Method for regulating immune function in primates using the Foxp3 protein

Abstract

ecules are provided which encode Fkh^sf, as well as mutant forms thereof. Also provided are expression vectors suitable for expressing such nucleic acid molecules, and host cells containing such expression vectors. Also provided are pharmaceutical compounds and methods of identifying such compounds that can modulate the immune system. In addition are provided methods for identifying proteins regulated by Scurfin and proteins that induce or inhibit Foxp3 expression.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the benefit of U.S. Provisional Patent Application No. 60/333,409 filed Nov. 26, 2001 and 60/289,654 filed May 8, 2001, where these applications are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002]1. Field of the Invention

[0003]The present invention relates generally to pharmaceutical products and methods and, more specifically, to methods for identifying compounds which can modulate the immune system, further, to methods for identifying proteins regulated by Scurfin and those that induce or inhibit Foxp3 expression.

[0004]2. Description of the Related Art

[0005]A number of autoimmune diseases, such as Inflammatory Bowel Disease, Multiple Sclerosis, rheumatoid Arthritis, and Asthma, involve immune dysregulation. In all these diseases, subsets of T cells are hyper-activated and contribute to an immune reaction towards self. In recent years, mice with mutations in CD95, CD95-ligand, CTLA-4 or TGF-β have proven useful for dissecting a number of pathways involved in T cell regulation and immune system homeostasis. Mice with mutations in any one if the above genes have profoundly altered immune responses, attributed to a failure to control T cell function.

[0006]T cell activation in the periphery involves signaling via the T cell receptor and CD28 costimulation (reviewed in Bluestone, J. A., Immunity 2:555-559 (1995); Jenkins, M. K., Immunity 1:443-448 (1994); Rudd, C. E., Immunity 4:527-534 (1996)). Down regulation of peripheral T cell responses involves several pathways. Some of these include apoptosis mediated by members of the TNFR family, including CD95 and its ligand, activation induced death due to cytokine withdrawal, and negative signaling through CTLA-4 (CD152) (Lenardo et al., Ann. Rev. Immun. 17:221-253 (1999); Oosterwegel et al., Curr. Opin. Immun. 11:294-300 (1999); Saito, T., Curr. Opin. Immun. 10:313-321 (1998); Wallach et al., Ann. Rev. Immun. 17:331-367 (1999)). Mutations or expression of dominant negative forms of some of these genes have proven their critical role in the regulation of peripheral T cell responses. Mutations in CD95, CD95L, TGF-β or CTLA-4 lead to progressive autoimmune lymphoproliferative disorders (Kulkarni et al., Proc. Nat'l. Acad. Sci. USA 90:770-774 (1993); Shull et al., Nature 359:693-699 (1992); Takahashi et al., Cell 76:969-976 (1994); Tivol et al., Immunity 3:541-547 (1995); Watanbe-Fukunaga et al., Nature 356:314-347 (1992); Waterhouse et al., Science 270:985-988 (1995)). More recent data suggests that regulation of T cell activity by CD4⁺CD25⁺ regulatory T cells is also important for maintaining peripheral T cell tolerance (Roncarolo et al., Curr. Opin. Immun. 12:676-683 (2000); Sakaguchi, S., Cell 101:455-458 (2000); Shevach, E. M., Ann. Rev. Immun. 18:423-449 (2000)). Depletion of such regulatory T cells from normal animals leads to development of various autoimmune diseases and the adoptive transfer of these regulatory cells can also prevent disease in vivo in a number of systems (Asano et al., J. Exp. Med. 184:387-396 (1996); Sakaguchi et al., J. Immun. 155:1151-1164 (1995); Suri-Payer et al., J. Immun. 160:1212-1218 (1998)).

[0007]The specific mechanism by which regulatory T cells (T-reg cells) mediate their suppressive effect is currently unclear. While TGFB and IL-10 can mediate suppressive effects, and blocking these cytokines eliminates suppression in some in vivo models, there is good evidence to indicate other molecules are also involved. Mounting evidence indicates a role for CD152 in the activation and/or function of CD4⁺CD25⁺ T cells (Read et al., J. Exp. Med. 192:295-302 (2000); Takahashi et al., J. Exp. Med. 192:303-310 (2000)). Intriguingly, several studies suggest that signaling through CD152 results in the induction of TGFB (Chen et al., J. Exp. Med. 188:1849-1857 (1998); Gomes et al., J. Immunol. 164:2001-2008 (2000); Kitani et al., J. Immunol. 165:691-702 (2000)), providing a potential link between TGFB-mediated inhibition and the inhibitory activity of CD4⁺CD25⁺ cells.

[0008]The X-linked lymphoproliferative disease observed in the scurfy (sf) mouse, a spontaneous mutant animal that shares many characteristics with the pathogenesis seen in targeted deletions of CTLA-4 (Tivol et al., Immunity 3:541-547 (1995); Waterhouse et al., Science 270:985-988 (1995)) as well as TGF-β (Kulkarni et al., Proc. Nat'l. Acad. Sci. USA 90:770-774 (1993); Shull et al., Nature 359:693-699 (1992)), including death by three weeks of age (Godfrey et al., Am. J. Pathol. 145:281-286 (1994); Godfrey et al., Proc. Nat'l. Acad. Sci. USA 88:5528-5532 (1991); Godfrey et al., Am. J. Pathol. 138:1379-1387 (1991); Kanangat et al., Eur. J. Immunol. 26:161-165 (1996); Lyon et al., Proc. Nat'l. Acad. Sci. USA 87:2433-2437 (1990)). In sf animals, disease is mediated by CD4⁺ T cells, and these cells exhibit an activated phenotype both in vivo and in vitro (Blair et al., J. Immunol. 153:3764-774 (1994)). The specific mutation responsible for the disease has been recently cloned and the gene shown to be a new member of the forkhead family of transcription factors (Brunkow et al., Nature Genetics 27:68-72 (2001)). The gene has been designated Foxp3 and the protein product, scurfin. Mutations in the orthologous human gene cause a similar lymphoproliferative disorder among affected male progeny, which if left untreated is generally fatal (Bennett et al., Nature Genetics 27:20-21 (2001); Chatila et al., JM2, J. Clin. Invest 106:R75-81 (2000); Wildin et al., Nature Genetics 27:18-20 (2001)).

[0009]The present invention discloses methods and compositions useful for diagnosing scurfy-related diseases, more specifically, to methods for identifying compounds which can modulate the immune system, further, to methods for identifying proteins regulated by Scurfin and those that induce or inhibit Foxp3 expression

BRIEF SUMMARY OF THE INVENTION

[0010]The present invention relates generally to the discovery of novel genes which, when mutated, results in a profound lymphoproliferative disorder. In particular, a mutant mouse, designated `Scurfy`, was used to identify the gene responsible for this disorder through backcross analysis, physical mapping and large-scale DNA sequencing. Analysis of the sequence of this gene indicated that it belongs to a family of related genes, all containing a winged-helix DNA binding domain.

[0011]Thus, within one aspect of the invention isolated nucleic acid molecules are provided which encode FKH^sf or Fkh^sf, including mutant forms thereof. Within certain embodiments, Fkh^sf of any type may be from a warm-blooded animal, such as a mouse or human. Within further embodiments, isolated nucleic acid molecules are provided wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule that encodes an amino acid sequence comprising SEQ ID NOs:2 or 4, (b) a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs:1 or 3, or its complement, and (c) a nucleic acid molecule that encodes a functional fragment of the polypeptide encoded by either (a) or (b). Preferably, the nucleic acid molecule is not JM2. Within related aspects, vectors (including expression vectors), and recombinant host cells are also provided, as well as proteins which are encoded by the above-noted nucleic acid molecules. Further, fusion proteins are also provided which combine at least a portion of the above-described nucleic acid molecules with the coding region of another protein. Also provided are oligonucleotide fragments (including probes and primers) which are based upon the above sequence. Such fragments are at least 8, 10, 12, 15, 20, or 25 nucleotides in length, and may extend up to 100, 200, 500, 1000, 1500, or, 2000 nucleotides in length.

[0012]Within other aspects methods of using the above noted expression vector for producing a Fkh^sf protein (of any type) are provided, comprising the general steps of (a) culturing recombinant host cells that comprise the expression vector and that produce Fkh^sf protein, and (b) isolating protein from the cultured recombinant host cells.

[0013]Also provided are antibodies and antibody fragments that specifically bind to Fkh^sf proteins. Representative examples of such antibodies include both polyclonal and monoclonal antibodies (whether obtained from a murine hybridoma, or derived into human form). Representative examples of antibody fragments include F(ab')₂, F(ab)₂, Fab', Fab, Fv, sFv, and minimal recognition units or complementarity determining regions.

[0014]Within yet other aspects, methods are provided for detecting the presence of a Fkh^sf nucleic acid sequence in a biological sample from a subject, comprising the steps of (a) contacting a Fkh^sf specific nucleic acid probe under hybridizing conditions with either (i) test nucleic acid molecules isolated from said biological sample, or (ii) nucleic acid molecules synthesized from RNA molecules, wherein the probe recognizes at least a portion of nucleotide sequence of SEQ ID NOs:1 or 3, and (b) detecting the formation of hybrids of the nucleic acid probe and (i) or (ii).

[0015]Within another related embodiment, methods are provided for detecting the presence of an Fkh^sf, or a mutant form thereof, in a biological sample, comprising the steps of: (a) contacting a biological sample with an anti-Fkh^sf antibody or an antibody fragment, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.

[0016]Within other aspects of the invention, methods are provided for introducing Fkh^sf nucleic acid molecules to an animal, comprising the step of administering a Fkh^sf nucleic acid molecule as described herein to an animal (e.g., a human, monkey, dog, cat, rat, or, mouse). Within one embodiment, the nucleic acid molecule is contained within and expressed by a viral vector (e.g., a vector generated at least in part from a retrovirus, adenovirus, adeno-associated virus, herpes virus, or, alphavirus). Within another embodiment the nucleic acid molecule is expressed by, or contained within a plasmid vector. Such vectors may be administered either in vivo, or ex vivo (e.g., to hematopoietic cells such as T cells).

[0017]Within other embodiments, transgenic non-human animals are provided wherein the cells of the animal express a transgene that contains a sequence encoding Fkh^sf protein.

[0018]In one preferred embodiment, a method is provided for regulating an immune function in a primate. The method comprises inserting a plurality of nucleic acid sequences that encode the Foxp3 protein into the lymphocytes of the primate; placing the nucleic acid sequence under the control of cytokine c; and activating expression of the nucleic acid sequences to increase the amount of the Foxp3 protein in the primate with cytokine c.

[0019]Accordingly, it is an object of the present invention to provide an assay for use in identifying agents that alter expression of Foxp3. Specifically, an assay is provided to measure the induction or inhibition of Foxp3 under varying conditions. The expression altering agents include small molecules, peptides, polynucleotides, cytokines, antibodies and Fab' fragments.

[0020]In one preferred embodiment, a method is provided for identifying a compound that modulates the level of expression of scurfin. The method comprises providing a composition comprising a reporter gene ligated to a scurfin promoter; contacting the composition with a test compound; determining the level of reporter gene expression; and comparing the level of reporter gene expression in (c) with the predetermined level of expression and thereby determining if the test compound modulates the expression of scurfin.

[0021]In a preferred embodiment, the compound decreases the level of scurfin expression.

[0022]In another embodiment, the compound increases the level of scurfin.

[0023]In one embodiment the test compound is selected from the group consisting of: a monoclonal antibody, a polyclonal antibody, a peptide, and a small molecule.

[0024]In another embodiment the test compound is selected from the group consisting of an organic molecule, a natural product, a peptide, an oligosaccharide, a nucleic acid, a lipid, an antibody or binding fragment thereof, and a cell.

[0025]In yet another embodiment, the test compound is from a library of compounds.

[0026]With other embodiments, the library is selected from the group consisting of a random peptide library, a natural products library, a combinatorial library, an oligosaccharide library and a phage display library.

[0027]In one preferred embodiment, a method is provided for suppressing an immune response comprising contacting T cells of the mammal with a compound that increases scurfin expression in the T cell, wherein an immune response is suppressed.

[0028]In one preferred embodiment, a method is provided for enhancing an immune response comprising contacting T cells with a compound that decreases scurfin expression in the T cell, wherein an immune response is enhanced.

[0029]Within another related embodiment, a method for inhibiting an autoimmune response in a subject, wherein the method comprises administering to the subject a compound which increases scurfin expression, thereby inhibiting an autoimmune response by the subject.

[0030]In a related embodiment the autoimmune response is selected from the group consisting of Inflammatory Bowel Disease, Psoriasis, Diabetes, Multiple Sclerosis, Rheumatoid Arthritis, and Asthma.

[0031]In one preferred embodiment, a method is provided for enhancing an immune response to a disease in a subject, wherein the method comprises administering to the subject a compound which decreases scurfin expression, thereby treating the disease in the subject.

[0032]In a related embodiment, a method is provided for enhancing an immune response to HIV or cancer in a subject, wherein the method comprises administering to the subject a compound which decreases scurfin expression, thereby treating HIV and cancer.

[0033]In one preferred embodiment, a method for inhibiting graft versus host disease in a subject wherein the method comprises administering to the subject a compound that increases scurfin expression, thereby inhibiting tissue transplant rejection by the subject.

[0034]In one preferred embodiment, a method is provided for inhibiting an autoimmune response in a patient comprising. The method comprising: isolating T cells from the patient; transducing the T cells with the scurfin gene; expanding the transduced T cells; and reintroducing the transduced T cells into said patient, wherein an autoimmune disease in the patient is inhibited.

[0035]In a related embodiment, a method is provided for inhibiting an autoimmune response in a patient comprising. The method comprising: isolating CD4⁺CD25+ regulatory T cells from the patient; transducing the CD4⁺CD25+ regulatory T cells with the scurfin gene; expanding the transduced CD4⁺CD25+ regulatory T cells; and reintroducing the transduced CD4⁺CD25+ regulatory T cells into the patient, wherein an autoimmune disease in the patient is inhibited.

[0036]In one preferred embodiment, a method is provided for inhibiting an autoimmune response in a patient, wherein the autoimmune disease is selected from the group consisting of Inflammatory Bowel Disease, Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis, Diabetes and Asthma. The method comprising: isolating T cells from the patient; transducing the T cells with the scurfin gene; expanding the transduced T cells; and reintroducing the transduced T cells into said patient, wherein an autoimmune disease in the patient is inhibited.

[0037]In one preferred embodiment, a method is provided for inhibiting an autoimmune response in a patient comprising. The method comprising: isolating T cells from the patient; transducing the T cells with the scurfin gene contained in a retroviral vector; expanding the transduced T cells; and reintroducing the transduced T cells into said patient, wherein an autoimmune disease in the patient is inhibited.

[0038]In yet another preferred embodiment, a method for enhancing an immune response to a disease in a patient is provided. The method comprising: isolating T cells from the patient; transfecting the T cells with a test compound that inhibits scurfin expression; expanding the transfected T cells; and reintroducing the transfected T cells into said patient, wherein an immune response to a disease in the patient is enhanced.

[0039]In yet another preferred embodiment, a method for enhancing an immune response to a HIV and cancer in a patient is provided. The method comprising: isolating T cells from the patient; transfecting the T cells with a test compound that inhibits scurfin expression; expanding the transfected T cells; and reintroducing the transfected T cells into said patient, wherein an immune response to HIV or cancer in the patient is enhanced.

[0040]These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0041]FIGS. 1A and 1B depict a nucleotide sequence of mouse Fkh^sf cDNA (SEQ ID NO:1); translation is predicted to initiate at position 259 and terminate at position 1546.

[0042]FIG. 2 depicts the amino acid sequence of mouse Fkh^sf (SEQ ID NO:2).

[0043]FIGS. 3A and 3B depict a nucleotide sequence of 1735 bp corresponding to human FKH^sf cDNA (SEQ ID NO: 3; including a 1293 bp coding region); translation is predicted to initiate at position 55 and terminate at position 1348.

[0044]FIG. 4 depicts the sequence of a 431 amino acid human FKH^sf protein (SEQ ID NO: 4).

[0045]FIG. 5 diagrammatically depicts a vector for generation of FKH^sf transgenic mice.

[0046]FIG. 6 is a photograph which demonstrates that the FKH^sf transgene corrects the defect in scurfy animals.

[0047]FIG. 7 is a diagram which shows that FKH^sf tg mice have reduced lymph node cells, as compared to normal cells.

[0048]FIG. 8 is a diagram which shows that FKH^sf transgenic mice respond poorly to in vitro stimulation.

[0049]FIG. 9 is a comparison of FKH^sf and JM2 cDNAs.

[0050]FIG. 10 compares homology in various regions of human FKH^sf and murine Fkh^sf.

[0051]FIG. 11A is a graph monitoring the weight of both scurfy and wild-type mice. The mice were monitored for weight loss at regular intervals for 10 weeks. Each data point is an average of 3 mice except after week 5 when one of the three mice died in sf CD4 transfer group (indicated by an arrow on the graph). The data is representative of more than 3 independent experiments.

[0052]FIG. 11B is a photograph of a tissue section. Large intestines from C3H/SCID mice receiving either sf T cells (left panel) or a mixture of WT and sf T cells (right panel) were fixed in formalin, sectioned and processed for hematoxylin and eosin staining.

[0053]FIG. 11C is a graph depicting IL-4 production from 5×10⁴ PBMC from C3H/SCID mice receiving either sf CD4⁺ T cells or a mixture of WT and sf CD4⁺ T cells or WT CD4⁺ T cells were stimulated with 5 μg/ml anti-CD3 and 1 μg/ml anti-CD28 immobilized onto round bottom plates. Supernatants were harvested at 48 h and IL-4 levels were measured by ELISA.

[0054]FIGS. 12A and 12B are graphs depicting the weight loss of mice treated with either CD4+CD25+ or CD4⁺CD25- T-regulatory cells. CD4+CD25+ T-regulatory subset mediates the suppression of disease caused by sf T cells in vivo. A mixture of 4×10⁶ sf T cells and varying numbers of wildtype CD4+CD25+ (a) or CD4+CD25- (b) T cells was transferred into C3/SCID mice via tail-vein injection. These mice were monitored for weight loss over a period of time. Each data point is an average of 3 mice except sf CD4 transfer group and sf CD4+1.1×10⁶ CD4+CD25- T cells which have 2 mice each in the group. Also, arrows on the graph indicate mice that died or were sacrificed due to disease progression.

[0055]FIG. 13 depicts a graph of a proliferation assay that determines the suppressor activity of CD4+CD25+ T regulatory cells. Sf CD4+ T cells can be inhibited by CD4+CD25+ T-regulatory cells in vitro. 5×10⁴ WT or sf CD4+ T cells were stimulated with anti-CD3 (1 μg/ml) and 5×10⁴ mitomycin C treated Thy-1^- APC. CD4⁺CD25⁺ T-regulatory cells were added at various ratios to the assay. The cells were cultured for 72 h and pulsed with [³H] thymidine for final 8 hrs of the culture. Data is mean of triplicates.

[0056]FIG. 14 A depicts a graph of a proliferation assay in which 5×10⁴ WT or sf CD4+ T cells were stimulated with immobilized anti-CD3. TGF-β was added at a final concentration of 2.5 ng/ml at the beginning of the assay. The cells were cultured for 72 h and pulsed with [³H] thymidine for final 8 hrs of the culture. Data is mean of triplicates.

[0057]FIG. 14B depicts a graph of a proliferation assay in which 5×10⁴ WT or sf CD4+ T cells were stimulated with immobilized anti-CD3 (varying concentrations) and anti-CD28 (1 μg/ml). TGF-β was added at a final concentration of 2.5 ng/ml at the beginning of the assay. The cells were cultured for 72 h and pulsed with [³H] thymidine for final 8 hrs of the culture. Data is mean of triplicates.

[0058]FIGS. 15A and 15B are graphs examining Foxp3 expression in cDNA samples from various cell subsets using a real-time RT-PCR method in which Dad1 served as an endogenous reference gene. Normalized Foxp3 values were derived from the ratio of Foxp3 expression to Dad1 expression.

[0059]FIG. 16 depicts the level of CD25 surface expression on CD4+ T cells from WT animals, Foxp3 transgenic animals, and scurfy animals. Lymph node cells from sf, Foxp3 transgenic or littermate controls were examined for the expression of CD25 expression on CD4⁺ T cells. Data is representative of six individual mice examined.

[0060]FIG. 17 is a graph depicting the level of proliferation in 5×10⁴ WT CD4+ T cells were stimulated with anti-CD3 (1 μg/ml) and 5×10⁴ mitomycin C treated Thy-1^- APC. CD4⁺CD25⁺ T-regulatory cells from WT or Foxp3 transgenics were added at various ratios to the assay. The cells were cultured for 72 h and pulsed with [³H] thymidine for final 8 hrs of the culture. Data is mean or triplicates.

[0061]FIG. 18 is a FACS plot evaluating the expression of surface markers associated with T regulatory cells and the suppressive activity of these cells.

[0062]FIG. 19 is a graph depicting the level of T cell inhibition in freshly isolated CD4⁺CD25^- T cells from Foxp3 transgenic as tested in T-reg assays.

DETAILED DESCRIPTION OF THE INVENTION

[0063]The invention relates to the discovery that the scurfin protein is involved in the generation and/or activity of the CD4⁺CD25⁺ subset of regulatory T cells. Foxp3 expression is directly correlated with cells of this regulatory phenotype and its expression is uniquely increased upon activation of this specific subset. Mutant (sf) animals appear to lack this subset, whereas Foxp3 transgenic animals appear to possess an increased percentage of CD4⁺CD25⁺ cells. Further, while the CD4⁺CD25⁺ subset from transgenic animals does not appear inhibitory on a per cell basis, the expression of Foxp3 is still elevated in this subset relative to their CD25- counterparts. Interestingly, overexpression of Foxp3 in CD4⁺CD25^- T cells confers suppressive activity on these cells, although they remain less effective than CD4⁺CD25⁺ T cells. Overall, the data suggest that the recently described transcription factor, scurfin, is a critical regulator of immune cell function and may work primarily through the generation and/or activity of CD4⁺CD25⁺ regulatory T cells.

[0064]The results from the Examples indicate that expression of scurfin (Foxp3 gene) can downregulate the immune system in part through regulatory T (T-reg) cell activity. Consequentially, if the expression of the endogenous Foxp3 gene can be induced in T cells it can be used to downregulate the immune response in a variety of autoimmune diseases such as Inflammatory Bowel Disease, Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis, Diabetes, and Asthma or in other scenarios such as Graft versus Host disease. Furthermore, scurfin expression can be down-regulated to activate the immune system in cancer or AIDS.

DEFINITIONS

[0065]Prior to setting forth the Invention in detail, it may be helpful to an understanding thereof to set forth definitions of certain terms and to list and to define the abbreviations that will be used hereinafter.

[0066]"Scurfy" refers to an inherited disease in mice which exhibit a severe lymphoproliferative disorder (see, e.g., Lyon et al., Proc. Natl. Acad. Sci. USA 87:2433, 1990). The responsible gene (mutant forms of which are responsible for the disease) is shown in Sequence I.D. Nos. 1 and 3.

[0067]"Foxp3" refers to the forkhead domain-containing gene, which is mutated in the scurfy mouse mutant. "Foxp3" refers to the protein encoded by the mouse Foxp3 gene. "FOXP3" refers to the human ortholog of the murine Foxp3 gene. "FOXP3" refers to the protein encoded by the human FOXP3 gene. The cDNA sequences for murine Foxp3 and human FOXP3 are disclosed in U.S. patent application Ser. No. 09/372,668 wherein the mouse scurfy gene is designated Fkh^sf and the human ortholog is designated FKH^sf. The genomic sequence for human FOXP3 is disclosed in Genbank Accession No. AF235087. Genbank Accession No. AF235097 and U.S. patent application Ser. No. 09/372,668 are incorporated by reference in their entireties for all purposes.

[0068]"Molecule" should be understood to include proteins or peptides (e.g., antibodies, recombinant binding partners, peptides with a desired binding affinity), nucleic acids (e.g., DNA, RNA, chimeric nucleic acid molecules, and nucleic acid analogues such as PNA), and organic or inorganic compounds.

[0069]"Nucleic acid" or "nucleic acid molecule" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acids can be composed of monomers that are naturally-occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), or analogs of naturally-occurring nucleotides (e.g., α-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid" also includes so-called "peptide nucleic acids," which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

[0070]"Isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that corresponds to a gene that has been separated from the genomic DNA of a eukaryotic cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.

[0071]"Promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.

[0072]"Vector" refers to an assembly which is capable of directing the expression of desired protein. The vector must include transcriptional promoter elements which are operably linked to the genes of interest. The vector may be composed of either deoxyribonucleic acids ("DNA"), ribonucleic acids ("RNA"), or a combination of the two (e.g., a DNA-RNA chimeric). Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase or hygromycin phosphotransferase. Additionally, depending on the host cell chosen and the vector employed, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences conferring inducibility of transcription, and selectable markers, may also be incorporated into the vectors described herein.

[0073]"Isolated" in the case of proteins or polypeptides, refers to molecules which are present in the substantial absence of other biological macromolecules, and appear nominally as a single band on SDS-PAGE gel with coomassie blue staining. "Isolated" when referring to organic molecules means that the compounds are greater than 90% pure utilizing methods which are well known in the art (e.g., NMR, melting point).

[0074]"Cloning vector" refers to nucleic acid molecules, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign nucleotide sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.

[0075]"Expression vector" refers to a nucleic acid molecule encoding a gene that is expressed in a host cell. Typically, gene expression is placed under the control of a promoter, and optionally, under the control of at least one regulatory element. Such a gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a promoter are operably linked if the regulatory element modulates the activity of the promoter.

[0076]"Recombinant host" refers to any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.

[0077]In eukaryotes, RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA. A nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA. The RNA transcript is termed an "anti-sense RNA" and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene." Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.

[0078]An "anti-sense oligonucleotide specific for Fkh^sf" or a "Fkh^sf anti-sense oligonucleotide" is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript. Similarly, an "anti-sense oligonucleotide specific for "Fkh^sf" or a "Fkh^sf anti-sense oligonucleotide" is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the Fkh^sf gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Fkh^sf gene.

[0079]A "ribozyme" is a nucleic acid molecule that contains a catalytic center. The term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions. A nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene."

[0080]Abbreviations: YAC, yeast artificial chromosome; PCR, polymerase chain reaction; RT-PCR, PCR process in which RNA is first transcribed into DNA at the first step using reverse transcriptase (RT); cDNA, any DNA made by copying an RNA sequence into DNA form. As utilized herein "Fkh^sf" refers to the gene product of the Fkh^sf gene (irrespective of whether the gene is obtained from humans, mammals, or any other warm-blooded animal). When capitalized "FKH^sf" the gene product (and gene) should be understood to be derived from humans.

[0081]As noted above, the present invention relates generally to pharmaceutical products and methods and, more specifically, to methods and compositions useful for diagnosing scurfy-related diseases, as well as methods for identifying compounds which can modulate the immune system.

[0082]Thus, as discussed in more detail below this discovery has led to the development of assays which may be utilized to select molecules which can act as agonists, or alternatively, antagonists of the immune system. Furthermore, such assays may be utilized to identify other genes and gene products which are likewise active in modulating the immune system.

Scurfy

[0083]Briefly, the present invention is based upon the unexpected discovery that a mutation in the gene which encodes Fkh^sf results in rare condition (scurfy) characterized by a progressive lymphocytic infiltration of the lymph nodes, spleen, liver and skin resulting in gross morphological symptoms which include splenomegaly, hepatomegaly, greatly enlarged lymph nodes, runting, exfoliative dermatitis, and thickened malformed ears (Godfrey et al., Amer. J. Pathol. 138:1379, 1991; Godfrey et al., Proc. Natl. Acad. Sci. USA 88:5528, 1991). This new member of the winged-helix family represents a novel component of the immune system.

[0084]Methods which were utilized to discover the gene responsible for scurfy are provided below in Example 1. Methods for cloning the gene responsible for murine scurfy, as well as the human ortholog, are provided below in Examples 2 and 3. Methods for confirmation of gene identity and correlation with gene function, as determined using transgenic mice, are also provided in the Examples.

[0085]Also provided by the present invention are methods for determining the presence of Fkh^sf genes and gene products. Within one embodiment, such methods comprise the general steps of (a) contacting a Fkh^sf specific nucleic acid probe under hybridizing conditions with either (i) test nucleic acid molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from RNA molecules, wherein the probe recognizes at least a portion of an Fkh^sf nucleotide sequence, and (b) detecting the formation of hybrids of said nucleic acid probe and (i) or (ii). A variety of methods may be utilized in order to amplify a selected sequence, including, for example, RNA amplification (see Lizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-02, 1989; Lomeli et al., Clinical Chem. 35(9):1826-31, 1989; U.S. Pat. No. 4,786,600), and nucleic acid amplification utilizing Polymerase Chain Reaction ("PCR") (see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse-transcriptase-PCR and CPT (see U.S. Pat. Nos. 4,876,187, and 5,011,769).

[0086]Alternatively, antibodies may be utilized to detect the presence of Fkh^sf gene products. More specifically, within one embodiment methods are provided for detecting the presence of an Fkh^sf peptide, or a mutant form thereof, in a biological sample, comprising the steps of (a) contacting a biological sample with an anti-Fkh^sf antibody or an antibody fragment, wherein said contacting is performed under conditions that allow the binding of said antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.

[0087]Such methods may be accomplished in a wide variety of assay formats including, for example, Countercurrent Immuno-Electrophoresis (CIEP), Radioimmunoassays, Radioimmunoprecipitations, Enzyme-Linked Immuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition or Competition assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, supra).

Nucleic Acid Molecules, Proteins, and Methods of Producing Proteins

[0088]Although various FKH^sf or Fkh^sf proteins and nucleic acid molecules (or portions thereof) have been provided herein, it should be understood that within the context of the present invention, reference to one or more of these proteins should be understood to include proteins of a substantially similar activity. As used herein, proteins are deemed to be "substantially similar" if: (a) they are encoded by a nucleotide sequence which is derived from the coding region of a gene which encodes the protein (including, for example, portions of the sequence or allelic variations of the sequence); (b) the nucleotide sequence is capable of hybridization to nucleotide sequences of the present invention under moderate, high or very high stringency (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, N.Y., 1989), or has at least 50%, 60%, 70%; 75%, 80%, 90%, 95%, or greater homology to the sequences disclosed herein, or, (c) the DNA sequences are degenerate as a result of the genetic code to the DNA sequences defined in (a) or (b). Further, the nucleic acid molecule disclosed herein includes both complementary and non-complementary sequences, provided the sequences otherwise meet the criteria set forth herein. Within the context of the present invention, high stringency means standard hybridization conditions (e.g., 5×SSPE, 0.5% SDS at 65° C., or the equivalent). For purpose of hybridization, nucleic acid molecules which encode the amino-terminal domain, zinc finger domain, middle domain, or forkhead domain (see Example 10) may be utilized.

[0089]The structure of the proteins encoded by the nucleic acid molecules described herein may be predicted from the primary translation products using the hydrophobicity plot function of, for example, P/C Gene or Intelligenetics Suite (Intelligenetics, Mountain View, Calif.), or according to the methods described by Kyte and Doolittle (J. Mol. Biol. 157:105-32, 1982).

[0090]Proteins of the present invention may be prepared in the form of acidic or basic salts, or in neutral form. In addition, individual amino acid residues may be modified by oxidation or reduction. Furthermore, various substitutions, deletions, or additions may be made to the amino acid or nucleic acid sequences, the net effect of which is to retain or further enhance or decrease the biological activity of the mutant or wild-type protein. Moreover, due to degeneracy in the genetic code, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence.

[0091]Other derivatives of the proteins disclosed herein include conjugates of the proteins along with other proteins or polypeptides. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins which may be added to facilitate purification or identification of proteins (see U.S. Pat. No. 4,851,341, see also, Hopp et al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteins (e.g., FKH or Fkh-luciferase or FKH or Fkh-GFP) may be constructed in order to assist in the identification, expression, and analysis of the protein.

[0092]Proteins of the present invention may be constructed using a wide variety of techniques described herein. Further, mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution, or deletion.

[0093]Alternatively, oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and Sambrook et al. (supra). Deletion or truncation derivatives of proteins (e.g., a soluble extracellular portion) may also be constructed by utilizing convenient restriction endonuclease sites adjacent to the desired deletion. Subsequent to restriction, overhangs may be filled in, and the DNA religated. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989).

[0094]Mutations which are made in the nucleic acid molecules of the present invention preferably preserve the reading frame of the coding sequences. Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, that would adversely affect translation of the mRNA. Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed mutants screened for indicative biological activity. Alternatively, mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution, or deletion. Mutations may be introduced for purpose of preserving or increasing activity of the protein, or, for decreasing or disabling the protein (e.g., mutant Fkh).

[0095]Nucleic acid molecules which encode proteins of the present invention may also be constructed utilizing techniques of PCR mutagenesis, chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83:3402-06, 1986), by forced nucleotide misincorporation (e.g., Liao and Wise Gene 88:107-11, 1990), or by use of randomly mutagenized oligonucleotides (Horwitz et al., Genome 3:112-17, 1989).

[0096]The present invention also provides for the manipulation and expression of the above described genes by culturing host cells containing a vector capable of expressing the above-described genes. Such vectors or vector constructs include either synthetic or cDNA-derived nucleic acid molecules encoding the desired protein, which are operably linked to suitable transcriptional or translational regulatory elements. Suitable regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, insect, or plant genes. Selection of appropriate regulatory elements is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of regulatory elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a transcriptional terminator, and a ribosomal binding sequence, including a translation initiation signal.

[0097]Nucleic acid molecules that encode any of the proteins described above may be readily expressed by a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, insect, or plant cells. Methods for transforming or transfecting such cells to express foreign DNA are well known in the art (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929-33, 1978; Murray et al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel et al., U.S. Pat. No. 4,766,075; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and Marton, Plant Physiol. 104:1067-71, 1994; and Paszkowski et al., Biotech. 24:387-92, 1992).

[0098]Bacterial host cells suitable for carrying out the present invention include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art. Representative examples of bacterial host cells include DH5α (Stratagene, LaJolla, Calif.).

[0099]Bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the β-lactamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studier et al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin et al., Gene 87:123-26, 1990), the trp promoter (Nichols and Yanofsky, Meth. in Enzymology 101:155, 1983) and the tac promoter (Russell et al., Gene 20:231, 1982). Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Many plasmids suitable for transforming host cells are well known in the art, including among others, pBR322 (see Bolivar et al., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth. in Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-68, 1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).

[0100]Yeast and fungi host cells suitable for carrying out the present invention include, among others, Saccharomyces pombe, Saccharomyces cerevisiae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349). Suitable expression vectors for yeast and fungi include, among others, YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-39, 1978), YEp13 (Broach et al., Gene 8:121-33, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-08, 1978) and derivatives thereof.

[0101]Preferred promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-34, 1982) or alcohol dehydrogenase genes (Young et al., Hollaender et al. (eds.), in Genetic Engineering of Microorganisms for Chemicals, Plenum, New York, 1982, p. 355; Ammerer, Meth. Enzymol. 101:192-201, 1983). Examples of useful promoters for fungi vectors include those derived from Aspergillus nidulans glycolytic genes, such as the adh3 promoter (McKnight et al., EMBO J. 4:2093-99, 1985). The expression units may also include a transcriptional terminator. An example of a suitable terminator is the adh3 terminator (McKnight et al., ibid., 1985).

[0102]As with bacterial vectors, the yeast vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected. Preferred selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include leu2 (Broach et al., ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3 (Struhl et al., ibid.). Another suitable selectable marker is the cat gene, which confers chloramphenicol resistance on yeast cells.

[0103]Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929-33, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-47, 1984), and Russell (Nature 301:167-69, 1983). The genotype of the host cell may contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.

[0104]Protocols for the transformation of yeast are also well known to those of ordinary skill in the art. For example, transformation may be readily accomplished either by preparation of spheroplasts of yeast with DNA (see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment with alkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163, 1983). Transformation of fungi may also be carried out using polyethylene glycol as described by Cullen et al. (Bio/Technology 5:369, 1987).

[0105]Viral vectors include those which comprise a promoter that directs the expression of an isolated nucleic acid molecule that encodes a desired protein as described above. A wide variety of promoters may be utilized within the context of the present invention, including for example, promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, adenoviral promoter (Ohno et al., Science 265:781-84, 1994), neomycin phosphotransferase promoter/enhancer, late parvovirus promoter (Koering et al., Hum. Gene Therap. 5:457-63, 1994), Herpes TK promoter, SV40 promoter, metallothionein Ila gene enhancer/promoter, cytomegalovirus immediate early promoter, and the cytomegalovirus immediate late promoter. Within particularly preferred embodiments of the invention, the promoter is a tissue-specific promoter (see e.g., WO 91/02805; EP 0,415,731; and WO 90/07936). Representative examples of suitable tissue specific promoters include neural specific enolase promoter, platelet derived growth factor beta promoter, human alpha1-chimaerin promoter, synapsin I promoter and synapsin II promoter. In addition to the above-noted promoters, other viral-specific promoters (e.g., retroviral promoters (including those noted above, as well as others such as HIV promoters), hepatitis, herpes (e.g., EBV), and bacterial, fungal or parasitic (e.g., malarial)-specific promoters may be utilized in order to target a specific cell or tissue which is infected with a virus, bacteria, fungus or parasite.

[0106]Mammalian cells suitable for carrying out the present invention include, among others: PC12 (ATCC No. CRL1721), N1E-115 neuroblastoma, SK-N-BE(2)C neuroblastoma, SHSY5 adrenergic neuroblastoma, NS20Y and NG108-15 murine cholinergic cell lines, or rat F2 dorsal root ganglion line, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281; BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314)), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and NS-1 cells. Other mammalian cell lines may be used within the present invention, including Rat Hep I (ATCC No. CRL 1600), Rat Hep II (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), Human lung (ATCC No. CCL 75.1), Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No. HB 8065), Mouse liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL 9.1), SP2/0-Ag14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777), Jurkat (ATCC No. Tib 152) and RINm 5AHT₂B (Orskov and Nielson, FEBS 229(1):175-178, 1988).

[0107]Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Viral promoters include the cytomegalovirus immediate early promoter (Boshart et al., Cell 41:521-30, 1985), cytomegalovirus immediate late promoter, SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-64, 1981), MMTV LTR, RSV LTR, metallothionein-1, adenovirus E1a. Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), a mouse V.sub.κ promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81:7041-45, 1983; Grant et al., Nucl. Acids Res. 15:5496, 1987) and a mouse V_H promoter (Loh et al., Cell 33:85-93, 1983). The choice of promoter will depend, at least in part, upon the level of expression desired or the recipient cell line to be transfected.

[0108]Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the peptide or protein of interest. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest. Suitable polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the Adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9:3719-30, 1981). The expression vectors may include a noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer. Expression vectors may also include sequences encoding the adenovirus VA RNAs. Suitable expression vectors can be obtained from commercial sources (e.g., Stratagene, La Jolla, Calif.).

[0109]Vector constructs comprising cloned DNA sequences can be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-45, 1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987). To identify cells that have stably integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. Other selectable markers include fluorescent proteins such as GFP (green fluorescent protein) or BFP (blue fluorescent protein). The selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.).

[0110]Mammalian cells containing a suitable vector are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable, selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels. Cells expressing the introduced sequences are selected and screened for production of the protein of interest in the desired form or at the desired level. Cells that satisfy these criteria can then be cloned and scaled up for production. Cells may also be selected for transfection based on their expression of GFP by sorting for GFP-positive cells using a flow cytometer.

[0111]Protocols for the transfection of mammalian cells are well known to those of ordinary skill in the art. Representative methods include calcium phosphate mediated transfection, electroporation, lipofection, retroviral, adenoviral and protoplast fusion-mediated transfection (see Sambrook et al., supra). Naked vector constructs can also be taken up by muscle cells or other suitable cells subsequent to injection into the muscle of a mammal (or other animals).

[0112]Numerous insect host cells known in the art can also be useful within the present invention, in light of the subject specification. For example, the use of baculoviruses as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28:215-24, 1990).

[0113]Numerous plant host cells known in the art can also be useful within the present invention, in light of the subject specification. For example, the use of Agrobacterium rhizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore) 11:47-58, 1987).

[0114]Within related aspects of the present invention, proteins of the present invention, may be expressed in a transgenic animal whose germ cells and somatic cells contain a gene which encodes the desired protein and which is operably linked to a promoter effective for the expression of the gene. Alternatively, in a similar manner transgenic animals may be prepared that lack the desired gene (e.g., "knockout" mice). Such transgenics may be prepared in a variety non-human animals, including mice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al., Nature 315:680-83, 1985, Palmiter et al., Science 222:809-14, 1983, Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-42, 1985, Palmiter and Brinster, Cell 41:343-45, 1985, and U.S. Pat. Nos. 5,175,383, 5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384). Briefly, an expression vector, including a nucleic acid molecule to be expressed together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs, for example, by microinjection. Integration of the injected DNA is detected by blot analysis of DNA from tissue samples. It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is passed on to the animal's progeny. Tissue-specific expression may be achieved through the use of a tissue-specific promoter, or through the use of an inducible promoter, such as the metallothionein gene promoter (Palmiter et al., 1983, ibid), which allows regulated expression of the transgene.

[0115]Animals which produce mutant forms of Fkh^sf other than the naturally occurring scurfy mutant ("sf"), or in genetic backgrounds different from the naturally occurring mutant, may be readily produced given the disclosure provided herein.

[0116]Proteins can be isolated by, among other methods, culturing suitable host and vector systems to produce the recombinant translation products of the present invention. Supernatants from such cell lines, or protein inclusions or whole cells where the protein is not excreted into the supernatant, can then be treated by a variety of purification procedures in order to isolate the desired proteins. For example, the supernatant may be first concentrated using commercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following concentration, the concentrate may be applied to a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support. Alternatively, anion or cation exchange resins may be employed in order to purify the protein. As a further alternative, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps may be employed to further purify the protein. Other methods of isolating the proteins of the present invention are well known in the skill of the art.

[0117]A protein is deemed to be "isolated" within the context of the present invention if no other (undesired) protein is detected pursuant to SDS-PAGE analysis followed by Coomassie blue staining. Within other embodiments, the desired protein can be isolated such that no other (undesired) protein is detected pursuant to SDS-PAGE analysis followed by silver staining.

Assays for Selecting Molecules which Modulate the Immune System

[0118]As noted above, the present invention provides methods for selecting and/or isolating molecules which are capable of modulating the immune system. Representative examples of suitable assays include the yeast and mammalian 2-hybrid systems (e.g., Dang et al., Mol. Cell. Biol. 11:954, 1991; Fearon et al., Proc. Natl. Acad. Sci. USA 89:7958, 1992), DNA binding assays, antisense assays, traditional protein binding assays (e.g., utilizing ¹²⁵I or time-resolved fluorescence), immunoprecipitation coupled with gel electrophoresis and direct protein sequencing, transcriptional analysis of Fkh^sf regulated genes, cytokine production and proliferation assays.

[0119]For example, within one embodiment proteins that directly interact with Fkh^sf can be detected by an assay such as a yeast 2-hybrid binding system (see, e.g., U.S. Pat. Nos. 5,283,173, 5,468,614, 5,610,015, and 5,667,973). Briefly, in a two-hybrid system, a fusion of a DNA-binding domain-Fkh^sf protein (e.g., GAL4-Fkh^sf fusion) is constructed and transfected into a cell containing a GAL4 binding site linked to a selectable marker gene. The whole Fkh^sf protein or subregions of Fkh^sf may be used. A library of cDNAs fused to the GAL4 activation domain is also constructed and co-transfected. When the cDNA in the cDNA-GAL4 activation domain fusion encodes a protein that interacts with Fkh^sf, the selectable marker is expressed. Cells containing the cDNA are then grown, the construct isolated and characterized. Other assays may also be used to identify interacting proteins. Such assays include ELISA, Western blotting, co-immunoprecipitations, in vitro transcription/translation analysis and the like.

[0120]Within another aspect of the present invention, methods are provided for determining whether a selected molecule is capable of modulating the immune system, comprising the steps of (a) exposing a selected candidate molecule to cells which express Fkh^sf, or, mutant Fkh^sf, and (b) determining whether the molecule modulates the activity of Fkh^sf, and thereby determining whether said molecule can modulate the immune system. Cells for such tests may derive from (a) normal lymphocytes, (b) cell lines engineered to overexpress the FKH^sf (or Fkh^sf) protein (or mutant forms thereof) or (c) transgenic animals engineered to express said protein. Cells from such transgenic mice are characterized, in part, by a hyporesponsive state including diminished cell number and a decreased responsiveness to various stimuli (e.g., Example 8).

[0121]It should be noted that while the methods recited herein may refer to the analysis of an individual test molecule, that the present invention should not be so limited. In particular, the selected molecule may be contained within a mixture of compounds. Hence, the recited methods may further comprise the step of isolating the desired molecule. Furthermore, it should be understood that candidate molecules can be assessed for their ability to modulate the immune system by a number of parameters, including for example, T-cell proliferation, cytokine production, and the like.

Candidate Molecules

[0122]A wide variety of molecules may be assayed for their ability to modulate the immune system. Representative examples which are discussed in more detail below include organic molecules, proteins or peptides, and nucleic acid molecules.

[0123]1. Organic Molecules

[0124]Numerous organic molecules may be assayed for their ability to modulate the immune system. For example, within one embodiment of the invention suitable organic molecules may be selected either from a chemical library, wherein chemicals are assayed individually, or from combinatorial chemical libraries where multiple compounds are assayed at once, then deconvoluted to determine and isolate the most active compounds.

[0125]Representative examples of such combinatorial chemical libraries include those described by Agrafiotis et al., "System and method of automatically generating chemical compounds with desired properties," U.S. Pat. No. 5,463,564; Armstrong, R. W., "Synthesis of combinatorial arrays of organic compounds through the use of multiple component combinatorial array syntheses," WO 95/02566; Baldwin, J. J. et al., "Sulfonamide derivatives and their use," WO 95/24186; Baldwin, J. J. et al., "Combinatorial dihydrobenzopyran library," WO 95/30642; Brenner, S., "New kit for preparing combinatorial libraries," WO 95/16918; Chenera, B. et al., "Preparation of library of resin-bound aromatic carbocyclic compounds," WO 95/16712; Ellman, J. A., "Solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support," U.S. Pat. No. 5,288,514; Felder, E. et al., "Novel combinatorial compound libraries," WO 95/16209; Lerner, R. et al., "Encoded combinatorial chemical libraries," WO 93/20242; Pavia, M. R. et al., "A method for preparing and selecting pharmaceutically useful non-peptide compounds from a structurally diverse universal library," WO 95/04277; Summerton, J. E. and D. D. Weller, "Morpholino-subunit combinatorial library and method," U.S. Pat. No. 5,506,337; Holmes, C., "Methods for the Solid Phase Synthesis of Thiazolidinones, Metathiazanones, and Derivatives thereof," WO 96/00148; Phillips, G. B. and G. P. Wei, "Solid-phase Synthesis of Benzimidazoles," Tet. Letters 37:4887-90, 1996; Ruhland, B. et al., "Solid-supported Combinatorial Synthesis of Structurally Diverse β-Lactams," J. Amer. Chem. Soc. 111:253-54, 1996; Look, G. C. et al., "The Indentification of Cyclooxygenase-1 Inhibitors from 4-Thiazolidinone Combinatorial Libraries," Bioorg and Med. Chem. Letters 6:707-12, 1996.

[0126]2. Proteins and Peptides

[0127]A wide range of proteins and peptides make likewise be utilized as candidate molecules for modulating the immune system.

a. Combinatorial Peptide Libraries

[0128]Peptide molecules which modulate the immune system may be obtained through the screening of combinatorial peptide libraries. Such libraries may either be prepared by one of skill in the art (see, e.g., U.S. Pat. Nos. 4,528,266 and 4,359,535, and Patent Cooperation Treaty Publication Nos. WO 92/15679, WO 92/15677, WO 90/07862, WO 90/02809), or purchased from commercially available sources (e.g., New England Biolabs® Phage Display Peptide Library Kit).

b. Antibodies

[0129]Antibodies which modulate the immune system may readily be prepared given the disclosure provided herein. Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., Fab, and F(ab')₂, F_v variable regions, or complementarity determining regions). As discussed above, antibodies are understood to be specific against Fkh^sf if they bind with a K_a of greater than or equal to 10⁷M, preferably greater than of equal to 10⁸M. The affinity of a monoclonal antibody or binding partner, as well as inhibition of binding can be readily determined by one of ordinary skill in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-72, 1949).

[0130]Briefly, polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Typically, Fkh^sf, or a unique peptide thereof of 13-20 amino acids (preferably conjugated to keyhole limpet hemocyanin by cross-linking with glutaraldehyde) is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, in conjunction with an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for reactivity to the protein or peptide. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity to the protein, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.

[0131]Monoclonal antibodies may also be readily generated using conventional techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).

[0132]Other techniques may also be utilized to construct monoclonal antibodies (see William D. Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-81, December 1989; see also L. Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl. Acad. Sci. USA 86:5728-32, August 1989; see also Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, January 1990).

[0133]A wide variety of assays may be utilized to determine the presence of antibodies which are reactive against the Fkh^sf (or the mutant forms of Fkh^sf described herein), including for example countercurrent immuno-electrophoresis, radioimmunoassays, radioimmunoprecipitations, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, western blots, immunoprecipitation, Inhibition or Competition Assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988).

[0134]Once suitable antibodies have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques.

[0135]Antibodies of the present invention may be utilized not only for modulating the immune system, but for diagnostic tests (e.g., to determine the presence of an FKH^sf or Fkh^sf protein or peptide), for therapeutic purpose, or for purification of proteins.

c. Mutant Fkh^sf

[0136]As described herein and below in the Examples, altered versions of Fkh^sf, may be utilized to inhibit the normal activity of Fkh^sf, thereby modulating the immune system (see generally, nucleic acid molecules and proteins above).

[0137]Further mutant or altered forms of FKH^sf or Fkh^sf may be utilized for a wide variety of in vitro assays (e.g., in order to examine the effect of such proteins in various models), or, for the development of antibodies.

[0138]3. Nucleic Acid Molecules

[0139]Within other aspects of the invention, nucleic acid molecules are provided which are capable of modulating the immune system. For example, within one embodiment antisense oligonucleotide molecules are provided which specifically inhibit expression of FKH^sf or Fkh^sf nucleic acid sequences, or, of mutant FKH^sf or Fkh^sf (see generally, Hirashima et al., in Molecular Biology of RNA: New Perspectives (M. Inouye and B. S. Dudock, eds., 1987 Academic Press, San Diego, p. 401); Oligonucleotides: Antisense Inhibitors of Gene Expression (J. S. Cohen, ed., 1989 MacMillan Press, London); Stein and Cheng, Science 261:1004-12, 1993; WO 95/10607; U.S. Pat. No. 5,359,051; WO 92/06693; and EP-A2-612844). Briefly, such molecules are constructed such that they are complementary to, and able to form Watson-Crick base pairs with, a region of transcribed Fkh^sf mRNA sequence. The resultant double-stranded nucleic acid interferes with subsequent processing of the mRNA, thereby preventing protein synthesis.

[0140]Within other aspects of the invention, ribozymes are provided which are capable of inhibiting FKH^sf or Fkh^sf, or mutant forms FKH^sf or Fkh^sf. As used herein, "ribozymes" are intended to include RNA molecules that contain anti-sense sequences for specific recognition, and an RNA-cleaving enzymatic activity. The catalytic strand cleaves a specific site in a target RNA at greater than stoichiometric concentration. A wide variety of ribozymes may be utilized within the context of the present invention, including for example, the hammerhead ribozyme (for example, as described by Forster and Symons, Cell 48:211-20, 1987; Haseloff and Gerlach, Nature 328:596-600, 1988; Walbot and Bruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature 334:585, 1988); the hairpin ribozyme (for example, as described by Haselhoff et al., U.S. Pat. No. 5,254,678, issued Oct. 19, 1993 and Hempel et al., European Patent Publication No. 0 360 257, published Mar. 26, 1990); and Tetrahymena ribosomal RNA-based ribozymes (see Cech et al., U.S. Pat. No. 4,987,071). Ribozymes of the present invention typically consist of RNA, but may also be composed of DNA, nucleic acid analogs (e.g., phosphorothioates), or chimerics thereof (e.g., DNA/RNA/RNA).

[0141]4. Labels

[0142]FKH^sf or Fkh^sf, (as well as mutant forms thereof), or, any of the candidate molecules described above and below, may be labeled with a variety of compounds, including for example, fluorescent molecules, toxins, and radionuclides. Representative examples of fluorescent molecules include fluorescein, Phycobili proteins, such as phycoerythrin, rhodamine, Texas red and luciferase. Representative examples of toxins include ricin, abrin diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Representative examples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. In addition, the antibodies described above may also be labeled or conjugated to one partner of a ligand binding pair. Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein.

[0143]Methods for conjugating or labeling the molecules described herein with the representative labels set forth above may be readily accomplished by one of ordinary skill in the art (see Trichothecene Antibody Conjugate, U.S. Pat. No. 4,744,981; Antibody Conjugate, U.S. Pat. No. 5,106,951; Fluorogenic Materials and Labeling Techniques, U.S. Pat. No. 4,018,884; Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Pat. No. 4,897,255; and Metal Radionuclide Chelating Compounds for Improved Chelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Jakoby and Wilchek (eds.), Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B, Academic Press, New York, 1974, p. 30; see also Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Anal. Biochem. 171:1-32, 1988).

Pharmaceutical Compositions

[0144]As noted above, the present invention also provides a variety of pharmaceutical compositions, comprising one of the above-described molecules which modulates the immune system, along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Preferably, the pharmaceutical composition (or, `medicament`) is provided in sterile, pyrogen-free form.

[0145]In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes. In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.

Methods of Treatment

[0146]The present invention also provides methods for modulating the immune system. Through use of the molecules described herein which modulate the immune system, a wide variety of conditions in warm blooded animals may be readily treated or prevented. Examples of warm-blooded animals that may be treated include both vertebrates and mammals, including for example humans, horses, cows, pigs, sheep, dogs, cats, rats and mice. Such methods may have therapeutic value in patients with altered immune systems. This would include such patients as those undergoing chemotherapy of those with various immunodeficiency syndromes, as well as patients with a T cell mediated autoimmune disease. Therapeutic value may also be recognized from utility as a vaccine adjuvant.

[0147]Therapeutic molecules, depending on the type of molecule, may be administered via a variety of routes in a variety of formulations. For example, within one embodiment organic molecules may be delivered by oral or nasal routes, or by injection (e.g., intramuscularly, intravenously, and the like).

[0148]Within one aspect, methods are provided for modulating the immune system, comprising the step of introducing into lymphoid cells a vector which directs the expression of a molecule which modulates the immune system, and administering the vector containing cells to a warm-blooded animal. Within other related embodiments, the vector may be directly administered to a desired target location (e.g., the bone marrow).

[0149]A wide variety of vectors may be utilized for such therapeutic purposes, including both viral and non-viral vectors. Representative examples of suitable viral vectors include herpes viral vectors (e.g., U.S. Pat. No. 5,288,641), adenoviral vectors (e.g., WO 94/26914, WO 93/9191 WO 99/20778; WO 99/20773; WO 99/20779; Kolls et al., PNAS 91(1):215-19, 1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-07, 1993; Zabner et al., Cell 75(2):207-16, 1993; Li et al., Hum Gene Ther. 4(4):403-09, 1993; Caillaud et al., Eur. J. Neurosci. 5(10):1287-91, 1993; Vincent et al., Nat. Genet. 5(2):130-34, 1993; Jaffe et al., Nat. Genet. 1(5):372-78, 1992; and Levrero et al., Gene 101(2):195-202, 1991), adeno-associated viral vectors (WO 95/13365; Flotte et al., PNAS 90(22):10613-617, 1993), baculovirus vectors, parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457-63, 1994), pox virus vectors (Panicali and Paoletti, PNAS 79:4927-31, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm. 193(2):653-60, 1993), and retroviruses (e.g., EP 0,415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218). Viral vectors may likewise be constructed which contain a mixture of different elements (e.g., promoters, envelope sequences and the like) from different viruses, or non-viral sources. Within various embodiments, either the viral vector itself, or a viral particle which contains the viral vector may be utilized in the methods and compositions described below.

[0150]Within other embodiments of the invention, nucleic acid molecules which encode a molecule which modulates the immune system (e.g., a mutant Fkh^sf, or, an antisense or ribozyme molecule which cleaves Fkh^sf) may be administered by a variety of alternative techniques, including for example administration of asialoosomucoid (ASOR) conjugated with poly-L-lysine DNA complexes (Cristano et al., PNAS 92122-126, 1993), DNA linked to killed adenovirus (Curiel et al., Hum. Gene Ther. 3(2):147-54, 1992), cytofectin-mediated introduction (DMRIE-DOPE, Vical, California), direct DNA injection (Acsadi et al., Nature 352:815-18, 1991); DNA ligand (Wu et al., J. of Biol. Chem. 264:16985-987, 1989); lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-17, 1989); liposomes (Pickering et al., Circ. 89(1):13-21, 1994; and Wang et al., PNAS 84:7851-55, 1987); microprojectile bombardment (Williams et al., PNAS 88:2726-30, 1991); and direct delivery of nucleic acids which encode the protein itself either alone (Vile and Hart, Cancer Res. 53: 3860-64, 1993), or utilizing PEG-nucleic acid complexes.

[0151]Representative examples of molecules which may be expressed by the vectors of present invention include ribozymes and antisense molecules, each of which are discussed in more detail above.

[0152]As will be evident to one of skill in the art, the amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth. Typically, the compositions may be administered by a variety of techniques, as noted above.

[0153]The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

Example 1

Identification of the Gene Responsible for the Scurfy Mutant

A. Cloning of a Scurfy Gene

[0154]The original scurfy mutation arose spontaneously in the partially inbred MR stock at Oak Ridge National Laboratory (ORNL) in 1949. Backcross analysis was used to fine map the peri-centromeric region of the X chromosome containing the mouse Scurfy mutation. A physical map covering the same region was generated concurrently through the isolation of overlapping yeast and bacterial artificial chromosomes (YACs and BACs). Once the candidate region was narrowed down to ˜500 kilobase pairs (kb), large-scale DNA sequencing was performed on 4 overlapping BAC clones. All the transcription units in this 500 kb region were identified through a combination of sequence database searching and the application of computer exon prediction programs. Candidate genes were then screened for Scurfy-specific mutations by comparing the sequences of cDNAs obtained by the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) procedure from normal and Scurfy-derived RNA samples. In one gene, referred to here as Fkh^sf, a two base pair (bp) insertion was found in the coding region of the Scurfy cDNA, relative to the normal cDNA. The insertion was confirmed by comparing the DNA sequences of PCR products derived from the genomic DNA of several mouse strains, including the Scurfy mutant. Again, the two bp insertion was found only in the Scurfy sample, establishing this as the probable cause of the Scurfy defect.

[0155]The mouse Fkh^sf gene is contained within the BAC clone 8C22, and has been completely sequenced. It spans ˜14 kb and contains 11 coding exons. The locations of exon breaks were initially identified by computer analysis of the genomic DNA sequence, using the GenScan exon prediction program; exon locations were then confirmed by direct comparison of cDNA sequences derived from normal mouse tissues to the genomic sequence.

[0156]The length of cDNA obtained is 2160 bp; the coding region spans 1287 bp of that, encoding a protein of 429 amino acids. FIG. 1 shows the nucleotide sequence of the mouse Fkh^sf cDNA; translation is predicted to initiate at position 259 and terminate at position 1546. FIG. 2 shows the amino acid sequence of mouse Fkh^sf.

B. Generation of Fkh^sf Transgenic Mice.

[0157]The identity of the Fkh^sf gene as the true cause of the Scurfy phenotype was confirmed in transgenic mice. Briefly, a 30 kb fragment of the normal genomic DNA, including the ˜7 kb coding region of the Fkh^sf gene, as well as ˜20 kb of upstream flanking sequences and ˜4 kb of downstream sequences (FIG. 5) was microinjected into normal mouse one-cell embryos. Five individual founder animals were generated, each with distinct integrations, and a male animal from each transgenic line was crossed to a female sf carriers. Male offspring carrying both the transgene (normal Fkh^sf) and sf mutation (mutant Fkh^sf) were analyzed.

[0158]Analysis consisted of examination of animals for runting, scaly skin, fur abnormalities and other hallmarks of the scurfy phenotype. In addition, lymphoid tissues (thymus, spleen and nodes) were harvested and their size and cell number examined and compared to both normal animals as well as scurfy mice. For all five transgenic lines, male sf progeny that carried the transgene were normal in size and weight and appeared healthy in all respects. Lymph node size in these transgenic mice was similar to (or smaller than) that of normal animals (FIG. 6) and there was no sign of activated T cells. These parameters are extremely different from sf mice and indicate that addition of the normal Fkh^sf gene can overcome the defect found in scurfy mice, thus confirming that the mutation in the Fkh^sf gene is the cause of Scurfy disease.

Example 2

Generation of FKH^sf cDNA

[0159]A complementary DNA (cDNA) encoding the complete mouse Fkh^sf protein may be obtained by a reverse-transcriptase polymerase chain reaction (RT-PCR) procedure. More specifically, first-strand cDNA is generated by oligo dT priming 5 ug of total RNA from a suitable source (eg., mouse spleen) and extending with reverse transcriptase under standard conditions (eg., Gibco/BRL SuperScript kit). An aliquot of the first-strand cDNA is then subjected to 35 cycles of PCR (94° C. for 30 sec, 63° C. for 30 sec, 72° C. for 2 min) in the presence of the forward and reverse primers (Forward primer: GCAGATCTCC TGACTCTGCC TTC; Reverse primer: GCAGATCTGA CAAGCTGTGT CTG) (0.2 mM final concentration), 60 mM Tris-HCl, 15 mM ammonium sulfate, 1.5 mM magnesium chloride, 0.2 mM each dNTP and 1 unit of Taq polymerase.

Example 3

Generation of the Human Ortholog to Murine Fkh^sf

[0160]A human FKH^sf cDNA encoding the complete FKH^sf protein may be obtained by essentially the same procedure as described in Example 2. In particular, starting with total spleen RNA, and utilizing the following oligonucleotide primers (Forward primer: AGCCTGCCCT TGGACAAGGA C; Reverse primer: GCAAGACAGT GGAAACCTCA C), and the same PCR conditions outlined above, except with a 60° C. annealing temperature.

[0161]FIG. 3 shows the nucleotide sequence of the 1869 bp cDNA obtained to date (including an 1293 bp coding region); translation is predicted to initiate at position 189 and terminate at position 1482. FIG. 4 shows the sequence of the 431 amino acid human FKH^sf protein. Comparison of the predicted coding region of the human gene to the mouse cDNA sequence reveals nearly identical exon structure and 86.1% amino acid sequence identity across the entire protein.

Example 4

Methods for Detecting Scurfy Mutations

[0162]As noted above, the Scurfy mutation was originally discovered by directly sequencing cDNAs derived by RT-PCR of sf and normal mouse RNA samples, and confirmed by sequencing the same region from genomic DNA. The nature of the mutation (i.e., a 2 bp insertion) lends itself to a number of different mutation detection assays. The first is based on differential hybridization of oligonucleotide probes. Such a hybridization-based assay could allow quantitative analysis of allele-specific expression.

[0163]As an example, a 360 bp DNA fragment is amplified from 1^st strand cDNA using the following oligos:

TABLE-US-00001 DMO5985 (forward): CTACCCACTGCTGGCAAATG (ntd. 825-844 of FIG. 1) DMO6724 (reverse): GAAGGAACTATTGCCATGGCTTC (ntd. 1221-1199)

[0164]The PCR products are run on an 1.8% agarose gel, transferred to nylon membrane and probed with end-labeled oligonucleotides that are complementary to the region corresponding to the site of the Scurfy-specific 2 bp insertion. Two separate hybridization reactions are performed to detect the normal and Scurfy PCR products, using the oligonucleotides below (the site of the 2 bp insertion is shown in bold):

TABLE-US-00002 Normal: ATGCAGCAAGAGCTCTTGTCCATTGAGG DMO7439 Scurfy: GCAGCAAGAGCTCTTTTGTCCATTGAGG DMO6919

[0165]The Scurfy mutation can also be detected by a cold Single-Strand Conformation Polymorphism (cSSCP) assay. In this assay, the same PCR products described above are run on 20% acrylamide (TBE) gels after strand denaturation. The Scurfy insertion causes a shift in strand mobility, relative to the normal sequence, and the separate strands are detected after staining with ethidium bromide.

Example 5

Fkh^sf Gene Expression

[0166]Semi-quantitative RT-PCR has been used to analyze the pattern of mouse and human Fkh^sf gene expression in a wide variety of tissues and cell lines. Levels of expression are normalized to the ubiquitously expressed DAD-1 gene. In short, the Fkh^sf gene is expressed, albeit at very low levels, in nearly every tissue examined thus far, including thymus, spleen, sorted CD4+ and CD4-CD8- T-lymphocytes, as well as kidney, brain, and various mouse and human T-cell lines and human tumors. Absence of expression, however, was noted in freshly sorted mouse B-cells.

[0167]As expected, no differences in level of expression were observed in normal vs. Scurfy tissues in the RT-PCR assays.

Example 6

In Vitro Expression of Fkh^sf

[0168]Full-length mouse and human Fkh^sf cDNAs, as well as various sub-regions of the cDNAs are cloned into vectors which allow expression in mammalian cells (such as the human Jurkat T-cell line), E. coli or yeast. The E. coli or yeast systems can be used for production of protein for the purpose of raising Fkh^sf-specific antibodies (see below).

Example 7

Generation of Anti-Fkh^sf Antibodies

[0169]Protein expressed from vectors described in Example 6 are used to immunize appropriate animals for the production of FKH^sf specific antibodies. Either full length or truncated proteins can be used for this purpose. Protein can be obtained, for example, from bacteria such as E. coli, insect cells or mammalian cells. Animal species can include mouse, rabbit, guinea pig, chicken or other. Rabbit antisera specific for FKH^sf has been generated, as determined by biochemical characterization (immunoprecipitation and western blotting).

Example 8

Assay for Function of an FKH^sf Gene

[0170]Since loss of function of the FKH^sf protein results in the phenotype observed in scurfy animals (wasting, hyperactive immune responsiveness and death), assays are described for assessing excessive expression of the FKH^sf protein. Transgenic animals (described in Example 1) are examined for their state of immune competence, using several different parameters. Animals are examined for the number of lymphoid cells present in lymph nodes and thymus (FIG. 7) as well as the responsiveness of T cells to in vitro stimulation (FIG. 8).

[0171]Scurfy mutant animals have roughly twice as many cells in their lymph nodes as normal animals, whereas mice which express excess levels of the normal FKH^sf protein contain roughly one-third as many cells (FIG. 7). Further, the number of thymocytes is normal (FIG. 7) as is their cell surface phenotype as assessed by flow cytometry using standard antisera (not shown), indicating that there is no developmental defect associated with excess FKH^sf protein.

[0172]Normal, scurfy and transgenic animals are further examined for their proliferative responses to T cell stimulation. CD4+ T cells are reacted with antibodies to CD3 and CD28 and their proliferative response measured using radioactive thymidine incorporation. Whereas only scurfy cells divide in the absence of stimulation, normal cells respond well following stimulation. FKH^sf transgenic cells also respond to stimulation, however the response is significantly less than that of normal cells (FIG. 8). This indicates that CD4+ T cells that express excess FKH^sf have a diminished capacity to respond to stimuli.

Example 9

Human FKH^sf cDNA Sequence is Related to JM2

[0173]A modified version of the human FKH^sf cDNA sequence exists in the GenBank public sequence database. This sequence is called JM2 (GenBank acc. # AJ005891), and is the result of the application of exon prediction programs to the genomic sequence containing the FKH^sf gene (Strom, T. M. et al., unpublished--see GenBank acc. # AJ005891). In contrast, the structure of the FKH^sf cDNA was determined experimentally. The GAP program of the Genetics Computer Group (GCG; Madison, USA) Wisconsin sequence analysis package was used to compare the two sequences, and the differences are illustrated in FIG. 9. The 5' ends of the two sequences differ in their location within the context of the genomic DNA sequence, the second coding exon of FKH^sf is omitted from JM2, and the last intron of the FKH^sf gene is unspliced in the JM2 sequence. These differences result in a JM2 protein with a shorter amino-terminal domain, relative to FKH^sf, and a large insertion within the forkhead domain (see below) at the carboxy-terminus.

Example 10

The FKH^sf Protein is Conserved Across Species

[0174]The FKH^sf protein can be divided into sub-regions, based on sequence motifs that may indicate functional domains. The two principal motifs in FKH^sf are the single zinc finger (ZNF) of the C₂H₂ class in the middle portion of the protein, and the forkhead, or winged-helix domain at the extreme carboxy-terminus of the protein. For the purposes of characterizing the degree of homology between FKH^sf and other proteins, we have split the protein up into four regions: [0175]Amino-terminal domain: residues 1-197 of FIG. 2 residues 1-198 of FIG. 4 [0176]Zinc finger domain: residues 198-221 of FIG. 2 residues 199-222 of FIG. 4 [0177]Middle domain: residues 222-336 of FIG. 2 residues 223-336 of FIG. 4 [0178]Forkhead domain: residues 337-429 of FIG. 2 residues 337-431 of FIG. 4

[0179]Using the Multiple Sequence Alignment program from the DNAStar sequence analysis package, the Lipman-Pearson algorithm was employed to determine the degree of similarity between the human FKH^sf and mouse Fkh^sf proteins across these four domains. The results are shown in FIG. 10. The similarity indices ranged from 82.8% to 96.4%, indicating that this protein is very highly conserved across species.

Example 11

Identification of Novel Fkh^sf-Related Genes

[0180]The unique features of the FKH^sf gene sequence may be used to identify other novel genes (and proteins) which fall into the same sub-class of forkhead-containing molecules. The FKH^sf protein is unique in its having a single zinc finger domain amino-terminal to the forkhead domain as well as in the extreme carboxy-terminal position of the forkhead domain. A degenerate PCR approach may be taken to isolate novel genes containing a zinc finger sequence upstream of a forkhead domain. By way of example, the following degenerate primers were synthesized (positions of degeneracy are indicated by parentheses, and "I" indicates the nucleoside inosine):

TABLE-US-00003 Forward primer: CA(TC)GGIGA(GA)TG(CT)AA(GA)TGG Reverse primer: (GA)AACCA(GA)TT(AG)TA(AGT)AT(CT)TC(GA)TT

[0181]The forward primer corresponds to a region within the zinc finger sequence and the reverse primer corresponds to a region in the middle of the forkhead domain. These primers were used to amplify first-strand cDNA produced as in Example 2 from a variety of human tissues (including liver, spleen, brain, lung, kidney, etc.). The following PCR conditions were used: forward and reverse primers at 0.2 mM final concentration, 60 mM Tris-HCl, 15 mM ammonium sulfate, 1.5 mM magnesium chloride, 0.2 mM each dNTP and 1 unit of Taq polymerase, subjected to 35 cycles (94° C. for 30 sec, 50° C. for 30 sec, 72° C. for 2 min). PCR products were visualized on a 1.8% agarose gel (run in 1×TAE) and sub-cloned into the TA cloning vector (Invitrogen, Carlsbad, Calif.); individual clones were sequenced and used for further characterization of full-length cDNAs.

[0182]Alternatively, the unique regions of the FKH^sf gene (i.e., the "Amino-terminal" and "Middle" domains) may be used to screen cDNA libraries by hybridization. cDNA libraries, derived from a variety of human and/or mouse tissues, and propagated in lambda phage vectors (eg., lambda gt11) were plated on agarose, plaques were transferred to nylon membranes and probed with fragments derived from the unique regions of the FKH^sf gene. Under high stringency conditions (eg., hybridization in 5×SSPE, 5×Denhardt's solution, 0.5% SDS at 65° C., washed in 0.1×SSPE, 0.1% SDS at 65 C) only very closely related sequences are expected to hybridize (i.e., 90-100% homologous). Under lower stringency, such as hybridization and washing at 45°-55° C. in the same buffer as above, genes that are related to FKH^sf (65-90% homologous) may be identified. Based on results obtained from searching public databases with the unique sequences of FKH^sf any genes identified through low- to mid-stringency hybridization experiments are expected to represent novel members of a "FKH^sf family".

Example 12

Overexpression of the Wild-Type Foxp3 Gene Results in Decreased Numbers of Peripheral T Cells

[0183]The original breeding stocks for scurfy mice were obtained from Oak Ridge National Laboratory (ORNL), with mice subsequently derived by caesarian section into SPF conditions. Transgenic mice were generated by oocyte microinjection by DNX Transgenic Services (Cranbury, N.J.), as described (Brunkow et al., Nat. Gen. 27:68-72, 2001). For the 2826 mouse line, a 30.8 kb cosmid construct was generated from mouse BAC K60 for injection. This cosmid contains the entire Foxp3 gene along with approximately 18 kbp of 5' sequence and 4 kbp of 3' sequence. Expression of the gene parallels that of the endogenous gene with respect to tissue distribution (Brunkow et al., Nat. Gen. 27:68-72, 2001). The Ick-Foxp3 transgenic animals were generated using the Ick pacmotor to drive expression (Garvin et al., Int. Immunol 2(2):173, 1990). Both transgenic and scurfy mice were backcrossed onto the C57B1/6 background (JAX) for 4-6 generations for all studies. No differences in responsiveness or phenotype were noted during backcrossing. Northern blot analysis was performed as described previously (Brunkow et al., Nat. Gen. 27:68-72, 2001).

[0184]Initial experiments involving the Foxp3 transgenic mice demonstrated that in 5/5 lines generated from distinct founder animals, the expression of the wild-type Foxp3 transgene prevented disease in sf/Y mutant mice (Brunkow et al., Nat. Gen. 27:68-72, 2001). Further analysis demonstrates that the copy number of the transgene is directly correlated to the expression of the gene at the mRNA level (Brunkow et al., Nat. Gen. 27:68-72, 2001). This is likely due to the fact that the transgene construct consisted of a large genomic fragment including a substantial portion of 5' sequence and much of the regulatory region. In analyzing the various transgenic lines, it also becomes clear that there was a direct relationship between the expression of the Foxp3 gene and the number of lymph node cells (Brunkow et al., Nat. Gen. 27:68-72, 2001). The relationship between transgene copy number and cell number is shown for three of the founder lines, with the scurfy mutant animal (sf/Y) and normal littermate controls (NLC) for comparison (see, Table 1 below). Lymphoid cell number from transgenic (lines 2826, 1292 and 2828), normal littermate control and scurfy mutant (sf/Y) mice were determined for various tissues from representative age-matched (4 week old) mice. The approximate transgene copy number was determined by Southern blot analysis and correlated well with Foxp3 gene expression (Brunkow et al., Nat. Gen. 27:68-72, 2001). Although there is a less dramatic, but consistent, difference in the number of splenic cells in the transgenic mice as well, the number of thymocytes is not significantly affected. For reasons of simplicity, except where noted, the remainder of the experiments utilized the 2826 transgenic line. Animals from this line are generally healthy and survive for greater than one year under SPF conditions. The line has approximately 16 copies of the transgene and by northern blot analysis is expressed at ten to twenty times the level of the endogenous gene in lymphoid tissues (Brunkow et al., Nat. Gen. 27:68-72, 2001). The transgene, like the endogenous gene, is only poorly expressed in non-lymphoid tissues, a likely consequence of its expression under the control of its endogenous promoter. Lymph node cell number in mice from this line range from 15-50 percent of normal, with the number of cells accumulating with age. Splenic cell number is less dramatically affected although generally decreased, with a range of 25-90 percent of normal.

TABLE-US-00004 TABLE 1 Transgene Copy Cell Number (×10⁶) Genotype Number Thymus Lymph Node Spleen NLC NA 121.4 1.5 84.4 2826 ~16 111.8 0.5 60.8 1292 ~9 98.6 1.0 76.4 2828 ~45 108.5 0.4 61.1 Scurfy NA 64.4 4.7 109.5

Example 13

Thymic Phenotype of Scurfin-Transgenic Mice

[0185]The role of the Foxp3 gene in thymic selection remains unclear. Deletion of superantigen-specific Vβ-bearing thymocytes appears normal in both sf/Y as well as 2826 transgenic mice. Consistent with this, overexpression of the Foxp3 gene using its own endogenous promoter (2826 line) also does not appear to result in any gross changes in thymic development or selection. The number of thymocytes (Table I) and their distribution amongst the major phenotypic subsets is indistinguishable from littermate control animals. Thymus, lymph node and splenic tissues were collected as described (Clark et al., Immunol 162:2546, 1999) and were resuspended in staining buffer (1% BSA, 0.1% sodium azide in PBS) at a cell density of 20×10⁶/mL. Cell aliquots were treated with 2% normal mouse serum (Sigma) to block non-specific binding then stained by incubation on ice for 30 minutes with combinations of the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs): CD3, CD8β, CD4, CD25, IgG2a control (Caltag Laboratories, Burlingame, Calif.); CD28, CD45RB, CD44, CD62L, CD69, CD95 (PharMingen, San Diego, Calif.). The fluorescence intensity of approximately 10⁵ cells was examined using a MoFlo® flow cytometer (Cytomation, Fort Collins, Colo.) with dead cell exclusion by addition of propidium iodide (10 μg/mL).

[0186]A more detailed examination of the CD4^-8- subset also reveals a normal distribution of gamma-delta cells and CD25⁺ cells. Importantly, the fraction of CD4^+8- thymocytes expressing the maturation markers CD69 and HSA is identical in 2826 and control animals, suggesting that the maturation process is normal.

[0187]Overexpression of the Foxp3 gene in the thymus alone has a significantly different phenotype from the 2826 mice noted above. Transgenic mice expressing Foxp3 selectively in the thymus (16.5 and 8.3) under control of the Ick proximal promoter were crossed to sf/+ carrier females. Male scurfy mice (sf/Y) that carried the thymus-specific transgene (16.5 and 8.3) succumbed to disease at the same time and in the same manner as non-transgenic littermates. Sf/Y transgenic animals expressing Foxp3 under its endogenous regulatory sequences (2826) did succumb to disease. Cell number is derived from mice that carried the transgene in addition to the wild-type Foxp3 gene.

[0188]Transgenic animals that express the Foxp3 gene exclusively in thymus (under the control of the Ick proximal promoter) are unable to rescue sf/Y mice from disease (see, Table 2 below). Two separate founder animals were crossed to scurfy carrier females in an attempt to prevent disease. In each case sf/Y mice carrying the Ick proximal promoter--Foxp3 transgene developed an acute lymphoproliferative disease that was identical both in severity and time course to that seen in non-transgenic sf/Y siblings. In each case expression of the transgene was restricted to the thymus with no detectable expression in peripheral organs, including spleen. The Northern Blot analysis was carried out as presented in Example 1. Further, thymic expression of the Ick-driven transgene was substantially greater than that of the gene in 2826 transgenic animals or of the endogenous gene in normal littermate control mice. Hence it appears that the fatal lymphoproliferative disease seen in sf/Y mice does not arise as a consequence of scurfin mediated developmental defects in the thymus.

TABLE-US-00005 TABLE 2 Disease in Cell Number (×10⁶) Genotype Sf/Y mice? Thymus Lymph Node NLC NA 79.0 2.9 2826 No 100.1 2.2 16.5 Yes 110.4 2.7 8.3 Yes 32.2 2.9

[0189]Although transgenic (non-sf) animals carrying the Ick-driven transgene appear generally normal, high level expression of the transgene within the thymus does have phenotypic consequence in normal (non-sf) animals. Significantly increased expression of the transgene in otherwise normal mice leads to a relative decrease in the percentage of double-positive thymocytes and a corresponding increase in the percentage of double-negative (DN) cells, as well as a decrease in overall thymic cell number (see, Table 2). T cell development still occurs in these animals as assessed by the generation of CD4 and CD8 single positive cells and by the presence of relatively normal numbers of peripheral T cells in both lymph node and spleen (see, Table 2). CD69 expression on CD4^+8- cells from the thymus is similar in transgenic and wild-type littermates, suggesting positive selection likely proceeds normally, whereas within the DN compartment, the fraction of cells expressing CD25 is diminished relative to wild-type animals. These transgenic animals indicate that overexpression of the Foxp3 gene within the thymic compartment specifically can alter thymic development, but this appears to have no effect on regulating peripheral T cell activity.

Example 14

Altered Phenotype of Peripheral T Cells from Scurfin-Transgenic Mice

[0190]In addition to a decrease in the number of peripheral T cells in 2826 mice, there is a slight reduction in the percentage of CD4^+8- cells in both the lymph node and spleen relative to NLC. Whereas the CD3 levels appear normal on peripheral T cells, there are a number of other surface markers with altered expression levels. For CD4^+8- cells in the transgenic mice, the most consistent changes are a small decrease in the expression of CD62L and CD45RB as well as an increase in the expression of CD95. By comparison, cells from sf mutant animals have a very different phenotype. CD4^+8- cells from these mice are large and clearly activated. They are predominantly CD44^H1, CD45RB^LO, CD62L^LO and partially CD69⁺ (Clark et al., Immunol 162:2546).

[0191]CD4^-8+ cell numbers were also reduced in both the spleen and lymph nodes of scurfin-transgenic mice. This decrease is typically more dramatic! (50-75%) than the decrease in the CD4^{+8compartment} (25-50%). CD4^-8+ T cells display relatively minor and variable changes in the level of CD62L, CD45RB and CD95 on the cell surface in comparison to NLC. In contrast to CD4^+8- T cells, there is a more pronounced increase in the percentage of CD4^-8+ T cells that were also CD44^H1. Overall, the CD4^-S⁺ cells do not express surface markers at levels that characterize them as specifically naive, activated or memory.

Example 15

Histological Analyses of Scurfin-Transgenic Mice

[0192]Whereas peripheral T cells in 2826 mice are clearly decreased in number, a determination was made whether the architecture of the lymphoid organs was also perturbed. Histological examination of the major lymphoid organs (thymus, lymph node and spleen) indicated that the most significant changes were found in the mesenteric and peripheral lymph nodes. Tissues for histological analysis were removed from mice approximately 8 weeks after birth and immediately fixed in buffered 10% formalin. Paraffin-embedded sections were processed for hematoxylin and eosin staining and comparative histopathology performed on representative mice. As expected, the thymus appears relatively normal, with a well-defined cortico-medullary junction, although there appears to be a slight reduction in the size of the thymic medulla. Transgenic animals had smaller peripheral lymph nodes, lack robust and normally distributed lympoid follicles, lack distinct margins between follicular and interfollicular areas and had more obvious sinuses than those found in the lymph nodes of the normal littermate control mice. Even though the spleen and Peyer's patches appear approximately normal in size and microarchitecture, there is a moderate decrease in total cell number and no or minimal evidence of germinal centers in these tissues. The changes noted here reflect a hypocellular state distinct from a number of other targeted mutations in which the lymph nodes fail to develop. Therefore, while T cells are capable of development in an apparently normal manner, their representation within the peripheral lymphoid tissues, particularly the lymph nodes, is substantially decreased.

Example 16

Decreased Functional Responses of CD4^+8- Cells from Scurfin-Transgenic Mice

[0193]The phenotypic and cell number data suggest that there are specific defects in the biology of CD4 T cells from 2826 transgenic animals. The functional responses of T cells from these animals to several stimuli were evaluated, including anti-CD3 and anti-CD28. Lymphocytes were isolated from various tissues from NLC, 2826 transgenic or scurfy (mutant) mice and CD4 cells were purified by cell sorting. Thymus, lymph node and splenic tissues were removed from appropriate animals, macerated between sterile microscope slides, filtered through a sterile 70 μm nylon mesh and collected by centrifugation. CD4⁺ T lymphocytes were sort purified from these tissues by positive selection using the MoFlo. Sort purities as determined by post-sort analysis were typically greater than 95%. Cells were cultured at 37° C. in complete RPMI (cRPMI) (10% fetal bovine serum, 0.05 mM 2-mercaptoethanol, 15 mM HEPES, 100 U/mL penicillin, 100 μg/mL each streptomycin and glutamine) in 96-well round-bottomed tissue culture plates. Culture wells were prepared for T cell activation by pre-incubation with the indicated concentrations of purified antibody to CD3 (clone 2C11) in sterile PBS for 4 hours at 37° C. Purified α-mouse CD28 (clone 37.51) or α-mouse KLH (control antibody) was co-immobilized at 1 μg/ml final concentration.

[0194]T cells were cultured at a cell density of 1 to 5×10⁴ cells/well in 200 μL of cRPMI for 72 hours. Supernatant (100 μl) was removed at 48 hours for analysis of cytokine production. Wells were pulsed with 1 μCi/well of ³[H]-thymidine (Amersham Life Science, Arlington Heights, Ill.) for the last 8-12 hours of culture and then harvested (Tomtec). Proliferation data reported are based upon mean value of triplicate wells and represent a minimum of 3 experiments. Cytokine levels were determined by ELISA assay according to the manufacturer's direction (Biosource International, Camarillo, Calif.).

[0195]To test for proliferation and IL-2 production, a single cell suspension of Balb/c spleen cells was generated to used as stimulator cells. These cells were irradiated (3300 rads) and incubated a 10:1 ratio (stimulator:effector) with scurfin-transgenic or NLC spleen cells. To some cultures, IL-2 was added at 100 U/ml. For proliferation assays, cells were pulsed after five days and harvested as above. Both proliferation and IL-2 production are significantly diminished in cells from the transgenic animals compared to their littermates. Although transgenic animals increase their responsiveness with increasing stimulation, they rarely reached the levels achieved by NLC. This is particularly true for IL-2 production, in which cells from 2826 mice consistently produce low to undetectable amounts of this cytokine. Similar results were seen whether the cells were derived from the spleen or the lymph nodes.

[0196]As expected, cells from scurfy animals were hyper-responsive to stimulation and produce increased amounts of IL-2. The effect of the transgene was independent of strain and have remained constant during the back-crossing of the animals onto C57BI/6 through at least generation N6. T cells from transgenic mice remained responsive to anti-CD28 in this assay whereas stimulation with anti-CD3 and control Ig results in generally poor responses that were lower than, but similar to NLC responses. Addition of high doses of IL-2 is able to partially overcome the proliferative defect in CD4^+8- T cells from 2826 mice, but generally fails to restore the response to that of wild-type animals.

[0197]In contrast to peripheral T cells, but consistent with the phenotypic data above, the proliferative response of thymic CD4^+8- cells is approximately comparable between transgenic and NLC mice. IL-2 production by thymic CD4^+8- cells however, is reduced substantially from the transgenic animals. The reduction in IL-2 production by thymocytes is somewhat more variable than that seen in lymph node or spleen and may suggest that the IL-2 produced is also consumed during the culture. Alternatively, thymocytes may produce other growth factors less affected by the expression of the Foxp3 gene. Nevertheless, the data generally support the conclusion that a major defect in the transgenic animals is in the ability of both thymic and peripheral T cells to produce IL-2.

Example 17

Altered Functional Responses of Scurfin-Transgenic CD4^-8+ T Cells

[0198]The ability of transgenic T cells to generate and function as cytotoxic T cells (CTL) was determined in an in vitro assay. A single cell suspension of Balb/c spleen cells was generated to use as stimulator cells. These cells were irradiated (3300 rads) and incubated a 10:1 ratio (stimulator:effector) with scurfin-transgenic or NLC spleen cells. To some cultures, IL-2 was added at 100 U/ml. For generation of CTL, splenic T cells were stimulated in a similar manner in the presence of 100 U/ml of IL-2. After five days, cells were either assayed in the JAM assay (Matzinger, P. J Immunol 145(1-2):185 (1991)) or re-stimulated on a new stimulator layer. Cells were approximately 95% CD4^-8+.

[0199]Transgenic T cells were stimulated in a mixed-lymphocyte culture containing increasing numbers of irradiated allogeneic stimulator cells in the presence or absence of IL-2. The proliferative response of either transgenic or NLC effector cells was then measured. T cells from the transgenic animals responded poorly in the absence of exogenous IL-2, consistent with the data for purified CD4^+8- cells (above). In the presence of exogenous IL-2, transgenic T cells displayed an increased proliferative response, but still required a higher number of stimulator cells to reach a similar level of proliferation as control cells. The ability of mixed T cell populations to respond to stimulation in this assay may reflect the presence of both CD4^+8- and CD4^-8+ T cells in these cultures.

[0200]As a direct indicator of CD4^-8+ activity, the cytotoxic ability of T cells were assayed in a standard target cell lysis assay. CD4^-8+ T cells were generated using allogeneic feeder cells in the presence of IL-2 and assayed to determine the ability of these cells to lyse target cells. Balb/c spleen cells were stimulated with PMA (10 ng/ml) in the presence of ionomycin (250 ng/ml) for 24 hours to allow for efficient loading of cells with ³[H]-thymidine. After 24 hours, ³[H]-thymidine (5 μCi/ml) was added to PMA+Ionomycin-stimulated Balb/c spleen cells. Cells were incubated at 37° C. for 18 hours and then washed. CD4^-8+ effector cells were plated with target Balb/c cells at increasing ratios ranging from 1.5:1 to 50:1 (effector:target) in a 96-well flat-bottom plate (experimental) in a final volume of 100 μl. The cells were pelleted by centrifugation and incubated at 37° C. for four hours. A plate containing labeled Balb/c cells alone was harvested immediately and used to determine total counts (TC). A second plate containing labeled Balb/c cells alone was also incubated at 37° C. for four hours to determine spontaneous release (SR). After four hours of incubation, cells were harvested onto glass fiber and counted in a scintillation counter.

[0201]Percent lysis was determined as follows: {[(Total-SR)-(Experimental-SR)]/(Total counts-SR)}*100=% lysis. At higher effector-to-target ratios (50:1 and 25:1), scurfin-transgenic CD4^-8+ cells were as effective at lysing target cells as cells generated from NLC, while at the intermediate ratios (12.5-3:1), transgenic cells were significantly reduced in their cytolytic function in comparison to NLC. However, the transgenic cells were still effective with 50-60% lysis at these intermediate ratios. Overall, these data suggest that scurfin-transgenic T cells possess cytolytic activity, but are less effective than NLC. In addition, exogenous IL-2 was required to generate functional CD4^-8+ T cells, presumably due to the poor endogenous production of this cytokine.

[0202]As a further indicator of T cell responsiveness, the functional responsiveness of 2826 transgenic animals to antigen in vivo was addressed. Contact sensitivity responses using Oxazalone as the challenging agent were carried out on 2826 mice and their littermate controls. Age-matched animals were treated on the left ear with 2% Oxazalone (diluted in olive oil/acetone), using a final volume of 25 μl. After 7 days, ear thickness was measured using spring-loaded calipers and mice were challenged on the right ear with 2% Oxazalone (8 μl per ear). Ear thickness was measured at 24 hours and is reported as change in ear thickness compared to pre-challenge. Control mice were challenged only. Thickness of ears following initial priming (prior to challenge) was no different from untreated ears. Mice were subsequently treated with PMA (10 ng/ml; 8 μl/ear) on the priming ear. Ear thickness was measured at 18 hours and is reported as thickness compared to pre-treatment.

[0203]In these studies, transgenic animals made a consistently poor response to Oxazalone at all times examined, whereas control animals responded normally. The transgenic animals however responded normally to challenge with PMA, indicating that they were capable of generating an inflammatory reaction to a strong, non antigen-specific challenge. Further studies using animals transgenic for both a TCR and Foxp3 will examine in vivo responses in greater detail.

Example 18

Scurfy T Cells can be Inhibited by Wildtype T Cells In Vivo

[0204]It has previously been reported that adoptive transfer of CD4^+8- T cells from sf mice into nude mice transfers disease as measured by the wasting and skin lesions characteristics of sf. However, grafting of sf thymus into normal mice does not transmit the disease suggesting immunocompetent mice are capable of inhibiting sf cells (Godfrey et al., Am. J. Pathol. 145:281-286, 1994). To better understand the mechanism of inhibition either 3×10⁶ sf CD4+ T cells or wildtype CD4⁺ T cells or a mix of sf and wildtype CD4⁺ T cells were adoptively transferred into syngeneic C3H-SCID mice.

[0205]C3H SCID mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). All animals were housed in specific pathogen free environment and studies were conducted following PHS guidelines. The original double mutant strain, sf (sf) and closely linked sparse-fur (Otc^spf), were obtained from Oak Ridge National Laboratory. The double mutants were backcrossed to Mus musculus castaneous to obtain recombinants carrying either (Otc^spf) mutation or sf mutation (Brunkow et al., Nat Gen 27:68-72, 2001). Prior to cloning of sf gene carrier females for sf mutation were identified by the amplification of genomic DNA with primers 5'-ATTTTGATT ACAGCATGTCCCC-3' (SEQ ID NO:15) and 5'-ACGGAAACACTCTTATGTGCG-3' (SEQ ID NO:16) (primers for microsatellite marker DXMit136 which was found to be inseparable from sf phenotype during backcrossing).

[0206]The single mutant sf strain was maintained by breeding carrier females to F1 males of (C3Hf/rI×101/RI) or (101/RI×C3H/RI). Sf males were used at age 15-21 days and wildtype control animals were used at 6-12 weeks of age. Scurfy or wildtype CD4⁺ T were purified by cell sorting. The cells were resuspended in 0.9% saline, pH 7.2 and mixed at different ratios in a final volume of 200 μl and injected into SCID mice via tail-vein. Mice were monitored weekly for weight loss. Approximately 50 μl of blood was collected by eye-bleeds. Red blood cells were lysed and leukocytes were stimulated at 5×10⁴ cells/well for 48 hours with immobilized anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml).

[0207]Mice that received sf T cells showed signs of wasting (seen as weight loss) 3-4 weeks post-transfer that became progressively worse, whereas the mice that received a mixture of sf and wildtype T cells showed a normal weight gain corresponding to their age (FIG. 11A). Mice that received only wildtype T cells showed a similar weight gain with age. In addition, mice receiving only sf T cells developed an inflammatory reaction around, but not within, the eye that persisted throughout the experiment. If the disease was allowed to progress, the recipients of sf T cells only died 8-16 weeks after transfer. Recipients of a mix of sf and wildtype T cells remained healthy throughout the experiment (experiments done up to 16 weeks).

[0208]Histological examination of the large intestine of mice receiving sf T cells showed crypt abscesses, thick epithelium, increased epithelial cellularity and cellular infiltrates in the colonic wall, consistent with proliferative colitis (FIG. 11B). In comparison, the intestine of mice receiving a mixture of sf and wildtype T cells (or wildtype cells alone) appeared normal, correlating with the lack of wasting in these mice.

[0209]For the histological examination, tissues were removed from C3H/SCID mice receiving either sf T cells, wildtype T cells or a mixture of sf and wildtype T cells. Intestines were flushed with cold PBS and immediately fixed in 10% formalin. Paraffin embedded sections were processed for hematoxylin and eosin staining and comparative histopathology (Applied Veterinary Pathobiology, Bainbridge Is., WA).

[0210]Cellular infiltration and inflammation was also noted in a number of other organs (including kidney, liver and skin) from mice that received sf T cells only and such cells were not found in animals that received wildtype cells. Further, the lymph nodes and spleen from sf-recipient animals were substantially enlarged compared to their controls, indicating a marked lymphoproliferative process. Lymph nodes were collected from 6-12 weeks old mice and macerated in DMEM+10% FBS in between the ground glass ends of sterile microscope slides. The cells were filtered through 70 μM nylon mesh, collected by centrifugation and resuspended at ˜50×10⁶ cells/ml in complete media.

[0211]CD4⁺ T cells from sf mice have been shown to be hyperproliferative and to secrete large amounts of cytokines such as IL-2, IL-4 and IFN-γ (Blair et al., J. Immunol. 153:3764-774 (1994); Kanangat et al., Eur. J. Immunol. 26:161-165 (1996)). To monitor the activation status of the CD4⁺ T cells that were transferred into the SCID animals, IL-4 secretion by PBMC of recipient mice was measured. PBMC from various recipients were stimulated with anti-CD3 and anti-CD28 in vitro for 48 hours and secreted IL-4 was detected by ELISA kit (BioSource International, Camarillo, Calif.) according to manufacturer's instruction. At an early time point post-transfer (8-10 days), IL-4 was produced by PBMC from all the recipients (FIG. 11c). At later time points (2 weeks or more), PBMC from recipients of sf T cells secreted significant amounts of IL-4 whereas the PBMC of mice receiving either wildtype T cells only or a mixture of sf and wildtype T cells secreted little IL-4. Lack of weight loss, tissue infiltrates and suppression of IL-4 secretion in mice receiving a mixture of sf and wildtype T cells indicated that wildtype T cells were inhibiting the activation and disease progression normally associated with the transfer of sf CD4+ T cells.

Example 19

Sf Cells are Regulated by CD4⁺ CD25⁺ T-Regulatory Cells

[0212]There have been numerous reports that the CD4⁺CD25⁺ subset of peripheral CD4⁺ T cells (T-reg cells) is involved in regulating other T cells, both in vivo and in vitro (Roncarolo et al., Curr. Opin. Immun. 12:676-683 (2000); Sakaguchi, S., Cell 101:455-458 (2000); Shevach, E. M., Ann. Rev. Immun. 18:423-449 (2000)). It was therefore of interest to determine if such T-reg cells were responsible for the inhibition of disease seen after co-transfer of sf and wildtype CD4⁺ T cells in vivo. Two million sf CD4⁺ T cells were mixed at different ratios either with wildtype CD4⁺CD25^- T cells or with wildtype CD4⁺CD25⁺ T-reg cells and injected into C3H/SCID mice. The recipients were monitored for weight loss and IL-4 secretion by PBMC as described in Example 1. For isolating T-reg cells these were stained with anti-CD4-FITC (Caltag Laboratories, Burlingame, Calif.) and anti-CD25-biotin (Caltag) for 30 min on ice. The cells were washed twice with PBS and stained with strepavidin-APC (Molecular Probes, Eugene, Oreg.) for 20 min on ice. Cells were washed twice and positive sorted for CD4⁺CD25⁺ T cells.

[0213]As before, mice receiving sf T cells alone showed signs of wasting (FIG. 12) and IL-4 production. However, mice that received a mixture of sf T cells and higher doses (110,000 or more) of wildtype CD4⁺CD25⁺ T-reg cells showed a marked reduction in signs of disease such as weight loss. In comparison, mice that received a mix of sf and CD4⁺CD25^- T cells showed signs of disease at all doses except when the number of CD4⁺CD25^- T cells was greater than 1.1 million. The small amount of suppression seen with higher numbers of CD4⁺CD25^- T cells may indicate that there are additional mechanisms of suppression or that CD4⁺CD25^- T cells give rise to CD4⁺CD25⁺ T-reg cells post-transfer. It seems unlikely that the mechanism of inhibition by CD4⁺CD25^- T-reg involves in vivo competition for lymphoid space, since as few as 1.1×10⁵ T-reg can inhibit the activity of 2×10⁶ sf T cells.

[0214]In order to better understand the mechanism of inhibition of sf CD4⁺ T cells by CD4⁺CD25⁺ T-reg cells, in vitro mixing experiments were conducted. Sf CD4⁺ T cells or wildtype CD4⁺CD25^- T cells were activated with anti-CD3 in presence or absence of CD4⁺CD25⁺ T-reg cells at various responder:suppressor ratios. FIG. 13 shows that the proliferative responses of wildtype CD4⁺ T cells stimulated with APC and anti-CD3 were suppressed significantly by the addition of CD4⁺CD25⁺ T-reg cells. These CD4⁺CD25⁺ T-reg cells also inhibited the proliferative responses of sf CD4⁺ T cells. However CD4+CD25+ T-reg cells were less effective in inhibiting sf CD4⁺ T cells than wildtype CD4⁺ T cells. This result, like that seen with co-transfer in vivo, indicates that the hyper-responsive state of sf T cells can be regulated by T-reg cells.

[0215]For APC, spleens were collected in a similar fashion as lymph nodes and stained with anti-Thy-1-FITC or anti-Thy1-PE (Caltag). Cells were washed and negative sorted for Thy-1. The cells were sorted using a MoFlo flow cytometer (Cytomation, Fort Collins, Colo.) and Cyclops (Cytomation) software at a rate of 10-20,000/min. Cell doublets and monocytic cells were eliminated on the basis of forward and side scatter gates, and dead cells were excluded by propidium iodide (10 μg/ml) stain. The purity of the sorted cell population was routinely 90-99%. Thy-1^- APC were treated with mitomycin C (Sigma, 50 μg/ml) for 20 min at 37° C. and washed three times with DMEM+10% FBS before using in proliferation assays. For regulatory T cell assays, CD4+ T cells were stimulated at 5×10⁴ cells/well in 200 ul DMEM+10% FBS with soluble anti-CD3 (2C11; Pharmingen) at 1 μg/ml and an equal number of mitomycin C treated Thy-1^- APC from spleens. For T-reg assays MoFlo sorted CD4⁺CD25⁺ T cells were added at various ratios.

[0216]Cultures were incubated 72 hour at 37° C. and pulsed with 1 μCi/well with [³H] thymidine (Amersham Life Sciences, Arlington, Ill.) for the last 8-12 hours of culture. For in vitro preactivation, CD4⁺CD25⁺ or CD4⁺CD25^- T cells were stimulated at 5×10⁴ cells/well in 200 μl DMEM+10% FBS with soluble anti-CD3 (1 μg/ml for wildtype cells or 10 μg/ml for Foxp3 transgenic cells), 4 ng/ml rIL-2 (Chiron) and an equal number of mitomycin-C treated Thy-1^- APC from spleens. The cells were harvested at 72 hours, stained with CD4-FITC or CD4-PE and positively sorted for CD4 using a MoFlo flow cytometer as described above. These cells were then added to the regulatory T cell assay as previously described at the same ratios as freshly isolated CD4⁺CD25⁺ T cells.

Example 20

TGF-β does not Inhibit SF CD4⁺ T Cells

[0217]Recent studies have implicated a critical role for CTLA-4 and secretion of TGF-β in regulatory function of CD4⁺CD25⁺ T-reg cells in vivo (Read et al., J. Exp. Med. 192:295-302, 2000); Takahashi et al., J. Exp. Med. 192:303-310, 2000). To test whether sf cells were sensitive to inhibition with TGF-β, CD4⁺ T cells were stimulated with or without the addition of exogenous TGF-β. For the TGF-β assays, anti-CD3 (at varying concentrations, Pharmingen) and anti-CD28 (1 μg/ml, Pharmingen) were immobilized on plastic. TGF-β (R&D) was added at a final concentration of 2.5 ng/ml. Cultures were incubated for indicated time periods at 37° C. Individual wells were pulsed with 1 μCi/well with [³H] thymidine (Amersham Life Sciences, Arlington, Ill.) for the last 8-12 hours of culture. Proliferation data are mean value of triplicate wells and represent a minimum of three experiments.

[0218]As expected, wildtype CD4⁺ T cells stimulated with either anti-CD3 alone (FIG. 14A) or with the combination of anti-CD3 and anti-CD28 were inhibited significantly by TGF-β (FIG. 14B). However, sf cells stimulated either with anti-CD3 or anti-CD3/CD28 were not sensitive to inhibition with TGF-β, regardless of the dose of anti-CD3 or TGF-β. The lack of TGF-β, inhibition was specific to T cells since proliferation and cytokine production by B cells as well as monocytes, both stimulated with LPS, from sf mice were sensitive to TGF-β inhibition. It is worth noting however, that high levels of exogenous IL-2 can largely overcome the inhibitory effect of TGF-β on T cells, potentially by downregulating TGF-β receptor expression (Cottrez et al., J. Immunol. 167:773-778, 2001). T cells from sf animals produce extremely high levels of IL-2 upon stimulation, and this may contribute to the lack of inhibition by TGF-β on T cell function of sf mice. Additionally, the role of TGF-β production by T-reg cells in in vitro assays is not clear. Most experimental systems do not point to a role for TGF-β in this system, although the in vivo data does indicate an important role for TGF-β in inhibitory activity of CD4⁺CD25⁺ T-reg cells (Read et al., J. Exp. Med. 192:295-302, 2000).

Example 21

Foxp3 Expression is Upregulated in CD4⁺CD25⁺ T-Reg Cells

[0219]The Foxp3 gene is expressed at highest levels in lymphoid tissues such as thymus, lymph node and spleen (Brunkow et al., Nature Genetics 27:68-72, 2001). The lymphoid expression of Foxp3 seems to be predominantly in CD4⁺ T cells, since the level of expression in CD8⁺ T cells as well as B cells was significantly lower or undetectable (Brunkow et al., Nature Genetics 27:68-72, 2001).

[0220]To assess if Foxp3 plays a role in CD4⁺CD25⁺ T-reg cells, the expression of Foxp3 transcript in CD4⁺CD25⁺ and CD4⁺CD25^- T cells from normal as well as Foxp3 transgenic mice (˜16 copies of Foxp3 transgene) was compared. CD4⁺CD25⁺ or CD4⁺CD25^- T cell populations were collected as described above. Oligo dT primed first-strand cDNA was synthesized from these cells using the SuperScript Preamplification System (Gibco-BRL, Rockville, Md.) and used as a template for real-time RT-PCR using an ABI Prism 7700 instrument. Foxp3 expression was measured using the primers 5'-GGCCCTTCTCCAGGACAGA-3' (SEQ ID NO:17) and 5'-GCTGATCATGGCTGGGTTGT-3' (SEQ ID NO:18) at a final concentration of 300 nM and with internal TaqMan probe 5'-FAM-AGCTTCATCCTAGCGGTTTGCCTGAG-AATAC-TAMRA-3' (SEQ ID NO:19) at a final concentration of 100 nM. Dad1 was used as an endogenous reference (Hong et al., 1997). Dad1 primers were 5'-CCTCTCTG-GCTTCATCTCTTGTGT-3' (SEQ ID NO:20) and 5'-CCGGAGAGATGCCTTGGAA-3' (SEQ ID NO:21), used at a final concentration of 50 nM and TaqMan probe 5'-6FAM-AGCTTCATCCTAGCGGTTTGCCTGAGAATAC-TAMARA-3' (SEQ ID NO:22) at a final concentration of 100 nM. Other components of the PCR mix were from the TaqMan Universal Master Mix (PE Applied Biosystems). PCR cycling conditions were 50° C. for 2 min; 95° C. for 10 min; and 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute.

[0221]Data was collected by ABI Prism 7700 Sequence Detection System Software, Version 1.6.4. A standard curve was generated with a dilution series (1×, 1:10, 1:100, 1:1000, 1:10,000) of a standard cDNA sample which was run at the same time as the unknown samples. The software determines the relative quantity of each unknown based by plotting a curve of threshold cycle (C_T) versus starting quantity and using the C_T to calculate the relative level of unknown sample. Each sample was run in duplicate and mean values used for calculations. Data is expressed as normalized Foxp3 expression, which was obtained by dividing the relative quantity of Foxp3 for each sample by the relative quantity of Dad1 for the same sample.

[0222]Interestingly, the level of Foxp3 expression in CD4⁺CD25^T cells was nearly undetectable whereas CD4⁺CD25⁺ T-reg cells expressed the highest amounts of Foxp3 so far described (FIG. 15A). The level of Foxp3 expression in T cell subsets of Foxp3 transgenic mice was also determined. These animals have ˜16 fold the amount of Foxp3 message found in wildtype animals. In Foxp3 transgenic mice, Foxp3 expression was detectable in both CD4⁺CD25^- as well as CD4⁺CD25⁺ T cells, but similar to wildtype cells, CD4⁺CD25⁺ T cells expressed significantly greater levels of Foxp3. A subset of CD4⁺ cells in sf mutant animals also expresses CD25, although this population is large in size and expresses CD69, indicating they are likely cells previously activated in vivo. Nonetheless, it was determined that these CD4⁺CD25⁺ cells from the sf mutant animals do not show enhanced amounts of Foxp3 message, indicating that these cells are likely not T-reg in nature.

[0223]CD4⁺CD25⁺ T-reg cells express certain markers such as CTLA-4, OX-40, GITR (McHugh, R. S. et al. Immunity 16: 311-23, 2002); Shimizu, J. et al. Nature Immunology 3: 135-42, 2002) that are characteristics of activated T cells. To assess if Foxp3 expression in CD4⁺CD25⁺ T-reg cells was due to activation of T cells, Foxp3 expression was measured in CD25+ and CD25- subsets of CD4 T cells before and after in vitro activation (FIG. 15B). CD4⁺CD25- T cells did not express any Foxp3 even after in vitro with anti-CD3 and IL-2. Interestingly, the expression of Foxp3 in CD4⁺CD25⁺ T-reg cells decreased slightly after activation. This indicated that Foxp3 unlike any other markers reported so far on CD4⁺CD25⁺ T-reg cells was specific to this subset and was unrelated to the activated/memory phenotype of these cells.

Example 22

Overexpression of Foxp3 Leads to an Increased Number of CD4⁺CD25⁺ Cells But does not Lead to an Increase in Regulatory Activity

[0224]The relatively exclusive expression of Foxp3 within the T-reg subset might indicate that this transcription factor is either required for the generation of this subset or is directly involved in its function. To determine if Foxp3 plays a role in CD4⁺CD25⁺ T-reg cell function, the functional activity of CD4⁺CD25⁺ and CD4⁺CD25^- T cell subset from Foxp3 transgenic mice was examined. These animals have 16 fold more Foxp3 message than found in wildtype animals, with very high amounts in the CD4⁺CD25⁺ subset. Additionally, there were fewer total CD4⁺ cells in these transgenic animals and those cells are hyporesponsive relative to their littermate controls. Whereas there were a slightly increased percentage of CD4+CD25+ T cells in the transgenic mice, the expression of CD25 was more diffuse and, unlike in wildtype animals, these cells did not comprise a distinct subset of cells (FIG. 16). A comparison of functional activity of CD4⁺CD25⁺ T-reg cells from wildtype and Foxp3 transgenic mice showed that although cells from the transgenic mice do display regulatory activity, there was no significant increase in suppressive ability relative to their wildtype counterparts on a per-cell basis (FIG. 17).

[0225]Under the T-reg assay conditions the CD4⁺CD25⁺ T-reg cells were activated at the same time as the responders. Since CD4⁺ T cells from Foxp3 transgenics were hyporesponsive to TCR stimulation it was likely that the Foxp3-Tg CD4+CD25+ T-reg cells were not getting activated to the same extent as the wildytpe CD4⁺CD25⁺ T-reg cells during the assay. This raised the possibility that if CD4⁺CD25⁺ T-reg cells from Foxp3 transgenics were activated to the same extent as wildtype cells they would exhibit higher regulatory activity.

[0226]To address the issue CD4⁺CD25⁺ T cells were pre-activated in vitro with anti-CD3 in the presence of APC and IL-2 for 72 hours according to the previously published protocol (Thornton et al., J. Immun. 164:183-190, 2000). Based on our previous observations the T cells from Foxp3 transgenic mice were activated with a higher dose of anti-CD3 in vitro to give comparable proliferation as the wildtype cells. These preactivated T cells were then tested in a T-reg assay. As reported by others, preactivation of CD4⁺CD25⁺ T cells in vitro made them much more potent suppressors. However, preactivation of Foxp3 transgenic T-reg cells gave them comparable suppressor activity as wildtype T-reg cells (FIG. 17). This suggested that there was no intrinsic defect in T-reg cells from Foxp3 transgenics however overexpression of Foxp3 beyond a threshold level did not further enhance T-reg activity.

Example 23

CD4⁺CD25^- T Cells from Foxp3 Transgenic Mice Show Regulatory Activity

[0227]Since CD4⁺CD25^- T cells from Foxp3 transgenics express Foxp3 at levels higher than wild-type CD4⁺CD25⁺ T cells, we next evaluated expression of surface markers associated with T regulatory cells and the suppressive activity of these cells. Interestingly, the CD4⁺CD25^- T cells from Foxp3 transgenics also expressed GITR (TNFRSF18) that has recently been shown to modulate T-reg activity (FIG. 18). These cells did not express other activation associated T cell markers such as OX40, CTLA4 or Ly-6A/E (data not shown). More importantly, when freshly isolated CD4⁺CD25^- T cells from Foxp3 transgenic were tested for function in T-reg assay they had significant suppressive activity (FIG. 19). This activity usually ranged from comparable to lower than that of CD4⁺CD25⁺ T cells from the same mice. As expected, such suppressive activity was never detected with wild-type CD4⁺CD25^- T cells. In contrast to the CD4⁺CD25⁺ T cells, the suppressive activity of CD4⁺CD25^- T cells from Foxp3 transgenic could not be enhanced by preactivation with anti-CD3 and IL-2 in vitro (data not shown). This further supports the idea that the expression of Foxp3 commits a T-cell to the T-reg lineage without a direct correlation with regulatory activity.

[0228]The gene mutated in sf mice (Foxp3) has a critical role in the regulation of peripheral T cell responses. Loss of function mutations in the gene leads to a potentially fatal T cell mediated autoimmune disease both in mice and humans (Bennett et al., Nature Genetics 27:20-21 (2001); Lyon et al., Proc. Nat'l. Acad. Sci. USA 87:2433-2437 (1990); Wildin et al., Nature Genetics 27:18-20 (2001)). Additionally, overexpression of scurfin in transgenic mice leads to decreased peripheral T cell numbers and inhibition of a variety of T cell responses including proliferation and IL-2 production. Inhibition of IL-2 production by scurfin is not the sole explanation for hyporesponsivess since addition of exogenous IL-2 does not completely restore normal T cell response in mice overexpressing scurfin. To better understand the immunoregulatory mechanisms that may be controlled by the Foxp3 gene, further studies were conducted on the expression of this gene and the biological role of scurfin-expressing cells.

[0229]As shown in this Example, wildtype T cells can inhibit disease caused by adoptive transfer of sf CD4+ T cells into SCID mice. These observations are very similar to those made by several other groups characterizing the activity of regulatory T cells. Similar to the observation made by Powrie et al., the disease caused by sf cells was inhibited by even a small number of CD4⁺CD25⁺ T cells. CD4⁺CD25^- T cells were less effective at inhibiting sf T cell activity in this model, which may be due to a subset of these cells developing into a T-reg cell subset and making the appropriate inhibitory factors or due to additional mechanisms of inhibition. In addition, the in vitro hyper-responsive state of sf T cells can be inhibited by the presence of wildtype CD4⁺CD25⁺ cells, but not by the addition of TGF-β. Generally, data from in vitro T-reg experiments suggest a direct cell-cell interaction is required with no involvement of cytokines such as TGF-β (Thornton et al., J. Exp. Med. 188:287-296 (1998); Thornton et al., J. Immun. 164:183-190 (2000)). Additionally, TGF-β has no inhibitory effect on activated T cells (Cottrez et al., J. Immunol. 167:773-778 (2001)) making it unlikely that CD4⁺CD25⁺ T-reg cell inhibition of sf cells in vivo is mediated by TGF-β.

[0230]To assess whether the Foxp3 gene product plays a role in CD4+CD25+ T-reg cell function we measured the expression of Foxp3 in CD4+CD25+ T-reg and CD4+CD25- T cells and measured the regulatory activity of CD4+CD25+ T-reg cells from mice overexpressing the Foxp3 gene. In both wildtype and Foxp3 transgenics, CD4+CD25+ T-reg cells expressed the highest level of Foxp3 mRNA of all different cell populations tested to date. A comparison of functional activity of CD4+CD25+ T-reg cells from wildtype and Foxp3 transgenic mice showed no increase in regulatory activity in cells from transgenic mice, even following an optimal stimulation of these cells. Importantly however, CD4+CD25- T cells from Foxp3 transgenic animals did have suppressive activity. While it is not possible to phenotypically identify a subset of T-reg cells in sf mutant mice (due to the high level of endogenous activation), CD4+CD25+ cells isolated from mutant animals neither expressed the Foxp3 gene nor did they display any suppressive activity in vitro.

[0231]These results indicate that although expression of Foxp3 can commit a T cell to the T-reg cell lineage, over expression of Foxp3 beyond a threshold level does not lead to further enhancement of regulatory activity. Furthermore, expression of Foxp3 by itself is likely not sufficient to generate T-reg cells, as CD4+CD25- from Foxp3 transgenic mice have comparable Foxp3 expression to wild-type T-reg cells but less suppressive activity. This effect on regulatory activity is unlikely due to an effect on CTLA-4 expression since there is no increase in CTLA-4 expression in Foxp3 transgenic mice and sf mutant animals express normal levels of CTLA-4.

Example 24

Modulation of Scurfin Expression

[0232]Antibodies or NCEs that modulate scurfin expression are identified using the following methods:

[0233]The scurfin promoter is cloned into commercially available Luciferase reporter vector (Promega, Madison, Wis.). This construct is then transfected into cells, such as a murine or human T cell line. Agents, such as antibodies generated against T cells, cytokines, receptors, or other proteins, in addition to small molecules, peptides, and cytokines, will be used to treat the transfected cells. The level of Luciferase activity is then determined using commercially available Luciferase assay systems (Promega) according to manufacturer's instruction to identify agents that either increase or decrease the expression of scurfin.

[0234]In an alternative approach, agents such as those described above are incubated with primary T cells under conditions that allow for the modulation of scurfin expression. The scurfin expression will be measured using the RT-PCR method described above in Example 21. Agents identified by either of the above methods will be used directly for the treatment of an autoimmune disease. Alternatively, T cells will be isolated from patients of an autoimmune disease, treated with the specific agents identified above to induce scurfin expression and transferred back into the patients to suppress the activation of other T cells.

[0235]In summary, the results of the Examples show that Foxp3 expression is predominantly seen in CD4+CD25+ T-reg subset and correlates with a basal level of regulatory activity. Over-expression of this gene can confer a regulatory function on CD4+ cells that lack CD25, indicating that this factor may be directly involved in commitment to this functional lineage.

[0236]All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

[0237]From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

[0238]From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Sequence CWU 1

2312160DNAMus musculus 1gctgatcccc ctctagcagt ccacttcacc aaggtgagcg agtgtccctg ctctccccca 60ccagacacag ctctgctggc gaaagtggca gagaggtatt gagggtgggt gtcaggagcc 120caccagtaca gctggaaaca cccagccact ccagctcccg gcaacttctc ctgactctgc 180cttcagacga gacttggaag acagtcacat ctcagcagct cctctgccgt tatccagcct 240gcctctgaca agaacccaat gcccaaccct aggccagcca agcctatggc tccttccttg 300gcccttggcc catccccagg agtcttgcca agctggaaga ctgcacccaa gggctcagaa 360cttctaggga ccaggggctc tgggggaccc ttccaaggtc gggacctgcg aagtggggcc 420cacacctctt cttccttgaa ccccctgcca ccatcccagc tgcagctgcc tacagtgccc 480ctagtcatgg tggcaccgtc tggggcccga ctaggtccct caccccacct acaggccctt 540ctccaggaca gaccacactt catgcatcag ctctccactg tggatgccca tgcccagacc 600cctgtgctcc aagtgcgtcc actggacaac ccagccatga tcagcctccc accaccttct 660gctgccactg gggtcttctc cctcaaggcc cggcctggcc tgccacctgg gatcaatgtg 720gccagtctgg aatgggtgtc cagggagcca gctctactct gcaccttccc acgctcgggt 780acacccagga aagacagcaa ccttttggct gcaccccaag gatcctaccc actgctggca 840aatggagtct gcaagtggcc tggttgtgag aaggtcttcg aggagccaga agagtttctc 900aagcactgcc aagcagatca tctcctggat gagaaaggca aggcccagtg cctcctccag 960agagaagtgg tgcagtctct ggagcagcag ctggagctgg aaaaggagaa gctgggagct 1020atgcaggccc acctggctgg gaagatggcg ctggccaagg ctccatctgt ggcctcaatg 1080gacaagagct cttgctgcat cgtagccacc agtactcagg gcagtgtgct cccggcctgg 1140tctgctcctc gggaggctcc agacggcggc ctgtttgcag tgcggaggca cctctgggga 1200agccatggca atagttcctt cccagagttc ttccacaaca tggactactt caagtaccac 1260aatatgcgac cccctttcac ctatgccacc cttatccgat gggccatcct ggaagccccg 1320gagaggcaga ggacactcaa tgaaatctac cattggttta ctcgcatgtt cgcctacttc 1380agaaaccacc ccgccacctg gaagaatgcc atccgccaca acctgagcct gcacaagtgc 1440tttgtgcgag tggagagcga gaagggagca gtgtggaccg tagatgaatt tgagtttcgc 1500aagaagagga gccaacgccc caacaagtgc tccaatccct gcccttgacc tcaaaaccaa 1560gaaaaggtgg gcgggggagg gggccaaaac catgagactg aggctgtggg ggcaaggagg 1620caagtcctac gtgtacctat ggaaaccggg cgatgatgtg cctgctatca gggcctctgc 1680tccctatcta gctgccctcc tagatcatat catctgcctt acagctgaga ggggtgccaa 1740tcccagccta gcccctagtt ccaacctagc cccaagatga actttccagt caaagagccc 1800tcacaaccag ctatacatat ctgccttggc cactgccaag cagaaagatg acagacacca 1860tcctaatatt tactcaaccc aaaccctaaa acatgaagag cctgccttgg tacattcgtg 1920aactttcaaa gttagtcatg cagtcacaca tgactgcagt cctactgact cacaccccaa 1980agcactcacc cacaacatct ggaaccacgg gcactatcac acataggtgt atatacagac 2040ccttacacag caacagcact ggaaccttca caattacatc cccccaaacc acacaggcat 2100aactgatcat acgcagcctc aagcaatgcc caaaatacaa gtcagacaca gcttgtcaga 21602429PRTMus musculus 2Met Pro Asn Pro Arg Pro Ala Lys Pro Met Ala Pro Ser Leu Ala Leu 1 5 10 15Gly Pro Ser Pro Gly Val Leu Pro Ser Trp Lys Thr Ala Pro Lys Gly 20 25 30Ser Glu Leu Leu Gly Thr Arg Gly Ser Gly Gly Pro Phe Gln Gly Arg 35 40 45Asp Leu Arg Ser Gly Ala His Thr Ser Ser Ser Leu Asn Pro Leu Pro 50 55 60Pro Ser Gln Leu Gln Leu Pro Thr Val Pro Leu Val Met Val Ala Pro65 70 75 80Ser Gly Ala Arg Leu Gly Pro Ser Pro His Leu Gln Ala Leu Leu Gln 85 90 95Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His Ala 100 105 110Gln Thr Pro Val Leu Gln Val Arg Pro Leu Asp Asn Pro Ala Met Ile 115 120 125Ser Leu Pro Pro Pro Ser Ala Ala Thr Gly Val Phe Ser Leu Lys Ala 130 135 140Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp Val145 150 155 160Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Arg Ser Gly Thr Pro 165 170 175Arg Lys Asp Ser Asn Leu Leu Ala Ala Pro Gln Gly Ser Tyr Pro Leu 180 185 190Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe Glu 195 200 205Glu Pro Glu Glu Phe Leu Lys His Cys Gln Ala Asp His Leu Leu Asp 210 215 220Glu Lys Gly Lys Ala Gln Cys Leu Leu Gln Arg Glu Val Val Gln Ser225 230 235 240Leu Glu Gln Gln Leu Glu Leu Glu Lys Glu Lys Leu Gly Ala Met Gln 245 250 255Ala His Leu Ala Gly Lys Met Ala Leu Ala Lys Ala Pro Ser Val Ala 260 265 270Ser Met Asp Lys Ser Ser Cys Cys Ile Val Ala Thr Ser Thr Gln Gly 275 280 285Ser Val Leu Pro Ala Trp Ser Ala Pro Arg Glu Ala Pro Asp Gly Gly 290 295 300Leu Phe Ala Val Arg Arg His Leu Trp Gly Ser His Gly Asn Ser Ser305 310 315 320Phe Pro Glu Phe Phe His Asn Met Asp Tyr Phe Lys Tyr His Asn Met 325 330 335Arg Pro Pro Phe Thr Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu 340 345 350Ala Pro Glu Arg Gln Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr 355 360 365Arg Met Phe Ala Tyr Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala 370 375 380Ile Arg His Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Ser385 390 395 400Glu Lys Gly Ala Val Trp Thr Val Asp Glu Phe Glu Phe Arg Lys Lys 405 410 415Arg Ser Gln Arg Pro Asn Lys Cys Ser Asn Pro Cys Pro 420 42531869DNAHomo sapiens 3gcacacactc atcgaaaaaa atttggatta ttagaagaga gaggtctgcg gcttccacac 60cgtacagcgt ggtttttctt ctcggtataa aagcaaagtt gtttttgata cgtgacagtt 120tcccacaagc caggctgatc cttttctgtc agtccacttc accaagcctg cccttggaca 180aggacccgat gcccaacccc aggcctggca agccctcggc cccttccttg gcccttggcc 240catccccagg agcctcgccc agctggaggg ctgcacccaa agcctcagac ctgctggggg 300cccggggccc agggggaacc ttccagggcc gagatcttcg aggcggggcc catgcctcct 360cttcttcctt gaaccccatg ccaccatcgc agctgcagct gcccacactg cccctagtca 420tggtggcacc ctccggggca cggctgggcc ccttgcccca cttacaggca ctcctccagg 480acaggccaca tttcatgcac cagctctcaa cggtggatgc ccacgcccgg acccctgtgc 540tgcaggtgca ccccctggag agcccagcca tgatcagcct cacaccaccc accaccgcca 600ctggggtctt ctccctcaag gcccggcctg gcctcccacc tgggatcaac gtggccagcc 660tggaatgggt gtccagggag ccggcactgc tctgcacctt cccaaatccc agtgcaccca 720ggaaggacag caccctttcg gctgtgcccc agagctccta cccactgctg gcaaatggtg 780tctgcaagtg gcccggatgt gagaaggtct tcgaagagcc agaggacttc ctcaagcact 840gccaggcgga ccatcttctg gatgagaagg gcagggcaca atgtctcctc cagagagaga 900tggtacagtc tctggagcag cagctggtgc tggagaagga gaagctgagt gccatgcagg 960cccacctggc tgggaaaatg gcactgacca aggcttcatc tgtggcatca tccgacaagg 1020gctcctgctg catcgtagct gctggcagcc aaggccctgt cgtcccagcc tggtctggcc 1080cccgggaggc ccctgacagc ctgtttgctg tccggaggca cctgtggggt agccatggaa 1140acagcacatt cccagagttc ctccacaaca tggactactt caagttccac aacatgcgac 1200cccctttcac ctacgccacg ctcatccgct gggccatcct ggaggctcca gagaagcagc 1260ggacactcaa tgagatctac cactggttca cacgcatgtt tgccttcttc agaaaccatc 1320ctgccacctg gaagaacgcc atccgccaca acctgagtct gcacaagtgc tttgtgcggg 1380tggagagcga gaagggggct gtgtggaccg tggatgagct ggagttccgc aagaaacgga 1440gccagaggcc cagcaggtgt tccaacccta cacctggccc ctgacctcaa gatcaaggaa 1500aggaggatgg acgaacaggg gccaaactgg tgggaggcag aggtggtggg ggcagggatg 1560ataggccctg gatgtgccca cagggaccaa gaagtgaggt ttccactgtc ttgcctgcca 1620gggcccctgt tcccccgctg gcagccaccc cctcccccat catatccttt gccccaaggc 1680tgctcagagg ggccccggtc ctggccccag cccccacctc cgccccagac acacccccca 1740gtcgagccct gcagccaaac agagccttca caaccagcca cacagagcct gcctcagctg 1800ctcgcacaga ttacttcagg gctggaaaag tcacacagac acacaaaatg tcacaatcct 1860gtccctcac 18694431PRTHomo sapiens 4Met Pro Asn Pro Arg Pro Gly Lys Pro Ser Ala Pro Ser Leu Ala Leu 1 5 10 15Gly Pro Ser Pro Gly Ala Ser Pro Ser Trp Arg Ala Ala Pro Lys Ala 20 25 30Ser Asp Leu Leu Gly Ala Arg Gly Pro Gly Gly Thr Phe Gln Gly Arg 35 40 45Asp Leu Arg Gly Gly Ala His Ala Ser Ser Ser Ser Leu Asn Pro Met 50 55 60Pro Pro Ser Gln Leu Gln Leu Pro Thr Leu Pro Leu Val Met Val Ala65 70 75 80Pro Ser Gly Ala Arg Leu Gly Pro Leu Pro His Leu Gln Ala Leu Leu 85 90 95Gln Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His 100 105 110Ala Arg Thr Pro Val Leu Gln Val His Pro Leu Glu Ser Pro Ala Met 115 120 125Ile Ser Leu Thr Pro Pro Thr Thr Ala Thr Gly Val Phe Ser Leu Lys 130 135 140Ala Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp145 150 155 160Val Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Asn Pro Ser Ala 165 170 175Pro Arg Lys Asp Ser Thr Leu Ser Ala Val Pro Gln Ser Ser Tyr Pro 180 185 190Leu Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe 195 200 205Glu Glu Pro Glu Asp Phe Leu Lys His Cys Gln Ala Asp His Leu Leu 210 215 220Asp Glu Lys Gly Arg Ala Gln Cys Leu Leu Gln Arg Glu Met Val Gln225 230 235 240Ser Leu Glu Gln Gln Leu Val Leu Glu Lys Glu Lys Leu Ser Ala Met 245 250 255Gln Ala His Leu Ala Gly Lys Met Ala Leu Thr Lys Ala Ser Ser Val 260 265 270Ala Ser Ser Asp Lys Gly Ser Cys Cys Ile Val Ala Ala Gly Ser Gln 275 280 285Gly Pro Val Val Pro Ala Trp Ser Gly Pro Arg Glu Ala Pro Asp Ser 290 295 300Leu Phe Ala Val Arg Arg His Leu Trp Gly Ser His Gly Asn Ser Thr305 310 315 320Phe Pro Glu Phe Leu His Asn Met Asp Tyr Phe Lys Phe His Asn Met 325 330 335Arg Pro Pro Phe Thr Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu 340 345 350Ala Pro Glu Lys Gln Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr 355 360 365Arg Met Phe Ala Phe Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala 370 375 380Ile Arg His Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Ser385 390 395 400Glu Lys Gly Ala Val Trp Thr Val Asp Glu Leu Glu Phe Arg Lys Lys 405 410 415Arg Ser Gln Arg Pro Ser Arg Cys Ser Asn Pro Thr Pro Gly Pro 420 425 430523DNAArtificial SequencePrimer for generation of mouse Fkh cDNA 5gcagatctcc tgactctgcc ttc 23623DNAArtificial SequencePrimer for generation of mouse Fkh cDNA 6gcagatctga caagctgtgt ctg 23721DNAArtificial SequencePrimer for generation of human Fkh cDNA 7agcctgccct tggacaagga c 21821DNAArtificial SequencePrimer for generation of human Fkh cDNA 8gcaagacagt ggaaacctca c 21920DNAArtificial SequencePrimer for PCR amplification of mouse Fkh cDNA 9ctacccactg ctggcaaatg 201023DNAArtificial SequencePrimer for PCR amplification of mouse Fkh cDNA 10gaaggaacta ttgccatggc ttc 231128DNAArtificial SequenceOligonucleotide for hybridization reaction 11atgcagcaag agctcttgtc cattgagg 281228DNAArtificial SequenceOligonucleotide for hybridization reaction 12gcagcaagag ctcttttgtc cattgagg 281322DNAArtificial SequencePrimer for amplification of Fkh cDNA 13catcggngag atgctaagat gg 221428DNAArtificial SequencePrimer for amplification of Fkh cDNA 14gaaaccagat tagtaagtat cttcgatt 281522DNAArtificial SequencePCR primer 15attttgatta cagcatgtcc cc 221621DNAArtificial SequencePCR primer 16acggaaacac tcttatgtgc g 211719DNAArtificial SequencePCR primer 17ggcccttctc caggacaga 191820DNAArtificial SequencePCR primer 18gctgatcatg gctgggttgt 201931DNAArtificial SequencePCR primer 19agcttcatcc tagcggtttg cctgagaata c 312024DNAArtificial SequencePCR primer 20cctctctggc ttcatctctt gtgt 242119DNAArtificial SequencePCR primer 21ccggagagat gccttggaa 192231DNAArtificial SequencePCR primer 22agcttcatcc tagcggtttg cctgagaata c 3123130788DNAHomo sapiens 23gatccaggtc tcccaaacct gtcatgttcc ccctttgggg acagtctgtg gtgtcacagt 60ggttacaatt gctagctcat ccttcaacgt tgttgttggc acgcgttctt cagggacacc 120ccttggatct cccctgactc tcctcttatt ctcccactcc accactcccc atgccggact 180gtactgtttt ccactctccc tcctccagcc agcacccagg gcttaccacc gaggtgttga 240gtcccgaggt cacagggtct ctcagctcct tgcatgtgtt ccctgtctgg cggcagacag 300gcatctcctt gataatgttc tctgggtctg tggccatctt cacatctgac agccccttgg 360cccatgccga tgagctaact agccacatga aggcgaacac agccgtggcc agaaagtcct 420aaggcaggca ggggtgagga agacagcact gtgagtggac tgcttcacgc taggccctgg 480ggcctcctcg gatcacaggt cccccagaag aggcctgtct ctcccctgcc ttttcagctc 540tcaagcccac aacactttca cctgaaggtg gccctcccag cttgagcttg tgtgtgtaca 600tgacacagag gatgtgggag tgtgattgtg atatgtggct gtgggtgttg gggggagggg 660aactctggga tgatttccga atctgtggaa agtaatgtgt ctttctactt ttgtctgcat 720gtgggaattt ttgtcatttt gtgtctaggc tgagggtatg tgagtacagt ggtgtaggaa 780tcagtgtgtc tgtgtggcat gatgacaggc atgactgtca cagcgaacct ctgtgggcct 840cttcctttga tctaggctgt gtagttgtgt gagagtggga aggttctcag gtgtgggcct 900gtctgggatt ctgcataggc tgtatgtctc cgtgactcta tgactctgtg tcccggatgc 960ctggtgtgga ttaatagcag cagcaactat atgtgtggct gttatggtgg ctgtttgcat 1020gtgggcctgt ctgggatttg gtgtggtctg tgagcatggc tgtgtccctg gggttccgtg 1080tccccatggt ccctacatgc aagtggctgt ggggactcac cagcatgggc cctttgttat 1140tctctcggta cttgttctgc aggaagatgt aggtggccag agcccccatg gagtagagga 1200aggcaaacac ggccacggtg acaaagaatt cggctgacga ggagtagtcc ccaactaaga 1260agaccttggt ggtgccccct cggcaggtgg gtgcatcaaa gtacacttgg tgcagcctgc 1320aaacagaggt gggcaggtgt ggcccagccc ctcagaacac cctcaaccct ccagcccaat 1380catgggtccc cccatttctg acaaggcccc aggaagagga ggaaataacc attcatcgag 1440cacctactat gtaccagtca catttcactg attgctcaaa acatcctggt gagataggtg 1500ttattcatcc ctcactccaa cccaagcact ctggcctctt gattatttct agaacttgcc 1560aggcatcctc cctccctagg atctttgttt gtactggctg ttccctttgc caggaatgct 1620tttcccctag atagcttcgg gtcttactcc ctcatctcct tttttttttt tttttttttt 1680ttttgagatg gagtcttgct ctgtcaccag gctggagtgc ggtggcacaa tcccggctca 1740ctgcaacctc tgcctcccgg gttcaagcga ttctcctgcc tcagcctcct gagtagctgg 1800gattacaggc atgcgccgct acgcccagct aatttttgta cttttaatag agacggggtt 1860tcaccatgtt ggccaggata gtcttgatct cttgaccttg tgatctgccc gccttggcct 1920cccagagtgc tgggattaca ggcgtgagcc actgcgcccg gcctccctca tctccttgaa 1980gctttgctca actgtcacct tctcattgag accttccctg atctccctac ttaaaattaa 2040attgaaacat tctctcaacc agcacttctt agcctggctt ccttgctcca cttttctcga 2100tagtagtggt acccatctga cgtaccaggt attttttttt tttttaatct tggtttttgc 2160tgtaactaca agagggcagg gctttttcct gtgttttcac ctccgtttat ctccaggccc 2220tggaacagtg ctggcaccta gcagacactc aagaaatgtt tatcaaatga aatgtacaga 2280ggcggaaaca gaggctcagg gaagtgcatt aacttgccta aagtcacaca gctggcaagg 2340gaatgtcaga gttgagatct gaaccttgtt ttctccctat ctcatgttga cttttctccc 2400ccaccccagt tcaggtcccc gagagaaaaa aatgtatgca tagcagagga gaggccagcg 2460actagataac aggaaaagac agacagaaag aaaggagggt aaagggatct tggaacaggg 2520tgtgtgtatg tgagggaaga ggggaggtgt cctgggacta aggcctttcc tgtggtgtga 2580tggaggagag gtgggggttg tagagagaaa atgtctgtgg gagaagttct ggagcacagt 2640ctcagattgg aggccaggat cctgagactg agcaaatggt gccaatgcct ttgtgtagca 2700aaggccctga gggaagaggc ttgcacttat tagtcaccta ctaagcagtt tacataaatg 2760tctcattaat gacagcctcc agcaattctc taaagaaggc attattagac cctttttaca 2820gatgaataaa cagaggctta gcaaggttat tacttgccca aggttgtaca gctcctaaat 2880gatggagcta ggagttgcat ctgggtttga gtttctgttt ctagtgggct gggtcaggag 2940agggggtccc taagagggaa tgagaggtcc ttgggtatta gggcagtttg ggagggattt 3000tgctcagggg gacccagggc caagactgtt tttctagggg acttgtgggc tagagcctta 3060ggacccaaag ttctagacat gggaggcgtg gggagggggc acaggggatg gttgtggtct 3120tgtggatccc caaaggactg gctctaagca gtgtcccttg ctgccagctc cccctccttc 3180cccagactct tttctccctt gcctttgtgg acccatggat acctggctgc ccctctgcct 3240ctcaggccac actttcccgg catctcctct cttttccatc attgctcagg gcctgtgcta 3300ggttttcttc tactttcact cttccatctc cccactgggt gatcacttga gcctacattt 3360accctcagta ctcgaagacc cccagtttcc aactcaaccc ctgtcctctt tcctgagtcc 3420cttaaattta ttttacttat tgaacagaca ctgatggagc acttattgtg tgccaggcac 3480tgtgctaagc acctgatgaa tattaactca ttgaatgcta accacgaccc tgtgaggtag 3540atattattag tattcctttt tacagatgag gaaattgaga cccagagtgg ttatgtgatt 3600tggccaaata gtgggggagt ctcagaaacc tgtgcgggga ttgtgtctgt gggtctctgt 3660catcccaccc tgcaggaggt aaacatcatt gtgagggtag tgcccatgcc agccgtgctc

3720attcttgtgc acctagcacc cagtgtattg cccggcacac agtgtgagct caataaatgt 3780cagaattgtt taattgggtt taatttttcc ctatatctaa gcagcacccc tcattattct 3840attctctacc ataaaaccct acatgaggat ctcctcgagt ttctctcagt ctgctattat 3900cctctttgtt aatttatttg ttataatgta ggctccatga aggtagggag ttgggtctgg 3960tttttttttt tttggagatg gagtttcgct cttgttgccc aggctggagt gcaatggcgc 4020gatttcggct cccaggttca agtgattctc ctgcttcagc ctcctgagta gccgggatta 4080caggcatgca tgaccacgcc tggctaattt tgtattttta gtagagacgg ggtttctcca 4140tgttggtcag gctggtctcg aactcctgac ctcaggtgat ccgcccacct cggcctctca 4200aagtgctggg attacaggcg tgagccactg tgcctggcgg gagttgggtc tgttttagtt 4260actatcatgc ttccagcact ttgagtagta ccagatataa aataagttct caaaaatgtg 4320tatagaatga atgaatgggg attaaataat aatattggct aacattaatt gggtacctac 4380tgggttaagg tatgtatata cacctgtctt acttttgtat cttgtttatt gcctgtccct 4440taccccttcg ctggaacaga gccagagggc cacagcctat gtctgccttg ctcaccacat 4500agtcctagag tccagagcag tgccaggtac aaggcaggtg ctcagtgatc ccagtggatg 4560tagcctcgag gtgggagctg ggcaggagct acttgcgggg ggaggtattg gccggttcca 4620ccctgcccct tcctctccca ggtctgttgg tatcccaggg tgggagcaca cctgaagggg 4680tactcgaact cgacctcgat gctgaggtca ctctcggtct tgttggcaca atccacgctc 4740agctggagct ccccactgta gctgccgcat gtggcaaagg cgaagatggc gaagaccttg 4800ggcagcaggg atggggatgg ccacagtgaa cctgtgtgca tgtgggaggg aggtgccctc 4860ctctggacag atcgggggcc cagcacaggc agcagcagat ggagcagtcc ccacccccac 4920cccctaatca gggctcatcg ggaccaagga caagctgatt acaagggtat ggctgtctct 4980tcctgtttct ctctctgtgt ctctctctgt ctctgccttt ccatgctgac ctccctcttc 5040tctgtttgtc tctgttgttc tctgtgtatc tcactgtgtc tctgtttctc tcggtatctc 5100tgtttctcta tgtgtctttg tctttctcag atgtctcttt gtctctctgt gtctctgatc 5160ctctctgcct ctgcctatct gatctgtcga tgtctctttt tgtctctttt ttctctgtct 5220gtgtaagtgt atgcctgtat gtctgtgtct ttctgtttct tggtctgttt gtctctgtct 5280ctctgtcctg tctctcactc ggtctccgga tggttctctc tctgtctccc tcttgcctct 5340gtgtgttggt ttctcttttt tctgagtgtc tctttctctc tgtgtctctc tctccctcac 5400cctgttttcc tgcctctctc cctgcgtctg tttctctttg catctgtttt ctgtgtctcc 5460tttttctttg tttctgtttg tctcttctct cttatgtctg tatctcttct catctccata 5520tttctttttt ttttctcagt atttccatct ctgtctttat cattccctgt ccctcattct 5580ctctgtcacc ttcactgtct ctgggtttct ctgactttgc atctctcacc cagcatctgc 5640tacaggctcc acctccccca cccctccctc acttcaagag aggaggtagg acgtcacagt 5700ctcccacttc acaacggggg taccctcagc cctatgtgac gtcacagctc atctgttttc 5760tggcttcaga cccctgaaac ctcaccctgg agcccaattc ccccatcccc tacaaccccc 5820gcactcatcc ctggctactc gcccctactc cacccctgct cttctggacc ccatgacctc 5880cctctctccc tgcactctgc cccaacagcc cggaccacac tcacccattg cagcaccttc 5940acaaagccga ggggctcctt gaccacccgg aactgacccc cagccaccag ctgaggagag 6000aaacagtggg gaaggggtgg ggttgttgga ggtacaggga ggtgagcggc aaggctgccc 6060atcaatgacc ccaggggatg gagcatgtga cctcggggag agggtgagaa ggagaaagtg 6120acaccttggg gatggtgtga gccccagggt ccaaggactg gtgggaaggt ctgaagagat 6180ggtgagtcag atggcagagg aagtcggggg tgagggagat ggggatgagg tcaggactag 6240ttaaggaggg ggagcaggtt tgaaacgtaa aggcgagtcg gatggggaag gggttcgggt 6300agagaatggg gatggagtgg ggtgcattcg ggtggggggg ttgtgatggt gaaggtgagt 6360cataacgggg caggagtcgg tttggctcag gtgggggaag ggtctgcagg ggtaaaggtg 6420tgttagacgg cggtggcggt agatatttga ggtgatgggg atggggaagg gttgggactg 6480attgggggtg agtttctgag gtggcaaaga cgagagacat ggcggagagg gtctggactg 6540attaaggtgg gggagggctg ggacacggct tcagtcgcta tgtgttgggg gtgaggaggc 6600tgggggtttt ggaaggagta aaaccggatt gtcccctagg tgtgctctgg gggaaggggt 6660ggtcgcctat gggggtacag tctctgtttg ggaggggcgg gtgccagccc tctcccccat 6720ccccgccccc acaccttctg tcgctctatt tcctagctta tccgcagcac cccccatctt 6780ccttccactc tcccctcccc ttgtcccttg ccctcactcc agcccccagg cccctattcc 6840tcccctcgcg gtcgccagta ataaacgggg ctccgggggc ccccgctgcg gcagtgagga 6900cggccctcgc tgcggcaaag agcgcactcc ttttctccac ctcccccttc ctccccttca 6960cctgccgcag acccccatcc ccagcgccgg ggaccgggtc tccgctgcca gaccctggcc 7020accacctccc agagtcggct ggcctgagcg acccggccta ggcagccggg cggcggcggg 7080ctctgtccac ggtgctggag cgcgtacctg attcaccacg tccatgtccg ccagcagcag 7140catcagcaat gcagggggcg ggaggctcgg ctagcaaggg cggcgcgggg cgcggcgggg 7200gcggcgcgcg ctcgttggaa cagcccaggc ggctgcagcc ccgcccctcg cgccccctcc 7260ttgcccgcca cgcgcctgcg cacagcgggt tgtaccacag tctcaccggc agccattccg 7320gcccggaggc tggagccgtg agagcgtgcg ggaatatgcg ttgggaaaac acaagtcgag 7380ggccggggag aggagacctc cctgctcggt cctacagtgt gacaactttg ccaggctctt 7440ttctgagttt gttaaacatg ttcactcatt taatgcttac agaagtccta cgttgtaggg 7500actattaggc cgtttttaca gacgaggaaa ataattcccc agagagctta agtgactctc 7560acaaggtcag ccagctacta gctggcagtg cccagagtcc ctgcagttta aacacggttc 7620aaggccttcc aggttccttt ttcatagtct gcattttaag cacaccctga aactcctgcc 7680cccatctagc catctccctt atttctttct tcctctttgt caaattaatg cctactcgaa 7740ctctaccact ttacctattt attccttagc tctctcttaa ttggaataac cctccttcct 7800gcgactaaaa ctaggcccat caaggtcaac agtgatggct atgatgcaaa atccaaaata 7860cagttggggc ttaagatgct caacttcggc caggcgcagt ggctcatgcc tgtaatccca 7920gcactttggg aggccgaagt ggacagtgga tcacttgaag tcaggagttt gagactagcc 7980tggccaacat agtgaaaccc catctctatt aaaaatacaa aaattagctg gctgtggtgg 8040caggcgcctg taatcccagc cacttgggag gctgaggcag gagaattgct tgaacccagg 8100aggcggaggt tgcactgagg cgagattgca ccactgcact ccagcctggg caacagagcg 8160agactccatc tcaaaaacaa acaagcaaac aaaaatactg agatactgtt tttcatcttt 8220cagggtggtg agtatcaaaa agtttgataa cgttccgtat tggtaggttg gccagaaaca 8280agcgttctca taaatcgcct gtggaagttt cattggtata acgtgtagaa ggcaatcggg 8340caaaatctaa aaaaaaaaaa taacaaattc atttactgtt tggttcacct tcatacttct 8400aggaattcat cttacagata gactcataca tgtgctaaaa gatatatgta taagataata 8460catgactggg ctgtttattt tttatttttt tttgagacgg agtctcactc tgttgcccag 8520gctggagtgc agtggcatga tctccgctta ctgcaacctc cgcctcccgg gttcaagcaa 8580ttctccctgc ctcaccctcc ctgagtagct gagattacag gcattcacca ccacgcccat 8640ctaatttttt tttttttttt tgtagtttta gtagagatgg ggtttcgcca tattggctag 8700ggtggtcatg aactcctgac ctcatgtgat ctgcctgcct cggcctccca aagtgctggg 8760attacaggtg tgagccactg ggccaggcca gggctgttta taataggata aagaatggac 8820actcccaaag tgctcattgt tggtagcctg tttaaataaa ctctgatact atgccgctgt 8880cattataata acatggaact atggaatgat tgccaagata tttagttaag taaaaaatgc 8940aagatgtcaa actgtgtggt aattgtgtac aaagagaaga gaagggttat atcagacaga 9000gaatagctaa catttaaatt gtggctgttt tctgtcaagt actctttaga gaactttgcc 9060tgtattactc acttaaattc ttactgtaac cctatgtgac aggtacgaca actatctttg 9120ttttacagct gaataaacta agaggcggag aggaattttc ctaagttcac acagctggta 9180aattggtaga accaggatat gaacccagga agtctggctc cagcacctgt tactcttagc 9240cactgtgcta acttatcagt acaccatctt ttttttttca gcacaccatc ttgtatgtct 9300gtgaaagata cctgataata ctatttgctt ccagagggta gggaaacttt tcactttata 9360cccctttgta ccttttgaga ctccatctca aataaaataa aaaaataaac agcctggtag 9420tgtatcatct tatacataca ccttttagca catatatgag tctacctgta agaaattcct 9480agaagtatga agctgaacaa aacagtaaat gaatttattt ttctagattt tgcccagttg 9540ccttctacac aggttatacc aatgaaactt ccacaagcga tttatgagaa cgcttgtttc 9600tggccaacct gccagtatgg aacattatca aactttttga tactcaccac cctgaaagat 9660gaaaaacagc atctcagtag ttttgttttg ttttgttttt gagacagagt ctcactctgt 9720tgcccaggct ggagtgcagt ggtgcaatct cagttcactg caacctccgc ctcctgggtt 9780caaacgattc tcctacctct gcctcctgag tagctgggat tacaagcaca aaccaccacg 9840tccggctatt tttttttttt tttttttttt tttttagtag agatggggtt ttgccatgtt 9900ggccaggctg gttgtgaact cctgacctta agtgatccac tgtccacttg gcctcccaaa 9960gtgctgggat tacaggtgtg agccactgca tctggcctac cttttgaatt ttgaaccatc 10020tgaatgaata tctttttttt gagacagggt ttccctgtgt tgcccaggct ggagtccagt 10080ggcatgaata tggctcactg cagcctcgac ctcctgggct caagcgatcc tcctgcctca 10140gcctcctatg tagctgggac cacaggcgtg agacaccatg cccagctaat ttttaaatat 10200ttttgtagag acgaggtctc actttgttgc ccaggctggt cttgaactcc tgggctcaag 10260tgatcctccc gcctcagcct cccaaagtgc tgagatgaca ggcatgagtc accatgccca 10320gctcaatacc tgttttttta aacatgaaaa ttcacacata tatgtaatgg atacttccct 10380gttgttgcct tattggggaa gcagtacaca ggaggggtta aaagcatgga tcttggcatc 10440aaactgcttg aatttgaatg ccacctctga tacttcttgt ttgaccctgg acaggtaaga 10500tggtttaaaa tgatacctat aaggttgttg cactgattca acgagatgat cactgtaatg 10560cagttagcac agggcccaat aaatggttca ttttacctcc tacacatctc tggaatccac 10620tcacttctca ccatctcctc tgccacctcc tctggctaag gcgaccatca ttcctgccaa 10680ttttagcatc tccagtctct tccccctccc ttctgttttc agcatagccg tcagaacatc 10740tatttaaaat gcaatgttaa ctatgtcact accctactca aatccctccc atggctcccc 10800actgcttata ggaaaaatcc cagagtttgg gacctggcat tcaaggttcc acctgatctg 10860gcctcagtgg cttctcccac ctcatccctc aacagtctcc cttcactctc tgtgccctgc 10920ccatgacttg ctgttctcac ctaactccag cccaaccact ttaagccttt gtttcaaggc 10980ctaatgtaac tcaatcttct cagtcctagt ctccaattct ggcttccctc aaatttcagt 11040cctggcttga ttggcgcaga ttatacaaat gagacacccc tgttctggga gctgggaaga 11100gttcctcccc gactggtggt ctcatcctgc accactccca atgctacctt caattcctct 11160ggagtggggc tgactcccac tttggatgag acctgtcccg tgtttcaacc agttcagttt 11220gccccgagag aaacccagag ccggtgcctg gcacatagta aatgcctgat aagccttcag 11280tgagcgaatg taaccccaaa ggctgggtga agagtaaagc tggggcaaga agcagagagg 11340acctctggga tctgtgtggg ataagagtag gttactgtta ggtggtctca ggaaagccag 11400gtgggccctg agtgggttcc ccagggctgg gagccctgac ctttgctgtc acctatgttg 11460tctactgatg gatttaggtt ttcttttctt tctcaggcag aaagtgggag atgggagaca 11520gagcgatctc aatttagcca ccctaaaatt cctgttgccg tcagaggacc cctctgccta 11580agatgcatca cataccaggc cgattccaac ctccaggcct ctcccctggc aggattcttc 11640tctgggtttc cttgtccttt ccttctcatc cgtctaccct tcctagtttg acctggctga 11700gccctggtga aatctctgcc tctcttcctc tctgggtcca agcacccctc acacccgctc 11760tctccccttt cctccatctc tgtccagtgc cctccccctc ccccctcctc cactctgcct 11820ccagcttttt ttggctgctg tctcttcctc ctcctcctcc tcctccctcc ttccctgacg 11880ctgaataatc aggcctggct gtcctggttt ctggaaatac ccgtgtgtgg gcagtggctc 11940agggctagaa caggggatgg atggatgggg gcccctgggc agccccagac ccccagtgca 12000aagacaacaa aatccaggga tgtggtccat gcctgcctcc tgctggggag gggagggcag 12060gaggtttatt gagcagttgg ggaggggtgg gaattcagag ggcgtggacg caggccatct 12120cgtctcccaa gtcctcctca tcgaagcggg agaggatgga ctcctcgtcg ctatagagag 12180agctggttcc ctgtgccagc aggtcactgg cagcattgtc catctcatcc agcgtcaggc 12240gacacgcatc tgcaatctcc tgcttggcca gggccacgaa acgtgggtct cgagcaaaga 12300ggcccagacc ctctgagata agcacctggg ggcacaggtg ggatcagtgt tgacggacgg 12360cacagtgtgc acagaaggtt cagtgtggat aaatgacgtg cccatgaggg catgtaggga 12420agtcagtggg ggacaggggt gagcagaggt cgtagggcaa agggatatct acatgcacaa 12480cacaggacct ggggactcct ttccgtcctc ctccagtacc cacctcctcc ccttctcccc 12540catccgtctt gtctatatgc cctctggact cacagcctcc accaagctgt cggcactgcc 12600cctcttccca tggctggggt ccgagtgggt tccaggcacg tgcagacagg tgaaggtgcg 12660cagtgggcca ctggatctgc cgaggtaccc ctcccccgct gcgccctctt ccaccaacaa 12720cagcggggca tacaggagcc gaccccgctg aggtggtgtt gcccaggaac cctgagccag 12780aataaacatc agaggggcag ggatggatga gtagggtatg agggtctagg actcaccgct 12840gtgcctactt ctttccctca gtatacaact ggagtgaaat tgaatgggta taagtcaggc 12900ttgagtttat ttatgtaaga gtgcaggagg cctgggttta aatgtcagct ctgccactta 12960ctagctgcat gatctggggt aaacctctca ggacctcagt ttcctcacaa gtacagtgag 13020cattgtgtgc attcattcat atgtgtcctg ttcttcagaa ggctctgagg ccaggcgcgg 13080tggcttatgc ctgtaatccc agcactttgg gaggctgaga caggcggatc acctgaggtc 13140aggggtttga gaccagcccg gccaacatgg tgaaacccca tctctactaa aaatgcaaaa 13200attagccagg catggtggtg agcacctgta atcccagcta cttgggaggc tgaggcagga 13260gaattgcttg gacccgggag gcaggggtta cagtgagcca agatctcaac actgtactcc 13320agcctgggca gcagagcaaa actctgtctc aaaaaaaaaa aaaaaaaaaa aaaaaaaagg 13380gcctgatatg tgctgtgttt gagaaattca gcctaggctg cccccatctc tccagacccc 13440agaccccagc accacctcca cagcctcacc tgagccctat tgggccctga atttcgccca 13500cgatgatagg tgcctgggat gggtaaatcc tcacaactgc cctggcgctg cagacactgg 13560atggtgaagg agggcttccg acctgggggt gggtggggca ccaggggcaa atagcaatgt 13620cagtgcttcc ccagatctct gtcctgcagg cccgtggggg ccccttggat ccatccctgc 13680tctcacctgc aggtgtgggg ggcagcagac ggcggcgtgg taccaggtgc ccatccatgt 13740atctctgagc tctgtgaggt ggcaaaagga ctgagcacgg gggagtccct gcctgctcat 13800ctaggtagga aagcctgtgt gggtgggagg gccaggggtg aacttgggtc tgggcctgga 13860cctgggcctg ggagatgggg cacaaacagt cctccccgac ccagttccca cccctcctcc 13920accccagctc atgggttcat cccatgtggc atggccagtg taccgatcag ggacttcctc 13980atcctcatct tgcttgtttt gccctttggt tcccttgggc tgagaatttc cttcttctgg 14040gatggtgaaa atgagagccc cagagcctct cctggggaaa gggaggcacg ttaggcagac 14100cagatccctc aatatgtggc cctggacccc tggttcctct catcccatgc cccaaccctt 14160acagaaacac tgggcttttg gatttttttg ttgaggacaa gtcacataaa attaaccatt 14220aaccatttta aagtgaccat gggcttttgt ttaatagatg ttcatctaat taggactgaa 14280agtttcaaaa aaatcaaagt tgacctgtag agagacacct tgaacacaaa actgggcatg 14340tattaggagt ggggggagcc tgtaaaagct aggcacttta tttttttatt tctatttttt 14400attttctgag acgaggtctt actctgtcac ttaggctgga gtgcagcagc atgatctcag 14460ctcactgcag cctcaacctc ccaggctcaa tttatcctcc cacctcagcc tcccaagtag 14520ctgggactac aggcatgcgc caccatgctc ggctattttt ttattttttg tatggacggg 14580gtctcattat gttgtgcagg ctggtcttgt actcctgggc tcaagcgatc ctcccacctt 14640ggcctcccaa agtgctggga ttacgagtgt gagccaccgt gcctaggcaa gctagacact 14700ctgaaatttt tttttaaaat ttttctttaa tctgcttatg tattgtctag cagctaagtg 14760tttgacatgc attacctcat ttcatccttt ctttatccct gtgaagtaag tgctttgtca 14820tcccccttaa acaggtgagg aaactgaggc tcatagaaac gaagtgacta ggcagggcac 14880ggtggctcaa gcttgtaatc ccagcacttt gggaggctga ggtgggcaga tcacctgagg 14940tcaggagttc gaaaccagcc tggccaacat ggcaaaacct cgtctctact gaaaaaaaaa 15000aaaaaaatta gcctggcatt atggcgcacg cctgtaatcc cagctactcg ggaggctcag 15060gcaggagaat cgcttgaacc cgggaggcag aggttgcagt gagccgaaat tgtgccactg 15120cactccagcc tgggagacag agcgggactc tgtctcaaaa aaaaaaaaaa aaaaaagtga 15180ttagctcaag gtcacttagc aaatggcaga ggcagaatgt gaactgaaac tcttggtttc 15240acacaatgtg ctttttgcat taaacctttg tattgccttt ctcatatgaa tctcttatca 15300ccctctattc ttacctgagc ccttgaaaca cctccattct tcccaaattt ctgcttgaag 15360gcctctaggc cctccctccc acccccctca acttcctgcc tcctgaattg cctctatcca 15420gtgtctagtc cttgccttcc ctcacaatct acttccagac acttgataat acctgtgggt 15480gttggatcca gcctggggca cactgggctg actggaggtg ggagtccccc tgtcatcatc 15540actgggccca aaggagagtg aatctggaag tctgtccccg acaggcagag acacagaaat 15600cccggagccc cggcgagctg agggctggga gaccatcgtg gcctgtggag agtcacagga 15660ctgagtgttg catgcacttt gtggctaagt catttggtca tataacatgt ctcccccaca 15720ggtccatgaa tgtgggcctt gccctccaac agcaatggta aatgtcaaat aaataaatac 15780gtaaataaat aaataaatat ttgcgatagg gtcttgtcct gtcaccaggc gggagtgcag 15840tggcatgatc acagctcact gtggcctcat cctcccaggt tcaagcaatc ctcctgcctc 15900agcctcccaa gtagctggga ctacaggctt gcaccaccac tcctagctaa tttttaaatt 15960tttttgtaga gaggcggttt cactattttg ccaggactgg tcttgaaccc tagggttcaa 16020gcgatccttc caccctggac tcccaaagtg ctgggattat aggcatgagc caccactccc 16080agcctcaaat aaatattttt ctattgcaga gggggcttct ctgggggagc ttcttcccag 16140aagcagtgtc aaaatcactc aggggattga cgaagtattt tacaatcaag ttcctgggag 16200agtgaaaact tgtccccact ttgttagttt ccaagtcctt ttcatcttcc tcctccactc 16260cctcctgccc ctcttcttcc tcctcctctg tgtcacaggt gagggcctgc cgcatctcag 16320gacccaagtc ctgcaggctc cgcagaccag cctgtggggg tggagaaata ggtaagcagg 16380cagctcaggg tctgaacttt ccctctgttt cccctgcagg cgggtagggt gggggaagga 16440gtccttcagc caccctggcc ctccgtgagg cctggggcct cagtttcctt atctgtgaca 16500tgggtttcac aagatcaaaa tgggggtcag cctcagccac agcactgcca cacgtgccca 16560tccacactag gccctgcccc gaagacctga agggcggaag aggtgctagg ggcggcgtcg 16620ttgcctagta gccctttttc tttcctccgc cggaatttgc ggaaatagtc ctggatcaga 16680aatgtggcgt agaatttgcc cacggtgacc tcctcctcta ggggcaaggg agagaaggca 16740agatcacaca ggggccctct gacccagccc acctcgccgc ctctctaccc accagcatgg 16800acttcccctc gccctctgcc cagcgctggt tttgtggtgg agagcgatca tctgccattc 16860aggggctggc gcggggaact ggggcgggga tagctcaccg tctggtgggg ggatgacctc 16920atctagcagc ttctgtttca tccgcttcca gatctttttg atgacaatcc gcagctcctg 16980gttggcttgc tccaggttcc ctgcgttgga ggaggatgtg gggcatgagc caataggaag 17040aacctccaca gctgccccct cactgttggg tggggggaac ttcacaagct gctaccgcag 17100atgcacaccg cccccattgg acggggtggg gggaaacagg cctggagaat cctgtcaccc 17160attctggggt tcagagctag gagtggggcc catgattcaa tgagtttgct ccttgcaccc 17220tccccaggaa cgatcccact cctgcccctc ccctacacct tctgttttga tcttcaggga 17280tgtccggacc agggcaaaga gtgtggcgtt gaatgtcacc gtcccatctg agttgagggg 17340catgttcatt gccacaagtc tctgcaaaca cgagagacat gaaacccact ctgggaaaat 17400gccgggtatg caggaacgaa tgtcaagggg cagaaacctc tgaggatgcg atcaaacacc 17460cctccccacc caaggcagat cccttcccac ccttgccatg tgatagacag ttctctcagg 17520agcctggggg tgggcaggtg cacagacctt gcaggccact cggtgtgggc acagcttccc 17580aaatcccaga gggggctgga tacgtctcag cagggcaacc acatccaagt gtttgatgcg 17640gcccctggag gagttgggga ggtaccactg gattggcgat gcccccacta gccctttctc 17700aaggccccac cagctcatca ctacctcctc ctaccaatac cccagagctt gccagggaag 17760aactggaggg cagtcagaga gggcagagga tggggcagtg gaaactttgg ggcatctatt 17820gactgggtaa tgagatgcag cagtcggagg tttgggggct aggggactgt gtagggcaat 17880acattgtccc ctggattcta ggtaagggta tagagggcat acttggcccc agggtcatat 17940tcagaccaga tcctcttgaa ttcatcaagg tgatgggggc ccaggatgga ccaatctctg 18000gtgagataat caaagttgtc catgatcaca gccacaaaga gatttatgat ctgtgggcca 18060gtgagcaagg gagcagttag gtgaagggca gaacactgtc atggacggat ggtgggaggc 18120tggggcgggc ttatatggtc atgagtctct gggttgatct tgtcatcaga ccagccctct 18180cacccactat gaagatctgg gctgcccacg tcacatccct gagcccctca gggccagccc 18240ccgtgacggt ggtggtggtt gtgaggaaat ggttctcacc aggaaggcac agagcatgaa 18300gaagctgatg aaataggcga tggcaaaatt gctaccacag gtaaactctt caccagggcc 18360gaagtcagac tcaggatcac accgatttcc gggaaggctg gcaagcatta tctcctgcca 18420tgcctcacca gtggcacacc tggtgggagt cacacagggg tagcatcctg aaggggaggc 18480tatgaagtca tggtaaatgg agttggggga ggactgggga atgaggagat gacctacttc 18540tcaccagggg ctacatctgg cacagataac atttcaatat tttaacagcc agtgtggcca 18600tgttgacgac agtcatgttt gcatcttaca gatgaggcac tgagacctga gaaggggagg 18660cgtcttacag tcagaaagaa aatggctgag gctagaatac tctaacagac tcctcacatc 18720atagccagga aacatctcgg gctgtatgga gccccatgct gccatattat tcccatagct

18780caagcagtct ggggctcaga gagggtgggt gggcacccac gggcataagg tggcagggga 18840gtgagtagat gtcacctgaa cagaagcagc acagcctgtg gaaaggtctg gaagttgttg 18900tttcggttta tctgtgtgcc atcctgaaga gccaccttgc cgaacatctg tggacacatc 18960aaggctgtgt cagtggggtg aacccatgcc ccacccattt cacacctcct aatacctaaa 19020ggagcctcat aaccctgaca aacagggcat cactgctgat ctcatttcac agatgtgtag 19080attgacccag ggccgtccag cttgtctgtc tacctgccac ccctactttg gggctattct 19140taacccatcc cctgccagca gaggagtgct taagggatgc ttcctagggt ccccacttgc 19200ctgcatgcca atgacggcat agatgaagaa tatcattgcg atgagaagag ccacataggg 19260caaggcctat agggatgggg gaggggggca gagaatagat gcacaggctc aggcaaagaa 19320ctataagccc cagaatgcac tgctttccct ggcctgggcc tactgggaga tgcagtccat 19380ctccaaagtg ccaaggactg tggtgtctct caaatgtgaa catagtgtta agtagataga 19440gtaaggtcag tgagaccaga ggtgtgttgg aaactgagag cccagaaaaa agagaaaatt 19500ggaataataa tttggaaatg ggtatggcat gttgggagat gtagttctga gggtggtaaa 19560ggggcaggct gagaagtgtg aaatgggggc gaggtatggt caaagggatg gggtagggga 19620ttacctggaa ggacttgatg aatgtccaga gcaatgtgcg gatcccttca cccttactga 19680gaagcttgac cagccgcata actcggaaga ggcgaaagaa ggtaatggaa atgcgggagc 19740tgtcctcaga gctctggggt gaggggtgca gtagggatgc tcagtttgca tttactccag 19800ctatctccct atctcatgct ttggccctcc cagtacccaa atcactcagg ccctagggta 19860atccggaggg atggagggac agagggacat gggaaaagaa gcagagagtc cctgttatta 19920gggtgggcta cctcgccaag gtggccacca ttctggaggg agatatggcc aagaaaaagg 19980tgatacagga gatgatgaca tcataggccc aaatgaacaa ggtgagatat gatggagcag 20040atataggggg tgatggtggg gtcattacac aggggacaat gggaaagttt atatggtagt 20100ggattaggat taggaaaaag tgggtaatgg gaatggaagg gagaggagag agtaaaggag 20160gtgatggatc agagacattg gagccaatag aggaaatgat ggaaccagtg gtgtcataga 20220gatgatggag atgagagaac cgatggagaa gccaatggag aagccagtga tggagatagg 20280gaaccagcag cgttgtcagt tgaggtgaca gaaccatgta gtacccaatg gaggtgatgg 20340agccaatgat atagtcaatg gtgatgacag agccatgaaa gagccagtgt cagagatgat 20400gaagttggtg gagaaatgaa gccaatgaag tcatcagaaa agatggagcc aaagcaggag 20460ccagtgaaca tgacggagcc aatggatggg ggatgtagag gcaatgaaag agataacaga 20520aataacagga gaggcatttc aggagatggt ggagccagta gataggttac aaaatggaat 20580gagaaagcca gtagaggggg acttttgctg aacccaacag ggactcagaa gagatagagc 20640tagaatgagg actgagtctc caggagtagg gaggatgtgc gtgggagcca cccacccacg 20700tgaaagggaa ccccgggata gaggtgaggg aaggtgtata gggcgagagt ggattagtgg 20760aaaggccagt tctcacattg acttcagtga cggcaatatc cactatgctg cccaccacaa 20820taagagcgtc aaacgtgttc caggcatcag tgaagtaatg ctgcaagtag gagaaaagca 20880gtgccctgtc ttctcaaagc tcgcaagcca gggtaggggg ccagtagtag tagggggcgc 20940cggagggggg cagggcttcg cttggatcac acgatgggcc tgcacctgag tccctctcac 21000tccctagact cacactccct ggctgctgcc cgcatcgcca accccagagc tctgcaatga 21060gctccactcc caaggaattc atccactggt gggtggggct agagaagggg ggccaccttg 21120ggcttgaagg cgatgatttt gagcaccatc tcaatagtga agaggccagt gaagaccatg 21180ttgaggatgt ccatggcata gttgaaggga gcagtctgct catagtgctg caggagaaaa 21240tcaggttgag atggagcctg aggcagagat gccgccacac ccaaccaaat attccctggc 21300ctgggctgag gcgagtctgg ggacttgatt gcactgtgta ttggtcaaac ctcttgcact 21360gtctgcctcc cattatggat actggaaagt ttagttcttg attactagct tttcttgtag 21420ttaggcaagg ccaggttgac tagtgagaca taaacataaa tctgctgttg gttagcagct 21480tgtgtaaaaa aaattttgct ttccgatgaa aggatcaaat aaggagaggc cccaggtgca 21540gctctccctg ccttgagtgt ggttttgatg cctggagctg tgatcatgaa acaacaatca 21600tgaaaatgaa tgccaacttg ctaagaatgg tgaagcagaa agatagagca tgggtcccgg 21660atgaacaatg aacaaacagc ctacctctgc acttctcaat acatgaggta attaccacag 21720ttcattgact ccaggaggca catcttttta cattttagaa tctctgaaat tggggcgcat 21780tttataacaa atggcaactt acaatggcca ctggctactt tttttttttt ttccacattc 21840ttacatctcc gaaatcagaa tgcaacttac aagtactggc catagactta aagaaatgtg 21900gcaaatgtct ttatagctta agttgctgtt agttggcagg gggtttgtta tttgcagcct 21960taaatgttcc caatggttcc caagggatta tgtggagata atagggtcag gagtctggcg 22020ggggtcaggc agggatctca gacctgcatg gctagggcaa ctgtgttgag caggatgagc 22080aggaacatca ggtactcaaa ggcagcagag ttcacagtgg cccacacacg atactgatgc 22140gggttcttgg ggatgtaacg gcggagtggc tgggccttga gggcatattc cacacattga 22200cgctgcatac cagggcaggg catgagtgag cagtggggtc ctgaggcaag gagggaaggg 22260ttggtgacca tccatagggg gtcaggggtc agaggtcacc tggttcttgt ccagctcaca 22320gttttggtac tcctgctcgc cctgggcacg gaaagtgatg atgacgaagc ccacgaagat 22380gttcatcatg aagaacgcaa tgatgatgat gtagacaatg aagaacactg agatctccac 22440acggtaatta tagatggggc catggtcctc tgcatatgca tcgatggcct tgtatagcag 22500tctgtgggag ccagaggggg ggtagaggtg ggggcaggtg aggggatatc ccagccccct 22560cagacctgcc cccagtctac ttatcacaga caccccagag tttcctgcct ggcaccccaa 22620agacccctaa atctcctgac ctaaccacct gggacctcca tacatactct aagagacctc 22680taccaggccc tcaaagagtc caaatcctct gtcccatgcc ctcagaaacc cccatagagt 22740cctccagaga atctccattc agaacctcaa agacccagaa atcccctaag tttccacaga 22800ccttcaaatc cccttctcca accacttgga gacccccatg cgcctcccca gaacttccac 22860acaagccccc aacgatctgt aaatttatcc ctgtcccatc aacttgaatc cccactccat 22920ctccccacag agccccacaa tgagtatccc aaagacctgc acagctttct agaaatcgct 22980gaatccctgc ttcatctctt cagagaccaa tcccagtccc ctcagacctc cactctgtcc 23040ccccaaacca tctccaatcc tttccaggct tctatctagc tacctagtcc cttagaaacc 23100gctgtccaat tcctggagac ccctcactca agtccccaaa gaacccccac ccagcttctg 23160ggaactccac ctctacagtc ctctgataca cccaccactc tccagaaaac cctatccagt 23220ctcgagagac cttcgtcaca tccccaaggt acactcccac ccattcccag gagatcccaa 23280cccaatcctg cagagacccc tgcctcatcc cctgataaac ccagggccct gtccctcacg 23340caggccagcc ttcaaaggtg gagacagtga acagggccat catggctgaa aggacattgt 23400caaagttgaa atcactgttg acccagagcc gctcccggac caggggccgt gacacgtctc 23460catctgggta taccaggaag gagcccctgt ggatgtgcaa acaggttaga tgggtgagga 23520ggggtgagga actggggaaa gggtctgggg tctccagcat ggaggcagca ggacatagtg 23580gtggagcaat atgttctagg ttcaaatcct gtccctccat gtcctggctg ggtgaccttg 23640cgcatttacc ctttctgtgc ttctgtttcc ccattctaag aaataaggat aataacagaa 23700tcgatttcct ggcttgtcac aaggattaat aaatgtcaca agtcaataaa tgagactagt 23760gtctggcaca tagttggcat catttcatca tcaccattat catcctcatc atcgcccacc 23820aggaaaaagt aaaacgaata atagccaacg tttattagca gtaactatat gccaggcact 23880gagctagatt ttctttttct tctcttcttt tctattctct cttttttttt gagacggagt 23940cttgaaaaag actctgccca ggctggagtg cagtggcatg atcttggctc actgcaacct 24000ccgcctcccg ggttcaagcg attctccctc ctcatcttcc agagtagctg ggattacagg 24060tgcacaacac caagcccagc taatttttgt atttttagtg gagacggggt ttcaccatgc 24120tgactaggct ggtgtcgaac tcccaacctc aggtgatctg cctgccttgg cctcccaaag 24180tgctgggatt acaggcgtga gccacagcgc ccggcccaag ctaggttttc tgtgtagcaa 24240ctttacattg tacaaagtac attttccatg tagtagctca tttactcttc acaacagcct 24300tgtgagatag gcactgttta gccccgtttt tcagttgagt gaaatggaac tgagtaagtt 24360taagaaatgt acctaagtct catgaagtgg atttgaaccg taatgtctga cttcacagcc 24420caggctttta gctactaccc tctacaggag tctcaagatg gaagctgggg gctcagggtg 24480ggaatggtga ttgctaatgg gtctggagtg gaatgtaggt cacttgggga atgcggagag 24540ggatttgggg gaggtatcgg gggccgccga agacttggtg agatctgagg gcctctgcag 24600ttcttgggac aattctggga ctatatcttt gggccttggt gagatctaga ggctctaaag 24660tctttgggag gggtcctgag ctccgtggac ggcagggtct tgggcactca cttgcattct 24720tgaggggtgt gtttggcctc gtccgtgcag gtgtagaatt tcccctgtag agaggatgtc 24780tgtcaagtag gttcaccctt catcacactc ccgcccagac ccctgcctgg cattccctcc 24840agtgtttgcc ccaccttgaa gagctgcacc ccgatgcagg cgaacataaa ttgcagaagt 24900gtggtgacaa tcatgatgtt tccgatggtc cggatggcca caaatacaca ctgcaccaca 24960tgctgcgggc acccaagcat atggctactg aacactacag gccacagtgg tcatggggca 25020gggactctgg tcatagatgc agctgaggga cttgggctgg ggacatgtgg tgatgggtca 25080gggatgtatg gttagcaaca tgtgttcaag aggcagtgtt atgggctaga gacgtgtggg 25140catccaccag gaataagtgt ttgccgggtg gggatctgtg gccacctgtc agggagctgg 25200gtctgagaaa tgtggccact ggtagaaaca tatggtcact tggaaggaac atgggtcagg 25260ccacatggct agtggtcaga gatgtgtatt tatgtgtcgg gtccaggccg tatggtcatg 25320catccgtgca aatggtcagt ttgatcagtg atcctggccc caggggcagg gcagggcctg 25380ggtcctgtgg gtttgggtgg ggtggggtta ccttgagtcc cttggccctg ttgatggctc 25440ggaggggccg cagtactcgg agtactcgca gaatcttcac caccgagatg gcgctggagc 25500tggggaaggg gcaatcctca ggcattggcc ctggctgggc cctggctgtg agaggaatgg 25560ggtgggctgg ggatctgtca cttactggat gccaaaggag atgagggaca cactgaccac 25620cagcagatcc aacatattaa accagctacg gcagaaggag ccgcggtgca ggaaggcccc 25680aaacactgtc atctggggac aggacaagag gctagactca gacctgggga tggtgggggt 25740tgggggaggt ggggatgggg acaggagagg tgggctgcaa gaaccggtgg ggagagtgcc 25800agggcaactg agggtaggac tggggtccca ttagtcacct ttagtagaat ctccacagtg 25860aaaatggagg tgaaggcata atcgaagtaa cccagaatct ggggtgtgag aaagagcggg 25920gagccactga gtcacggctg agggggatcc taggtgactt ctcaaaaggc ttggccatca 25980gaaagattag ggttcaaatt tggacccttc tgcctctacc cagctacatg ggcgcctttg 26040gcctgcttaa cctctctgag cacagcttcc tcctcagtca aatattgatc tcctccctgg 26100tgcttccccg tgtgttgcac atgccgtaaa ggctggcatt gcatcagctg cgttaatgag 26160cccctctctc ggctcaccta gggctcttct ccttatctcc accctgaccc agcttgtgcc 26220cactaggaca cccctgggag tgtcccctca gctcctagct cccagccaaa ggctcacatg 26280gttgcggaag gagtgggctc ggatggggtc ctcagcggcc agggacacac tgctgaggat 26340gatgaacacc aggataagat tggtgaagac atgatggtgg atgagggtgt ggcagccctt 26400cctcagcctg tggagaggga gtgggccatg gtcagaactc aggaccacag aattgttcag 26460ggattccaag ggatacagtt ctggccctga cacaacaccc atgcccccac tcccaccttg 26520gctgctcctc catgctcctc cacctgccct agcccctcct gcctcacccc ctgccacttc 26580cggcactcac gggttggttt ggctgaggca gaagaaggcg ctgccctcag ggatgggtac 26640caccttctcc ttgggtacaa cttcctgcag gagttccaca ccccctgcac cctcttcctc 26700ttcttcctct tcttcctctt cttcctcctc ctcctcctcc tccatgtctg gcaccagaga 26760aaagcaaaaa aaaattaatt gagcaagttg atgtaaagca cccagaacag tgtttgccac 26820gtggtcaatt cttcccattt ccctgctaga atgtcagctc ctccaggaag gtattttagt 26880ctgttttgta tgctgctatg tccacagcac ttagaacagt gcctgggaca cagtaggagc 26940tcagtaaatg tgtgttgaaa gaatgttgta tatacagact attttataat aaaggctctg 27000agaagttcag cagtaaacaa aacgtgtttc tcttcttctt gtttttcttt aaagataggg 27060tcttactctg ctcccccagg ctggacttca gtggtgccat catggctcac tgcaaccttg 27120aacttctggg ctcaagcaat cctcctacct tagcctcctg agtagctggg actacaggct 27180tgcatcatca tgcttggcta attaatttct tttttagaga tggggtcttg ctatgttgcc 27240caggctgcat ttcccttctt taacctcaat gttcccaaat gttgctgacc acataccaag 27300tctgttattt tcaggatgat cttggaaaag gctgatctct ggagaggagg gtctcagggg 27360tcaggagctg tctgcctgag ctctttcccc aaggtcccac acctgctcca cctggccctg 27420cccacttgcc tgctccttcc ctccttgcac cctcctcttc ctctttctcc acaccaggca 27480cctgaggaca ggaagacggg agtcatcaat ggtgagggag acacagggaa tggacacagg 27540ggaggcatgg aagaaatgta ataatagggg aaagaatagg cagaggatga aggttaaaga 27600catgcacata tggctgggcg cagtggctca tgcctgtaat cccagcactt tgggaggctg 27660aggcaggtgg atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc 27720ctgtctcttt caaaaataac aaaaattagc cgagtgtggt ggcgcatgcc tgtaaatccc 27780agctacttgg gaggctgagg caggagaatc tcttggaccc aggaggcaga ggttgcagtg 27840agccgagatg gcacaactgc actccagcct gggcgacaga gtgagactct atctcaaaat 27900aaaacaacaa caacaaaaaa acagatatgc acacatagac aggtagtggt gggagtggga 27960ggtgtagaca gtgcccaggc atctaactca ccaggccttc attctcctgt gggagatcct 28020tctcattgct cttctccctg tgcgaggaaa taggggtgat tgctgcagat gggctaaggg 28080ggatcactac agtcagaaca gacctttctt gggtcccata gaactttctt gcttccgctc 28140ttccccactg cccctcccaa cagtccgttc tcaaagcagc cagagggagc ctgttaaacc 28200ctgtctggga tatcccaact ctgcccacaa ccctcccatg tctccttaat tcaggaaaaa 28260ctacagtcct caccctggcc cagaatgccc tcaatctggc cctccatttc ctccactgac 28320cttcactcca taccactctc ctccttcctc actccattcc agcctcactg tttttatttt 28380tttgagacgg agtctcactc tattgctcag gctagagggc agtggtgcga tattggctca 28440ctgcaacctc cgcctcctgg gttcaagcga gcttgtccag ctaattttct tttgtatttt 28500tagtagagac agggtttcac catattggcc aggctggcct cgaactccta agctcaggtg 28560atccaccctc ctcggcctcc caaagtgctg ggattacagg cgtgagccac tgctcccggg 28620catcgctggt attttttgaa agtgctaaga atgtggtcag gtgcggtggc tcacgcctgt 28680aatctcagca ctttgggagg ctgaggcggg tggatcacct gaggttagga gttcaagacc 28740agccttgcca acatggtgaa accccatctc tactaaaaat acaaaaaaat ttagctgagc 28800atggtggtgt acgtctgtaa tcccagctac tcaggaggtt gaggcaggag aattgcttga 28860acctgggtgg cggaggttgc agtgagctga gatagcacca ctgcactcca gcctggatga 28920caagagtgaa actccatatc agaataaaaa aaaaaagtgc taagaacatt agcgccccag 28980aagctttgca cttgccactc cccttccttg aatactcttc ccccagatat ttgcatggct 29040cactccctca cttcattcag gtctctggtg aaatgtcact tcctcacaga gaccttcctt 29100gctgacctta accccaaaga gcaatcttca tttcttgcta tcctttcatt ctgttttctc 29160tttattacat attattaaca tatattatat attcatttgt tttatttttc ttgccttcct 29220caactagaat gttagttcca tgaaggcaag gactcttgtc tgccttgacc cctgttgtct 29280cctcagcatc tagaacagga cctggcacac agtaggcagt caatagatgc atgttgaatg 29340aatgaggact ggcagatggt atctggagat ggggattacc agggtgacta gaggcatctc 29400tggtggtggg ggtgtctgag gggttcccag tcagggatcc cagagagaga ctgtgttagg 29460ggtggagcca gatctcaccc gcccttgtcc ttggcagtgc ctgcatctcc actggccagg 29520ttgtccacag caatggcaag aaacacgttc aacaggatgt ctgggatgag cttaggctgc 29580aaaggatgga gaagcaccct gcctatagac cacccagttc cccttaccca ccccgactcc 29640accatcatcc agccccacaa agctccaagg atacagttgc cacagatgaa gagaatgatg 29700aaatagatgc acaccaacat tcctgggaag aaggggccac catatgccat gataccatca 29760tacatgacca cgttccagtc ctcacctgtc aggatctggg ggtgggaagt cactgtggag 29820ctcttggacc ccctccaact ccagggtctc cccgacccac ccatcccatg gtctccagat 29880cctacctgaa agacagtgag gagggcctgg gggaacgtgt caaaggtgct tcgcttggtg 29940tgggtctggt caaagttgaa cttgccccca aacagctgca tgccaagcag ggagaagata 30000atgatgaaga ggaagaggag aagcagcaag gatgcgatgg atttcattga attgagcagg 30060gatgccacca gattgctcag agaagcccag tgtctgcagc agaaatggga gggggcaggg 30120tcgatgccat gtccactgga catggctgga gcatcaggag ccagggcagg gctccagctt 30180gagatgcacc tcggcaggtt gggctcaaat aggctttggt gtcagggtta gaaatgattt 30240tggagttggg gtgagatgag agtaagaagt gggggaagct gggccctgtg gtggctcacg 30300cctgtaatcc cagcactttg ggaggccaag gcgtgtgggt catgaggtca ggagactgag 30360accatcctgg ctaacacggt gaaaccccgt ctctactaaa aaaaaattag ccaggcatgg 30420tggcaggtgc ctgtagtccc agctactcag gaggctgagg cagaagaatg gcgtgaaccc 30480aggaggcgga gcttgcagtg agccaggatt gcactgcact ccagcctggg cgatagacag 30540agactctgtc tcaaaaaaaa aaaaaaaaaa aaaaaaaaaa agaagaagaa gtaaagaggt 30600aggggaaagt tgagagtttg gggttagggt tggtggtagg ggtataatgg gagactggga 30660gatgaggtta ggatggagtt tttttttttc ttatcttttt tttttttttg agatgaagtc 30720tcactgtgtt gcccaagctg gagtgcagtg gcacaatctc gtctcactgc aacctccacc 30780ttccaggttc aagcgattct cctgcctcag cctcccgagt agctgggact acagtcactt 30840gccaccactc ccggctaatt tttttgtatt tttagtagag acggggtttc actacattgg 30900ccaggctggt cttgaactcc tgaccttgtg atccacccgc ctcagcctcc caaagtgctg 30960ggattacagg cgtgagccac cgcgtccagc ctaggatgga ggtttaatgg aagttggtga 31020agctagaatt gggtttcagc ttgggactgg gcctggtgtt ggggatgggg ttgtgatgta 31080tttggggttg gagatggagt taagggtaag ttgtggttga gggctgggat agaggagggg 31140atgggatgag actggggctg aggctggaaa tggagttgca gttgaagata gggtgatggt 31200taagggttag ggacgtggga gttctctttg gggattgagg atggtattat ggttggtggt 31260tagtgacagg gatggggctg ggagttaggg atgaggtggg agatggagtt gggcttggga 31320atgggtgtta gcgttgtaaa tgggctcata ggtgacctgt ggttgggggt tgggatcaga 31380gatggtattg tggttggggg ttgggatggg gtttgggctg ggaatagtat ggcattgaaa 31440tcggagacag ggttggggaa aaacgttaga gcaggagttg aggttgagga aggcaatgga 31500aatggagtag acattctccc cagtggatga agacaggaac ttgtgtctgc tttgattcct 31560ggtgtttccc tagggcttgg tagggtgcct ggcacacagc aggcactcaa tggatgtctg 31620ctcaatgaat ggtgaagcag atgaagttat cattggagct tgggtggggg tgttgaggct 31680gtgtttgagg cccttgtctt ccccctcccc taatacaaac ctggtgacct taaagatcct 31740gaggaggcgc acacatcgga gcactgagat gcccaagggc tgcatggcgc ccacctccac 31800caaggtggtc tctaggatgc ccccacagac cacaaagcag tcaaagcggt tgaagaagga 31860agacacatag gcagaggggc ccagaccgta caatttgaga agcatctcca ccgtgaacag 31920acagagcaac actttgttgg catactctgt ggggagagag gcagggatga tcgggctggc 31980cagtttctgt ggtgacatgt ggggaggtaa ctagggacca tggggtgacc cacggtagct 32040ggtatcaggc caggtgctgg ctgagggaag gcttagggct agggattggg gtgagtttgt 32100aggtcagagt aggtgaagtt ttgaggggct tggagatggg gatgaggggc agagtcgggg 32160tgggggccag agtcagtgtc tatgagtcca gaagacccaa attcacaagt agagttagag 32220tctagggttt cttcgggact ggggctggtt tgtgggttat gggttagaaa tgtgctcggt 32280attcaggctg gggctgagat tataatgaat aatagtaaga gctgacactt ataaagtgtt 32340agctatatgg tgggccatac aatgtcctaa gcacttttca ctttacatca tttactcctc 32400ttcgctgctt catgaagtgg atactactat ctatcatctc cattttgtag atgtggagaa 32460cttttccaga cagtgtgatg ataagagcct ggattctgga gccaggctgc ctgggttcta 32520attctggctc tgccactcgc tagctgggtc ccgagattag tcgcttaacc cctcagtgcc 32580ttgatgctca gtgttctgtg ccttagtatt taattcgcat aaggttgtgg tgaagaaatt 32640gtcagtacac ataaagaaag gtgctgagaa aggagcttga ggcacacagc aagtgctacc 32700tgtgtaagct acaaggtggg atgggacagg ctaagggctc tggatttatt tgggtagggg 32760ggaaggcagt gtcttggggg ctgaggcgcc ctagaggttt tggatttaag cttctggggt 32820agaaggaata ggaggctggg ggagacgact agcaggctgg gggtgtaccc tggatctggg 32880tgagccacac aggctgcccg tggtgctcag aggcgatggt caacgtgttg aggaagacga 32940gcaacagcac agcccagtag caggcattgg acttcactgc ccgacggcag cgtgcccgaa 33000ggacccggtt ggctcggcgg aggcggcggc tgggggaagg ggagcccaca ggctgagatc 33060acctacctaa gcctgccctg gggggctcag gtgggggatc caaaggtcat gagggcttgg 33120aggtgggggg tttcaaagtt atggagtcaa ggatttgcac aactttccca actcaccaga 33180ctctggtttt catgatcttg tttctgaaaa agaaggggga tgggaggtgt gtgtcgtaaa 33240gggcagaagg ggtgtcagtg actggggcca gaggtcaagg actgagatcg aggcttgagg 33300ctaaagaaag gacttgagtc agggtttgga ctggcttctg ggctgggtca ggggctgggg 33360gccctcacag gcagcgtgta cagctggcca gagccccctc ctcctcatcc tcatcgcctt 33420gggtctctgt catggaaccg gtgtcactgg ctgggaggct ggctgtaggc aaggtgggga 33480tagtggtcag gacctgaaga ccccgaggtc caatctttgg ctttcacctc tgcctaccca 33540cccccacccc agcctggctg gacccccacc atggctgctg gtggagtgtg tggagcgagt 33600agaatgactg aaccagcgca gacgtccacg cctcctattg gtcagctcgg ccagctgtgg 33660ccctgcaggg agagaaagga caattaggga ggacatactg ggggcagggt tagggccatg 33720tctagggtag gggtgtcatt tggagtcttt atagctgagg ctggtcttga ttctgcattt 33780tgagccataa cttgggggct ggggctagag ttgttgcaga tcaggcctgg gtgggtctca

33840ggagggcaga ccacatctaa ggcatgcagg gaatgggcca gattggagtt gcctacgatg 33900gcccgcccgg ccctcttcag ccatagaacc aaggttgtca tcggcggagg ggtcctccat 33960gtccagctct tcggcttgag tgatccagtc caggtagccc cgcaggtctt cctccatctg 34020ctgcttctcc cgctgcttct ggaagtcccc gcgagctttc gctttctctc tctccttgga 34080gaactccctg agggaggagg atagagggct agggggagga gccaatctga acaccttgct 34140ttcatcactc cagccacact agcctcctag ctactctttg aatatgccaa gcacattcta 34200gcctcagggc ctttgcactg accattctct ttgcttggaa tgcttttccc tcagataacc 34260acatggtgca cccacctcat ctcttttttt ttcttttccc cagatacagg gtctcactct 34320atcactcagg gtggagtgca gtggtgtgat catagctcac cgcagcctcg acctcctggg 34380ctcaagcgat ccttccacct cagcttcctg agtagctagg actacaggtg tgcgccacca 34440tgcccagcta tttttgttca ttttttgtaa agatgaagtc tcactatgtt gcccaggctg 34500gtctcgaact cctgacctca agccatcctc ctgccttggc ctcccatagt gctgggatta 34560caggcatgag ccactgcacc tggcctcttt cacattttta ctcaaataac accttgtcaa 34620gacagccttt cctgacatcc ctatttataa tcacaagatc tctccattct tatcttttat 34680ctttcctttc tttccacagt acatattcca tataaaacac tatatatatg tgtgtgccaa 34740tatataaatg cacacactta ttttattgca tatgttgcta ctttctgtct ctccccatca 34800gaatgtaaaa ccaggaaggc agagattttt gtcttttgtt ccactcctaa aatggtgcct 34860cacatacaat agatgctcat gattgaatga aggagtattt cagatgggtt ttgaaggatg 34920tctaggagtt tgccaggcac aaagaagtgg gcagtggctg aggggtggga agcagggagt 34980gtctaggtcc ctcacccact caggacgcca agcacaaggt tgaggacgaa gaaggaccca 35040aagatgacaa ggctcacaaa gtacacccag ggcagttcat accccatggc atcttgcatc 35100tggggagaga aaaggcatgc atccctcagg gtaggcagac actcctgcgt gagtgtcagg 35160gaggaaaggc aggtgcagcc tttgagctct gtgcccacag tccacctcac ccagtagagc 35220acatcggtcc agccttccat ggtgacacac tggaagactg tcagcatggc gaagaagaag 35280ttgtcaaagt tggtgatgcc tccattgggc cctggccagc gcccgcggca ctcagtctgg 35340ttcagcgtgc acgcacgccc tgatcccgaa gacgcacagg gcgatgggtc ctcctccgct 35400tccatgtctg caggaagact ggagcttggg gcttcgaagc aggcccactc tcagtcgctg 35460ccaccctgaa gccccgccca cttaggaagc tcgacttggg tcctagattt ttctctttct 35520ctctttcttt ctttcttttt ttgagacggg gtctccctct gtcgcccagg ctggagtgca 35580gtggcacgat ctcggctcac cacaacttcc gcctcccggg ttcaagcaat tctcctgcct 35640cagcctcctg agtagctggg attaccggcg tgtgccacaa cgcccggcta attttttttg 35700tatttttagt agagacgggg gtttcaccat attggtcagg ttggtctcga actcctgacc 35760ttgtgatccg cccgcctcgg cctcccaaag tgctgggatt acaggcgtga gccaccgtgc 35820ccggccagtc ctagattttt ctaaggcaac acttagctcc acttctaggg cagaaccctg 35880cgagctccgt attctgaacc actgggaagc tagggatccc accaaccggg atttaaatcc 35940tggcgtggag tcttgtaaat aacaatgagg caagagcctg ggggcggagt cttgtgaatt 36000tggacgcgtc tcttgaaaat aggggagggg caaggcatgg gcgggagtct gggtaaatgg 36060tctgagtgta tgagaacagt cgtggagaat aaagcttttt tttggtttgt ttttgttttg 36120tttttgagac cgagtctcac tctgtcgccc aggctggagc gcagtgagtg gtgcgatccg 36180atctcagctc actgcaacct ccatctccca ggctcaagtg attctcatgc cgcagcctcc 36240cgagtagctg ggattacaga catgcaccac cacgcccggc tcaactttgt atttttagta 36300gagatggggt ttcactatgt tggccaggct ggtctcgaac tcctgacctc aagtgatcca 36360tctgcctcgg cctcccaaag tgctgggatt acaggcgtga accacggcgc ccagccgagg 36420ataaagtttt ttaaaattgt ttttgttatt attttttgga gactggatct cactattttg 36480cacaggctgg tagcaaactc ctgggcgtaa gtgatcctcc cgcctcggcc tcccaaatgc 36540cgggaataca ggtgtaagcc aaggcgcaga gtttttgttt tgttttgttt tgtttttgag 36600acggagtctc gctctgtcgc ccaggctgga gtgcagtggc gcatctcggc tcactgcaag 36660ctccgcctcc tgggttcacg ccattctcct gcctcagcct caggttcacg ccattctcct 36720gcctcagcct cccgagtagc tgggactaca ggcgcccgcc accatgccca gctaattttt 36780tgtatttttt tagtagaggc agggtttcac cgtgttagcc aggatggtct cgatctcctg 36840acctcgtgat ccgcccgcct cggcctccca aagtgctggg attacaggcg cagagtcttg 36900tcttaagtgc ggggcacttg cacttaagtc ttaagtgcgg ggcaggacct tagcctgtgg 36960gtacagtctc gtgatggggc ggagctatta gcgaggtggg gttgtagtct gatttagggc 37020agagtcttga atatatgggt gggacttggt tatctgggga tggaacctga gcctgggggc 37080cgaatcttgt gcatttcagt gggtttccct gagtgcagca ttggatctag gaaccgaagg 37140agggggcatg ttctgcctag gaggggcggg cagactaacc ggatcccagg aagtagcacg 37200tcttgtgcat tcgtccaagg aacagctcga gcccaatgat ggcataaatg atgatgacga 37260agagcacgag cagtgcaatg tgcagcagcg gcaccagagc cttcatgatg gaattgagca 37320ctatgtgcag gcctgcgggg agggaggggg aggcagaaat aagggcgggg tcagctcagc 37380cccaccccca ccctccacct ccgacctcgg gggatgtgcc actcactcgg gaccccagac 37440accagcctca gtggccgcag cacccgaaac gccctcaatg ccttcacatc gaagcctcct 37500ggctttcccc cggtgtgcgg ggcgtcgcct ggccgtccgg ggccctgctc cagcagaacg 37560ctgaacagcc tgatggggga gcaccgggca gggaggcgga ggtcaggcct tgggattccc 37620ccctccaccc taaccttctc agaaaagcac aagtccctgt aactagaaat agaaatgggt 37680tttaataata ttcctgattc tggctgccga atgggggcgg tctgaaagag tcgctttcct 37740gagcccagaa ttcagcgggc ctgacggtag gaaggcgact agggtggggt gcctcccgca 37800gacgcgcacc cgaccacgac gatgatgaag tcgagtaggt tccagccatt gcggatgtag 37860gcgctggggt ggagcaccag cccgtaggcc acgatcttga gcaccgtctc cacagtgaaa 37920atcaccagga atacgtactc cacctgctcc tgggggtggg accggggggc gggtcgggaa 37980gtcggggagt tatttgaagg cgaggtttga cgggggcgtg gggagcatag tcaggaccga 38040gggtggggct gagccaggca gggccatccg ggtcagagag ggggcggggt ctggctggaa 38100ggagtgagct ctactggggc tctatttggc tgggaactgg ctggggcggg gcgggcctta 38160ccaggttgtg gttggcagtg ttggagtcgt cctcagggaa ggggatgtaa actcccaggg 38220ccacgcagtt ggcaaagatg gtcagcagga tgaggatgtc gaagggcctc aggtggacac 38280agtcaaggac cctggcagag tggcattgga acccccctac tcccttggga acctcccacc 38340cagtgctgac ccttgacgcc caaggtgaca ggtcctggca ttctcgaccc ctgccctgac 38400aaggccttga ctttgtccct cctggaaacc ttaaccatcc ccaactcttg ctgtgattca 38460gtccttgact cttgaaccct ggtgaccttt gcctttctga acctgccccc caacccagtt 38520cttgactctt gaccttgatg atctcagccc tcctctgttc cagccctgac ccaattcttt 38580actcctggta ccctgatgac ccttacccct gggccctgcc cctgccttcc aggatgcttc 38640cactccacga tgctgatgca ggaccgtcgc agaggattgg ccagggtgag gcagaagagt 38700gcccgaggtg accgctgggc actggccact gccactgtct tgtgcttgct gtgctggttt 38760cttcgcttag gggtccctag gcctgatgcc ccactgcttt caccttccac agctgggggc 38820ccggggcaca gcccccattc gggaccaggg cctgccccat tggctggact gggctctggg 38880gtggtgtcta tatggagaaa ctgtgtcagg gagggacagg gtattggggc aattgtggct 38940gcttcctacc tgatcctgtc cctctgtttg aaggaaggag agaagagcat ggaaaattag 39000ggagaactgg gagtagctgg gttaaggact gggaagtaag agtgttcgaa ttaagattag 39060agtgagagtt tataggggag tgaaggtgag attacatttc caagtgaagt agggtttggt 39120gtttaagttg ggggtagagt ttaaattgcc atggggtggg cgaatttaag gttaaatttg 39180agtggtgggt tttgggttag ggtggggatg ggtttagaca taggtaggag tggagttttg 39240tctaagggtt aggactgggt attttgaatt ggaattagaa ttaggataca ggttagggtc 39300gaagttgagt tttgagtggg acttgaattt tgcattgggg ttttcagcta aggccactcc 39360cagttcccat ggtctcgttt cagcctcaga gggcggtaga ttggcctggt taagattccc 39420agctttggat gaaacaagct gcctgagttt aaatcctgac tttaccactt ggtagctatg 39480tgttgccttg ggcaaattat gtaatctctt tgagcctcag tttccttatc tgtataataa 39540ggatgataat aacagtacct aaaatatagc ggtgttatga ggattaaatg agttcagaat 39600caaaagaatg cctggcacat agagttattc ccattttgca gatgagaaaa ttgagaagaa 39660aaagttaagt gacttgctga agtggctgaa tggggagtca cactcagtct aactctagag 39720ccctcatggc ttactaccac catcaagtat gtcttctctc tctttctctc ttttgttttt 39780gttttttttt ttaaagggtc tgtgcacttt ctggaaagtc agagactgca atcactaaca 39840gtattgacat cccggggttg aggttgaggt taggtcactt taggtctgta tctggggtgg 39900ggtttagact ctaacagggg tttgccttct ggctggtaga gtttgtgtta ggtttcaggt 39960gggcgtttgg ataggggtca gagccaggat tgaggttggg tttcctttgg gaactaaggc 40020tgtggatgag gctgagaggc catggggggt ggtgtctggg tcaggctgag cgttagggct 40080ctggtggcag agaggctgtt tggggctggc tgggatgggg aacagggtga tgtggttctt 40140gtcccctaga ggctctctgg gggtggggtc tgggtgggca atgggtgggt cagaggggct 40200ctaccttgct ccaagggtct gcagggatgg aaggattctc tcacctttcc cgccttcaga 40260ttccgacatc tttctttcga gattgaaggg ccatctgcac acaacccccc tctcttcccc 40320cagctttgga gggattaagg ccgcagggcg gggcaataac agtgttgtat gggagggtgg 40380cggtgggggt gcaaatggga cgtttgtggg ccagagcctg gcaacagatg aaatatttac 40440caaatatata cctgcagcaa acagtcaaac taaaataaga aaaacgatgt cattttccag 40500tagtatatgg aattcgaagt atataggaaa aacctaatat atgtgcaata tctctatgga 40560aaaaagatca aatattatca attattatta tttttagaga cagggtcttg ctctgtcacc 40620caggctggag tgcagtggcg tgatcatagc tcacagtagc gttgaattcc tgtgctcaag 40680tgatcctctg acctcagcct ctgtagctgg gactgcaggt gtgcactacc atgcccaact 40740attttttttt taaattttgg gttgcttcga actttatttg agaacaacag aagataaacc 40800tatcaaaaga acacacaggt gggtgcgggg gcacggctag tggcggcggc cgggggcatc 40860cgggctaagg cttttacttg gctgcagact ggtcggattt cgcagctcct aggcccccaa 40920gctgggcccg tgactccaag gtgatctcgt tggactgcgt ggctagcttg ggcatggggg 40980cctccacggt cagtgtgccc tcaggggaca gggaggagga gaccttggtg gggtccacac 41040cggggggcag cgtgtatttc cgggtgaagc atcgggagat gtagccatgc tcgtcctgcc 41100gctcctcgtg cttgctggtg atctccacca tgccatcctt ggtcttgacc gtcagctcgt 41160cggaggcgaa gtagttgatg tccagggata cgcgccagcg gtccgctgtg ggccgattct 41220ccgagacccc gccgctgagc tgccggctga gcgcgtagct gtaggcgggc gcggccaccg 41280cggggctctc gaccgcggcg ggggcagggg acgcacgtag cggggccagc agctggtgcc 41340caaccactgc gaccagcccg aaggcctggt caaagaggcg actgtgcggg taccagtcgc 41400ggaaggggtc ccagctgggg tcccgcagga gcgagaaggg gacgcggcgc tcggtcatgc 41460tggctggctc tgctggggac gtctgcttgg acaagtgtca attttttttt ttttttttaa 41520gagatggtgt attgctgtat tgcccatgcc ggtctcaaac tcctgacctc aagcgatcct 41580cccgccttgg cctccaaaac tgctgggatt gcaggtgcgc gtcggctgtt caagaaatgg 41640ttacatagat ttcttttgct ttatttattt atttagagac ggagtctcgc tctgttgccc 41700aggctggagt gtaatggcgc aatctcagct cactgccacc ttcgcttccc cagttcaagt 41760gattctcctg cctaagcctc ccgagtagct gggactacag gcatgtgcca ccatgcctgg 41820ctaatttttc tcattttagt agagaggggg tttcaccatg ttggccaggc tggtctcgaa 41880ctcctgacct caggtgatcc acccgactcg gcctcccaaa gtgctgggat tacaggcgtg 41940agccactgca cccagccggt tacatagata tttgctgtgt aattattctt tggagtgtac 42000aaatgtgtat catattaaag catttattga atgcatggac actagtaact ataacttaca 42060tagtgctttc ttatcttatg tgtcaggcac atagtacaac gtgctttata taaattaatt 42120ctgttttaat cctaaaaagc aggtactgtc aatagtccca ttttacagat gaggagacta 42180agacacaggg aagtagtcgc tttgtgttaa aattctgccc ttaatgtgta cacttttata 42240gcctagaaga tgaatgccgc cttaaacgaa tgagtcagga agcccctttc tgtccggccc 42300ctccactccc caccacacgg gttgttcctt tttcccttgg aagagcccac tggactgcag 42360gtggaagaac tacagttccc agcagctatt gcaagctcaa cctccgtgca cactcacccc 42420aggcctcaca tccggcatgc gccgtgctcg ctcacagaac tacactttcc aactctcccc 42480acacgacccg tgacactctg tggaccgcga gcacggagca gggtttctac agctgctccc 42540cactttctcg gacccggtcc tggacccagc ccccgactcc gacacggctc caccatggag 42600gaggcggacc gaatcctcat ccattcgctg cgccaggccg gcacgtaagg acagagcccc 42660cgcccacccc cgaagcccac atccgggact ctaaagccca ggaccccgtt tcccgggaac 42720cttaaaaccc gggatcctga cattcagggc tccaactcca ggatcctaag accctcaccc 42780ccttacacac acacacacac acacacacac acacacgcac acacacacac gcacacacac 42840accgccctcc ctgacaccga catcagaggc ctcccaaatc ctttaaccat gatatttggt 42900acacccaaag ctctgggacc cagacacctt gagacgttac aaccttgaaa cctcaagacc 42960cggaaccctg taatctgggg aatcttaaca tcagttccct gagaccctgt tatctggaga 43020ccctaagaca cctgtgccct aagaaccagg gagcgtaaaa ccccaggacc ctggcactcg 43080gggactccaa aaaatccctg gaccagatac ttgggatcct tcaaactcca gttccccaaa 43140cacctggggc ttaaaaaaac ccaggattct tttttttttt ttccttttcc gagatggagt 43200ctcgctctgt cgcctaggct ggagtgcagt ggggtgatct cagctcactg cagcctccgc 43260ctcccaggtt caagtgattc tcctgcctca gcctcccaag tagctggtat tacagccgtg 43320cgccaccttg cccggctaat ttttgtattt ttagcagaga cagggtttca ccatgttggt 43380caggctggtc tccaactcct gacctcaagt gatctgcccg cctccgcctc ccaaagtgct 43440gggattacag gtgtgagcca ccacgcccag ccaaaaaaac ccagaattct aaaatctgac 43500atcagcattt ggtcaccctg acgcacgaag actcctccgt tcagaggccc taaaaaccca 43560ggatgccaac atctagggag tctgattttt gtattgcttg aaactaggtg gtcttgatat 43620tcagggatct tgatacccag ggaccctgac atttggaact tctgaaaaca gcagcctcct 43680aaaatctagt cttctcaaaa ctctagcagc tcaatatgta tgcaaagaac atactatctg 43740ggaaccaaga aacccagaga ttgtgccacc tggacccttt caccttccca gactcccaaa 43800ttttgatatt tttctaaact gagttctctt ccacccacca ttctcctact ccaagatctt 43860aggacttgag gatttcccta gttcctggtt ttccaaatgg aagtcacagt caccaacatc 43920caggatctgt ctgaactgta ggcatcctcc ctgccccgac cctggtacta gtgtttccaa 43980aaggctgcag ggctcagcct gatccctgtt gcctgactat tcctgtgatc aagctggtcc 44040ccttcttcag ggcagttcct ccagatgtgc agaccttgcg cgccttcacc actgagctgg 44100ttgtagaggc tgtggtccgc tgcctgcgtg tgatcaaccc tgcggtgggc tctggcctca 44160gccctctgct gcctcttgcc atgtctgccc ggttccgcct ggccatgagc ctggctcagg 44220cctgcatggt gagtggccct cctcctaatg cacacatcct ctttcttcct cttttacaaa 44280gtaaccaagt tcctttgcaa cacttgcctt tcctctgcaa cacttgcctt tcctctgcaa 44340cacttgcctt tcctctgtca tttttcctgc ggcttagttt tcattagtgg ttaaggctgc 44400tgactgtact gccaaactgc ctgagtagtt tagatcctag ccccaccact tggtaactgt 44460gtgaccctaa cccttctggg ccccagtttt cctatttgtg aaatggagat gataaatgta 44520gtgcttttat gaggagcaaa tgagtttatc cagtttgcac ctgggctgct cctggcttct 44580atgttttaaa tttagctgca aaataccacc cccaccccat agtatccatt tcctagttcc 44640agaaaatact ctgtctgggt catatactcc taaactaatc actctgtgct ctgattggcc 44700cagtctggat gacatggtca ttcctttttt tttttttttt ttgagatgga gtctcacttt 44760gtcgcccaag ctggagtgca gtggtgcgat cttggctcac tgcaacctct gcctcccggg 44820ttcaagcaat tctcctgcct cagcctcccg agtagctggg actacaggcg tgtgccacca 44880cacccaacta attttcatgt ttttagtaga gacggggttt caccatgttg gccaggatgg 44940ttttaatctc ttgacctcat gatccacctg ccttggcctc ccaaagtgct gggattacag 45000gagtgagcca ccgtgcccag ccttttcttt tctttttttc tttttttttt ttttttgatg 45060tgaagtctct ctctgtcacc caagctggaa tgcagtggcg tgatctcagc tcacggctca 45120ctgcaacctc cgtctcctgg gttcaagtga ttctcctgcc tcagcctccc gagtagctgg 45180gactacaggc atgcaccacc atgctcagtt aatttttgta tttttagtac agacggggtt 45240tcactatgtt ggctaggctg gtcttgaact cctgacctcg tgatctgccc acctcggcct 45300cccaaagtgc tgagattaca ggcgtgagcc actgcgcccg gccgacacgg tcattcctat 45360aggacactgt gattagccac tccttcagga tcctgtggag ttgggataag gatgattacc 45420caaagaaagg catgctggtt acccaaaaga gtgtctgcta tattacctct gtgggaacca 45480catatcctgc ctctgctaag agcaattcca acaatgtctc tgtgacagaa taaataaagt 45540gcttttcttt taaaaaatat tttaattttt ttagaggtaa gtgtcttgct atattgccca 45600ggctggtctt gaactcctgg cctcaagaga ttctcctgcc tctgcctcac tagtagctag 45660gactataggc acatgccacc ctgcccgaga atttttaaat ttttcgtaga gatggggtct 45720cgcttttgta gagatgttgc ccaggctggt cttaagctcc tggcctcaag caatcttccc 45780gccttggcct cctgagtagt tgggactaca ggcgtgcacc actgtgcctg gtaaagagcc 45840attctgatga aacactcacc cattcccagt atagagctga gtccaggagt ccagttcctg 45900tattcaagag ctgcaagtaa tgccactccc cctggctcac tcactctgtg actttgggcc 45960agtctttttt ttttttgaag cccaggctgg agtgcagtgg cacaatctcg gcttactgca 46020acctcctccg cctcctgggt tccagcaatt ctcctgcctc agcctgccga gtggctggga 46080ttacaggtgt ctgccactgt gcccggctaa tttttttgta tttttagtgg agacagggtt 46140tcaccatctt ggccaggctg gtctcgaact cctgaccttg tgatccaccc acctcagcct 46200cccaaagtgt tgggattaca ggtgtgagcc accgcgtctg gctctcttaa cctttttgag 46260gctaagtttc cacatgtgta aaatgggtat aagaattgta gctactgtat agggttgctg 46320tgaggattaa acatgagtta atgtgtgaaa agctggttat aataagcttt gcataaatgg 46380gattactatt attggatagg tccgatctgg aacctgtgaa tacatagtga atggaaacac 46440tttgaactga cccaggaagt atatggtggt ggagggacga tagagtaact accgtgaaaa 46500ctttcattta gatatagggg actgggtggc tagagttgtt aaatttgggc cttgcttatg 46560cagtttctgt ctcttagcaa caggtctcag agctccatcc atcccttcgc tctcaggttc 46620acccagctct caggagttgt cacattgttc tctctggggc tcttggtggc cttatgaggc 46680aggcagtctg tcccctggcc caggactgta tgtattctta aggttagcac ttaatagggg 46740ggaagttatg tcttctgttt gcagaggaga gtacacagca ggaggtgttg aggtgggggc 46800tcaggcttcc tgcagttctc tgttcttccc tcagtgctgt ctctcttgga ttttgttcac 46860ctgcttttgc ttacattgat tttagtgggg gttagtgact atggcttttc cagtggccag 46920gaggtacatg tgggctgggc acgttggctc ttgcatgtaa tcccagcact ttgggagtct 46980gaggtgggag gatcagttga gcccaggagc tcgagatcag cctgggcaac atagtgagac 47040ccccatctct acaaaaaata aaaaaaaaat agctgggcat ggtggcacac gcctgtggtc 47100ccagctatgt gggaggctga ggtaggagga ttgcttgagc ctgggaggtc caggctgcag 47160tgggctgtga ttgcgccact gcactctagc ttgggaaaca gagtgaaacc ccatctccaa 47220aaaaaaaaaa aaaaaaaaag actggggacg gtggctcctg taattccagc actttgggag 47280gtggaggcgg gcagatctca ccagaggtca ggagtttgag accagcctgg ccaacctggc 47340gaaaccctgt ctctactaaa aatacaaaaa ttagcctggt gtggtggtgt gtgcctgtaa 47400tcccagctac tcaggaggct gaggcaggag aatcgcttga acccaggagg cggaggttcc 47460agtgagtcga gattgcacca ctgcactcca gcctgagcga cagagcaaga ctcttgtttc 47520caaaaaaata aaaaacaggt acatatggtt gtctggcccc cagcagcctt ggtttatcag 47580cagcaggcaa aaggagttct cttaatccag ctgtgtgctg tccctgtagc ccccccgcaa 47640ctcagcactg ccatgttctg gcatcttttc ttcatatgcc ctgctcctgg caacagtttc 47700tgcaccttag ccacctccat attttggcac cttccccact cctggaatga gtttctatat 47760cagtcacagc tctttgattg catattgtgg aaaaaatcca aagtaaaata ccttaaaagc 47820cagtggttat acttatttga ccttgagcct ccaaaataaa tatatcttgc attgtgaccc 47880agtatactcc taagtataga ataatgatgg cttctaagtg tactctgtgc caggccctgt 47940ggtaagtaac atggtgttaa ctcatttaat tctcaggaca accttctgct atatgtactg 48000ttggtatacc ctctttcaga tgaagaaagt gaagcacaga gggattaagt gatctgcctg 48060aggtcgtata gttggtaaga ggcaaagctt gggtttgaat ccaggaagtc tgctttcaga 48120gtctatgcag ttccaaagca gtggcaactc tggaagaaaa aggctttcct aggccgggtg 48180tggtgattcc cacctgtaat cccagcactt tgggtggctg aggtgggcag attgcttgag 48240cccaggagtt tgagaccagc ctggccaaca tggtgtagcc ctgtctacac tgaaaataca 48300aaaattagcc gggtgtggtg gcatgcacct gtaatcccag ctactcggga ggctgcgtgg 48360gaggatcact tgagcccagg aggcagaggt tccagtgagc caagatcacg ctactgcact 48420ccagcctgag cgacagagcg agaccctgtc tcaaaaaaaa aaaaaaaaaa aggaaaaagg 48480ctttctttct ctcctggcct gtttcctcag tccccatcag aaccctcatg agtccttttg 48540ttctgagcta ttgtgcttct ggtctttttg tcctgtttat ttttaggcac tctttctata 48600ataggtagat ttgctcttta cctatgatat gggctccaaa tattttttcc caacttgtca 48660ttaacttttg actttgctta ttctattttc tttaaatatt ttctttcttt tcatgtattc 48720aaaagtaccc atcttttctt ttatggcctc tggattttga gtcatagaaa ggccagagct 48780ttaatcttgt ttcttgttct ctgtagcgtt gactatcatc actctgtgac tccccccagg 48840gcccagaggc cttggggagc tgggggaggt tgggagggtg gtggttagtg agaaagtggg

48900agcgttttca gcctaggccc aagtctccca gggcaggagg accctgcctg cttcctgata 48960gccgcccacc aaccctcagg acctgggcta tcccttggag cttggctatc agaacttcct 49020ctaccccagt gagcctgacc tccgagacct gcttctcttc ttggctgagc gtctgcccac 49080cgatgcctct gaggatgcag accagcctgc aggtactggg tgtctgggat gtgggcgggg 49140gcggtgaggg gagaggaggg ccttctaggg gctgtagggc tgagagaggt agaggtggta 49200aaaaggttga tgggtagggg tggcggtatg tgccttcagg agagctaggc aggaggatta 49260ggcatcagag aggggctaca gggcaggggg cagggggctg aggttgagct gaccccggtg 49320gtgcgttggt gcggggaggg gcctccctga ctcaacccct gtgccctccc tctctctcac 49380ttcgctctaa ccacttgggc ctcctgggtg ctactcaaac ataccccagt acactcccgc 49440ctctgcacct ttgtatcgtt ggttaccttt cccctaggta ccctcatggt tcactccctt 49500acctctttga ggttaacctc ctctccgcag ccctctcctg atccccacct ggctgcagtc 49560tggatggtat tcaggatcct attcatgatc tctctcctct gctagcttgc cggctccctg 49620agggcaggga cctttatcca gtttgttcca cgaagtagcc ctagtgtcta gaacagcgac 49680ctgtacatag taggtgctcg gtgtttgttg aatgactgaa cgtgagaacg caagctggat 49740agatgtgtat ctctggatgg gggtcaggag tggaggctgg tctcctaggg tatgcccttt 49800tggatttgca gcattgacat ctgattcact tcctccctat cccccatagg tgactcagct 49860attctcctcc gggccattgg gagccaaatt cgggaccagc tggcactgcc ttgggtcccg 49920ccccaccttc gcactcccaa gctgcagcac ctccaggtga gacccctgac tcccatggat 49980cttctcttgt ccccgtctgg gtgcccaggg ttttggcccc ctacccctgg caaccctcat 50040cccacttcac cctggagatt ctgagcctgc tctcccacca gggctcggcc ctccagaagc 50100ctttccatgc cagcaggctg gtcgtgccag aattgagttc cagaggtggt gagcatgagg 50160ctgtggggag gggtgaggag gaaggtgggg gggaacctca tagcgttgcc atgcggcagg 50220gccagctgac tctgttcctg cctccagagc cacgggagtt ccaggcgagt cccctgctgc 50280ttccagtccc tacccaggtg cctcagcctg ttggaagggt ggcctcgctc ctcgaacacc 50340atgccctgca gctctgccag cagacgggcc gggaccggcc aggggatgag gactgggtcc 50400accggacatc ccgcctccca ccccaggtac agccagatgc ctggctccct gctgtctggg 50460ctgctgctca ctgacactcc cgctggtcct ctgctctcct tccccacttt gtccctccct 50520tccattgttt cccctctgtg tgtgcttacc tagctcccca cctgaaagaa cactggagtc 50580agaaaaaagg agaacctggg acaagtcagt atccctcctc agagccttgg tttcctgtgc 50640taaaatttgg ggtagtaata gtgctctcct ctcagggcag tagttagact gaataatgtg 50700cttgagattc ctggcaactg gagcaatcca gattggctac ctgccttcat cattcattaa 50760ttcattcatt catttggagt cttgctctgt cacccaggct ggagtgcagt ggcgtgatct 50820cagcacactg caacctccat ctcccgggtt caagcgattc tcctgcctca gcctcccaag 50880tagctgggat tacaggcttg caccaccaca cctggctaat ttttatattt ttagtagaga 50940cagggttttg ccatgttggc taggctggtc tcgaattcct gacctcaggt gatctgccca 51000ccttggcctc ccaaagtgtt gggattacag gcatgagcca ccgcacccgg ccctaatctt 51060tctctgtttc tgcacttgtc actgatgttc atttttctgg tttctaggtt tttgcacaat 51120ttctgcctgt ccccattatt ctagttctgt gtttttgctg ctcctctttc ttacctctct 51180gtctcctcgt ttctgtactg aattcctctc ttgctctgtc tatctatctg ttcccccttc 51240tcctctgtct acttctttat ctgtcccctc ttcttctctg tgcacctttt tatctgtgtc 51300cccttctgct ctgtccacct ctgtatctct ccccttcctt gctgtccacc tatatatctg 51360tcacccctct gcctctgttt acttcttaat ctctctcctc ttcctctctg tccacctctg 51420tatctgttgc cccttccctc tgtcccctta tctctccctt cttcctctct gtccacctct 51480gtatttgtcc ctcctcccct tctgtccatc tctttatctg tcctctcttc ttcttttttt 51540ttgagacggt gtctcgctct gtcacccagg ctggactaca atggcatgat cagggctcaa 51600ggcagcctca aattcccggg ctcaagcaat cttcccacct caggcatctg agtagctggg 51660tctacaggtg cgtgcgcccg gctaattttt gtattttttg tagagatggg gttttgccat 51720gttgaccagg ctggtctcga actcctgacc tgaagcaatc cacccacctt ggccttccaa 51780agtgctggga ttacaggcat gagccaccat gccgagcccc ctcttcttct ctgtcaacct 51840ctttgtcccc tcttcttctc tatccatctc tgtatctgtc ccctcttccc ctctgtccac 51900ttatctgtcc cctcttcctc tgtccacctc tgcatctgtc ccctccttct ctctgtccac 51960ctctctgtca ccctctccct ctgtccattt ctttatctgt ccccttttcc tctctgtcca 52020cttctctttt tccctccctc cattctgctc tcctatttct gttccctctt cctctgtgtt 52080cacccaggta tcagtacccc tccccttctg tccaccttta catctgtccc ctcttcctct 52140ctgtccacct ctgtatttgg cccccctccc cttctgtctg cctctttatc tgtctcctct 52200tcctctgtgt ccacatctct gtctgcccct ctttttctcc acctctgtgt cggccccctc 52260ctggtctgtc cactttttta tctgtcctct ctttctgtct tcacctctgt gtctttttac 52320atttttattt ttttatcatt attattattt tttgagatgg agtctcgctc tgtcacccag 52380gctggagtac agtggtacga tctcagctca ctgcaacctc tgcctcctgg gttcaagtga 52440ttctcctgcc tcagcctccc acatagctag gactacaggc atgcaccacc acgcccagct 52500aatttttgta tttttagtag agacagggtt tcaccatgtt ggccaggatg gtctcgatct 52560cttgacctca tgatccatct gcctcggcct cccaaagtgt tgtgattaca ggcgtgagcc 52620accacgcctg gctgtctgtt tttgttttta tatctgtagt aattttcaaa cataaatgta 52680gagagaatat tctagtgaat cctatgtacc attttgccaa cttttcttca tcttttctct 52740cccaactttt tctttgttgc tgtattattt taaagcaaat ctcagacatc atgtcatttc 52800agctctaaat acttaggact acatctctta actcataagg acattcagtt ttcaaggtaa 52860ccactggacc attttcatgg ctaatgaagt taacaataat atcttgtggg tttttttttg 52920ttttgttttg ttttgttttt tgtttttttt tgttttgttt ttgagacgga gtctctctct 52980gttgcccagg ctggagtgca atggcgtgat gtcgcttcac tgcaacctcc acttcctggg 53040ttcaagccat tctcctgcct cagcccccaa gtagctggaa ttacaggagc acactaagtt 53100ttgtattttt agtagagtcg gggttttacc atgttggcca ggctggtctt gatctcctga 53160cctcaggtga tctacctacc ttggcctttc aaagggctag gattataggc atgagccact 53220gtacccggcc aacaatgata tcttaatacc atccaatact tgagttcata atcagatttt 53280cccattatct tgaaactctg ttgtccctct cttgcctctc tctctctgcc tctttctgtg 53340cttggtacct ttgattccct gtctctgccc tgtcccccga tatcctgtct atgcttctct 53400tggatttggg ggctctaggc ccacccccct ctcttcccac atccttctcc agcatgggga 53460tctagtgggt ggagagaagt atgtctgtga gcaagaggag acccctgtcc tcgaggagat 53520cccaggctgg tggagcagga gagtagagca gggcctgcct tagtgggaag gctggagggt 53580gggggtgact tgtctgtata ctcttgtcag ggggtccttg gaggaggcag ggccttgggt 53640gctaggtggg cccctacacc ttcctgcttc ccccgccttt tctccccagg aggacacacg 53700ggctcagcgg cagcggctgc agaagcaact gactgagcat ctgcgccaaa gctggggcct 53760gcttggggcc cccatacaag cccgggacct gggagaactg ctgcaggcct ggggtgctgg 53820ggccaagact ggtgctccta agggctcccg cttcacgcac tcagagaagt tcaccttcca 53880tctggtgggt gcgcctgagg acatgagatg tgtggatggg cgtggcaggc ttggagggtg 53940gctttttgtg tcagcaccac acctttatcc acacaggttc ctgtgttccc actggacagg 54000ccctcgcccc actgtgggat gggtacccag aggggtcccc tagcttatta gggacttgac 54060tggagaaaat gtggataggt gggaaccatg caaaggtgtg ggggtgttct tggggcaaca 54120ccccctttcc tcaggcagtt tcctttgagc atatcttctg tcctaagatg ttcactctga 54180gggcccccca tccttctcat gggcatttga ggcttgagag atggggcagg tgggtaggag 54240ctgtgacagg gtcagggaga tttctccacg agcaagcact ctggcccgag gttgcagatg 54300gtgcctttac ccacatagtc acagtctggc caccatcggt gcttcagtgg gcatgcatgc 54360cgcactgggg gcagttctca ggggaggctg aggctgggcc acgtgaggaa gggccttccc 54420tggcagccag gatgcccctc gtcactcccc ttaggagccc caggcccagg ccactcaggt 54480gtcagatgtg ccagccacct cccggcggcc tgaacaggtg agcagagtgg tttggagggg 54540ggtgtcccag gcccttgctt gtctactggg cctgacaccc caaccctgac tggcctgggc 54600ctcccaggtc acgtgggcag ctcaggaaca ggagctcgag tcccttcggg agcagctgga 54660aggagtgaac cgcagcattg aggaggttga ggccgacatg aagaccctgg gcgtcagctt 54720tgtgcaggta aggggcggag gaggggctgc gcgttgggct aggtcagaag gagggcctcg 54780gggtgtgagg gactagatgg ggcaagaggt gctctgtaga ggtctgcaca tggcagaagg 54840gttcctggga gccattaggg atctgtgggc ctcttgaggg tggctatgag aatcaggcca 54900gggtgagggt ctgtgggcat ctatggggca ccgcgggtct gcatgggcag gggattgagg 54960ggtcctcgga gggtctgtgg gcatctgtgg ggtacccacg ggtctgcatg gacaggggat 55020tagggggtcc tcggggtcct tggcaccagc gtggagctgt tagagaggcc tgtgggggcc 55080acaggggtgt acagtcatct gtggagctcc atgggggctg tggcatgtga ctgggtatcc 55140accggccagg cagagtctga gtgccggcac agcaagctca gtacagcaga gcgtgagcag 55200gccctgcgcc tgaagagccg cgcggtggag ctgctgcccg atgggactgc caaccttgcc 55260aagctgcagg tggggttggg gctgtagctg ggcggagagg ggcagggtgg ggtggggtgg 55320ggttggaggg cccagcctgt gtgacatgta cccatccccc accagcttgt ggtggagaat 55380agtgcccagc gggtcatcca cttggcgggt cagtgggaga agcaccgggt cccactcctc 55440gctgagtacc gccacctccg aaagctgcag gattgcagag aggtaagcag tggggccctg 55500ggctgtgggc gggccagggc aggctcggtc cctctctagg gggccatccc tatgctctgc 55560tcactgtctt ctgcctgtgg gctcatggca gctggaatct tctcgacggc tggcagagat 55620ccaagaactg caccagagtg tccgggcggc tgctgaagag gcccgcagga aggaggaggt 55680ctataagcag ctggtaaggc ctgtgtgagg gacctgggta gcttaggagg gtggggggat 55740ggtcctgggg cagtgcctgc tatatccctg cctagatgtc agagctggag actctgccca 55800gagatgtgtc ccggctggcc tacacccagc gcatcctgga gatcgtgggc aacatccgga 55860agcagaagga agagatcacc aaggtacact gccagggcca tggagggtgg gtcatgtggg 55920ctgtcaggca tagtgtggcc gcacagggac ctcacaccct caggcagagc tgtccagtca 55980cactctaaca cagaatagtc acacacaatc catcccagtc acccctgaca cagtgacaca 56040gtccctgtct ggtacacatg aggaccctcc actgctagcc agcctgcccc aggcaggggc 56100tcatggctgc catggtgtct gccagatctt gtctgatacg aaggagcttc agaaggaaat 56160caactcccta tctgggaagc tggaccggac gtttgcggtg actgatgagc ttgtgttcaa 56220ggtgtggggc aggttgggcg ggggtgagtg gggtgaggct gggctgctgc cttgtgcatc 56280tgctaattgg ctggctgggg tccagaccca ggccctgtgc gaggctggag gtgcactgat 56340acccagggct ggctttgttt catggaggat gaagctagtg gggtggtggg agagggtggc 56400cttcttaggg catggagatg gtcaagggca gcccactgat acctttgagg tccctgtgtc 56460tggtcaggat gccaagaagg acgatgctgt tcggaaggcc tataagtatc tagctgctct 56520gcacgaggtg aggggagaca tgtgcctggg gtggggctgc tgggggtggg tgggactggg 56580tgcaagcctt ctgctcctgt tgtccccaga actgcagcca gctcatccag accatcgagg 56640acacaggcac catcatgcgg gaggttcgag acctcgagga gcaggtgagg cctgggggca 56700ggatggggag ccaaggcggg ccggggggac agttcctcag gttatgctga cagaggctgt 56760ggagccacac acagccgatg gctggacacc cagccctgcc ccttagtgcc tgtgacctgg 56820gacaggcaag tggcctactg tgagccccag cttccacccc aagggccctc ctgtctgcct 56880cccagggcca tgggcagagg cttcagctta aagatgtagg gggaatcctg ccacatggcg 56940aaggatgctt tgggtagagg gaacaccaca cgaggcctgg ccatgggaca gagcaggctg 57000ttggagttgg tgggaggggc ccagagtggc tgtgatgggg gctggtgagc aggagctggg 57060aaaggggctg tgtgtgctga gggggcatgt gttcacattg cctcagatcg agacagagct 57120gggcaagaag accctcagca acctggagaa gatccgggag gactaccgag ccctccgcca 57180ggagaacgct ggcctcctag gccgggtccg ggaggcctga ggagccgccg gcagaggtct 57240ctccccagcc tcaggcaggg atttggggtg ctggaggcag tggccaagca catgccctag 57300ctacttcctc cgctgtccag ttcctcctgc tgcggccttg gacccagacc cctgcccact 57360gaccgcaacc cttatatggg gtgatagtcc agcatgtggg gagctcggct gcagtttatt 57420ggggacggta ctgtgggttg ggggccttgg atcccaaata aatgagtagt tcctctgcag 57480tctaagctga ggcatggatc agggctcagg gaatgggagt gaggtgagtg gcaggggaga 57540cacggggtat ttttggcaag gcagtgtgtg tggctgtgtg tgtctgcacg ggactcaaga 57600gacccactgg ggggctgtgc gtgtgcatat gcgtgagata cacaggtgaa ttctaacagg 57660ccgtgtgtgt gagcgagcac gtgttgggac ctcagatcct gagggtactg acgctgcttc 57720tgtgtaggcc tctgggcaca cccctgtgtt gacagtgccc ctgtgggccc tgaggctggc 57780tgtgggtgcg tgccttgggg tgtgtgggtt gtcagggctg tgcttgtgtg tgattgtgtg 57840atgatgcagc tttgaggttg tttgagtgta ctgaggcagg ctctctgtgt tttggggttt 57900gtgttgagtg agggacagga ttgtgacatt ttgtgtgtct gtgtgacttt tccagccctg 57960aagtaatctg tgcgagcagc tgaggcaggc tctgtgtggc tggttgtgaa ggctctgttt 58020ggctgcaggg ctcgactggg gggtgtgtct ggggcggagg tgggggctgg ggccaggacc 58080ggggcccctc tgagcagcct tggggcaaag gatatgatgg gggagggggt ggctgccagc 58140gggggaacag gggccctggc aggcaagaca gtggaaacct cacttcttgg tccctgtggg 58200cacatccagg gcctatcatc cctgccccca ccacctctgc ctcccaccag tttggcccct 58260gttcgtccat cctcctttcc ttgatcttga ggtcaggggc caggtgtagg gttggaacac 58320ctgctgggcc tctggctccg tttcttgcgg aactccagct catccacggt ccacacagcc 58380cccttctcgc tctccacccg cacaaagcac ttgtgcagac tcaggttgtg gcggatggcg 58440ttctgtggaa ggccggggac agggagcagg tgggcgtcaa cctctgaggc cagcagccac 58500caccaacaac ccacatcccg ttcctcccca atgtgcctat gagcccagac ccaggcctgc 58560ccactttgag ctgcgatggc acttgaggcc atcccagtca ccgccacctc agaggagctc 58620accttccagg tggcaggatg gtttctgaag aaggcaaaca tgcgtgtgaa ccagtggtag 58680atctcattga gtgtccgctg cttctctgga gcctccagga tggcctggaa gttggggggt 58740ggggtaaggg gcacattccc caaacttggg gtttagaggg gctatgacct accccgagcc 58800atctgacatg ggggcggaat tgtaggggca ggtaatcagg gacaggacta gatgtggggt 58860gaagcatggg gtcaaagatg gggttagaaa aggggtgaag tgtcgggttg ggcagggtta 58920gatgggtgtg ggtatggttg ttctgggatt aggtaaggga tcaggactga ggttgggagt 58980ggggtcttgt tcagggctag ggctgaagtg aggtgaaagg tctgggatgg agttggagtt 59040ggggttctct gtggaggtga catttcaggg ttggggaagg tgaagggtca gaagtggggt 59100caaggatttg gaaggggtaa agggccaggc caacttaagg gtcagggaag gggtgggtta 59160agagtcaggc tggggtggac tcaggtgggg ggtctagggg tgagggatgg gatgacttgg 59220ctttaggtca gaagccagag atggtttgaa ttatcgagta tcttacgtgt cagggacatg 59280gttaggtggt taggctcagg gcaaggatga ggttagttgt ggggttcggt gtggagtgag 59340gctgagggtc agggaatttg atcagtttgg attcaggaat gggattacag agtcagcgat 59400gatgattgca gtgaggctat cagtcaggat ggggctaggt cagggtttca gttcagagac 59460agtcggggaa tatctggtat catgtagggg tgaggttcag gtttggggtt aggtgtggcg 59520ctaggatgaa ggttctgaga aggcattggg gaggctgaga tgaaggagtt gggatggggt 59580gatgaagtta aggatcaaat gggtgttaca aggaaaggtt gggaatggtg cccagttggg 59640ggtgtattga catactgggg tacgttgggg ccagggaagg agatggggtt gggttggcat 59700aaaggctggg gaaggagtta gagtaagagc tggggtacgt ttagggcaag gtgcagagat 59760ggggttagct ttaggctatt ttatgggtcc aggagagggt taggtatggg gctgaggtgg 59820acatctggaa aggggtaggt ttagggtcag aatttggcat gctctggcct ggatggtggt 59880ttaggtttgg atttgcggac aggtttgggg tgaagccagc atgaggggtc acatttgagg 59940cacggcttgg ggatccttgg ggctggggct tgggaatgga ggaacccact ctgagggcac 60000tcagagggag acaggagttt gggaggcagg tcccccaccc catctttgtc ttcctcctcc 60060ttggggccga gctgccctgc ttacccagcg gatgagcgtg gcgtaggtga aagggggtcg 60120catgttgtgg aacttgaagt agtccatgtt gtggaggaac tctgtcagag ggtggggatg 60180aatcaagccc catgcaggac ctcctagcta gctccctgtc ccctccccct acaaggtgag 60240tctacaggcc tgagatctca ccgtcaacac ccgtgtccac gggcacaggt actgtttgct 60300gagcacctga cataagttgt atcatttatt ctttgcacca cttctgccaa aatagttctc 60360cccgaggttg aaaagaagcg gagtaacttg cacaccaaag gatgcaagag gttaaatggc 60420agagccagga tgacagtcaa ggtctctgat ccctgctaag cccacaggcc aggcctggtg 60480gagagcagtg gataggtgag ctcgggcgaa tccaccccga ttttccttgg tcaggggagg 60540aaaggaggtg ctcctggaat tacttagcag ggtccctccc ttctgatggc cgaatatagt 60600agctggagtc cagagtgggt gaggcatggc cccaatcccc aagggagtca gggctagggg 60660cccgacactc gagaccatat ggggggcttt caggccacgg acatcccgaa aggaagcttt 60720tgtgagcgga tgcattttcc caaaggctga gtggcggcag ctgcagtggt ggtggtggtg 60780ggaaggggca gcatggagct cctttgcacc ctccacccag agcctgtcag gattaggagc 60840ttgggggcac cgtgtagtgc aaggaccatt cttacctggg aatgtgctgt ttccatggct 60900accccacagg tgcctccgga cagcaaacag gctgtcaggg gcctcccggg ggccagacca 60960ggctgggacg acagggcctt ggctgccagc agctacgatg cagcaggagc ccttgtcgga 61020tgatgcctgg gtgaggggga gaggctggtg acccagaggc ttaaacttcc cactttttac 61080tttctatttt atgtttttta ttttttttga tactgagtct tgctctgtcg cccaggctgg 61140agtgcagtgg tgcaatctcg gctcactgca acccccacct cccagattca agcaagtctt 61200ctgcctcagc ctcccgagta gctgggacta caggtgcccg tcactacgcc cagctaattt 61260ttgtattttt ggtagagatg gggtttcacc atgttagcca ggctggtctc gaactcctga 61320cctcatgtga tccacccgct tcagcctccc aaagtgctgg gattacaggc atgagccact 61380gcgcctggct ccactttatt tttaaatcag tgtttttcaa agcaaggacg ccctcttcta 61440aattccagtc tgaggtggaa tcccacaaaa cagcatgagc cgtatttatt agagcacagg 61500tgcggatgtc gtatgtggcc actgatgctg ggaccatgaa ctggggttga atagggctct 61560ttaccaccca actgtgacct tgggaaagtc acctaaaccc ccctggcctc aatattcctc 61620atctgtacat tcgcatcatg agaaataaat taccaccagc aaagcgctca gaaacagtgc 61680ctggcctcca gggctggctt catgggcgtg tgacctatgt ggttatgtgg caccctgtgc 61740tttgtttaat gctctgtggt cgccatcttg aaatctgaac aaggggccct gcaggttaca 61800tagctggtcc tgctggcaca cagcctgcat ctggcaagtc tggctatttg catttgcttt 61860aacaactcag gatcacagtg tttggggatc ttagagtcag agggtttttt tttttttttt 61920ttcttgagac ggagtcttga tctgtcgcct aggctggagt gcagtggtat gatctcagct 61980cactgcaaac tccgcctccc aggttcaagc aattctctgc ctcagcctcc caagtagttg 62040ggactacaga cacctgccac cacgcctggc taattttttg tacttttagt agacacaggg 62100tttcaccatc ttggcgaggc tggtcttgaa ctcctgacct cgtgatccac ccgcctcggc 62160ctcccaaagt gctgggatta caggcgtgag ccaccacgcc cagccggggt cagagggttt 62220gtaagtagaa ggggacagat ttccaggtct gggcatgttt ggagctgggg acaggggccc 62280ctagctctca ggacctgaat gtgaggttag gttccctgca ccgtgcagac ctcctccctg 62340ccccccagca gtctgagtct gccaccacca gtcctggggt cgctcaccac agatgaagcc 62400ttggtcagtg ccattttccc agccaggtgg gcctgcatgg cactcagctt ctccttctcc 62460agcaccagct gtgaaatggc acaaacatga ggcctcagcc tggcccttct ctgccacatc 62520tttgcccagg ctacggtctt ccctgggagt gcccgctcct cttccttcct ttataccagc 62580cctcgtccca ggtgaacttg gtttctggca catggtggac agaaggtttt gcgcactatc 62640cctatccctt accctccacc gccctggcat tacctgctgc tccagagact gtaccatctc 62700tctctggagg agacattgtg ccctgccctt ctcatccaga agatggtccg cctggcagtg 62760cctaagtagg gagaagattc catgcaggtg accacgacag gcctggtctg gctcaatgct 62820ctgaatgggg agggcccaga ccctctggga gttctctcct ctgagcccca gctcccctcc 62880cctctctacc tcagtctccc tctcacaccc ctcgttccct taacacatgc ccctcagcac 62940ctactgcatg tcaggcctga actcaccact tgagcctggc cagagtgctg gagataatgt 63000tggaagtgtg gtgagttgag aatgggccag ggaggtgaga gtgggcaaag cattctgggt 63060ggagggacgg cctgtgcaaa ggcctggctg caaggaggac aggacatgtg gggttgctgc 63120taagggttgt gtgtagtgtg gtgtgtattt gtgtgtgtgt gtgtgtgaga gagagagaaa 63180gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga catatggggg 63240gactgagggg aggcagcagc agccatctag aggagctaga actttgggaa cagtggaata 63300aggctggcat gttggcatga ggagtagcag ggcaaagcag gagtgcagat tctagagcct 63360ggctacatgg gttcaaatcc cagctctgtc actcaggaac tgggagattt tgatcaagac 63420acttaacctc tttggcctca gtctccttgt ctgtaaaatg ggggtaaata acagcacaaa 63480cgtttcttat tatacatacg agaaaactga ggtcgagaga agctaagtaa tttgtccaag 63540gtcacacagc cagtcaggga tggagctggg atttgaaccc acagtctcag agtttagctc 63600ttgcatctta ctacttattg ggatgaagcc tgagctgaga tctgcaccct agacctctcc 63660ccacaagcca gggccggtag actggcacag gcctgggcca ctcacttgag gaagtcctct 63720ggctcttcga agaccttctc acatccgggc cacttgcaga caccatttgc cagcagtggg 63780taggagctct ggggcacagc cgaaagggtg ctggggggac agagggtgtc aggggagggg 63840ataggagggc gaggatcctt cccagccctg tccactgacc tgtccttcct gggtgcactg 63900ggatttggga aggtgcagag cagtgccggc tccctggaca cccattccag gctggccacg

63960ttgatccctg tgggtgggga cagggcacct atggaggctg tgggctgggc tctggagctt 64020ggcccacaag gcctctcatt ttgagcttcc caccctcctg agcctcgaaa accctgactc 64080ccagggggct ctgctgtccc caaagtccca ggcttctggc agagaagctt aaagacggcc 64140attcgcaggt gctgacattt tgactagctt tgtaaagctc tgtggttttg tgattttgac 64200attctgcatc tttaaggttc tgcacctgac gttttttgga gggtggagtt tccaagcctc 64260tgagacctga cacctttgac ccccagagta ctgcaattca gaatagccta cactgctcac 64320agccaaggat ctggggactt gggggttctg tgaagccatg gggtacgggc tgaggtgtta 64380ccaggtggga ggccaggccg ggccttgagg gagaagaccc cagtggcggt ggtgggtggt 64440gtgaggctga tcatggctgg gctctccagg gggtgcacct gcagcacagg ggtccgggcg 64500tgggcatcca ccgttgagag ctggggggca catgtgggct gtggttcagc ctgactcggg 64560gcccctcccc acagttctcc cacctgctcc ctcctccctg cccattcacc gtccatacct 64620ggtgcatgaa atgtggcctg tcctggagga gtgcctgtaa gtggggcaag gggcccagcc 64680gtgccccgga gggtgccacc atgactaggg gcagtgtggg cagctgggca gaaaggcagg 64740tgggtgagag gccatcctga tcctcactgt tctgtgtcta attcaaatac tctgcactgc 64800aagcccacat ggtagatgct atgatcatcc cccttttaca cgtatggaaa ctgaggctca 64860tggagatcga gtaacttttt aaagatcaaa cagctaataa gttgcagagc tggcctcagc 64920cctgtcacct cacctacttg gccccagtcc tcttctcttg tcacatgggg atggggacac 64980atagctatgc tcatgggact acaatacggc ctcctcctct cctgagacag ggattgggag 65040gtcggggaga gcctccaatc tctgaggcct ggcaggtggg gattttcttg gccctgcaac 65100atctgcataa gtcacagact tgcctgggac ccagaaacca cttcctgtgc cccagccagc 65160ccccctcccg cccagtgcca cagtaaaggt cggcacctgt aggtccaggt accccaccct 65220gcctgcccca tcctgggccc agggcctcac ctgcagctgc gatggtggca tggggttcaa 65280ggaagaagag gaggcatggg ccccgcctcg aagatctcgg ccctggaagg ttccccctgg 65340gccccgggcc cccagcaggt ctgaggcttt gggtgcagcc ctccagctgg gcgaggctcc 65400tggggatggg ccaagggcca aggaaggggc cgagggcttg ccaggcctgg ggttgggcat 65460cgggtccttg tccaagggca ggctgcgtag acaatagggg aaaggagtca cacgtgtgct 65520agggcggtat gagatactcg accacctgag ccacgtggac actcctctgg tcaaagcagg 65580cattggctgg gacatgtccc gaggggcccc atagttgcac cccagctcta gacacacaca 65640cacacacaca cacacacaca caccaagaac acaggtacat gtacataccc acacatgccc 65700cacgtgcaga ggtccagcac ctggcttgcc tgcccacgct agcacagccc tggtgtggat 65760gtgtcctcta tgagggcaat ggttgtttcc ccctccactt gagagctgtt tcaagcctca 65820ggcctctagc cctccctgca ccctgcacag gctgtgtttg ctcatcttgc cggagctggt 65880ctcggacttt ctcctcggag tcctattttg ccccagtgac taggcatgga ctcaaaagat 65940tcatctggct gctgtgagtg gggctagtga ggaggctatt gtaacagtcc tggcaagtga 66000tgatgctggc acagaatggg ctggtggcag tggagaaggc gagaagtggg tagattctga 66060gacttaatct gaagctggat caggagcagt gctagcagct tggatgtagt gggcaagagg 66120gagagtcaaa gtgacatggg ttttagcttg agcagctgga aggaccgagc tgacattacc 66180tgagatgggg gacatggcgg ggagttggat tgggtgcaaa agtgcaggtg tagatagaca 66240tgaagagtct ggcattaaat atgtgagtgg aggagctgag ggggcagctg aatacggggg 66300tctggatctc agggcaagga ggcgagtcca ggagtgtgat catgcacgga tccagcatgg 66360caagtgacag agaggaggag agatggggtc tcttgagctg gggcctgtag aagcttctct 66420acccagcccc catcagagtt caccccaatt tctggccctc aagcctggct catgctacac 66480cccctgcctc aaatgcccac tccttctcct ctttccctgt ccaagccacg caagacctgc 66540tcttctattg tcctcacctt gaaagccctc cacaatggct ccgggccccc tgctgagccc 66600cagaaccttc cactccctga gggaagcact ggcttttcag gatcctataa tcctggtctg 66660agaggaagcc agagctggaa gggactgccc agccaacccc attatacaga aggggatgct 66720cagatgccga gttccgtagt cccatagtga cttgagaact ccacttcttt ctttaggaag 66780tgtttccgtg tgcacatttt ataaactctc tggtgtgtgt gtgtgtgtgt ctccatctcc 66840ccgttccacc tcacagcact gagttgggca cacagctgct gagagcagcc cgggggagta 66900tagaagggtt ctgggggagc agttgctcct tcctttcttt gctgtcacct cctgggggtg 66960gttgtcagag ctgtggtgct gagggagatg agtgtgagaa tccaggtatt aagttcttag 67020tctcctgggg gcttagaaca ttactgcgtg agaaacagga gtgtgggtct gtggaggctc 67080cgaacaaggg cctgggagag cactggtgag atgagaaggt gagtgaatga atgaagccag 67140agatggggtg atgctccttc aagccaagaa ataacaaaga ttgccagcaa ccaccagaag 67200ctgggggaga ggcctggagc agcttctccc ttatggcccc cagaaggaac caaccctggc 67260aacaccttga tcttggactc tggcctccag aactgtgaga tgatcaattt ctgttgctga 67320agccactcag ttgtggtact ttgttatagc agccagaaca aactaatacc gatttcggtg 67380caaatggatg ttttccacca ctctggcctg gcccatgtgg ctggcctgtg gtcacttctg 67440aagctgcctg gacacttggc cagagctaag aattctcccc aaacacatgt gggatggcct 67500gactcagcaa agcatagata cattctcaga cagggacatg gagatgatct gtctgggggt 67560agaggaccta gagggccggg ctgggcagcc ggcttcctgc actgtctgtt gggacgtccc 67620tttctgactg ggtttctcag aagctgaatg ggggatgttt ctgggacaca gattatgttt 67680tcatatcggg gtctgcatct gggccctgtt gtcacagccc ccgacttgcc cagatttttc 67740cgccattgac gtcatggcgg ccggatgcgc cgggcttcat cgacaccacg gaggaagaga 67800agagggcaga taccccaccc cacaggtttc gttccgagaa ctggctgccc tgtcctgcag 67860caggcttggc ccaggtgggg tgacatgggt gctggtggat gtggtaggtg atgtccatct 67920ggccactatg acaagcccct agctctgaag acctggccct tcttgggttg tggagaggac 67980ccaggtttga agctctgaga gtgccaggca ggctccacag atactgggac ccctggggtc 68040ttcaaatagt ataacaccag gacctcagaa tcatagaaca gcatttctta gatttaaagg 68100atcctagaat ctcaaaacca cagactgtgg ggtctacggt cccaaagtct cagtatgtgt 68160aggccagtgt ccctggtgtc caaactcctt ggaaccatct gaaagtcagg cagcttgctg 68220cttcataggg cctcttgcct accaggcctg gaaacacagc cagcctccct agggcctcag 68280tctccctgtc cactctggaa caacgttccc aaatacatgg ccactccgcc agagatggca 68340acagggggag gaggaggttg aggctggtgt gcctttggtc tgggcctcat ggggggagct 68400ggaaaagcct cagccttcgc caatacagag cccatcatca gactctctag aggggcccca 68460caatcaaggt tttcggggac cagcaccagc tctggggaca cacagcctga ctgactgaca 68520tgcctccatc atcaccacgc tctggccaac taggcctcct gacctatgga gtccggggcc 68580tcacctagcc cagctcttgt gaggctgggc cccacactgt gatcgtggat cgtccaacct 68640gtgggaagtt ggggtccaac gtgtgagaag gcagaagggg gaatggtagc ccaggttccc 68700cttccccctt ctgggtgctg aggggtaaac tgaggccttc agttggggag agagccagaa 68760ccagggtccc acctagagtc ctgagatcta ggcttggatt tcaactctgc cgctgcattt 68820cggtgaggcc ctgagatctc tggtcttcaa tttgcccttc tacactgagc acggagaggc 68880gtggagtaga caagggccag ggcccttcta cgctgtctgg ttaagtcatt aggtgtctgc 68940agggcttcaa gttgacaatt gcccctctat ccaggggact ggctgagaga tagggataca 69000tagagacaaa gagacacaca caaagagcga gcaagagaga acaagagata gtgagagaca 69060ttgagagaaa tggacacatg catggagagc cagagtgcat gtgtgcgaga ggaggattgc 69120ctcaaataag aacatttgct ggtctctggc tggttcaact gatgctgcct gaaataatca 69180agaataaaga agggcaaggt gccagggaca cccatggctg ggtattgaat tgtattgcaa 69240agcaacaatc agcaaaacag tgtggccctg gtataagaac agatactggg gaagagaaca 69300gaatagaaag cttggacatg gacccacata tggagagaac tgaatttgtg atgaatgtgg 69360catttcaaac tggaggacca tggagtatgg tttaacaaat gtgtctgaga taattaggga 69420gaagataaag ttattgagtg aatagtcagt ccattatccc aacaacccct ccctgcccag 69480tttgaaatgt caccatcatc atatacccta aaatgcccag atccattcaa gatataagtt 69540ttaacaccta atgctgatct tgggtttatt gtgtgtcagg ccttgtgcta agtatttact 69600gtggttaaaa attttaatct aaacaaagac tcctgaggaa ggtactatta taaccattgc 69660agtacatatg aggaaatgga ggtatggaga ggttaagtgc ctggctaaaa atcacacata 69720gggcttgggg tgacgctggg tttgtcccag acagtctggc tccagtaccc acactcttaa 69780cctctatagt aaatggaaaa aatgaagcca taaaagagac tagaagccaa cataggtgaa 69840catttatctc attttcaagt aggaaaggac tttctaagca caaaaccaga gacagaagcc 69900acagaaaaaa agactaacct atttgactgt ataaaaccat cataagcatc acaaaaaaca 69960ccataaacaa atagaaaaaa agcaaatgat gaattgggga aaatatttgc tatatatgta 70020atggctgatg aaaggttaat aaccattcca aataaagagc tgtggcaaat caataaggga 70080aaaataagat gaacacccta ttaggagtaa ggacatgacc agacaaccaa aaaaaaaaaa 70140aaaaaaaagc atgaatggcc aatgaatagt aaaagtaatc acaagatgca aatttcaaca 70200atgtgataat gtgtttttct tacctgtcat gttgggaaat aattaaaaca taataatact 70260cacctagggt tagcttaagt agagggagca taaaatagga caaccttttg gaaggagaat 70320tagcagagag ggtcataaac tttaaaaatg cacgccccct ttgccccagc aactcccttt 70380tcaggaatcc aaggaagcag tcagggatgt ttatagaaaa aaacgagaaa caaccggaat 70440gtccaacaat cggcacttgg tcaaatcaat caaagttcat gctgatgtga ggacagtctt 70500gtccatcatg aaagatcatg tgttcaaaca atttttagta agcttcaaaa acactactgt 70560taattgaata aagcaggaac aaaaccaata tatagcatga ttctaatttg gttacagaaa 70620tactaatagc taacacttcg tgagcactta ctttgtgcca aacgctgtgc taagcctgca 70680gaatcgagct caccccagcc ctgaacaacc tgtttgcttc ctgaatatgg actctggtca 70740cacacatgca gtcctggggt aggtccacac agctaaacta cggttgacaa tggtgtgaag 70800tgctccctgc ccccccgccc caagggtctc ctctaaagcg atacaagcaa agttcagtta 70860agtgctcagc ttgccccggc accttgcaat cctcctgcta ctagggtgaa cagaactgat 70920gctcactctc ataaaatgta aaggtcctcg gcgacattac tattattaaa cgccagctgt 70980gtacaaagct ctaggctgga tgctggctgg gaaggcaggt gggggaaggc aagaaaagag 71040agcgggagag atggaggaaa ggagatcgat ggagtgtggt caagatggag gagacagaga 71100taggggagat ggtcagaggc caggagagat gcggggaaag agagtctgag tgtagcgaca 71160gacagatggc gggagaaaga gaggcagaga aacatgtaaa agagcaagac agggtgagca 71220gagagacaga gaaggatgag aggcatcaag agctaagaga caaagagatg agagagatgc 71280agttgaaatt ttcagttgca cctggacagc atttcaagtt gttcaaagct ctgaaatcca 71340taaagactgg cagctgacat attttaaaaa tcctatccat ctacgtatca attgatgaat 71400tcatttattt ttgcccctgc ccatgcatta agtacttcac ctttaagtct tctgccattt 71460attctattat tattttttta aagaccttac ctggctggaa tcacggtagc tgggtacatc 71520ccactgtacc agagggcccc tgaccccccc gccgtgccta cctccctgcc atctcctcca 71580atggggccca catctggtag gggagagcag ggacactcac cttggtgaag tggactgaca 71640gaaaaggatc agcctggctt gtgggaaact gtcacgtatc aaaaacaact ttgcttttat 71700accgagaaga aaaaccacgc tgtacggtgt ggaagccgca gacctctctc ttctaataat 71760ccaaattttt ttcgatgagt gtgtgcgctg ataatcacgg ggtggggggg ggttctcata 71820gttttttttt tctctctctc tgtgtttctc cttttcttga ttatgagact taaacggaaa 71880ttttgaaatt ttgggttttt ttctttgccc tttacgagtc atctgaaaat atgatttctt 71940cccctcacca cagaggtgag aggtatcaat gagataatag ggctcatgag aaaccacagt 72000ttttaaaaca aaagtgtata gagtttgaaa aaaaaaaaac aagggaaaag aactaaaata 72060aatcacaggg ccaacccgag gcaggcagag acaccattct gtgagtgaga ggatatttga 72120gggtctctgg ggaaagaaag agaatctgaa gctctatgtg tggatgggaa atgccaggga 72180aagagacaga atagggctgg gttcccagag gcctcgccct actccaggcc tcagtttccc 72240tatagatgga attgatatgg tccccattgc ttgaactacc cggcgagggg agttctgcac 72300cccctgcaac acccctccca cagagggtga ggggcatgta agttacttca tggaggagaa 72360agcggagagc ccactgtcat cccctaaaat tcatggactt gaagagtcgg agaatccctg 72420gctcccagaa tctactgaca gttactgaat gaggaaagag agaaaagtct cgcctcatca 72480cgttttaggg cttgagtgcc ctgacccagc cactgtccca ctgggaaggt ccctagcagg 72540tccccaatat taatgcatcc atcctcacga tgaaccccag aggcccattg ggaccctgac 72600cctcttgttt caggaacatc atggcctgat gcttctgagg ccctcagcct ttggggtctt 72660ttacttcaaa aagccccagt ttccaaggat ttaggattcc aaatatccgc catcatctca 72720gtagctgatg tttatcttga attttccatg ggtcagatac agtgctgaac atgtctcaca 72780agtgatcatg ttcaatcctc accatggccc cttgagctcc atctcatcat tattgtattc 72840ctgttataca gatgagggaa acggaggtat aagtagtcaa gcaacttgcc caagatcaca 72900gagctggcga gtgtgggtcc cgctttcttt ggtgctgggc tttgaaatcc ccaagctttc 72960ctgaacttga tgttctcagg ttttaaattc taggatgtga tggcagggag atcctgggat 73020ttggagagtc cttggagact tgaagcttgt gaggctttta ggttgtccag gaattggcgg 73080ctgcatctct tgacctcagc aggcactcta ttcaaccact ggtcagcatg gtagaccagc 73140ccccaggggc ttctcctgac aggccatggt gaagacttcc agctcccagc accctgcaaa 73200gcccagggtt ctagtcgtcc aacaaccact ccagccctct acaagactgg cttcagacct 73260ggggaagaga aattggaaag ggctttttat ttacatggta caaatctggt ggccaagatc 73320caccccttca cccctttgcc ccctttcctc ccccaacctt ctaagccctc gtaaaaagga 73380gggcctcttt cctcaactca gactcaccct tctggaaaag cccagaatgg gcctgatggc 73440ctggctttgt gaagacaaga aaaaaaaaaa aaaagaatag aaaaaaaaag accttaaaaa 73500aatcagccaa caacaattaa aaaaaaaaaa aaaacaccca accctcccca ccattagttc 73560aaaacaaaac tttcgctggt tgaggcttct gagttggtgg gacacatctg agacccaaaa 73620atggcttggg tgtgttggac agccagggga ggcaccaagg tgggatgagg tcccctgctt 73680ggaggcctgc agggaccctg tgaacagcag gtgtttaaca gatgtcacgg catgtgggag 73740ggagcaaggg agggggtgag gagcatgcat gtcaggaagg agtcctgggg agctgattct 73800agtgatggcc ccaaggttaa tgaccccttg cagtgatggt cctaaggtta atgatccctt 73860gcactgatgt tcccatagtt aataacccct tgctccctgc agacttgggt cggaatgttc 73920cctacacctc ctgcgtaatt ataaaccagg ccagttctat cagcagaggc attgttccct 73980gccccagcac ctgtccccct cctggaaaag ggggctagtt ttctctctca ggatcagagg 74040tgtgggctgc attctcctta taccttcccc agaccatctg tcccttactg tcactcactc 74100catgaaagag agtggccaga gatataggag gcaaacgaag tgaggatggt ggccgagaat 74160gtgagctggc cccttgggca ggcccagggt gtgagaggga aatgctacgg ctgctgaaag 74220ccaagatcct tgcagaatgg gggctggggt caggagggca gagaggggta gaggtcgcta 74280cctcccctct atactggact atatcctctc tcatggaggg cttctcggga aggagactgg 74340gagggctggg agttgggggt gagtaggcaa gattaaacgg gtcaggaaaa gtccccagag 74400acaggggcct ctgacaaaga cttgaaggag atgagggagg gagccagcta gatatctgag 74460gaactccaaa ggaaacagcc agggcaaagg tcctgaggtg ggaatgtacc tgatttgttc 74520aaggtctaca agggggctgg tgtggttagt gtggagagag caagaagaga agctggaggt 74580gaggtcagaa aggggactcg gaggttagac cacgcaggac cttgtaggcc atggtaagga 74640ctttggcttc aacgctgagt gaggaggaag gcatgacagg gttgcaagca gaggagaaac 74700acaaaccgtg ttaggattta acaggaagcc cctgaatgct gagtgtacaa tggggtctag 74760ggcctgaatg tggaagcagg ggaccagcaa ggagctggct actgcagtag tctaggtgac 74820tggtgatagt gctggcagtg gaggtggtga gaagaggatg gatttggtaa atatttttaa 74880tagacagcca aaaggatttg ttgaagactg agagtcttgg agccagatcg ggacagacaa 74940tgcccaaacc aggtgggcag gaaggagtcc ctggactatt tgccagcttg aacttttagg 75000tgagaaaaaa ataaagttct gtcttgctta agcccccatt attttatgat ttttctgtta 75060cttgcagctg aacatattca tttctaactc atggacatcc tgaacatgga gacctgggga 75120gaggggctgc tggaaggatc acctccctcc taggatgctg tgaggttcta atgagataac 75180agcatgctca gaacctggca ctcagtaggt gttcagtaaa tcttcccagg atggagaagg 75240aatgtatgaa tggatgtgtt cagcggacaa aagctgcagt tgagttccca cagaaggtga 75300gaccccaaag ggagagcgaa aggagtttac tagatggaag cagcagaagg aacagcactc 75360ttgggagagg gaacagtaga agcagagatg gggggacaga aagaagggag gtgaggcccc 75420tgtggggtgt ggagtgtcag gttgggcaag aaggacaggg ctttgattgt gattccatta 75480gtcagaagct acccatatgc ccaatcaact tctggtagca cacagtcagg tcagtgtttg 75540ctgagctgaa atttaaaaaa gtgaatcctt cagaaagtta aatttcctat tcggccattc 75600attcaacaat agtatcaagt cctggagagt gcttgcggct aacaatcagg ggcattcttg 75660gcttgtggca ctgctcaaga aaaggttgta gttgggcagg gaggacccag taagtaacta 75720actgcaacag gacagtcatt ttcactttcc tagttcacac accaaggtgt ggtctaggcc 75780ttcaggggct ctgggtattc ctggggcaat ggggagtcac ggagggttag ggagagggta 75840accccatccc tgagaagcac atttttccta atggctgcct gcgttctatg gttctgctgt 75900ccggccaccc aatacctcag actctgtcta cacagcgtcc tcctctccag cctccttatt 75960ggctactcat gcctcccttc acaccaccta cactgccccc gtcggctctg cttttttccc 76020catcttgcct ccaagtcctc cctccgtccc gctgcactat ctagcctcgg cattggctca 76080tcccatacta gtactgtcct ttgaggtctc tacgccttca ccctggctcc ctcattggct 76140atccgccagc tctcgtcctc gctcccgcct tcagcctcca cctccattgg ctcggctccc 76200tacacacccg cctcttggtc tccttttagc cttcgtattg gctagctcca ccttggcacc 76260accccttcga agccgcagtg cacgctcaca agcctcccca ttggctcgct gcagccattg 76320tcgtcatccc acgcgtggct ctccattggc tgttctctct ctctagctac cgttccaccg 76380cctacgcaac ccgcgggatc tcccactttt tgggcctctg cgttcgttcc cggctgccct 76440catcgcctgt agccattcca cttttcccac cgcccacatg cctctctcgc tcagacttcc 76500gcattagctg tctgcttctg ttttcttcat catggatttc gcaccacccc cattccggcc 76560tctccattga ttcctcgatc atcccgcccc ctacaccgcc cactcccggg catccccatt 76620ggctgcgtgc ttctccggct ctcaattcgc tgtacgtcat ccgtgcatgg ctgcccattg 76680gccctctgca atacttgtct tcatctcacc gcctatgccc ccgtgagccg tacccctccg 76740cgctggcctt cccattggct gcccgcccct tcaggccctg cccccgccgg tcccgccgcc 76800ggtgccgtcg gtgccgccgc cgccgccgat atggcgcgta cggcccctgt ggagcccccg 76860ctgcggcatt ccgcgccccc ctcgccggcc gcgggtgagc cccgcacctc ggtcgaggcg 76920gcggtggccc cgcggagggt gctgttcgcc gacgaggcct tggggctgcc gctggcgcag 76980ttgcgccgct accggccgtg gggcgggccc ggggcgggca agatggcggc ggcggccggg 77040caagatggcg gcggcggcgg cggggccgac gaggacgacg atggcgagga tggggatgaa 77100ggggaggagg aagaggaggc ttgccccgag ccctcaccgc tgtgccccgt ccccgctggc 77160ggggggtttt acctggtccc cacattttcg ctgccgcccg cgccgggccg tctggagcgc 77220ttggggcgcg tcatggtgga gctggaggcg ctgctgccgc ctcccggagc ggtccccggg 77280ggtgccgggg tgtgggtgcc tgggggccgc ccgccggtgc tgcgcgggtt ggtacgcgtg 77340ctgaaccgct ccttcgagaa ggcggtgcac gtgcgggcct cacacgacgg ctgggcttcc 77400ttttgcgacc acccagcgcg ctacgtcccg cgcagcccgc cgtgggcagg agcgggagga 77460acaggagcag gagatcccat cctggatccg gggctcggcc tgggtcccgg ccaggcatcc 77520gcctcctcgc ccgacgacgg cggccgcacc gaccgctttg ccttccagct gccctttgct 77580gagggcgcgg gcgatggggc gcgcctcgac ttcgtggtgc gctatgagac ccctgagggc 77640actttctggg ccaacaacca cggccgcaac tacacagtcc tgctccggat cgcacccgct 77700cccacaccca ctgatgccga agggctgccc cagcagcagc agctgccgca gctggagcca 77760cagcccgagt gccagggtcc cgtggaggct gaggccaggc agctgaagag ctgcatgaag 77820ccggtgaggc gcaggtaatg tcagccagcg ccacctccgc caacgcaggg ctgtgtctgg 77880gatggaggac aagcattccg gcccaggaac ccctcaggcc tgctctccag gacagggagg 77940agggtgcatt gagtcagtca gtcaaagagt gatggaggtc aaacacgtgc taggcactgt 78000tttaactggg gttttattcc tccatttatt gagttccact gtaaagccct gaacaagaca 78060gacatctacc ctaccctcaa ggattcatac aggctcctat gggagacagt ttgaaaaaac 78120aagcaagcaa gcagatagga taaataggga caaagttagc tgctgaaggg agtaagggga 78180tagattggga gggaatgact gtgtcagaca tgctgttaaa gggagggcct ttgaggaggt 78240gagtcccgaa tgatgataaa gaagagttat gcagatacgg tggggaggtg ggggagagtt 78300tgtaggaaaa ggaacaggga ggacttttct gaggaggttt catttaaact aagtcctgga 78360tgatgaataa cagttatgtg catatatgga acaagggtgt tctaggtaga ggaaacagca 78420agtgcaaagt cctggggtgg aaatgagctt ggtgtgaatg atcagcagtg tgctggagtg 78480gagagaactg caatgacata gtgtggacca agggctttgc cagtgggggg tagagcagat 78540tctctttctg acagagcagg aaaccaggat tccagaggtg gagaaactgg ttgaggcagt 78600gatggcacct ggctcttcca gttcacccat gcctgacctt gaagcctgtg cactctactg 78660ttaacgctgt aatctacttt tgagtcctgt ggaggcgagg ggctgagccc atgctctcac 78720aagaaagcag tggctctagc agagagacag aaccagatgg gtactggact tgaagttttt 78780gtcccttgtt tttttgccac accacctgtg atcatttagg gacagggaga aataatcagt 78840tataccccag ggggccccag aagatttccc taggtttcct ctatcaagct atgagttctt 78900taagggcagc agctagtcct gattcttttt gcggtcacct aagtctggcc caggatcact 78960agattctccc taaaatgtat acatttccct gttcctggga ggactgggaa tggaaatggg

79020aaaatgcttt ttggaccttg tatgtatacg cttggtcaga agcccctggc tgcagtaaga 79080catttgtggg ttctgctggt tctggtaagg ccttaggctg tgagtggttc agaaaggtct 79140agctccggtc tggtgggcag atgtgcctgg gtaactgagc aggcaggcag gtacccactc 79200acccacctgt gagccccacc ataatctgag acttcattta aagggggcag gcagcatggt 79260ttttcttaca gcattaagag tgtggattct ggcatagatt gcctgggttc aaatcctggc 79320gctgccactt actggccttg tgaccttggc caacgcattt aactacacca tgcctcagga 79380ccctcacctg taaaacagaa ttatagcatc tactttacgg gataagagta aaggtgctca 79440gaaccgagtc tggctcaaag tagtatcata caggtgttag ctagtgtcag tatttaggga 79500gccacaccta cagtttcctg gattcctctc ctaaattcgg ctccgcccat cagctgtgtg 79560gccttgcctc cgtttccttc tctgctaagg gaaatgacag tcactttgca tggttggatt 79620gaatgcagca aacatgggca aggtaactga agcatggtgg taactgcaaa agataactgg 79680ccagcattta ctgatttctg tgctatgaac atcagatcca ttctctccgt gaggcctgct 79740gtgcatgctg tgttgtgtag gtgctatgat tatccccatt tagtagaggg gagacaggta 79800gctcagagag atgagccagc ttgctcaaag cctctcagct agtgagtgac agggccaggg 79860aagaaatgct agggacattt atttttttct attttttttt ttttaaactg gtcctctaga 79920aagcgtggca tgattcaggg caaattctgg atttcactct tggtgttccc agcatgtcgt 79980gctttccttt tacctttttt tttttttttg ctttatcgag gtatacctac ataaagtgta 80040ctgatattct gtgtatagcc cagtggattt gtacatatgt atgcacttgt gtagccacca 80100cccagatgaa gatactgagc aggtccagta ccccagagct accttcattt ctatcacttt 80160cccaagctgt catccccgcc ggctgtcatg ggaaccctgt ctgtaagatg cgacagtttg 80220ggtaaaggag tttggtcatt ttaaagagtg tgaaaggcag agaacagaga aatcaaaacc 80280ttgcagggcc aaggtgggtg gagagggtgt ttttctttta acatacatgg gcggttttaa 80340ggagaaattg aagcagcctg ttcagacaat tgttttggta tctggcccca ggtctgtggt 80400tcctaacatg acttgtgata ttattttaag tgggcagatg gctttttgat agcttcttta 80460tctttcgatc tcagctcttg caaaggggag gttggtgctc attgcaagat cagcgataag 80520ggtttctttg taggtcggtg gctttcttgg tgagtacatt tcaacatatt attgttttag 80580aacctgtgtg ctgccagtga cttgcagcac tgttgaagac tagccaccct ttgtgaccta 80640gccctcttgg gaaatggcgg aggatctcag ggtatatccc ttacctgtgg gagccctatc 80700agagggcttc ctgttgagga aatgttggct gtaggccctc tgtgcactga gcacagccac 80760atcaggtgag ggcatgggag aagtccgtgg tagccatcag gatagagttc agaaacccaa 80820atggctgctt tccctgggtt gctggaccag gcatttggta actctaaagc ttagagatga 80880ttcatcgaca agcatttatt gactgcctac tgtgtgctgg gcacagtgct aggttccagc 80940agggaaagag atgcagagtg gtcctaagat gtcacagggc ttgtgggatg gagtacagag 81000gatttgttgt atgagacctg gctgagcccc tgctcttcgc caggcacttc actgagtact 81060atattttcat gcaaggtctg gtttaattcc gacaacattc cctgtgaagt agagggtttt 81120aaatttctct ttcctggatg agcaaactga ggctcccaga gtccaaagtc ctagggctgg 81180tgtggggaaa agccctcctg atcttccttg gcacttgaca ctaccctttg gaagtgtcct 81240attcttctta aaagtaaaga ccaagagagc ctcaccatgg tcttcaagca ccctcccatc 81300ccattcccca atctggcctc cactggctca tagtcttaac accagtggtc tttctgagct 81360cctgtcacca tggtctttct gttactccaa agggcctcgc tcctttctgc tgcagggcct 81420ttgcccctgc tgtttccttc accaggaaca tttatcccgt accccttgca gtctcctcca 81480gtgttgatcc ctcagttcta agaggacttc cccaggggag cctgtccacc ccagtgtcct 81540cgggcccctg gctttttcat agcactcatc agcccagcac gtgctaagct ttgttagaga 81600atgtcccatg gcagagcaga tgaacatcgc caccagtcga ggggtcatcg gccaccctgc 81660ctagcagttc tactatattt ttttgatcca ctcgtttctc ctctgaagtg actctttcaa 81720atgttcttta ccttcagact ttctctcctg cttcaaaaaa caaaacagaa gccataagaa 81780tggaactccc tataacttgt ggcccccaag tctgcacgct gatttccctc ccctgtgtta 81840cagatgacga ggagtccctt cttgctcagg ccacttctcc ctcctgtgct ctgggtccca 81900gtttctgcta cttgctctag aatctgacat catcagtcac cttttctctc ctggcaggtt 81960ttctgctgcc ctccttgctt gatcctctat tcacctttgc ctgtgccagc atcccttctg 82020tctccgctga ccccacgttc tctccctcct gcttctggcc agcacctcat caccctcccc 82080ttcactgctg acccctgcag agttgcctgt acttgccttc tgcacttgct cacccccgta 82140agtgttagcc cccggcatct gccagattct gcaggtggtt ttttagtttt cctcttgaag 82200tggttgacca ctcctactta aaacactttg tcttcccttg ccttgagacc acactctgct 82260ggttttcctc ttaactctct ggcccttctc tttctcctgg ctcttccctt agcttcatgt 82320atccaaactc ggcatctcca cttgactgat ggcccaaata attcctcaaa ctcaccttgt 82380cggggactgc attcatcacc tctcccacag tgttcccaag cagcctctca ctcccctacc 82440tccccacttg ctcgttttgc ttttcagcat ccttaagtaa gccctgcctg accctgtaga 82500ttgggccagg gacccttatt ctagggcatt ctttcctttt ctaaaggcac tgatcgcagt 82560ttgtagttat atctttatta gggggttgct ggatgactct ctgccccgct agactgtaag 82620ctccgtgagg ggtagggaca tggtctgctt tcattcacca ctgtattcct gcaggcctgc 82680cacagtgcct ggcacttagc aggtgctcaa taaatgtttg ttgaaataat gagtttgagt 82740atttttttaa tgtcttcctc catactgaat tgtaagcttc ctagttgggt ttgctcacca 82800tggtgtctcc aggatccgtc actggggctg atgcacagga aggcccactg tctttcagtg 82860cacctataac tgaagggatg gggatccaac ctgctatctg agttggcagc ctcaggggaa 82920accagaataa ctggagttga acgggacaag tcaggggcat cttagttttc tgtagagtga 82980ggaattaatg tgaacgtgat cctcttttat gccacagaaa acttggtagg gcattatggt 83040taaggacatg ggcctgaggc cacgtgctaa gaaagtggca gagctgggat ctgagcctgt 83100gtggtcctgc tctagtgctc cagctcttag ctactgtacc aggctgggtg atgcatttta 83160ccagcagggc aacactgggc aagttgcttt cctttcagag cttcagtttc cctcaatgga 83220aatttggagg gggttactgt taagtgcttt ataggtactt ttcaaatatt tctttgatct 83280gctgaacaac tcactgatgt aggtattatt atcccctcct tagagatgag gtaactgagg 83340cacagagagg ttaagtaact tgccccaggt cacacagctg ataggtggca gagctggaat 83400tgttcaaaat tgcatatcct aaccctttat tgagttgtga aatcaattta ctgggtttca 83460acaagcatta aaaagaaaaa ggaaatagca taagaaatgc cagtgtatca ccacatgcag 83520taaaggtaag tattgtttca ctcgaagaac aaccattttt cagttattta tatgtctgtt 83580tacacgtgtg ggtgtgctgg gttatgatgt agaatggatt tcttactatg ggtcgtggcc 83640aaagaatgaa gtcatggctg ttgaagaagc tcatggcacg agaaactgct ctccctctac 83700cccactggat cacccgcaag tcccagagtt gaggctgaca cacttgttgg ggaaggcaaa 83760gcagtgcccc acataacttg gtggctttca cagccacctt caaaccctgt tcttttcaga 83820aagcagatgg ctcagggtaa ctgcaattct gagtatcgtg gggcaggttt ctgaagccac 83880tcatcccccc ggaaagtcag cgttatcttc aggttgacta catgggagcc ggggtctgct 83940gagtttccct ttgggtttca gtgcctgacc cactttgggc tggcaaaatt ctttgaaaac 84000atgaatgctg ggggtgctcc agaggactcg ggtggtggtg gcaatggcag tctgtgactg 84060ctctgaaagt ctgctttgct tttctccaca gggcttgtca gccctcaccc gctcgcttac 84120tctgtgactg gcgaatcacc tttcttggct ttcttggctg gcctaggccg gggccaacac 84180cacctctttc caaatcctca ccctctggtc tcctcggggc ctatcagctt ggcagccatt 84240gtgtttcctg atggccggag gaatttgcac gcccaggaga ctggcgtgca ggcctgagat 84300ggcccctttt agtcgacacc agcttgacta gtgctcacta gcacccaaat gatgcatgtc 84360caagattttc cagatctgtg tgccctggcc cctatggctc actgcccttg aggggatgcc 84420acgtggtact tgtggggctg gtgccaaaag aacaggtttc cttcttgaaa acgagcaggc 84480atactgcagg tacagttttg ttcttaatct tctccctccc catttttcta agaacccctc 84540ttctctgtta ccgatcagtg agtcagtata catttgtact tgatttctct tactatcctc 84600atgttgattg aagtcatagc tgcccttgag tttttactgt gaaagacggt tcaaagataa 84660cttgtttctt tttaagccca caatttcaaa ctctcttcaa agtggagccc tcctggagtg 84720tttgttacca gcgtggttgt gtagtcagtg agtgtagaga tgcagttcct tgagttttag 84780tttttgcatt tgtaaaaagg aagggtgttg ttttgaagga tagatgtgaa ggttttcaaa 84840tgccttggtg tgtcaatgag aggggcccat ggtggaggag gtgaacaata catgcttgtg 84900ctttctgctt tcatatctga ctttggagaa cgacttgttt gcttctgtcg atgttgtgga 84960tcttgggatt ggctcaatgg cgtgacctgc tttttggatg ttctcgccct cctcagccat 85020ggaaagggtg ctctgggggc tgaaggattg attgtgtatt tgtttttctt tctccttcct 85080ccaactgaat tgtggagtcc tttacctgct ggctagctga ttcctgagtg ttctcctttt 85140tctgtctcac atctatgact gcagtggctt ttagaagcct gtttgtaata tatgtccgga 85200ctaggccaga tggaggagaa ggcttgcctg ctactgcgca caggtgggag ggctggcttt 85260ctgtctgtct gtgggccttc ttgaagaggc ttggtttaga agatcctagg aggaggatgt 85320tttctgtcat gaaggactat ggtaacaaaa agaagtaagt tagtgcagcc tggcagaaat 85380tgtgttgaaa caaaagtcca aagacctgga ttttaggacc acggagggga ttggtgtgag 85440accaagctgg tttgctctga atctctcttt ctcatctgtg atgtgtggga ggtggcagct 85500gggctcccag agtcacccac cctaggccct gtagtattct gattcaagta cctctggtgg 85560gatttgtgag tcctagacag tagagaaaga cagacggtcc cttctagact ctagatccgt 85620atatatggtt caaatgttct ccccgagggt gtttatagaa agcagttctt gcagccattc 85680tgtgtctagt ggcctcctag ccacgcatgt cccaggccca tggaggtagg gaaatgggga 85740atgaatatgt gggcaaaggc aggtccaaag aaaagcctgc atgaaggagg agggagggat 85800cttgtctagt attgatgcct ccctctatct acttctagaa aggatggaga tgggttgggt 85860aagaccccag tctccctcat ccaagaagca gcagctgact gggtaagagc caaaaggcct 85920gggttgtgat gtgaagtgta tgaccttgag ccatttactc agcctctgtt ttatcctctg 85980taaaatggag acaatagtac tgcctgcttc agaggattac aagtacctgt ctcatagggt 86040gattgtgagg attgaattaa cacatgttgc ccaggcaccg tggctcatgc ctgtagtact 86100aggacttcgg gaggctgaga tgggaggatc gcttgagtcc aggagttcac gaccagcctg 86160gtcaacgtag gaagacccca tctctaaaaa caaaatacaa aaaaacaaaa acaaaaacca 86220aaaaacccca aaaacttctt taaaaaaatg tgaagcactt agaacagtgt gtaatgccat 86280agtaggtact atgtatgggt cagatggtga tactcgtttc tgtgaaggta attggttatg 86340ggttttcaaa aagtttcaac caggccgggc acggtggctc atgcctgtaa ccccagcaca 86400ttgggaggcc gaggagggtg gatcacctga ggtcaggagt ttgagaccag cctggccaac 86460atggcgaaac cccgtctcta ttaaaaatac aaaaattagc tggctgtgat ggtgggtacc 86520tgtaatccct gatacttgag aggctgaggc aggagaatca cttgaacctg ggaggcagag 86580gttgcagtga gccgagatca tgctattgca ctccagcctg gcgacagaac gagactccat 86640ctcaaaaaaa aaaatttttc aaccattgtc agaagtatca agaatgtcac attgcctctt 86700aggaagaaaa aggtgtgggc attcgcccca gctcctctca ggttttagtc aatgcttccc 86760tcatttacct taatctgtag acacttgata ctcattttca gaataagaac agtgacttct 86820ccatgatgag atgccacccc tgggaggacc cacctgaaat gcactcggct tatgccactc 86880agggatctga ctggcctggt gaaggaggag caagacccat aaatatctac agttgattca 86940atcccagctg tgtgtcaggc cctgatttct ggctgtccat cctcattcct cttgactgcc 87000ctgtgcttag tgcacatcct gactccccgg ggctctgttt tatgagtgag gaggtgaagc 87060gacctgccag gctgatacgg ggacgagagg gttagcatag ggacccaggc ctctgtgacc 87120tccctcccag tctccatttg cattccttcc tctgcctcac agtcttgtct acctgccatt 87180tcttagaaaa ggtgtttttg ttttgttttg ttttgttttg tttttgtgag ataatctctc 87240actctgtcgc ccaggctgga gtgcagtggt gtgatttcgg ctcactgcaa tctctacctc 87300ttgggttcaa gcgattttcc tgcctcagcc tcccaggtag ttgggattac aggcgcccac 87360caccacgccc agctaatttt tatattttta gtagagacgg agtttcacca tgttggccag 87420gctggtcttg aattcctgac ctcaggtgat ccacctgcct cggcctccca aactgttggg 87480attacagatg tgagccaccg cgtctggccc tttttttttt tttgagactg agtttcactc 87540ttgtccccta ggctggagtg caatggcgcg atcttggctc actgcaacct ccgcctccca 87600ggttcaaatg attctcctgc ctcggcctcc caagtagttg ggactatggg cgtgcaccgc 87660catgcccagc taattttttg tatttttagt agagacaggg tttcatcatg ttgcccaggc 87720tggtcttgaa ctcctaacct caagtggtct ccctgcctcg gcctcccaaa gtgctggaat 87780tacaggtgta agccaccacg cccagccgaa aagatgggtt cttctatctt ttctctttcc 87840atagccagga atgtatccta aagaaatgat acaagtgtcg ataaaactat gcccaatgct 87900gtttgctggt ttgctttgca acctctaaat gaaagaactt agctgctcag taggaaggga 87960atggtatgat atgagtatta agcagccatt tgaaatgttt gcaaaagtgt ataatgatgt 88020ttcttactgt taaatggaca aagctagctg tgaaattatg tctgtagaat ctaaatgatt 88080tcgtagaata tctggaagga aattttccag aatggtaaca gtagttgtct ctggctgatg 88140tgtgattgtt tttcttgctt ctttagactt tcctgtattt tgcgaatttt ctataatgag 88200gctgtaggac ttttaggatg aggggataaa caaaggcagc agaagagaaa acagcagctg 88260tcaggtgaag gtgggctagc acagaggctc agaaggtgcc agtgggtggc agaagtgacc 88320tctcagggta ttattagatc catgtactct ctcctctgcc ctgcaggcct gccgaggagg 88380aactgaagac gaagaacatg gatgataaca cctttgccat gggtaagcaa ttggcaagct 88440tcggaagttt tagcttgtat ttaccatgtc tttcttccta ctgtttttct ttctataaaa 88500atgaaaaagg ctgggctcag tggctcatgc ctgtaatccc agcactttgg gaggctgagg 88560caggaggatt gcttgaggcc aggagttcga gatgagcctg ggcaacatag tgagacctca 88620tctcttaaaa aaaaaaaaaa aagacgggca tgctggcatg catccgtagt ccctgctact 88680cgggaggctg agctgggagg atcatttgag cctgggaggt tgatgctgca gtgagtcatg 88740attgtgccac tgcaccccag ccttggtgac acatcgagac cctatctcaa aaaaaaaatg 88800aaggggtacc ctctgcttta gattgtcaca gaaagcttca gaggcaaaac tgccagttgg 88860ctaggatggc agattttctt aacaaaacca tacctacttg tatgcctgtc ttgcccaatg 88920ctgcctttct cttccctcta gcagagcatc ctgatgtcca ggagtcagtg ggtccactgg 88980tagcccccac ccctctccgt ccatggcccc agatgacact tcaggtaagt gggtccttgc 89040agccttggag tagacagccc aataggaggc tcacagtgtg actattgctg ggctgggtgg 89100tggggcccac tagctctggg tcctccctgg gttggaggga gggcacaaaa ctcagaacct 89160gatgggggag ggggagaatg cagttactca gctgggcttg tagaaatgtc atttgttaat 89220ttagcaaaaa tttatggagt gcttgttctg tgctaagcac tgttacaaac atgcaaaaac 89280gctctttgag ggggcttatg tgttagtagg gtgagacatg ataaagtaat agaatatatg 89340gtactttagg tgatgatgag tgataaggaa aaatagaaag gaatagggag tgggtgagag 89400actctgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgttgtgggc 89460gggggtcggt tgcaacttta gattgggtgg cctgggaagg ctttagtgat tagtgagatc 89520tgagcgaaga tctgaggaag gtgagagagt gagccacgtg gataactaag ggaagaacat 89580cccaggcgag tgtgaagttc ctgaggtgag gccatgacta acgtgtgtga gggacagtgt 89640agaagccagc gcagcagggg cagaacgagg gagggggaga gtggtgcggg gtgagggaga 89700gaggtggctg aggacagatt gggtggtgcc tcacaggcca tggtgaagac tttggctttt 89760actccgagtg agggggagcc gcaagagcat tgtggacgga ggagggatgt aatctgattt 89820aggatttaac aggctccctc tggctgctgc ttggaaaata gacgtcagag taagggtgga 89880aacaggcgac tcgtgaaggg gaggccactg cagtggtcca ggcgagagtg atggtaggta 89940ggggaggtag agacattttg gagggtgtgt gtgtgtgtgt gtgtgtgtgt gtgaactttt 90000taattacaaa aagtttcaaa catacaaaag taaaatagta taatgaacct acgtacacca 90060tcccccagct tcagtaaata tcaacacgtg ctcattctta ttttatctgt accccgtctc 90120ctccctccca ttccctctgg aattattttg gaagacatcc agacatcatc ccatccataa 90180atacttctgt ttgtatctca gaaaattaag aactcttttt tgtttttttt tttgagacag 90240agtctcactc tgtcacctag gctggagtgc agtggtgtga tctcagctta ctgcaacctc 90300tgcctcctgg gttcaagcaa gtacatccag ctaatttttg tatttttagt agagacggga 90360tccgttgcct tggcctccca aagtgctggg attacaggtg tgagccatca cgtctggcca 90420gaaaattaag aattcttaac cccacaatac tatcatcaca ccttagaaaa taaacggtaa 90480ttcctcagta tcatcaagta tttggtcagt gttcaagttt tcttctctca taagtgttta 90540ggatccaaac aaagttgaca tgttgcattt gattgatgtg tctctttaga tttttaaaat 90600ctcccccact ttgtttcttt gcaatttgtt ggttgaagaa actttgcttg tcctgtagaa 90660ttttccagtc tgggtctggc tgattacatc caggtggaaa tacatttctt actccatttt 90720tattaattta ttaattatat taattaatgt aatcttatta aatttattta atttatcaaa 90780ttgtattaaa cgaatgtatt taatctttta ttattaaatt aactagaatt aactctattt 90840aattctatta ttcccacttc ctttattagc tagaattctt ctctaaagaa aaactgtccc 90900acaccagcta tttggctact ttgagatata gtttgtgcag aaaaagcagt gtaaatattt 90960aattttttcc attttattta ccagttttca gagaagtact actacaaagg tgaccaataa 91020aaggattttt ggttttgtgt cattatgaac tcatgaattc taaacatatt tgaatgagtt 91080tcaatccatt gcacttattt gtttgctttt tttgatgctt aaattgaact atccctggct 91140agtaggagct tattgagatg ggctactctg tccttttgac acgccctcat cattctgtga 91200gcctttctca catcttccat gacaatatgt gctaggttca tcctgtactc tccctgcccc 91260aagcctggaa acagccattt ctttcttgct tttcttttct ttattttctt tttctttttc 91320tttcttttct ttcctttcct ttctttcctt acctttcctt tctttctttc ctttcctttg 91380tctctctctt tctttctttt cttttacttt atttttcttt cctttctttc ctctctctct 91440ctctttcttt cttttttctt tttctttctt tttttttttt ttttttttga gtcagagtct 91500ttctctgtcg cccaggctgg agtgcagtgg cacaatctcg gttcactgca acctctgcct 91560tctgggttca agcaattctc ctgtctcagc ctcttgagta gctgggatta caggcacatg 91620ccaccacacc cggctgattt ttgtattttt gcagagatgg ggtttcacca tgttggccag 91680gctggtcttg aactcctgac ctgcctggtg atccacctgc cttggcctcc caaagtgctg 91740ggattacagg cgtgagccac tgagcctggc cagaaacagc tatttcttta aggagcactg 91800attcctttca gtggagagtg gcattcaaag gccaagattt gggggctcgg catgcttgct 91860gctactagat tggtcattgt ttctaggccc tttcaaaaga tggagctagg aaataattgt 91920tttttaaaga gaagatgata catcatgggt tcatatggac atcctggcta tgttttggga 91980tttttttgca ttttattttt tttaaaattt ttttaatttt taatttttga ggggacatag 92040taagtgtata tatttatgga gatgttttga tataggcatg cattgtgtaa taatcacatc 92100atggagaatg aggtatgcat ctcctcaagc atttatcctg tgtgttacaa acaatccact 92160tacactcttt attttttttt tttttgagac aaagtctctg tcgcccaggc tgcagtgcag 92220tggcacagtc tcggctcact gcaatctctg ccctctgggt tcaagtgatt ctcctgcctc 92280agcctcttga gattacaggc gcacaacacc acgcccagct aatttttgta tttttggtag 92340agacggggtt tcaccatgtt ggccaggctg gtctcgaact cctagtttca tgtgatccgc 92400ccgccttggc ttcctaaagt gcttggatta caggtgagag ccactgtgcc tggctactta 92460cacttttagt tattttaact ggctctgttt tgaaggtaag ccagcagtat ttagtgatga 92520tcattcacca aatattcaca cccaccgaat tcctcagctt tataatctgc aagaagccct 92580gttttactgc atccaattgt gttattttac catagatgac agtctttgga gtctagacag 92640tgacagaggg cgctttaagg catttttata tagaaacccc tgtggggctg gctgtgggct 92700gtcacacttc ttactgtctc tgcttgttcc atcccctcct ctgcttcatt cccttgcttt 92760ttctctgccc ccttgccccg gccatggctc caggtttctg acgttccgat gactggcaac 92820cccgcagaag aaggtgatgt ccccagaagc agtccacctg tggcttttac agaggtcctc 92880caggcaccgg ccatcaggat tcccccctcc tcccctctct gtggcctggg tggctccccc 92940agagaccagg cctcagggcc cgatgcgagc gagggggcca ccgggccttt cctggagccc 93000agtcagcagc aggcagaggc cacatgggga gtatcgagtg agaatggagg ggggctggag 93060gctgtgagtg ggtcagagga gctgctcggt gaggacacca tcgaccagga gctggagcag 93120ctctacctgt ctcacctgag ccgcctacgg gctgctgtgg ctgcgggtgg ggcagggggt 93180ggtggggagg gctccacaga tggagggatg tcccccagcc atcccctggg catactgacg 93240gaccgcgacc tgatcttgaa gtggcctggc cctgagcggg ccctgaacag cgccctggct 93300gaggagatca cgctgcacta tgcccggctg gggcgtggcg tggagctcat caaggacacc 93360gaagaccctg atgatgaagg ggagggtgaa gaggggctct ctgtcacacc ctccagccca 93420gaaggggaca gccccaagga atcgcctcca gaaatcctct ccggggcccg ttctgtggta 93480gccacgatgg gagatgtgtg gctccccatg ggcagagggc tcaggatgtg acggccctgt 93540ggttctgggt acagagggtc agttcattgg ggatcctgag aaagggatgg gcaaggacac 93600cagctctttg cacatgaata gggtgatagc tggggtgact gagtccctgg gggaggccgg 93660gacagaagcc cagatagagg tcaccagtga gtgggcaggc agcttggatc ccatatctgg 93720caaggagcca gcctctcccg tccttctgca ggggcaaaat cccaccctcc tcagtccctt 93780gggggccgaa gtctgtctct ctagtgtagc caggcctcat gtgagctccc aggatgaaaa 93840ggatgcaggc ccaagccttg aacccccaaa gaagtctccc accctagcag tccctgcaga 93900atgtgtgtgt gcactgcctc ctcagctccg ggggcccttg acccagactc tgggggtcct 93960ggccgggcta gtggtggtcc ctgtggctct gaacagcggt gtgtccctcc tggtgcttgc 94020gctgtgcctc tctctggctt ggttctcata ggctctgctt gtgggatcag cagaggctta

94080agatgggata catggcctgt gcagtgaggg gacctgggtc ctttgcttct gagaatgctc 94140aactgaaaga gaggccttct catccccaag ctctccagtc aacacagggc tccctgtggt 94200gacaccagtg gagatgaggg aacgggtaga tggtgtgagt gaggggaact tttagagtgg 94260aactgggcat gtcctccgcc taccccccga gcctgtattt atttttgtat aattctctgg 94320atgagggaga gtggtcgtga gctggtcttg gggcacaatt acccagagat atatttatta 94380acagccaacc tgtgcaacct gctggagctt tatttttaat ttaatttata tagagtacct 94440attattatat gccacaatag agctctatga gaaacagtgt cttgcggtgt agtgttctcc 94500tgtttgggca tgagtgtgca gggtggtcac tttctgtggg aggatcacag tggggagttg 94560ggggtgggac gtggtcgcct gctgctgctt caacatgtct ttccttgaag atgtgtgtct 94620cctcgtctcg tggtcctaat ccatatggtt ctttgtcttt tccacattct gcctgtggga 94680ccctacaggt gtgtatttgg atggtggtgg tgggagccag ggaggaagag tggcagccac 94740atgagggttt ggtgtcagtc acatggttgc agtggtagct gtggtctcct gtggatgtgg 94800ggacatcagt tgtgaatcag ccacaaggtt ttgaggttac tgaaaaaaca gcctttgaca 94860ccagcaggga gaccccttag tccctgagat aaggaaggcc tcagaaagga aagaggagtt 94920aatgtactgc agtacttggt agcacagttg ctgtccacag acatcacatt tctactaaaa 94980acaggaagcc cagaagcttt gaaagaaaga tatatattta ttgcatgcaa ataaaaaact 95040gctcaacaaa ataaaacacc acatatttat ttacttccct ttacagcaaa acctattgga 95100agaaatatct atacgagctt tcttcagttc ttctatgatg cctttctgaa atagccccaa 95160tcagatcttt tttgcccaac catttcagca aaactgctga agatattcac attgttatag 95220ccatggttaa gtctcagatg tcatcttact tgggatattg gcagcatttg acatagttga 95280tcatctctac cttgcaaccc tttcttcact tggtttccaa gataccacac tctcctcatt 95340tcccttcttc ctcactggcc actgtgcaaa gctgtcagtt ctcttgaaac tgctttgtag 95400gttcaatgca aatccaacca caattccaat agagttttgc ctgtgtgtag atcatgacac 95460attaattctt aaatttatgt agaagtgcca agactagcca ggacactgat acttattgta 95520atggtatatt cagatggcac gatatttttg caagagtaga caaatcgacc aataaaaaaa 95580cagtgatctc ggaaacagtg tagacacact cgtgtagaca cagaactggt gaccaaggaa 95640gtgtgttaga ccagtgggga aatgattgga ttttgaataa atagaacaat tggttacccg 95700cattgaaaaa tatgaacttg gatccctact ttacaacaca aataaaaata attctaagtg 95760ggtttaaagc ctaaatgcga agggcaaaac caaagcgctt taagaataca atgaggcagg 95820gccgggtgct gtggctcact cttgtaatcc cagcactttg ggaggtctag atgggcggat 95880caggagtttg agaccaccag cctggccaac atggtgaaac cccgtctcta ctaaaaatac 95940aaaaattagc tgggcatcca cctgtagtcc cagctgtttg ggaggctgag gcgggacaat 96000cgtttgaact cagggggcag aggttgcagt gagccgagat ggcaccactg cactccagcc 96060tgggccacag atcgagactc catctcaaaa acaaaacaaa acaaaaaaca aggcaaaaca 96120ccttcgtata ctcatggtag caaagttttt cttcatcacg acacagaaac aaccataaat 96180gaaaattacg aaacattcag tgaccttaaa attaggaatg tccattgatc aaaagacacc 96240ataaagagag tgaaaacctt atgtagtagg ttgctatgaa taaaagcaaa acaaaaagga 96300gtgaaaacac aagtgaaaaa ttgggaggag ataatggaat cacatataac tgatgaaggg 96360ctcaaacctt agatgccaat aagcatctaa ataaattgtg gtatattcgc agaatggaat 96420attgtataat gagaatggaa gatccataac tgcacgaaag catacagatg aatcacaaaa 96480agccaaaaaa aagcttccac actgatccat ttatgcaaag ttcacaagca ggcaaaactc 96540atgaattacc acctaaatgg tgacattgta aagaaaagcg aggggctgat taccagagtc 96600aggatagtgt tcacctctgg cagagggagg gatgtggctg agaaggggcc cagaggactt 96660ctgggatgtc aataatggtt cactgcttga cctgggagac tgttcatgga tgtgcaattt 96720attcatcatc attaaaacag acttatgggc tgggcgcggt ggctcacacc tataatccca 96780gcactttggg aggctgaggc aggcatatca cttgaggtca ggagttcgag accagcctgg 96840ccaatgtggg gaaaccccgt ctgtactaaa agtataaaaa ttagccgggc gtggtggtgg 96900gcgcctgtaa tcccagctac ttgggaggct gaggcgggag aatcactaga acccaggagg 96960tggaggttgc agtgggccga gatcgcacca ctgcactcta gtctgggcaa cagagcgaga 97020ctcttgtctc aaaaaaacaa aacaaccccc cccatacatt taggttttat acactcttat 97080gcaaagacct cacaagtgaa gcagagtgca gtgggaagtg ggagtagcat gagtgtccga 97140gactcctgca cacaggcttg cagagactcc agcctcctgg tgtgaaccac agtgtaggcg 97200gcctccttcc tgcagaacaa tccctgggct ccgggtcttg ccttctgttt gtggagatcg 97260actagtgggg aatcctctat cgtgtgcccc tgacaccagc catagagtgg ggcctcttgg 97320gatggacagg acctgcccat attgtcctca gatgttgggg tccatctccc ctcaggcctc 97380attcacaccc acaatagccc aggacacatg ggctttgggg cctggtgggc ctcataaaag 97440cctggaaccc cgagccctgg tggtgtgacc ttgggcaagt cacccgtcct agaccagcct 97500cagttcccca ttcataaagg gggctaatgc ccatcaagca gggtaggtgc aggctttggg 97560gaaaacgttg tactgccctc ctcctgagcc catgttagac aggtcttata gtcaaggcag 97620ggtaggaggg tggacaggaa caggggctgg agcgacacag tttgggacta aattgcccag 97680ggtcacacag tgggcaagtg gcttcagccc tctgtgcctc cctttgctcg tctgtcatca 97740gggtaataac ttttcctcct cacagggtta agtgaggaag aggtgagatg actcttcagt 97800cactgagcag tgccaggcac agggcaagac tgagtacacg cccgttgtca ttccccctgg 97860ctggagctct tgcaatcact gttatgcccc caccctgccc tctcccatta atctgttaat 97920gtggtgaatt acattgataa gtttgtacat ctgtctttta aaattgagac gcaatcttgt 97980tctgtcaccc aagctggagt gcagtggcac aatcacggct cactgcagcc tcaacctcct 98040gggttcaagt gatcctccga cctcagcctc ccgagtagct gggactacag gcacatgcca 98100ccatgcccag ctaattttta tatttttact agagacaggg ttttgccatg ttgcccaggc 98160tggtctcgag ctcctcagct caagcaatcc acctgcctca gcctcccaaa gttttgggat 98220tacaggcatg agcacgtcca gcctactttt ctctctctct ctctctctct ctctctctct 98280ctctctctct ctgtctctct cctttctttc ttgacggagt ctcgctcttt ctcccaggct 98340ggagtgaagt ggcacattct cagctcactg taacctctgc cccctgggtt caagagattc 98400tcctgcctca gccttctgag tagctaggat tacaggcatg caccaccatg cccgactagt 98460ttttgtgttt ttagtagaga cagggtttca ccatgttggc caggctggtc tcgaactcct 98520gacctcaggt gatccacccg cctcagcctc ccaaagtgct aggattacag gtatgagcca 98580ccacgactgg ccctactttt cttttttaaa attttagtgg tcagttacta gataaactct 98640atttttatca cccatttaca ggtgagggcc ttccatgttc ttatcagccc tgagctcagc 98700cctttgccga ggccttcctg gtccacctct ccctcctcct tgtgactagg gccagcatct 98760tagtctcaca gtggcccctt ctctctctgt cctcagcctt cagccctgct atcacccacc 98820cgcatcctac aacccctctg cttcccctct tcagtgtttt ttgtccagga tgagaccaag 98880tccaaacctg agcttcctgc taagtcaaag tgactttttc ctttcagtca gggaagcctg 98940actaaagcgt ctggtcccac ggtgaggtct tggtgtctcc aacagcgcag ccatggacgg 99000ccaccctaca gtgatacctg cctttgccag cagggggcat cttatctcca agacagggaa 99060agcctcagag aagacgggaa cagtcttaaa cgttaactgc ttgtagggtc acaggcctca 99120gggtgcatga gctccaggct gggtgtccct tggagcctcc ctctctgcat gtggacaggt 99180gggggcagcc gagggcctgg cttttactgt tgttcggggt cttctacaga tgactctcct 99240cctcctcctc ttcctctttt tccttctacc aggaactcat ttccatcact aaagagaggt 99300tctcatattt ttaatcctct ttatctcact taaagggtcc attataggat ttttccacaa 99360gatgcactgg ctctgaggcc tgtgcccagg gcaggtcagg ctatagattg gccaggttgg 99420ccaggttggc caggttggct gagggcacca gcctggatcc tgaagacact ggagacagcc 99480ccaacagagc ctggggctca ggaaaaggcc accaaccccc cagccagatc ctggccagct 99540gccagctccg cctccaccca gtcctctccc agggcctgaa cgccgggtct agaagctcca 99600ggcctggtgg tctgggggag acacgggccg ggaggatccc tcgaaagagg aggcaggaga 99660caatctctgt atggcttgag ggatgcacag ctgtggggtg aggcacgccc cacctagcac 99720aggcccactg acactgctgt gcagctgcag gcccagctgc tgccctctat ggggctcagt 99780ctcctcacct gtgcaatggg catcattaca cccccaacgt gcaggggatg ctcgtgaatg 99840gaggggagcc gaggcgttga gaaagtagat ttggctcaag aggaggagga gcaggtcagg 99900gttggtgggg aaggatggat gcggccacct caggcatggc cggggctgcc ttcctcaaag 99960tgctcaggac agtcaggtgg gggaagttct gagtctgtga gcagaaccgt gggctgggga 100020ccaggtgagg ttgtctctgt agtgttctgg ggtggccagc cactgtgcca ctattctttc 100080ctgttggtcc tcatctctgc ccacggggag ccccaagatg ctccaagagc tcacagtgag 100140ctcatgtgag ttactgtgac ccacatgcag tcattacagc catgtgtcat catcacaaca 100200gccaccaggt taacacttgg ataatttatc caaataacga ctcacctgat gtgatttgca 100260tgatatgcca tcatttatta ttatactcct attggatgtt taggtagtgt tcgatttgct 100320tttttgccac tataaactat ctgtggtgag cattgtttcc catatatgta aacttgggag 100380agacacagga atgtttatat agtaaaacta tttgtaatag caaaatactg gaaacaaccc 100440aattatacat catcaggaaa tgcataagta aattatgctg tattgacatc atggaatact 100500acacagctat gagatggagt gaactaaggt acacaaagac acaatgttag gtgaaagaag 100560ccagatacaa aagaggagat gatatatgct tttcatatat ggctcaaaac caaacaaaac 100620tagtatttag gcatacatac acctgtgtta aaacagtttt acagaaaaca aggattctgg 100680ccagtcgcgg tgactcacgt ctgaaacccc agcactttgg gaggccgagg tgggaggatc 100740acttgagtcc aggagtttga gagcagcctg agcaacatgg agaaccccat ctctacaaaa 100800aatacaaaaa ttagctaggc atggtggcat gcgcccatag tcccagctac tcagaggcgg 100860aggagggagg atcacttgag cctgggaggc agaggttgca gtgagccaag atcgggccac 100920tgcactccag cctgggtgaa ggtatgagac cctgtctcaa aacaacaaca acaaacaacc 100980aaacaagcaa aaccccacac aaggattttc aactgcttga ttgataccat ttaaaaaatt 101040tatttaaaaa attgtcacag tagaatttac tttgggggag tttctatgaa ttttaatatg 101100tgtagattca tgtaaccacc actgtaatta gtattcagaa cagttctatt accctaaaga 101160actccattgt gcctcccctt agagtcactc cctctcctca ccccctcacc cacgcaacac 101220tccatcacta catttttttt tttttgagat agcggctcac tgcagccttg acctcctggg 101280ctcaagtgat tctcccacct cagcctcccg agtagatggc actacagaca catgtgccac 101340cacacccagc taaattttgt attttttgta gagatggggt ttcgccgtgt tgcccagggg 101400tctcgaactc cggagctcaa gtgatccgcc aacctcggcc tctcaaagtg ttgggagtac 101460aggcgtgagc cactgcagct ggtcaggcct ttcaattaca atgttgaata gcaggagtaa 101520gagtggacat tcttgcctca tttctgatct tagggagaaa gcatttagtt tttctctatt 101580aagtatgatc taagccaggc agggtggctc acgcctgtaa tcccagcact ttgagaggct 101640gagggggggt ggatcatgag gtcaggagat cgagaccatc ctggctaaca cggtgaaacc 101700ccgtctctac taaaaataca aaaaattagc caggcgtggt ggcaggcgcc tgtagtccca 101760gctactcggg aggctgaggc tgagaatggt gtgaactcgg gaggtggagc ttgcagtgag 101820ctaagatcgt gccactgcac tccagcctgg gcaacagagc gagaatatgt ctcaaaaaaa 101880aaaaaaaaaa gtatgatcta agctgtaggt tttttttttt ttttggtgga tgctttatat 101940caggttaaga aagttatctt cttgtttgct aagagttttt tttaagatca tgaatcaatg 102000ttgaattttg tcagaagctt tttctgcatg acatgatacg attatgtggt tcagttactt 102060tcgaatgtca gtatggtaga ttgcatttat tgatttttat tttattttat tttaatttgt 102120tttacttttt tgagacagag tctctctgtc accaaggccg gagtgtggtt gtgctgtatc 102180agctcactgc aactgcctcc tgggttcaag caattctcgt gtctcagcct cccaagtagc 102240tgggactata ggtgtgtgtc accacacctg actaattttt gtatttttag tagagatggg 102300gtttccccat gttggccagg ctcatctcga actcctgggc tcaagcgatc cccccacctc 102360agcctcccaa agtgctggga ttacaggcat gagccactgc acccggtcac attgattgat 102420ttttgaatat tgtgtcagtt ttgcatcccc aggataagcc tcacttggtc atggtgtctt 102480atttttgtat attggatttg aattggtaat attatgttga ggattttttt ttcatctatg 102540ttcatgaagg aagttgggct gtacttttcc catactttct ttttttctgg ttttggtatc 102600agggtaatgc tatttgagaa gtgttcctgt cttatttttt gaagagattt tgtagaattg 102660gtattatttc ttcttagatg tttgacacaa tttgccagtg aaaccatctg gacctggcaa 102720ctttatcttc ttctttttaa aaaagacttt ttttttttta aagagcagtt ttaggtttac 102780agtaaacctg agaggaaggt acagacatat cccatatacc cctcacctcc acacatgcac 102840agcctcccca ttatcagcaa ccctcaccag cgtggtgcat ttgttacaat tgatcattat 102900caccccaatg atacatgagg tatcatattg gcctgaaatt ttcttttttt gttgtgtctc 102960tgccaggttt tggtatcagg atgatgctgg cctcataaaa tgagttagga aggagtccct 103020ctttttctat tgtttggaat agtttcagaa gcaatggtac tagcttctct ttgtaactct 103080ggtagaattt ggctgtgaat ccatctggtc ctgggcttct tttggttggt aggtttttaa 103140ttactgcctc aatttcagaa cttgttattg gtctattcag ggattcgaat gcttcctggt 103200ttagtcttgg gagggtgtat gtatccagga atttatccat ttcttctaga ttttctagtt 103260tatttgtgta gaggtatgta tagtattctt tgatggtagt ttgtatttct gtgggatcag 103320tgctgatatc ccttttattg ttttttattg tgtctatttg attcttctct cttttcttct 103380ttattaatct ggctagcagt ctatctattt tgttaatctt ttaaaaaaac cagctcctgg 103440atttgttgat tttttgaagg gtttttcacc tctctgtctc cttcagttct gctctgatct 103500tagttatttc ttgtcttttg ctagcatttg aatttgtttg ctcttgcttc cctagttctt 103560ttaattgtga tattagggtg tcgattttag atctttcccg ctttctcctg tgggcgttta 103620gtgctataaa tttccttcta aacactgctt tagctgtgtc ccagagattc tggtacattg 103680tgtctttgtt ctcattggtt tcaaataact ttatttctgc cttaatttca ttatttaccc 103740agtagtcatt caggagtagg ttgttcagtt tccatgtagt tgtgtggttt tgagtgagtt 103800tcttaattct gagctctaat ttgattgcac tgttgtctga gagactgttt gttatgattt 103860ctgttctttt gcatttgctg aggagtgttt tacttcaaat tatgtggtca attttagaat 103920aagtgcgatg tggtgctgag aagaatgtat attctgttga tgtggggtgg agagttctgt 103980agatgtctat taggcccgct tggtccagag ctgagttcaa gtcctggata tccttgttaa 104040ttttctgtct cactgatctg tctaatattg ccagtgaggt gttaaagtct cccactatta 104100ttgtgtggga gtctaagtct ctttgtaggt ctctaagaac ttgctttatt tttatttatt 104160tatttattta tttatttatt tatttattta tttatttatt ttttgagaca gagtctcgct 104220ctgtcaccca ggctggagtg cagtggcgca atctcggctc acttcaagct ccatctcctg 104280ggttcacgcc attctcctgc ctcagcctcc cgagtagctg ggactacagg tgcctgccac 104340tatgcctggc taattttttt gtatttttag tagagacggg gtttcaccgt gttagccagg 104400atggtctcga tctccagacc tcgtgatccg cccgcctcgg cctctcaaag tgcggggatt 104460acaggcgtga gccaccgcgc ctggccaatt ttttgtattt tttagtagag gcagggtttc 104520accatgttag ccaggatggt ttcgatcttc tgaccttgtg atctacccgc ctcggcctcc 104580caaagtgctg ggattagagg catgagccaa cgcgcctgac caagaacttg ctttataaat 104640ctgggtgctc ctgtattggg tgcatatata tttaggatag ttacctcttc ttgttgcatt 104700gatcccttta ccattatgta atgcccttct ttgtctcttt tgatctttgt ctcttttggt 104760ttaaagtctg ttttatcaga gactaggatt gcaacccctg cttttttttt tttttttttt 104820ttttttttgc tttccatttg cttggtaagt attccttcat ccttttattt ttgagcctat 104880gtgtgtcttt gcatgtgaga tggctctcct gaatatagcc caccgatggg tcttgactct 104940atccaatttg ccagtctatg tcttttaatt ggggcattta gcccgtttac atttaaggtt 105000aatattgtta tgtgtgaatt tgatcctgtc attatgatgc tagctggtta ttttgcccat 105060tcgttcatgc agtttcatag tgtcaatggt ctttacaatt tggtatgttt ttgcagtggc 105120tggtactggt ttttcctttg catatttagt gcttccttca ggagctcttg taaggcaggc 105180ctggtggcga gaaaatctct cagcatttgc ttgtctgtaa aggattttat ttctcctttg 105240cttataaagc ttagtttggc tggatatgaa attctgggtt gaaaattctt ttctttaaga 105300atgttgaata ttggccccca ctctcttctg gcttgtaggg tttctgcaga gatccactct 105360tagtctgatg ggcttcccta tgtgggtaac ccgacctttc tctctggctg cccttaacat 105420tttttctttg atttcaacct tggtgaatct gatgattatg tgtcttgggg ttgctcttct 105480caagaagtat ctttgtgctg ttctctgtat ttcctgaatt tgaatcttgg cctgtcttgc 105540taggttgggg aagttctact ggataatatc ctgaagggtg ttatccaact tggttccatt 105600ctccccgtca ctttcaggta caccagtcaa atgtaggttt ggtcttttca catagtccca 105660tatttcttgg aggctttgtt cgttcctttt tattctaatc ttgttttcat gctttatttc 105720attaagttga tcttcagtct ctgatatcct ttcttccgct tgatcgattt ggctattgat 105780acttgtgtat acttcatgaa gttctcgtgc tgtgtttttc agctctatct ggtcgtttat 105840gttcttctct aaattagtta ttctagttag caatttctct aacctttttt caaggctctt 105900agtttccttg cattgggtaa gaacatgctc ctttagctcg aaggagtttg ttattaccca 105960ccttctgaag cctacttctg acagttcgtc aaactcattc tcggtccagt tttatttcct 106020tgctggcaag gagttgtgat cctttggagg agaagatgca ttctgggttt tgcaattttc 106080agcctttttg tgctggattt tcctcatctt tgtggattta tctccctttg gtctttgatg 106140ttggtgacct tcagatgggg tttctgtgtg gatgtccttt ttgttgatgt tgatactact 106200cctttctgtt tgttagtttt ccttctaaca gtcaggcccc tctgctgcag gtctgctgga 106260gtttgttgga gttccactcc agaccctgtt tgcctgggta tcaccagcag aggctgcaga 106320acagcaaaga ttgctgcctt ttccttcctc tgggaagctt cgtcccagag gggcacctgc 106380cagatgccag ccggagctct tgtatatgag gtatgtgtca gcccctgctt gaaggtgtct 106440cccagtcagg aggcatgggg gtcagggacc cacttgagga ggcagtctat cccttagcag 106500agcttgagcg ctgtgctggg agatccgctg ctctcttgag agccagcagg caggaacatt 106560taagtctgct gaagctgtgc ccacagccgc cccttccccc aggtgctctg tcccagaaag 106620atgggagttt tatctataag cccctgcctg gggctgctgc ctttctttca gagatgccct 106680tcccagagag gtggaatcta gagaggcagt ctggctacag cgggtttgcc aagcggccgt 106740gggctctgcc cagtttgaac ttcccggagg ctttgttcat aatgtgaggg gaaaactgcc 106800tactcaagcc tcagtaatga caaacgcccc tccccccacc aagcttgagc atcccaggtc 106860aacttcagac tgctgtgctg gcagtgagaa tttcaagtca gtggatctta gtttgctggg 106920ctccgtggtg gtgggacctg ctcagctaga ccacatggct ccctggcttt agcccccttt 106980ccagtggagt gaacggttct gtcttgctgg cattccaggc accactgggg tatgaaaaac 107040aactcctgca gcaagctcca tatctgccca aacggttgcc cagttttgtg tttgaaacct 107100agggccctgg tggcataggc acaggaggga atctcctggt ctgtgggttg agaagaccat 107160gggaaaagca tagtatctgg gcctggatgc accgttcctc acagcacagt tcctcataac 107220ttcccttggc taggggaagg agttccccga cccctactgc ttccccggta aggcagtgcc 107280ccactctact tcggcttgcc ttccatgggc tgcacccact gtctaatgag tcccagtgag 107340atgagccggg tacctcagct ggaaatgcag aaatcaccca ccttctgcct tgatctcact 107400gggagctgca gaccagaggt attactattt ggccatcttg ctattgctgc tgtttttttt 107460tttttttttt tttttttttt tttaagacag agtctcactc tgtcacccag gcgggagtgc 107520agtggtacaa cttcagctca ctgcaccctc tgactcccag gctcaagcga tcatcccatc 107580tcagcctctt gagtagctgg aactataggc acaagccacc gtggcccgat taattttgtt 107640tttttgatat aagttttgta gagatgaggt ctcactatgt tgcccaggat gggattctct 107700ggctcttaat aaataattgc tttttaaatc tttcacaaag gaaaccttga gtgagtgaat 107760aatcaaaagg tgatagattg tttagtttct attttctgtg gcatgaaggt cagtgatgct 107820caggatgggt gtgagtaaga tgcttgtgct aagcatgctc cctgccccac agtcagtctg 107880catgagccac tgtttctaat aagactgtgg atagagtgat ataatcacct ctaaccatat 107940caaatgttac acgtaagttt cagattttga gacatgagtt gataagattt gaagttcaaa 108000gaccatgact ttagtacttc ctgagtaatc aactgaaata tgttttacat atgtgttttc 108060caaattgctg accattcatt ataagtgctt ctgaatttaa aggaggtact tgatgtatag 108120gtaagaaatt acctttaaat tctggaggtc taccctcaaa gtgtatacag aggtttaatt 108180ggatgtaaga cacaggatca cctttagggt tctgtttttt tgtctattta ataaaaccca 108240aactgtagta tgctttacat gcctttagaa tcatataaat aaactgctgt taagtaatgt 108300tcccagttgt tatgtttctg ttacaggtga aaagcaatca cggagttaaa agaagacaag 108360ctgaaatgat gcaggctgct cctatgttgg aaatttgttc attaaaattc tcccaataaa 108420gctttacagc cttctgcaaa gtagtcttgc gcatcttttg tgaattttat ttctagcttt 108480ctgatgctgt gaaatatgta tcattctttg aaattttata ttctaactgt ttcagctggt 108540atgcagagac atcattcctt tttttttttt ctttttttct ctttccagac agagtctcac 108600tctgttgctt aggctagagt gcagtggtgt gatctctgct cactgcaacc cccgcctcct 108660ggatgcaagc aatttctgcc tcagcctccc gagtagctgg gattacagga gcccaccacc 108720tacccagcca gattttgtat ttttagtaga gacggggttt cgccatcttg gccggggtgc 108780tcttgaactc ctgacctcgt gatccaccca cctcggcctc ccaaagtgtt gggattacag 108840gcgtgagcca ccgcgcccgg gcgagacata attcttatat attgattttc tatccagcag 108900ccttgtgaaa tatgcttatg aattctaaaa gtttacttct agatggtttt cagtcttcaa 108960catacagaat cataccatcc ttgaataaga acaattttgt ttctgccatt ttttttttct 109020ttttcctttt gtattttttg tagagacggg gttttgccat gtttcccggg ctgttcttga 109080acttttgagt gcaagtgatg cacccgcctc acctcccaca gtgctgagat tactgacgtg

109140ggccaccgtg ccgggcctgt tgttgccatt gtaaagagtt ttatttcctt ttctgatttt 109200atggcattgt gcagacccac ctgttaaaat ggtgacagtc aatatccttg tcttatccct 109260gatgagaaac cgaaaaattt caacatttca ccatcctatt tactgtcctt tttttgtaga 109320tggactttat cagagtaagt cattccattc tgttccaaat ttgctgagag tattcatttg 109380aatatatgtt gagtttcatc agtgcatcta ttttgtttat aacagcattt ttttcccatt 109440catctgttaa tgtagtgaat tagattgata actttgtaca tttttatctt ctatttttaa 109500aaatcgagac agggtctcat tctatcaccc aggctggagt gcagtggtgt tatcagagct 109560cactgcatcc ttgaccttct tggctcaagt gatcctccca cctcaggctt ctaagtagct 109620gtgactatag gtacatgtaa ccattcccag ctaatttttc ttcttctttt tttttttttt 109680tgtagtgatg agattttctc atgttgctta ggctggtctc gaacacctga gctcaggcaa 109740tctgcccagc tcagcctcca aaaatactag tactacaggc atgagtcttg gcctggccag 109800tttttcttat acaagggtct tctctatgta aagactaaac ttatctgtat ctttgtgagg 109860gtgtgctaag ggcatgatga aatttatcat tctattgatt taaagaaaac tatccttgac 109920tttccagtgt gtaagtccat gaaagcataa ttatgttgaa agcatatatt gttatgggtg 109980ttgagaaccc tgcactttct gctgctgtgg gagcatgtcc ttggaggtac ctttcgtctg 110040ttttctcaac tccaaacatc ttaggaccat gggttgtgac tggtaaggaa tgtgtcttgc 110100gagtttcaag atggagttga ttttcacatg gtgtcactct ggctctcctg tttctctaat 110160actggcactt ctctttctgt gattctgatg ctacaaatga tagatatcgt tttagtattt 110220tcttatgggt cctagagatt gtattcattt ttctttcagt ctctttctct gacttgttca 110280catttaacaa tttctttttg gggtaggttg ctatttctgt tttcgcaggt ggtttacctg 110340tcttctcagg cagtcaccgt ggtccttgtc cccatggtgg ggccggggca agagagggcc 110400ctggagggga ggggggttta gttgaagatg gagtgagttt tgaggggagc actacttgag 110460tcccagaggc ataggaaaca gcagagggag gtcagattcc attatcctca gtggggatgg 110520gaatcgaggg ttgggggcgt ggggctggga acggcagcct tccccaaccc gcagctgtac 110580atgctccttg gctcccgcct cagtgcgcat gtccactggg cgtcttctgc tcagccgctt 110640tacccacgtg gagaacgcca gggagctgtg agggtgtgtg gtctcgttcc tgtcgtctgg 110700aatatttttc ctctactgag attcatctgg taggtctgca ggccagtcct cccggggtct 110760gaagtgtgag tgagggtgaa gagcaggcag tgtgcttcgg gtgagtccgt tgggtcctgc 110820ttcgtggtct gtggcctctg agggagaagg gcctcgaggc ttctgaaagg gaaggggctc 110880ctggcctctg aaagagaagg gccttgaggt cgtcctcctt ctctcaaagg ctgtgaggcc 110940accatctgct ttgtggtcgt gaaggggcca ggacaaggag gaaggtgggc catggagggg 111000aggcggtcag gggctcaggt gaagaagggg cgattgctgg gtgtgctgtc agagggatgg 111060aagtccggag gtgccaggaa tacccgatac aggggagatc cctgaatgag gtcccggaca 111120ggtgcgagga gggcgataaa gaagggacct ggcacctggg aagactgcgg gctggtgagt 111180gcccctgagc tttgtggagt ggggagcccc gagtgagaag catcgcaaga tctcacctcc 111240gccatggaag gtctggcaac agtgggaagg actgggagag gctgtgcggt ccaatcaaac 111300ttgatttgag agaagtgaat ggctctagta agtgggagtg tgcccaaagt agcaatcacg 111360agaattatga ttcactaatg ttttcatgtg gagtccactt gtgaaactaa acctcatcag 111420aaatgacctc tgccagtggg gcgccatggc ctgcgcctgt agtcccagtt actcgggacg 111480ctgaggtggg aggatccctt gagcaggagg tcgatgctgc agtgagctgt gatcacgccg 111540ctgcactctg ccagagcaac acagggagac tgcgggacca aaagaaattt agaaaaaaaa 111600tgtcctctgc gttttgtcac acgccttaag atgattgctc tgccagcttg gccagcataa 111660gtggctttgt aggcactcag aaaaggtaca cacatatgct taactctggg acttattttg 111720aaagtatttt caaaattaaa acgacaagtt aacatttatc catggaggtg atggaatata 111780gcagcccttt cgagggcatg ctcccaatca cggttgtctg ttttcagtgt gaaatatgag 111840ttggcgagga agatcgacct atcggtctag accaagactc tatgtagagc cccctgaaat 111900gattgggcct atgctggtga gtgcttaaac gttaattcgt tgttttctat tagcagaaat 111960taatttttgt gacagtattg ttgcattagt atggaaatgc tgataaaggt ctttcctgct 112020cataaaaaat gatgatggcg tctcatgaag gaaacattga ttctggagaa ttttttttcc 112080tctagtgttc ttcagctttt gcccatgact tctttctcag gctttgtttg ttaatgacag 112140attgtacaca tgtattccaa cactgagtat aatagcctcc aaagtcctcg tgcgtcactt 112200ttctcatagt aaccttcctg tgggtcgagt aaccttattg ggcatagagc atagagttgg 112260agaaatgtct ttaggcttag ttaggaccag aaatagctat gtattctgtg tatatatgta 112320aaattttgta tcaataacga aacttatttt ttatttgcac acccacacgt attccccagc 112380ccgagcagtt cagtgatgaa gtggaaccag caacacctga agaaggggaa ccagcaactc 112440aacgtcagga tcctgcagct gctcaggagg gagaggatga gggagcatct gcaggtcaag 112500gtgagggaaa gggaagaaga acgtctgctg gtgtgtgcgt gtgtgtgtgt tcgtgtgtgt 112560gtgtgtgcac gtgtgtgtgt gtgtgtgtta ggcattgtca cataggagga agaggaggaa 112620agaaaacaat ggaaagaatg cctgaaattg actggaaaag cgaggaggct atgtagtttg 112680cagcttagct taggcaaatc cctcactatg ataaaagttc tcatctttat gaatgagaaa 112740atggaggcgc tgggattgtg ttttatccaa gagcccttga ctggtgaata caaaatttgt 112800attttgttcc aaggtttgtg tcttcctacc atctatgttg ctgtaaaaac ggaaatgatt 112860ttgctgaaaa tgcttaaaac tcaaaggctt tactgtaagg tagcttagta ctgacccaag 112920aatagaccca gttcagagga gcaggagcag ctccaaaaac cgagtcactg aatgtcagcc 112980actgtttcct ttgattgatg tttttatatg gtacatttga taaaagctgg ataaatgagg 113040atactgccat acaggtagct ggtttagtta ttttctaagt ggcttttagg aggtgattaa 113100atccttttat ggttagaaaa agcaaaaaag gaattatcct gagattaaca ttgagataga 113160aataatttct cctagataaa atattttcaa acaaaacatt tatgtaactg aggtcatgga 113220ttattccagg gatgcactgt taaaaatttc tagaatctga ctgacaacaa tgcccattaa 113280ttgctgtcct cccactccct tattctcagt gtgggacagt atattttctg tgattcacaa 113340acaatgttat atttggtgct ttgttcttca cggggttcat ttatggaata ttacatttag 113400gacctttgga cctaaatata actttatttg aacaaaatga agtttctatt tacctcaata 113460agtaatgggt gtcatgactg taagattttc catagtcctc aaatccattc agctaatcga 113520tccttcagaa attgacattg taattgtaac cgaaatccta tccatgtggt agacttgaga 113580tttcttagct gatgcacact gctctcggta ctctatggct gaatataagc attatacatg 113640tcctgtggtt tatccttaga ttgtcattta ggagaaaggt ctcaagctgg gctgaatgct 113700gtgcacgcat agtcccagct acttgggagt ctgaggtgag aggattgctt gagccctgga 113760gttgaagccc agcctgggaa acatagcaag accttatcac taataaataa ataaacaaac 113820aaacaaacaa acagataact aaaggtttca tggtatagga aaacacagat gcaaagtttg 113880tgcctagttg ctggtaatgt tgcaaacata actccttagt gaactgtacc acttaaaaat 113940agttaagatg gtaaatttta gaatatgtgt attttttacc ataattaaaa aaaacctcct 114000gtcttcctaa agttcagtgt aattgtcata tattctttta aatttttact gtatctattt 114060tcaaggcata acattatgga aaatttgcaa gaatagtaca atgaactcct atacttttca 114120cctagattca ccaattgtta atagctttcg cttcataggt ttcatatcac ttccctctct 114180taccctgctt cccacacact cacacacaca cacatacaga tatatgttta ctgttattaa 114240tgctgaattg tttcgataaa ttttcaggtg ttatgaccct ttacaccaag tacttgaggg 114300tgtgtacatc atcagaacaa agaaaaagta attccttggt catcactgca gaaaaatcaa 114360aatcaggaaa tttaacaatg agaaaatgca gtcatttatt acacagtgta tactcaaatt 114420tcgccagttc tccagacaat ttcttttttc cttttttttt ttttcctttg ttgagacgga 114480gtctctctct gttgcccagg ttggagtgca gtagtgcaat cttggctcac tgcaacctac 114540acctcccagg ttgaagggat tctcttgcct cagcctccca tgtagctagg actacagggc 114600ctggccaccg tgcctgagta atgtttcctt ttttttgttt gtttgtattt ttagtagaga 114660tagggttcgc cctgttgtcc aggctggtct tgaacttctg acctcacctg atctgcccac 114720cttggcatcc caaaatgttt ggattacagt tgtgatggaa caggaattaa aagaaattaa 114780agaatgtgta agcaaaaact cagttgtatg taagaaaacc caatttcccc tgaggaagag 114840aaagagctgg agtcctttaa aattaactgc ctgtttttcc ttctgtggct agtgagtctt 114900atctctccct ttccgaggca ttgtgaagac cctgtttctc tagttgtgca gctgcaaggt 114960cactagacag ataatctcaa gtggtaaaac atgttgttcc ttgaaaagta agaaataatg 115020taatgcatgt ttcaattgag taactgtatt tgtttcccac ttctgtaata tgcttcccct 115080gcacagatct ccccctgccc cacgaaatgc ttaaaagata gcttgactct ttgtttgggg 115140ctcagtcctt tggatgttaa tctgactagg tcggtgcatc taaataatta aataattcct 115200cctcaacccc tcggtctctc tgattcctta attatcctgc agcatttctg gtgacccgga 115260cagggattgg agatggcaga tttactgtct cctttgtctg tgggactaga gccccacggc 115320caggggagac ctggtatcca aggcgtgccg taggggagct tcacctggat ggagactggc 115380tctcccggca tcccagtggc ctagctggct gcacaacgga actggagacg gggctgcagc 115440atgataccag cacttcaggt accacagtaa ggagaaaggg cccaaggcag gaaagcccat 115500cccataggga tgaaggggag cttgatcacc tcccggggac cgaccactaa tccaacccag 115560agtggctggg gggcagcagg agtggcctgc caatttggat gaacctcatg tccccctaat 115620aaagtgaaag tggctcagtg gtggagaaaa tgggccaata gagtggcaag tgcagcaagg 115680aagagcttgc tggcagggtg caagagtggc ttgccagccc aactgggagt gtgtgggtgt 115740gtgtgtggac ctacccagga catgagagag gctcgtttcg tctgatgagg agtcctgggg 115800taggagtggt gtgtaggtgt gtgaatgtgg gagcctaact aggctcaccc aggacacggg 115860agaggcccgt tttgtccgat gaggagtcct ggggcagggg acatgtgtga aagtgtgtga 115920aagagacggt ctcgggagag gccaatgcgg ggagtgacgt ggggaagcac agatccctta 115980gcgtgggctg tgtgctctga ggcaagtgcg ggggaaatca gacctaggac gttgtgtaca 116040gctgatagga ccagctccat ggccacagca ggctgtgaga ggggaaggca cgttcctggc 116100taagcagcct ctgaaactcc cataatagga cccagtctag tggacccgag agtgaaagtg 116160agagtgaaag tgcgccacaa gggaggaaat gggaggaaaa gtgttgaaac caactccttt 116220ggagtgcatg atgaaaaaat ttaaaaaaag atttagaggt gattatggga tgaaactgga 116280tgctcaaaag ttaaggacat actgtgaatt agaatagccc tcttttagtg tcagatggcg 116340ggccgaaggc actacagaga aattggccat gtgttttaag gtggtgacta gggtcagagg 116400acagccaggg cattcagacc tagtctttac attgatgact aaatgaatgc agccctgcct 116460agcagtttat tgtagaatgc tcgcagctca tgccaagaga aaccagctgc tctgacggct 116520acagagttaa agggaaagtc acagaggctt gtaactccaa aagtaaaaag tgaaaagtaa 116580aagccaaaag tgaaagtaaa aatcagctgc cctggcagct atggaaacaa aagaaaagtc 116640tgaggaagga gaaaactggt tttgcaagaa ccacaggagg gaatagagac ccctcctccc 116700tacattccaa tctacccccc tttactaagg ctaactgccc ctaaggagtt aagttcaaag 116760agatacaggc tcccagtctc acagaaggag aaatcagagc tccaggaagt taaagtgaaa 116820agctagaaaa gtcaggtagg ccgtctcagg tctggccatg ccgaagttat gcttacgcct 116880cttacgagga caaaaggacc cccactagga cccagatgat gcagtctggc ttcaacacct 116940acgaaggtgc cgagaaacac ttctgcaaaa gctaaaggat agtaaaagaa aaagacaacc 117000aatataaaaa atctcagagg tgctccaggg tgcagataaa agcaccagcc agttttaaga 117060aagactttgt gaggcatttt gggtgtacac tccgtttaac cccaaggctg ctgaaaatca 117120gtgcatggtg aatacaacat ttgtaagtca gggccaagga gatattaggc ataaattgca 117180gaagttagaa gctccgtagg tgtgaatgct actcagctta ttaaagtggc taccaaggtg 117240tacattaact gagatcagga ggcaaagaag aaagctgatc ggaggcttaa gaaaggctaa 117300tttactagca gcagccctta caggaagaga agctggctct ttgcaaggag acatggacgc 117360gggtgtgaac gcagtcatgg aaaaggctag tctggacagg agtttgaaag ccggccgagg 117420ctagagagag attaatgtgc acggtgcaaa aggaaaggac actagaagga taaatgtcaa 117480aagagtaagg agaatggtca atggcctaac acccaagagc ggcgttcggt tgctagttgt 117540ggtgcttccg aggcagatcc tgatctgatc ggcttagcgg ggggccgaga atttagcaga 117600ctgagacaga ccgggctcca tccttttagg ccccggggag cctatggtct ctatggaagt 117660agggggccga tcaatggatt tttttggtcg atattggtgc tgatttctct gtggtaaccc 117720acccgattag ccccccccca caaagaactg tgctactatc gtagggggta caggggccaa 117780agaaaagaga cccttttgca aatccaggag atgtattatt aggggacaag aagtgcagca 117840tgagtttcta tatatgccaa attgtcgagt gcccttgtta ggaagagact tactccagaa 117900actgcaggca caaatttcct ttacacctaa agggaatatg acactggagt ttggaaagtc 117960taaggcaatg gtattgactc taactgtcac aaaggctgag gaatggcggc tctgtgaact 118020gtgtgccaga aggctaccgg agccagacct acacaataag tggggaatgc ttttcaagga 118080accaggtgta taggctgagg acaacccccc tggacttgct gcaaacagac ccctggtggt 118140agtagagctt aaccctcatg ctgccctggt acgagtccgt caatatccct acccagagag 118200gcaattgatg gcataacaaa acatttaaat cggctgtatg aacataggat tatagtgaaa 118260tgcaagtcct tctggaatac tcctctgctg cctgtgcgca agccaaatgg tgaatacagg 118320ccagtttaca aagtgtccag ggacaaggca aaagtctgtt ttcgggaggt tggatatcta 118380ggattcatgg tatcccaagg ccagcgcagg cttggaagtg catgcaagga ggctgtatgt 118440gcattgccca ccccagtttc aaggcagcag gtcaagaaat ttctgggtgc agtgggattc 118500tgccgaatct ggattccaaa cttctccctt acagcaaggc ccttatatga ggctaccaaa 118560ggaaaagaaa gagagcccct cgtataggaa aaggaacagc aaaaggcctt caaagatata 118620aaggaagctc tcatccaagc cctggcgcta gggttgccag atgtaaaaag cccttctttt 118680gatatgtgga tgaacggaag ggaatggcag ctggagtctt cactcagttg ttgggctctt 118740gacattggcc ggtagcatac ttatccaagc gactggactt ggtggcctta ggttggcccc 118800actgcctcag ggcgctggca gctatggcaa tccttataga agatgccaac aagctagccc 118860taggtcagaa gacaatagtc tgggtgccac acgctatagt caccttaatg gagcaaagag 118920gacatcgttg gctgtccaac tctagaatgc taaagtatca agggcttctg tgtgaaaatc 118980cccagataac actggaaact ataaatacgg cctgtggagg accctgattg gaatgatggt 119040gggttgcctc actgctggca ggaccttccc cactgttgca taaatacggt gaacgagccg 119100ggaagatctc agagatacca ccttggagag cccagatgtt gaatacttca ctgatggtag 119160cagtttcata acagatgagg tgtgatatgc agggtatgca gtagtgaccc aatactcggt 119220ggttgaggct caagccttac cttctgggac ttctgcttag aaggctgaat taatagcatt 119280aaccagagca ctgttattgg ccaaggggaa gaaagtaaac atatgtaatg attcaagata 119340tgcttttgca accctgcatg cccatggggc aatacacaaa gagagaggac tattggctac 119400tgaaggaaaa gaaataaaaa ataaagagga aattttgcaa ttattagaag ccatatgggc 119460tccagagaag gtggctgtta ttcattgcaa aggacaccaa atcgggaaga gctatgaggt 119520gcccagcaac agaaaggcag accaagaggc taggcaggca gcaatgagaa aggctttacc 119580tgaagaaaga actctagcaa tgcctctcct tatagagccc cctttattgg aggtaaccaa 119640ttactcttca agggaaaaag cttggtttgg tcaggaaaca ggaaaatata ttaaaaatgg 119700atggtggctg ttctctgacg ggaggctagc tgtcccagaa acaaaagccc caaggtttgt 119760gaagcagatc catcaaggaa cacacattgg aaggacgact agaaactttg ataggtcggc 119820atttctatgt gccacggctc tctgccatcg cccatgctgt ttttgaacaa tgtctatcct 119880gtgcccggaa taatccaaaa caaggaccta ctcgacttcc cggaattcag gaagcaggaa 119940ctgttccttg tgagaacctg cttgtagagt tcactgagtt acctctagca ggaggttact 120000ggtatatgct agtgtttgtt tacaccttct ctgggtaggc tgaggccttc cccaccagaa 120060ctgaaagggc acgagaggtg acaaaggtgc tactaaaaga catcatacca agatttgggt 120120tgcctttaac cctaggatca gacagtggtc ctgcatttgt ggcagaagta gtacaaaagc 120180tgactcaact tttaaagatc aaatggaaac tgcacacagc ctactcacca cagagttcag 120240ggaaggtgga acggatgaac cggacactca aacagctact aaaaaagttt agccaggaaa 120300ctcacttacg atgggatcag gtcttgccca tggtcctcct ccaggtcagg tgtacaccta 120360caaaacaaac tgggtactca ccctatgaaa tattgttcgg aaggccaccc ccaattatta 120420atcaaattag aggggattta aaggagttag gagagttaac ccttaggaga cagatgcagg 120480ctttaggagt gggaatgtag gaggtgcata gctgggtaag ggaaaggata cctgtaagtc 120540taacagaccc agtgcatcca cataagccag aggactctgt ctgggttaaa aggtggaatc 120600caacaacctt ggggccctta tgggatgggc cccatattgt gatcatgtct acttccactg 120660ctgttaaagt tgcaggtgtc acaccttgga ttcaccatag ccgcctgaaa ccagtggcag 120720cagtgactcc cgacgatgac cagtggatta gccaacaaga cccagattgt cccacccgaa 120780tgctcctacg gcaaaaccca accaccggta agaaggacga ctgccctgct ctgaccacac 120840cagaggctgg tcagtctaca tatggctgaa gcttgaggat cctacaagct ctgctctagt 120900cacatcctgg aagctgacta gtctatgcat ggccgaagct aagaggacca tctccggata 120960attaaatgta aatacaattt ataagcctag ttataattct gtcaatactg attgttctgt 121020tgttattact gcaaatgctg caaatgtcta tgctcagaag aaggtttgcc atgcccatgt 121080gtagtgtaaa catgtttcta ttacgtacac tgatgttgtt accatttctg cctatactaa 121140aaggggagaa atcttgagaa ggatgcccac attgtgtaca cactacctgg gtaaaaaata 121200ccatagttaa aattctactg taccatacct actataaatg tacaggaatc aagttaagaa 121260cctacacata caaccagaaa cagtctgcaa tggtttaaca caagagaggc ttagcaggac 121320cagccctaaa catctgtatg gagaaccaca aatcagatgc cctgactgta acattcagtg 121380gtctacacta acacagctct aacacttata ttcaggaagg actgctctgc taaggagtat 121440gtcaaccaaa ccaaattgta agacaaggac atgcaatcct ttaaatttta ctatcttaaa 121500gccagagcta cctttctcgt ctacaggaca gacagcacta ttacaggtaa acagacaagg 121560agcaggcctt ggagttccac tactaattgt caaaaagact agaaggactc aaatgcgtcc 121620aaccccgcaa tttcgggtcc gtaagtcatt ctataagcat tttgttcagt cagtgcctga 121680gcttccccca tcaaccaaaa acttatttgc ccaatcagct gcaaacatag ctggcagctt 121740atgaatttcc tcatgctatg tatgtggagg aaataatatg ggggaccagt ggccatggga 121800ggcaaaggaa ttaatgccac aagataactt cactttgcct aaccctgcca gtgaaccaac 121860agcctcagcc agtgtttggt tgttaaaaac ctccataatt gaaaaatact gtattgcccg 121920ttggggaaag gctttcacag agagagtagg agaaacaacc tgcctagggc aacagtatta 121980tgataagact aaaaacaaaa ctctatggag aaatgcccag aatgactcct acttaccaga 122040tccaaaccct ttctctcggt tctctactct aagccactct tggcatctct agaggctcca 122100aatgcttgga aagcaccctc tggcctatat tggatctgtg gcacataggc atattggcaa 122160ctgctggcta aatggacagg ggcgtgtgtg ttaggaacaa tcaagccatc cttctttcta 122220attcctctaa agcaaaggga actcttaggg tatccagttt atgaggaaaa taaaagaaga 122280actagaagaa gcatattcac aaaaatagac acaaatgtca aaaaggatgt agacatagga 122340gactggaagg ataatgaatg gcctcatgaa acaatcacta aatattatgg gccaactacc 122400tgggcacagg atgggtcatg gggatatcac accacaatct atatgctcaa ctgcgtcata 122460aggttgcagg cagtccttga aattataacc aatgaagcat caagggcact agatttattg 122520gcaatacaag caacacaaat gagaaatgct atacatcaaa atagattggc tttagattac 122580ctcttagcct cagaaggagg agtatgtgga aaatttaatt taaccaactg ttgcctagaa 122640atcgatgata atggctgagc tgtcatggaa atcacagcta gaatgcgcaa gttggcccat 122700gttccagttc agacttggtc cggatggtcc ttggatttgt tgtttggagg atagttctca 122760acctttggag gattcaaaac tctcattggt gggtttttgt ttaatcttgg catctgcctc 122820atcctccctt ttattttacc cctgattatt aggagtattc agtcaactat aaaggcaata 122880gtaacccgac acactacctc acagttgatg gcattaacca aacatcagct gctgccagta 122940gaagaagaag cccagctcca cgaagaggtg gcaaataatg gtgcttgcta tgaacacctt 123000tgttatgaaa agcaccaaag gggggaaatg aaacaggaat tagaagaaat taaagaatgt 123060gtaagcaaaa actcagttgt ttgtaagaaa acccaactcc ccctgaggaa gagaaagagc 123120tggagtcctt taaaattaac tgcctgtttt tccttctgtg gctagtgagc cttatctccc 123180cctttcccag gcattgtgaa gactgtttct ctagctgtgc agcagtaagg tcactagaca 123240gataatctca agtcgcaaaa catgttgttc cttgaaaagt aagaaatgat gtaatgcatg 123300ttttaattga ataactgcct ttgtttcttg cttctgtaat acgcttcccc tgcacagatc 123360tcacctgccc cacgaaatgc ttaaaaggta gcttcactct ttgtttgggg ctcagtcctt 123420tggatgtaat ccaactgggt cggtgcacct aaataattaa ataattcctc ctcaacccct 123480tggtctctct gattccttaa ttatcccgca gaagtgagac cccacgccca acgcagataa 123540ttttattgat agaattcctt tttctgatcc ggagtcaagt tcaggatcac atcttgcatg 123600tgcttttcag gtgtttttag tttcctttaa tctggaatgt ttccttaatt tgtctttgtc 123660attcatgata cagacatttt tgaagaggat agaccagttg gtttccagaa tgttctgcag 123720tttgggcttt ttcatgtctt ttttaaagac cttttttaaa ctcagcattt attgctggct 123780agtcatgcca tataacagtc taagtgctag gagtgtaagt gctgtgagag acaggatttc 123840agccttgaat catttaatat gagaaggaca atcagaggta gaataacaaa gtgcaaagga 123900ggcagcagag ttgtctgagg gcagtctctg gaaaggaaga gggtaatatt tggaacacct 123960tgttttcctg ttttctgcta acagactcct gaaataatgt tcatgggatt cttatcaaca 124020tatttattat tatactagct aaagctttta tataataaca ccgagagcat gaatattatt 124080ttcttattca tatttcatgt tttactgctt aaattgatat gtatttttta tttttaatgg 124140ccgaagcctg aagctgatag ccaggaacag gttcacccaa agactgggtg tgagtgtgga

124200gatggtcctg atggccagga gatgggcctg ccaaatccag aggaggtgaa aaggcctgaa 124260gaaggtaggg aatccattag gcatgcacat tgtagggtgt ctgtttccac agtatcgtat 124320cataattatt attacatttt tgagatggag tcttgctctg tccaccaggc tggagtgcag 124380tggtggcatc tcggctcatt ggaaattccg ccttctgggt tcaagtgatt ctcctgtatg 124440agcctctcgc ggagctgggc ttatggacat acaccaatgt gcccagctaa tttttgtatt 124500tttagtagag acagggtttc attatgttgc acaggttgtt cccgaactcc tgacctcagg 124560tgatccactt aacttaacct ttgaaattgc caggattaca tgcgagagcc accatacgcg 124620accaaggcat tatattttta ataacacagg taacaatact gcctctttag taagagagtt 124680cttatatgaa ggttatttga aacgtagttc aggccccagc acccgactga tagactgtca 124740gatagggaaa caaactgagt caaagctatg ttgaattaaa agttttgagt ataaatcctt 124800aaaccagtag ctcacaattt tcagatgctt ttgtaaaggt ctgcttttaa tcaatacata 124860acacgtttgt aacacccatc acttggtgtg aaaaatgctg aagcactcat gcgggttcta 124920ataccagctc ttacagcctt ggcgagattc tgagtgagtc ctttcccttc taaacctatc 124980tttggttctt atgaaaatag tgagtttaag tcagagattt taaaaccatt ttgcattccg 125040tttctttcat actctgatcc tgttgcatag aatgcgtggg acacagagat catctgcttc 125100gcatggtttg ttaatcacaa atcatgaaac cctggcccga gtcatctgaa aatctctgaa 125160ttgagatttc attgtcagta agacagtgag cgggccctct gcttcatcct agtttttccg 125220tgtggagagc tgaatacgta gtgtaagatc ttgtgaaatt gtgaattctc cctcttcttg 125280gtttgtttgt ttgtttggga cagagtctca gtgtgtcacc caggctggag tgcagtgatg 125340caatttcagc tcactgcaac ttctggctcc caggctaaag ccgtcctccc acctcagcct 125400cccgagtggc tggaactaca tgcacaagcc accgtgcctg actacatttt tttgttttca 125460tttttgtaga gatgaggtct cactgtgttg cccaggcagg gtttctctgg cttttaatga 125520acaattgctt cttttttttc cttttattta tttattatac tttaagtttt agggtacatg 125580tgcacgttgt gcaggttagt tacatacgta tacatgtgcc atgctggtgc gctgcaccca 125640ctatctcatc atctagcatt aggtacatct cccagtgcta tccctccccc ctccccccac 125700ccgacaacag tccccagggt gtgatattcc ccttcctctg tccatgtgat ctcattgttc 125760agttcccacc tatgagtgag aatatgcggt gtttggtttt ttgttcttgc gatagtttac 125820tgagaatgat gatttccagt ttcatccatg tccctacaaa ggacatgaac tcatcatttt 125880ttagggctgc atagtattcc atggtgtata tgtgtcacat tttcttaatc cagtctatcg 125940ttgttggaca tttgggttgg ttccaagtct ttgctatcgt gaataatgcc gcaataaaca 126000tacgtgtgca tgtgtcttta tagcagcatg atttatagtc ctttgggtat atacccagta 126060atgggatggc tgggtcaaat ggtacaattg cttcttaaat ctttccccac ggaaaccttg 126120agtgactgaa ataaatatca aatggcgaga gaccgtttag ttcctatcat ctgtggcatg 126180taggtcagtg atgctcagca tgggtgtgag taagatgcct gtgctatgca tgctccctgc 126240cccactgtca gtcttcatga gccactattt ctaataagac ggtagacaca catacgatat 126300aatcatctct aatcatatca aatgttacat gtaagtttca gctttagaga catgaattga 126360taagatttaa agttgaaaga ccatgactct agtacttcct gagtaatcaa ctgaagtatg 126420ctttacacat gtgttttcca aattgctgac tgttaattgt aagtgcttgt gacttgaaag 126480gaagcacttg atgttcaggg gggaaattcc ttttaaattc tgcaggtcta cgctcaaagt 126540ttatgcagag gttcaattgc gtgtaagaca cgggatcacc catagggttc tgtttttagt 126600ccatttaata aaacccaaac tgtagtgtgc tttgtatgcc tttagggtca tctgaataat 126660ctgttgctaa gtcatgttcc caatcgttgt gtttctgtta caggtgaaaa gcaatcacag 126720tgttaaaaga agacacgttg aaatgatgca ggctgctcct atgttggaaa tttgttcatt 126780aaaattctcc caataaagct ttacagcctt ctgcaaagaa gtcttgcgca tcttttgtga 126840agtttatttc tagctttttg atgctgtgaa atatgtatca ttctttgaaa tcgtgtattg 126900taactctctg agctggtatg tagagacatc gttctttttt ttttctttct ttctttgtcc 126960tcttttgaga cggagtcttg ctctgtcgcc caggctggag tgcagtggcg cgatctctgc 127020tcactgcaac cccgcctccc ggattcaagc aattgtctgc ctcagcctcc cgagtagctg 127080ggattatagg cacccaccag cacgcctggc taagttttgt gtttttacta gagatgggct 127140ttcgccatct tggccggggt gctcttgaac tcctgacctc gtgattcacc tgccttggcc 127200tcccaaagtg ctgggattac aggcatgagc ctccgcgccc ggtggagaca taattcttac 127260atattggttt tctatccagt ggccttgtga aatatgcttg tgaattctaa agtttacttc 127320taggtcgttt tcagtcttca atatacagaa acatatcatc ctggaataag agcagttttg 127380tttccgccat ttttttttct tttccctttt gtattttttt gtagagacgg ggttttgcca 127440tgtttcccgg gctgttgttg aacttttgag tgcaagtgat gcacccacgt catctcccac 127500agtgctggga ttactggcgt gggccaccgt ggcgggcccg tcgttgccat tgtaaagagt 127560tttatttcct tttctgattt tatggcattg cgcagaccca cccgttacaa tggtgacagt 127620ggacatcctt gtcttatccc tgatgagaaa ccgaaaaatt tcaacatttc accatcctat 127680tcactctcct ttttttgtag atggacttta tcagagtgag tcattccatt ctgttccaaa 127740tttgctgaga gtattcattt gaatatatgt tgattttcat caaacagtgc atctatttcg 127800attaccacag cgttttttcc cattcatgtg ttaatatagt gaattcgatt gataaatttg 127860tacgttttta ggttcgatta ttaaaacttg agacagcgtc tcactctgtc accgaggctg 127920gagtgcagtg gtgttatcag agctcgctgc agccttgacc tcctgggctc aggcgctcct 127980cccacctcag cctcctgagt agctgtgagt ataggtacat gccaccatgc ccagctaatt 128040tttcgatggt tttttgtttg ttttttgtag tgatgagatt ttctgatgtt gcttaggctg 128100gtctcgaagt cctgagctca ggtgatctgg ccagctcagc ctcccaaaat actaggatta 128160caggcgtgag ccttggcctg gtctggtttt tcttatatag gggtcttatc tatataaaga 128220ctaaagttaa tctgtgcctt tgtgcgggtg ggctaagagc atgatgactt ttatcattct 128280attgatttaa agaaaactat ccttgactta ccagtgtgta aggccatgaa agcataattc 128340tgttgaaagc atatattgtt aatgggtgtt gggaaccgtg cactttccgc tgctgtggga 128400gcatgtcctt ggaggtacct ttcatctgtt ttctcaactc caaacatctt aggaccatgg 128460gttgtgactg gtaggactat gtatcttgct gctttcaaga cggagtatat tttcacgtgg 128520tttcactctg gctgtcctgt ttccctaata ctgtcacttc accctctgtg attctgatgc 128580tacaaatgat agatatcgtt ttagcatttt cttacgggtc ctagcgattc tattcatttt 128640tctttcagtc tctttctctg acttgttcac aatgaacaat ttccttttgg gataggttgc 128700tatttctgtt ttcgcaggtg gtttacctgt cttcccagcc agtcacagtg gtccttgtcc 128760ccatggtggg tccggggcaa gagagggccc tgggttgggg gtggggttca gttgaagatg 128820gggtgagttt tgaggggagc actacttgag tcccagaggc ataggaaaca gcagagggag 128880gtgggattcc cttatcctca atgaggatgg gcatggaggg tttggggcgt ggcgctggga 128940acggcagccc tccccagccc acagccgcgc atgctccctg ggctcccgcc tcagtgcgca 129000tgttcactgg gcgtcttctg cccggcccct tcgcccacgt gaagaacgcc agggagctgt 129060gaggcagtgc tgtgtggttc ctgccgtccg gactcttttt cctctactga gattcatctg 129120gtaggtctgc aggccagtca tcccgggggc tgaagtgtga gtgagggtgg agagggcctt 129180gggtgggtca ggcgggtccc gcttcctggt ctgtggcctc cgagggagaa gggccacgag 129240gtcgtcctcc ttcccttcac aggctgcgag gccaccggcg gcttcgtggt cgtgaagggg 129300cctggacggg gaggaaggtg ggccgtggag gggaggcggt caggggctca ggtgaagatg 129360gggtgagtgc tgttgggggg atggaagtcc cgaggtgccg ggaacccccg acgacacagg 129420gcagattccc tgaatggggc ctccggcggg ggcgaggcgg gcggtgaaga aggggcctgg 129480cacctgggaa ggctgcggcc tggtgagcgc cccccccagc ggtgtggagt gcggagcgcc 129540tgagtgagaa gcactgcaag gtctcacctc cgccatggaa ggtccgaaaa cagtgggaag 129600gagtgggcga ggcagtgcgg tccaaccaaa cttgttgtga gggggggtga atggctctag 129660gaagtgggag tgtgcccaaa gcagcaatca cgagaattgt gattcactag ggttttcgtg 129720gggagtgcac ttgtgaaact aaacctcatc agaaatgacc tctgtctgcg gggcgcagtg 129780gcgctcgcct acgtagtccc agttactcgg gacactgagg tgggaggatc ccttgagcgg 129840gaggtcgagg ctgcagtgag ctgtgatcac gccgctgcac tccagcctga gcaacacagc 129900gagaccgcgt gtccaaaaga aatttagaaa aaaatgtcct ctgccttttg ccacacgcct 129960taagatgatt gctctgccag cctggccagc agaagtggct ttgtaggcac tcagacagcg 130020tacacacgta tgcttaactc tgggacttat cttgagagta ttttcaaaag taaaacggca 130080agtttacatt tatccatgga agtgatcgaa tatagcagcc ctgtggagcg cacgttccca 130140atcacggttg tctgttttca gtgtgaaata tgagttggcg aggaagatcg acctatcggc 130200ctagaccaag acgctacgta gagcctcctg aaatgattgg gcctatgcgg gtgagtgctt 130260aaacgttaat tcgatgtttt ctattagtag aaattaattt ttgtgatagc gttgttgcat 130320tagtgtggaa atgctgataa aggtctttcc tgctcataaa aaatgatgat ggcatctcat 130380gaaggaaaca ttgattctgg aggatttttt ttttcctctc gtgttcttca gcttttgccc 130440atgacttctt tctccggctt tgtttgttaa tgacagattg tacacatgta ttccaacaca 130500gagtacaata gcctccaaag tcctcgtgcg tcacttttct cacagtaacc tccctgtggg 130560tggagtaacc ttattgggca tagagcatag agttggagaa atgtctttag gcttagttat 130620gaccagaaat agctatgtat tctgtgtata tatgtaaaat tttgtatcaa taacgaaact 130680tattttctat ttgcacaccc acacgtattc cccagcccga gcagttcagt gatgaagtgg 130740aaccagcaac acctgaagaa ggggaaccag caactcaacg tcaggatc 130788

Claims

1. A method for identifying a compound that modulates the level of expression of scurfin comprising the steps of:(a) providing a composition comprising a reporter gene ligated to a scurfin promoter;(b) contacting the composition with a test compound;(c) determining the level of reporter gene expression; and(d) comparing the level of reporter gene expression in (c) with the predetermined level of expression and thereby determining if the test compound modulates the expression of scurfin.

2. The method of claim 1, wherein the level of scurfin expression is decreased.

3. The method of claim 1, wherein the level of scurfin expression is increased.

4. The method of claim 1, wherein the test compound is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a peptide, a small molecule, an organic molecule, a natural product, a peptide, an oliciosaccharide, a nucleic acid, a lipid, and an antibody or binding fragment thereof.

5. (canceled)

6. The method of claim 1, wherein the test compound is from a library of compounds.

7. The method of claim 6, wherein the library is selected from the group consisting of a random peptide library, a combinatorial library, an oligosaccharide library and a phage display library.

8. A compound identified according to the method of claim 1.

9. A method for suppressing an immune response in a mammal comprising contacting T cells of the mammal with a compound that increases scurfin expression in the T cell, wherein an immune response is suppressed.

10. A method for enhancing an immune response in a mammal comprising contacting T cells of the mammal with a compound that decreases scurfin expression in the T cell, wherein an immune response is enhanced.

11. A method of claim 9 wherein said immune response is an autoimmune response, and wherein said contacting results from administering said compound to said mammal which increases scurfin expression in said T cells, thereby inhibiting an autoimmune response by the subject and wherein the autoimmune response is selected from the group consisting of Inflammatory Bowel Disease, Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis, Diabetes, and Asthma.

12. (canceled)

13. A method of claim 10 wherein said contacting results from administering to said mammal said compound which decreases scurfin expression, thereby enhancing an immune response to a disease in the subject.

14. The method of claim 13, wherein the disease is selected from the group consisting of AIDS and cancer.

15. A method of claim 9 wherein said immune response contributes to graft versus host disease wherein said contacting comprises administering to the mammal said compound that increases scurfin expression in said T cells, thereby inhibiting graft versus host disease in the subject.

16. A method of claim 9 wherein said method comprises:(a) isolating T cells from said mammal;(b) transducing the T cells with the scurfin gene;(c) expanding the transduced T cells; and(d) reintroducing the transduced T cells into said mammal,wherein an autoimmune response in the patient is inhibited.

17. The method of claim 16 wherein the T cells are CD4+CD25+ regulatory T cells.

18. The method of claim 16, wherein the autoimmune disease is selected from the group consisting of Inflammatory Bowel Disease, Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis, Diabetes, and Asthma.

19. The method of claim 16, wherein the transduction vector is a retroviral vector.

20. A method of claim 10 wherein said method comprises:(a) isolating T cells from said mammal;(b) transfecting the T cells with a compound that inhibits scurfin expression;(c) expanding the transfected T cells; and(d) reintroducing the transfected T cells into said mammal, wherein an immune response to a disease in the patient is enhanced.

21. The method of claim 20 wherein the compound is an anti-sense molecule.

22. The method of claim 20, wherein the disease is selected from the group consisting of AIDS and cancer.

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