ggcctgggct gggccaggtt ttctgggctt tttcctgtgc tccgagtagg 180tgggttgtat tttacccagt aggagtggaa gactccttgg cgcttggtgc ttcaaccgga 240ctgacttcct gggcctggag ttggcgatta gaggtctgac atggctcacc tgatgactgt 300gcagttgttg ctcctggtga tgtggatggc cgaatgtgct cagtccagag ctactcgggc 360caggactgaa cttctcaatg tctgcatgga tgccaaacac cacaaa 4061591303DNAMus musculus 159gctgacggct ctagaagtcc ccaacctgtt gtgatcttca gtagacaaac actcctggtg 60tgtcacagga ttcaggccac taaacctcgg ccggctgtct cctggaatga agaaagcaaa 120ggaagcctag agtggagaca aagaagcccg aggcactctg agagctgcca tcttttcctt 180gtttgccgcc tgacacttct cagcaggatc cacataccct aagggagtgg agagaggcct 240gggctgggcc aggttttctg ggctttttcc tgtgctccga gtaggtgggt tgtattttac 300ccagtaggag tggaagactc cttggcgctt ggtgcttcaa ccggactgac ttcctgggcc 360tggagttggc gattagaggt ctgacatggc tcacctgatg actgtgcagt tgttgctcct 420ggtgatgtgg atggccgaat gtgctcagtc cagagctact cgggccagga ctgaacttct 480caatgtctgc atggatgcca aacaccacaa agaaaaaccg ggccctgagg acaatttaca 540cgaccagtgc agcccctgga agacgaattc ctgctgttcc acgaacacaa gccaggaagc 600acataaggac atttcctacc tgtaccggtt caactggaac cactgcggaa ctatgacatc 660ggaatgcaaa cggcacttta tccaagacac ctgcctctat gagtgttccc cgaacttggg 720accctggatc cagcaggtgg accagagctg gcgcaaagag cggatccttg atgttcccct 780gtgcaaagag gactgtcagc agtggtggga ggactgccag agctctttta cctgcaagag 840caattggcac aagggatgga actggtcctc ggggcataac gagtgtcctg tgggagcctc 900ctgccatccc ttcaccttct acttccccac atctgctgct ctgtgtgagg aaatctggag 960tcactcctac aagctcagca actacagtcg agggagcggc cgctgcattc agatgtggtt 1020cgacccagcc cagggcaacc ccaacgagga agtggcgagg ttctatgccg aggccatgag 1080tggagctggg tttcatggga cctggccact cttgtgcagc ctgtccttag tgctgctctg 1140ggtgatcagc tgagctcctg ttttaccttc agttgtctgg agcgccaccc tgcttggctc 1200agcctcccag ctcccagcct cctttgtggt ggggctctga cagcctcttt aataaaccag 1260acattccaca tgtgccttat gaattaaaaa aaaaaaaaaa aaa 130316070DNAMus musculus 160cccgttaaag gatttaaagt ggacctcatc caattacagg gccttgaaag aatcctgtat 60tgttatattt 70161822DNAMus musculusmodified_base(719) (781)N = A, C, G, or T/U 161ataaggcaca tgtggaatgt ctggttgatt aaagaggctg tcagagcccc accacaaagg 60aggctgggag ctgggaggct gagccaagca gggtggcgct ccagacaact gaaggtaaaa 120caggagctca gctgatcacc cagagcagca ctaaggacag gctgcacaag agtggccagg 180tcccatgaaa cccagctcca ctcatggcct cggcatagaa cctcgccact tcctcgttgg 240ggttgccctg ggctgggtcg aaccacatct gaatgcagcg gccgctccct cgactgtagt 300tgctgagctt gtaggagtga ctccagattt cctcacacag agcagcagat gtggggaagt 360agaaggtgaa gggatggcag gaggctccca caggacactc gttatgcccc gaggaccagt 420tccatccctt gtgccaattg ctcttgcagg taaaagagct ctggcagtcc tcccaccact 480gctgacagtc ctctttgcac aggggaacat caaggatccg ctctttgcgc cagctctggt 540ccacctgctg gatccagggt cccaagttcg gggaacactc atagaggcag gtgtcttgga 600taaagtgccg tttgcattcc gatgtcatag tttcgcaggg ttccagttga accggtacag 660gtaggaaatg tccctatgtg cttcctggct ttgtgtcgtg aacagcagga atcgtcttnc 720aggggctgcc actgtcgtgt aaattgcctc angggcccgt tttttctttg tgtggtgcat 780ncatgcagac aatttgaaat cagtcctggc cgagtagctc tg 822162185DNAMus musculus 162aaggcctggt aattaaaaag gctgcaaagc cccacccaaa ggaggttggg agctgggagg 60ttgacccaac cagggtggcc ctccaaacaa ctgaaggtaa aacaggagct cagttgatca 120cccaaagcag cattaaggac aggcttgcca aaagtggcca ggtcccatga aacccagttc 180cattc 185163347DNAMus musculus 163gtagttgctg agcttgtagg agtgactcca gatttcctca cacagagcag cagatgtggg 60gaagtagaag gtgaagggat ggcaggaggc tcccacagga cactcgttat gccccgagga 120ccagttccat cccttgtgcc aattgctctt gcaggtaaaa gagctctggc agtcctccca 180ccactgctga cagtcctctt tgcacagggg aacatcaagg atccgctctt tgcgccagct 240ctggtccacc tgctggatcc agggtcccaa gttcggggaa cactcataga ggcaggtgtc 300ttggataaag tgccgtttgc attccgatgt catagttccg cagtggt 34716443DNAMus musculus 164taccacaacc aaagaaagca ttacacgcgc atattaccca ctg 43165556DNAMus musculusmodified_base(501)N = A, C, G or T/U 165gagagttgaa cttgccaccc acttcaggga tctctggtac cacaaggtct tgtttctctc 60tctctcttgg aggcaggcta ctcaggtcta gctactggcg gctctccaca cctgtagctc 120atagaagctg aaggctgata aaagggcagt gggtggagcg ccctcagccc gctcacctct 180ttggcatcag gaggagcaac aggagggccc tgccttgaag gtcatggcac agtggtggca 240gatcctcttg gggttgtggg cagtcctacc caccttggca ggggacaaac tgctcagcgt 300ctgcatgaat tccaagcgcc acaagcaaga acctggccca gaagacgaac tctaccagga 360gtgcaggcct tgggaggaca

atgcctgctg cacacgttcc acaagttggg aagcccacct 420tgaggagccc ttgctcttta acttcagcat gatgcactgt ggactgctga ccccggcctg 480tcgcaaagca ctcattccag nccatttgtt tccatgatgt tcccccaacc tggggccctg 540gatcccaccc gtgtcc 556166353DNAMus musculus 166acacagagca gcagatgtgg ggaagtagaa ggtgaaggga tggcaggagg ctcccacagg 60acactcgtta tgccccgagg accagttcca tcccttgtgc caattgctct tgcaggtaaa 120agagctctgg cagtcctccc accactgctg acagtcctct ttgcacaggg gaacatcaag 180gatccgctct ttgcgccagc tctggtccac ctgctggatc cagggtccca aagttcggga 240acactcatag aggcaggtgt cttggataag tgccgttgca ttccgatgtc atagttccgc 300agtggttcag ttgacccgta cggtaggaat gtcctatgtg cttctggctg tgt 35316758DNAMus musculus 167cccccggggc cggaaggggg aaatttgccc cccggcgccc ttcctgggag ggggaacc 5816849DNAHomo sapiens 168cggcgaacac catcgaaagt taatagggca gacgttcaaa taggtcgtc 49169448DNARATmodified_base(387)N = A, C, G or T/U 169cggccgctcc ctcggctgta gttgctgagc ttgtaggaat gactccagat tttctcacac 60agaacagcag gtgtagggaa gtagaaagtg aagggatggc aggaggctcc cacagggcac 120tcattatgcc ccgaggtcca gttccatccc ttgtgccagt tgctcttgca ggtaaaagag 180ctcttgcagt cctcccacca cagcacacag tcttctttgc acaggggaac atcaaggatc 240cgctctttgc gccagctctg gtccacctgc tggatccagg gtcccaagtt cggggaacac 300tcatagaggc aagtgtcttg gataaagtga cgtttgcact ccggggtcat agttccacag 360tgattccagt tgaatcggta caggtangaa atgtccttat gtgcttggca tccatgcaga 420cattgagaag ttcgcctcgt gccgaatt 448170435DNARATmodified_base(378)N = A, C, G, or T/U 170tttttttttt tttttttaga tgtgtcatag atttattgaa aatgattgtt gtagaaaccc 60caagccccaa catggatcag gaactggatg gcagaggaga ggggtggaag caggctcaag 120acttccttat tctttgaaga gttgaaccaa ccgagaccaa gggtagctag aggagaagac 180tggcctcagt cagggagcca taatgacagc actggggcca ggctgggcac aagaagtatt 240gctgcatggt acacagtccc agatgtcata aaggaagcat agaacttcac cacttcctca 300ttgggattgc cctgggttga gtcaaaccac atctggatgc actggccact ccctctgcta 360tagttgctga ccttgtanga gtgactccag agaccctcac aaaggctggc tggtgtcggg 420aaatagtact gaaat 435171429DNARATmodified_base(387)N = A, C, G or T/U 171cggccgctcc ctcggctgta gttgctgagc ttgtaggagt gactccagat tttctcacac 60agaacagcag gtgtagggaa gtagaaagtg aagggatggc aggaggctcc cacagggcac 120tcattatgcc ccgaggtcca gttccatccc ttgtgccagt tgctcttgca ggtaaaagag 180ctcttgcagt cctcccacca cagcacacag tcttctttgc acaggggaac atcaaggatc 240cgctctttgc gccagctctg gtccacctgc tggatccagg gtcccaagtt cggggaacac 300tcatagaggc aggtgtcttg gataaagtga cgtttgcact ccggggtcat agttccacag 360tgattccagt tgaatcggta caggtangaa atgtccttat gtgcttcctg gcttgtgttg 420gtggagcag 429172427DNARAT 172cggccactcc ctcggctgta gttgctgagc ttgtaggagt gactccagat tttctcacac 60agaacagcag gtgtagggaa gtagaaagtg aagggatggc aggaggctcc cacagggcac 120tcattatgcc ccgaggtcca gttccatccc ttgtgccagt tgctcttgca ggtaaaagag 180ctcttgcagt cctcccacca cagcacacag tcttctttgc acaggggaac atcaaggatc 240cgctctttgc gccagctctg gtccacctgc tggatccagg gtcccaagtt cggggaacac 300tcatagaggc aggtgtcttg gataaagtga cgtttgcact ccggggtcat agttccacag 360tgattccagt tgaatcggta caggtaggaa atgtccttat gtgcttcctg gcttgtgttg 420gtggagc 427173382DNARAT 173cggccgctcc ctcggctgta gttgctgagc ttgtaggagt gactccagat tttctcacac 60agaacagcag gtgtagggaa gtagaaagtg aagggatggc aggaggctcc cacagggcac 120tcattatgcc ccgaggtcca gttccatccc ttgtgccagt tgctcttgca ggtaaaagag 180ctcttgcagt cctcccacca cagcacacag tcttctttgc acaggggaac atcaaggatc 240cgctctttgc gccagctctg gtccacctgc tggatccagg gtcccaagtt cggggaacac 300tcatagaggc aggtgtcttg gataaagtga cgtttgcact ccggggtcat agttccacag 360tgattccagt tgaatcggta ca 38217452DNAHomo sapiens 174aaggcccggg aactcccatc aaaagttgtt agggcaaact ttcaaatggg tc 52175847DNAMus musculusmodified_base(520)..(717)N = A, C, G or T/U 175aattcggatc catgggctga tctggaagta taaacaagaa aggaggctga cggctctaga 60agtccccaac ctgttgtgat cttcagtata caaacactcc tggtgtgtca caggattcag 120ctctgtttcc taggccacta aacctcggcc ggctgtctcc tggaatgaag aaagcaaagg 180aagcctatag tggagacaaa gaagcccgag gcactctgag agctgccatc ttttccttgt 240ttgccgcctg acacttctca gcaggatcca cataccctaa ggagtggaag actccttggc 300gcttggtgct tcaaccggac tgacttcctg tgcctggagt tggcgattag actctgcctt 360cagggtctga catggctcac ctgatgactg tgcagttgtt gctcctggtg atgtggatgg 420ccgaatgtgc tcagtccata gctactcggg ccaggactga acttctcaat gtctgcatgg 480atgcctaaca ccacaaagat aaaccgtccc tgaggacatn tacacgacca gtgcagcccc 540tgcaagacaa ttactgctgt tccactaaca caagccagga agcacataat gacatttcct 600acctgtaccg tttcactgga accactgctg aactatgaca tcggaatgca tacggcacta 660tatccaagac acttgctcta tgagtgttcc cccgacttgt gaccctgtat tcagcangtg 720gaacatgact tgcgcatata cggatccttg atgttcccct gtgcaaagag gactgtcagc 780attgatgtga tgactgccat agctctttac ctgtcagaac atttgtccat ggtatgtaac 840tgttcct 847176326DNAHomo sapiens 176gggtcatttc cacatgcttt attccagcaa tcaaaataat taaaaacatc tcaaattatt 60atacacatac aaaataggta cagagtcttt tgcttcctcc cacccctagg gggaaaaact 120gctttgtgct ttgggaagtt gtctctgaaa cccggggaca gaggacgcag gacagactag 180gagggagccg ggaggatggg ctgcagctgt ggaggagggt ttcagaggag agaggtcgga 240gagcagaggc ctgagaagcc tgattccccg tcacccgtgg tcaccatggt aggcacggca 300actaccatcg aaagttgatg ggcaga 3261774409DNAHomo sapiens 177actagttgtc tgttgctgca taacaaatca ttccataatt ttgtggtgta ttgctgcaga 60caatgttaaa ctaagtggat gaaaaggata ttcacatagt ctcagagtgt ctccctacaa 120ggtaggatta ctaacaaagg gaaactaata attatatagt aaggaaatct ccttaaccca 180ataatcacca gcaataagat gcagcaaccc tcatcatgta cctcttgata tgatgcactg 240acaaaagcac ctctcttctc taattttctt gccaaaatcg ataagctcaa gctaattaca 300ggaaaatata gacaaaccca aattgaggga cattctgcaa aataactgaa cagtaattct 360ccaaaagtgt caaggtcata aaagacaaag acattgagga ctgtcacaga ttggagggag 420actaagggga catgacaact acatgcaacc tggaatcatg gactgaatcc tgggccagag 480aaggacattg ggggggaact ggtgtaaagg gcataaagct tgtagattag ttaacagtat 540tgcctcaata ttaatttcct gattttttta agaactgggc tttggttaca taagatgcca 600atatttgggg aagttgcata aaaacatacg ggaaatcttt tgacgatgtt ttgcagtttt 660tctgcaaatc taaaattatt tcaaaacaaa aagtttaaaa atcaaataca catagttgct 720tgaaatagta actattttat tatattccaa gatgttgtga gtcaggaatt tggccaaaac 780tcaggtgggc gattcttctg caaagacccc cacaacacat tcaaagtcac aggcagaggt 840tgttggggga gggcattgaa aagaagagaa gagtcatagg tgggtgcaat ggagggaggg 900cagagggctg ctgactatgt gcaggactca tccataatgg agccctgggg aggcaagggc 960ttcataacta gacactggtc ttgtcacctc agactcacct gtagcaggac cagatactga 1020ggtcagactg aaaacacagg ctctgcctca ggagaggctc tctactagct gagtaaatga 1080tgacagtatt ggaaatgttc ccaacatcat aatgggaaaa catcacttca cactacataa 1140gcaatacaca ggggcagtgc cggtcgtctt cccaggttag tagcagttct actgcctcca 1200agagtgttgg agaaatacaa accaagcatt aggcactttt aacttgaaaa catgaagttc 1260tctttcctaa ctttctttgt ttccttattt cttcttcttc ttcttcttct tcttcttctt 1320cttcttcttc ttcttcttct tcttcttctt cttcttcttc ttcctcttct tcttcttctt 1380cttctttctt cttcttctcc ttctcctttt ccttcttctt tttttgctga gacagggtct 1440cactctgaca gtacagtggt gccatcacag ctcactgcag cctcgacctc cagggctcaa 1500gcaatcctcc cagctcaccc tcccaaatgg ctgaaactac aagctcgcac caccatacgt 1560ggctaatttt tctatttttg tgtgcagatg aagttttcct atgttgccca agtggtctca 1620aactcctggg atcaagtgat ccatccacct caacctccca aaacgctggg attacaggtg 1680taagccacca cacccagccc actaactttt ttatatcggc taatgaaata gttttaagtt 1740tagaccctac gaggcataaa gaaataattt tagttatgtt atcagatgta cagtaatact 1800caagtgtgca actgtggata acttgagttc atgaggtttt tgtttttttg tcaaaagaat 1860aaatttatag tgaaactacc caaaaaagca aagtacagaa cagtatgcta ccatttgtgc 1920acagaaatgg gatatatatg gtgtaactgc atcgaattta ctggatgtat gtccagggac 1980cagaactctt ggtggcttca tgttcatact tttgcaagca catgtgtagt atccttaact 2040taaaggtact gttgtataca ttctagtgtt atcaaaattt acatacatat tatcaagtca 2100gagaggtcat tctgtgtctt agtattttca cttcatattt ggtatattta tgtatgtata 2160cacacatacc tatatgtatt taaataagat ttatagtcac atggtccaaa aatcaaaaca 2220atgtggaaag gtttacagag aaaagtctca agcctaatcc tgttctctac tgccaggtga 2280ccatgttatt aatttctttt cataccttgc cacagaattt tcacctgcaa acacagatat 2340tcttttcttt tttaatgaca gagtcacgtt ctgttatcca ggctggagtg cagtggcgtg 2400atcttggctc actgcaaact cctcccgggt tcaagtgatt ctcctgtctc agcctcctga 2460gtagctggga ttacaggcat gtgccaccac acccagctaa tttttgtatt tttagtacag 2520atggggtttt atcatattga ccaggctgat gtcgaactcc tgacctcaag tgatccgcct 2580gcctcggcct cccacagtgc tgggattaca ggcgtgagcc accacgccca gtcaacacag 2640acattcttac tcctttttta cagagaattt attattatta ttttttacat agcatttttc 2700tgcacctttc tttttccact taacaatgca cttgaagatt tttccatatt tgtacatcag 2760gagctttctc tttctttgtt accacattaa attccactgg gtagatgtac cataatttaa 2820ctgggtcctt attgaaagac aattgagctg tctcctagac aaagccttgt gcaccttccc 2880gaacagaggg tctaaccaag caggcaggat ggggttataa agtaggtggg gaggtgggag 2940agactccacc ttcccaggtg ggctgagaat ggaggtaagg ccctgcaaca ggacagaggg 3000aaaagtgggg atgagaggtg ggaggcgaga tagcgcccac tgttctcgct cagccccctc 3060ctccgtttgc cgctgacctg ttggcctccc ccaacctctg agcctgcctc tgcctaggta 3120atttcccaag acccagaagg ggtgaagggt gaggtgtgat tgcccccacc tccttgcctc 3180ccgcagcatc tgctccggga ccatgaacaa tagctgacag ctccatggcc cttgctgtcc 3240ccatctcagc ttccctgggc atctaaacct cagctgccat ggggtaggag gacaggctga 3300ggaagcagaa gcctgaggct gtctagagtc tcactcctgc atcagcaggc caccacctgt 3360ggttcctcct tgtgcaaatt tgaaaagaat tgcataaaac actggagaaa tccaagaggg 3420gaagtccaca agggcggtgg ctccctacaa ggtcacagag caagctggtg tcagagcctg 3480gacctacagc gctgttggtg gaggtcctgc ctccaggtag gggaagggct ccctctcacc 3540tctacacgca gcgcatttct tggctcagct gccctgtagg ggatgcaggg tggggacagc 3600agagatctgg gcctgggagg gagagagtac acaatcacat ggctgttgcc cctgtctcag 3660gccttgtcta cctctgactg tggctctctg gcaggaatag atggacatgg cctggcagat 3720gatgcagctg ctgcttctgg ctttggtgac tgctgcgggg agtgcccagc ccaggagtgc 3780gcgggccagg acggacctgc tcaatgtctg catgaacgcc aagcaccaca agacacagcc 3840cagccccgag gacgagctgt atggccaggt gagggcagcc tggtgtagga cagcatgcac 3900acaggtcaga gggtgatggc acgagcaatg gcaggtccag tgtggtcaga accaagggtg 3960ccgctgctga caaggaaggg gaggggcggc cagggccacc atgccacagg taaggccact 4020gaggcagctt ggggaatatg agctccaatt tgaactccag gctcaggagt gtgcttgtat 4080ttcattcctc tggtctcctg gcctgctccc tacaaggttt cacattccca gagggctggg 4140gatgtgccta gggagagact gtggcgtgga cacaatctgt gggttaaagc gaagacagga 4200cagcctggaa gccccatgac atctgagtca ctcccaacat tccatttgct tatttttaaa 4260tcggggttaa aaaaaaaaaa caaatacata acatacattt tccactttgg ccatttttaa 4320ctgtacggtt cagtggcatt aggtatgctc atgtggttgt gcaaccatca ccaccatcca 4380tctcctgacc tctgtgattc tccaaaact 4409178847DNAMus musculusmodified_base(645) (712)N = A, C, G, or T/U 178aattcggatc catgggctga tctggaagta taaacaagaa aggaggctga cggctctaga 60agtccccaac ctgttgtgat cttcagtaga caaacactcc tggtgtgtca caggattcag 120ctctgtttcc taggccacta aacctcggcc ggctgtctcc tggaatgaag aaagcaaagg 180aagcctagag tggagacaaa gaagcccgag gcactctgag agctgccatc ttttccttgt 240ttgccgcctg acacttctca gcaggatcca cataccctaa ggagtggaag actccttggc 300gcttggtgct tcaaccggac tgacttcctg ggcctggagt tggcgattag actctgcctt 360cagggtctga catggctcac ctgatgactg tgcagttgtt gctcctggtg atgtggatgg 420ccgaatgtgc tcagtccaga gctactcggg cccagactga acctctcatg tctgatggat 480gccaaacacc acatagaata accgggccct gaggacaatt tacacgacca gtgcagcccc 540tggaagacga aatcctgctg ttccacgaac acaagccagg aagcacataa ggacatttcc 600tacctgtacc ggttcaactg gaaccactgc ggaactatga catcngcaat gcanacggca 660ctttatccaa gacacctgcc tctatgagtg ttccccgaac ttgggacact gnatccagca 720ggtgggacca aagcttgcgc caaagagcgg atcccttgat gtttcccctg ggcaaagagg 780actgtccagc agttgtgggg aggactgcca gaagctcttt tacctgccag agcaatttgc 840accaggg 847179541DNAMus musculus 179gtagttgctg agcttgtagg agtgactcca gatttcctca cacatagcag cagatgtggg 60gaagtagaag gtgaagggat ggcaggaggc tcccacagga cactcgttat gccccgagga 120ccagttccat cccttgtgcc aattgctctt gcaggtaaaa gagctctggc agtcctccca 180ccactgctga cagtcctctt tgcacagggg aacatcaagg atccgctctt tgcgccagct 240ctggtccacc tgctggatcc agggtcccaa gttcggggaa cactcataga ggcaggtgtc 300ttggataaag tgccgtttgc attccgatgt catagttccg cagtggttcc agttgaaccg 360gtacaggtag gaaatgtcct tatgtgcttc ctggcttgtg ttcgtggaac agcaggaatt 420cgtcttccag gggctgcact ggtcgtgtaa attgtcctca gggcccggtt tttctttgtg 480gtgtttggca tccatgcaga cattgagaag ttcagtcctg gcccgagtag ctctggactg 540a 541180525DNAMus musculus 180acacagtagt tttcagatgt ggggaagtag aaggtgaagg gagggcagga tgctcccaca 60ggacactcgt tatgccccga ggaccagttc catcccttgt gccaattgct cttgcaggta 120aaagagctct ggcagtcctc ccaccactgc tgacagtcct ctttgcacag gggaacatca 180aggatccgct ctttgcgcca gctctggtcc acctgctgga tccagggtcc caagttcggg 240gaacactcat agaggcaggt gtcttggata aagtgccgtt tgcattccga tgtcatagtt 300ccgcagtggt tccagttgaa ccggtacagg taggaaatgt ccttatgtgc ttcctggctt 360gtgttcgtgg aacagcagga attcgtcttc caggggctgc actggtcgtg taaattgtcc 420tcagggcccg gtttttcttt gtggtgtttg gcatccatgc agacattgag aagttcagtc 480ctggcccgag tagctctgga ctgagcacat tcggccatcc acatc 525181805DNAMus musculusmodified_base(535)..(740)N = A, C, G or T/U 181gtggacgaag actgtagaga ctacccagag tctgacctag ggagaggcca actcggatac 60ccctatgtgc gctcccagaa gctaaggaca ttgagacaga aagacatggc ctggaaacag 120acaccactct tgcttttggt ctacatggtc acaacaggca gtggccggga cagaacagac 180ctactcaacg tttgcatgga tgccaaacac cataagacaa agccgggccc cgaggacaag 240ctgcatgacc agtgtagtcc atggaagaaa aatgcctgtt gctcagtcaa caccagccag 300gagctacaca aggctgactc ccgtctgtac ttcaactggg atcactgtgg caagatggag 360cctgcctgta agagtcactt catccaagac tcctgcctgt atgagtgctc ccccaacctt 420gggccttgga tccagcaagt ggaccagagt tggcgtaaag agcgttttct ggatgtgccc 480ctatgcaaag aggactgtca ccagtggtgg gaagcctgtc gtacctcctt taccntgcag 540agagactggc atanaggctg ggactggtcc tcaggcatta acaagtgccc anacacagca 600ccctgtcaca cgtntgagta ctacttcccg acaccagcca gcctttgcga gggtctctgg 660agtcactcct acaaggtcag caaactacag cagaggagtg gccgctgcat ccagatgtgg 720ttgactcacc ccanngcann tcgaaatgag acgtggtgaa gtttatgctt ctttatacat 780ctgggatgtg cccatgcaca gtact 805182585DNAMus musculusmodified_base(511)..(513)N = A, C, G or T/U 182acacagagca gcagatgtgg ggaagtagaa ggtgaaggga tggcaggagg ctcccacagg 60acactcgtta tgccccgagg accagttcca tcccttgtgc caattgctct tgcaggtaaa 120agagctctgg cagtcctccc accactgctg acagtcctct ttgcacaggg gaacatcaag 180gatccgctct ttgcgccagc tctggtccac ctgctggatc cagggtccca agttcgggga 240acactcatag aggcaggtgt cttggataaa gtgccgtttg cattccgatg tcatagttcc 300gcagtggttc cagttgaacc ggtacaggta ggaaatgtcc ttatgtgctt cctggcttgt 360gttcgtggaa cagcaagaat tcgtcttcca ggggctgcac tggtcgtgta aattgtgctc 420atggccctgg tcttctttag tgtgtttagc atccatgcag acatcgagaa gatcagtcct 480ggtccgagta gctctggact gagcacagtc ngncattcac atcatccaga gcaacaactg 540cacagtcatc aggtgagcca tgtcagaccc tgatgcagag tctaa 585183584DNAMus musculusmodified_base(557)N = A, C, G or T/U 183tgggtcataa attgattgaa aatgattgta gaaaccccaa cccccaacat ggatcaggaa 60ctggatgaca gaggagaggg gtggaagcag gctcaaaact tccttattct ttgaagagtt 120gaaccagacc aagggtagat agaggagaaa tctaaagagg aagactgacc tctcagccag 180ggagccataa tgacagcact ggggccaggc tgggcacaag aagtactgct gcatggggca 240cagtcccaga tgtcataaag gaagcataaa acttcaccac gtcctcattc ggattgccct 300gggttgagtc aaaccacatc tggatgcagc ggccactccc tctgctgtag ttgctgacct 360tgtaggagtg actccagaga ccctcgcaaa ggctggcttg tgtcgggaag tagtactcaa 420acgtgtgaca gggtgctgtt gttgggcacc ttgttaatgc ctgaggacca gtcccagcct 480tattgcaatc tttcttgcag gtaaaggagg acgacaggct tccaccactg gtgcagtcct 540ctttgataag ggacatncag aaacgctctt acgccactct ggtc 58418485DNAMus musculus 184ctatccattc gaacgtgtgc catatcatct tctgatgtac caacccgtgc ctaccatgtg 60gaccacgggt gactggcaat ccaga 8518570DNAMus musculusmodified_base(9)N = A, C, G or T/U 185attccccgnc ccccggggtc accaggggag gcgcggggac taccattaaa agttgatagg 60gcaaactttt 70186576DNAManduca sextamodified_base(187)..(530)N = A, C, G or T/U 186gaactagggc ggtatctaat cgccttcgaa cctctaactt tcgttcttga ttgatgaaaa 60cacctttggc aaatgctttc gctgatgttc gtcttgcgac gatccaagaa tttcacctct 120aacgtcgcaa tacgaatgcc cccagttatc cctattaatc attacctcgg agttctgaaa 180accaacnaaa tagaaccgag atcatattct attattccat gcacgaaata ttcaagcagc 240attttgagcc cgctttgagc actctaattt gttcaaagna aaattgtcgg cccatctcga 300cactcaccga agagcaccgc gataggattt tgatattgaa ccgacgtttg ttacaacgcc 360ggctcaccga cnatatgctc cgcagacgtg tcagtatcac cgcggatgcg gtgcaccgac 420agcncggcgc acaaatgcan ctacnagctt tttaaccgca acaattttag tatacgctat 480tggagctggg aattaccgcg gctgctggca ccagacttgc cctcaattgn cctcgttaaa 540atatttaaag tgtctcattc cgattacgaa gcctcg 576187295DNAMus musculusmodified_base(154)..(195)N = A, C, G or T/U 187cagcgagcct ttgcgggggt gtctggagtg actcctacga ggtgagcgac tacagcagag 60ggagtggccg ctgcgtccag atgtggtttg agtcagccca gggcgatccc aatgaggacg 120tggtggagtt ttatgcttcc tttatgacat ctgngactgt gccccatgca gcagtagttc 180ttgtgcccag cctnngccca gtgctgtcat tatagctccc tggctgagag gtcagtgttc 240ctctctagat ttcgtcctct atctaccctt ggtgctggtt cagctcttca gagaa 29518885DNAMus musculus 188cagctcacct cctgttttac cttcacttct ctccacgccc caccctcgct tcgcgctcac 60gcctcccagc tcccacgcct ccttt

8518988DNAMus musculus 189cctcccggct cctgcccgag ggtcgggcgc ctgcggcttt ggtgacttta gattacctcg 60ggccgatcgc acgccccccg tggcggcg 88190369DNAMus musculusmodified_base(93)..(353)N = A, C, G or T/U 190gtctctctct ctcttctctt gcttcgctct cttgcttttc tctctctctt gctttttcgc 60tctcttgctt ctcgctctct cttgcttctt gcnctctttt cctgaagatg taagaataaa 120gctttgccgc agaagattct ggtctgtggt gttcttcctg gccggtcgtg anaacgcgtc 180taataacaat tggtgccgaa ttccgggang anaaaatccg ggacgagaaa aaaactccgg 240antggcgcag gagggatact tcattccagg aancagaact gcgaatcaag gttanaangg 300atcncgtnac acagattgat tgagaagnnn tccnactggc cgaattcnag aanctcatcg 360cttggggaa 369191332DNAMus musculusmodified_base(309)N = A, C, G, or T/U 191ggtttttcga gacagggttt ctctgtgtag ccctggctgt ccttgaactc actttgtaga 60ccaggctggc ctcgaactca gaaatccgcc tgcctctgcc tcccaagtgc tgggattaaa 120ggcattcgcc accaccaacc ggcgataaac aaattttata cgaaagaaaa gaagcaagta 180agattatgag aaacataagc tattttaaga gagtttagag aagatccttc aaatatttta 240aaagagatct gaataaatca gaaagcatta ttcctggata aataatgggg agagaaataa 300tagattaana tacaacctat caaaatttaa tc 332192331DNAMus musculusmodified_base(295)..(308)N = A, C, G or T/U 192cgagacaggg tttctctgtg cagtcctgga actcactctg tagaccaggc tggccttgaa 60ctcagaaatc cacctgcctc tgcctcccaa gtgctgggat tgcaggcatg cgccaccact 120gcctggctgc ctggtttttt aattactggc tttagcctaa atggcaaatt ctataactag 180gttataagaa tagttttaaa agaaagagcc tcaggagagt gggaacagga acatggagaa 240gtaagaggac acctgggctt tagtcaagat cctgtctaaa acaaaacaga ggggncggna 300gagctngngc aatggctcag ttggttagag c 33119367DNAHomo sapiens 193ccgccacggg ggggtcgcga tcggtccgag gttatctaga gtcaccaaag ccgccggcgt 60cgtcccc 67194593DNAMus musculusmodified_base(489)..(578)N = A, C, G or T/U 194ctctctccag gtattcctac ctaaccttaa cttttcctcg ggttcaagac ccttggaaag 60gcctgtatac ttattttgtg aaccatattt tctctttgtt cctactcttt cttcccgctt 120tacttctgat agcttgtcct gaatttcctc tagaattttc agccctatct taaccactat 180ataacatgtg aaaaggaaca aaagggcttc taacactaga aaaaattcaa ggccaaacat 240aacttgtaaa gccattttcc actttacttc tgatagactg tcttgaattt ccttagaaag 300ttcaagatca gacttacctc gttccccagc tgaaaagttc tgaattcata cagttgaatc 360ctcttaacag tctggcttta cgggaacctt atcaccgtcg ttccccagct ggatgagttc 420tgaatcggca gttgaatcct tctcaacagt ctgtgttacg ggaaccttat aacctggatt 480cgcagttcng ggttctggga aggaaagtaa tcccctcctg gcggccagtn ccgggagntt 540ttttcctcgg tcccgggatt tttcctcggt ccccgggnaa ttcgggcacc caa 59319558DNAHomo sapiens 195tgggtccgtt cctaaaacaa aaaaaaaaaa acagcggtcc tattccaata ttcctagc 5819622DNAHomo sapiens 196tgggcagacg ttcgaatggg tc 22197324DNAMus musculus 197gacatcgagc tcactcagtc tccagcttct ttggctgtgt ctctagggca gagggccatc 60atctcctgca aggccagcca aagtgtcagt tttgctggta ctagtttaat gcactggtac 120caccagaaac caggacagca acccaaactc ctcatctatc gtgcatccaa cctagaagct 180ggggttccta ccaggtttag tggcagtggg tctaagacag acttcaccct caatatccat 240cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtaggga atatccgtac 300acgttcggag gggggacaaa gttg 324198351DNAMus musculus 198caggtgcagc tgcagcagtc tggagctgag ctggtgaagc ctggggcttc agtgaagata 60tcctgcaagg cttctggtta ctcatttact ggctacttta tgaactgggt gaagcagagc 120catggaaaga gccttgagtg gattggacgt attcatcctt acgatggtga tactttctac 180aaccagaact tcaaggacaa ggccacattg actgtagaca aatcctctaa cacagcccac 240atggagctcc tgagcctgac atctgaggac tttgcagtct attattgtac aagatacgac 300ggtagtcggg ctatggacta ctggggccaa gggaccacgg tcaccgtctc c 351199360DNAHomo sapiens 199caggtgcagc tggtggagtc tgggggaggc ttggtacagc cagggcggtc cctgagactc 60tcctgcacaa cttctggatt cacttttggt gattatgcta tgatctgggc ccgccaggct 120ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac 180gcagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagaacga 300tacgattttt ggagtggaat ggacgtctgg ggcaaaggga ccacggtcac cgtgtcgagt 360200333DNAHomo sapiens 200cagtctgccc tgactcagcc tgcctcagtg tccgggtctc ctggacagtc cgtctccatc 60tcctgcactg gaaccatcaa tgatgttggt ggatataggt ttgtctcctg gtaccaacga 120cgccccggca aagcccccaa actcatcatt tctgatgtca ttaggcggcc atcaggggtc 180cctgatcgct tctctagttc caagtctgac aacacggcct acctgaccat ctctgggctc 240caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cactctctat 300gtcttcggaa ctgggaccaa ggtcaccgtc cta 333201330DNAHomo sapiens 201cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgatgttggg agttataacc ttgtctcctg gtaccaacag 120cacccaggca aagcccccaa actcatgatt tatgagggca gtaagcggcc ctcaggggtt 180tctaatcgct tctctggctc caagtctggc aacgcggcct ccctgacaat ctctgggctc 240caggctgagg acgaggctga ttattactgc cagtcctatg acagcagcct gagtgtggta 300ttcggcggag ggaccaagct gaccgtccta 330202349DNAMus musculus 202acacagagca gcagatgtgg ggaagtagaa ggtgaaggga tggcaggagg ctcccacagg 60acactcgtta tgccccgagg accagttcca tcccttgtgc caattgctct tgcaggtaaa 120agagctctgg cagtcctccc accactgctg acagtcctct ttgcacaggg gaacatcaag 180gatccgctct ttgcgccagc tctggtccac ctgctggatc cagggtccca agttcgggga 240acactcatag aggcaggtgt cttggataaa gtgccgtttg cattccgatg tcatagttcc 300gcagtggttc cagttgaacc ggtacaggta ggaaatgtcc tcctcgtgc 349203276DNAHomo sapiens 203ttttttttta aattcatgtt tttaattggc ttaatacaaa ggtcccccag gaggccctgg 60gaggaggggg acagcctggg agaggcagag attcatggcc agcagcccac ccccacctgc 120cacccactcc ccaacaaggg tcccagactc tttcaataat cctaaaaaaa ccgacgagag 180cgcaggcaga tgaagagccc cttcatcctc aaacggcgac taccatcgaa agttgatagg 240gcagacgttc gaatgggtcg tcgccgccac gggggg 276204668DNAMus musculus 204gatccttcga ctccttggcg cttggtgctt caaccggact gacttcctgg gcctggagtt 60ggcgattaga ggtctgacat ggctcacctg atgactgtgc agttgttgct cctggtgatg 120tggatggccg aatgtgctca gtccagagct actcgggcca ggactgaact tctcaatgtc 180tgcatggatg ccaaacacca caaagaaaaa ccgggccctg aggacaattt acacgaccag 240tgcagcccct ggaagacgaa ttcctgctgt tccacgaaca caagccagga agcacataag 300gacatttcct acctgtaccg gttcaactgg aaccactgcg gaactatgac atcggaatgc 360aaacggcact ttatccaaga cacctgcctc tatgagtgtt ccccgaactt gggaccttgg 420atccagcagg tggaccagag ctggcgcaaa gagcggatcc ttgattgttc ccctgtgcaa 480agaggactgt catcagtggt gggaggactt gcagagctct tttccctgca agagcaattt 540ggacaaggga tggaacttgg tctcggggca taacgagtgt cctgtggggc ctccttgcaa 600tccttcacgt tttatttccc agattggttg gtcttgttgt gaggaatctg gggttcactc 660ttacagct 66820573DNAMus musculus 205ccagctccaa taacgtatat gagagttgca gcagataagg ggcaagtagt agagtatgga 60gagagggaga gcg 73206542DNAMus musculus 206ctgtcaccag tggtgggaag cctgtcgtac ctcctttacc tgcaagagag actggcataa 60aggctgggac tggtcctcag gcattaacaa gtgcccaaac acagcaccct gtcacacgtt 120tgagtactac ttcccgacac cagccagcct ttgcgagggt ctctggagtc actcctacaa 180ggtcagcaac tacagcagag ggagtggccg ctgcatccag atgtggtttg actcaaccca 240gggcaatccc aatgaggacg tggtgaagtt ttatgcttcc tttatgacat ctgggactgt 300gccccatgca gcagtacttc ttgtgcccag cctggcccca gtgctgtcat tatggctccc 360tggctgagag gtcagtcttc ctctctagat ttctcctcta tctacccttg gtctggttca 420actcttcaaa gaataaggaa gtcttgagcc tggttccacc cctctcctct gtcatccagt 480tcctgatcca tgttggggga tggggtttct acatcatttc aataaactat gaacatctgg 540gc 542207269DNAMus musculus 207gacatttcct aactgtaacg ggtcaactgg aagcactgcg gaaatatgac atcggaatgc 60aaacgggact tttttcaaga cacctgcctc tatgagtgtt ccccgaattt ggaccttgat 120tcagcaggtg gagcaaaact tgcgcaagaa ggggttcctg aagttcccct gtgcaaaaag 180gactttcaca attggttgga ggatttccaa agctctttta cccgcaagag gaatttgcac 240aagggtttga acatgtcctc ggggaataa 269208785DNAMus musculus 208attcggatcc ttcaaacctc ggccggctgt ctcctggaat gaagaaagca aaggaagcct 60agagtggaga caaagaagcc cgaggactct gagagctgcc atcttttcct tgtttgccgc 120ctgacacttc tcagcaggat ccacataccc taaggagtgg aagactcctt ggcgcttggt 180gcttcaaccg gactgacttc ctgggcctgg agttggcgat tagaggtctg acatggctca 240cctgatgact gtgcagttgt tgctcctggt gatgtggatg gccgaatgtg ctcagtccag 300agctactcgg gccaggactg aacttctcaa tgtctgcatg gatgccaaac accacaaaga 360aaaaccgggc cctgaggaca atttacacga ccagtgcagc ccctggaaga cgaattcctg 420ctgttccacg aacacaagcc aggaagcaca taaggacatt tcctacctgt accggttcaa 480ctggaaccac tgcggaacta tgacatcgga atgcaaacgg cactttatcc aagacacctg 540cctctatgag tgttccccga acttgggacc ctggatccag caggtggacc agagctggcg 600caaagagcgg atccttgatg ttcccctgtg caagaggact gtcagcagtg gtgggaggac 660tgccagagct cttttaccct gcagagcaat tggcacaagg gtggaatggt cccccgggca 720taacgatttc ccgtggaggc ttctggaatc ccttaacctc taattcccaa tctgcggcct 780gtgtg 785209718DNAMus musculus 209attcggatcc ttcctggaag tataaacaag aaaggaggct gacggctcta gaagtcccaa 60cctgttgtga tcttcagtag acaaacactc ctggtgtgtc acaggattca ggccactaaa 120cctcggccgg ctgtctcctg gaatgaagaa agcaaaggaa gcctagagtg gagacaaaga 180agcccgaggc actctgagag ctgccatctt ttccttgttt gccgcctgac acttctcagc 240aggatccaca taccctaagg agtggaagac tccttggcgc ttggtgcttc aaccggactg 300acttcctggg cctggagttg gcgattagag gtctgacatg gctcacctga tgactgtgca 360gttgttgctc ctgctgatgt ggatggccga atgtgctcag tccagagcta ctcgggccag 420gactgaactt ctcaatgtct gcatggatgc caaacaccac aaagaaaaac cgggccctga 480ggacaattta cacgaccagt gcagcccctg gaagacgaat tcctgctgtt tcacgaacac 540aagccaggaa gcacataagg acagttccta cctgtaccgg ttcaactggg accactgcgg 600aactatgaca tcggaatgca aacggcactt tatccagaaa cctgcctcta ttagtgttcc 660cccacattgg gaccctggat tcaccagtgg gacaaagatg gcgcgaaaaa acgggtcc 718210323DNAMus musculus 210attcggatcc ttcggaacta tgacatcgga atgcaaacgg cactttatcc aagacacctg 60cctctatgag tgttccccga acttgggacc ctggatccag caggtggacc agagctggcg 120caaagagcgg atccttgatg ttcccctgtg caaagaggac tgtcagcagt ggtgggagga 180ctgccagagc tcttttacct gcaagagcaa ttggcacaag ggatggaact ggtcctcggg 240gcataacgag tgtcctgtgg gagcctcctg ccatcccttc accttctact tccccacatc 300tgctgctctg tgtgaggaaa tct 323211450DNAMus musculus 211attcggatcc ttcctggaag tataaaccag aaaggaggct gacggctcta gaagtcccca 60acctgttgtg atcttcagta gacaaacact cctggtgtgt cacaggattc aggccactaa 120acctcggccg gctgtctcct ggaatgaaga aagcaaagga agcctagagt ggagacaaag 180aagcccgagg cactctgaga gctggcatct tttccttgtt tgccgcctga caattctcag 240cagggtccac atatcctaag taagagtggg agactccttt gcgcttggtg cttcaaccgg 300actgaattcc tgggcctgga attggcgatt agaggtccga catggctcaa ctgatgacct 360tgcaattgtt ggccccggtg atgtggatgg gcgaaagtgc ttcagttcaa gaagctactt 420cgggccaagg actgaaactt tctcaaatgt 450212682DNAMus musculus 212gaagactcct tggcgcttgg tgcttcaacc ggactgactt cctgggcctg gagttggcga 60ttagaggtct gacatggctc acctgatgac tgtgcagttg ttgctcctgg tgatgtggat 120ggccgaatgt gctcagtcca gagctactcg ggccaggact gaacttctca atgtctgcat 180ggatgccaaa caccacaaag aaaaaccggg ccctgaggac aatttacacg accagtgcag 240cccctggaag acgaattcct gctgttccac gaacacaagc caggaagcac ataaggacat 300ttcctacctg taccggttca actggaacca ctgcggaact atgacatcgg aatgcaaacg 360gcactttatc caagacacct gcctctatga gtgttccccg aacttgggac cctggatcca 420gcaggtggac cagagctggc gcaaagagcg gatccttgat gttcccctgt gcaaagagga 480ctgtcagcag tggtgggagg actgccagag ctcttttacc tgcaagagca attggcacaa 540gggatggaac tggtcctcgg ggcataacga gtgtcctgtg ggagcctcct gccatccctt 600caccttccta cttcccaaca tctgctgctc tgtgtgagga aatctggagt cactcctcaa 660gctcagcaac tacagttcga gg 682213411DNAMus musculus 213cgggccctga ggacaattta cacgaccagt gcagcccctg gaagacgaat tcctgctgtt 60ccacgaacac aagccaggaa gcacataagg acatttccta cctgtaccgg ttcaactgga 120accactgcgg aactatgaca tcggaatgca aacggcactt tatccaagac acctgcctct 180atgagtgttc cccgaacttg ggaccctgga tccagcaggt ggaccagagc tggcgcaaag 240agcggatcct tgatgttccc ctgtgcaaag aggactgtca gcagtggacg gaggactgcc 300agagctcttt tacctgcaag agcaattggc acaagggatg gaactggtcc tctgggcata 360acgagtgtcc tgtgggagcc tcctgccatc ccttcacctt ctacttcccc a 411214653DNAMus musculus 214ctggagctga gcacacactt ggaggttcca cttaccttag ctctgccttc agggtctgac 60atggctcacc tgatgactgt gcagttgtgg ctgctggtga tgtggatggc cgaatgtgct 120cagtccagag ctactcgggc caggactgaa cttctcaatg tctgcatgga tgccaaacac 180cacaaagaaa aaccgggccc tgaggacaat ttacacgacc agtgcagccc ctggaagacg 240aattcctgct gttccacgaa cacaagccag gaagcacata aggacatttc ctacctgtac 300cggttcaact ggaaccactg cggaactatg acatcggaat gcaaacggca ctttatccaa 360gacacctggc tctatgagtg ttccccgaac ttgggaccct ggattcagca ggtggaccaa 420agctggcgca agagagggat cctttatgtt cccctggtgc aaagaggact tgtcagcagt 480tggtgggagg actgccagaa ctcgtgtacc tgccaggagc aattggcaca agggatggaa 540ttggttcttc ggggcataac gaagtgctct gtgtggagcc tcctgcagtc ctgtaacgtc 600taattcccac atttggcggt ctgtgtaatg aatctcgggc actccacagg ctc 653215256DNAHomo sapiens 215acacctgcct ctacgagtgc tcccccaact tggggccctg gatccagcag gtggatcaga 60gctggcgcaa agagcgggta ctgaacgtgc ccctgtgcaa agaggactgt gagcaatggt 120gggaagattg tcgcacctcc tacacctgca agagcaactg gcacaagggc tgcaactgga 180cttcagggtt taacaagtgc gcagtgggag ctgcctgcca acctttccat ttctacttcc 240ccacacccat tgcccg 2562161282DNAHomo sapiens 216gtgtccccag aagtggcctt gaaccgaata tctccaatgg acagggctgg ggagcccaca 60gggctggtgc ggcgggagtc agtggaggcg aagatgcaga gtgccagctg gaaggtcaga 120atacgctcca ccaccatggc ctggccctgc gttgtgttgt tggtagagcg cgttgtctac 180cctgtaccga agacagaggc tgtggggaca gcctaggggc cctggatcta ttgcctactt 240agagagaggc caactcagac acagccgtgt atgctcccag cagcaacgga ggttcagcac 300cgcctgcagg gacagaaaga catggtctgg aaatggatgc cacttctgct gcttctggtc 360tgtgtagcca ccatgtgcag tgcccaggac aggactgatc tcctcaatgt ctgtatggat 420gccaagcacc acaagacaaa gccaggtcct gaggacaagc tgcatgacca atgcagtccc 480tggaagaaga atgcctgctg cacagccagc accagccagg agctgcacaa ggacacctcc 540cgcctgtaca actttaactg ggaccactgc ggcaagatgg agcccgcctg cagcgccact 600tcatccagga cacctgtctc tatgagtgct caccaacctg gggccctgga tccagcaggt 660gaatcagagc tggcggcaaa gaacgcttcc tggatgtgcc cttatgcaaa gagcactgtc 720agcgctggtg ggaggattgt cacacctccc acacgtgcaa gagcaactgg cacagaggat 780gggactggac ctcaggagtt aacaagtgcc cagctggggc tctctgccgc acctttgagt 840cctacttccc cactccagct gccctttgtc aaggcctctg gagtcactca tacaaggtca 900gcaactacag ccgagggagc ggccgctgca tccagatgtg gtttacttca gcccagggca 960accccaacga ggaagtggcg aggttctatg ctgcagccat gcatgtgaat gctggtgaga 1020tgcttcatgg gactgggggt ctcctgctca gtctggccct gatgctgacc ctctggctcc 1080tcggctgcgt tcagtcctcc cagactacct gccctcagct tggataacca ggctgggctc 1140agctcagctc ccacaaatga cagcccctta agcatgcttc tattagtcac ctaaccctct 1200gtcacccagt ctgttgctgc tccatggtgg ggccaagagt cacttctaat aaacagactg 1260ttttctaata aaaaaaaaaa aa 1282217211DNAMus musculus 217aggattctat gccgaggcca tgagtggagc tgggcttcat gggacctggc cactcttgtg 60cagcctgtcc ttagtgctgc tctgggtgat cagctgagtt cctgttttac cttcagttgt 120ctggagcgcc accctgcttg gctcagcctc ccagctccca gcctcctttg tggtggggct 180ctgacagcct ctttaataaa ccagacattc c 211218559DNAMus musculus 218attaggatcc ttccttctca gcaggatcca cataccctaa ggagtggaag actccttggc 60gcttggtgct tcaaccggac tgacttcctg ggcctggagt tggcgattag aggtctgaca 120tggctcaact gatgactgtg cagttgttgc tcctggtgat gtggatggcc gaatgtgctc 180agtccagagc tactcgggcc aggactgaac ttctcaatgt ctgcatggat gccaaacacc 240acaaagaaaa accgggccct gaggacaatt tacacgacca gtgcagcccc tggaagacga 300attcctgctg ttccacgaac acaagccagg aagcacataa ggacatttcc tacctgtacc 360ggttcaactg gaaccactgc ggaaatatga aatcggaatg caaacggcac tttatccaag 420aaaccttgac tcaatgagtg ttacacgaaa cttgggacac tggataagca agtggaacag 480agatgggcga aaagagcgga tacattgatg taaccctgtg acaagaggac tgttcagcag 540tggtgggagg actgccaga 559219661DNAMus musculus 219aattcggatc catgatctgg aagtataaac aagaaaggag gctgacggct ctagaagtcc 60ccaacctgtt gtgatcttca gtagacaaac actcctggtg tgtcacagga ttcaggccac 120taaacctcgg ccggctgtct cctggaatga agaaagcaaa ggaagcctag agtggagaca 180aagaagcccg aggcactctg agagctgcca tcttttcctt gtttgccgcc tgacacttct 240cagcaggatc cacataccct aagcagggag tggagagagg cctgggctgg gccaggtttt 300ctgggctttt tcctgtgctc cgagtaggtg ggttgtattt tacccagtag gagtggaaga 360ctccttggcg cttggtgctt caaccggact gacttcctgg gcctggagtt ggcgattaga 420ggtctgacat ggctcacctg atgactgtgc agttgttgct cctggtgatg tggatggccg 480aatgtgctca gtccagagct actcgggcca ggactgaact tctcaatgtc tgcatggatg 540ccaaacacca caaagaaaaa ccgggccctg aggacaattt acacgaccag tgcagcccct 600ggaagacgaa ttcctgctgt tcaacgacac aagcaggaag cactaaggac ttttctactg 660t 661220695DNAMus musculus 220ttcggatcct tctctggaag tataaacaag aaaggaggct gacggctcta gaagtcccca 60acctgttgtg atcttcagta gacaaacact cctggtgtgt cacaggattc aggccactaa 120acctcggccg gctgtctcct ggaatgaaga aagcaaagga agcctagagt ggagacaaag 180aagcccgagg cactctgaga gctgccatct tttccttgtt tgccgcctga cacttctcag 240caggatccac ataccctaag gagtggaaga ctccttggcg cttggtgctt caaccggact 300gacttcctgg gcctggagtt ggcgattaga ggtctgacat ggctcacctg atgactgtgc 360agttgttgct cctggtgatg tggatggccg aatgtgctca gtccagagct actcggggcc 420aggactgaac ttctcaatgt ctgcatggat gccaaacacc acaaagaaaa accgggccct 480gaggacaatt tacacgacca gtgcagcccc tggaagacga

attcctgctg ttccacgaac 540acaagccagg aagcacataa ggacatttcc tacctgtacc ggttcaactg gaaccactgc 600ggaactatga catcggaatg caaacggcac tttatccaag acacctgcct ctatgagtgt 660tccccgaact tgggactgga ttcagcaggt ggacc 695221567DNAMus musculus 221tggaagactc cttggcgctt ggtgcttcaa ccggactgac ttcctgggcc tggagttggc 60atttagaggt ctgacatggc tcacctgatg actgtgcagt tgttgctcct ggtgatgtgg 120atggccgaat gtgctcagtc cagagctact cgggccagga ctgaacttct caatgtctgc 180atggatgcca agcaccacaa agaaaaaccg ggccctgagg acaatttaca cgaccagtgc 240agcccctgga agacgaattc ctgctgttcc acgaacacaa gccaggaagc acataaggac 300atttcctacc tgtaccggtt caactggaac cactgcggaa ctatgacatc ggaatggcaa 360cggcactttt atcaaagaca cctgcctcta tgagtgttcc ccgaactttg ggaacctgga 420ttccagaagt tggacagagc ctgcgcaaaa gagcggattc ttgatggttc cctgtgcaaa 480gaggactgtc agcagtggtg ggagactgcc aagctcttta cctgcaagag cattggcaca 540aggatggaat ggtcctctgg caaacga 567222600DNAMus musculus 222atggctccct gatgactgtg cagttgttgc tcctgctgat gtggatggcc gaatgtgctc 60agtccagagc tactcgggcc aggactgaac ttctcagtgt ctgcatggat gccagacacc 120acaaagagaa accgggccct gaggacaatt tacacgacca gtgcagcccc tggaagacga 180attcctgctg ttccacgaac acaagccagt aagcacataa ggacatttcc tacctgtacc 240ggttcaactg gaaccactgc ggaactatga catcggaatg caaacggcac tttatccaag 300acagctgcct ctatgagtgt tccccgaact tgggagcctg tatgcagcag gtggacgaga 360gctgtcgcaa agagcggatc cttgatgtgc ccctgtgcaa agaggactgt cagcagtggt 420gcgagtgctg cggagctctt gtacctgcag agaggaattt gcacagggga tggaactggt 480tccctggggc ataacaagtg tcctgtggta gcctgccggc aggccgttag cgttgtagtt 540tcgcggatcg gctggtcggg tgaagaagtt gtggggcatg ccacatgtca gtagtttgtt 600223582DNAMus musculus 223aattcgcatc cttcataaac aagacaggag gctgacggct ctagaagtcc ccaacctgtt 60gtgatcttca gtagacaaac actcctggtg tgtcacagga ttcaggccac taaacctcgg 120ccggctgtct cctggaatga agaaagcaaa ggaagcctag agtggagaca aagaagcccg 180aggcactctg agagctgcca tcttttcctt gtttgccgcc tgacacttct cagcaggatc 240cacataccct aaggagtgga agactccttg gcgcttggtg cttcaaccgg actgacttcc 300tgggcctgga gttggcgatt agaggtctga catggctcac ctgatgactg tgcagttgtt 360gctcctggtg atgttgatgg ccgaatgtgc tcagtccaga gctactcggg ccaggactga 420acttctcaat gtctgcatgg atgccaaaca ccacaaagaa aaaccgggcc ctgaggacaa 480tttacacgac cagtgcagcc cctggaagac gaatttctgc tgttccacga acacaagcca 540ggaagcacat aaggacattt cctaactgta acggttcaat gg 582224372DNAMus musculus 224tcccatttcc tacctgtacc ggttcaactg gaaccactgc ggaactatga catcggaatg 60caaacggcac tttatccaag acacctgcct ctatgagtgt tccccgaact tgggaccctg 120gatccagcag gtggaccaga gctggcgcaa agagcggatc cttgatgttc ccctgtgcaa 180agaggactgt cagcagtggt gggaggactg ccagagctct tttacctgca agagcaattg 240gcacaaggga tggaactggt cctcggggca taacgagtgt cctgtgggag cctcctgcca 300tcccttcacc ttctacttcc ccacatctgc tgctctgtgt gaggaaatct ggagtcactc 360ctacgagctc ag 372225375DNAMus musculus 225ctatcccatt tcctacctgt accggttcaa ctggaaccac tgcggaacta tgacatcgga 60atgcaaacgg cactttatcc aagacacctg cctctatgag tgttccccga acttgggacc 120ctggatccag caggtggacc agagctggcg caaagagcgg atccttgatg ttcccctgtg 180caaagaggac tgtcagcagt ggtgggagga ctgccagagc tcttttacct gcaagagcaa 240ttggcacaag ggatggaact ggtcctcggg gcataacgag tgtcctgtgg gagcctcctg 300ccatcccttc accttctact tccccacatc tgctgctctg tgtgaggaaa tctggagtca 360ctcctacaag ctcag 375226440DNAMus musculus 226tatccctgag agctgccatc ttttccttgt ttgccgcctg acacttctca gcaggatcca 60cataccctaa gggagtggag agaggcctgg gctgggccag gttttctggg ctttttcctg 120tgctccgagt cagtgggttg tattttaccc agtaggagtg gaagactcct tggcgcttgg 180tgcttcaacc ggaactgact tcctgggcct ggagttggcg attagaggtc ctacatggct 240cacctgatga ctgtgcaagt tgtgcccccg gtgatgttga atggcggatg tgctcagtcc 300agaagtaatt tgggccaaga ctggacttct ccatggctgc attgatggca aacaccccaa 360aggaaaacgg ggccttgggg caattatcac ggccctgtaa cccttggaaa ccaattcccg 420ggttccgaaa cacagccgga 440227544DNAMus musculus 227aattcggatc catgggctga tctggaagta taaacaagaa aggaggctga cggctctaga 60agtccccaac ctgttgtgat cttcagtaga caaacactcc tggtgtgtca caggattcag 120gccactaaac ctcggccggc tgtctcctgg aatgaagaaa gcaaaggaag cctagagtgg 180agacaaagaa gcccgaggac tctgagagct gccatctttt ccttgtttgc cgcctgacac 240ttctcagcag gatccacata ccctaaggga gtggagagag gcctgggctg ggcaggtttt 300ctgggctttt tcctgtgctc cgagtaggtg ggttgtattt tacccagtag gagtggaaga 360ctccttggcg cttggtgctt caaccggact gacttcctgg gcctggagtt ggcgattaga 420ggtctgacat ggctcacctg atgactgtgc agttgttgct cctggtgatg tggatggccg 480aatgtgctca gtccagagct actcgggcca ggactgaact tctcaatgtc tgcatggatg 540ccaa 544228350DNAMus musculus 228ttggcatcca tgcagacatt gagaagttca gtcctggccc gagtagctct ggactgagca 60cattcggcca tccacatcac caggagcaac aactgcacag tcatcaggtg agccatgtca 120gacctctaat cgccaactcc aggcccagga agtcagtccg gttgaagcac caagcgccaa 180ggagtcttcc actcctactg ggtaaaatac aacccaccta ctcggagcac aggaaaaagc 240ccagaaaacc tggcccagcc caggcctctc tccactccct tagggtatgt ggatcctgct 300gagaagtgtc aggcggcaaa caaggaaaag atggcagctc tcagagtgcc 350229563DNAMus musculus 229ttcggatcca tggtgctccg agtaggtggg ttgtatttta cccagtagga gtggaagact 60ccttggcgct tggtgcttca accggactga cttcctgggc ctggagttgg cgattagagg 120tctgacatgg ctcacctgat gactgtgcag ttgttgctcc tggtgatgtg gatggccgaa 180tgtgctcagt ccagagctac tcgggccagg actgaacttc tcaatgtctg catggatgcc 240aaacaccaca aagaaaaacc gggccctgag gacaatttac acgaccagtg cagcccctgg 300aagacgaatt cctgctgttc cacgaacaca agccaggaag cacataagga catttcctac 360ctgtaccggt tcaactggaa ccactgcgga actatgacat cggaatgcaa acggcacttt 420atccaagaca cctgcctcta tgagtgttcc ccgaacttgg gaccctggat ccagcaagtg 480gaccagagct ggcgcaagag cggatccttg aatgtccctg tgcaagagga ctgtcagcag 540tggtgggaga ctgcagagct ctt 563230587DNAMus musculus 230aattcgggat ccatgggctg atctggaagt ataaacaaga aaggaggctg acggctctag 60aagtccccaa cctgttgtga tcttcagtag acaaacactc ctggtgtgtc acaggattca 120gctctgtttc ctaggccact aaacctcggc cggctgtctc ctggaatgaa gaaagcaaag 180gaagcctaga gtggagacaa agaagcccga ggcactctga gagctgccat cttttccttg 240tttgccgcct gacacttctc agcaggatcc acatacccta aggagtggaa gactccttgg 300cgcttggtgc ttcaaccgga ctgacttcct gggcctggag ttggcgatta gaggtctgac 360atggctcacc tgatgactgt gcagttgttg ctcctggtga tgtggatggc cgaatgtgct 420cagtccagag ctactcgggc caggactgaa cttctcaatg tctgcatgga tgccaaacac 480cacaaagaaa aaccgggccc tgaggacaat ttacacgacc agtgcagccc ctggaagacg 540aattcctgct gttccacgaa cacaagccag gaagcacata aggacat 587231557DNAMus musculus 231attcggatcc acgtataaac aagaaaggag gctgacggct ctagaagtcc ccaacctgtt 60gtgatcttca gtagacaaac actcctggtg tgtcacagga ttcagctctg tttcctaggc 120cactaaacct cggccggctg tctcctggaa tgaagaaagc aaaggaagcc tagagtggag 180acaaagaagc ccgaggcact ctgagagctg ccatcttttc cttgtttgcc gcctgacact 240tctcagcagg atccacatac cctaagggag tggagagagg cctgggctgg gcaggttttc 300tgggcttttt cctgtgctcc gagtaggtgg gttgtatttt acccagtagg agtggaagac 360tccttggcgc ttggtgcttc aaccggactg acttcctggg cctggagttg gcgattagag 420gtctgacatg gctcacctga tgactgtgca gttgttgctc ctggtgatgt ggatggccga 480attggctcat tccaaagcta ctcgggccgg aactgaactc ctcaaggtct gcatggatgc 540aaacgccaca aagaaaa 557232613DNAMus musculus 232gttcggatcc atgggctgat ctggaagtat aaacaagaaa ggaggctgac ggctctagaa 60gtccccaacc tgttgtgatc ttcagtagac aaacactcct ggtgtgtcac aggattcagc 120tctgtttcct aggccactaa acctcggccg gctgtctcct ggaatgaaga aagcaaagga 180agcctagagt ggagacaaag aagcccgagg cactctgaga gctgccatct tttccttgtt 240tgccgcctga cacttctcag caggatccac ataccctaag ggagtggaga gaggcctggg 300ctgggccagg ttttctgggc ttttcctgtg ctccgagtag gtgggttgta ttttacccag 360taggagtgga agactccttg gcgcttggtg cttcaaccgg actgacttcc tgggcctgga 420gttggcgatt agaggtctga catggctcac ctgatgactg tgcagttgtt gctcctggtg 480atgtggatgg cgaatgtgct cagtccagag ctactcgggc caagactgaa cttctcaatg 540tctgcatgga tgccaacacc acaagaaaaa cggggcttga gacaatttca cgacagtgca 600gccctggaaa aga 6132331230DNAPig 233gaattcgcgg ccgctccggg aaggggggaa gggcacaact ccctcgggaa gctcgccgct 60gcctcctgga gcagaaggca gacaaagcca tgccctggaa gctgacagcc cttctgctct 120ttctggccgg ggtggtctcc gtgtgccgcg cccgggccag gacggacctg ctcaacgtct 180gcatggatgc caagcaccac aaggtagagc caggccctga ggacgagctg cacgaccagt 240gcgtcccctg gaagaagaac gcctgctgct ccgccagagt cagccacgag ctgcaccggg 300acaagtcctc cctgtataac ttttcctggg agcactgcgg caggatggag ccggcctgca 360agcgccactt cattcagaac aactgtctgt acgagtgctc gcccaacctg gggccctggt 420tccaggaggt gaaccagaag tggcgcaaag agcggttcct gaacgtgccc ctctgcaaag 480aggactgtct ggactggtgg gaagactgcc gcacctccta cacctgcaag agcagctggc 540acaagggctg gaactggagc tcaggatcta accagtgtcc cacggggacc acctgcgaca 600catttgagtc cttcttcccc acacccgcag cgctgtgtga gggcatctgg aatcacgatt 660ataagttcac caactacagc cggggcagcg gccgctgcat ccagatgtgg tttgacgcgg 720ccgagggcaa ccccaacgag gaggtagcga ggttctacgc cttggccttg agtgcgggga 780ccatgtccct tgggaccggg cctctcctgc tcagcgcagc cctgatgctg ccacttgggc 840tccttgactg agtccggcgt ctccagacgg tccttctgcc tgtccccagc tttgatgacc 900aggctggtct caactcagct cccaccaatg agggagccct aagcccgcct catctgttac 960ccatccctct gtcatcaagt tcctgccgta gggtgggcct tggggtctct ctgacagcca 1020gttctaacag gcagattaac agcactgtgt ctgatgggct gttttggttg tgagctggtg 1080tgtggcagag gacagagccc atagcttttg gattccttca gcttagagaa atgagacctg 1140ggtttgaatt ccagctctgc cactcactat gtcaagtgaa gcagttgcgc gacggctcta 1200aaccataggc tcctcctcaa taaaatgaag 1230234317DNAMus musculus 234aatctggagt cactcctaca agctcagcaa ctacagtcga gggagcggcc gctgcattca 60gatgtggttc gacccagccc agggcaaccc caacgaggaa gtggcgaggt tctatgccga 120ggccatgagt ggagctgggt ttcatgggac ctggccactc ttgtgcagcc tgtccttagt 180gctgctctgg gtgatcagct gagctcctgt tttaccttca gttgtctgga gcgccaccct 240gcttggctca gcctcccagc tcccagcctc ctttgtggtg gggctctgac agcctcttta 300ataaaccaga cattcca 317235529DNAMus musculus 235cacgaacaca agccaggaag cacataagga catttcctac ctgtaccggt tcaactggaa 60ccactgcgga actatgacat cggaatgcaa acggcacttt atccaagaca catgcctcta 120tgagtattcc ccgaacttgg gaccctggat ccagcaggtg gaccagagct ggcgcaaaga 180gcggatcctt gatgttcccc tgtgcaaaga ggactgtcag cagtggtggg aggactgcca 240gagctctttt acctgcaaga gcaattggca caagggatgg aactggtcct cggggcataa 300cgagtgtcct gtgtgagcct cctgccatcg cttcaccttc tacttcccca catctgctgc 360tctgtgtgaa gaaatctgga gtcactccta caagcttaac aactacagtc gagggaagcg 420gccgctgcag tcagatgtgg ttcgacccag ccatggcaaa cccagcgagg aagttgcgag 480gtctatgccg aggcaatagt gagctggtgt ctgggactgg gcactttgt 529236557DNAMus musculus 236aagactgtag agactaccca gagtctgacc tagggacagg ccaactcgga tacccctatg 60tgcgctccca gaagctaagg acattgagac agaaagacat ggcctggaaa cagacaccac 120tcttgctttt ggtctacatg gtcacaacag gcagtggcgg gacagaacag acctactcaa 180cgtttgcatg gatgccaaac accataagac aaagccgggc cccgaggaca agctgcatga 240ccagtgtagt ccatggaaga aaaatgcctg ttgctcagtc aacaccagcc aggagctaca 300caaggctgac tcccgtctgt acttcaactg ggatcactgt ggcaagatgg agcctgcctg 360taagagtcac ttcatccaag actcctgcct gtatgattgt ttcccaaacc ttggcccttg 420attcagtcaa gtggatcaag attgggctta aaaaggtttt cctgatgtgc ccctaatgca 480agaagacctg tcaccagtgt tggaaagctt gtggtacctc ctttactggc agaagagact 540ggcataaagc tcggact 557237616DNAMus musculus 237attcggatcc atgggctgat ctggaagtat aaacaagaaa ggaggctgac ggctctagaa 60gtcccaacct gttgtgatct tcagtagaca aacactcctg gtgtgtcaca ggattcagct 120ctgtttccta ggccactaaa cctcggccgg ctgtctcctg gaatgaagaa agcaaaggaa 180gcctagagtg gagacaaaga agcccgaggc actctgagag ctgccatctt ttccttgttt 240gccgcctgac acttctcagc aggatccaca taccctaagg agtggaagac tccttggcgc 300ttggtgcttc aaccggactg acttcctggg cctggagttg gcgattagac tctgccttca 360gggtctgaca tggctcacct gatgactgtg cagttgttgc tcctggtgat gtggatggcc 420gaatgtgctc agtccagagc tactcgggcc aggactgaac ttctcaatgt ctgcatggat 480gccaaacacc acagagaaag accgggccct gaggacaatt ttacacgaca gtgcagcccc 540tggaagacga attcctgttg ttcacgaaca caagcaggat gacataggac atttctactg 600taccgttcac tggaac 616238648DNAMus musculusmodified_base(605)N = A, C, G, or T/U 238aattcggatc catgggctga tctggaagta taaacaagaa aggaggctga cggctctaga 60agtccccaac ctgttgtgat cttcagtaga caaacactcc tggtgtgtca caggattcag 120ctctgtttcc taggccacta aacctcggcc ggctgtctcc tggaatgaag aaagcaaagg 180aagcctagag tggagacaaa gaagcccgag gcactctgag agctgccatc ttttccttgt 240ttgccgcctg acacttctca gcaggatcca cataccctaa ggagtggaag actccttggc 300gcttggtgct tcaaccggac tgacttcctg ggcctggagt tggcgattag actctgcctt 360cagggtctga catggctcac ctgatgactg tgcagttgtt gctcctggtg atgtggatgg 420ccgaatgtgc tcagtccaga gctactcggg ccaggactga acttctcaat gtctgcatgg 480atgccaaaca ccacaaagaa aaaccgggcg ctgaggacaa tttacacgac cagtgcagca 540cctggaagac gaattcctgg ctgttcacga gcacaagcta ggaagcacat aaggacattt 600tctanctgta ccggttcaac tggacccact gcggactatg acatcgga 648239636DNAMus musculus 239attcggatcc atgcagctta gaagggcctc cagctttagg ctttatagat acctggccca 60cccttcccca gtcagcaggc tgatctggaa gtataaacaa gaaaggaggc tgacggctct 120agaagtcccc aacctgttgt gatcttcagt agacaaacac tcctggtgtg tcacaggatt 180caggccacta aacctcggcc ggctgtctcc tggaatgaag aaagcaaagg aagcctagag 240tggagacaaa gaagcccgag gcactctgag agctgccatc ttttccttgt ttgccgcctg 300acacttctca gcaggatcca cataccctaa gtaggagtgg aagactcctt ggcgcttggt 360gcttcaaccg gactgacttc ctgggcctgg agttggcgat tagaggtctg acatggctca 420cctgatgact gtgcagttgt tgctcctggt gatgtggatg ggcgaatgtg ctcagtccag 480agctactcgg gccaggactg aacttctcaa tgtctgcatg gatgtcaaac accacaaaga 540aacaccgggc ctgaggacaa tttacacgac cagtgcagcc cctggaagac gaatcctgct 600gttccagaaa caagcaggag cacataggcc attcct 636240660DNAMus musculus 240attcggatcc atgggctgat ctggaagtat aaacaagaaa ggaggctgac ggctctagaa 60gtccccaacc tgttgtgatc ttcagtagac aaacactcct ggtgtgtcac aggattcagg 120ccactaaacc tcggccggct gtctcctgga atgaagaaag caaaggaagc ctagagtgga 180gacaaagaag cccgaggcac tctgagagct gccatctttt ccttgtttgc cgcctgacac 240ttctcagcag gatccacata ccctaaggag tggaagactc cttggcgctt ggtgcttcaa 300ccggactgac ttcctgggcc tggagttggc gattagaggt ctgacatggc tcacctgatg 360actgtgcagt tgttgctcct ggtgatgtgg atggccgaat gtgctcagtc cagagctact 420cgggccagga ctgaacttct caatgtctgc atggatgcca aacaccacaa agaaaaaccg 480ggccctgagg acaatttaca cgaccagtgc atgccctgga agacgaattc ctgctgttcc 540acgaacacaa gccaggaagc acatagagac atttcctgct gtaccggttc aactggacca 600ctgcggaact atgacatcga atgcagacgc actttgccag gacactggct ctatgagtgt 660241578DNAMus musculus 241aattcggatc catgggctct agaagtcccc aacctgttgt gatcttcagt agacaaacac 60tccgtggtgt gtcacaggat tcaggccact aaacctcggc cggctgtctc ctggaatgaa 120gaaagcaaag gaagcctaga gtggagacaa agaagcccga ggcactctga gagctgccat 180cttttccttg tttgccgcct gacacttctc agcaggatcc acatacccta aggagtggaa 240gactccttgg cgcttggtgc ttcaaccgga ctgacttcct gggcctggag ttggcgatta 300gaggtctgac atggctcacc tgatgactgt gcagttgttg ctcctggtga tgtggatggc 360cgaatgtgct aagtccagag ctactcgggc caggactgaa ctcctaaatg tctgcatgga 420tgccaaacac cacaaggaaa aacgggcccc tgaggacaat tacacgacca gtgcaagccc 480tggaagacga aattctgctg ttcaagacca caagccagta gcacataggg acattccaac 540ctgtaccgtt caacttgaac actgcggaat atgactcg 578242403DNAMus musculus 242ccactaacca cataaggaca tttcctacct gtaccggttg acctgcaacg actgccgaac 60tatgacatcg caatgcacac gccactttat cgaccacacc tgcctctatg agtgttaccc 120gaacttcgca ccctccatcc accaggtgca cgacagctgg cccacagagc gcatccttca 180tgttcccctg tccacagacg actgtcagca gtcgtcccag cactcccaca gctctcttac 240ctgcaacacc aattcccaca acggatggaa ctcgtcctcg cggcatcacg agtgtcctgt 300agcaccctcc tgccatccct tcaccttcta cttccgcaca tctcgtgctc tgtgtgatga 360actctggagt cactcctaga cactcagcaa ctacagtcga cgg 403243477DNAMus musculus 243aattcggatc catgcatgga tccggatcca tggcccctgg aagacgaatt cctgctgttc 60cacgaacaca agccaggaag cacataagga catttcctac ctgtaccggt tcaactggaa 120ccactgcgga actatgacat cggaatgcaa acggcacttt atccaagaca cctgcctcta 180tgagtgttcc ccgaacttgg gaccctggat ccagcaggtg gaccagagct ggcgcaaaga 240gcggatcctt gatgttcccc tgtgcaaaga ggactgtcag cagtggtggg aggactgcca 300gagctctttt acctgcaaga gcaattggca caagggatgg aactggtcct cggggcataa 360cgagtgtcct gtgggagcct cctgccatcc cttcaccttc tacttcccac atctgctgct 420ctgtgtgagg aatctggagt cactctacaa gctcagcact acagtcgagg agccgcc 477244643DNAMus musculus 244ctgagtctga ggccagctgg tcgacaaggg tctgacatgg ctcacctgat gactgtgcag 60ttgttgctcc tggtgatgtg gatggccgaa tgtgctcagt ccagagctac tcgggccagg 120actgaacttc tcaatgtctg catggatgcc aaacaccaca aagaaaaacc gggccctgag 180gacaatttac acgaccagtg cagcccctgg aagacgaatt cctgctgttc cacgaacaca 240agccaggaag cacataagga catttcctac ctgtaccggt tcaactggaa ccactgcgga 300actatgacat cggaatgcaa acggcacttt atccaagaca cctgcctcta tgagtgttcc 360ccgaacttgg gaccctggat ccagcaggtg gaccagagct ggcgcaaaga gcggatcctt 420gatgttcccc tgtgcaaaga ggactgtcag cagtggtggg aggactgcca gagctctttt 480acctgcaaga gcaattggca caagggatgg aactggtcct cggggcataa cgagtgtcct 540gtgggagcct cctgccatcc gttcacttct acttcgcaca tctgctgtct gtgtgaggaa 600tctggagtca ctctacaagt ctagaataca gtcgaggacc ggc 643245351DNAMus musculus 245aaccactgcg gaactatgac atcggaatgc aaacggcact ttatccaaga cacctgcctc 60tatgagtgtt ccccgaactt gggaccctgg atccagcagg tggaccagag ctggcgcaaa 120gagcggatcc ttgatgttcc cctgtgcaaa gaggactgtc agcagtggtg ggaggactgc 180cagagctctt

ttacctgcaa gagcaattgg cacaagggat ggaactggtc ctcgggggca 240taacgagtgt cctgtgggag cctcctggca tcccttcagc ttctacttcc ccacatctgg 300ctgctcctgt gttaggaaaa tcttggattc actcctacca agcttcagca a 351246423DNAMus musculus 246aattcggcac taggggaggc tgacggctct agaagtcccc aacctgttgt gatcttcagt 60agacaaacac tcctggtgtg tcacaggatt cagctctgtt tcctaggcca ctaaacctcg 120gccggctgtc tcctggaatg aagaaagcaa aggaagccta gagtggagac aaagaagccc 180gaggcactct gagagctgcc atcttttcct tgtttgccgc ctgacacttc tcagcaggat 240ccacataccc taaggagtgg aagactcctt ggcgcttagt gctgctctgg gtgatcagct 300gagctcctgt tttaccttca gttgtctgga gcgccaccct gcttggctca gcctcccagc 360tcccagcctc ctttgtggtg gggctctgac agcctcttta ataaaccaga cattccaaaa 420aag 423247638DNAMus musculus 247gtggacgaag actgtagaga ctacccagag tctgacctag ggagaggcca actcggatac 60ccctatgtgc gctcccagaa gctaaggaca ttgagacaga aagacatggc ctggaaacag 120acaccactct tgcttttggt ctacatggtc acaacaggca gtggccggga cagaacagac 180ctactcaacg tttgcatgga tgccaaacac cataagacaa agccgggccc cgaggacaag 240ctgcatgacc agtgtagtcc atggaagaaa aatgcctgtt gctcagtcaa caccagccag 300gagctacaca aggctgactc ccgtctgtac ttcaactggg atcactgtgg caagatggag 360cctgcctgta agagtcactt catccaagac tcctgcctgt atgagtgctc ccccaacctt 420gggccttgga tccagcaagt ggaccagagt tggcgtaaag agcgtttcct ggatgtgccc 480ttatgcagag aggactgtca ccagtggtgg gaagcctgtc gtacctcctt tacctgcaag 540agagactggc ataaaggctg ggaatggtcg tcaggcatgt acaagtgcgc aacacagcac 600ctgtacacgt gtgagtactc ttccgaacca gcagcttt 638248638DNAMus musculus 248gggctgtgga cgaagactgt agagactacc cagagtctga cctagggaga ggccaactcg 60gataccccta tgtgcgctcc cagaagctaa ggacattgag acagaaagac atggcctgga 120aacagacacc actcttgctt ttggtctaca tggtcacaac aggcagtggc cgggacagaa 180cagacctact caacgtttgc atggatgcca aacaccataa gacaaagccg ggccccgagg 240acaagctgca tgaccagtgt agtccatgga agaaaaatgc ctgttgctca gtcaacacca 300gccaggagct acacaaggct gactcccgtc tgtacttcaa ctgggatcac tgtggcaaga 360tggagcctgc ctgtaagagt cacttcatcc aagactcctg cctgtatgag tgctccccca 420accttgggcc ttggatccag caagtggacc agagttggcg taaagagcgt ttcctggatg 480tgccttatgc aaagaggact gtcaccagtg gtgggaagcc tgtcgtacgt cctttacctg 540caagagagac tggcataaag gctgggactg gtctcaggca ttaccagtgc caaacacagg 600accctgtaaa cgttgagtac tattccgaaa cagcagcc 638249502DNAMus musculus 249ttcggcacag ggggctgtgg acgaagactg tagagactac ccagagtctg acctagggag 60aggccaactc ggatacccct atgtgcgctc ccagaagcta aggacattga gacagaaaga 120catggcctgg aaacagacac cactcttgct tttggtctac atggtcacaa caggcagtgg 180ccgggacaga acagacctac tcaacgtttg catggatgcc aaacaccata agacaaagcc 240gggccccgag gacaagctgc atgaccagtg tagtccatgg aagaaaaatg cctgttgctc 300agtcaacacc agccaggagc tacacaaggc tgactcccgt ctgtacttca actgggatca 360ctgtggcaag atggagcctg cctgtaagag tcacttcatc caagactcct gcctgtatga 420gtgctccccc aaccttgggc cttggatcca gcaagtggac cagagttggc gtaaagagcg 480tttcctggat gtgcccttat gc 5022501325DNAHomo sapiens 250ggaaaggatt ttctcagccc ccatctccag cactgtgtgt tggccgcacc catgagagcc 60tcagcactct gaaggtgcag ggggcaaagg ccaaaagagc tctggcctga acttgggtgg 120tccctactgt gtgacttggg gcatggcctc atctgtgctg aaatgattcc acaaagatta 180aactggctat catttgttga tttccccctt cttacattta atccttgcag gagaaagcta 240agcctcaaga tagtttgctt ctctttcccc caaggccaag gagaaggtgg agtgagggct 300ggggtcggga caggttgaac gggaaccctg tgctctaaca gttagggccc gccgaggaac 360tgaacccaaa ggatcacctg gtattccctg agagtacaga tttctccggc gtggccctca 420agggacagac atggctcagc ggatgacaac acagctgctg ctccttctag tgtgggtggc 480tgtagtaggg gaggctcaga caaggattgc atgggccagg actgagcttc tcaatgtctg 540catgaacgcc aagcaccaca aggaaaagcc aggccccgag gacaagttgc atgagcagtg 600tcgaccctgg aggaagaatg cctgctgttc taccaacacc agccaggaag cccataagga 660tgtttcctac ctatatagat tcaactggaa ccactgtgga gagatggcac ctgcctgcaa 720acggcatttc atccaggaca cctgcctcta cgagtgctcc cccaacttgg ggccctggat 780ccagcaggtg gatcagagct ggcgcaaaga gcgggtactg aacgtgcccc tgtgcaaaga 840ggactgtgag caatggtggg aagattgtcg cacctcctac acctgcaaga gcaactggca 900caagggctgg aactggactt cagggtttaa caagtgcgca gtgggagctg cctgccaacc 960tttccatttc tacttcccca cacccactgt tctgtgcaat gaaatctgga ctcactccta 1020caaggtcagc aactacagcc gagggagtgg ccgctgcatc cagatgtggt tcgacccagc 1080ccagggcaac cccaatgagg aggtggcgag gttctatgct gcagccatga gtggggctgg 1140gccctgggca gcctggcctt tcctgcttag cctggcccta atgctgctgt ggctgctcag 1200ctgacctcct tttaccttct gatacctgaa aatccctgcc ctgttcagcc ccacagctcc 1260caactatttg gttcctgctc catggtcggg cctctgacag ccactttgaa taaaccagac 1320accgc 1325251819DNAHomo sapiens 251cgcaggaata gatggacatg gcctggcaga tgatgcagct gctgcttctg gctttggtga 60ctgctgcggg gagtgcccag cccaggagtg cgcgggccag gacggacctg ctcaatgtct 120gcatgaacgc caagcaccac aagacacagc ccagccccga ggacgagctg tatggccagt 180gcagtccctg gaagaagaat gcctgctgca cggccagcac cagccaggag ctgcacaagg 240acacctcccg cctgtacaac tttaactggg atcactgtgg taagatggaa cccacctgca 300agcgccactt tatccaggac agctgtctct atgagtgctc acccaacctg gggccctgga 360tccggcaggt caaccagagc tggcgcaaag agcgcattct gaacgtgccc ctgtgcaaag 420aggactgtga gcgctggtgg gaggactgtc gcacctccta cacctgcaaa agcaactggc 480acaaaggctg gaattggacc tcagggatta atgagtgtcc ggccggggcc ctctgcagca 540cctttgagtc ctacttcccc actccagccg ccctttgtga aggcctctgg agccactcct 600tcaaggtcag caactatagt cgagggagcg gccgctgcat ccagatgtgg tttgactcag 660cccagggcaa ccccaatgag gaggtggcca agttctatgc tgcggccatg aatgctgggg 720ccccgtctcg tgggattatt gattcctgat ccaagaaggg tcctctgggg ttcttccaac 780aacctattct aatagacaaa tccacatgaa aaaaaaaaa 819252283DNAHomo sapiensmodified_base(259)N = A, C, G, or T/U 252catgagcagt gtcgaccctg gaggaagaat gcctgctgtt ctaccaacac cagccaggaa 60gcccataagg atgtttccta cctatataga ttcaactgga accactgtgg agagatggca 120cctgcctgca aacggcattt catccaggac acctgcctct acgagtgctc ccccaacttg 180gggccctgga tccagcaggt ggatcagagc tggcgcaaag agcgggtact gaacgtgccc 240ctgtgcaaag aggactgtna gcaaatggtg gggaagattg tcg 283253834DNAHomo sapiens 253gaattccgga caaggattgc atgggccagg actgagcttc tcaatgtctg catgaacgcc 60aagcaccaca aggaaaagcc aggccccgag gacaagttgc atgagcagtg tcgaccctgg 120aggaagaatg cctgctgttc taccaacacc agccaggaag cccataagga tgtttcttac 180ctatatagat tcaactggaa ccactgtgga gagatggcac ctgcctgcaa acggcatttc 240atccaggaca cctgcctcta cgagtgctcc cccaacttgg ggccctggat ccagcaggtg 300gatcagagct ggcgcaaaga gcgggtactg aacgtgcccc tgtgcaaaga ggactgtgag 360caatggtggg aagattgtcg cacctcctac acctgcaaga gcaactggca caagggctgg 420aactggactt cagggtttaa caagtgcgca gtgggagctg cctgccaacc tttccatttc 480tacttcccct ctcccactgt tctgtgcaat gaaatctgga ctcactccta caaggtcagc 540aactacagcc gagggagtgg ccgctgcatc cagatgtggt tcgacccagc ccagggcaac 600cccaatgagg aggtggcgag gttctatgct gcagccatga gtggggctgg gccctgggca 660gcctggcctt tcctgcttag cctggcctaa tgctgctgtg gctgctcagc tgacctcctt 720ttaccttctg atacctggaa atccctgccc tgttcagccc cacagctccc aactatttgg 780ttcctgctcc atggtcgggc ctctgacagc cattttgaat aaaccagaca ccgc 8342541067DNAHomo sapiens 254cctgtgtctt cccgcatcca gtgtagtctc tggagaaaga atgcctgagc tttaccagca 60ccacccagga agcccataag aatattccca tctatatgga ttcaactgga accactgtgg 120agagatggta cctgcctgca aacggcactt tatccaggac acctgccttt acgagtgacc 180ccccaacttg gggccctgga tccagcaggt atgcatggct tcctggcatc caagagctag 240cagaggagct gaattttcca ggcgtctctg caggcagcaa ccccagctcc acttctattc 300agggctgggt tcctgggatt cttgagcctg agcccttctt ttctaccaaa atctcccagg 360tggatcagag ctggtgcaaa gagtgggtgc tgaatgtgcc cctgtgcaaa gaggactgtg 420agcaatggtg ggaagattgt cgcacctcct acacctgcaa gagcaatggg cacaagggct 480ggaactggac ctcaggtgag ggctggggtg ggcaggaaag gagggatttg gaagtgaagg 540tgtgttgggt gtggaacagg tgtgtgacat tttggggttg tagggctggc agaatcagag 600acctttgggg cccagtggct aaaggtcttc cctcttccta cagggtctaa caagtgccag 660gtggcagctg cctgactacc tttccatctc tactttctca cacccactgc tctgtgcagt 720gaaatctgga ctcactccta cagggtcagc aactacaacc gagggagcag ccgctgcatc 780cagatgtggt tcgacctggc ccagggcaac cccaatgagg aggtggcaag gttctatgct 840gcagctctga gtggggctgg gccctgggca gcctggcctc tcctgctcaa cctggcccta 900atgctgctgt ggctgctcag ctgacctcct tttaccttct gatacttgga catccctgcc 960ctgtttagcc ccacagctcc caactatttg gttcctcttc tatggtcttg tctctgacag 1020ccactttgaa taaaccacac accacacatg tatcttgaga attattt 1067255900DNAchicken 255gaattcctct agggagaagt ctcacccaga aggacagcaa aagaggaaaa gaagggaaca 60acaatgctga ggtttgccat caccctcttt gctgtcatca catcatctac ctgccagcag 120tatggatgtc tggaagggga cacccacaaa gcgaagccaa gtcctgagcc aaacatgcat 180gaatgcactc tgtattctga atcttcctgt tgctatgcaa acttcacaga gcaattggct 240cattccccaa taattaaagt aagcaacagc tactggaaca gatgtgggca gctcagtaaa 300tcctgtgaag atttcacaaa gaaaatcgag tgcttttacc ggtgttctcc gcacgctgct 360cgctggatcg atcccagata tactgctgct attcagtctg ttccactgtg tcagagcttc 420tgtgatgact ggtatgaagc ctgcaaagat gattccattt gtgctcataa ctggctgacg 480gactgggaac gggatgaaag tggagaaaac cactgtaaga gtaaatgcgt accatacagt 540gagatgtatg caaatgggac cgacatgtgc cagagtatgt ggggggaatc ctttaaggtg 600agcgaatcct cctgcctctg cttgcaaatg aacaagaagg acatggtggc aatcaagcac 660ctcctctccg aaagctcaga ggaaagctcc agtatgagca gcagtgagga gcacgcctgc 720caaaagaaac tcctgaagtt tgaggcactg cagcaagagg aaggggaaga gagaagatga 780attttggtgg atgaatatca ggaggagagg aatcattgtg gaggttgtgc tcggggcatc 840acagcagcct gtcttatccc tcacttctga gaacacaata aatcaatggt tggctatatt 9002561223DNAMus musculus 256gctttagagg cagatcaggg tgtagttttc agctagcgcc gtgccttccc caccatgttc 60cttgccatga tgataatgta ctagacctct gaaactgtag cttctttgtt acagagtctc 120cgtgaatctg gaattcacca attcggcgag tctgaaagcc tcagtgatct ctcaggctcc 180atctgtctcc actccccagt ggaaggcttg cagctgtgtc accgctccag acttcacaca 240ggtgctggaa gactgaacta agacagaaag acatggcctg gaaacagaca ccactcttgc 300ttttggtcta catggtcaca acaggcagtg gccgggacag aacagaccta ctcaacgttt 360gcatggatgc caaacaccat aagacaaagc cgggccccga ggacaagctg catgaccagt 420gtagtccatg gaagaaaaat gcctgttgct cagtcaacac cagccaggag ctacacaagg 480ctgactcccg tctgtacttc aactgggatc actgtggcaa gatggagcct gcctgtaaga 540gtcacttcat ccaagactcc tgcctgtatg agtgctcccc caaccttggg ccttggatcc 600agcaagtgga ccagagttgg cgtaaagagc gtttcctgga tgtgccctta tgcaaagagg 660actgtcacca gtggtgggaa gcctgtcgta cctcctttac ctgcaagaga gactggcata 720aaggctggga ctggtcctca ggcattaaca agtgcccaaa cacagcaccc tgtcacacgt 780ttgagtacta cttcccgaca ccagccagcc tttgcgaggg tctctggagt cactcctaca 840aggtcagcaa ctacagcaga gggagtggcc gctgcatcca gatgtggttt gactcaaccc 900agggcaatcc caatgaggac gtggtgaagt tttatgcttc ctttatgaca tctgggactg 960tgccccatgc agcagtactt cttgtgccca gcctggcccc agtgctgtca ttatggctcc 1020ctggctgaga ggtcagtctt cctctctaga tttctcctct atctaccctt ggtctggttc 1080aactcttcaa agaataagga agtcttgagc ctgcttccac ccctctcctc tgtcatccag 1140ttcctgatcc atgttggggg ttggggtttc tacaatcatt ttcaataaat ctatgacaca 1200tctgggccta atgaaaaaaa aaa 12232571565DNAMus musculus 257ggatccaaga gattttatac tgtccttcag cactgtcctt cagttctttt tgtttttttg 60ttttttgttt tgttttgttt ttggtttttc gagacagggt ttctctgtgt agccctggct 120gtcctggaac tcactctgta gaccaggctg gcctcgaact cagaaatcca cctgcctctg 180cctcccaagt gctgggttta aaggcatacg ccaccacagc ccggctcttc ggttctttag 240gtcattattt tttggggtag ggggacaaac aaattctcac tatgtatcac agattggcct 300agaccccaca agccttcccc cttcccgtcc tccatgtcct ggggttgcag gcgtgtctca 360ccaattgcag ctgggcttgt tttgtgtgtt tccttttgag aggtttcggt cgggtcgggt 420gcttttgctg cagatgccgc tgtcaggatg ggctgtcagg gcagaatggc ttttggagaa 480caggaaagga aaatactgag gaagcaaaac tttacaaagc agcactcttt cttgtgtacc 540ctctaaccac accatcctgt gggctgtcac ttggtcctcc tgccaatctg gagaacttgg 600cagggctggg tcaccacctc cctcagggct aacaggactt ctaggctgac atgatgaccc 660agctgataca gagtggaatg ccgagaacct cctgtgacag gatgaaggat ctgtgtgtcc 720ctggcccttg tcaaggtagc aagcagcagg aacctgaact atttaactat gtgtcataaa 780gtctggaaat aagatgaaag catggggcat cccatcttct ctaggttgga aagctttgct 840tcttttataa cccccctccc caatgccatg gggccatggg ataaaagagt ctccttgctg 900acctctattc cagcttcagg gagcctgagg acatgaatgc tgaaggagaa gggactgatc 960taatctttca ctatagggac agagagtctg agtcagggaa taaatgaagt ccctcccccc 1020tctggtctag gtctccctaa ctttagctcc ctctgcacag acagaaagac atggcctgga 1080aacagacacc actcttgctt ttggtctaca tggtcacaac aggcagtggc cgggacagaa 1140cagacctact caacgtttgc atggatgcca aacaccataa gacaaagccg ggccccgagg 1200acaagctgca tgaccaggtt ctgtgccagt gtggtcctga tgggagggtg atagagggca 1260gggtggggtt agtgagcagc cagacacacc cacaccctga gctcttgttg gcagagatgg 1320cttggtggaa agtagtgagg tgattttctg agggctgtcc ccagaagagg acacagtagt 1380ggcaatgaag cagttgatca ttagaagcct ctaattagag gccacgtgag gtcatgtgat 1440gataatctct atatctctca aataagggcc cgtggaagca cagggactca ctctcacagg 1500ttagacacac ctgatttttt tttttttgag agcattggtg ttttgcctac atatgtgttg 1560gatcc 15652581000DNAMus musculus 258tctagaattt tcagccctat cttaagcact atataacatg tgaaaaggaa caaaagggct 60tctaacacta gaaaaaattt aaggccaaac ataacttgta aagccatttt ccactttact 120tctgatagac tgtcttgaat ttccttagaa agttcaagat cagacttacc tcgttcccca 180gctgaaaagt tctgaattca tacagttgaa tccttcttaa cagtctgctt tacgggaacc 240tttatcaccg tcgttcccca gctgatgagt tctgaattcg gcagttgaat ccttctcaac 300agtctgtgtt acgggaacct tataaccttg attcgcagtt ctggttctgg aatgagggat 360cttccttgcg ccagtcccga gttttttctc gtcccggatt ttctcgtccc ggaattcggc 420accaattgtt attcgacgcg ttctcacgac cggccaggaa gaacaccaca gaccagaatc 480ttctgcgaca aagctttatt cttacatctt caggaaaaga gagcaagaag caagagagag 540caagaagcaa gagagggaag caagagagag caagaagcaa gagagggaag caagagagag 600caaagcaaga gagagagaaa aacgaaaccc cttctatttt aaagagaaca accattgcct 660agggcgcatc actccctgat tggctgcagc ccatggccga gctgacgttc acgggaaaaa 720cagagtacaa gtagtcgtaa atacccttgg ctcatgcgca gattatttgt ttaccaactt 780agaacacagg atgtcagcgc catcttgtga cggcgaatgt gggggcggct tcccacaagg 840ctccacccac tggagctgag cacacacttg gaggttccac ttaccttagc tctgccttca 900gggtctgaca tggctcacct gatgactgtg cagttgttgc tcctggtgat gtggatggcc 960gaatgtgctc agtccagagc tactcgggcc aggactgaac 10002591391DNAMus musculus 259actagttgtg tctagatcct attgcactga tggtcatgaa gttgaaacat gggggaaaat 60gaactttata cccttcttca tgacttctgt ccttttgcct gcctcctttc tcatctccta 120atattacagt cttggtttcc tctctaaatt tttagacttt taacccacac ctaaacctgt 180atcagctttt ataaaaatct tttcaaaact tcacactgaa gcatctgcct ccaaaggttt 240tgaatgtgaa cgtgggtaaa ctctgttttt gcaaatggcc catctcttat tttttaattg 300ccctgtgtga gtctcaggac cactaagtct aacaggctgt gaccagtgat tgtctctagg 360gcatctgagc ctcacagagt ctgggaagac tgacaggagg aggtgaccca aggtctgtga 420gtgcaggctc cacccactgg agctgagcac acacttggag gttccactta ccttagctct 480gccttcaggg tctgacatgg ctcacctgat gactgtgcag ttgttgctcc tggtgatgtg 540gatggccgaa tgtgctcagt ccagagctac tcgggccagg actgaacttc tcaatgtctg 600catggatgcc aagcaccaca aagaaaaacc gggccctgag gacaatttac acgaccaggt 660aggacgaagg gtgatgtgtg gctgactaag ggctcgtggg tcaggagaaa gaagtatcta 720gtcccagttt atggtggagg tggtcagacc tacctgagga gaccttcggt tctctctagt 780gtgggtgact ttgacagtac atattggctg ccaactgcca gtgtgatatt atcagctcat 840cttcctggta gctgaatttt gacgttgcat aagtaaggaa gtagattcaa ggaggaactt 900gggaatggaa caggcaaacc attgtgatgg ttttagattt aaactgattg gggaggacgc 960ctctgggagt ctcaggggag ggactgtatg ctgcccagtc acttttctgc cagcctttga 1020agacttgaga aggagactct catatctgag aagcctttgg aggcaggcat ctagcgaaca 1080cttggactgt ggtcctcagc ttgagggctg gagggcttga gggctctgtg ttataacagt 1140tgtttgccat agtgctttta gtatcccaaa gctcactaaa catttaataa aatcagtgtg 1200atgcaacaac tatgaagtca accagcagca ggtctgctat tggggaggta caatcagtgc 1260agacaacaaa gtgggagggg ggtctcaaaa aagccaagat gagggctgga gagttggctc 1320agtggttaaa agcacttgtt gagcttgcag aataccaagg tctgatccac aacatccaag 1380gtggtggatc c 1391260979DNAMus musculus 260tggagctgag cacacacttg gaggttccac ttaccttagc tctgccttca gggtctgaca 60tggctcacct gatgactgtg cagttgttgc tcctggtgat gtggatggcc gaatgtgctc 120agtccagagc tactcgggcc aggactgaac ttctcaatgt ctgcatggat gccaagcacc 180acaaagaaaa accgggccct gaggacaatt tacacgacca gtgcagcccc tggaagacga 240attcctgctg ttccacgaac acaagccagg aagcacataa ggacatttcc tacctgtacc 300ggttcaactg gaaccactgc ggaactatga catcggaatg caaacggcac tttatccaag 360acacctgcct ctatgagtgt tccccgaact tgggaccctg gatccagcag gtggaccaga 420gctggcgcaa agagcggatc cttgatgttc ccctgtgcaa agaggactgt cagcagtggt 480gggaggactg ccagagctct tttacctgca agagcaattg gcacaaggga tggaactggt 540cctctgggca taacgagtgt cctgtgggag cctcctgcca tcccttcacc ttctacttcc 600ccacatctgc tgctctgtgt gaggaaatct ggagtcactc ctacaagctc agcaactaca 660gccgagggag cggccgctgc attcagatgt ggtttgaccc agcccagggc aaccccaacg 720aggaagtggc gaggttctat gccgaggcca tgagtggagc tgggcttcat gggacctggc 780cactcttgtg cagcctgtcc ttagtgctgc tctgggtgat cagctgagtt cctgttttac 840cttcagttgt ctggagcgcc accctgcttg gctcagcctc ccagctccca gcctcctttg 900tggtggggct ctgacagcct ctttaataaa ccagacattc cacatgtgcc ttatgaatta 960aaaaaaaaaa aaaaaaaaa 97926166DNAHomo sapiens 261acaaggattg catgggccag gactgagctt ctcaatgtct gcatgaacgc caagcaccac 60aaggaa 662621108DNAHomo sapiens 262ctggaggcct ggctggtgct cacatacaat aattaactgc tgagtggcct tcgcccaatc 60ccaggctcca ctcctgggct ccattcccac tccctgcctg tctcctaggc cactaaacca 120cagctgtccc ctggaataag gcaaggggga gtgtagagca gagcagaagc ctgagccaga 180cggagagcca cctcctctcc cagggacaga catggctcag cggatgacaa cacagctgct 240gctccttcta gtgtgggtgg ctgtagtagg ggaggctcag acaaggattg catgggccag

300gactgagctt ctcaatgtct gcatgaacgc caagcaccac aaggaaaagc caggccccga 360ggacaagttg catgagcagt gtcgaccctg gaggaagaat gcctgctgtt ctaccaacac 420cagccaggaa gcccataagg atgtttccta cctatataga ttcaactgga accactgtgg 480agagatggca cctgcctgca aacggcattt catccaggac acctgcctct acgagtgctc 540ccccaacttg gggccctgga tccagcaggt ggatcagagc tggcgcaaag agcgggtact 600gaacgtgccc ctgtgcaaag aggactgtga gcaatggtgg gaagattgtc gcacctccta 660cacctgcaag agcaactggc acaagggctg gaactggact tcagggttta acaagtgcgc 720agtgggagct gcctgccaac ctttccattt ctacttcccc acacccactg ttctgtgcaa 780tgaaatctgg actcactcct acaaggtcag caactacagc cgagggagtg gccgctgcat 840ccagatgtgg ttcgacccag cccagggcaa ccccaatgag gaggtggcga ggttctatgc 900tgcagccatg agtggggctg ggccctgggc agcctggcct ttcctgctta gcctggccct 960aatgctgctg tggctgctca gctgacctcc ttttaccttc tgatacctgg aaatccctgc 1020cctgttcagc cccacagctc ccaactattt ggttcctgct ccatggtcgg gcctctgaca 1080gccactttga ataaaccaga caccgcac 1108263991DNAHomo sapiens 263ggagagccac ctcctctccc aggaactgaa cccaaaggat cacctggtat tccctgagag 60tacagatttc tccggcgtgg ccctcaaggg acagacatgg ctcagcggat gacaacacag 120ctgctgctcc ttctagtgtg ggtggctgta gtaggggagg ctcagacaag gattgcatgg 180gccaggactg agcttctcaa tgtctgcatg aacgccaagc accacaagga aaagccaggc 240cccgaggaca agttgcatga gcagtgtcga ccctggagga agaatgcctg ctgttctacc 300aacaccagcc aggaagccca taaggatgtt tcctacctat atagattcaa ctggaaccac 360tgtggagaga tggcacctgc ctgcaaacgg catttcatcc aggacacctg cctctacgag 420tgctccccca acttggggcc ctggatccag caggtggatc agagctggcg caaagagcgg 480gtactgaacg tgcccctgtg caaagaggac tgtgagcaat ggtgggaaga ttgtcgcacc 540tcctacacct gcaagagcaa ctggcacaag ggctggaact ggacttcagg gtttaacaag 600tgcgcagtgg gagctgcctg ccaacctttc catttctact tccccacacc cactgttctg 660tgcaatgaaa tctggactca ctcctacaag gtcagcaact acagccgagg gagtggccgc 720tgcatccaga tgtggttcga cccagcccag ggcaacccca atgaggaggt ggcgaggttc 780tatgctgcag ccatgagtgg ggctgggccc tgggcagcct ggcctttcct gcttagcctg 840gccctaatgc tgctgtggct gctcagctga cctcctttta ccttctgata cctggaaatc 900cctgccctgt tcagccccac agctcccaac tatttggttc ctgctccatg gtcgggcctc 960tgacagccac tttgaataaa ccagacaccg c 9912641230DNAHomo sapiens 264gaatcaattc ctccaaaccg caagaacagt aacatttatt attcaaaaaa acaaaaacca 60gattatagga tatgacattt ggtataacaa taatgttatt gaaaaatgga aaaatgatcc 120attaatggct tgggctaaaa attcggggga cagcctaggg gcctggatct attgcctact 180tagagagagg ccaactcaga cacagccgtg tatgctccca gcagcaacgg aggttcacgt 240ccgcctgcag ggacagaaag acatggtctg gaaatggatg ccacttctgc tgcttctggt 300ctgtgtagcc accatgtgca gtgcccagga caggactgat ctcctcaatg tctgtatgga 360tgccaagcac cacaagacaa agccaggtcc tgaggacaag ctgcatgacc aatgcagtcc 420ctggaagaag aatgcctgct gcacagccag caccagccag gagctgcaca aggacacctc 480ccgcctgtac aactttaact gggaccactg cggcaagatg gagcccgcct gcaagcgcca 540cttcatccag gacacctgtc tctatgagtg ctcacccaac ctggggccct ggatccagca 600ggtgaatcag acgtggcgaa aagaacgctt cctggatgtg cccttatgca aagaggactg 660tcagcgctgg tgggaggatt gtcacacctc ccacacgtgc aagagcaact ggcacagagg 720atgggactgg acctcaggag ttaacaagtg cccagctggg gctctctgcc gcacctttga 780gtcctacttc cccactccag ctgccctttg tgaaggcctc tggagtcact catacaaggt 840cagcaactac agccgaggga gcggccgctg catccagatg tggtttgatt cagcccaggg 900caaccccaac gaggaagtgg cgaggttcta tgctgcagcc atgcatgtga atgctggtga 960gatgcttcat gggactgggg gtctcctgct cagtctggcc ctgatgctgc aactctggct 1020ccttggctga gttcagtcct cccagactac ctgccctcag cttggataac caggctgggc 1080tcagctcagc tcccacaaat gacagcccct taagcatgct tctattagtc acctaaccct 1140ctgtcaccca gtctgttgct gctccatggt ggggccaaga gtcacttcta ataaacagac 1200tgttttctaa taaaaaaaaa aaaaaaaaaa 1230265819DNAHomo sapiens 265cgcaggaata gatggacatg gcctggcaga tgatgcagct gctgcttctg gctttggtga 60ctgctgcggg gagtgcccag cccaggagtg cgcgggccag gacggacctg ctcaatgtct 120gcatgaacgc caagcaccac aagacacagc ccagccccga ggacgagctg tatggccagt 180gcagtccctg gaagaagaat gcctgctgca cggccagcac cagccaggag ctgcacaagg 240acacctcccg cctgtacaac tttaactggg atcactgtgg taagatggaa cccacctgca 300agcgccactt tatccaggac agctgtctct atgagtgctc acccaacctg gggccctgga 360tccggcaggt caaccagagc tggcgcaaag agcgcattct gaacgtgccc ctgtgcaaag 420aggactgtga gcgctggtgg gaggactgtc gcacctccta cacctgcaaa agcaactggc 480acaaaggctg gaattggacc tcagggatta atgagtgtcc ggccggggcc ctctgcagca 540cctttgagtc ctacttcccc actccagccg ccctttgtga aggcctctgg agccactcct 600tcaaggtcag caactatagt cgagggagcg gccgctgcat ccagatgtgg tttgactcag 660cccagggcaa ccccaatgag gaggtggcca agttctatgc tgcggccatg aatgctgggg 720ccccgtctcg tgggattatt gattcctgat ccaagaaggg tcctctgggg ttcttccaac 780aacctattct aatagacaaa tccacatgaa aaaaaaaaa 8192661123DNAHomo sapiens 266gaggagggta tggggaggca cttagttcct gtgtcttccc cacccagtgc agtccctgga 60agaagaatgc ctgctgcaca gccagcacca gccaggagct gcacaaggac acctcccgcc 120tgtacaactt taactgggac cactgcggca agatggagcc cgcctgcaag cgccacttca 180tccaggacac ctgtctctat gagtgctcac ccaacctggg gccctggatc cagcaggtag 240ggtgtctccc ccccacccac cccagcagac tgccatcccc ctcagtcact tcaaggcgat 300ggctgccagc atccctggct gagaggagcc ctgcctcccc acctcccacc caggtgaatc 360agacgtggcg caaagaacgc ttcctggatg tgcccttatg caaagaggac tgtcagcgct 420ggtgggagga ttgtctcacc tcccacacgt gcaagagcaa ctggcacaga ggatgggact 480ggacctcagg tgagggtgat tgagttgggg ttaggaaaaa ggagattgag gtagggtttg 540gaaaatcctc aaggatttgg ggtggggtga agatttctgg gggtggccag aaatgagctt 600tgggcccagg ggctgaaagt ctgtgtccac catgcctctc cctgcaggag ttaacaagtg 660cccagctggg gctctctgcc gcacctttga gtcctacttc cccactccag ctgccctttg 720tgaaggcctc tggagtcact catacaaggt cagcaactac agccgaggga gcggccgctg 780catccagatg tggtttgatt cagcccaggg caaccccaac gaggaagtgg cgaggttcta 840tgctgcagcc atgcatgtga atgctggtga gatgcttcat gggactgggg gtctcctgct 900caggctggcc ctgatgctgc aactctggct ccttggctga gttcagtcct cccagactac 960ctgccctcag cttggataac caggctgggc tcagctcagc tcccacaaat gccagcccct 1020taagcatgct tctattagtc acctaaccct ctgtcaccca gtctgttgct gctccatggt 1080ggggccaaga gtcacttcta ataaacagac tgttttctaa taa 1123267374DNAHomo sapiens 267agcttcaggg ccccagcatc gaaggaacag ggtctgacct catttgccac cgtagggatg 60gggagactga ggcaggaggt gaatggctcc cagcttggag ccctttcccc tcaggacttg 120gtttccctac cctacgtccg cctgcaggga cagaaagaca tggtctggaa atggatgcca 180cttctgctgc ttctggtctg tgtagccacc atgtgcagtg cccaggacag gactgatctc 240ctcaatgtct gtatggatgc caagcaccac aagacaaagc caggtcctga ggacaagctg 300catgaccaag tacggctgga gtgtgcctct gctaaggagg ggcttgttct aacagggagg 360agaaagtcag gatg 3742689PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 268Glu Ile Trp Thr His Ser Tyr Lys Val 1 52699PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 269Leu Leu Ser Leu Ala Leu Met Leu Leu 1 52704PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 270Ser Tyr Lys Val 12719PRTArtificial SequenceDescription of Artificial Sequence Synthetic Peptide 271Phe Ile Trp Thr Phe Ser Thr Lys Val 1 5

Claims

1-22. (canceled)

23. A pharmaceutical composition comprising at least one peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof, and a pharmaceutically acceptable excipient.

24. The pharmaceutical composition of claim 23, wherein the composition comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5.

25. The pharmaceutical composition of claim 23, wherein the composition comprises an adjuvant.

26. The pharmaceutical composition of claim 24, wherein the composition comprises an adjuvant.

27. The pharmaceutical composition of claim 23, wherein the composition is formulated for intradermal, subcutaneous or intravenous administration.

28. The pharmaceutical composition of claim 24, wherein the composition is formulated for intradermal, subcutaneous or intravenous administration.

29. A method of inducing immunity against a cancer expressing folate binding protein (SEQ ID NO: 10) in an individual, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising at least one peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof, and a pharmaceutically acceptable excipient.

30. The method of claim 29, wherein the pharmaceutical composition comprises an adjuvant.

31. The method of claim 29, comprising administering an effective amount of a second pharmaceutical composition comprising a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 268, and a pharmaceutically acceptable excipient.

32. The method of claim 31, wherein the second pharmaceutical composition comprises an adjuvant.

33. The method of claim 31, wherein the second pharmaceutical composition is formulated for intradermal, subcutaneous or intravenous administration.

34. The method of claim 29, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, brain cancer, sarcomas, or a combination thereof.

35. The method of claim 31, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, brain cancer, sarcomas, or a combination thereof.

36. The method of claim 29, further comprising administering a booster composition comprising a peptide consisting of an amino acid sequence selected from the group consisting SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof.

37. The method of claim 29, further comprising administering a booster composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 268.

38. The method of claim 31, further comprising administering a booster composition comprising a peptide consisting of an amino acid sequence selected from the group consisting SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof.

39. The method of claim 31, further comprising administering a booster composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 268.

40. A method of inducing immunity against a cancer expressing folate binding protein (SEQ ID NO: 10) in an individual, the method comprising administering to the individual an effective amount of a first pharmaceutical composition comprising at least one peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof, a pharmaceutically acceptable excipient and an adjuvant; and a second pharmaceutical composition comprising a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 268, a pharmaceutically acceptable excipient and an adjuvant.

41. The method of claim 40, further comprising administering a booster composition comprising at least one peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof, and an adjuvant; or administering a booster composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 268 and an adjuvant.