Compositions and Methods for Induced Brown Fat Differentiation

Abstract

The invention provides methods and compositions for inducing brown fat cell differentiation through modulation of both Prdm1β and C/EBPβ activity and/or expression. Also provided are methods for preventing or treating obesity or an obesity related disorder in a subject through stimulation of both Prdm1β and C/EBPβ expression and/or activity. Further provided are methods for identifying compounds that are capable of modulating both Prdm1β and C/EBPβ expression and/or activity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional Application of U.S. patent application Ser. No. 13/143,645, filed on Jul. 7, 2011, which is the U.S. National Stage Application of International Application No. PCT/U.S. Pat. No. 10/20480, filed on Jan. 8, 2010, and which claims the benefit of priority to U.S. Provisional Application No. 61/204,607, filed on Jan. 8, 2009, the entire contents of each of which application are expressly incorporated in their entirety.

BACKGROUND OF THE INVENTION

[0003] Obesity represents the most prevalent of body weight disorders, affecting an estimated 30 to 50% of the middle-aged population in the Western world. Obesity, defined as a body mass index (BMI) of 30 kg/²m or more, contributes to diseases such as coronary artery disease, hypertension, stroke, diabetes, hyperlipidemia and some cancers. (See, e.g., Nishina, P. M. et al. (1994), Metab. 43:554-558; Grundy, S. M. & Barnett, J. P. (1990), Dis. Mon. 36:641-731). Obesity is a complex multifactorial chronic disease that develops from an interaction of genotype and the environment and involves social, behavioral, cultural, physiological, metabolic and genetic factors.

[0004] Generally, obesity results when energy intake exceeds energy expenditure, resulting in the growth and/or formation of adipose tissue via hypertrophic and hyperplastic growth. Hypertrophic growth is an increase in size of adipocytes stimulated by lipid accumulation. Hyperplastic growth is defined as an increase in the number of adipocytes in adipose tissue. It is thought to occur primarily by mitosis of pre-existing adipocytes caused when adipocytes fill with lipid and reach a critical size. An increase in the number of adipocytes has far-reaching consequences for the treatment and prevention of obesity.

[0005] Adipose tissue consists primarily of adipocytes. Vertebrates possess two distinct types of adipose tissue: white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores and releases fat according to the nutritional needs of the animal. This stored fat is used by the body for (1) heat insulation (e.g., subcutaneous fat), (2) mechanical cushion (e.g., surrounding internal organs), and (3) as a source of energy. BAT burns fat, releasing the energy as heat through thermogenesis. BAT thermogenesis is used both (1) to maintain homeothermy by increasing thermogenesis in response to lower temperatures and (2) to maintain energy balance by increasing energy expenditure in response to increases in caloric intake (Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419). BAT is also the major site of thermogenesis in rodents and plays an important role in thermogenesis in human infants. In humans, and to a lesser extent rodents, brown fat diminishes with age, but can be re-activated under certain conditions, such as prolonged exposure to cold, maintenance on a high fat diet and in the presence of noradrenaline producing tumors.

[0006] Fat metabolism is regulated by two pathways, lipogenesis and lipolysis. Lipogenesis is the deposition of fat which occurs in the liver and in adipose tissue at cytoplasmic and mitochondrial sites. This process allows the storage of energy that is ingested which is not needed for current energy demands. Lipolysis is the chemical decomposition and release of fat from adipose and/or other tissues. This process predominates over lipogenesis when additional energy is required by the body.

[0007] Any treatment for obesity has to reduce energy intake, increase energy expenditure or combine both effects. Current therapies for obesity predominantly lead to decreased energy intake by acting at satiety centers in the brain or by reducing the efficiency of intestinal absorption. To date, no safe and reliable molecular mechanism for treating and/or preventing obesity by increasing energy expenditure or metabolic activity has been identified. Given the severity and prevalence of obesity related disorders, there exists a great need for the identification of an anti-obesity therapeutic.

SUMMARY OF THE INVENTION

[0008] The present invention is based in part on the discovery that Prdm16 and CCAAT/enhancer binding protein beta (C/EBPβ) can cooperatively induce brown fat differentiation in non-adipocyte mammalian cells (e.g., myoblasts and fibroblasts) including activating a distinct set of target genes (including, for example, cidea, adiponectin, type II deiodinase, cig30, pgc1α, elov3, and ucp1) characteristic of brown fat cells. In particular, functional brown fat cells can be differentiated from fibroblastic cells (e.g., embryonic and skin fibroblasts) upon expression and/or activity of both Prdm16 and C/EBPβ. Increased brown fat differentiation in mammals induces the expression of mitochondrial genes (including, for example, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5) and cellular respiration (i.e., total and uncoupled respiration). Densely packed mitochondria are a characteristic of brown fat cells. Increased respiration (both total and uncoupled respiration) results in increased heat dissipation and increased energy expenditure by the mammal. The increases in heat dissipation and increased energy expenditure stimulate the metabolic rate of the mammal. Through the stimulation of the metabolic rate, expression and/or activity of both Prdm16 and C/EBPβ may be used to treat and/or prevent obesity or an obesity related disorder. Moreover, the induced brown fat cells of the present invention exhibit heightened expression of biochemical characteristics, metabolic rate, and thermogenesis relative to authentic brown fat cells. In addition, the novel compositions and methods of the present invention can be used to differentiate cells (e.g., autologous cells) that can be easily obtained from subjects (e.g., skin fibroblasts) into functional brown fat cells for use in transplantation. Such methods allow for "custom made" brown fat cells from any individual for use in autologous transplantation.

[0009] Accordingly, the present invention provides methods for preventing or treating obesity or an obesity-related disorder in a subject comprising inducing both Prdm16 and C/EBPβ expression and/or activity sufficient to activate brown fat cell differentiation, wherein the differentiated brown fat cells increase energy expenditure to thereby prevent or treat obesity or an obesity-related disorder in the subject. In one embodiment, the obesity-related disorder is selected from the group consisting of: insulin resistance, type II diabetes, hypertension, hyperuricemia, fatty liver, non-alcoholic fatty liver disease, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia. In another embodiment, the subject is a human.

[0010] In another aspect, methods are provided for increasing energy expenditure in a mammal comprising inducing both Prdm16 and C/EBPβ expression and/or activity sufficient to activate brown fat cell differentiation in the mammal, wherein the differentiated brown fat cells promote energy expenditure thereby increasing energy expenditure in the mammal. In one embodiment, the mammal is a human.

[0011] In still another aspect, methods are provided for treating obesity or an obesity-related disorder in a subject comprising administering to the subject an agent that induces expression and/or activity of both Prdm16 and C/EBPβ, wherein Prdm16 and C/EBPβ expression and/or activity increases respiration and energy expenditure to thereby treat obesity or an obesity-related disorder in the subject. In one embodiment, respiration is total respiration and/or uncoupled respiration. In another embodiment, the obesity-related disorder is selected from the group consisting of: insulin resistance, type II diabetes, hypertension, hyperuricemia, fatty liver, non-alcoholic fatty liver disease, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia. In still another embodiment, the subject is a human.

[0012] In yet another aspect, methods are provided for inducing brown fat cell differentiation in a mammal comprising expressing both Prdm16 and C/EBPβ in cells; delivering the cells expressing both Prdm16 and C/EBPβ into the mammal; and monitoring the differentiation of brown fat cells in the mammal. In one embodiment, the cells are selected from the group consisting of fibroblasts and myoblasts (e.g., skin fibroblasts, dermal fibroblasts, primary embryonic fibroblasts, immortalized embryonic fibroblasts, and human foreskin fibroblasts). In another embodiment, the cells are autologous, allogeneic, syngeneic, xenogeneic, or HLA compatible with the mammal. In still another embodiment, the cells are delivered by a subcutaneous injection or an intravenous injection. In yet another embodiment, the differentiation of brown fat cells is monitored by measuring the expression of a marker selected from the group consisting of: cidea, adiponectin, adipsin, otopetrin, type II deiodinase, cig30, ppar gamma 2, pgc1α, ucp1, elovl3, cAMP, Prdm16, cytochrome C, cox4i1, coxIII, cox5b, cox7a1, cox8b, glut4, atpase b2, cox II, atp5o, ndufb5, Table 1, Table 2, and Table 3. In another embodiment, the differentiation of brown fat cells is monitored by measuring the degree of cellular locularity. In still another embodiment, the differentiation of brown fat cells is monitored by measuring respiration within the cell expressing Prdm16 and C/EBPβ (e.g., total respiration or uncoupled respiration, as measured by oxygen consumption or positron emission tomography, for example). In another embodiment, the mammal is a human.

[0013] In another aspect, methods are provided for inducing brown fat cell differentiation in a mammal comprising: obtaining cells from the mammal; expressing both Prdm16 and C/EBPβ in the cells; delivering the cells expressing both Prdm16 and C/EBPβ into the mammal; and monitoring the differentiation of brown fat cells in the mammal. In still another embodiment, the cells are delivered by a subcutaneous injection or an intravenous injection. In another embodiment, the differentiation of brown fat cells is monitored by measuring the expression of a marker selected from the group consisting of: cidea, adiponectin, adipsin, otopetrin, type II deiodinase, cig30, ppar gamma 2, pgc1α, ucp1, elovl3, cAMP, Prdm16, cytochrome C, cox4i1, coxIII, cox5b, cox7a1, cox8b, glut4, atpase b2, cox II, atp5o, ndufb5, Table 1, Table 2, and Table 3. In still another embodiment, the differentiation of brown fat cells is monitored by measuring the degree of cellular locularity. In yet another embodiment, the differentiation of brown fat cells is monitored by measuring respiration within the cell expressing Prdm16 and C/EBPβ (e.g., total respiration or uncoupled respiration, as measured by oxygen consumption or positron emission tomography, for example). In another embodiment, the cells are selected from the group consisting of fibroblasts and myoblasts (e.g., skin fibroblasts, dermal fibroblasts, primary embryonic fibroblasts, immortalized embryonic fibroblasts, and human foreskin fibroblasts).

[0014] In still another aspect, a non-human transgenic animal model of PRDM16 and C/EBPβ expression in cells of the animal model is provided comprising PRDM16 operably linked to a promoter specifically expressed in the cells and C/EBPβ operably linked to a promoter specifically expressed in the cells. In one embodiment, the cells are selected from the group consisting of fibroblasts, myoblasts, preadipocytes, white adipose tissue cells, epithelial, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B lymphocytes, T lymphocytes, macrophages, monocytes, mononuclear cells, cardiac muscle cells, and skeletal muscle cells. In another embodiment, the non-human transgenic animal is a mouse.

[0015] In yet another aspect, the present invention provides for an isolated complex comprising (a) a PRDM16 polypeptide and a C/EBPβ polypeptide; (b) a PRDM16 polypeptide and a fragment of a C/EBPβ polypeptide; (c) a fragment of a PRDM16 polypeptide and a C/EBPβ polypeptide; (d) a fragment of an PRDM16 polypeptide and a fragment of a C/EBPβ polypeptide; or (e) a PRDM16 polypeptide and a polypeptide from Table 1 or Table 3. In one embodiment, the PRDM16 polypeptide comprises an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6. In another embodiment, the C/EBPβ polypeptide fragment comprises an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO: 8 or 10. In still another embodiment, the complex is at least about 75% pure by weight as compared to the weight of the total protein in the sample. In yet another embodiment, at least one polypeptide or fragment is a fusion protein or labeled. In another embodiment, the complex is generated within a host cell. In still another embodiment, the PRDM16 polypeptide or polypeptide fragment and said C/EBPβ polypeptide or polypeptide fragment are covalently linked.

[0016] It will be appreciated that specific sequence identifiers (SEQ ID NOs) have been referenced throughout the specification for purposes of illustration and should therefore not be construed to be limiting. Any marker of the invention, including, but not limited to, the markers described in the specification and markers presented in Tables 1, 2, and 3, or fragments thereof, can be used in the embodiments of the invention.

[0017] In another aspect, a host cell or composition comprising a recombinant amino acid encoding at least one of the polypeptides or polypeptide fragments of the complex is provided.

[0018] In still another aspect, an isolated antibody that has a higher binding affinity for the complex than for the uncomplexed PRDM16 or C/EBPβ polypeptides or polypeptide fragments is provided. In one embodiment, the isolated antibody has the ability to stabilize the complex.

[0019] In yet another aspect, methods are provided for identifying a compound that modulates a PRDM16/C/EBPβ complex comprising: contacting a complex comprising (a) a PRDM16 polypeptide and a C/EBPβ polypeptide; (b) a PRDM16 polypeptide and a fragment of a C/EBPβ polypeptide; (c) a fragment of a PRDM16 polypeptide and a C/EBPβ polypeptide; or (d) a fragment of an PRDM16 polypeptide and a fragment of a C/EBPβ polypeptide, with a test compound; and assaying the amount or activity of the complex, wherein a change in the amount or activity of the complex in the presence of the test compound as compared to the amount or activity of the complex in the absence of the test compound is indicative of a compound that modulates an PRDM16-C/EBPβ complex.

[0020] Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF FIGURES

[0021] FIG. 1 includes 6 panels, identified as panels A, B, C, D, E, and F, which show that C/EBPβ is a critical binding partner in the PRDM16 transcriptional complex. Panel A shows the strategy for identifying key PRDM16 binding partners. Panel B shows the results of C2C12 myoblasts expressing indicated viral vectors stained with Oil Red O 6 days after inducing adipocyte differentiation. Panel C shows PRDM16 transcriptional complexes immunopurified from brown fat cells expressing full-length or deletion mutants of PRDM16. Panel D shows gene expression of known or predicted transcription factors identified in the PRDM16 complex in BAT and WAT. n=6. Panel E shows endogenous C/EBPβ was detected in the PRDM16 complex by Western blotting. Input is shown to the left. IB, immunoblot; IP, immunoprecipitate. Panel F shows transcriptional activity of the Pgc1a promoter in response to PRDM16 and/or C/EBP-β. n=3; all error bars are s.e.m.; *P<0.05, **P<0.01.

[0022] FIG. 2 includes 7 panels, identified as panels A, B, C, D, E, F, and G, which show that C/EBPβ is required for initiation of the myoblast to brown fat conversion by PRDM16. Panel A shows Western blot analysis for C/EBPβ and PRDM16 in C2C12 myoblasts expressing scr, shβ-1 or shβ-2, with PRDM16 or vector. Panel B shows Pparg2 gene expression. n=3. Panel C shows cells stained with Oil Red O 6 days after inducing adipocyte differentiation. Panel D shows BAT-selective gene expression. n=4. Panel E shows microarray analysis of undifferentiated C2C12 myoblasts expressing scr or shβ-1 with PRDM16 or vector. n=3. Panel F shows haematoxylin and eosin (H&E) staining of BAT from wild-type (WT) and C/EBPβ knockout (KO) mice, as well as immunohistochemistry to detect UCP1 expression. Scale bars, 20 μm. Panel G shows mRNA expression of BAT and skeletal-muscle-selective genes in BAT from E17.5 embryos. n=5-8; all error bars are s.e.m.; *P<0.05, **P<0.01.

[0023] FIG. 3 includes 6 panels, identified as panels A, B, C, D, E, and F, which show that PRDM16 and C/EBPβ can reconstitute the brown fat gene program in fibroblasts. Panel A shows Pparg2 expression in undifferentiated MEFs expressing indicated viral vectors. n=3. Panel B shows immortalized MEFs or skin fibroblasts expressing indicated viral vectors stained with Oil Red O 6-8 days after inducing adipocyte differentiation. Panel C shows BAT-selective gene expression. Panel D shows thermogenic gene expression. The cells were treated with cAMP for 4 h. n=4; ctrl, control. Panel E shows BAT-selective gene expression in primary skin fibroblasts expressing vector or PRDM16 and C/EBP-β. n=3. Adipsin is also known as Cfd. Panel F shows total and uncoupled cellular respiration in differentiated brown fat cells and the MEFs expressing vector or PRDM16 and C/EBP-β. The cells were treated with dibutyryl-cAMP for 12 h. n=3; all error bars are s.e.m.; *P<0.05, **P<0.01.

[0024] FIG. 4 includes 5 panels, identified as panels A, B, C, D, and E, which show generation of functional brown adipose tissue in vivo by expression of PRDM16 and C/EBP-β. Panel A shows fat pads from transplanted MEFs expressing indicated viral vectors were stained by H&E. Scale bar, 500 μm. Panel B shows high magnification images of H&E staining in the transplants expressing PRDM16 and C/EBP-β and endogenous BAT. Arrowheads show multilocular fat cells. Scale bar, 50 μm. Panel C shows immunohistochemistry to detect UCP1 expression in the transplant (left, anti-UCP1; middle, negative control) and BAT (right). Scale bar, 50 μm. Panel D shows PET/computed tomography image of mice with engineered BAT (eBAT) and engineered WAT (eWAT). Panel E shows computed tomography (CT) image (left) and PET image (right) of mouse skin with the eBAT and eWAT.

[0025] FIG. 5 includes 2 panels, identified as panels A and B, which show that myoblasts express low levels of PPAR-γ. mRNA levels of two PPAR-γ isoform Ppar-γ1 (Panel A) and PPAR-γ isoform Ppar-γ2 (Panel B) were measured in white pre-adipocyte cell lines (3T3-F442A and 3T3-L1), immortalized brown preadipocytes, primary myoblasts and C2C12 myoblast cell line. n=3-4. Data are presented as mean and s.e.m.

[0026] FIG. 6 includes 2 panels, identified as panels A and B, which show that the ZF-1 region of PRDM16 is required for the induction of a brown fat gene program from C2C12 myoblasts. Panel A shows wild type (WT), PR-domain deletion mutant (ΔPR), ZF-1 deletion mutant (ΔZF-1) of PRDM16 were expressed in C2C12 myoblasts at similar levels. PRDM16 (upper panel) and β-actin (lower panel) were detected by Western blotting. Panel B shows that these cells were differentiated and mRNA expression of adipocyte markers (Adiponectin and Adipsin) and BAT-selective genes (Ucp1, Cox7a1, Cidea and Otopetrin) were analyzed by real-time PCR. n=4. Data are mean and s.e.m. *P<0.05, **P<0.01.

[0027] FIG. 7 shows that C/EBP-β protein is enriched in BAT. C/EBP-β protein was detected in the WAT and BAT from mice at ambient temperature, and BAT from mice kept at 4° C. for 5 hours by Western blotting. RNA polymerase II (Pol II) protein is shown as a loading control (lower panel).

[0028] FIG. 8 shows that C/EBP-β is abundantly expressed in myoblasts. mRNA levels of three members of the C/EBP family (C/EBP-α, C/EBP-β, and C/EBP-δ) were measured in white pre-adipocyte lines (3T3-F442A and 3T3-L1), immortalized brown pre-adipocyte, primary myoblasts and C2C12 myoblast cell line. n=3-4. Data are presented as mean and s.e.m.

[0029] FIG. 9 includes 2 panels, identified as panels A and B, which show that PRDM16 does not interact with LIP. Flag-tagged full length PRDM16 was transiently expressed with several deletion mutants of C/EBPβ in HEK293 cells. Panel A shows that PRDM16 was immunoprecipitated using flag M2 agarose, separated by SDS-PAGE, and C/EBPβ was detected by Western blotting. The inputs are also shown. Panel B shows schematic illustrations of C/EBPβ deletion mutants. The far left box represents the activation domain of C/EBP-β. R1 and R2 domains represent repression domain-1 and -2, respectively.

[0030] FIG. 10 includes 3 panels, identified as panels A, B, and C, which show that PRDM16 physically interacts with three members of the C/EBP family. Panel A shows that Flag-tagged full length PRDM16 was transiently expressed with C/EBPβ in HEK293 cells. PRDM16 or C/EBPβ was immunoprecipitated using flag M2 agarose or C/EBPβ antibody, respectively. The immunoprecipitants were separated by SDS-PAGE, and C/EBPβ or PRDM16 was detected by Western blotting. The inputs are also shown. Panel B and Panel C show that Flag-tagged PRDM16 was transiently expressed with C/EBP-α (Panel B) or C/EBP-δ (Panel C) in HEK293 cells. Interaction of PRDM16 with C/EBP-α or C/EBP-δ was analyzed as described in Example 1.

[0031] FIG. 11 shows that PRDM16 directly interacts with C/EBPβ through the two zinc finger domains. Full length C/EBPβ (top), C/EBP-δ (middle) or CtBP1 (bottom) were ³⁵S-labeled by in vitro translation and incubated with various GST-fusion fragments of PRDM16. GST-beads were washed, separated by 4-20% gradient SDS-PAGE, and analyzed by autoradiography.

[0032] FIG. 12 includes 3 panels, identified as panels A, B, and C, which show that expression of a dominant-negative form of C/EBPβ (LIP) blunts the PRDM16-induced brown fat gene program. Panel A shows that GFP or a dominant-negative form of C/EBP-β (LIP) was co-expressed with PRDM16 or vector control in C2C12 myoblasts. Protein expressions of PRDM16 (top) and LIP (bottom) were detected by Western blotting. Panel B shows that these cells were stained with Oil-Red-O 6 days after inducing adipocyte differentiation. Panel C shows that mRNA expression of BAT-selective genes were analyzed by real-time PCR. n=3. Data are mean and s.e.m. *P<0.05, ** P<0.01.

[0033] FIG. 13 shows validation of microarray analysis by real-time PCR. Undifferentiated C2C12 myoblasts were transduced with a scrambled control shRNA (scr) or shRNA targeting C/EBPβ (sh-CEBP-β) together with PRDM16 or vector control. Genes identified by the microarray analysis were individually measured by real-time PCR. n=3. Data are mean and s.e.m.

[0034] FIG. 14 shows a schematic model of the myoblast-brown fat conversion through PRDM16 and C/EBP-β. PRDM16 acts in myf5-positive myoblastic precursors, at least in part, by coactivation of C/EBPβ to induce the expression of PPAR-γ and PGC-1α. PRDM16 coactivates PPAR-γ and PGC-1α by direct binding, which drives a complete brown fat differentiation program.

[0035] FIG. 15 includes 2 panels, identified as panels A, and B, which show that PRDM16 and C/EBPβ induces brown fat gene program in human skin fibroblasts through PRDM16 and C/EBP-β. Panel A shows that primary skin fibroblasts from human newborn foreskin were transduced with vector control, PRDM16, C/EBP-β, or combination of PRDM16 and C/EBP-β. These cells were differentiated using a standard adipocyte differentiation protocol, and stained with Oil-Red-O. Panel B shows that mRNA expression of BAT-enriched genes (Elovl3, Cidea, and Cyt-c) and thermogenic genes (Ucp1 and Pgc-1α) were analyzed by realtime PCR. The cells were treated with or without cAMP (forskolin, 10 μM) for 4 hours prior to RNA isolation. n=3. Data are mean and s.e.m. *P<0.05. **P<0.01.

[0036] FIG. 16 includes 2 panels, identified as panels A and B, which show induction of brown fat gene program by PRDM16 and C/EBP-β in MEFs used for transplantation. Panel A shows that immortalized MEFs were transduced with combination of PRDM16 and C/EBPβ or PPAR-γ alone. These cells were implanted subcutaneously into 7-9 week-old male nude mice (n=6). After 4-6 weeks, the fat pads with similar sizes were carefully dissected. Panel B shows that mRNA expression of BAT-selective genes (Ucp1, Pgc-1α, Cox7a1, Cox8b, Cidea, Elovl3, and Glut4) and general adipose marker genes (Adiponectin and Adipsin) were analyzed by real-time PCR. n=6. Data are mean and s.e.m. **P<0.01.

BRIEF DESCRIPTION OF TABLES

[0037] Table 1 shows proteins identified in the LC-MS/MS analyses described herein.

[0038] Table 2 shows the primer sequences used in the RT-PCR analyses described herein.

[0039] Table 3 shows nucleic acid and amino acid sequences referenced herein.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention is based in part on the discovery that Prdm16 and CCAAT/enhancer binding protein beta (C/EBPβ) can form an isolated complex and cooperatively induce brown fat differentiation in non-adipocyte mammalian cells (e.g., myoblasts and fibroblasts). The compositions of the present invention are capable of activating a distinct set of target genes (including, for example, cidea, adiponectin, type II deiodinase, cig30, pgc1α, elov3, and ucp1) characteristic of brown fat cells. For example, functional brown fat cells can be differentiated from fibroblastic cells (e.g., embryonic and skin fibroblasts) upon expression and/or activity of both Prdm16 and C/EBPβ. Increased brown fat differentiation in mammals induces the expression of mitochondrial genes (including, for example, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5) and cellular respiration (i.e., total and uncoupled respiration).

[0041] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0042] The term "amino acid" is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. The names of the natural amino acids are abbreviated herein in accordance with the recommendations of IUPAC-IUB.

[0043] The term "antisense" nucleic acid refers to oligonucleotides which specifically hybridize (e.g., bind) under cellular conditions with a gene sequence, such as at the cellular mRNA and/or genomic DNA level, so as to inhibit expression of that gene, e.g., by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.

[0044] The term "binding" or "interacting" refers to an association, which may be a stable association, between two molecules, e.g., between a polypeptide of the invention and a binding partner, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions. Exemplary interactions include protein-protein, protein-nucleic acid, protein-small molecule, and small molecule-nucleic acid interactions.

[0045] The term "biological sample" when used in reference to a diagnostic assay is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[0046] The term "complex" refers to an association between at least two moieties (e.g. chemical or biochemical) that have an affinity for one another. "Protein complex" or "polypeptide complex" refers to a complex comprising at least one polypeptide. In one embodiment, a complex comprises PRDM16 and C/EBPβ, PRDM16 and C/EBPδ, or PRDM16 and CtBP1. In another embodiment, a complex comprises a fragment of PRDM16 and C/EBPβ, a fragment of PRDM16 and C/EBPδ, or a fragment of PRDM16 and CtBP1. In an exemplary embodiment, a complex comprises a fragment of PRDM16 having amino acid residues from about 224-454 and/or a fragment of PRDM16 having amino acid residues from about 881-1038 and C/EBPβ. In another exemplary embodiment, a complex comprises a fragment of PRDM16 having amino acid residues from about 224-454 and/or a fragment of PRDM16 having amino acid residues from about 881-1038 and C/EBPδ. In yet another exemplary embodiment, a complex comprises a fragment of PRDM16 having amino acid residues from about 680-880 and CtBP1. These embodiments may encompass other molecules (e.g., polypeptides) that can bind to the complex, such as an antibody.

[0047] The term "isolated polypeptide" refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found within nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.

[0048] The terms "label" or "labeled" refer to incorporation or attachment, optionally covalently or non-covalently, of a detectable marker into a molecule, such as a polypeptide. Various methods of labeling polypeptides are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes, fluorescent labels, heavy atoms, enzymatic labels or reporter genes, chemiluminescent groups, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). Examples and use of such labels are described in more detail below. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0049] The term "PRDM16 binding domain" or "PRDM16-BD" refers to a region on a PRDM16 polypeptide that is capable of interacting with a C/EBPβ polypeptide, or fragment thereof. In an exemplary embodiment, the term PRDM16-BD refers to a region comprising amino acids 224-454 and/or 881-1038.

[0050] The term "PRDM16-C/EBPβ complex polypeptide" refers to a polypeptide that may be found in a complex comprising PRDM16 and C/EBPβ. In one embodiment, the term PRDM16-C/EBPβ complex polypeptide includes PRDM16 polypeptides, and fragments thereof, and/or C/EBPβ polypeptides, and fragments thereof, as described further herein. In another embodiment, the term PRDM16-C/EBPβ complex polypeptide may encompass other polypeptides that can bind to a PRDM16-C/EBPβ complex, such as, for example, an antibody.

[0051] The terms "metabolic disorder" and "obesity related disorders" are used interchangeably herein and include a disorder, disease or condition which is caused or characterized by an abnormal metabolism (i.e., the chemical changes in living cells by which energy is provided for vital processes and activities) in a subject. Metabolic disorders include diseases, disorders, or conditions associated with aberrant thermogenesis or aberrant adipose cell (e.g., brown or white adipose cell) content or function. Metabolic disorders can be characterized by a misregulation (e.g., downregulation or upregulation) of PGC-1 activity. Metabolic disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, or migration, cellular regulation of homeostasis, inter- or intra-cellular communication; tissue function, such as liver function, muscle function, or adipocyte function; systemic responses in an organism, such as hormonal responses (e.g., insulin response). Examples of metabolic disorders include obesity, insulin resistance, type II diabetes, hypertension, hyperuricemia, fatty liver, non-alcoholic fatty liver disease, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia.

[0052] As used herein, "obesity" refers to a body mass index (BMI) of 30 kg/²m or more (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)). However, the present invention is also intended to include a disease, disorder, or condition that is characterized by a body mass index (BMI) of 25 kg/²m or more, 26 kg/²m or more, 27 kg/²m or more, 28 kg/²m or more, 29 kg/²m or more, 29.5 kg/²m or more, or 29.9 kg/²m or more, all of which are typically referred to as overweight (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)). The obesity described herein may be due to any cause, whether genetic or environmental. Examples of disorders that may result in obesity or be the cause of obesity include overeating and bulimia, polycystic ovarian disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's syndrome, Type II diabetics, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g., children with acute lymphoblastic leukemia.

[0053] "Treatment" refers to reducing the BMI of the mammal to less than about 25.9, and maintaining that weight for a period of time, e.g., for at least about 6 months. The treatment suitably results in an increase in metabolic activity.

[0054] "Prevention" refers to preventing obesity or an obesity related disorder from occurring if the treatment is administered prior to the onset of the obese condition. Moreover, if treatment is commenced in subjects already suffering from or having symptoms of obesity or an obesity related disorder, such treatment is expected to prevent, or to prevent the progression of obesity or the obesity related disorder, and the medical sequel of obesity, such as, e.g., arteriosclerosis, Type II diabetes, polycystic ovarian disease, cardiovascular diseases, osteoarthritis, dermatological disorders, hypertension, insulin resistance, hypercholesterolemia, hypertriglyceridemia, and cholelithiasis.

[0055] As used herein, a "graft" refers to a cell, tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect. A graft may further comprise a scaffold. The tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: "autograft", "autologous transplant", "autologous implant" and "autologous graft". A graft comprising cells from a genetically different individual of the same species is referred to herein by the following interchangeable terms: "allograft", "allogeneic transplant", "allogeneic implant" and "allogeneic graft". A graft from an individual to his identical twin is referred to herein as an "isograft", a "syngeneic transplant", a "syngeneic implant" or a "syngeneic graft". A "xenograft", "xenogeneic transplant" or "xenogeneic implant" refers to a graft from one individual to another of a different species.

[0056] As used herein, the term "prdm16" refers to a PR-domain containing protein-16 and is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof. Two spliced variants encoding distinct isoforms of Prdm16 have been previously described (Nishikata, et al. (2003) Blood 102(9):3323-32; Mochizuki, N. et al., (2000) Blood 96(9):3209-14). The nucleic acid sequence of human Prdm16 corresponding to spliced transcript variant 1 (Genbank Accession number NM_022114) is provided herein as SEQ ID NOs: 1 (see Table 3). The nucleic acid sequence of human Prdm16 corresponding to spliced transcript variant 2 (Genbank Accession number NM_199454) is provided herein as SEQ ID NOs: 3 (see Table 3). Variant 1 encodes a longer isoform of Prdm16. Variant 2 uses an alternative splice site for the 3' exon compared to variant 1, and thus, lacks an internal region compared to variant 1. The amino acid sequences of human Prdm16 corresponding to spliced transcript variants 1 and 2 are provided herein as SEQ ID NOs: 2 and 4, respectively (Genbank Accession numbers NP_071397 and NP_955533, respectively).

[0057] The nucleotide and amino acid sequences of mouse Prdm16, which correspond to Genbank Accession number NM_027504 and NP_081780 respectively, are set forth in SEQ ID NO: 5 and 6 (see Table 3).

[0058] The nucleotide and amino acid sequences of human C/EBPβ, which correspond to Genbank Accession number NM_005194 and NP_005185 respectively, are set forth in SEQ ID NO: 7 and 8 (see Table 3). In addition, the nucleotide and amino acid sequences of mouse C/EBPβ, which correspond to Genbank Accession number NM_009883 and NP_034013 respectively, are set forth in SEQ ID NO: 9 and 10 (see Table 3). LAP is full-length C/EBPβ.

[0059] As used herein, the term "PGC-1" refers to a PPARγ Coactivator 1 protein and is intended to include any of its' derivatives, including PGC-1α and PGC-1β. PGC-1 has been described previously (Puigserver, P. et al. (1998) Cell 92(6):829-39; U.S. Pat. No. 6,166,192; and PCT International Publication Nos. WO 98/54220; the contents of all of which are incorporated herein by reference). The term PPARγ1 is well known in the art and a representative sequence corresponds to Genbank Accession number NM_001127330. The term PPARγ2 is well known in the art and a representative sequence corresponds to Genbank Accession number NM_011146.

[0060] It will be appreciated that specific sequence identifiers (SEQ ID NOs) have been referenced throughout the specification for purposes of illustration and should therefore not be construed to be limiting. Any marker of the invention, including, but not limited to, the markers described in the specification and markers presented in Tables 1, 2, and 3, or fragments thereof, can be used in the embodiments of the invention.

I. Nucleic Acids of the Invention

[0061] One aspect of the invention pertains to methods utilizing isolated nucleic acid molecules that encode prdm16 or biologically active portions thereof and/or C/EBPβ or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify prdm16-encoding or C/EBPβ-encoding nucleic acid (i.e., prdm16 or C/EBPβ mRNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (i.e., cDNA or genomic DNA) and RNA molecules (i.e., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated prdm16 or C/EBPβ nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (i.e., a brown adipocyte). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

[0062] A nucleic acid molecule of the present invention, i.e., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, and 9 or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more (e.g., about 98%) homologous to the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, and 9 or a portion thereof (i.e., 400, 450, 500, or more nucleotides), can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a human prdm16 and/or C/EBPβ cDNA can be isolated from a human liver, heart, kidney, or brain cell line (from Stratagene, LaJolla, Calif., or Clontech, Palo Alto, Calif.) using all or portion of SEQ ID NOs: 1, 3, 5, 7, and 9 as a hybridization probe and standard hybridization techniques (i.e., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NOs: 1, 3, 5, 7, or 9 or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, or 9 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NOs: 1, 3, 5, 7, or 9 or the homologous nucleotide sequence. For example, mRNA can be isolated from liver cells, heart cells, kidney cells, brain cells, or brown adipocytes (i.e., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, or 9 or to the homologous nucleotide sequence. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a prdm16 and/or C/EBPβ nucleotide sequence can be prepared by standard synthetic techniques, i.e., using an automated DNA synthesizer.

[0063] Probes based on the prdm16 and/or C/EBPβ nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, i.e., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which express a prdm16 and/or C/EBPβ protein, such as by measuring a level of a prdm16-encoding and/or C/EBPβ-encoding nucleic acid in a sample of cells from a subject, i.e., detecting prdm16 and/or C/EBPβ mRNA levels.

[0064] Nucleic acid molecules encoding other prdm16 family and/or C/EBPβ members and thus which have a nucleotide sequence which differs from the prdm16 and/or C/EBPβ sequences of SEQ ID NOs: 1, 3, 5, 7, or 9 are intended to be of the invention. Moreover, nucleic acid molecules encoding Prdm16 and/or C/EBPβ proteins from different species, and thus which have a nucleotide sequence which differs from the prdm16 and/or C/EBPβ sequences of SEQ ID NOs:1, 3 5, 7, or 9 are intended to be within the scope of the invention. For example, rat or monkey Prdm16 and/or C/EBPβ cDNA can be identified based on the nucleotide sequence of a human and/or mouse Prdm16 and/or C/EBPβ.

[0065] In one embodiment, the nucleic acid molecule(s) of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 such that the protein or portion thereof maintains or, in complex, modulates (e.g., enhance), one or more of the following biological activities: 1) it can modulate the expression of cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1; 2) it can modulate the expression of mitochondrial genes including, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5; 3) it can increase or stimulate total respiration of a cell; 4) it can increase or stimulate uncoupled respiration of a cell; 5) it can increase or stimulate heat dissipation; 6) it can modulate thermogenesis; 7) it can increase or stimulate energy expenditure; 8) it can treat diseases or disorders characterized by increased PGC-1 expression or activity, e.g., diabetes or obesity.

[0066] As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in SEQ ID NO: 2, 4, 6, 8, or 10) amino acid residues to an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 such that the protein or portion thereof maintains or, in complex, modulates (e.g., enhance) one or more of the following biological activities: 1) it can modulate the expression of cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1; 2) it can modulate the expression of mitochondrial genes including, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5; 3) it can increase or stimulate total respiration of a cell; 4) it can increase or stimulate uncoupled respiration of a cell; 5) it can increase or stimulate heat dissipation; 6) it can modulate thermogenesis; 7) it can increase or stimulate energy expenditure; 8) it can treat diseases or disorders characterized by increased PGC-1 expression or activity, e.g., diabetes or obesity.

[0067] In another embodiment, the protein is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the entire amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.

[0068] Portions of proteins encoded by the Prdm16 and/or C/EBPβ nucleic acid molecule of the invention are preferably biologically active portions of the Prdm16 and/or C/EBPβ protein. As used herein, the term "biologically active portion of Prdm16 and/or C/EBPβ" is intended to include a portion, e.g., a domain/motif, of Prdm16 and/or C/EBPβ or, in complex, modulates (e.g., enhance) that has one or more of the following activities: 1) it can modulate the expression of cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1; 2) it can modulate the expression of mitochondrial genes including, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5; 3) it can increase or stimulate total respiration of a cell; 4) it can increase or stimulate uncoupled respiration of a cell; 5) it can increase or stimulate heat dissipation; 6) it can modulate thermogenesis; 7) it can increase or stimulate energy expenditure; 8) it can treat diseases or disorders characterized by increased PGC-1 expression or activity, e.g., diabetes or obesity.

[0069] Standard binding assays, e.g., immunoprecipitations and yeast two-hybrid assays, as described herein, can be performed to determine the ability of a Prdm16 protein and/or C/EBPβ or a biologically active portion thereof or complex thereof to interact with a target of interest. To determine whether a Prdm16 and/or C/EBPβ family member of the present invention modulates cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1 expression, in vitro transcriptional assays can be performed. To perform such an assay, the full length promoter/enhancer region of the gene of interest (e.g., cidea, type II deiodinase, cig30, pgc-1α, elov3, and ucp1) can be linked to a reporter gene such as chloramphenicol acetyltransferase (CAT) or luciferase and introduced into host cells (e.g., liver cells such as Fao hepatoma cells, or COS cells). The same host cells can then be transfected a nucleic acid molecule encoding the Prdm16 molecule and/or C/EBPβ molecule. In some embodiments, nucleic acid molecules encoding PPARγ can also be transfected. The effect of the Prdm16 and/or C/EBPβ molecule can be measured by testing CAT or luciferase activity and comparing it to CAT or luciferase activity in cells which do not contain nucleic acid encoding the Prdm16 molecule and/or C/EBPβ molecule. An increase or decrease in CAT or luciferase activity indicates a modulation of expression of the gene of interest. In another embodiment, because cidea, adiponectin, adipsin type II deiodinase, cig30, pgc-1α, elov3, and ucp1 expression is known to be a critical component in the cascade of events leading to elevated thermogenesis, this assay can also measure the ability of the Prdm16 and/or C/EBPβ molecule or complex thereof to modulate thermogenesis in adipocytes.

[0070] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9 (and portions thereof) due to degeneracy of the genetic code and thus encode the same Prdm16 and/or C/EBPβ protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10 or a protein having an amino acid sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.

[0071] It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of Prdm16 and/or C/EBPβ may exist within a population (e.g., a mammalian population, e.g., a human population). Such genetic polymorphism in the Prdm16 and/or C/EBPβ gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a Prdm16 and/or C/EBPβ protein, preferably a mammalian, e.g., human, Prdm16 and/or C/EBPβ protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the Prdm16 and/or C/EBPβ gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in Prdm16 and/or C/EBPβ that are the result of natural allelic variation and that do not alter the functional activity of Prdm16 and/or C/EBPβ are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding Prdm16 and/or C/EBPβ proteins from other species, and thus which have a nucleotide sequence which differs from the human or mouse sequences of SEQ ID NO: 1, 3, 5, 7, or 9, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the human or mouse Prdm16 and/or C/EBPβ cDNAs of the invention can be isolated based on their homology to the human or mouse Prdm16 and/or C/EBPβ nucleic acid sequences disclosed herein using the human or mouse cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions (as described herein).

[0072] In addition to naturally-occurring allelic variants of the Prdm16 and/or C/EBPβ sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9 thereby leading to changes in the amino acid sequence of the encoded Prdm16 and/or C/EBPβ protein, without altering the functional ability of the Prdm16 and/or C/EBPβ protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1, 3, 5, 7, or 9. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of Prdm16 and/or C/EBPβ (e.g., the sequence of SEQ ID NO: 2, 4, 6, 8, or 10) without altering the activity of Prdm16 and/or C/EBPβ, whereas an "essential" amino acid residue is required for Prdm16 and/or C/EBPβ activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved between mouse and human) may not be essential for activity and thus are likely to be amenable to alteration without altering Prdm16 and/or C/EBPβ activity. Furthermore, amino acid residues that are essential for Prdm16 and/or C/EBPβ functions related to thermogenesis and/or adipogenesis, but not essential for Prdm16 and/or C/EBPβ functions related to gluconeogenesis, are likely to be amenable to alteration.

[0073] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding Prdm16 and/or C/EBPβ proteins that contain changes in amino acid residues that are not essential for Prdm16 and/or C/EBPβ activity. Such Prdm16 and/or C/EBPβ proteins differ in amino acid sequence from SEQ ID NO: 2, 4, 6, 8, or 10 yet retain at least one of the Prdm16 and/or C/EBPβ activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 and is capable of inducing brown fat differentiation. Preferably, the protein encoded by the nucleic acid molecule is at least about 70% homologous, preferably at least about 80-85% homologous, still more preferably at least about 90%, and most preferably at least about 95% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.

[0074] "Sequence identity or homology", as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared ×100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified "loop out regions", e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.

[0075] The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. Preferably, the alignment can be performed using the Clustal Method. Multiple alignment parameters include GAP Penalty=10, Gap Length Penalty=10. For DNA alignments, the pairwise alignment parameters can be Htuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For protein alignments, the pairwise alignment parameters can be Ktuple=1, Gap penalty=3, Window=5, and Diagonals Saved=5.

[0076] In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0) (available online), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0077] An isolated nucleic acid molecule encoding a Prdm16 and/or C/EBPβ protein homologous to the protein of SEQ ID NO: 2, 4, 6, 8, or 10 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9 or a homologous nucleotide sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO: 1, 3, 5, 7, or 9 or the homologous nucleotide sequence by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in Prdm16 and/or C/EBPβ is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a Prdm16 and/or C/EBPβ coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a Prdm16 and/or C/EBPβ activity described herein to identify mutants that retain Prdm16 and/or C/EBPβ activity. Following mutagenesis of SEQ ID NO: 1, 3, 5, 7, or 9, the encoded protein can be expressed recombinantly (as described herein) and the activity of the protein can be determined using, for example, assays described herein.

[0078] Prdm16 and/or C/EBPβ levels may be assessed by any of a wide variety of well known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0079] In preferred embodiments, Prdm16 and/or C/EBPβ levels are ascertained by measuring gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Expression levels can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.

[0080] In a particular embodiment, the Prdm16 and/or C/EBPβ mRNA expression level can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).

[0081] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding Prdm16 and/or C/EBPβ. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that Prdm16 and/or C/EBPβ is being expressed.

[0082] In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array, e.g., an Affymetrix® gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of the Prdm16 and/or C/EBPβ mRNA expression levels.

[0083] An alternative method for determining the Prdm16 and/or C/EBPβ mRNA expression level in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[0084] For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to the Prdm16 and/or C/EBPβ mRNA.

[0085] As an alternative to making determinations based on the absolute Prdm16 and/or C/EBPβ expression level, determinations may be based on the normalized Prdm16 and/or C/EBPβ expression level. Expression levels are normalized by correcting the absolute Prdm16 and/or C/EBPβ expression level by comparing its expression to the expression of a non-Prdm16 and/or C/EBPβ gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, e.g., a normal sample, or between samples from different sources.

[0086] The level or activity of a Prdm16 and/or C/EBPβ protein can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The Prdm16 and/or C/EBPβ polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express Prdm16 and/or C/EBPβ.

[0087] In addition to the nucleic acid molecules encoding Prdm16 and/or C/EBPβ proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, i.e., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire Prdm16 and/or C/EBPβ coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding Prdm16 and/or C/EBPβ. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding Prdm16 and/or C/EBPβ. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).

II. Recombinant Expression Vectors and Host Cells

[0088] Another aspect of the invention pertains to the use of vectors, preferably expression vectors, containing a nucleic acid encoding Prdm16 and/or C/EBPβ (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. In one embodiment, adenoviral vectors comprising a Prdm16 and/or C/EBPβ nucleic acid molecule are used.

[0089] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[0090] The recombinant expression vectors of the invention can be designed for expression of Prdm16 and/or C/EBPβ in prokaryotic or eukaryotic cells. For example, Prdm16 and/or C/EBPβ can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0091] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the Prdm16 and/or C/EBPβ is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-Prdm16 and/or similar constructs for C/EBPβ. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant Prdm16 and/or C/EBPβ unfused to GST can be recovered by cleavage of the fusion protein with thrombin.

[0092] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[0093] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0094] In another embodiment, the Prdm16 and/or C/EBPβ expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

[0095] Alternatively, Prdm16 and/or C/EBPβ can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0096] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0097] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0098] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to Prdm16 mRNA and/or C/EBPβ mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.

[0099] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0100] A host cell can be any prokaryotic or eukaryotic cell. For example, Prdm16 and/or C/EBPβ protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Fao hepatoma cells, primary hepatocytes, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[0101] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[0102] A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. A PRDM16-C/EBPβ complex, a PRDM16 polypeptide or fragment thereof, and/or a C/EBPβ polypeptide or fragment thereof, may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, PRDM16-C/EBPβ complex, a PRDM16 polypeptide or fragment thereof, and/or a C/EBPβ polypeptide or fragment thereof, may be retained cytoplasmically and the cells harvested, lysed and the protein or protein complex isolated. A PRDM16-C/EBPβ complex, a PRDM16 polypeptide or fragment thereof, and/or a C/EBPβ polypeptide or fragment thereof, may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and inmmunoaffinity purification with antibodies specific for particular epitopes of PRDM16, C/EBPβ or a complex thereof. In one embodiment, the components of a PRDM16-C/EBPβ complex may be purified separately and then mixed together to form a complex. In another embodiment, the PRDM16-C/EBPβ complex may be purified from a source (e.g., a host cell, composition, cell lysate, etc.) comprising both PRDM16 and C/EBPβ polypeptide or fragments thereof.

[0103] Thus, a nucleotide sequence encoding all or a selected portion of a PRDM16 polypeptide and/or a C/EBPβ polypeptide may be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant PRDM16-C/EBPβ complex polypeptides by microbial means or tissue-culture technology in accord with the subject invention.

[0104] In another variation, protein production may be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are well known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used. Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors. In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.

[0105] In certain embodiments, the PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide, may be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell. Chemical synthesis may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules. (see e.g., U.S. Pat. Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M. Sclmolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord, "Chemical Approaches to Protein Engineering", in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91: 12544-12548; M. Schnlzer, et al., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).

[0106] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding Prdm16 and/or C/EBPβ or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0107] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) Prdm16 and/or C/EBPβ protein. Accordingly, the invention further provides methods for producing Prdm16 and/or C/EBPβ protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding Prdm16 and/or C/EBPβ has been introduced) in a suitable medium until Prdm16 and/or C/EBPβ is produced. In another embodiment, the method further comprises isolating Prdm16 and/or C/EBPβ from the medium or the host cell.

[0108] The host cells of the invention can also be used to produce nonhuman transgenic animals. The nonhuman transgenic animals can be used in screening assays designed to identify agents or compounds, e.g., drugs, pharmaceuticals, etc., which are capable of ameliorating detrimental symptoms of selected disorders such as glucose homeostasis disorders, weight disorders or disorders associated with insufficient insulin activity. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which Prdm16-coding and/or C/EBPβ-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous Prdm16 and/or C/EBPβ sequences have been introduced into their genome or homologous recombinant animals in which endogenous Prdm16 and/or C/EBPβ sequences have been altered. Such animals are useful for studying the function and/or activity of Prdm16 and/or C/EBPβ and for identifying and/or evaluating modulators of Prdm16 and/or C/EBPβ activity. As used herein, a "transgenic animal" is a nonhuman animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include nonhuman primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a nonhuman animal, preferably a mammal, more preferably a mouse, in which an endogenous Prdm16 and/or C/EBPβ gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0109] A transgenic animal of the invention can be created by introducing Prdm16-encoding and/or C/EBPβ-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The human Prdm16 and/or C/EBPβ cDNA sequence can be introduced as a transgene into the genome of a nonhuman animal. Alternatively, a nonhuman homologue of the human Prdm16 and/or C/EBPβ gene can be used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the Prdm16 and/or C/EBPβ transgene to direct expression of Prdm16 and/or C/EBPβ protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the Prdm16 and/or C/EBPβ transgene in its genome and/or expression of Prdm16 and/or C/EBPβ mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding Prdm16 and/or C/EBPβ can further be bred to other transgenic animals carrying other transgenes.

[0110] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a Prdm16 and/or C/EBPβ gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the Prdm16 and/or C/EBPβ gene. The Prdm16 and/or C/EBPβ gene can be a human gene (e.g., from a human genomic clone isolated from a human genomic library screened with the cDNA of SEQ ID NO: 1 or 3), but more preferably, is a nonhuman homologue of a human Prdm16 and/or C/EBPβ gene. For example, a mouse Prdm16 and/or C/EBPβ gene can be used to construct a homologous recombination vector suitable for altering an endogenous Prdm16 and/or C/EBPβ gene, respectively, in the mouse genome. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous Prdm16 and/or C/EBPβ gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous Prdm16 and/or C/EBPβ gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous Prdm16 and/or C/EBPβ protein). In the homologous recombination vector, the altered portion of the Prdm16 and/or C/EBPβ gene is flanked at its 5' and 3' ends by additional nucleic acid of the Prdm16 and/or C/EBPβ gene to allow for homologous recombination to occur between the exogenous Prdm16 and/or C/EBPβ gene carried by the vector and an endogenous Prdm16 and/or C/EBPβ gene in an embryonic stem cell. The additional flanking Prdm16 and/or C/EBPβ nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced Prdm16 and/or C/EBPβ gene has homologously recombined with the endogenous Prdm16 and/or C/EBPβ gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

[0111] In another embodiment, transgenic nonhuman animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0112] Clones of the nonhuman transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

III. Isolated PRDM16-C/EBPβ Complexes, PRDM16-C/EBPβ Complex Polypeptides, and Antibodies Thereto

[0113] The present invention contemplates polypeptide complexes. For example, PRDM16-C/EBPβ complexes comprising (a) a full length PRDM16 polypeptide and a full length C/EBPβ polypeptide, (b) a fragment of PRDM16 and a full length C/EBPβ , (c) a full length PRDM16 and a fragment of C/EBPβ, or (d) a fragment of PRDM16 and a fragment of C/EBPβ.

[0114] The present invention makes available in a variety of embodiments soluble, purified and/or isolated forms of the PRDM16-C/EBPβ complexes or the PRDM16-C/EBPβ complex polypeptides.

[0115] In one aspect, a PRDM16-C/EBPβ complex polypeptide may comprise (a) a full-length PRDM16-C/EBPβ complex polypeptide amino acid sequence, (b) a full-length PRDM16-C/EBPβ complex polypeptide amino acid sequence with 1 to about 20 conservative amino acid substitutions, (c) a polypeptide amino acid sequence that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to an PRDM16-C/EBPβ complex polypeptide sequence of interest or (d) a fragment of the PRDM16-C/EBPβ complex polypeptide of interest (e.g., a polypeptide having less than the full-length sequence). In another aspect, the present invention contemplates a composition comprising an isolated PRDM16-C/EBPβ complex or PRDM16-C/EBPβ complex polypeptide and less than about 25%, or alternatively 15%, or alternatively 5%, contaminating biological macromolecules or polypeptides. In an exemplary embodiment, the PRDM16 fragment comprises from about amino acids 224-454, 680-880, or 881-1038 of PRDM16.

[0116] The present invention further provides compositions related to producing, detecting, or characterizing a PRDM16-C/EBPβ complex, a PRDM16 polypeptide or fragment thereof, or a C/EBPβ polypeptide or fragment thereof, such as nucleic acids, vectors, host cells, and the like. Such compositions may serve as compounds that modulate a PRDM16-C/EBPβ complex, an PRDM16 polypeptide or fragment thereof, or C/EBPβ polypeptide or fragment thereof, such as antisense nucleic acids.

[0117] In certain embodiments, a PRDM16-C/EBPβ complex polypeptide of the invention may be a fusion protein containing a domain which increases its solubility and/or facilitates its purification, identification, detection, and/or structural characterization. Exemplary domains, include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His-Asp or FLAG fusion proteins and tags. Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type III secretion system-targeting peptides, transcytosis domains, nuclear localization signals, etc. In various embodiments, a PRDM16-C/EBPβ complex polypeptide of the invention may comprise one or more heterologous fusions. Polypeptides may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains. The fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. It is also within the scope of the invention to include linker sequences between a polypeptide of the invention and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein. In another embodiment, the polypeptide may be constructed so as to contain protease cleavage sites between the fusion polypeptide and polypeptide of the invention in order to remove the tag after protein expression or thereafter. Examples of suitable endoproteases, include, for example, Factor Xa and TEV proteases.

[0118] In another embodiment, provided are fusions between PRDM16 and C/EBPβ polypeptides, and nucleotide sequences encoding the fusion polypeptides. The fusion polypeptides may comprise all or a part of a PRDM16 polypeptide and all or a part of a C/EBPβ polypeptide. In one embodiment, the fusion proteins may optionally contain a linker sequence between the PRDM16 and C/EBPβ sequences. In another embodiment, the fusion proteins may contain a protease cleavage site between the PRDM16 and C/EBPβ sequences (as described further above). In an exemplary embodiment, the fusion proteins will be capable of carrying out at least one biological activity of a PRDM16-C/EBPβ complex and may be useful for identifying a modulator of the activity and/or formation of a PRDM16-C/EBPβ complex. The fusion proteins may optionally contain other heterologous sequences such as polypeptide tags or labels. In certain embodiments, the fusion proteins may be formed by chemically or enzymatically linking two separate sequences together or may be formed by expressing or synthesizing a single polypeptide sequence comprising both PRDM16 and C/EBPβ sequences.

[0119] In still another embodiment, a PRDM16-C/EBPβ complex polypeptide of the invention may be labeled with a fluorescent label to facilitate their detection, purification, or structural characterization. In an exemplary embodiment, a PRDM16-C/EBPβ complex polypeptide of the invention may be fused to a heterologous polypeptide sequence which produces a detectable fluorescent signal, including, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).

[0120] Another aspect of the invention pertains to the use of isolated Prdm16 and/or C/EBPβ proteins, and biologically active portions thereof, as well as peptide fragments suitable for use as immunogens to raise anti-Prdm16 and/or anti-C/EBPβ antibodies. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of Prdm16 and/or C/EBPβ protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of Prdm16 and/or C/EBPβ protein having less than about 30% (by dry weight) of non-Prdm16 and/or non-C/EBPβ protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-Prdm16 and/or non-C/EBPβ protein, still more preferably less than about 10% of non-Prdm16 and/or non-C/EBPβ protein, and most preferably less than about 5% non-Prdm16 and/or non-C/EBPβ protein. When the Prdm16 and/or C/EBPβ protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of Prdm16 and/or C/EBPβ protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of Prdm16 and/or C/EBPβ protein having less than about 30% (by dry weight) of chemical precursors of non-Prdm16 and/or non-C/EBPβ chemicals, more preferably less than about 20% chemical precursors of non-Prdm16 and/or non-C/EBPβ chemicals, still more preferably less than about 10% chemical precursors of non-Prdm16 and/or non-C/EBPβ chemicals, and most preferably less than about 5% chemical precursors of non-Prdm16 and/or non-C/EBPβ chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same animal from which the Prdm16 and/or C/EBPβ protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a human Prdm16 and/or C/EBPβ protein in a nonhuman cell.

[0121] An isolated Prdm16 and/or C/EBPβ protein or a portion thereof of the invention has one or more of the following biological activities or, in complex, modulates (e.g., enhance) one or more of the following biological activities: 1) it can modulate the expression of cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1; 2) it can modulate the expression of mitochondrial genes including, cytochrome c, cox 4i1, cox III, cox 5b, cox8b atpase b2, cox II, atp5o and ndufb5; 3) it can increase or stimulate total respiration of a cell; 4) it can increase or stimulate uncoupled respiration of a cell; 5) it can increase or stimulate heat dissipation; 6) it can modulate thermogenesis; 7) it can increase or stimulate energy expenditure; 8) it can treat diseases or disorders characterized by increased PGC-1 expression or activity, e.g., diabetes or obesity.

[0122] In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 such that the protein or portion thereof maintains one or more of the following biological activities or, in complex, modulates (e.g., enhance) one or more of the following biological activities: 1) it can modulate the expression of cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1; 2) it can modulate the expression of mitochondrial genes including, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5; 3) it can increase or stimulate total respiration of a cell; 4) it can increase or stimulate uncoupled respiration of a cell; 5) it can increase or stimulate heat dissipation; 6) it can modulate thermogenesis; 7) it can increase or stimulate energy expenditure; 8) it can treat diseases or disorders characterized by increased PGC-1 expression or activity, e.g., diabetes or obesity. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, the Prdm16 and/or C/EBPβ protein has an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10, respectively, or an amino acid sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10. In yet another preferred embodiment, the Prdm16 and/or C/EBPβ protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, or 9 or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9. The preferred Prdm16 and/or C/EBPβ proteins of the present invention also preferably possess at least one of the Prdm16 and/or C/EBPβ biological activities, or activities associated with the complex, described herein. For example, a preferred Prdm16 and/or C/EBPβ protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, or 9 and which can maintain one or more of the following biological activities or, in complex, modulates (e.g., enhance) one or more of the following biological activities: 1) it can modulate the expression of cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1; 2) it can modulate the expression of mitochondrial genes including, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5; 3) it can increase or stimulate total respiration of a cell; 4) it can increase or stimulate uncoupled respiration of a cell; 5) it can increase or stimulate heat dissipation; 6) it can modulate thermogenesis; 7) it can increase or stimulate energy expenditure; 8) it can treat diseases or disorders characterized by increased PGC-1 expression or activity, e.g., diabetes or obesity.

[0123] In other embodiments, the Prdm16 and/or C/EBPβ protein is substantially homologous to the amino acid sequence of SEQ ID NO: 2, 4 or 6 and retains the functional activity of the protein of SEQ ID NO: 2, 4, 6, 8, or 10 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the Prdm16 and/or C/EBPβ protein is a protein which comprises an amino acid sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10.

[0124] Biologically active portions of the Prdm16 and/or C/EBPβ protein include peptides comprising amino acid sequences derived from the amino acid sequence of the Prdm16 and/or C/EBPβ protein, e.g., the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10 or the amino acid sequence of a protein homologous to the Prdm16 and/or C/EBPβ protein, which include fewer amino acids than the full length Prdm16 and/or C/EBPβ protein or the full length protein which is homologous to the Prdm16 and/or C/EBPβ protein, and exhibit at least one activity of the Prdm16 and/or C/EBPβ protein, or complex thereof. Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif, e.g., PR domain (PRDI-BF1-RIZ homology region). In a preferred embodiment, the biologically active portion of the protein which includes one or more the domains/motifs described herein can modulate differentiation of adipocytes and/or thermogenesis in brown adipocytes. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of the Prdm16 and/or C/EBPβ protein include one or more selected domains/motifs or portions thereof having biological activity.

[0125] Prdm16 and/or C/EBPβ proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the Prdm16 and/or C/EBPβ protein is expressed in the host cell. The Prdm16 and/or C/EBPβ protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a Prdm16 and/or C/EBPβ protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native Prdm16 and/or C/EBPβ protein can be isolated from cells (e.g., brown adipocytes), for example using an anti-Prdm16 antibody and/or anti-C/EBPβ antibody (described further below).

[0126] The invention also provides Prdm16 and/or C/EBPβ chimeric or fusion proteins. As used herein, a Prdm16 and/or C/EBPβ "chimeric protein" or "fusion protein" comprises a Prdm16 and/or C/EBPβ polypeptide operatively linked to a non-Prdm16 and/or non-C/EBPβ polypeptide, respectively. A "Prdm16 and/or C/EBPβ polypeptide" refers to a polypeptide having an amino acid sequence corresponding to Prdm16 and/or C/EBPβ, whereas a "non-Prdm16 and/or non-C/EBPβ polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the Prdm16 and/or C/EBPβ protein, respectively, e.g., a protein which is different from the Prdm16 and/or C/EBPβ protein and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the Prdm16 and/or C/EBPβ polypeptide and the non-Prdm16 and/or non-C/EBPβ polypeptide are fused in-frame to each other. The non-Prdm16 and/or non-C/EBPβ polypeptide can be fused to the N-terminus or C-terminus of the Prdm16 and/or C/EBPβ polypeptide, respectively. For example, in one embodiment the fusion protein is a GST-Prdm16 and/or GST-C/EBPβ fusion protein in which the Prdm16 and/or C/EBPβ sequences, respectively, are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant Prdm16 and/or C/EBPβ. In another embodiment, the fusion protein is a Prdm16 and/or C/EBPβ protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of Prdm16 and/or C/EBPβ can be increased through use of a heterologous signal sequence.

[0127] Preferably, a Prdm16 and/or C/EBPβ chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A Prdm16-encoding and/or C/EBPβ-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the Prdm16 and/or C/EBPβ protein.

[0128] The present invention also pertains to homologues of the Prdm16 and/or C/EBPβ proteins which function as either a Prdm16 and/or C/EBPβ agonist (mimetic) or a Prdm16 and/or C/EBPβ antagonist. In a preferred embodiment, the Prdm16 and/or C/EBPβ agonists and antagonists stimulate or inhibit, respectively, a subset of the biological activities of the naturally occurring form of the Prdm16 and/or C/EBPβ protein. Thus, specific biological effects can be elicited by treatment with a homologue of limited function. In one embodiment, treatment of a subject with a homologue having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the Prdm16 and/or C/EBPβ protein.

[0129] Homologues of the Prdm16 and/or C/EBPβ protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the Prdm16 and/or C/EBPβ protein. As used herein, the term "homologue" refers to a variant form of the Prdm16 and/or C/EBPβ protein which acts as an agonist or antagonist of the activity of the Prdm16 and/or C/EBPβ protein. An agonist of the Prdm16 and/or C/EBPβ protein can retain substantially the same, or a subset, of the biological activities of the Prdm16 and/or C/EBPβ protein. An antagonist of the Prdm16 and/or C/EBPβ protein can inhibit one or more of the activities of the naturally occurring form of the Prdm16 and/or C/EBPβ protein, by, for example, competitively binding to a downstream or upstream member of the Prdm16 and/or C/EBPβ cascade which includes the Prdm16 and/or C/EBPβ protein. Thus, the mammalian Prdm16 and/or C/EBPβ protein and homologues thereof of the present invention can be, for example, either positive or negative regulators of adipocyte differentiation and/or thermogenesis in brown adipocytes.

[0130] In an alternative embodiment, homologues of the Prdm16 and/or C/EBPβ protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the Prdm16 and/or C/EBPβ protein for Prdm16 and/or C/EBPβ protein agonist or antagonist activity. In one embodiment, a variegated library of Prdm16 and/or C/EBPβ variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of Prdm16 and/or C/EBPβ variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential Prdm16 and/or C/EBPβ sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of Prdm16 and/or C/EBPβ sequences therein. There are a variety of methods which can be used to produce libraries of potential Prdm16 and/or C/EBPβ homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential Prdm16 and/or C/EBPβ sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

[0131] In addition, libraries of fragments of the Prdm16 and/or C/EBPβ protein coding can be used to generate a variegated population of Prdm16 and/or C/EBPβ fragments for screening and subsequent selection of homologues of a Prdm16 and/or C/EBPβ protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a Prdm16 and/or C/EBPβ coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the Prdm16 and/or C/EBPβ protein.

[0132] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of Prdm16 and/or C/EBPβ homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify Prdm16 and/or C/EBPβ homologues (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Engineering 6(3):327-331).

[0133] A variety of antibodies directed to PRDM16-C/EBPβ complexes, PRDM16 polypeptides or fragments thereof, or C/EBPβ polypeptides or fragment thereof, are also provided. In one embodiment, the present invention provides an isolated antibody that has a higher binding affinity for a PRDM16-C/EBPβ complex than for the any of the components of the complex alone, including a PRDM16 polypeptide or fragment thereof, or a C/EBPβ polypeptide or fragment thereof. In an exemplary embodiment, an antibody, or antibody fragment may be capable of binding to a PRDM16-C/EBPβ complex with less than 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, or less cross-reactivity with a component of the complex when not in the complex. In another embodiment, the invention provides an isolated antibody that binds to an interaction site on a PRDM16-C/EBPβ complex polypeptide (for example, a site on a PRDM16 polypeptide or fragment thereof that is capable of interacting with a C/EBPβ polypeptide or fragment thereof, or a site on a C/EBPβ polypeptide or fragment thereof that is capable of interacting with a PRDM16 polypeptide or fragment thereof). In still other embodiments, the isolated antibodies of the invention may disrupt or stabilize a PRDM16-C/EBPβ complex.

[0134] In one embodiment, an isolated Prdm16 and/or C/EBPβ protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind Prdm16 and/or C/EBP, respectively, or a complex thereof, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length Prdm16 and/or C/EBPβ protein can be used or, alternatively, antigenic peptide fragments of Prdm16 and/or C/EBPβ, or peptides in complex, can be used as immunogens. A Prdm16 and/or C/EBPβ immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed Prdm16 and/or C/EBPβ protein or a chemically synthesized Prdm16 and/or C/EBPβ peptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic Prdm16 and/or C/EBPβ preparation induces a polyclonal anti-Prdm16 and/or anti-C/EBPβ antibody response.

[0135] Accordingly, another aspect of the invention pertains to the use of anti-Prdm16 and/or anti-C/EBPβ antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as prdm16 and/or C/EBPβ. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind Prdm16 and/or C/EBPβ. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of Prdm16 and/or C/EBPβ. A monoclonal antibody composition thus typically displays a single binding affinity for a particular Prdm16 and/or C/EBPβ protein with which it immunoreacts.

[0136] Polyclonal anti-Prdm16 and/or C/EBPβ antibodies can be prepared as described above by immunizing a suitable subject with a Prdm16 and/or C/EBPβ, or complex thereof, immunogen. The anti-Prdm16 and/or anti-C/EBPβ antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized Prdm16 and/or C/EBPβ. If desired, the antibody molecules directed against Prdm16 and/or C/EBPβ can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, i.e., when the anti-Prdm16 and/or anti-C/EBPβ antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a Prdm16 and/or C/EBPβ immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds Prdm16 and/or C/EBPβ.

[0137] Any of the many well-known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-Prdm16 and/or anti-C/EBPβ monoclonal antibody (see, i.e., G. Galfre et al. (1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, i.e., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind Prdm16 and/or C/EBPβ, i.e., using a standard ELISA assay.

[0138] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-prdm16 and/or anti-C/EBPβ antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with Prdm16 and/or C/EBPβ to thereby isolate immunoglobulin library members that bind Prdm16 and/or C/EBPβ. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP® Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[0139] Additionally, recombinant anti-Prdm16 and/or anti-C/EBPβ antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0140] An anti-Prdm16 and/or anti-C/EBPβ antibody (e.g., monoclonal antibody) can be used to isolate Prdm16 and/or C/EBPβ by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-Prdm16 and/or anti-C/EBPβ antibody can facilitate the purification of natural Prdm16 and/or C/EBPβ from cells and of recombinantly produced Prdm16 and/or C/EBPβ expressed in host cells. Moreover, an anti-Prdm16 and/or anti-C/EBPβ antibody can be used to detect Prdm16 and/or C/EBPβ protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the Prdm16 and/or C/EBPβ protein. Anti-Prdm16 and/or anti-C/EBPβ antibodies can be used to monitor protein levels in a cell or tissue, e.g., adipose cells or tissue, as part of a clinical testing procedure, e.g., in order to monitor a safe dosage of an uncoupling agent. Detection can be facilitated by coupling (e.g., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0141] In vivo techniques for detection of Prdm16 and/or C/EBPβ protein include introducing into a subject a labeled antibody directed against the protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

IV. Identification of Compounds That Modulate a PRDM16-C/EBPβ Complex

[0142] The PRDM16-C/EBPβ complexes and/or PRDM16-C/EBPβ complex polypeptides described herein may be used to design modulators of one or more of biological activities of the complex or complex polypeptides. In particular, information useful for the design of therapeutic and diagnostic molecules, including, for example, the protein domain, structural information, and the like for polypeptides of the invention is now available or attainable as a result of the ability to prepare, purify and characterize the complexes and complex polypeptides, and domains, fragments, variants and derivatives thereof.

[0143] In another aspect, modulators, inhibitors, or antagonists against the polypeptides of the invention, biological complexes containing them, or orthologues thereto, may be used to treat any disease or other treatable condition of a patient (including humans and animals), including, for example, obesity or obesity-related disorders.

[0144] Modulators of PRDM16-C/EBPβ complexes, other structurally related molecules, and PRDM16-C/EBPβ complex polypeptides, may be identified and developed as set forth below and otherwise using techniques and methods known to those of skill in the art. The modulators of the invention may be employed, for instance, to inhibit and treat PRDM16-C/EBPβ-mediated diseases or disorders. The modulators of the invention may elicit a change in one or more of the following activities: (a) a change in the level and/or rate of formation of a PRDM16-C/EBPβ complex, (b) a change in the activity of a PRDM16-C/EBPβ complex, (c) a change in the stability of a PRDM16-C/EBPβ complex, (d) a change in the conformation of a PRDM16-C/EBPβ complex, or (e) a change in the activity of at least one polypeptide contained in a PRDM16-C/EBPβ complex. A number of methods for identifying a molecule which modulates a PRDM16-C/EBPβ complex, or a PRDM16-C/EBPβ complex polypeptide, are known in the art. For example, in one such method, a PRDM16-C/EBPβ complex, or a PRDM16-C/EBPβ complex polypeptide, is contacted with a test compound, and the activity of the PRDM16-C/EBPβ complex, or a PRDM16-C/EBPβ complex polypeptide, in the presence of the test compound is determined, wherein a change in the activity of the PRDM16-C/EBPβ complex, or a PRDM16-C/EBPβ complex polypeptide, in the presence of the compound as compared to the activity in the absence of the compound (or in the presence of a control compound) is indicative that the test compound modulates the activity of the PRDM16-C/EBPβ complex, or a PRDM16-C/EBPβ complex polypeptide.

[0145] Compounds to be tested for their ability to act as modulators of PRDM16-C/EBPβ complexes, or PRDM16-C/EBPβ complex polypeptides, can be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly. Compounds for use with the above-described methods may be selected from the group of compounds consisting of lipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleic acids (PNAs), small molecules, natural products, aptamers and polynucleotides. In certain embodiments, the compound is a polynucleotide. In some embodiments, said polynucleotide is an antisense nucleic acid. In other embodiments, said polynucleotide is an siRNA. In certain embodiments, the compound comprises PRDM16-C/EBPβ complex polypeptide.

[0146] A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein may nevertheless be comprehended by one of ordinary skill in the art based on the teachings herein. Assay formats for analyzing PRDM16-C/EBPβ complex formation, PRDM16-C/EBPβ complex activity, and/or activity of a PRDM16-C/EBPβ complex polypeptide, may be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Simple binding assays can also be used to detect agents which modulate a PRDM16-C/EBPβ complex, for example, by enhancing the formation of a PRDM16-C/EBPβ complex, by enhancing the binding of a PRDM16-C/EBPβ complex to a substrate, and/or by enhancing the binding of a PRDM16-C/EBPβ complex polypeptide to a substrate. Another example of an assay useful for identifying a modulator of a PRDM16-C/EBPβ complex is a competitive assay that combines one or more PRDM16-C/EBPβ complex polypeptides with a potential modulator, such as, for example, polypeptides, nucleic acids, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. PRDM16-C/EBPβ complex polypeptides can be labeled, such as by radioactivity or a colorimetric compound, such that PRDM16-C/EBPβ complex formation and/or activity can be determined accurately to assess the effectiveness of the potential modulator.

[0147] Assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof. Assays may also employ any of the methods for isolating, preparing and detecting PRDM16-C/EBPβ complexes, or complex polypeptides, as described above.

[0148] Complex formation between a PRDM16 polypeptide or a C/EBPβ polypeptide and a binding partner may be detected by a variety of methods. Modulation of the formation of PRDM16-C/EBPβ complexes may be quantified using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners, by immunoassay, or by chromatographic detection. Methods of isolating and identifying PRDM16-C/EBPβ complexes described above may be incorporated into the detection methods.

[0149] In certain embodiments, it may be desirable to immobilize a PRDM16 polypeptide and/or C/EBPβ polypeptide to facilitate separation of PRDM16 and/or C/EBPβ complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a PRDM16 and/or C/EBPβ polypeptide to a binding partner may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein may be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the binding partner, e.g. an ³⁵S-labeled binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes may be dissociated from the matrix, separated by SDS-PAGE, and the level of PRDM16 and/or C/EBPβ polypeptides found in the bead fraction quantified from the gel using standard electrophoretic techniques such as described in the appended examples.

[0150] Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, either a PRDM16 and/or C/EBPβ polypeptide may be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated polypeptide molecules may be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide may be derivatized to the wells of the plate, and polypeptide trapped in the wells by antibody conjugation. As above, preparations of a binding partner and a test compound are incubated in the polypeptide presenting wells of the plate, and the amount of complex trapped in the well may be quantified. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with the PRDM16 and/or C/EBPβ polypeptide and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme may be chemically conjugated or provided as a fusion protein with the binding partner. To illustrate, the binding partner may be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of PRDM16 and/or C/EBPβ polypeptide trapped in the PRDM16-C/EBPβ complex may be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-1-napthol. Likewise, a fusion protein comprising the PRDM16 and/or C/EBPβ polypeptide and glutathione-S-transferase may be provided, and PRDM16 and/or C/EBPβ complex formation quantified by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

[0151] For processes that rely on immunodetection for quantitating one of the PRDM16-C/EBPβ complex polypeptides trapped in the PRDM16-C/EBPβ complex, antibodies against the PRDM16-C/EBPβ complex polypeptide, such as anti-polypeptide antibodies, may be used. Alternatively, the PRDM16 and/or C/EBPβ polypeptide to be detected in the PRDM16-C/EBPβ complex may be "epitope-tagged" in the form of a fusion protein that includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST fusion proteins described above may also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N.J.).

[0152] In certain in vitro embodiments of the present assay, the protein or the set of proteins engaged in a protein-protein, protein-substrate, or protein-nucleic acid interaction comprises a reconstituted protein mixture of at least semi-purified proteins. By semi-purified, it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins. For instance, in contrast to cell lysates, the proteins involved in a protein-substrate, protein-protein or nucleic acid-protein interaction are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and more preferably are present at 90-95% purity. In certain embodiments of the subject method, the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-substrate, protein-protein interaction, or nucleic acid-protein interaction.

[0153] In one embodiment, the use of reconstituted protein mixtures allows more careful control of the protein-substrate, protein-protein, or nucleic acid-protein interaction conditions. Moreover, the system may be derived to favor discovery of modulators of particular intermediate states of the protein-protein interaction. For instance, a reconstituted protein assay may be carried out both in the presence and absence of a candidate agent, thereby allowing detection of a modulator of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.

[0154] Assaying biological activity resulting from a given protein-substrate, protein-protein or nucleic acid-protein interaction, in the presence and absence of a candidate modulator, may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.

[0155] In yet another embodiment, a PRDM16-C/EBPβ complex polypeptide may be used to generate an two-hybrid or interaction trap assay (see also, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696), for subsequently detecting agents which disrupt binding of the interaction components to one another.

[0156] In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator may be fused in frame to the coding sequence for a "bait" protein, e.g., a PRDM16 and/or C/EBPβ polypeptide of sufficient length to bind to a potential interacting protein. The second hybrid protein encodes a transcriptional activation domain fused in frame to a gene encoding a "fish" protein, e.g., a potential interacting protein of sufficient length to interact with the protein-protein interaction component polypeptide portion of the bait fusion protein. If the bait and fish proteins are able to interact, e.g., form a protein-protein interaction component complex, they bring into close proximity the two domains of the transcriptional activator. This proximity causes transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene may be detected and used to score for the interaction of the bait and fish proteins. The host cell also contains a first chimeric gene which is capable of being expressed in the host cell. The gene encodes a chimeric protein, which comprises (a) a DNA-binding domain that recognizes the responsive element on the reporter gene in the host cell, and (b) a bait protein (e.g., a PRDM16 and/or C/EBPβ polypeptide). A second chimeric gene is also provided which is capable of being expressed in the host cell, and encodes the "fish" fusion protein. In one embodiment, both the first and the second chimeric genes are introduced into the host cell in the form of plasmids. Preferably, however, the first chimeric gene is present in a chromosome of the host cell and the second chimeric gene is introduced into the host cell as part of a plasmid.

[0157] The DNA-binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein may be derived from transcriptional activators having separable DNA-binding and transcriptional activation domains. For instance, these separate DNA-binding and transcriptional activation domains are known to be found in the yeast GAL4 protein, and are known to be found in the yeast GCN4 and ADR1 proteins. Many other proteins involved in transcription also have separable binding and transcriptional activation domains which make them useful for the present invention, and include, for example, the LexA and VP16 proteins. It will be understood that other (substantially) transcriptionally-inert DNA-binding domains may be used in the subject constructs; such as domains of ACE1, λcI, lac repressor, jun or fos. In another embodiment, the DNA-binding domain and the transcriptional activation domain may be from different proteins. The use of a LexA DNA binding domain provides certain advantages. For example, in yeast, the LexA moiety contains no activation function and has no known affect on transcription of yeast genes. In addition, use of LexA allows control over the sensitivity of the assay to the level of interaction (see, for example, the Brent et al. PCT publication WO94/10300).

[0158] In certain embodiments, any enzymatic activity associated with the bait or fish proteins is inactivated, e.g., dominant negative or other mutants of a protein-protein interaction component can be used.

[0159] Continuing with the illustrative example, formation of a complex between the bait and fish fusion proteins in the host cell, causes the activation domain to activate transcription of the reporter gene. The method is carried out by introducing the first chimeric gene and the second chimeric gene into the host cell, and subjecting that cell to conditions under which the bait and fish fusion proteins and are expressed in sufficient quantity for the reporter gene to be activated. The formation of a complex results in a detectable signal produced by the expression of the reporter gene.

[0160] In still further embodiments, the PRDM16-C/EBPβ complex, or complex polypeptide, of interest may be generated in whole cells, taking advantage of cell culture techniques to support the subject assay. For example, the PRDM16-C/EBPβ complex, or complex polypeptide, may be constituted in a prokaryotic or eukaryotic cell culture system. Advantages to generating the PRDM16-C/EBPβ complex, or complex polypeptide, in an intact cell includes the ability to screen for modulators of the level and/or activity of the PRDM16-C/EBPβ complex, or complex polypeptide, which are functional in an environment more closely approximating that which therapeutic use of the modulator would require, including the ability of the agent to gain entry into the cell. Furthermore, certain of the in vivo embodiments of the assay are amenable to high through-put analysis of candidate agents.

[0161] The PRDM16-C/EBPβ complexes and PRDM16-C/EBPβ complex polypeptides can be endogenous to the cell selected to support the assay. Alternatively, some or all of the components can be derived from exogenous sources. For instance, fusion proteins can be introduced into the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the fusion protein itself or mRNA encoding the fusion protein. Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of the protein-protein interaction.

[0162] The amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain, western blots or an intrinsic activity. In certain embodiments, the product of the reporter gene is detected by an intrinsic activity associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.

[0163] In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays of the present invention which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target. Accordingly, potential modulators of PRDM16-C/EBPβ complexes may be detected in a cell-free assay generated by constitution of a functional PRDM16-C/EBPβ complex in a cell lysate. In an alternate format, the assay can be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.

[0164] The activity of a PRDM16-C/EBPβ complex or a PRDM16-C/EBPβ complex polypeptide may be identified and/or assayed using a variety of methods well known to the skilled artisan. For example, the activity of a PRDM16-C/EBPβ complex or a PRDM16-C/EBPβ complex polypeptide may be determined by assaying for the level of expression of RNA and/or protein molecules. Transcription levels may be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product. Translation levels may be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product (e.g., fluorescence, luminescence, enzymatic activity, etc.). Depending on the particular situation, it may be desirable to detect the level of transcription and/or translation of a single gene or of multiple genes.

[0165] In other embodiments, the biological activity of a PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide, may be assessed by monitoring changes in the phenotype of a targeted cell. For example, the detection means can include a reporter gene construct which includes a transcriptional regulatory element that is dependent in some form on the level and/or activity of a PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide. The PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide, may be provided as a fusion protein with a domain that binds to a DNA element of a reporter gene construct. The added domain of the fusion protein can be one which, through its DNA-binding ability, increases or decreases transcription of the reporter gene. Which ever the case may be, its presence in the fusion protein renders it responsive to a PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide. Accordingly, the level of expression of the reporter gene will vary with the level of expression of a PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide.

[0166] Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. A reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable. The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. For instance, the product of the reporter gene can be an enzyme which confers resistance to an antibiotic or other drug, or an enzyme which complements a deficiency in the host cell (i.e. thymidine kinase or dihydrofolate reductase). To illustrate, the aminoglycoside phosphotransferase encoded by the bacterial transposon gene Tn5 neo can be placed under transcriptional control of a promoter element responsive to the level of a PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide, present in the cell. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of inhibition of the PRDM16-C/EBPβ complex, or PRDM16-C/EBPβ complex polypeptide.

V. Methods of the Invention

[0167] The methods of the invention relate to the expression and/or activity of both Prdm16 and C/EBPβ sufficient to activate brown fat cell differentiation, wherein the differentiated brown fat cells increase energy expenditure to thereby treat obesity or an obesity related disorder, e.g., Type II diabetes.

[0168] The invention also relates to methods for increasing energy expenditure in a mammal comprising inducing expression and/or activity of both Prdm16 and C/EBPβ sufficient to activate brown fat cell differentiation in the mammal, wherein the differentiated brown fat cells promote energy expenditure thereby increasing energy expenditure in the mammal.

[0169] The term "sufficient to activate" is intended to encompass any increase in expression and/or activity of both Prdm16 and C/EBPβ that promotes, activates, stimulates, enhances, or results in brown fat differentiation.

[0170] In another aspect, the invention relates to methods for treating obesity or an obesity-related disorder, e.g., Type II diabetes, in a subject comprising administering to the subject an agent that induces expression and/or activity of both Prdm16 and C/EBPβ, wherein expression and/or activity of both Prdm16 and C/EBPβ increases respiration and energy expenditure to thereby treat obesity or an obesity-related disorder. In one embodiment, total respiration is increased following the expression and/or activity of both Prdm16 and C/EBPβ. In another embodiment, uncoupled respiration is increased following the expression and/or activity of both Prdm16 and C/EBPβ. Uncoupled respiration dissipates heat and thereby increases energy expenditure in the subject.

[0171] As used herein, the term "agent" and "therapeutic agent" is defined broadly as anything that cells from a subject with obesity or an obesity-related disorder may be exposed to in a therapeutic protocol.

[0172] The term "administering" is intended to include routes of administration which allow the agent to perform its intended function of increasing expression and/or activity of both Prdm16 and C/EBPβ. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. Further the agent may be coadministered with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.

[0173] The term "effective amount" of an agent that induces expression and/or activity of both Prdm16 and C/EBPβ is that amount necessary or sufficient to promote expression and/or activity of both Prdm16 and C/EBPβ in the subject or population of subjects. The effective amount can vary depending on such factors as the type of therapeutic agent(s) employed, the size of the subject, or the severity of the disorder.

[0174] It will be appreciated that individual dosages may be varied depending upon the requirements of the subject in the judgment of the attending clinician, the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, a number of additional factors may be considered by the attending clinician, including, but not limited to: the pharmacodynamic characteristics of the particular respiration uncoupling agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment; and other relevant circumstances. U.S. Pat. No. 5,427,916, for example, describes a method for predicting the effectiveness of antineoplastic therapy in individual subjects, and illustrates certain methods which can be used in conjunction with the treatment protocols of the instant invention.

[0175] Treatment can be initiated with smaller dosages which are less than the effective dose of the compound. Thereafter, in one embodiment, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

[0176] The effectiveness of any particular respiration uncoupling agent to treat obesity or obesity-related disorders can be monitored by comparing two or more samples obtained from a subject undergoing anti-obesity or obesity-related disorder treatment. In general, it is preferable to obtain a first sample from the subject prior to beginning therapy and one or more samples during treatment. In such a use, a baseline of expression of cells from subjects with obesity or obesity-related disorders prior to therapy is determined and then changes in the baseline state of expression of cells from subjects with obesity or obesity-related disorders is monitored during the course of therapy. Alternatively, two or more successive samples obtained during treatment can be used without the need of a pre-treatment baseline sample. In such a use, the first sample obtained from the subject is used as a baseline for determining whether the expression of cells from subjects with obesity or obesity-related disorders is increasing or decreasing.

[0177] Another aspect of the invention relates to a method for inducing brown fat cell differentiation in a mammal comprising expressing both Prdm16 and C/EBPβ in cells; delivering the cells expressing both Prdm16 and C/EBPβ into the mammal; and monitoring the differentiation of brown fat cells in the mammal. The cells can be delivered by well-known methods in the art (e.g., grafting, subcutaneous or intravenous injection). Increased brown adipose tissue in the mammal will warm up the body and blood of the mammal resulting in an increased energy expenditure from the cells. The increased energy expenditure will increase the metabolic rate of the subject and may be used for the treatment and/or prevention of obesity and obesity related disorders. The induction of brown fat cells may be monitored by 1) an increase or stimulation of the expression of cidea, adiponectin, adipsin, type II deiodinase, cig30, pgc-1α, elov3, and ucp1; 2) an increase or stimulation of the expression of mitochondrial genes including, cytochrome c, cox 4i1, cox III, cox 5b, cox8b, atpase b2, cox II, atp5o and ndufb5; 3) an increase or stimulation of total respiration of a cell; 4) an increase or stimulation of uncoupled respiration of a cell; 5) an increase or stimulation of heat dissipation; 6) an increase or stimulation of thermogenesis; and/or 7) an increase or stimulation of energy expenditure. The cells that can be used are described in more detail below. However, the method is suitable for obtaining cells from the mammal itself (i.e., autologous cells).

[0178] Mammalian cells (e.g., human) cells may be obtained by well known methods. Representative examples of mammalian cells useful in the present invention include, without limitation, fibroblasts (skin fibroblasts, dermal fibroblasts, primary embryonic fibroblasts, immortalized embryonic fibroblasts, and human foreskin fibroblasts), myoblasts, preadipocytes, white adipocytes, epithelial, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), macrophages, monocytes, mononuclear cells, cardiac muscle cells, skeletal muscle cells, hepatocytes and other muscle cells, etc. Moreover, the mammalian cells may be obtained from different organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, etc.

[0179] Clinical use of autologous subject-derived sources of cells (e.g., skin fibroblasts) is advantageous to avoid potential adverse allogeneic immune reactivity. In other embodiments, the cells may be allogeneic, syngeneic, xenogeneic, or HLA compatible with the subject. When a combination of cell types is administered, some or all of the cell types may be autologous, allogeneic, syngeneic, xenogeneic or HLA compatible with the subject, whereas in other embodiments one or some cell types may be autologous and the other cell type(s) allogeneic, syngeneic, xenogeneic, or HLA compatible with the subject.

[0180] Methods of isolating and culturing cells for use in the methods of the present invention are well-known in the art (see, for example, Culture of Animal Cells; A manual of Basic Technique (2nd edition), Freshney, copyright 1987, Alan R. Liss, Inc., New York). In one embodiment, fibroblast or fibroblast-like cells are used. Fibroblast cells are a useful cell type because they can be obtained from developing fetuses and adult animals in large quantities. Importantly, these cells can be easily propagated in vitro with a rapid doubling time and can be clonally propagated for use in genetic engineering procedures. Confirmation of cell type can be conducted by numerous methods well-known in the art. For example, immunocytochemical staining with antibodies directed against cell type-specific markers (e.g., cytoskeletal filaments vimentin for fibroblasts or cytokeratin for epithelial cells) can be performed.

VI. Gene Therapy

[0181] Any means for the introduction of a polynucleotide into mammals, human or non-human, or cells thereof may be adapted to the practice of this invention for the delivery of the various constructs of the invention into the intended recipient. In one embodiment of the invention, the DNA constructs are delivered to cells by transfection, i.e., by delivery of "naked" DNA or in a complex with a colloidal dispersion system. A colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a lipid-complexed or liposome-formulated DNA. In the former approach, prior to formulation of DNA, e.g., with lipid, a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e.g., inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, may then be effected using known methods and materials and delivered to the recipient mammal. See, e.g., Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No. 5,679,647 by Carson et al.

[0182] The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

[0183] The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject (see below).

[0184] Nucleic acids can be delivered in any desired vector. These include viral or non-viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.

[0185] The nucleic acids encoding a protein or nucleic acid of interest may be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well known and any can be selected for a particular application. In one embodiment of the invention, the gene delivery vehicle comprises a promoter and a demethylase coding sequence. Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters which are activatable by infection with a virus, such as the α- and β-interferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter. A promoter may be constitutive or inducible.

[0186] In another embodiment, naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Pat. No. 5,580,859. Such gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles which can optionally be used include DNA-ligand (Wu et al., J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA combinations (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc. Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectiles (Williams et al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

[0187] A gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment, the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, U.S. Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805).

[0188] Other viral vector systems that can be used to deliver a polynucleotide of the invention have been derived from herpes virus, e.g., Herpes Simplex Virus (U.S. Pat. No. 5,631,236 by Woo et al., issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway, "Mammalian expression vectors," In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey of molecular cloning vectors and their uses. Stoneham: Butterworth; Baichwal and Sugden (1986) "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene, 68:1-10), and several RNA viruses. Preferred viruses include an alphavirus, a poxivirus, an arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244:1275-1281; Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al., 1988; Horwich et al. (1990) J. Virol., 64:642-650).

[0189] In other embodiments, target DNA in the genome can be manipulated using well-known methods in the art. For example, the target DNA in the genome can be manipulated by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposable elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear DNA. Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis. Methods for generating transgenic cells typically include the steps of (1) assembling a suitable DNA construct useful for inserting a specific DNA sequence into the nuclear genome of a cell (e.g., PRDM16 and C/EBPβ); (2) transfecting the DNA construct into the cells; (3) allowing random insertion and/or homologous recombination to occur. The modification resulting from this process may be the insertion of a suitable DNA construct(s) into the target genome; deletion of DNA from the target genome; and/or mutation of the target genome. DNA constructs can comprise a gene of interest as well as a variety of elements including regulatory promoters, insulators, enhancers, and repressors as well as elements for ribosomal binding to the RNA transcribed from the DNA construct. Due to the effective recombinant DNA techniques available in conjunction with DNA sequences for regulatory elements and genes readily available in data bases and the commercial sector, a person of ordinary skill in the art can readily generate a DNA construct appropriate for establishing transgenic cells using the materials and methods known in the art.

VII. Pharmaceutical Compositions

[0190] In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of an agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

[0191] The phrase "therapeutically-effective amount" as used herein means that amount of an agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex, or composition comprising an agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex, which is effective for producing some desired therapeutic effect, e.g., weight loss, at a reasonable benefit/risk ratio.

[0192] The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0193] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0194] The term "pharmaceutically-acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex encompassed by the invention. These salts can be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0195] In other cases, the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex. These salts can likewise be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting the purified respiration uncoupling agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).

[0196] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0197] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0198] Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0199] Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a respiration uncoupling agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0200] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a respiration uncoupling agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

[0201] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0202] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

[0203] Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0204] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0205] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0206] Suspensions, in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0207] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more respiration uncoupling agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.

[0208] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0209] Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0210] The ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0211] Powders and sprays can contain, in addition to an agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0212] The agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.

[0213] Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

[0214] Transdermal patches have the added advantage of providing controlled delivery of a respiration uncoupling agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.

[0215] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

[0216] Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more respiration uncoupling agents in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0217] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0218] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0219] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0220] Injectable depot forms are made by forming microencapsule matrices of an agent that modulates (e.g., enhances) Prdm16 and/or C/EBPβ expression and/or activity, or expression and/or activity of the complex in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

[0221] When the respiration uncoupling agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0222] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

[0223] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Exemplification

[0224] This invention is further illustrated by the following examples, which should not be construed as limiting.

EXAMPLE 1

Materials and Methods for Examples

A. Cell Culture

[0225] Immortalized brown fat cells have been described in Uldry, M. et al. (2006) Cell Metab. 3, 333-341. Mouse embryonic fibroblasts were isolated from E13.5 C57/B16 embryos (Jackson Laboratory), and immortalized according to the methods described in Todaro, G. J. and Green, H. (1963) J. Cell Biol. 17, 299-313. Mouse dermal fibroblasts were obtained from Millipore. R2F primary skin fibroblasts isolated from human newborn foreskin from J. G. Rheinwald were cultured as per methods described in Rheinwald, J. G. et al. (2002) Mol. Cell. Biol. 22, 5157-5172. HEK293 cells and C2C12 cells were obtained from ATCC. Adipocyte differentiation in C2C12 cells was induced by treating confluent cells in DMEM containing 10% FBS, 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5 μM dexamethasone, 850 nM insulin, 1 nM T3 and 1 μM rosiglitazone. Two days after induction, cells were switched to the maintenance medium containing 10% FBS, 850 nM insulin, 1 nM T3 and 1 μM rosiglitazone. Adipocyte differentiation in fibroblasts was induced with medium containing 5 μM dexamethosone, 850 nM insulin, 1 nM T3 and 1 μM rosiglitazone. For cAMP treatment, cells were incubated with 10 μM forskolin or 0.5 mM dibutyryl-cAMP. All chemicals for cell culture were obtained from Sigma unless otherwise indicated.

B. DNA Constructs and Viruses Production

[0226] Deletion mutants of Flag-tagged PRDM16 were amplified by PCR using full-length PRDM16 as a template, and subcloned into pMSCV-puro retroviral vector (Stratagene). Various fragments of GST-fused PRDM16 fragments (1-223, 224-454, 455-680, 680-880, 881-1038 and 1039-1176) were described in Kajimura, S. et al. (2008) Genes Dev. 22, 1397-1409. Myc-tagged C/EBPβ constructs (Bezy, O. et al. (2007) Mol. Cell. Biol. 27, 6818-6831) were from S. R. Farmer. The sequences used for retroviral shRNA expression vectors targeting C/EBPβ were 5'-GCCCTGAGTAATCACTTAAAG-3' (shβ-1) and 5'-CCGGGCCCTGAGTAATCAC-3' (shβ-2). The corresponding double-stranded DNA sequences were ligated into pSUPER-Retro (Oligoengine) for retroviral expression. For retrovirus production, Phoenix packaging cells (Kinsella, T. M. and Nolan, G. P. (1996) Hum. Gene Ther. 7, 1405-1413) were transfected at 70% confluence by calcium phosphate method with 10 μg retroviral vectors. After 48 h, the viral supernatant was collected and filtered. Cells were incubated overnight with the viral supernatant, supplemented with 8 μg ml^-1 polybrene. Subsequently, puromycin (PRDM16), hygromycin (C/EBP-β) or G418 (shRNAs) were used for selection. Fibroblasts expressing both PRDM16 and C/EBPβ were selected by puromycin and hygromycin to ensure expression of both constructs.

C. Affinity Purification of PRDM16 Transcriptional Complex

[0227] Immortalized brown fat cells stably expressing Flag-tagged wild-type, PRΔ mutant, and ZF-1Δ, mutant of PRDM16 or an empty vector were grown to confluence. The cells were homogenized to prepare nuclear extracts (Kajimura, S. et al. (2008) Genes Dev. 22, 1397-1409). The nuclear extracts were incubated overnight with Flag M2 agarose (Sigma), washed in a binding buffer (180 mM KCl), and then eluted by incubating with 1× Flag peptide (0.2 mg ml^-1). The eluted materials were TCA precipitated, separated in a 4-20% acrylamide gradient gel, and visualized by silver staining, as described in Kajimura, S. et al. (2008) Genes Dev. 22, 1397-1409.

D. Mass Spectrometry

[0228] The immunoprecipitated proteins were precipitated with methanol and chloroform, and precipitates were dissolved in 50 mM Tris-HCl, pH 7.5, containing 8 M urea, 50 mM EDTA and 0.005% n-dodecyl β-d-maltoside (DDM). Proteins were reduced with dithiothreitol (DTT) and alkylated with iodoacetamide. After diluting urea concentration to 1 M with 50 mM Tris-HCl, pH 7.5, containing 0.005% DDM, trypsin was added and proteins were digested in solution at 37° C. for 12 h. The reaction was stopped with formic acid, and the resultant peptides were desalted with StageTips (Rappsilber, J. et al. (2007) Nat. Protocols 2, 1896-1906). Desalted peptides were subjected to reverse-phase LC-MS/MS using a high-resolution hybrid mass spectrometer (LTQ-Orbitrap, Thermo Scientific) with TOP10 method as described in Haas, W. et al. (2006) Mol. Cell. Proteomics 5, 1326-1337. The obtained data were searched against the International Protein Index (IPI) mouse database (Kersey, P. J. et al. (2004) Proteomics 4, 1985-1988). Proteins were identified with at least two unique valid peptides, and the false discovery rate was estimated to be 0% using target-decoy approach (Elias, J. E. and Gygi, S. P. (2007) Nat. Methods 4, 207-214).

E. Protein Interaction Analysis

[0229] HEK293 cells expressing PRDM16 or C/EBPs were collected 24 h after transfection. Total cell lysates were incubated overnight at 4° C. with Flag M2 agarose, washed and eluted with Flag peptide. The eluted materials were analysed by western blot using antibodies against C/EBP-α, C/EBP-β and C/EBP-δ (Santa Cruz). For in vitro binding assays, various fragments of the GST-fusion PRDM16 fragments were purified as described in Kajimura, S. et al. (2008) Genes Dev. 22, 1397-1409. [³⁵S]-labelled proteins were made with a TNT reticulocyte lysate kit (Promega). Equal amounts of GST-fusion proteins (2 μg) were incubated overnight at 4° C. with in vitro translated proteins in a binding buffer containing 20 mM HEPES, pH 7.7, 300 mM KCl, 2.5 mM MgCl₂, 0.05% NP40, 1 mM DTT and 10% glycerol. The sepharose beads were then washed five times with the binding buffer. Bound proteins were separated by SDS-PAGE and analysed by autoradiography.

F. Gene Expression Analysis

[0230] Total RNA was isolated from cells or tissues using Trizol (Invitrogen). Reverse transcriptase reactions were performed using a cDNA reverse transcription kit (Applied Biosystems). The primer sequences are listed in Table 2. Quantitative real-time PCR was performed with SYBR green fluorescent dye using an ABI9300 PCR machine. TATA-binding protein acted as an internal control.

G. Microarray Analysis

[0231] Total RNA was isolated from undifferentiated C2C12 cells transduced with scr or shβ together with PRDM16 or vector control. Array hybridization and scanning were performed by the Dana-Farber Cancer Institute Core Facility using Affymetrix GeneChip Mouse Genome 430 2.0 arrays according to established methods (Lockhart, D. J. et al. (1996) Nat. Biotechnol. 14, 1675-1680). The array data were analysed using the DNA-Chip Analyser software (Li, C. and Wong, W. H. (2001) Proc. Natl Acad. Sci. USA 98, 31-36). The statistical significance of differences in gene expression was assessed using an unpaired t-test (P<0.05).

H. Reporter Gene Assay

[0232] The PGC1A (-2 kb) promoter linked to a luciferase reporter was transiently co-transfected with PRDM16 and/or C/EBPβ expression plasmids in brown preadipocytes using Lipofectamine 2000 (Invitrogen). Forty-eight hours after the transfection, cells were collected and reporter gene assays were carried out using the Dual Luciferase Kit (Promega). Transfection efficiency was normalized by measuring expression of Renilla luciferase.

I. Cellular Respiration Assay

[0233] Immortalized brown fat cells or MEFs transduced with retroviral PRDM16 and C/EBPβ or an empty vector were grown to confluence and induced to differentiate. At day 6 or 7 of differentiation, oxygen consumption was measured as described in Kajimura, S. et al. (2008) Genes Dev. 22, 1397-1409 and Seale, P. et al. (2007) Cell Metab. 6, 38-54. For cAMP-induced respiration assays, fully differentiated fat cells were incubated with 0.5 mM dibutyryl-cAMP for 12 h before measuring oxygen consumption.

J. Animals

[0234] All animal experiments were performed according to procedures approved by Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee. C/EBP-β-null mice (CebpbtmlVpo/J) were obtained from the Jackson Laboratory. For transplantation studies, male NCR-nude mice (NCr-Foxn1^nu) were obtained from Taconic.

K. Cell Transplantations

[0235] Immortalized MEFs (3×10⁷) were transduced with retroviral PRDM16, C/EBP-β, vector control, or a combination of PRDM16 and C/EBP-β, and implanted subcutaneously into 7-9-week-old male nude mice (n=6 mice per group), according to methods described in Seale, P. et al. (2007) Cell Metab. 6, 38-54 and Green, H. and Kehinde, O. (1979) J. Cell. Physiol. 101, 169-171. For PET scanning studies, MEFs expressing retroviral PPARγ alone were implanted as a control. After 4-6 weeks, fat pads were carefully dissected and fixed in 4% paraformaldehyde for histological analysis. For immunohistochemistry, paraffin-embedded sections were incubated with anti-UCP1 antibody (Chemicon), followed by detection using the ABC Vectastain-Elite kit (Vector Labs) according to the manufacturer's instructions.

L. PET/CT Imaging

[0236] ¹⁸FDG (100 μCi) was injected intravenously to animals acclimated for at least 48 h to room temperature. Animals were imaged or euthanized at 1 h after injection in the Longwood small animal imaging facility of Harvard Medical School. PET/CT imaging was performed using a Minerve anaesthesia bed moved between a Philips Mosaic HP small animal scanner and a Bioscan CT scanner, and co-registered using custom fiducial markers. The acquired data was reconstructed by InVivoScope software (Bioscan).

EXAMPLE 2

PRDM16 Forms a Transcriptional Complex with Active Forms of C/EBP-β by Direct Interaction and Regulates Their Transcriptional Activity

[0237] Because of the importance of brown adipose tissue (BAT) as a natural defense against hypothermia and obesity (Cannon, B. and Nedergaard, J. (2004) Physiol. Rev. 84, 277-359), and its demonstrated presence in adult humans (Nedergaard, J. et al. (2007) Am. J. Physiol. Endocrinol. Metab. 293, E444-E452; Cypess, A. M. et al. (2009) N. Engl. J. Med. 360, 1509-1517; van Marken Lichtenbelt, W. D. et al. (2009) N. Engl. J. Med. 360, 1500-1508; Virtanen, K. A. et al. (2009) N. Engl. J. Med. 360, 1518-1525), understanding its formation in mechanistic detail is critical for developing new therapeutics for metabolic diseases such as obesity and type-2 diabetes. PRDM16, a 140-kDa zinc finger protein, functions as a bidirectional switch in brown fat cell fate by stimulating the development of brown fat cells from white preadipocytes (Kajimura, S. et al. (2008) Genes Dev. 22, 1397-1409; Seale, P. et al. (2007) Cell Metab. 6, 38-54) and from Myf5-positive myoblastic precursors (Seale, P. et al. (2008) Nature 454, 961-967) in vitro and in vivo. At a molecular level, PRDM16 works as a transcriptional co-regulatory protein by co-activating PPARγ (peroxisome proliferator-activated receptor γ), which is considered the `master` gene of fat cell differentiation (Tontonoz, P. et al. (1994) Cell 79, 1147-1156; Tontonoz, P. and Spiegelman, B. M. (2008) Annu. Rev. Biochem. 77, 289-312). However, both isoforms of PPARγ are expressed at very low levels in primary and immortalized myoblasts, whereas they are abundantly expressed in white and brown preadipocytes (FIGS. 5A and 5B). Hence, PRDM16 initiates the process of myoblast to brown fat conversion by complexing with other DNA-binding factors, well before the co-activation of PPARγ.

[0238] FIG. 1A illustrates the strategy described herein to identify such DNA-binding factors. Briefly, proteomic analyses of transcriptional complexes formed with wild-type PRDM16 or different mutant alleles that were differentiation-competent or -incompetent were performed. Transcription factors that co-purified preferentially with differentiation-competent PRDM16 proteins were identified; their expression in white and brown fat was then analysed and compared to that of PRDM16. Subsequently, their function was examined in the process of myoblast to brown fat conversion through PRDM16.

[0239] As shown in FIG. 1B, wild type PRDM16 and a mutant protein lacking the PR (PRD1-BF1-RIZ1 homologous) domain (ΔPR; amino acids 91-223) that shares homology to the SET chromatin remodelling domain (Mochizuki, N. et al. (2000) Blood 96, 3209-3214; Shing, D. C. et al. (2007) J. Clin. Invest. 117, 3696-3707), induced brown fat cell differentiation from myoblasts. In contrast, a mutant allele lacking zinc finger domain-1 (ΔZF-1; amino acids 224-447) completely lost its adipogenic function. The brown fat gene program was also induced by both wild-type and PR, but not by ΔZF-1 (FIGS. 6A and 6B). To avoid comparing proteomic analyses of complexes from cells of very different phenotypes, all three PRDM16 forms were expressed in bona fide brown fat cells. PRDM16 complexes were then immunopurified to apparent homogeneity (FIG. 1C), and subjected to high-resolution `shotgun` sequencing by liquid chromatography with tandem mass spectrometry (LC-MS/MS; Haas, W. et al. (2006) Mol. Cell. Proteomics 5, 1326-1337). In total, 49 proteins were identified in differentiation-competent PRDM16 complexes, but only eight of these (Bclaf1, Zfp655, p53 (also known as Trp53), Cebpb, Zcchc8, Zkscan3, Zfp143 and Vezf1) are known or predicted transcription factors (Table 1).

[0240] Because the expression of a key initiating transcription factor was presumed not to be extinguished during the brown fat cell adipogenesis, and as PRDM16 is highly enriched in BAT relative to white adipose tissue (WAT; Seale, P. et al. (2007) Cell Metab. 6, 38-54), it was determined whether any of these factors were similarly enriched in BAT. As shown in FIG. 1D, the expression of only Cebpb (C/EBP-β) was co-enriched with PRDM16 in BAT versus WAT. In addition, C/EBP-β protein was enriched in BAT, and further induced by cold exposure (FIG. 7). Notably, both primary and immortalized myoblasts express C/EBPβ at similar levels to those seen in preadipocytes (FIG. 8), where this factor is thought to have a very important role in adipogenesis (Wu, Z. et al. (1995) Genes Dev. 9, 2350-2363; Farmer, S. R. et al. (2006) Cell Metab. 4, 263-273).

[0241] Brown fat cells express three forms of C/EBP-β, two active forms, named LAP (liver-enriched transcriptional activator protein) and a dominant-negative form, LIP (liver-enriched transcriptional inhibitory protein) (FIG. 1E; Descombes, P. and Schibler, U. (1991) Cell 67, 569-579). Notably, PRDM16 preferentially bound to LAP, but not to LIP (FIGS. 1E, 9A, and 9B). Independent co-expression assays in HEK293 cells confirmed the physical binding of PRDM16 and C/EBP-β. Furthermore, PRDM16 interacts with other C/EBP family members, C/EBP-α and -δ (FIGS. 10A, 10B, and 10C). This interaction is believed to be direct through the two zinc finger domains, because the zinc finger domains of the purified glutathione S-transferase (GST)-fused PRDM16 bound to in vitro translated C/EBPβ (FIG. 11).

[0242] In addition, PRDM16's effects on C/EBPβ transcriptional activity were assessed. A luciferase reporter assay was performed using the -2 kilobase (kb) Pgc1a (also known as Ppargc1a) promoter where the C/EBP-binding sites have been characterized, since C/EBP-β is known to induce Pgc1a (Wang, H. et al. (2008) Mol. Endocrinol. 22, 1596-1605). FIG. 1F shows that PRDM16 and C/EBPβ synergistically stimulated Pgc1a promoter activity. Taken together, these data indicate that PRDM16 forms a transcriptional complex with active forms of C/EBP-β by direct interaction, and regulates their transcriptional activity.

EXAMPLE 3

The PRDM16-C/EBP-β Transcriptional Complex Specifically Initiates Myoblast to Brown Fat Switch

[0243] To examine the functional role of the interaction between PRDM16 and C/EBPβ in the myoblast to brown fat conversion, retroviruses expressing a short hairpin (sh) scrambled control RNA (scr), or shRNAs targeting C/EBPβ (shβ-1 and shβ-2) were transduced together with PRDM16 or an empty vector into C2C12 myoblasts (FIG. 2A). Knockdown of C/EBPβ significantly blunted the induction of Pparg2 expression by PRDM16 in undifferentiated C2C12 myoblasts (FIG. 2B). Consistent with this result, Oil Red O staining showed that depletion of C/EBPβ blunted the adipogenesis induced by PRDM16 (FIG. 2C). Furthermore, induction of brown-fat-selective genes including Pgc1a, Ucp1, Elov13 and Cox7a1 were completely or partially blocked by knockdown of C/EBP-β, correlating with the knockdown efficacy (FIG. 2D). In addition, ectopic expression of LIP, a dominant-negative form of C/EBP-β, also significantly blunted PRDM16-induced adipogenesis and brown-fat-selective gene expression (FIGS. 12A, 12B, and 12C).

[0244] Next, a systematic approach was taken to determine what fraction of the PRDM16-regulated genes requires C/EBPβ at the initiating step of the myoblast to brown fat conversion. RNAs from undifferentiated C2C12 myoblasts expressing PRDM16 or control together with scr or shβ-1, maintained under conditions non-permissive for differentiation, were subjected to Affymetrix microarray analysis. As shown in FIG. 2E, 316 genes were significantly increased or reduced by PRDM16 (>two-fold, P<0.05), which were clustered into four groups: (1) genes increased by PRDM16 in a C/EBP-β-dependent manner, (2) genes increased by PRDM16 in a C/EBP-β-independent manner, (3) genes repressed by PRDM16 in a C/EBP-β-dependent manner, and (4) genes repressed by PRDM16 in a C/EBP-β-independent manner. The expression of a subset of genes identified by microarray analyses was validated by PCR with reverse transcription (RT-PCR; FIG. 13). Notably, most genes activated by PRDM16 before differentiation (62 out of 95, 65.3%) indeed required C/EBP-β, whereas most of the repressed genes (210 out of 221, 95.0%) were not grossly altered by C/EBPβ depletion.

[0245] The genetic requirement for C/EBPβ in brown fat development was further investigated by analysing C/EBP-β-deficient embryos. Defects in BAT of C/EBP-β-null newborn or adult mice have been described, although the reported phenotype was inconsistent (Tanaka, T. et al. (1997) EMBO J. 16, 7432-7443; Carmona, M. C. et al. (2005) Biochem. J. 389, 47-56). Because a large number of these embryos died within the first 24 h after birth (Tanaka, T. et al. (1997) EMBO J. 16, 7432-7443; Screpanti, I. et al. (1995) EMBO J. 14, 1932-1941), analyses were performed at late gestation (stage embryonic day (E18.5) so as to permit a clear separation of developmental changes in the BAT, as opposed to those that might occur secondarily to abnormalities in other tissues after birth. Haematoxylin and eosin staining showed that brown fat cells in knockout embryos contained significantly less lipid droplets than those in wild-type embryos, indicating defects in brown fat development per se (FIG. 2F). Moreover, UCP1 expression was severely reduced in knockout embryos (FIG. 2F), consistent with the results of Tanaka, T. et al. (1997) EMBO J. 16, 7432-7443. A definitive molecular characterization of the BAT from wild-type and knockout embryos was also conducted. Notably, BAT from C/EBP-β-knockout mice nearly phenocopied that from PRDM16-knockout mice at the gene expression level; that is, a broad reduction of BAT-selective gene expression, and a broad induction of the skeletal muscle gene expression (FIG. 2G). Together, these data indicate that the PRDM16-C/EBPβ transcriptional complex specifically has a critical role in the initiation of myoblast to brown fat switch. This further indicates that PRDM16 acts in Myf5-positive myoblastic precursors, at least in part, by co-activation of C/EBPβ to induce the expression of Pparg and Pgc1a. Subsequently, PRDM16 co-activates PPARγ and PGC-1α by direct binding events, which drives a complete brown fat differentiation program (FIG. 14).

EXAMPLE 4

The PRDM16-C/EBP-β Transcriptional Complex is Sufficient to Reconstitute a Near Complete Brown Fat Program

[0246] The mechanistic model presented in FIG. 14 raises the critical question of whether the two factors are sufficient to reconstitute a brown fat program in naive cells. To this end, PRDM16 and C/EBPβ were ectopically expressed in mouse embryonic fibroblasts (MEFs) or primary skin fibroblasts with no inherent adipose or brown fat character. As shown in FIG. 3A, Pparg2 messenger RNA expression was synergistically induced by PRDM16 and C/EBPβ in a dose-dependent manner in undifferentiated fibroblasts. After 6-8 days under adipogenic conditions, both MEFs and skin fibroblasts expressing these two factors uniformly differentiated into lipid-filled adipocytes, as shown by Oil Red O staining (FIG. 3B). The single factors alone were not sufficient to robustly stimulate the differentiated state. Gene expression studies showed that PRDM16 and C/EBPβ powerfully induced mRNA levels of brown fat genes including Cox7a1 (70-fold), Cox8b (260-fold), Elovl3 (16-fold) and Cidea (170-fold) to levels comparable with or even higher than those seen in bona fide immortalized brown fat cells (FIG. 3C). Notably, as in authentic brown fat cells, mRNA level of thermogenic genes such as Pgc1a and Ucp1 were further enhanced by cyclic AMP treatment (FIG. 3D). The mechanism underlying the augmentation of cAMP effects in the engineered brown fat cells remains unknown. The mRNA levels of those genes at the basal state were activated to levels seen in cAMP-stimulated brown fat cells. Furthermore, the two factors were able to induce the brown fat gene program from primary mouse skin fibroblasts (FIG. 3D) and human skin fibroblasts isolated from newborn foreskin (FIGS. 15A and 15B).

[0247] An important characteristic of brown fat cells is their extraordinarily high rates of respiration, particularly uncoupled respiration in response to cAMP. As shown in FIG. 3F, engineered brown fat cells induced by these two factors have significantly higher levels of total and uncoupled respiration than control cells, by 4.4- and 6.5-fold, respectively, at the basal state. Notably, the engineered cells have greater basal respiration, both total and uncoupled, than bona fide brown fat cells. However, whereas the bona fide brown fat cells can increase both total and uncoupled respiration further (by 85% and 90%, respectively) in response to cAMP, engineered brown fat cells were already at their maximal respiration. That these cells are responsive to cAMP is shown by the fact that expression of thermogenic genes, such as such as Pgc1a and Ucp1, are induced by cAMP treatment (FIG. 3D).

EXAMPLE 5

The PRDM16-C/EBP-β Transcriptional Complex Can Generate Functional Brown Adipose Tissue in vivo

[0248] The finding that the combination of PRDM16 and C/EBPβ is sufficient to reconstitute a near complete brown fat program offers an opportunity for controlling brown fat levels and function in vivo. Accordingly, transplantation studies were conducted (Green, H. and Kehinde, O. (1979) J. Cell. Physiol. 101, 169-171) using undifferentiated MEFs expressing vector, PRDM16, C/EBP-β, or a combination of the two factors. As shown by haematoxylin and eosin staining (FIG. 4A), the cells expressing vector or PRDM16 or C/EBPβ alone did not form visible fat tissues. In contrast, the cells expressing both PRDM16 and C/EBPβ formed very distinct fat pads in vivo. At high magnification, the engineered fat tissue induced by the two factors contained `multilocular` fat cells, a morphological characteristic of brown fat in vivo (FIG. 4B). The population of multilocular fat cells (area 1) is mixed with regions of `unilocular` fat cells (area 2). Notably, immunohistochemical analyses showed that the engineered adipose tissue was UCP1-positive in both the multilocular and unilocular fat cells (FIG. 4C).

[0249] To characterize the activity of engineered brown fat tissue in vivo further, positron emission tomography (PET) with fluorodeoxyglucose (¹⁸FDG) recently used to detect active BAT in adult humans (Nedergaard, J. et al. (2007) Am. J. Physiol. Endocrinol. Metab. 293, E444-E452; Cypess, A. M. et al. (2009) N. Engl. J. Med. 360, 1509-1517; van Marken Lichtenbelt, W. D. et al. (2009) N. Engl. J. Med. 360, 1500-1508; Virtanen, K. A. et al. (2009) N. Engl. J. Med. 360, 1518-1525), was used. This technique measures glucose uptake, with brown fat functioning in vivo as an active `sink` for glucose. To this end, two adipose tissues with similar sizes were engineered in the same nude mice: a `brown` fat tissue induced by PRDM16 and C/EBP-β, and a `white` fat tissue induced by PPARγ alone as a control (FIG. 16A). The induction of BAT-selective genes by PRDM16 and C/EBP-β was confirmed in the cultured cells by RT-PCR (FIG. 16B). As shown in FIG. 4D, PET scanning detected a signal in mice from the engineered BAT. To enhance the sensitivity and specificity of the PET signal from the engineered fat tissues, the skin with these fat tissues attached was removed and scanned. The combination of computed tomography image and PET image (FIG. 4E) clearly showed that the PET signal was detected from the engineered BAT, but not from the engineered WAT.

[0250] These results indicate that the engineered brown fat cells function as a sink for active glucose disposal. Given the incredible capacity of BAT to dissipate stored chemical energy and thus counteract obesity, the PRDM16 pathway can be used to drive brown fat development in vivo in a therapeutic setting. Natural or synthetic compounds that can induce PRDM16 in white fat precursors or in myoblastic cells now have great value in human metabolic disease. Alternatively, as demonstrated herein, engineered brown fat induced by PRDM16 and C/EBPβ in amounts that are both clinically acceptable and therapeutically useful can be autologously transplanted.

INCORPORATION BY REFERENCE

[0251] The contents of all references, patent applications, patents, and published patent applications, as well as the Figures and the Sequence Listing, cited throughout this application are hereby incorporated by reference.

Equivalents

[0252] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence CWU 1

1

13613828DNAHomo sapiens 1atgcgatcca aggcgagggc gaggaagcta gccaaaagtg acggtgacgt tgtaaataat 60atgtatgagc ccaaccggga cctgctggcc agccacagcg cggaggacga ggccgaggac 120agtgccatgt cgcccatccc cgtggggcca ccgtccccct tccccaccag cgaggacttc 180acccccaagg agggctcgcc gtacgaggcc cctgtctaca ttcctgaaga cattccgatc 240ccagcagact tcgagctccg agagtcctcc atcccagggg ctggcctggg ggtctgggcc 300aagaggaaga tggaagccgg ggagaggctg ggcccctgcg tggtggtgcc ccgggcggcg 360gcaaaggaga cagacttcgg atgggagcaa atactgacgg acgtggaagt gtcgccccag 420gaaggctgca tcacaaagat ctccgaagac ctgggcagtg agaagttctg cgtggatgca 480aatcaggcgg gggctggcag ctggctcaag tacatccgtg tggcgtgctc ctgcgatgac 540cagaacctca ccatgtgtca gatcagtgag cagatttact ataaagtcat taaggacatt 600gagccaggtg aggagctgct ggtgcacgtg aaggaaggcg tctaccccct gggcacagtg 660ccgcccggcc tggacgagga gcccacgttc cgctgtgacg agtgtgacga actcttccag 720tccaagctgg acctgcggcg ccataagaag tacacgtgtg gctcagtggg ggctgcgctc 780tacgagggcc tggctgagga gctcaagccc gagggccttg gcggtggcag cggccaagcc 840cacgagtgca aggactgcga gcggatgttc cccaacaagt acagcctgga gcagcacatg 900gtcatccaca cggaggagcg cgagtacaaa tgcgaccagt gtcccaaggc cttcaactgg 960aagtccaacc tcatccgcca ccagatgtcc cacgacagcg gcaaacgctt cgaatgtgaa 1020aactgcgtga aggtgttcac ggaccccagc aaccttcagc ggcacatccg ctcgcagcac 1080gtgggcgctc gggcccacgc ctgccccgac tgcgggaaga ccttcgccac gtcctccggc 1140ctcaagcagc acaagcatat ccacagcacg gtgaagcctt tcatatgtga ggtctgccac 1200aagtcctaca cgcagttctc caacctgtgc cggcacaagc ggatgcacgc cgactgccgc 1260acgcagatca agtgcaagga ctgtggccag atgttcagca ctacctcctc cctcaacaag 1320caccggcgct tctgcgaggg caagaaccat tacacgccgg gcggcatctt tgccccgggc 1380ctgcccttga cccccagccc catgatggac aaggcaaaac cctcccccag cctcaatcac 1440gccagcctgg gcttcaacga gtactttccc tccaggccgc acccggggag cctgcccttc 1500tccacggcgc ctcccacgtt ccccgcactc acccccggct tcccgggcat cttccctcca 1560tccttgtacc cccggccgcc tctgctacct cccacatcgc tgctcaagag ccccctgaac 1620cacacccagg acgccaagct ccccagtccc ctggggaacc cagccctgcc cctggtctcc 1680gccgtcagca acagcagcca gggcacgacg gcagctgcgg ggcccgagga gaagttcgag 1740agccgcctgg aggactcctg tgtggagaag ctgaagacca ggagcagcga catgtcggac 1800ggcagtgact ttgaggacgt caacaccacc acggggaccg acctggacac gaccacgggg 1860acgggctcgg acctggacag cgacgtggac agcgaccctg acaaggacaa gggcaagggc 1920aagtccgccg agggccagcc caagtttggg ggcggcttgg cgcccccggg ggccccgaac 1980agcgtggccg aggtgcctgt cttctattcc cagcactcat tcttcccgcc acccgacgag 2040cagctgctga ctgcaacggg cgccgccggg gactccatca aggccatcgc atccattgcc 2100gagaagtact ttggccccgg cttcatgggg atgcaggaga agaagctggg ctcgctcccc 2160taccactcgg cgttcccctt ccagttcctg cccaacttcc cccactccct ttaccccttc 2220acggaccgag ccctcgccca caacttgctg gtcaaggccg agccaaagtc accccgggac 2280gccctcaagg tgggcggccc cagtgccgag tgcccctttg atctcaccac caagcccaaa 2340gacgtgaagc ccatcctgcc catgcccaag ggcccctcgg cccccgcatc cggcgaggag 2400cagccgctgg acctgagcat cggcagccgg gcccgtgcca gccaaaacgg cggcgggcgg 2460gagccccgca agaaccacgt ctatggggaa cgcaagctgg gcgccggcga ggggctgccc 2520caggtgtgcc cggcgcggat gccccagcag cccccgctcc actacgccaa gccctcgccc 2580ttcttcatgg accccatcta cagggtagaa aagcggaagg tcacagaccc cgtgggagcc 2640ctgaaggaga agtacctgcg gccgtccccg ctgctcttcc acccccagat gtcagccata 2700gagaccatga cagagaagct ggagagcttt gcagccatga aggcggactc gggcagctcc 2760ctgcagcccc tcccccacca ccccttcaac ttccggtccc cacccccaac gctctccgac 2820cccatcctca ggaagggcaa ggagcgatac acgtgcaggt actgtgggaa gatcttcccc 2880agatcagcca atctcaccag acacctgagg acgcacactg gggagcagcc gtacaggtgt 2940aagtactgcg accgctcctt cagcatctct tcgaacctcc agcggcacgt ccggaacatc 3000cacaacaagg agaagccttt caagtgccac ctgtgcaacc gctgcttcgg gcagcagacc 3060aacctggacc ggcacctcaa gaagcacgag cacgagaacg caccagtgag ccagcacccc 3120ggggtcctca cgaaccacct ggggaccagc gcgtcctctc ccacctcaga gtcggacaac 3180cacgcacttt tagacgagaa agaagactct tatttctcgg aaatcagaaa ctttattgcc 3240aatagtgaga tgaaccaagc atcaacgcga acagagaaac gggcggacat gcagatcgtg 3300gacggcagtg cccagtgtcc aggcctagcc agtgagaagc aggaggacgt ggaggaggag 3360gacgacgatg acctggagga ggacgatgag gacagcctgg ccgggaagtc gcaggatgac 3420accgtgtccc ccgcacccga gccccaggcc gcctacgagg atgaggagga tgaggagcca 3480gccgcctccc tggccgtggg ctttgaccac acccgaaggt gtgctgagga ccacgaaggc 3540ggtctgttag ctttggagcc gatgccgact tttgggaagg ggctggacct ccgcagagca 3600gctgaggaag catttgaagt taaagatgtg cttaattcca ccttagattc tgaggcttta 3660aaacatacac tgtgcaggca ggctaagaac caggcatatg caatgatgct gtccctttcc 3720gaagacactc ctctccacac cccctcccag ggttctctgg acgcttggtt gaaggtcact 3780ggagccacgt cggagtctgg agcatttcac cccatcaacc acctctga 382821275PRTHomo sapiens 2Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 Val Val Asn Asn Met Tyr Glu Pro Asn Arg Asp Leu Leu Ala Ser His 20 25 30 Ser Ala Glu Asp Glu Ala Glu Asp Ser Ala Met Ser Pro Ile Pro Val 35 40 45 Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 Pro Ala Asp Phe Glu Leu Arg Glu Ser Ser Ile Pro Gly Ala Gly Leu 85 90 95 Gly Val Trp Ala Lys Arg Lys Met Glu Ala Gly Glu Arg Leu Gly Pro 100 105 110 Cys Val Val Val Pro Arg Ala Ala Ala Lys Glu Thr Asp Phe Gly Trp 115 120 125 Glu Gln Ile Leu Thr Asp Val Glu Val Ser Pro Gln Glu Gly Cys Ile 130 135 140 Thr Lys Ile Ser Glu Asp Leu Gly Ser Glu Lys Phe Cys Val Asp Ala 145 150 155 160 Asn Gln Ala Gly Ala Gly Ser Trp Leu Lys Tyr Ile Arg Val Ala Cys 165 170 175 Ser Cys Asp Asp Gln Asn Leu Thr Met Cys Gln Ile Ser Glu Gln Ile 180 185 190 Tyr Tyr Lys Val Ile Lys Asp Ile Glu Pro Gly Glu Glu Leu Leu Val 195 200 205 His Val Lys Glu Gly Val Tyr Pro Leu Gly Thr Val Pro Pro Gly Leu 210 215 220 Asp Glu Glu Pro Thr Phe Arg Cys Asp Glu Cys Asp Glu Leu Phe Gln 225 230 235 240 Ser Lys Leu Asp Leu Arg Arg His Lys Lys Tyr Thr Cys Gly Ser Val 245 250 255 Gly Ala Ala Leu Tyr Glu Gly Leu Ala Glu Glu Leu Lys Pro Glu Gly 260 265 270 Leu Gly Gly Gly Ser Gly Gln Ala His Glu Cys Lys Asp Cys Glu Arg 275 280 285 Met Phe Pro Asn Lys Tyr Ser Leu Glu Gln His Met Val Ile His Thr 290 295 300 Glu Glu Arg Glu Tyr Lys Cys Asp Gln Cys Pro Lys Ala Phe Asn Trp 305 310 315 320 Lys Ser Asn Leu Ile Arg His Gln Met Ser His Asp Ser Gly Lys Arg 325 330 335 Phe Glu Cys Glu Asn Cys Val Lys Val Phe Thr Asp Pro Ser Asn Leu 340 345 350 Gln Arg His Ile Arg Ser Gln His Val Gly Ala Arg Ala His Ala Cys 355 360 365 Pro Asp Cys Gly Lys Thr Phe Ala Thr Ser Ser Gly Leu Lys Gln His 370 375 380 Lys His Ile His Ser Thr Val Lys Pro Phe Ile Cys Glu Val Cys His 385 390 395 400 Lys Ser Tyr Thr Gln Phe Ser Asn Leu Cys Arg His Lys Arg Met His 405 410 415 Ala Asp Cys Arg Thr Gln Ile Lys Cys Lys Asp Cys Gly Gln Met Phe 420 425 430 Ser Thr Thr Ser Ser Leu Asn Lys His Arg Arg Phe Cys Glu Gly Lys 435 440 445 Asn His Tyr Thr Pro Gly Gly Ile Phe Ala Pro Gly Leu Pro Leu Thr 450 455 460 Pro Ser Pro Met Met Asp Lys Ala Lys Pro Ser Pro Ser Leu Asn His 465 470 475 480 Ala Ser Leu Gly Phe Asn Glu Tyr Phe Pro Ser Arg Pro His Pro Gly 485 490 495 Ser Leu Pro Phe Ser Thr Ala Pro Pro Thr Phe Pro Ala Leu Thr Pro 500 505 510 Gly Phe Pro Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro Leu 515 520 525 Leu Pro Pro Thr Ser Leu Leu Lys Ser Pro Leu Asn His Thr Gln Asp 530 535 540 Ala Lys Leu Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val Ser 545 550 555 560 Ala Val Ser Asn Ser Ser Gln Gly Thr Thr Ala Ala Ala Gly Pro Glu 565 570 575 Glu Lys Phe Glu Ser Arg Leu Glu Asp Ser Cys Val Glu Lys Leu Lys 580 585 590 Thr Arg Ser Ser Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Val Asn 595 600 605 Thr Thr Thr Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser Asp 610 615 620 Leu Asp Ser Asp Val Asp Ser Asp Pro Asp Lys Asp Lys Gly Lys Gly 625 630 635 640 Lys Ser Ala Glu Gly Gln Pro Lys Phe Gly Gly Gly Leu Ala Pro Pro 645 650 655 Gly Ala Pro Asn Ser Val Ala Glu Val Pro Val Phe Tyr Ser Gln His 660 665 670 Ser Phe Phe Pro Pro Pro Asp Glu Gln Leu Leu Thr Ala Thr Gly Ala 675 680 685 Ala Gly Asp Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr Phe 690 695 700 Gly Pro Gly Phe Met Gly Met Gln Glu Lys Lys Leu Gly Ser Leu Pro 705 710 715 720 Tyr His Ser Ala Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His Ser 725 730 735 Leu Tyr Pro Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val Lys 740 745 750 Ala Glu Pro Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro Ser 755 760 765 Ala Glu Cys Pro Phe Asp Leu Thr Thr Lys Pro Lys Asp Val Lys Pro 770 775 780 Ile Leu Pro Met Pro Lys Gly Pro Ser Ala Pro Ala Ser Gly Glu Glu 785 790 795 800 Gln Pro Leu Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln Asn 805 810 815 Gly Gly Gly Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg Lys 820 825 830 Leu Gly Ala Gly Glu Gly Leu Pro Gln Val Cys Pro Ala Arg Met Pro 835 840 845 Gln Gln Pro Pro Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met Asp 850 855 860 Pro Ile Tyr Arg Val Glu Lys Arg Lys Val Thr Asp Pro Val Gly Ala 865 870 875 880 Leu Lys Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His Pro Gln 885 890 895 Met Ser Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe Ala Ala 900 905 910 Met Lys Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His His Pro 915 920 925 Phe Asn Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile Leu Arg 930 935 940 Lys Gly Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile Phe Pro 945 950 955 960 Arg Ser Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Gln 965 970 975 Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser Ser Asn 980 985 990 Leu Gln Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro Phe Lys 995 1000 1005 Cys His Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu Asp 1010 1015 1020 Arg His Leu Lys Lys His Glu His Glu Asn Ala Pro Val Ser Gln 1025 1030 1035 His Pro Gly Val Leu Thr Asn His Leu Gly Thr Ser Ala Ser Ser 1040 1045 1050 Pro Thr Ser Glu Ser Asp Asn His Ala Leu Leu Asp Glu Lys Glu 1055 1060 1065 Asp Ser Tyr Phe Ser Glu Ile Arg Asn Phe Ile Ala Asn Ser Glu 1070 1075 1080 Met Asn Gln Ala Ser Thr Arg Thr Glu Lys Arg Ala Asp Met Gln 1085 1090 1095 Ile Val Asp Gly Ser Ala Gln Cys Pro Gly Leu Ala Ser Glu Lys 1100 1105 1110 Gln Glu Asp Val Glu Glu Glu Asp Asp Asp Asp Leu Glu Glu Asp 1115 1120 1125 Asp Glu Asp Ser Leu Ala Gly Lys Ser Gln Asp Asp Thr Val Ser 1130 1135 1140 Pro Ala Pro Glu Pro Gln Ala Ala Tyr Glu Asp Glu Glu Asp Glu 1145 1150 1155 Glu Pro Ala Ala Ser Leu Ala Val Gly Phe Asp His Thr Arg Arg 1160 1165 1170 Cys Ala Glu Asp His Glu Gly Gly Leu Leu Ala Leu Glu Pro Met 1175 1180 1185 Pro Thr Phe Gly Lys Gly Leu Asp Leu Arg Arg Ala Ala Glu Glu 1190 1195 1200 Ala Phe Glu Val Lys Asp Val Leu Asn Ser Thr Leu Asp Ser Glu 1205 1210 1215 Ala Leu Lys His Thr Leu Cys Arg Gln Ala Lys Asn Gln Ala Tyr 1220 1225 1230 Ala Met Met Leu Ser Leu Ser Glu Asp Thr Pro Leu His Thr Pro 1235 1240 1245 Ser Gln Gly Ser Leu Asp Ala Trp Leu Lys Val Thr Gly Ala Thr 1250 1255 1260 Ser Glu Ser Gly Ala Phe His Pro Ile Asn His Leu 1265 1270 1275 33771DNAHomo sapiens 3atgcgatcca aggcgagggc gaggaagcta gccaaaagtg acggtgacgt tgtaaataat 60atgtatgagc ccaaccggga cctgctggcc agccacagcg cggaggacga ggccgaggac 120agtgccatgt cgcccatccc cgtggggcca ccgtccccct tccccaccag cgaggacttc 180acccccaagg agggctcgcc gtacgaggcc cctgtctaca ttcctgaaga cattccgatc 240ccagcagact tcgagctccg agagtcctcc atcccagggg ctggcctggg ggtctgggcc 300aagaggaaga tggaagccgg ggagaggctg ggcccctgcg tggtggtgcc ccgggcggcg 360gcaaaggaga cagacttcgg atgggagcaa atactgacgg acgtggaagt gtcgccccag 420gaaggctgca tcacaaagat ctccgaagac ctgggcagtg agaagttctg cgtggatgca 480aatcaggcgg gggctggcag ctggctcaag tacatccgtg tggcgtgctc ctgcgatgac 540cagaacctca ccatgtgtca gatcagtgag cagatttact ataaagtcat taaggacatt 600gagccaggtg aggagctgct ggtgcacgtg aaggaaggcg tctaccccct gggcacagtg 660ccgcccggcc tggacgagga gcccacgttc cgctgtgacg agtgtgacga actcttccag 720tccaagctgg acctgcggcg ccataagaag tacacgtgtg gctcagtggg ggctgcgctc 780tacgagggcc tggctgagga gctcaagccc gagggccttg gcggtggcag cggccaagcc 840cacgagtgca aggactgcga gcggatgttc cccaacaagt acagcctgga gcagcacatg 900gtcatccaca cggaggagcg cgagtacaaa tgcgaccagt gtcccaaggc cttcaactgg 960aagtccaacc tcatccgcca ccagatgtcc cacgacagcg gcaaacgctt cgaatgtgaa 1020aactgcgtga aggtgttcac ggaccccagc aaccttcagc ggcacatccg ctcgcagcac 1080gtgggcgctc gggcccacgc ctgccccgac tgcgggaaga ccttcgccac gtcctccggc 1140ctcaagcagc acaagcatat ccacagcacg gtgaagcctt tcatatgtga ggtctgccac 1200aagtcctaca cgcagttctc caacctgtgc cggcacaagc ggatgcacgc cgactgccgc 1260acgcagatca agtgcaagga ctgtggccag atgttcagca ctacctcctc cctcaacaag 1320caccggcgct tctgcgaggg caagaaccat tacacgccgg gcggcatctt tgccccgggc 1380ctgcccttga cccccagccc catgatggac aaggcaaaac cctcccccag cctcaatcac 1440gccagcctgg gcttcaacga gtactttccc tccaggccgc acccggggag cctgcccttc 1500tccacggcgc ctcccacgtt ccccgcactc acccccggct tcccgggcat cttccctcca 1560tccttgtacc cccggccgcc tctgctacct cccacatcgc tgctcaagag ccccctgaac 1620cacacccagg acgccaagct ccccagtccc ctggggaacc cagccctgcc cctggtctcc 1680gccgtcagca acagcagcca gggcacgacg gcagctgcgg ggcccgagga gaagttcgag 1740agccgcctgg aggactcctg tgtggagaag ctgaagacca ggagcagcga catgtcggac 1800ggcagtgact ttgaggacgt caacaccacc acggggaccg acctggacac gaccacgggg 1860acgggctcgg acctggacag cgacgtggac agcgaccctg acaaggacaa gggcaagggc 1920aagtccgccg agggccagcc caagtttggg ggcggcttgg cgcccccggg ggccccgaac 1980agcgtggccg aggtgcctgt cttctattcc cagcactcat tcttcccgcc acccgacgag 2040cagctgctga ctgcaacggg cgccgccggg gactccatca aggccatcgc atccattgcc 2100gagaagtact ttggccccgg cttcatgggg atgcaggaga agaagctggg ctcgctcccc 2160taccactcgg cgttcccctt ccagttcctg cccaacttcc cccactccct ttaccccttc 2220acggaccgag ccctcgccca caacttgctg gtcaaggccg agccaaagtc accccgggac 2280gccctcaagg tgggcggccc cagtgccgag tgcccctttg atctcaccac caagcccaaa 2340gacgtgaagc ccatcctgcc catgcccaag ggcccctcgg cccccgcatc cggcgaggag 2400cagccgctgg acctgagcat cggcagccgg gcccgtgcca gccaaaacgg cggcgggcgg 2460gagccccgca agaaccacgt ctatggggaa cgcaagctgg gcgccggcga ggggctgccc 2520caggtgtgcc cggcgcggat gccccagcag cccccgctcc actacgccaa gccctcgccc 2580ttcttcatgg accccatcta cagggtagaa aagcggaagg tcacagaccc cgtgggagcc 2640ctgaaggaga agtacctgcg gccgtccccg ctgctcttcc acccccagat gtcagccata 2700gagaccatga cagagaagct ggagagcttt gcagccatga

aggcggactc gggcagctcc 2760ctgcagcccc tcccccacca ccccttcaac ttccggtccc cacccccaac gctctccgac 2820cccatcctca ggaagggcaa ggagcgatac acgtgcaggt actgtgggaa gatcttcccc 2880agatcagcca atctcaccag acacctgagg acgcacactg gggagcagcc gtacaggtgt 2940aagtactgcg accgctcctt cagcatctct tcgaacctcc agcggcacgt ccggaacatc 3000cacaacaagg agaagccttt caagtgccac ctgtgcaacc gctgcttcgg gcagcagacc 3060aacctggacc ggcacctcaa gaagcacgag cacgagaacg caccagtgag ccagcacccc 3120ggggtcctca cgaaccacct ggggaccagc gcgtcctctc ccacctcaga gtcggacaac 3180cacgcacttt tagacgagaa agaagactct tatttctcgg aaatcagaaa ctttattgcc 3240aatagtgaga tgaaccaagc atcaacgcga acagagaaac gggcggacat gcagatcgtg 3300gacggcagtg cccagtgtcc aggcctagcc agtgagaagc aggaggacgt ggaggaggag 3360gacgacgatg acctggagga ggacgatgag gacagcctgg ccgggaagtc gcaggatgac 3420accgtgtccc ccgcacccga gccccaggcc gcctacgagg atgaggagga tgaggagcca 3480gccgcctccc tggccgtggg ctttgaccac acccgaaggt gtgctgagga ccacgaaggc 3540ggtctgttag ctttggagcc gatgccgact tttgggaagg ggctggacct ccgcagagca 3600gctgaggaag catttgaagt taaagatgtg cttaattcca ccttagattc tgaggcttta 3660aaacatacac tgtgcaggca ggctaagaac cagggttctc tggacgcttg gttgaaggtc 3720actggagcca cgtcggagtc tggagcattt caccccatca accacctctg a 377141256PRTHomo sapiens 4Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 Val Val Asn Asn Met Tyr Glu Pro Asn Arg Asp Leu Leu Ala Ser His 20 25 30 Ser Ala Glu Asp Glu Ala Glu Asp Ser Ala Met Ser Pro Ile Pro Val 35 40 45 Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 Pro Ala Asp Phe Glu Leu Arg Glu Ser Ser Ile Pro Gly Ala Gly Leu 85 90 95 Gly Val Trp Ala Lys Arg Lys Met Glu Ala Gly Glu Arg Leu Gly Pro 100 105 110 Cys Val Val Val Pro Arg Ala Ala Ala Lys Glu Thr Asp Phe Gly Trp 115 120 125 Glu Gln Ile Leu Thr Asp Val Glu Val Ser Pro Gln Glu Gly Cys Ile 130 135 140 Thr Lys Ile Ser Glu Asp Leu Gly Ser Glu Lys Phe Cys Val Asp Ala 145 150 155 160 Asn Gln Ala Gly Ala Gly Ser Trp Leu Lys Tyr Ile Arg Val Ala Cys 165 170 175 Ser Cys Asp Asp Gln Asn Leu Thr Met Cys Gln Ile Ser Glu Gln Ile 180 185 190 Tyr Tyr Lys Val Ile Lys Asp Ile Glu Pro Gly Glu Glu Leu Leu Val 195 200 205 His Val Lys Glu Gly Val Tyr Pro Leu Gly Thr Val Pro Pro Gly Leu 210 215 220 Asp Glu Glu Pro Thr Phe Arg Cys Asp Glu Cys Asp Glu Leu Phe Gln 225 230 235 240 Ser Lys Leu Asp Leu Arg Arg His Lys Lys Tyr Thr Cys Gly Ser Val 245 250 255 Gly Ala Ala Leu Tyr Glu Gly Leu Ala Glu Glu Leu Lys Pro Glu Gly 260 265 270 Leu Gly Gly Gly Ser Gly Gln Ala His Glu Cys Lys Asp Cys Glu Arg 275 280 285 Met Phe Pro Asn Lys Tyr Ser Leu Glu Gln His Met Val Ile His Thr 290 295 300 Glu Glu Arg Glu Tyr Lys Cys Asp Gln Cys Pro Lys Ala Phe Asn Trp 305 310 315 320 Lys Ser Asn Leu Ile Arg His Gln Met Ser His Asp Ser Gly Lys Arg 325 330 335 Phe Glu Cys Glu Asn Cys Val Lys Val Phe Thr Asp Pro Ser Asn Leu 340 345 350 Gln Arg His Ile Arg Ser Gln His Val Gly Ala Arg Ala His Ala Cys 355 360 365 Pro Asp Cys Gly Lys Thr Phe Ala Thr Ser Ser Gly Leu Lys Gln His 370 375 380 Lys His Ile His Ser Thr Val Lys Pro Phe Ile Cys Glu Val Cys His 385 390 395 400 Lys Ser Tyr Thr Gln Phe Ser Asn Leu Cys Arg His Lys Arg Met His 405 410 415 Ala Asp Cys Arg Thr Gln Ile Lys Cys Lys Asp Cys Gly Gln Met Phe 420 425 430 Ser Thr Thr Ser Ser Leu Asn Lys His Arg Arg Phe Cys Glu Gly Lys 435 440 445 Asn His Tyr Thr Pro Gly Gly Ile Phe Ala Pro Gly Leu Pro Leu Thr 450 455 460 Pro Ser Pro Met Met Asp Lys Ala Lys Pro Ser Pro Ser Leu Asn His 465 470 475 480 Ala Ser Leu Gly Phe Asn Glu Tyr Phe Pro Ser Arg Pro His Pro Gly 485 490 495 Ser Leu Pro Phe Ser Thr Ala Pro Pro Thr Phe Pro Ala Leu Thr Pro 500 505 510 Gly Phe Pro Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro Leu 515 520 525 Leu Pro Pro Thr Ser Leu Leu Lys Ser Pro Leu Asn His Thr Gln Asp 530 535 540 Ala Lys Leu Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val Ser 545 550 555 560 Ala Val Ser Asn Ser Ser Gln Gly Thr Thr Ala Ala Ala Gly Pro Glu 565 570 575 Glu Lys Phe Glu Ser Arg Leu Glu Asp Ser Cys Val Glu Lys Leu Lys 580 585 590 Thr Arg Ser Ser Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Val Asn 595 600 605 Thr Thr Thr Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser Asp 610 615 620 Leu Asp Ser Asp Val Asp Ser Asp Pro Asp Lys Asp Lys Gly Lys Gly 625 630 635 640 Lys Ser Ala Glu Gly Gln Pro Lys Phe Gly Gly Gly Leu Ala Pro Pro 645 650 655 Gly Ala Pro Asn Ser Val Ala Glu Val Pro Val Phe Tyr Ser Gln His 660 665 670 Ser Phe Phe Pro Pro Pro Asp Glu Gln Leu Leu Thr Ala Thr Gly Ala 675 680 685 Ala Gly Asp Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr Phe 690 695 700 Gly Pro Gly Phe Met Gly Met Gln Glu Lys Lys Leu Gly Ser Leu Pro 705 710 715 720 Tyr His Ser Ala Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His Ser 725 730 735 Leu Tyr Pro Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val Lys 740 745 750 Ala Glu Pro Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro Ser 755 760 765 Ala Glu Cys Pro Phe Asp Leu Thr Thr Lys Pro Lys Asp Val Lys Pro 770 775 780 Ile Leu Pro Met Pro Lys Gly Pro Ser Ala Pro Ala Ser Gly Glu Glu 785 790 795 800 Gln Pro Leu Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln Asn 805 810 815 Gly Gly Gly Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg Lys 820 825 830 Leu Gly Ala Gly Glu Gly Leu Pro Gln Val Cys Pro Ala Arg Met Pro 835 840 845 Gln Gln Pro Pro Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met Asp 850 855 860 Pro Ile Tyr Arg Val Glu Lys Arg Lys Val Thr Asp Pro Val Gly Ala 865 870 875 880 Leu Lys Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His Pro Gln 885 890 895 Met Ser Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe Ala Ala 900 905 910 Met Lys Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His His Pro 915 920 925 Phe Asn Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile Leu Arg 930 935 940 Lys Gly Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile Phe Pro 945 950 955 960 Arg Ser Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Gln 965 970 975 Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser Ser Asn 980 985 990 Leu Gln Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro Phe Lys 995 1000 1005 Cys His Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu Asp 1010 1015 1020 Arg His Leu Lys Lys His Glu His Glu Asn Ala Pro Val Ser Gln 1025 1030 1035 His Pro Gly Val Leu Thr Asn His Leu Gly Thr Ser Ala Ser Ser 1040 1045 1050 Pro Thr Ser Glu Ser Asp Asn His Ala Leu Leu Asp Glu Lys Glu 1055 1060 1065 Asp Ser Tyr Phe Ser Glu Ile Arg Asn Phe Ile Ala Asn Ser Glu 1070 1075 1080 Met Asn Gln Ala Ser Thr Arg Thr Glu Lys Arg Ala Asp Met Gln 1085 1090 1095 Ile Val Asp Gly Ser Ala Gln Cys Pro Gly Leu Ala Ser Glu Lys 1100 1105 1110 Gln Glu Asp Val Glu Glu Glu Asp Asp Asp Asp Leu Glu Glu Asp 1115 1120 1125 Asp Glu Asp Ser Leu Ala Gly Lys Ser Gln Asp Asp Thr Val Ser 1130 1135 1140 Pro Ala Pro Glu Pro Gln Ala Ala Tyr Glu Asp Glu Glu Asp Glu 1145 1150 1155 Glu Pro Ala Ala Ser Leu Ala Val Gly Phe Asp His Thr Arg Arg 1160 1165 1170 Cys Ala Glu Asp His Glu Gly Gly Leu Leu Ala Leu Glu Pro Met 1175 1180 1185 Pro Thr Phe Gly Lys Gly Leu Asp Leu Arg Arg Ala Ala Glu Glu 1190 1195 1200 Ala Phe Glu Val Lys Asp Val Leu Asn Ser Thr Leu Asp Ser Glu 1205 1210 1215 Ala Leu Lys His Thr Leu Cys Arg Gln Ala Lys Asn Gln Gly Ser 1220 1225 1230 Leu Asp Ala Trp Leu Lys Val Thr Gly Ala Thr Ser Glu Ser Gly 1235 1240 1245 Ala Phe His Pro Ile Asn His Leu 1250 1255 53828DNAMus musculus 5atgcgatcca aggcgagggc gaggaagcta gccaaaagtg acggtgacgt tgtaaataat 60atgtatgaac ctgacccgga cctgctggcc ggccagagtg ccgaggagga gaccgaagac 120ggcatcctgt cccccatccc catggggcca ccgtccccct tccccaccag cgaggacttc 180actcccaagg agggctcgcc ctatgaggct cctgtctaca ttcctgaaga cattccaatc 240ccaccagact tcgagctacg agagtcctcc ataccaggag ctggcctggg gatctgggcc 300aagcggaaga tggaaatcgg ggagaggttt ggcccctacg tggtgacgcc ccgggccgca 360ctgaaggagg ccgactttgg atgggagatg ctgacggata cagaggtgtc atcccaggag 420agctgcatca aaaagcagat ctctgaagac ttgggtagcg agaagttctg cgtggatgcc 480aatcaggcgg ggtctggcag ctggctcaag tacatccgtg tagcgtgttc ctgtgatgac 540caaaacctcg ccatgtgtca gatcaacgaa cagatttact ataaagtcat taaggacatc 600gagcctggag aggaactgtt ggtgcatgtg aaagaaggtg cctactcctt gggtgtcatg 660gcccccagct tggatgagga ccccacattc cgctgtgatg agtgtgatga gctcttccag 720tgcaggctgg acctgaggcg ccacaagaag tacgcgtgca gctctgcagg agcccagctc 780tacgagggcc taggggagga actcaagccc gagggccttg gcgtgggcag cgacgggcaa 840gcgcatgagt gcaaggattg cgagcggatg ttccccaaca agtacagctt ggagcaacac 900atgatcgtcc acacggaaga gcgtgagtac aaatgtgacc agtgtcccaa ggccttcaac 960tggaagtcca acctcatccg ccaccagatg tctcacgaca gtggcaagcg cttcgaatgt 1020gaaaactgtg tcaaggtgtt cacggacccc agcaacctcc agcgtcacat ccgctcacag 1080catgtcggtg cccgggccca tgcctgccct gactgtggca agaccttcgc cacatcctct 1140ggcctcaaac agcacaagca tatccacagc acggtgaagc cattcatatg cgaggtctgc 1200cacaagtcct acacgcagtt ctccaacctg tgccggcaca agcggatgca cgccgactgc 1260aggacgcaga tcaagtgcaa ggactgtggg cagatgttca gcactacctc ctccctcaac 1320aagcatcgga gattctgcga gggcaagaac cattacacgc ctggcagcat cttcacccca 1380ggcctgccct tgacccccag ccccatgatg gacaagacaa aaccctcccc gaccctcaac 1440cacgggggcc taggcttcag cgagtacttc ccctccagac ctcatcctgg gagcctgccc 1500ttctcggctg ctcctccggc cttccccgca ctcactccgg gcttcccggg catctttcct 1560ccatccctgt acccacgacc acctctgcta cctcccacgc cgctgctcaa gagccccctg 1620aaccacgcgc aggacgccaa gctacccagc ccgctgggaa acccagccct gccccttgtc 1680tccgcggtca gcaatagcag ccagggtgcc acagcggcca ccgggtcaga ggagaaattt 1740gatggccgct tggaagacgc atatgcggag aaggtcaaaa ataggagccc tgacatgtcg 1800gatggcagtg actttgagga tatcaacacc acgaccggga cagacttgga cactaccacg 1860ggcacggggt cagacctgga cagcgacctg gacagtgaca gagacaaagg caaggacaag 1920gggaagccag tggagagcaa acctgagttt gggggtgcat ctgtgccccc tggggccatg 1980aacagtgtgg ccgaggtacc ggccttctac tcacagcatt ccttcttccc gccacccgag 2040gaacagctgc tgacggcctc gggagctgcc ggcgactcca tcaaggccat cgcgtccatc 2100gcggagaaat acttcggtcc tggcttcatg agcatgcagg agaagaagct gggctcacta 2160ccctaccact ccgtgttccc cttccagttc ctgcctaact ttccccactc cctctacccc 2220tttacggacc gagccctcgc ccacaacttg ctggtcaagg ctgagccaaa gtcaccccgg 2280gatgccctca aggtgggcgg ccccagtgcg gagtgcccct tcgacctcac caccaaacca 2340aaagaggcca aacccgccct gctcgcaccc aaggtccccc tcatcccctc atctggcgag 2400gaacagccac tggacctgag catcggcagc agggccaggg caagccagaa cggaggtggc 2460cgtgagccgc ggaagaacca cgtctacggt gaacggaagc cgggggtcag cgaggggctg 2520cctaaggtgt gcccagcaca gctgccccag cagccctcct tgcattatgc taagccttca 2580ccgttcttca tggatcccat ctacagggta gaaaagcgga aggtggcaga ccctgtggga 2640gtcctgaaag agaagtacct gcggccgtcc ccacttctgt tccaccccca gatgtcagcc 2700atagaaacca tgacggagaa gctggagagc tttgcagcca tgaaggccga ctcaggcagc 2760tccctgcagc ccctgcctca ccacccgttc aacttccgct ccccaccccc aacgctctcg 2820gatcccatcc tcaggaaggg gaaggagaga tacacgtgca ggtactgtgg caagatcttc 2880cccagatctg caaatctcac aagacatctg aggacacaca caggggagca gccatacagg 2940tgcaagtact gtgaccggtc attcagcatc tcctccaacc tccagcggca cgtgaggaac 3000atccacaaca aagagaagcc gttcaagtgc catctgtgca accgctgctt cgggcagcag 3060accaacctag accggcacct gaagaagcac gaacacgagg gcgcaccagt gagccagcac 3120tccggggtgc tcacgaacca cctgggcacc agcgcctcct cccccacctc cgagtcggac 3180aaccatgcac ttttagatga gaaggaagat tcttacttct ccgagatccg aaacttcatc 3240gccaacagcg agatgaacca ggcatccact cgaatggaca aacggcctga gatccaagac 3300ctggacagca acccaccgtg tccaggctca gccagtgcaa agccagagga cgtagaggag 3360gaggaagagg aggagctgga ggaagaggat gatgacagct tagccgggaa gtcacaggag 3420gacacggtgt cccccacacc tgagccccaa ggagtctatg aagatgaaga ggatgaggaa 3480ccacccagcc tgaccatggg ctttgaccat acccggaggt gtgttgagga gcgaggaggc 3540ggcctgttag ctttggagcc gacgccgacc tttgggaagg ggctggatct ccgcagagca 3600gctgaggaag catttgaagt taaagatgtg cttaattcca ccttagattc tgaggtttta 3660aaacaaaccc tgtacaggca ggctaagaac caggcatatg caatgatgct gtccctctct 3720gaagacactc ctctccacgc cccctcccag agctcactgg atgcttggtt gaacatcaca 3780ggaccctcgt cagagtccgg agcctttaac cccatcaacc acctctga 382861275PRTMus musculus 6Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 Val Val Asn Asn Met Tyr Glu Pro Asp Pro Asp Leu Leu Ala Gly Gln 20 25 30 Ser Ala Glu Glu Glu Thr Glu Asp Gly Ile Leu Ser Pro Ile Pro Met 35 40 45 Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 Pro Pro Asp Phe Glu Leu Arg Glu Ser Ser Ile Pro Gly Ala Gly Leu 85 90 95 Gly Ile Trp Ala Lys Arg Lys Met Glu Ile Gly Glu Arg Phe Gly Pro 100 105 110 Tyr Val Val Thr Pro Arg Ala Ala Leu Lys Glu Ala Asp Phe Gly Trp 115 120 125 Glu Met Leu Thr Asp Thr Glu Val Ser Ser Gln Glu Ser Cys Ile Lys 130 135 140 Lys Gln Ile Ser Glu Asp Leu Gly Ser Glu Lys Phe Cys Val Asp Ala 145 150 155 160 Asn Gln Ala Gly Ser Gly Ser Trp Leu Lys Tyr Ile Arg Val Ala Cys 165 170 175 Ser Cys Asp Asp Gln Asn Leu Ala Met Cys Gln Ile Asn Glu Gln Ile 180 185 190 Tyr Tyr Lys Val Ile Lys Asp Ile Glu Pro Gly Glu Glu Leu Leu Val 195 200 205 His Val Lys Glu Gly Ala Tyr Ser Leu Gly Val Met Ala Pro Ser Leu 210 215 220 Asp Glu Asp Pro Thr Phe Arg Cys Asp Glu Cys Asp Glu Leu Phe Gln 225 230 235 240 Cys Arg Leu Asp Leu Arg Arg His Lys Lys Tyr Ala Cys Ser Ser Ala 245 250 255 Gly Ala Gln Leu Tyr Glu Gly Leu Gly Glu Glu Leu Lys Pro Glu Gly 260 265 270 Leu Gly Val Gly Ser Asp Gly Gln Ala His Glu Cys Lys Asp Cys

Glu 275 280 285 Arg Met Phe Pro Asn Lys Tyr Ser Leu Glu Gln His Met Ile Val His 290 295 300 Thr Glu Glu Arg Glu Tyr Lys Cys Asp Gln Cys Pro Lys Ala Phe Asn 305 310 315 320 Trp Lys Ser Asn Leu Ile Arg His Gln Met Ser His Asp Ser Gly Lys 325 330 335 Arg Phe Glu Cys Glu Asn Cys Val Lys Val Phe Thr Asp Pro Ser Asn 340 345 350 Leu Gln Arg His Ile Arg Ser Gln His Val Gly Ala Arg Ala His Ala 355 360 365 Cys Pro Asp Cys Gly Lys Thr Phe Ala Thr Ser Ser Gly Leu Lys Gln 370 375 380 His Lys His Ile His Ser Thr Val Lys Pro Phe Ile Cys Glu Val Cys 385 390 395 400 His Lys Ser Tyr Thr Gln Phe Ser Asn Leu Cys Arg His Lys Arg Met 405 410 415 His Ala Asp Cys Arg Thr Gln Ile Lys Cys Lys Asp Cys Gly Gln Met 420 425 430 Phe Ser Thr Thr Ser Ser Leu Asn Lys His Arg Arg Phe Cys Glu Gly 435 440 445 Lys Asn His Tyr Thr Pro Gly Ser Ile Phe Thr Pro Gly Leu Pro Leu 450 455 460 Thr Pro Ser Pro Met Met Asp Lys Thr Lys Pro Ser Pro Thr Leu Asn 465 470 475 480 His Gly Gly Leu Gly Phe Ser Glu Tyr Phe Pro Ser Arg Pro His Pro 485 490 495 Gly Ser Leu Pro Phe Ser Ala Ala Pro Pro Ala Phe Pro Ala Leu Thr 500 505 510 Pro Gly Phe Pro Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro 515 520 525 Leu Leu Pro Pro Thr Pro Leu Leu Lys Ser Pro Leu Asn His Ala Gln 530 535 540 Asp Ala Lys Leu Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val 545 550 555 560 Ser Ala Val Ser Asn Ser Ser Gln Gly Ala Thr Ala Ala Thr Gly Ser 565 570 575 Glu Glu Lys Phe Asp Gly Arg Leu Glu Asp Ala Tyr Ala Glu Lys Val 580 585 590 Lys Asn Arg Ser Pro Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Ile 595 600 605 Asn Thr Thr Thr Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser 610 615 620 Asp Leu Asp Ser Asp Leu Asp Ser Asp Arg Asp Lys Gly Lys Asp Lys 625 630 635 640 Gly Lys Pro Val Glu Ser Lys Pro Glu Phe Gly Gly Ala Ser Val Pro 645 650 655 Pro Gly Ala Met Asn Ser Val Ala Glu Val Pro Ala Phe Tyr Ser Gln 660 665 670 His Ser Phe Phe Pro Pro Pro Glu Glu Gln Leu Leu Thr Ala Ser Gly 675 680 685 Ala Ala Gly Asp Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr 690 695 700 Phe Gly Pro Gly Phe Met Ser Met Gln Glu Lys Lys Leu Gly Ser Leu 705 710 715 720 Pro Tyr His Ser Val Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His 725 730 735 Ser Leu Tyr Pro Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val 740 745 750 Lys Ala Glu Pro Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro 755 760 765 Ser Ala Glu Cys Pro Phe Asp Leu Thr Thr Lys Pro Lys Glu Ala Lys 770 775 780 Pro Ala Leu Leu Ala Pro Lys Val Pro Leu Ile Pro Ser Ser Gly Glu 785 790 795 800 Glu Gln Pro Leu Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln 805 810 815 Asn Gly Gly Gly Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg 820 825 830 Lys Pro Gly Val Ser Glu Gly Leu Pro Lys Val Cys Pro Ala Gln Leu 835 840 845 Pro Gln Gln Pro Ser Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met 850 855 860 Asp Pro Ile Tyr Arg Val Glu Lys Arg Lys Val Ala Asp Pro Val Gly 865 870 875 880 Val Leu Lys Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His Pro 885 890 895 Gln Met Ser Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe Ala 900 905 910 Ala Met Lys Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His His 915 920 925 Pro Phe Asn Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile Leu 930 935 940 Arg Lys Gly Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile Phe 945 950 955 960 Pro Arg Ser Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu 965 970 975 Gln Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser Ser 980 985 990 Asn Leu Gln Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro Phe 995 1000 1005 Lys Cys His Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu 1010 1015 1020 Asp Arg His Leu Lys Lys His Glu His Glu Gly Ala Pro Val Ser 1025 1030 1035 Gln His Ser Gly Val Leu Thr Asn His Leu Gly Thr Ser Ala Ser 1040 1045 1050 Ser Pro Thr Ser Glu Ser Asp Asn His Ala Leu Leu Asp Glu Lys 1055 1060 1065 Glu Asp Ser Tyr Phe Ser Glu Ile Arg Asn Phe Ile Ala Asn Ser 1070 1075 1080 Glu Met Asn Gln Ala Ser Thr Arg Met Asp Lys Arg Pro Glu Ile 1085 1090 1095 Gln Asp Leu Asp Ser Asn Pro Pro Cys Pro Gly Ser Ala Ser Ala 1100 1105 1110 Lys Pro Glu Asp Val Glu Glu Glu Glu Glu Glu Glu Leu Glu Glu 1115 1120 1125 Glu Asp Asp Asp Ser Leu Ala Gly Lys Ser Gln Glu Asp Thr Val 1130 1135 1140 Ser Pro Thr Pro Glu Pro Gln Gly Val Tyr Glu Asp Glu Glu Asp 1145 1150 1155 Glu Glu Pro Pro Ser Leu Thr Met Gly Phe Asp His Thr Arg Arg 1160 1165 1170 Cys Val Glu Glu Arg Gly Gly Gly Leu Leu Ala Leu Glu Pro Thr 1175 1180 1185 Pro Thr Phe Gly Lys Gly Leu Asp Leu Arg Arg Ala Ala Glu Glu 1190 1195 1200 Ala Phe Glu Val Lys Asp Val Leu Asn Ser Thr Leu Asp Ser Glu 1205 1210 1215 Val Leu Lys Gln Thr Leu Tyr Arg Gln Ala Lys Asn Gln Ala Tyr 1220 1225 1230 Ala Met Met Leu Ser Leu Ser Glu Asp Thr Pro Leu His Ala Pro 1235 1240 1245 Ser Gln Ser Ser Leu Asp Ala Trp Leu Asn Ile Thr Gly Pro Ser 1250 1255 1260 Ser Glu Ser Gly Ala Phe Asn Pro Ile Asn His Leu 1265 1270 1275 71038DNAHomo sapiens 7atgcaacgcc tggtggcctg ggacccagca tgtctccccc tgccgccgcc gccgcctgcc 60tttaaatcca tggaagtggc caacttctac tacgaggcgg actgcttggc tgctgcgtac 120ggcggcaagg cggcccccgc ggcgcccccc gcggccagac ccgggccgcg cccccccgcc 180ggcgagctgg gcagcatcgg cgaccacgag cgcgccatcg acttcagccc gtacctggag 240ccgctgggcg cgccgcaggc cccggcgccc gccacggcca cggacacctt cgaggcggct 300ccgcccgcgc ccgcccccgc gcccgcctcc tccgggcagc accacgactt cctctccgac 360ctcttctccg acgactacgg gggcaagaac tgcaagaagc cggccgagta cggctacgtg 420agcctggggc gcctgggggc cgccaagggc gcgctgcacc ccggctgctt cgcgcccctg 480cacccaccgc ccccgccgcc gccgccgccc gccgagctca aggcggagcc gggcttcgag 540cccgcggact gcaagcggaa ggaggaggcc ggggcgccgg gcggcggcgc aggcatggcg 600gcgggcttcc cgtacgcgct gcgcgcttac ctcggctacc aggcggtgcc gagcggcagc 660agcgggagcc tctccacgtc ctcctcgtcc agcccgcccg gcacgccgag ccccgctgac 720gccaaggcgc ccccgaccgc ctgctacgcg ggggccgcgc cggcgccctc gcaggtcaag 780agcaaggcca agaagaccgt ggacaagcac agcgacgagt acaagatccg gcgcgagcgc 840aacaacatcg ccgtgcgcaa gagccgcgac aaggccaaga tgcgcaacct ggagacgcag 900cacaaggtcc tggagctcac ggccgagaac gagcggctgc agaagaaggt ggagcagctg 960tcgcgcgagc tcagcaccct gcggaacttg ttcaagcagc tgcccgagcc cctgctcgcc 1020tcctccggcc actgctag 10388345PRTHomo sapiens 8Met Gln Arg Leu Val Ala Trp Asp Pro Ala Cys Leu Pro Leu Pro Pro 1 5 10 15 Pro Pro Pro Ala Phe Lys Ser Met Glu Val Ala Asn Phe Tyr Tyr Glu 20 25 30 Ala Asp Cys Leu Ala Ala Ala Tyr Gly Gly Lys Ala Ala Pro Ala Ala 35 40 45 Pro Pro Ala Ala Arg Pro Gly Pro Arg Pro Pro Ala Gly Glu Leu Gly 50 55 60 Ser Ile Gly Asp His Glu Arg Ala Ile Asp Phe Ser Pro Tyr Leu Glu 65 70 75 80 Pro Leu Gly Ala Pro Gln Ala Pro Ala Pro Ala Thr Ala Thr Asp Thr 85 90 95 Phe Glu Ala Ala Pro Pro Ala Pro Ala Pro Ala Pro Ala Ser Ser Gly 100 105 110 Gln His His Asp Phe Leu Ser Asp Leu Phe Ser Asp Asp Tyr Gly Gly 115 120 125 Lys Asn Cys Lys Lys Pro Ala Glu Tyr Gly Tyr Val Ser Leu Gly Arg 130 135 140 Leu Gly Ala Ala Lys Gly Ala Leu His Pro Gly Cys Phe Ala Pro Leu 145 150 155 160 His Pro Pro Pro Pro Pro Pro Pro Pro Pro Ala Glu Leu Lys Ala Glu 165 170 175 Pro Gly Phe Glu Pro Ala Asp Cys Lys Arg Lys Glu Glu Ala Gly Ala 180 185 190 Pro Gly Gly Gly Ala Gly Met Ala Ala Gly Phe Pro Tyr Ala Leu Arg 195 200 205 Ala Tyr Leu Gly Tyr Gln Ala Val Pro Ser Gly Ser Ser Gly Ser Leu 210 215 220 Ser Thr Ser Ser Ser Ser Ser Pro Pro Gly Thr Pro Ser Pro Ala Asp 225 230 235 240 Ala Lys Ala Pro Pro Thr Ala Cys Tyr Ala Gly Ala Ala Pro Ala Pro 245 250 255 Ser Gln Val Lys Ser Lys Ala Lys Lys Thr Val Asp Lys His Ser Asp 260 265 270 Glu Tyr Lys Ile Arg Arg Glu Arg Asn Asn Ile Ala Val Arg Lys Ser 275 280 285 Arg Asp Lys Ala Lys Met Arg Asn Leu Glu Thr Gln His Lys Val Leu 290 295 300 Glu Leu Thr Ala Glu Asn Glu Arg Leu Gln Lys Lys Val Glu Gln Leu 305 310 315 320 Ser Arg Glu Leu Ser Thr Leu Arg Asn Leu Phe Lys Gln Leu Pro Glu 325 330 335 Pro Leu Leu Ala Ser Ser Gly His Cys 340 345 9891DNAMus musculus 9atgcaccgcc tgctggcctg ggacgcagca tgcctcccgc cgccgcccgc cgcctttaga 60cccatggaag tggccaactt ctactacgag cccgactgcc tggcctacgg ggccaaggcg 120gcccgcgccg cgccgcgcgc ccccgccgcc gagccggcca ttggcgagca cgagcgcgcc 180atcgacttca gcccctacct ggagccgctc gcgcccgccg cggacttcgc cgcgcccgcg 240cccgcgcacc acgacttcct ctccgacctc ttcgccgacg actacggcgc caagccgagc 300aagaagccgg ccgactacgg ttacgtgagc ctcggccgcg cgggcgccaa ggccgcgccg 360cccgcctgct tcccgccgcc gcctcccgcc gcgctcaagg cggagccggg cttcgaaccc 420gcggactgca agcgcgcgga cgacgcgccc gccatggcgg ccggtttccc gttcgccctg 480cgcgcctacc tgggctacca ggcgacgccg agcggcagca gcggcagcct gtccacgtcg 540tcgtcgtcca gcccgcccgg cacgccgagc cccgccgacg ccaaggccgc gcccgccgcc 600tgcttcgcgg ggccgccggc cgcgcccgcc aaggccaagg ccaagaagac ggtggacaag 660ctgagcgacg agtacaagat gcggcgcgag cgcaacaaca tcgcggtgcg caagagccgc 720gacaaggcca agatgcgcaa cctggagacg cagcacaagg tgctggagct gacggcggag 780aacgagcggc tgcagaagaa ggtggagcag ctgtcgcgag agctcagcac cctgcggaac 840ttgttcaagc agctgcccga gccgctgctg gcctcggcgg gccactgcta g 89110296PRTMus musculus 10Met His Arg Leu Leu Ala Trp Asp Ala Ala Cys Leu Pro Pro Pro Pro 1 5 10 15 Ala Ala Phe Arg Pro Met Glu Val Ala Asn Phe Tyr Tyr Glu Pro Asp 20 25 30 Cys Leu Ala Tyr Gly Ala Lys Ala Ala Arg Ala Ala Pro Arg Ala Pro 35 40 45 Ala Ala Glu Pro Ala Ile Gly Glu His Glu Arg Ala Ile Asp Phe Ser 50 55 60 Pro Tyr Leu Glu Pro Leu Ala Pro Ala Ala Asp Phe Ala Ala Pro Ala 65 70 75 80 Pro Ala His His Asp Phe Leu Ser Asp Leu Phe Ala Asp Asp Tyr Gly 85 90 95 Ala Lys Pro Ser Lys Lys Pro Ala Asp Tyr Gly Tyr Val Ser Leu Gly 100 105 110 Arg Ala Gly Ala Lys Ala Ala Pro Pro Ala Cys Phe Pro Pro Pro Pro 115 120 125 Pro Ala Ala Leu Lys Ala Glu Pro Gly Phe Glu Pro Ala Asp Cys Lys 130 135 140 Arg Ala Asp Asp Ala Pro Ala Met Ala Ala Gly Phe Pro Phe Ala Leu 145 150 155 160 Arg Ala Tyr Leu Gly Tyr Gln Ala Thr Pro Ser Gly Ser Ser Gly Ser 165 170 175 Leu Ser Thr Ser Ser Ser Ser Ser Pro Pro Gly Thr Pro Ser Pro Ala 180 185 190 Asp Ala Lys Ala Ala Pro Ala Ala Cys Phe Ala Gly Pro Pro Ala Ala 195 200 205 Pro Ala Lys Ala Lys Ala Lys Lys Thr Val Asp Lys Leu Ser Asp Glu 210 215 220 Tyr Lys Met Arg Arg Glu Arg Asn Asn Ile Ala Val Arg Lys Ser Arg 225 230 235 240 Asp Lys Ala Lys Met Arg Asn Leu Glu Thr Gln His Lys Val Leu Glu 245 250 255 Leu Thr Ala Glu Asn Glu Arg Leu Gln Lys Lys Val Glu Gln Leu Ser 260 265 270 Arg Glu Leu Ser Thr Leu Arg Asn Leu Phe Lys Gln Leu Pro Glu Pro 275 280 285 Leu Leu Ala Ser Ala Gly His Cys 290 295 111190PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 11Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Leu Glu Arg Ser Lys 1 5 10 15 Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp Val Val Asn Asn 20 25 30 Met Tyr Glu Pro Asp Pro Asp Leu Leu Ala Gly Gln Ser Ala Glu Glu 35 40 45 Glu Thr Glu Asp Gly Ile Leu Ser Pro Ile Pro Met Gly Pro Pro Ser 50 55 60 Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu Gly Ser Pro Tyr 65 70 75 80 Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile Pro Pro Asp Phe 85 90 95 Glu Leu Arg Glu Ser Ser Ile Pro Gly Ala Gly Leu Gly Ile Trp Ala 100 105 110 Lys Arg Lys Met Glu Ile Gly Glu Arg Phe Gly Pro Tyr Val Val Thr 115 120 125 Pro Arg Ala Ala Leu Lys Glu Ala Asp Phe Gly Trp Glu Gln Met Leu 130 135 140 Thr Asp Thr Glu Val Ser Ser Gln Glu Ser Cys Ile Lys Lys Gln Ile 145 150 155 160 Ser Glu Asp Leu Gly Ser Glu Lys Phe Cys Val Asp Ala Asn Gln Ala 165 170 175 Gly Ser Gly Ser Trp Leu Lys Tyr Ile Arg Val Ala Cys Ser Cys Asp 180 185 190 Asp Gln Asn Leu Ala Met Cys Gln Ile Asn Glu Gln Ile Tyr Tyr Lys 195 200 205 Val Ile Lys Asp Ile Glu Pro Gly Glu Glu Leu Leu Val His Val Lys 210 215 220 Glu Gly Ala Tyr Ser Leu Gly Val Met Ala Pro Ser Leu Asp Glu Asp 225 230 235 240 Pro Thr Phe Arg Cys Asp Glu Cys Asp Glu Leu Phe Gln Cys Arg Leu 245 250 255 Asp Leu Arg Arg His Lys Lys Tyr Ala Cys Ser Ser Ala Gly Ala Gln 260 265 270 Leu Tyr Glu Gly Leu Gly Glu Glu Leu Lys Pro Glu Gly Leu Gly Val 275 280 285 Gly Ser Asp Gly Gln Ala His Glu Cys Lys Asp Cys Glu Arg Met Phe 290 295 300 Pro Asn Lys Tyr Ser Leu Glu Gln His Met Ile Val His Thr Glu Glu 305 310 315 320 Arg Glu Tyr Lys Cys Asp Gln Cys Pro Lys

Ala Phe Asn Trp Lys Ser 325 330 335 Asn Leu Ile Arg His Gln Met Ser His Asp Ser Gly Lys Arg Phe Glu 340 345 350 Cys Glu Asn Cys Val Lys Val Phe Thr Asp Pro Ser Asn Leu Gln Arg 355 360 365 His Ile Arg Ser Gln His Val Gly Ala Arg Ala His Ala Cys Pro Asp 370 375 380 Cys Gly Lys Thr Phe Ala Thr Ser Ser Gly Leu Lys Gln His Lys His 385 390 395 400 Ile His Ser Thr Val Lys Pro Phe Ile Cys Glu Val Cys His Lys Ser 405 410 415 Tyr Thr Gln Phe Ser Asn Leu Cys Arg His Lys Arg Met His Ala Asp 420 425 430 Cys Arg Thr Gln Ile Lys Cys Lys Asp Cys Gly Gln Met Phe Ser Thr 435 440 445 Thr Ser Ser Leu Asn Lys His Arg Arg Phe Cys Glu Gly Lys Asn His 450 455 460 Tyr Thr Pro Gly Ser Ile Phe Thr Pro Gly Leu Pro Leu Thr Pro Ser 465 470 475 480 Pro Met Met Asp Lys Thr Lys Pro Ser Pro Thr Leu Asn His Gly Gly 485 490 495 Leu Gly Phe Ser Glu Tyr Phe Pro Ser Arg Pro His Pro Gly Ser Leu 500 505 510 Pro Phe Ser Ala Ala Pro Pro Ala Phe Pro Ala Leu Thr Pro Gly Phe 515 520 525 Pro Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro Leu Leu Pro 530 535 540 Pro Thr Pro Leu Leu Lys Ser Pro Leu Asn His Ala Gln Asp Ala Lys 545 550 555 560 Leu Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val Ser Ala Val 565 570 575 Ser Asn Ser Ser Gln Gly Ala Thr Ala Ala Thr Gly Ser Glu Glu Lys 580 585 590 Phe Asp Gly Arg Leu Glu Asp Ala Tyr Ala Glu Lys Val Lys Asn Arg 595 600 605 Ser Pro Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Ile Asn Thr Thr 610 615 620 Thr Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser Asp Leu Asp 625 630 635 640 Ser Asp Leu Asp Ser Asp Arg Asp Lys Gly Lys Asp Lys Gly Lys Pro 645 650 655 Val Glu Ser Lys Pro Glu Phe Gly Gly Ala Ser Val Pro Pro Gly Ala 660 665 670 Met Asn Ser Val Ala Glu Val Pro Ala Phe Tyr Ser Gln His Ser Phe 675 680 685 Phe Pro Pro Pro Glu Glu Gln Leu Leu Thr Ala Ser Gly Ala Ala Gly 690 695 700 Asp Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr Phe Gly Pro 705 710 715 720 Gly Phe Met Ser Met Gln Glu Lys Lys Leu Gly Ser Leu Pro Tyr His 725 730 735 Ser Val Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His Ser Leu Tyr 740 745 750 Pro Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val Lys Ala Glu 755 760 765 Pro Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro Ser Ala Glu 770 775 780 Cys Pro Phe Asp Leu Thr Thr Lys Pro Lys Glu Ala Lys Pro Ala Leu 785 790 795 800 Leu Ala Pro Lys Val Pro Leu Ile Pro Ser Ser Gly Glu Glu Gln Pro 805 810 815 Leu Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln Asn Gly Gly 820 825 830 Gly Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg Lys Pro Gly 835 840 845 Val Ser Glu Gly Leu Pro Lys Val Cys Pro Ala Gln Leu Pro Gln Gln 850 855 860 Pro Ser Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met Asp Pro Ile 865 870 875 880 Tyr Ser Arg Val Glu Lys Arg Lys Val Ala Asp Pro Val Gly Val Leu 885 890 895 Lys Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His Pro Gln Met 900 905 910 Ser Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe Ala Ala Met 915 920 925 Lys Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His His Pro Phe 930 935 940 Asn Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile Leu Arg Lys 945 950 955 960 Gly Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile Phe Pro Arg 965 970 975 Ser Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Gln Pro 980 985 990 Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser Ser Asn Leu 995 1000 1005 Gln Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro Phe Lys 1010 1015 1020 Cys His Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu Asp 1025 1030 1035 Arg His Leu Lys Lys His Glu His Glu Gly Ala Pro Val Ser Gln 1040 1045 1050 His Ser Gly Val Leu Thr Asn His Leu Gly Thr Ser Ala Ser Ser 1055 1060 1065 Pro Thr Ser Glu Ser Asp Asn His Ala Leu Leu Asp Glu Lys Glu 1070 1075 1080 Asp Ser Tyr Phe Ser Glu Ile Arg Asn Phe Ile Ala Asn Ser Glu 1085 1090 1095 Met Asn Gln Ala Ser Thr Arg Met Asp Lys Arg Pro Glu Ile Gln 1100 1105 1110 Asp Leu Asp Ser Asn Pro Pro Cys Pro Gly Ser Ala Ser Ala Lys 1115 1120 1125 Pro Glu Asp Val Glu Glu Glu Glu Glu Glu Glu Leu Glu Glu Glu 1130 1135 1140 Asp Asp Asp Ser Leu Ala Gly Lys Ser Gln Glu Asp Thr Val Ser 1145 1150 1155 Pro Thr Pro Glu Pro Gln Gly Val Tyr Glu Asp Glu Glu Asp Glu 1160 1165 1170 Glu Pro Pro Ser Leu Thr Met Gly Phe Asp His Thr Arg Arg His 1175 1180 1185 Met Gln 1190 12223PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 12Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 Val Val Asn Asn Met Tyr Glu Pro Asp Pro Asp Leu Leu Ala Gly Gln 20 25 30 Ser Ala Glu Glu Glu Thr Glu Asp Gly Ile Leu Ser Pro Ile Pro Met 35 40 45 Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 Pro Pro Asp Phe Glu Leu Arg Glu Ser Ser Ile Pro Gly Ala Gly Leu 85 90 95 Gly Ile Trp Ala Lys Arg Lys Met Glu Ile Gly Glu Arg Phe Gly Pro 100 105 110 Tyr Val Val Thr Pro Arg Ala Ala Leu Lys Glu Ala Asp Phe Gly Trp 115 120 125 Glu Gln Met Leu Thr Asp Thr Glu Val Ser Ser Gln Glu Ser Cys Ile 130 135 140 Lys Lys Gln Ile Ser Glu Asp Leu Gly Ser Glu Lys Phe Cys Val Asp 145 150 155 160 Ala Asn Gln Ala Gly Ser Gly Ser Trp Leu Lys Tyr Ile Arg Val Ala 165 170 175 Cys Ser Cys Asp Asp Gln Asn Leu Ala Met Cys Gln Ile Asn Glu Gln 180 185 190 Ile Tyr Tyr Lys Val Ile Lys Asp Ile Glu Pro Gly Glu Glu Leu Leu 195 200 205 Val His Val Lys Glu Gly Ala Tyr Ser Leu Gly Val Met Ala Pro 210 215 220 13232PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 13Met Ser Leu Asp Glu Asp Pro Thr Phe Arg Cys Asp Glu Cys Asp Glu 1 5 10 15 Leu Phe Gln Cys Arg Leu Asp Leu Arg Arg His Lys Lys Tyr Ala Cys 20 25 30 Ser Ser Ala Gly Ala Gln Leu Tyr Glu Gly Leu Gly Glu Glu Leu Lys 35 40 45 Pro Glu Gly Leu Gly Val Gly Ser Asp Gly Gln Ala His Glu Cys Lys 50 55 60 Asp Cys Glu Arg Met Phe Pro Asn Lys Tyr Ser Leu Glu Gln His Met 65 70 75 80 Ile Val His Thr Glu Glu Arg Glu Tyr Lys Cys Asp Gln Cys Pro Lys 85 90 95 Ala Phe Asn Trp Lys Ser Asn Leu Ile Arg His Gln Met Ser His Asp 100 105 110 Ser Gly Lys Arg Phe Glu Cys Glu Asn Cys Val Lys Val Phe Thr Asp 115 120 125 Pro Ser Asn Leu Gln Arg His Ile Arg Ser Gln His Val Gly Ala Arg 130 135 140 Ala His Ala Cys Pro Asp Cys Gly Lys Thr Phe Ala Thr Ser Ser Gly 145 150 155 160 Leu Lys Gln His Lys His Ile His Ser Thr Val Lys Pro Phe Ile Cys 165 170 175 Glu Val Cys His Lys Ser Tyr Thr Gln Phe Ser Asn Leu Cys Arg His 180 185 190 Lys Arg Met His Ala Asp Cys Arg Thr Gln Ile Lys Cys Lys Asp Cys 195 200 205 Gly Gln Met Phe Ser Thr Thr Ser Ser Leu Asn Lys His Arg Arg Phe 210 215 220 Cys Glu Gly Lys Asn His Tyr Thr 225 230 14227PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 14Met Pro Gly Ser Ile Phe Thr Pro Gly Leu Pro Leu Thr Pro Ser Pro 1 5 10 15 Met Met Asp Lys Thr Lys Pro Ser Pro Thr Leu Asn His Gly Gly Leu 20 25 30 Gly Phe Ser Glu Tyr Phe Pro Ser Arg Pro His Pro Gly Ser Leu Pro 35 40 45 Phe Ser Ala Ala Pro Pro Ala Phe Pro Ala Leu Thr Pro Gly Phe Pro 50 55 60 Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro Leu Leu Pro Pro 65 70 75 80 Thr Pro Leu Leu Lys Ser Pro Leu Asn His Ala Gln Asp Ala Lys Leu 85 90 95 Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val Ser Ala Val Ser 100 105 110 Asn Ser Ser Gln Gly Ala Thr Ala Ala Thr Gly Ser Glu Glu Lys Phe 115 120 125 Asp Gly Arg Leu Glu Asp Ala Tyr Ala Glu Lys Val Lys Asn Arg Ser 130 135 140 Pro Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Ile Asn Thr Thr Thr 145 150 155 160 Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser Asp Leu Asp Ser 165 170 175 Asp Leu Asp Ser Asp Arg Asp Lys Gly Lys Asp Lys Gly Lys Pro Val 180 185 190 Glu Ser Lys Pro Glu Phe Gly Gly Ala Ser Val Pro Pro Gly Ala Met 195 200 205 Asn Ser Val Ala Glu Val Pro Ala Phe Tyr Ser Gln His Ser Phe Phe 210 215 220 Pro Pro Pro 225 15202PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 15Met Pro Glu Glu Gln Leu Leu Thr Ala Ser Gly Ala Ala Gly Asp Ser 1 5 10 15 Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr Phe Gly Pro Gly Phe 20 25 30 Met Ser Met Gln Glu Lys Lys Leu Gly Ser Leu Pro Tyr His Ser Val 35 40 45 Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His Ser Leu Tyr Pro Phe 50 55 60 Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val Lys Ala Glu Pro Lys 65 70 75 80 Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro Ser Ala Glu Cys Pro 85 90 95 Phe Asp Leu Thr Thr Lys Pro Lys Glu Ala Lys Pro Ala Leu Leu Ala 100 105 110 Pro Lys Val Pro Leu Ile Pro Ser Ser Gly Glu Glu Gln Pro Leu Asp 115 120 125 Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln Asn Gly Gly Gly Arg 130 135 140 Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg Lys Pro Gly Val Ser 145 150 155 160 Glu Gly Leu Pro Lys Val Cys Pro Ala Gln Leu Pro Gln Gln Pro Ser 165 170 175 Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met Asp Pro Ile Tyr Ser 180 185 190 Arg Val Glu Lys Arg Lys Val Ala Asp Pro 195 200 16159PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 16Met Val Gly Val Leu Lys Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu 1 5 10 15 Phe His Pro Gln Met Ser Ala Ile Glu Thr Met Thr Glu Lys Leu Glu 20 25 30 Ser Phe Ala Ala Met Lys Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu 35 40 45 Pro His His Pro Phe Asn Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp 50 55 60 Pro Ile Leu Arg Lys Gly Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly 65 70 75 80 Lys Ile Phe Pro Arg Ser Ala Asn Leu Thr Arg His Leu Arg Thr His 85 90 95 Thr Gly Glu Gln Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser 100 105 110 Ile Ser Ser Asn Leu Gln Arg His Val Arg Asn Ile His Asn Lys Glu 115 120 125 Lys Pro Phe Lys Cys His Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr 130 135 140 Asn Leu Asp Arg His Leu Lys Lys His Glu His Glu Gly Ala Pro 145 150 155 17141PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 17Met Val Ser Gln His Ser Gly Val Leu Thr Asn His Leu Gly Thr Ser 1 5 10 15 Ala Ser Ser Pro Thr Ser Glu Ser Asp Asn His Ala Leu Leu Asp Glu 20 25 30 Lys Glu Asp Ser Tyr Phe Ser Glu Ile Arg Asn Phe Ile Ala Asn Ser 35 40 45 Glu Met Asn Gln Ala Ser Thr Arg Met Asp Lys Arg Pro Glu Ile Gln 50 55 60 Asp Leu Asp Ser Asn Pro Pro Cys Pro Gly Ser Ala Ser Ala Lys Pro 65 70 75 80 Glu Asp Val Glu Glu Glu Glu Glu Glu Glu Leu Glu Glu Glu Asp Asp 85 90 95 Asp Ser Leu Ala Gly Lys Ser Gln Glu Asp Thr Val Ser Pro Thr Pro 100 105 110 Glu Pro Gln Gly Val Tyr Glu Asp Glu Glu Asp Glu Glu Pro Pro Ser 115 120 125 Leu Thr Met Gly Phe Asp His Thr Arg Arg His Met Gln 130 135 140 18435DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polynucleotide" 18atggcggccg gtttcccgtt cgccctgcgc gcctacctgg gctaccaggc gacgccgagc 60ggcagcagcg gcagcctgtc cacgtcgtcg tcgtccagcc cgcccggcac gccgagcccc 120gccgacgcca aggccgcgcc cgccgcctgc ttcgcggggc cgccggccgc gcccgccaag 180gctaaggcca agaagacggt ggacaagctg agcgacgagt acaagatgcg gcgcgagcgc 240aacaacatcg cggtgcgcaa gagccgcgac aaggccaaga tgcgcaacct ggagacgcag 300cacaaggtgc tggagctgac ggcggagaac gagcggctgc agaagaaggt ggagcagctg 360tcgcgagagc tcagcaccct gcggaacttg ttcaagcagc tgcccaagcc gctgctggcc 420tcggccggtc actgc 43519145PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic polypeptide" 19Met Ala Ala Gly Phe Pro Phe Ala Leu Arg Ala Tyr Leu Gly Tyr Gln 1 5 10 15 Ala Thr Pro Ser Gly Ser Ser Gly Ser Leu Ser Thr Ser Ser Ser Ser 20 25 30 Ser Pro Pro Gly Thr Pro Ser Pro Ala Asp Ala Lys Ala Ala Pro Ala 35 40 45 Ala Cys Phe Ala Gly Pro Pro Ala Ala

Pro Ala Lys Ala Lys Ala Lys 50 55 60 Lys Thr Val Asp Lys Leu Ser Asp Glu Tyr Lys Met Arg Arg Glu Arg 65 70 75 80 Asn Asn Ile Ala Val Arg Lys Ser Arg Asp Lys Ala Lys Met Arg Asn 85 90 95 Leu Glu Thr Gln His Lys Val Leu Glu Leu Thr Ala Glu Asn Glu Arg 100 105 110 Leu Gln Lys Lys Val Glu Gln Leu Ser Arg Glu Leu Ser Thr Leu Arg 115 120 125 Asn Leu Phe Lys Gln Leu Pro Lys Pro Leu Leu Ala Ser Ala Gly His 130 135 140 Cys 145 2064DNAUnknownsource/note="Description of Unknown sh-Beta-1 shRNA sequence" 20gatccccgcc ctgagtaatc acttaaagtt caagagactt taagtgatta ctcagggctt 60ttta 642164DNAUnknownsource/note="Description of Unknown sh-Beta-1 shRNA sequence" 21agcttaaaaa gccctgagta atcacttaaa gtctcttgaa ctttaagtga ttactcaggg 60cggg 642260DNAUnknownsource/note="Description of Unknown sh-Beta-2 shRNA sequence" 22gatccccccg ggccctgagt aatcacttca agagagtgat tactcagggc ccggttttta 602360DNAUnknownsource/note="Description of Unknown sh-Beta-2 shRNA sequence" 23agcttaaaaa ccgggccctg agtaatcact ctcttgaagt gattactcag ggcccggggg 60242910DNAUnknownsource/note="Description of Unknown DeltaZF-1 sequence" 24atg cga tcc aag gcg agg gcg agg aag cta gcc aaa agt gac ggt gac 48Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 gtt gta aat aat atg tat gaa cct gac ccg gac ctg ctg gcc ggc cag 96Val Val Asn Asn Met Tyr Glu Pro Asp Pro Asp Leu Leu Ala Gly Gln 20 25 30 agt gcc gag gag gag acc gaa gac ggc atc ctg tcc ccc atc ccc atg 144Ser Ala Glu Glu Glu Thr Glu Asp Gly Ile Leu Ser Pro Ile Pro Met 35 40 45 ggg cca ccg tcc ccc ttc ccc acc agc gag gac ttc act ccc aag gag 192Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 ggc tcg ccc tat gag gct cct gtc tac att cct gaa gac att cca atc 240Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 cca cca gac ttc gag cta cga gag tcc tcc ata cca gga gct ggc ctg 288Pro Pro Asp Phe Glu Leu Arg Glu Ser Ser Ile Pro Gly Ala Gly Leu 85 90 95 ggg atc tgg gcc aag cgg aag atg gaa atc ggg gag agg ttt ggc ccc 336Gly Ile Trp Ala Lys Arg Lys Met Glu Ile Gly Glu Arg Phe Gly Pro 100 105 110 tac gtg gtg acg ccc cgg gcc gca ctg aag gag gcc gac ttt gga tgg 384Tyr Val Val Thr Pro Arg Ala Ala Leu Lys Glu Ala Asp Phe Gly Trp 115 120 125 gag cag atg ctg acg gat aca gag gtg tca tcc cag gag agc tgc atc 432Glu Gln Met Leu Thr Asp Thr Glu Val Ser Ser Gln Glu Ser Cys Ile 130 135 140 aaa aag cag atc tct gaa gac ttg ggt agc gag aag ttc tgc gtg gat 480Lys Lys Gln Ile Ser Glu Asp Leu Gly Ser Glu Lys Phe Cys Val Asp 145 150 155 160 gcc aat cag gcg ggg tct ggc agc tgg ctc aag tac atc cgt gta gcg 528Ala Asn Gln Ala Gly Ser Gly Ser Trp Leu Lys Tyr Ile Arg Val Ala 165 170 175 tgt tcc tgt gat gac caa aac ctc gcc atg tgt cag atc aac gaa cag 576Cys Ser Cys Asp Asp Gln Asn Leu Ala Met Cys Gln Ile Asn Glu Gln 180 185 190 att tac tat aaa gtc att aag gac atc gag cct gga gag gaa ctg ttg 624Ile Tyr Tyr Lys Val Ile Lys Asp Ile Glu Pro Gly Glu Glu Leu Leu 195 200 205 gtg cat gtg aaa gaa ggt gcc tac tcc ttg ggt gtc atg gcc ccc agc 672Val His Val Lys Glu Gly Ala Tyr Ser Leu Gly Val Met Ala Pro Ser 210 215 220 ttg gat gag gac ccc aca ttc cgc tgt gat cgg aga ttc tgc gag ggc 720Leu Asp Glu Asp Pro Thr Phe Arg Cys Asp Arg Arg Phe Cys Glu Gly 225 230 235 240 aag aac cat tac acg cct ggc agc atc ttc acc cca ggc ctg ccc ttg 768Lys Asn His Tyr Thr Pro Gly Ser Ile Phe Thr Pro Gly Leu Pro Leu 245 250 255 acc ccc agc ccc atg atg gac aag aca aaa ccc tcc ccg acc ctc aac 816Thr Pro Ser Pro Met Met Asp Lys Thr Lys Pro Ser Pro Thr Leu Asn 260 265 270 cac ggg ggc cta ggc ttc agc gag tac ttc ccc tcc aga cct cat cct 864His Gly Gly Leu Gly Phe Ser Glu Tyr Phe Pro Ser Arg Pro His Pro 275 280 285 ggg agc ctg ccc ttc tcg gct gct cct ccg gcc ttc ccc gca ctc act 912Gly Ser Leu Pro Phe Ser Ala Ala Pro Pro Ala Phe Pro Ala Leu Thr 290 295 300 ccg ggc ttc ccg ggc atc ttt cct cca tcc ctg tac cca cga cca cct 960Pro Gly Phe Pro Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro 305 310 315 320 ctg cta cct ccc acg ccg ctg ctc aag agc ccc ctg aac cac gcg cag 1008Leu Leu Pro Pro Thr Pro Leu Leu Lys Ser Pro Leu Asn His Ala Gln 325 330 335 gac gcc aag cta ccc agc ccg ctg gga aac cca gcc ctg ccc ctt gtc 1056Asp Ala Lys Leu Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val 340 345 350 tcc gcg gtc agc aat agc agc cag ggt gcc aca gcg gcc acc ggg tca 1104Ser Ala Val Ser Asn Ser Ser Gln Gly Ala Thr Ala Ala Thr Gly Ser 355 360 365 gag gag aaa ttt gat ggc cgc ttg gaa gac gca tat gcg gag aag gtc 1152Glu Glu Lys Phe Asp Gly Arg Leu Glu Asp Ala Tyr Ala Glu Lys Val 370 375 380 aaa aat agg agc cct gac atg tcg gat ggc agt gac ttt gag gat atc 1200Lys Asn Arg Ser Pro Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Ile 385 390 395 400 aac acc acg acc ggg aca gac ttg gac act acc acg ggc acg ggg tca 1248Asn Thr Thr Thr Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser 405 410 415 gac ctg gac agc gac ctg gac agt gac aga gac aaa ggc aag gac aag 1296Asp Leu Asp Ser Asp Leu Asp Ser Asp Arg Asp Lys Gly Lys Asp Lys 420 425 430 ggg aag cca gtg gag agc aaa cct gag ttt ggg ggt gca tct gtg ccc 1344Gly Lys Pro Val Glu Ser Lys Pro Glu Phe Gly Gly Ala Ser Val Pro 435 440 445 cct ggg gcc atg aac agt gtg gcc gag gta ccg gcc ttc tac tca cag 1392Pro Gly Ala Met Asn Ser Val Ala Glu Val Pro Ala Phe Tyr Ser Gln 450 455 460 cat tcc ttc ttc ccg cca ccc gag gaa cag ctg ctg acg gcc tcg gga 1440His Ser Phe Phe Pro Pro Pro Glu Glu Gln Leu Leu Thr Ala Ser Gly 465 470 475 480 gct gcc ggc gac tcc atc aag gcc atc gcg tcc atc gcg gag aaa tac 1488Ala Ala Gly Asp Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr 485 490 495 ttc ggt cct ggc ttc atg agc atg cag gag aag aag ctg ggc tca cta 1536Phe Gly Pro Gly Phe Met Ser Met Gln Glu Lys Lys Leu Gly Ser Leu 500 505 510 ccc tac cac tcc gtg ttc ccc ttc cag ttc ctg cct aac ttt ccc cac 1584Pro Tyr His Ser Val Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His 515 520 525 tcc ctc tac ccc ttt acg gac cga gcc ctc gcc cac aac ttg ctg gtc 1632Ser Leu Tyr Pro Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val 530 535 540 aag gct gag cca aag tca ccc cgg gat gcc ctc aag gtg ggc ggc ccc 1680Lys Ala Glu Pro Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro 545 550 555 560 agt gcg gag tgc ccc ttc gac ctc acc acc aaa cca aaa gag gcc aaa 1728Ser Ala Glu Cys Pro Phe Asp Leu Thr Thr Lys Pro Lys Glu Ala Lys 565 570 575 ccc gcc ctg ctc gca ccc aag gtc ccc ctc atc ccc tca tct ggc gag 1776Pro Ala Leu Leu Ala Pro Lys Val Pro Leu Ile Pro Ser Ser Gly Glu 580 585 590 gaa cag cca ctg gac ctg agc atc ggc agc agg gcc agg gca agc cag 1824Glu Gln Pro Leu Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln 595 600 605 aac gga ggt ggc cgt gag ccg cgg aag aac cac gtc tac ggt gaa cgg 1872Asn Gly Gly Gly Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg 610 615 620 aag ccg ggg gtc agc gag ggg ctg cct aag gtg tgc cca gca cag ctg 1920Lys Pro Gly Val Ser Glu Gly Leu Pro Lys Val Cys Pro Ala Gln Leu 625 630 635 640 ccc cag cag ccc tcc ttg cat tat gct aag cct tca ccg ttc ttc atg 1968Pro Gln Gln Pro Ser Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met 645 650 655 gat ccc atc tac agc agg gta gaa aag cgg aag gtg gca gac cct gtg 2016Asp Pro Ile Tyr Ser Arg Val Glu Lys Arg Lys Val Ala Asp Pro Val 660 665 670 gga gtc ctg aaa gag aag tac ctg cgg ccg tcc cca ctt ctg ttc cac 2064Gly Val Leu Lys Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His 675 680 685 ccc cag atg tca gcc ata gaa acc atg acg gag aag ctg gag agc ttt 2112Pro Gln Met Ser Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe 690 695 700 gca gcc atg aag gcc gac tca ggc agc tcc ctg cag ccc ctg cct cac 2160Ala Ala Met Lys Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His 705 710 715 720 cac ccg ttc aac ttc cgc tcc cca ccc cca acg ctc tcg gat ccc atc 2208His Pro Phe Asn Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile 725 730 735 ctc agg aag ggg aag gag aga tac acg tgc agg tac tgt ggc aag atc 2256Leu Arg Lys Gly Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile 740 745 750 ttc ccc aga tct gca aat ctc aca aga cat ctg agg aca cac aca ggg 2304Phe Pro Arg Ser Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly 755 760 765 gag cag cca tac agg tgc aag tac tgt gac cgg tca ttc agc atc tcc 2352Glu Gln Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser 770 775 780 tcc aac ctc cag cgg cac gtg agg aac atc cac aac aaa gag aag ccg 2400Ser Asn Leu Gln Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro 785 790 795 800 ttc aag tgc cat ctg tgc aac cgc tgc ttc ggg cag cag acc aac cta 2448Phe Lys Cys His Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu 805 810 815 gac cgg cac ctg aag aag cac gaa cac gag ggc gca cca gtg agc cag 2496Asp Arg His Leu Lys Lys His Glu His Glu Gly Ala Pro Val Ser Gln 820 825 830 cac tcc ggg gtg ctc acg aac cac ctg ggc acc agc gcc tcc tcc ccc 2544His Ser Gly Val Leu Thr Asn His Leu Gly Thr Ser Ala Ser Ser Pro 835 840 845 acc tcc gag tcg gac aac cat gca ctt tta gat gag aag gaa gat tct 2592Thr Ser Glu Ser Asp Asn His Ala Leu Leu Asp Glu Lys Glu Asp Ser 850 855 860 tac ttc tcc gag atc cga aac ttc atc gcc aac agc gag atg aac cag 2640Tyr Phe Ser Glu Ile Arg Asn Phe Ile Ala Asn Ser Glu Met Asn Gln 865 870 875 880 gca tcc act cga atg gac aaa cgg cct gag atc caa gac ctg gac agc 2688Ala Ser Thr Arg Met Asp Lys Arg Pro Glu Ile Gln Asp Leu Asp Ser 885 890 895 aac cca ccg tgt cca ggc tca gcc agt gca aag cca gag gac gta gag 2736Asn Pro Pro Cys Pro Gly Ser Ala Ser Ala Lys Pro Glu Asp Val Glu 900 905 910 gag gag gaa gag gag gag ctg gag gaa gag gat gat gac agc tta gcc 2784Glu Glu Glu Glu Glu Glu Leu Glu Glu Glu Asp Asp Asp Ser Leu Ala 915 920 925 ggg aag tca cag gag gac acg gtg tcc ccc aca cct gag ccc caa gga 2832Gly Lys Ser Gln Glu Asp Thr Val Ser Pro Thr Pro Glu Pro Gln Gly 930 935 940 gtc tat gaa gat gaa gag gat gag gaa cca ccc agc ctg acc atg ggc 2880Val Tyr Glu Asp Glu Glu Asp Glu Glu Pro Pro Ser Leu Thr Met Gly 945 950 955 960 ttt gac cat acc cgg agg cat atg caa tga 2910Phe Asp His Thr Arg Arg His Met Gln 965 25969PRTUnknownsource/note="Description of Unknown DeltaZF-1 sequence" 25Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 Val Val Asn Asn Met Tyr Glu Pro Asp Pro Asp Leu Leu Ala Gly Gln 20 25 30 Ser Ala Glu Glu Glu Thr Glu Asp Gly Ile Leu Ser Pro Ile Pro Met 35 40 45 Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 Pro Pro Asp Phe Glu Leu Arg Glu Ser Ser Ile Pro Gly Ala Gly Leu 85 90 95 Gly Ile Trp Ala Lys Arg Lys Met Glu Ile Gly Glu Arg Phe Gly Pro 100 105 110 Tyr Val Val Thr Pro Arg Ala Ala Leu Lys Glu Ala Asp Phe Gly Trp 115 120 125 Glu Gln Met Leu Thr Asp Thr Glu Val Ser Ser Gln Glu Ser Cys Ile 130 135 140 Lys Lys Gln Ile Ser Glu Asp Leu Gly Ser Glu Lys Phe Cys Val Asp 145 150 155 160 Ala Asn Gln Ala Gly Ser Gly Ser Trp Leu Lys Tyr Ile Arg Val Ala 165 170 175 Cys Ser Cys Asp Asp Gln Asn Leu Ala Met Cys Gln Ile Asn Glu Gln 180 185 190 Ile Tyr Tyr Lys Val Ile Lys Asp Ile Glu Pro Gly Glu Glu Leu Leu 195 200 205 Val His Val Lys Glu Gly Ala Tyr Ser Leu Gly Val Met Ala Pro Ser 210 215 220 Leu Asp Glu Asp Pro Thr Phe Arg Cys Asp Arg Arg Phe Cys Glu Gly 225 230 235 240 Lys Asn His Tyr Thr Pro Gly Ser Ile Phe Thr Pro Gly Leu Pro Leu 245 250 255 Thr Pro Ser Pro Met Met Asp Lys Thr Lys Pro Ser Pro Thr Leu Asn 260 265 270 His Gly Gly Leu Gly Phe Ser Glu Tyr Phe Pro Ser Arg Pro His Pro 275 280 285 Gly Ser Leu Pro Phe Ser Ala Ala Pro Pro Ala Phe Pro Ala Leu Thr 290 295 300 Pro Gly Phe Pro Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro 305 310 315 320 Leu Leu Pro Pro Thr Pro Leu Leu Lys Ser Pro Leu Asn His Ala Gln 325 330 335 Asp Ala Lys Leu Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val 340 345 350 Ser Ala Val Ser Asn Ser Ser Gln Gly Ala Thr Ala Ala Thr Gly Ser 355 360 365 Glu Glu Lys Phe Asp Gly Arg Leu Glu Asp Ala Tyr Ala Glu Lys Val 370 375 380 Lys Asn Arg Ser Pro Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Ile 385 390 395 400 Asn Thr Thr Thr

Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser 405 410 415 Asp Leu Asp Ser Asp Leu Asp Ser Asp Arg Asp Lys Gly Lys Asp Lys 420 425 430 Gly Lys Pro Val Glu Ser Lys Pro Glu Phe Gly Gly Ala Ser Val Pro 435 440 445 Pro Gly Ala Met Asn Ser Val Ala Glu Val Pro Ala Phe Tyr Ser Gln 450 455 460 His Ser Phe Phe Pro Pro Pro Glu Glu Gln Leu Leu Thr Ala Ser Gly 465 470 475 480 Ala Ala Gly Asp Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr 485 490 495 Phe Gly Pro Gly Phe Met Ser Met Gln Glu Lys Lys Leu Gly Ser Leu 500 505 510 Pro Tyr His Ser Val Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His 515 520 525 Ser Leu Tyr Pro Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val 530 535 540 Lys Ala Glu Pro Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro 545 550 555 560 Ser Ala Glu Cys Pro Phe Asp Leu Thr Thr Lys Pro Lys Glu Ala Lys 565 570 575 Pro Ala Leu Leu Ala Pro Lys Val Pro Leu Ile Pro Ser Ser Gly Glu 580 585 590 Glu Gln Pro Leu Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln 595 600 605 Asn Gly Gly Gly Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg 610 615 620 Lys Pro Gly Val Ser Glu Gly Leu Pro Lys Val Cys Pro Ala Gln Leu 625 630 635 640 Pro Gln Gln Pro Ser Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met 645 650 655 Asp Pro Ile Tyr Ser Arg Val Glu Lys Arg Lys Val Ala Asp Pro Val 660 665 670 Gly Val Leu Lys Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His 675 680 685 Pro Gln Met Ser Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe 690 695 700 Ala Ala Met Lys Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His 705 710 715 720 His Pro Phe Asn Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile 725 730 735 Leu Arg Lys Gly Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile 740 745 750 Phe Pro Arg Ser Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly 755 760 765 Glu Gln Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser 770 775 780 Ser Asn Leu Gln Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro 785 790 795 800 Phe Lys Cys His Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu 805 810 815 Asp Arg His Leu Lys Lys His Glu His Glu Gly Ala Pro Val Ser Gln 820 825 830 His Ser Gly Val Leu Thr Asn His Leu Gly Thr Ser Ala Ser Ser Pro 835 840 845 Thr Ser Glu Ser Asp Asn His Ala Leu Leu Asp Glu Lys Glu Asp Ser 850 855 860 Tyr Phe Ser Glu Ile Arg Asn Phe Ile Ala Asn Ser Glu Met Asn Gln 865 870 875 880 Ala Ser Thr Arg Met Asp Lys Arg Pro Glu Ile Gln Asp Leu Asp Ser 885 890 895 Asn Pro Pro Cys Pro Gly Ser Ala Ser Ala Lys Pro Glu Asp Val Glu 900 905 910 Glu Glu Glu Glu Glu Glu Leu Glu Glu Glu Asp Asp Asp Ser Leu Ala 915 920 925 Gly Lys Ser Gln Glu Asp Thr Val Ser Pro Thr Pro Glu Pro Gln Gly 930 935 940 Val Tyr Glu Asp Glu Glu Asp Glu Glu Pro Pro Ser Leu Thr Met Gly 945 950 955 960 Phe Asp His Thr Arg Arg His Met Gln 965 263138DNAUnknownsource/note="Description of Unknown DeltaPR sequence" 26atg cga tcc aag gcg agg gcg agg aag cta gcc aaa agt gac ggt gac 48Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 gtt gta aat aat atg tat gaa cct gac ccg gac ctg ctg gcc ggc cag 96Val Val Asn Asn Met Tyr Glu Pro Asp Pro Asp Leu Leu Ala Gly Gln 20 25 30 agt gcc gag gag gag acc gaa gac ggc atc ctg tcc ccc atc ccc atg 144Ser Ala Glu Glu Glu Thr Glu Asp Gly Ile Leu Ser Pro Ile Pro Met 35 40 45 ggg cca ccg tcc ccc ttc ccc acc agc gag gac ttc act ccc aag gag 192Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 ggc tcg ccc tat gag gct cct gtc tac att cct gaa gac att cca atc 240Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 cca cca gac ttc gag cta cga gag tcc tcc agc ttg gat gag gac ccc 288Pro Pro Asp Phe Glu Leu Arg Glu Ser Ser Ser Leu Asp Glu Asp Pro 85 90 95 aca ttc cgc tgt gat gag tgt gat gag ctc ttc cag tgc agg ctg gac 336Thr Phe Arg Cys Asp Glu Cys Asp Glu Leu Phe Gln Cys Arg Leu Asp 100 105 110 ctg agg cgc cac aag aag tac gcg tgc agc tct gca gga gcc cag ctc 384Leu Arg Arg His Lys Lys Tyr Ala Cys Ser Ser Ala Gly Ala Gln Leu 115 120 125 tac gag ggc cta ggg gag gaa ctc aag ccc gag ggc ctt ggc gtg ggc 432Tyr Glu Gly Leu Gly Glu Glu Leu Lys Pro Glu Gly Leu Gly Val Gly 130 135 140 agc gac ggg caa gcg cat gag tgc aag gat tgc gag cgg atg ttc ccc 480Ser Asp Gly Gln Ala His Glu Cys Lys Asp Cys Glu Arg Met Phe Pro 145 150 155 160 aac aag tac agc ttg gag caa cac atg atc gtc cac acg gaa gag cgt 528Asn Lys Tyr Ser Leu Glu Gln His Met Ile Val His Thr Glu Glu Arg 165 170 175 gag tac aaa tgt gac cag tgt ccc aag gcc ttc aac tgg aag tcc aac 576Glu Tyr Lys Cys Asp Gln Cys Pro Lys Ala Phe Asn Trp Lys Ser Asn 180 185 190 ctc atc cgc cac cag atg tct cac gac agt ggc aag cgc ttc gaa tgt 624Leu Ile Arg His Gln Met Ser His Asp Ser Gly Lys Arg Phe Glu Cys 195 200 205 gaa aac tgt gtc aag gtg ttc acg gac ccc agc aac ctc cag cgt cac 672Glu Asn Cys Val Lys Val Phe Thr Asp Pro Ser Asn Leu Gln Arg His 210 215 220 atc cgc tca cag cat gtc ggt gcc cgg gcc cat gcc tgc cct gac tgt 720Ile Arg Ser Gln His Val Gly Ala Arg Ala His Ala Cys Pro Asp Cys 225 230 235 240 ggc aag acc ttc gcc aca tcc tct ggc ctc aaa cag cac aag cat atc 768Gly Lys Thr Phe Ala Thr Ser Ser Gly Leu Lys Gln His Lys His Ile 245 250 255 cac agc acg gtg aag cca ttc ata tgc gag gtc tgc cac aag tcc tac 816His Ser Thr Val Lys Pro Phe Ile Cys Glu Val Cys His Lys Ser Tyr 260 265 270 acg cag ttc tcc aac ctg tgc cgg cac aag cgg atg cac gcc gac tgc 864Thr Gln Phe Ser Asn Leu Cys Arg His Lys Arg Met His Ala Asp Cys 275 280 285 agg acg cag atc aag tgc aag gac tgt ggg cag atg ttc agc act acc 912Arg Thr Gln Ile Lys Cys Lys Asp Cys Gly Gln Met Phe Ser Thr Thr 290 295 300 tcc tcc ctc aac aag cat cgg aga ttc tgc gag ggc aag aac cat tac 960Ser Ser Leu Asn Lys His Arg Arg Phe Cys Glu Gly Lys Asn His Tyr 305 310 315 320 acg cct ggc agc atc ttc acc cca ggc ctg ccc ttg acc ccc agc ccc 1008Thr Pro Gly Ser Ile Phe Thr Pro Gly Leu Pro Leu Thr Pro Ser Pro 325 330 335 atg atg gac aag aca aaa ccc tcc ccg acc ctc aac cac ggg ggc cta 1056Met Met Asp Lys Thr Lys Pro Ser Pro Thr Leu Asn His Gly Gly Leu 340 345 350 ggc ttc agc gag tac ttc ccc tcc aga cct cat cct ggg agc ctg ccc 1104Gly Phe Ser Glu Tyr Phe Pro Ser Arg Pro His Pro Gly Ser Leu Pro 355 360 365 ttc tcg gct gct cct ccg gcc ttc ccc gca ctc act ccg ggc ttc ccg 1152Phe Ser Ala Ala Pro Pro Ala Phe Pro Ala Leu Thr Pro Gly Phe Pro 370 375 380 ggc atc ttt cct cca tcc ctg tac cca cga cca cct ctg cta cct ccc 1200Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro Leu Leu Pro Pro 385 390 395 400 acg ccg ctg ctc aag agc ccc ctg aac cac gcg cag gac gcc aag cta 1248Thr Pro Leu Leu Lys Ser Pro Leu Asn His Ala Gln Asp Ala Lys Leu 405 410 415 ccc agc ccg ctg gga aac cca gcc ctg ccc ctt gtc tcc gcg gtc agc 1296Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val Ser Ala Val Ser 420 425 430 aat agc agc cag ggt gcc aca gcg gcc acc ggg tca gag gag aaa ttt 1344Asn Ser Ser Gln Gly Ala Thr Ala Ala Thr Gly Ser Glu Glu Lys Phe 435 440 445 gat ggc cgc ttg gaa gac gca tat gcg gag aag gtc aaa aat agg agc 1392Asp Gly Arg Leu Glu Asp Ala Tyr Ala Glu Lys Val Lys Asn Arg Ser 450 455 460 cct gac atg tcg gat ggc agt gac ttt gag gat atc aac acc acg acc 1440Pro Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Ile Asn Thr Thr Thr 465 470 475 480 ggg aca gac ttg gac act acc acg ggc acg ggg tca gac ctg gac agc 1488Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser Asp Leu Asp Ser 485 490 495 gac ctg gac agt gac aga gac aaa ggc aag gac aag ggg aag cca gtg 1536Asp Leu Asp Ser Asp Arg Asp Lys Gly Lys Asp Lys Gly Lys Pro Val 500 505 510 gag agc aaa cct gag ttt ggg ggt gca tct gtg ccc cct ggg gcc atg 1584Glu Ser Lys Pro Glu Phe Gly Gly Ala Ser Val Pro Pro Gly Ala Met 515 520 525 aac agt gtg gcc gag gta ccg gcc ttc tac tca cag cat tcc ttc ttc 1632Asn Ser Val Ala Glu Val Pro Ala Phe Tyr Ser Gln His Ser Phe Phe 530 535 540 ccg cca ccc gag gaa cag ctg ctg acg gcc tcg gga gct gcc ggc gac 1680Pro Pro Pro Glu Glu Gln Leu Leu Thr Ala Ser Gly Ala Ala Gly Asp 545 550 555 560 tcc atc aag gcc atc gcg tcc atc gcg gag aaa tac ttc ggt cct ggc 1728Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr Phe Gly Pro Gly 565 570 575 ttc atg agc atg cag gag aag aag ctg ggc tca cta ccc tac cac tcc 1776Phe Met Ser Met Gln Glu Lys Lys Leu Gly Ser Leu Pro Tyr His Ser 580 585 590 gtg ttc ccc ttc cag ttc ctg cct aac ttt ccc cac tcc ctc tac ccc 1824Val Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His Ser Leu Tyr Pro 595 600 605 ttt acg gac cga gcc ctc gcc cac aac ttg ctg gtc aag gct gag cca 1872Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val Lys Ala Glu Pro 610 615 620 aag tca ccc cgg gat gcc ctc aag gtg ggc ggc ccc agt gcg gag tgc 1920Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro Ser Ala Glu Cys 625 630 635 640 ccc ttc gac ctc acc acc aaa cca aaa gag gcc aaa ccc gcc ctg ctc 1968Pro Phe Asp Leu Thr Thr Lys Pro Lys Glu Ala Lys Pro Ala Leu Leu 645 650 655 gca ccc aag gtc ccc ctc atc ccc tca tct ggc gag gaa cag cca ctg 2016Ala Pro Lys Val Pro Leu Ile Pro Ser Ser Gly Glu Glu Gln Pro Leu 660 665 670 gac ctg agc atc ggc agc agg gcc agg gca agc cag aac gga ggt ggc 2064Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln Asn Gly Gly Gly 675 680 685 cgt gag ccg cgg aag aac cac gtc tac ggt gaa cgg aag ccg ggg gtc 2112Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg Lys Pro Gly Val 690 695 700 agc gag ggg ctg cct aag gtg tgc cca gca cag ctg ccc cag cag ccc 2160Ser Glu Gly Leu Pro Lys Val Cys Pro Ala Gln Leu Pro Gln Gln Pro 705 710 715 720 tcc ttg cat tat gct aag cct tca ccg ttc ttc atg gat ccc atc tac 2208Ser Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met Asp Pro Ile Tyr 725 730 735 agc agg gta gaa aag cgg aag gtg gca gac cct gtg gga gtc ctg aaa 2256Ser Arg Val Glu Lys Arg Lys Val Ala Asp Pro Val Gly Val Leu Lys 740 745 750 gag aag tac ctg cgg ccg tcc cca ctt ctg ttc cac ccc cag atg tca 2304Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His Pro Gln Met Ser 755 760 765 gcc ata gaa acc atg acg gag aag ctg gag agc ttt gca gcc atg aag 2352Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe Ala Ala Met Lys 770 775 780 gcc gac tca ggc agc tcc ctg cag ccc ctg cct cac cac ccg ttc aac 2400Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His His Pro Phe Asn 785 790 795 800 ttc cgc tcc cca ccc cca acg ctc tcg gat ccc atc ctc agg aag ggg 2448Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile Leu Arg Lys Gly 805 810 815 aag gag aga tac acg tgc agg tac tgt ggc aag atc ttc ccc aga tct 2496Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile Phe Pro Arg Ser 820 825 830 gca aat ctc aca aga cat ctg agg aca cac aca ggg gag cag cca tac 2544Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Gln Pro Tyr 835 840 845 agg tgc aag tac tgt gac cgg tca ttc agc atc tcc tcc aac ctc cag 2592Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser Ser Asn Leu Gln 850 855 860 cgg cac gtg agg aac atc cac aac aaa gag aag ccg ttc aag tgc cat 2640Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro Phe Lys Cys His 865 870 875 880 ctg tgc aac cgc tgc ttc ggg cag cag acc aac cta gac cgg cac ctg 2688Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu Asp Arg His Leu 885 890 895 aag aag cac gaa cac gag ggc gca cca gtg agc cag cac tcc ggg gtg 2736Lys Lys His Glu His Glu Gly Ala Pro Val Ser Gln His Ser Gly Val 900 905 910 ctc acg aac cac ctg ggc acc agc gcc tcc tcc ccc acc tcc gag tcg 2784Leu Thr Asn His Leu Gly Thr Ser Ala Ser Ser Pro Thr Ser Glu Ser 915 920 925 gac aac cat gca ctt tta gat gag aag gaa gat tct tac ttc tcc gag 2832Asp Asn His Ala Leu Leu Asp Glu Lys Glu Asp Ser Tyr Phe Ser Glu 930 935 940 atc cga aac ttc atc gcc aac agc gag atg aac cag gca tcc act cga 2880Ile Arg Asn Phe Ile Ala Asn Ser Glu Met Asn Gln Ala Ser Thr Arg 945 950 955 960 atg gac aaa cgg cct gag atc caa gac ctg gac agc aac cca ccg tgt 2928Met Asp Lys Arg Pro Glu Ile Gln Asp Leu Asp Ser Asn Pro Pro Cys 965 970 975 cca ggc tca gcc agt gca aag cca gag gac gta gag gag gag gaa gag 2976Pro Gly Ser Ala Ser Ala Lys Pro Glu Asp Val Glu Glu Glu Glu Glu 980 985 990 gag gag ctg gag gaa gag gat gat gac agc tta gcc ggg aag tca cag 3024Glu Glu Leu Glu Glu Glu Asp Asp Asp Ser Leu Ala Gly Lys Ser Gln

995 1000 1005 gag gac acg gtg tcc ccc aca cct gag ccc caa gga gtc tat gaa 3069Glu Asp Thr Val Ser Pro Thr Pro Glu Pro Gln Gly Val Tyr Glu 1010 1015 1020 gat gaa gag gat gag gaa cca ccc agc ctg acc atg ggc ttt gac 3114Asp Glu Glu Asp Glu Glu Pro Pro Ser Leu Thr Met Gly Phe Asp 1025 1030 1035 cat acc cgg agg cat atg caa tga 3138His Thr Arg Arg His Met Gln 1040 1045 271045PRTUnknownsource/note="Description of Unknown DeltaPR sequence" 27Met Arg Ser Lys Ala Arg Ala Arg Lys Leu Ala Lys Ser Asp Gly Asp 1 5 10 15 Val Val Asn Asn Met Tyr Glu Pro Asp Pro Asp Leu Leu Ala Gly Gln 20 25 30 Ser Ala Glu Glu Glu Thr Glu Asp Gly Ile Leu Ser Pro Ile Pro Met 35 40 45 Gly Pro Pro Ser Pro Phe Pro Thr Ser Glu Asp Phe Thr Pro Lys Glu 50 55 60 Gly Ser Pro Tyr Glu Ala Pro Val Tyr Ile Pro Glu Asp Ile Pro Ile 65 70 75 80 Pro Pro Asp Phe Glu Leu Arg Glu Ser Ser Ser Leu Asp Glu Asp Pro 85 90 95 Thr Phe Arg Cys Asp Glu Cys Asp Glu Leu Phe Gln Cys Arg Leu Asp 100 105 110 Leu Arg Arg His Lys Lys Tyr Ala Cys Ser Ser Ala Gly Ala Gln Leu 115 120 125 Tyr Glu Gly Leu Gly Glu Glu Leu Lys Pro Glu Gly Leu Gly Val Gly 130 135 140 Ser Asp Gly Gln Ala His Glu Cys Lys Asp Cys Glu Arg Met Phe Pro 145 150 155 160 Asn Lys Tyr Ser Leu Glu Gln His Met Ile Val His Thr Glu Glu Arg 165 170 175 Glu Tyr Lys Cys Asp Gln Cys Pro Lys Ala Phe Asn Trp Lys Ser Asn 180 185 190 Leu Ile Arg His Gln Met Ser His Asp Ser Gly Lys Arg Phe Glu Cys 195 200 205 Glu Asn Cys Val Lys Val Phe Thr Asp Pro Ser Asn Leu Gln Arg His 210 215 220 Ile Arg Ser Gln His Val Gly Ala Arg Ala His Ala Cys Pro Asp Cys 225 230 235 240 Gly Lys Thr Phe Ala Thr Ser Ser Gly Leu Lys Gln His Lys His Ile 245 250 255 His Ser Thr Val Lys Pro Phe Ile Cys Glu Val Cys His Lys Ser Tyr 260 265 270 Thr Gln Phe Ser Asn Leu Cys Arg His Lys Arg Met His Ala Asp Cys 275 280 285 Arg Thr Gln Ile Lys Cys Lys Asp Cys Gly Gln Met Phe Ser Thr Thr 290 295 300 Ser Ser Leu Asn Lys His Arg Arg Phe Cys Glu Gly Lys Asn His Tyr 305 310 315 320 Thr Pro Gly Ser Ile Phe Thr Pro Gly Leu Pro Leu Thr Pro Ser Pro 325 330 335 Met Met Asp Lys Thr Lys Pro Ser Pro Thr Leu Asn His Gly Gly Leu 340 345 350 Gly Phe Ser Glu Tyr Phe Pro Ser Arg Pro His Pro Gly Ser Leu Pro 355 360 365 Phe Ser Ala Ala Pro Pro Ala Phe Pro Ala Leu Thr Pro Gly Phe Pro 370 375 380 Gly Ile Phe Pro Pro Ser Leu Tyr Pro Arg Pro Pro Leu Leu Pro Pro 385 390 395 400 Thr Pro Leu Leu Lys Ser Pro Leu Asn His Ala Gln Asp Ala Lys Leu 405 410 415 Pro Ser Pro Leu Gly Asn Pro Ala Leu Pro Leu Val Ser Ala Val Ser 420 425 430 Asn Ser Ser Gln Gly Ala Thr Ala Ala Thr Gly Ser Glu Glu Lys Phe 435 440 445 Asp Gly Arg Leu Glu Asp Ala Tyr Ala Glu Lys Val Lys Asn Arg Ser 450 455 460 Pro Asp Met Ser Asp Gly Ser Asp Phe Glu Asp Ile Asn Thr Thr Thr 465 470 475 480 Gly Thr Asp Leu Asp Thr Thr Thr Gly Thr Gly Ser Asp Leu Asp Ser 485 490 495 Asp Leu Asp Ser Asp Arg Asp Lys Gly Lys Asp Lys Gly Lys Pro Val 500 505 510 Glu Ser Lys Pro Glu Phe Gly Gly Ala Ser Val Pro Pro Gly Ala Met 515 520 525 Asn Ser Val Ala Glu Val Pro Ala Phe Tyr Ser Gln His Ser Phe Phe 530 535 540 Pro Pro Pro Glu Glu Gln Leu Leu Thr Ala Ser Gly Ala Ala Gly Asp 545 550 555 560 Ser Ile Lys Ala Ile Ala Ser Ile Ala Glu Lys Tyr Phe Gly Pro Gly 565 570 575 Phe Met Ser Met Gln Glu Lys Lys Leu Gly Ser Leu Pro Tyr His Ser 580 585 590 Val Phe Pro Phe Gln Phe Leu Pro Asn Phe Pro His Ser Leu Tyr Pro 595 600 605 Phe Thr Asp Arg Ala Leu Ala His Asn Leu Leu Val Lys Ala Glu Pro 610 615 620 Lys Ser Pro Arg Asp Ala Leu Lys Val Gly Gly Pro Ser Ala Glu Cys 625 630 635 640 Pro Phe Asp Leu Thr Thr Lys Pro Lys Glu Ala Lys Pro Ala Leu Leu 645 650 655 Ala Pro Lys Val Pro Leu Ile Pro Ser Ser Gly Glu Glu Gln Pro Leu 660 665 670 Asp Leu Ser Ile Gly Ser Arg Ala Arg Ala Ser Gln Asn Gly Gly Gly 675 680 685 Arg Glu Pro Arg Lys Asn His Val Tyr Gly Glu Arg Lys Pro Gly Val 690 695 700 Ser Glu Gly Leu Pro Lys Val Cys Pro Ala Gln Leu Pro Gln Gln Pro 705 710 715 720 Ser Leu His Tyr Ala Lys Pro Ser Pro Phe Phe Met Asp Pro Ile Tyr 725 730 735 Ser Arg Val Glu Lys Arg Lys Val Ala Asp Pro Val Gly Val Leu Lys 740 745 750 Glu Lys Tyr Leu Arg Pro Ser Pro Leu Leu Phe His Pro Gln Met Ser 755 760 765 Ala Ile Glu Thr Met Thr Glu Lys Leu Glu Ser Phe Ala Ala Met Lys 770 775 780 Ala Asp Ser Gly Ser Ser Leu Gln Pro Leu Pro His His Pro Phe Asn 785 790 795 800 Phe Arg Ser Pro Pro Pro Thr Leu Ser Asp Pro Ile Leu Arg Lys Gly 805 810 815 Lys Glu Arg Tyr Thr Cys Arg Tyr Cys Gly Lys Ile Phe Pro Arg Ser 820 825 830 Ala Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Gln Pro Tyr 835 840 845 Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser Ser Asn Leu Gln 850 855 860 Arg His Val Arg Asn Ile His Asn Lys Glu Lys Pro Phe Lys Cys His 865 870 875 880 Leu Cys Asn Arg Cys Phe Gly Gln Gln Thr Asn Leu Asp Arg His Leu 885 890 895 Lys Lys His Glu His Glu Gly Ala Pro Val Ser Gln His Ser Gly Val 900 905 910 Leu Thr Asn His Leu Gly Thr Ser Ala Ser Ser Pro Thr Ser Glu Ser 915 920 925 Asp Asn His Ala Leu Leu Asp Glu Lys Glu Asp Ser Tyr Phe Ser Glu 930 935 940 Ile Arg Asn Phe Ile Ala Asn Ser Glu Met Asn Gln Ala Ser Thr Arg 945 950 955 960 Met Asp Lys Arg Pro Glu Ile Gln Asp Leu Asp Ser Asn Pro Pro Cys 965 970 975 Pro Gly Ser Ala Ser Ala Lys Pro Glu Asp Val Glu Glu Glu Glu Glu 980 985 990 Glu Glu Leu Glu Glu Glu Asp Asp Asp Ser Leu Ala Gly Lys Ser Gln 995 1000 1005 Glu Asp Thr Val Ser Pro Thr Pro Glu Pro Gln Gly Val Tyr Glu 1010 1015 1020 Asp Glu Glu Asp Glu Glu Pro Pro Ser Leu Thr Met Gly Phe Asp 1025 1030 1035 His Thr Arg Arg His Met Gln 1040 1045 282651DNAMus musculus 28attcgcgacc cgaagctgcg cgggcgcgag ccagttgggg cactgggtgg gcggcggcga 60cagcggcgcc acgcgcaggc tggaggccgc cgaggctcgc catgccggga gaactctaac 120tcccccatgg agtcggccga cttctacgag gtggagccgc ggcccccgat gagcagtcac 180ctccagagcc ccccgcacgc gcccagcaac gccgcctttg gctttccccg gggcgcgggc 240cccgcgccgc ccccagcccc acctgccgcc ccggagccgc tgggcggcat ctgcgagcac 300gagacgtcta tagacatcag cgcctacatc gacccggccg ccttcaacga cgagttcctg 360gccgacctct tccagcacag ccgacagcag gagaaggcca aggcggcggc gggccccgcg 420ggtggcggcg gtgactttga ctacccggga gccccggcgg gccccggcgg cgcggtcatg 480tccgcggggg cgcacgggcc ccctcccggc tacggctgtg cggcggccgg ctacctggac 540ggcaggctgg agcccctgta cgagcgcgtc ggggcgcccg cgctacggcc gctggtgatc 600aaacaagagc cccgcgagga ggacgaggcg aagcagctgg cgctggccgg cctcttcccc 660taccagccac cgccgccacc gccaccgccg cacccgcacg cgtctcccgc gcacctggcc 720gccccccact tgcagttcca gatcgcgcac tgcggccaga ccaccatgca cctgcagcct 780ggccacccca caccgccgcc cacgcccgtg cccagcccgc acgctgcgcc cgccttgggt 840gctgcgggcc tgcctggccc cgggagcgcg ctcaagggct tggccggtgc gcaccccgac 900ctccgcacgg gaggcggcgg cggtggcagc ggtgccggtg cgggcaaagc caagaagtcg 960gtggacaaga acagcaacga gtaccgggta cggcgggaac gcaacaacat cgcggtgcgc 1020aagagccgag ataaagccaa acaacgcaac gtggagacgc aacagaaggt gctggagttg 1080accagtgaca atgaccgcct gcgcaagcgg gtggaacagc tgagccgtga actggacacg 1140ctgcggggca tcttccgcca gctgcctgag agctccttgg tcaaggccat gggcaactgc 1200gcgtgaggcg cgcggctgcg ggaccgcctt gggccggccc cctggctgga gacccagagg 1260atggtttcgg gtcgctggat ctctaggctg cccgggccgc gcaagccagg actaggagat 1320tccggtgtgg cctgaaagcc tggcctgctc cgcgtgtccc ctcccttcct ctgagccgga 1380ctcggtgcgt ctaagatgag ggagtcaggc cgtggtggtt tctccttgag accgagagac 1440tttccgcgga gctgagctgg gggcccggca gtactagtat taaggaagta accttgtgcc 1500ttggatactc aaaactcgct ccttttccta ccgagtaggg ggagcaaaaa tgtgccttga 1560tattttattt ggaggattcc tgcttcctct cgggcctcag ctggccccgt gagaaaaatg 1620aagggtgcag gcccagggca ggaggaagat acaggaagct gagatcccgg cagtgccctg 1680agctgcccct cagtccctgt ctttagaggg gagggactta ggtgttgggg atttgagtct 1740gtgtcctcac ccccagctac agggaggtgg agggctccta atcccttgct ttttgcacct 1800ccacctacat cccccccccc ccactcagct tacaacaggc caggtttcct gggtgagttc 1860atggagaatg ggggcaccac ccccagtcag accagaaagc tgagttgtga gttagccatg 1920tggtaggaga cagagaccta ggtttctggg ctttgtgggg tgggggatag gaggacacgg 1980ggaccattag ccttgtgtgt actgtatgtc gccagccgct gttgctgaag gaacttgaag 2040cacaatcgat ccatcccaga gggactggag ttatgacaag cttcccaaat attttgcttt 2100atcatccgat atcaacactt gtatctggtc tctgtgtccc agcggtgcct tgtgcaatgg 2160cagtgtgcac gtctatgcta aaccaccatt ttatttggtc ttttgttttg ttttggtttt 2220gctctgattc ttgccaaact gagactcttc actaacggct gggggaagga gctgagtgag 2280gctctcattc tttttggttt agggatgttt gggttttttc gtctgcctcc cagaggacca 2340atgaaatgaa gtgggcttcc ccctctcccc tagttgtcca agggtgtatg tagtagtggg 2400tcttagcttc ctccggctaa gacttaggct tccccaccca cccaacccca tccccaacgg 2460ccctggctct gggtctggaa agaaggccac ctccagccag ttcatacaca cacccctgtg 2520gctgggagca gggctggacc gcttccttct cttctttttt ttgggggggg gggacacaaa 2580gtttcatgct agatgtcgta tgtattatat ctataatata aacatatcaa actcaaaaaa 2640aaaaaaaaaa a 265129359PRTMus musculus 29Met Glu Ser Ala Asp Phe Tyr Glu Val Glu Pro Arg Pro Pro Met Ser 1 5 10 15 Ser His Leu Gln Ser Pro Pro His Ala Pro Ser Asn Ala Ala Phe Gly 20 25 30 Phe Pro Arg Gly Ala Gly Pro Ala Pro Pro Pro Ala Pro Pro Ala Ala 35 40 45 Pro Glu Pro Leu Gly Gly Ile Cys Glu His Glu Thr Ser Ile Asp Ile 50 55 60 Ser Ala Tyr Ile Asp Pro Ala Ala Phe Asn Asp Glu Phe Leu Ala Asp 65 70 75 80 Leu Phe Gln His Ser Arg Gln Gln Glu Lys Ala Lys Ala Ala Ala Gly 85 90 95 Pro Ala Gly Gly Gly Gly Asp Phe Asp Tyr Pro Gly Ala Pro Ala Gly 100 105 110 Pro Gly Gly Ala Val Met Ser Ala Gly Ala His Gly Pro Pro Pro Gly 115 120 125 Tyr Gly Cys Ala Ala Ala Gly Tyr Leu Asp Gly Arg Leu Glu Pro Leu 130 135 140 Tyr Glu Arg Val Gly Ala Pro Ala Leu Arg Pro Leu Val Ile Lys Gln 145 150 155 160 Glu Pro Arg Glu Glu Asp Glu Ala Lys Gln Leu Ala Leu Ala Gly Leu 165 170 175 Phe Pro Tyr Gln Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala 180 185 190 Ser Pro Ala His Leu Ala Ala Pro His Leu Gln Phe Gln Ile Ala His 195 200 205 Cys Gly Gln Thr Thr Met His Leu Gln Pro Gly His Pro Thr Pro Pro 210 215 220 Pro Thr Pro Val Pro Ser Pro His Ala Ala Pro Ala Leu Gly Ala Ala 225 230 235 240 Gly Leu Pro Gly Pro Gly Ser Ala Leu Lys Gly Leu Ala Gly Ala His 245 250 255 Pro Asp Leu Arg Thr Gly Gly Gly Gly Gly Gly Ser Gly Ala Gly Ala 260 265 270 Gly Lys Ala Lys Lys Ser Val Asp Lys Asn Ser Asn Glu Tyr Arg Val 275 280 285 Arg Arg Glu Arg Asn Asn Ile Ala Val Arg Lys Ser Arg Asp Lys Ala 290 295 300 Lys Gln Arg Asn Val Glu Thr Gln Gln Lys Val Leu Glu Leu Thr Ser 305 310 315 320 Asp Asn Asp Arg Leu Arg Lys Arg Val Glu Gln Leu Ser Arg Glu Leu 325 330 335 Asp Thr Leu Arg Gly Ile Phe Arg Gln Leu Pro Glu Ser Ser Leu Val 340 345 350 Lys Ala Met Gly Asn Cys Ala 355 302260DNAMus musculus 30gacagcccaa cttggacgcc aggtccggcc gacgccgcca tgagcgccgc gcttttcagc 60ctggacagcc cggtgcgcgg cacaccctgg cccacagaac ccgcggcctt ctacgagcca 120ggcagggtgg acaagcccgg ccgagggccc gagccagggg atctggggga gctgggctcc 180acgactcctg ccatgtacga cgacgagagc gccatcgact tcagcgccta cattgactcc 240atggccgccg tgcccaccct agagctgtgc cacgacgaac tcttcgccga cctcttcaac 300agcaaccaca aagcggccgg cgcgggcggc ctggagctgc tgcagggcgg ccctacgcga 360cccccgggtg tggggtctgt cgctaggggg ccgctcaagc gcgaacccga ctggggcgac 420ggcgacgcgc cgggctccct gctgccggcg caagtggcgg tgtgcgcgca gacagtggtg 480agcttggcgg ccgcggctca gcccactcca cccacttcgc cggagcctcc tcgaggcagc 540ccggggccga gcctcgcgcc cggcacagtc cgagaaaagg gcgcgggcaa gaggggtccg 600gaccgcggca gcccggagta ccggcagcgg cgcgagcgca acaacatcgc tgtgcgcaag 660agccgcgaca aggccaagcg ccgcaaccag gagatgcagc agaagctggt ggagttgtcg 720gccgagaacg agaagctgca tcagcgcgtg gagcagctca cccgggacct ggctggcctc 780cggcagttct tcaaaaaact gcccagcccg cctttcctgc cgcccaccgg cgccgactgc 840cggtaacgcg cggcgtgggc ctttgagact ctgaacgacc tatacctcag accccgacag 900cggggagcag acgccgcccg aatcgctagt ttctttggga cctgcgagcg acaggaagct 960gcagcttggg cactggactg cgagagaagc tatattaatc tttcccctta aattattttt 1020tataatggta gcattttcta cgtcttatta ccattgcagc taaggtacat ttgtagaaaa 1080gacatttccg acagactttt gtagataaga ggaagagact gcgcatgctt tttatattca 1140tttttacagt atttgtaaga ataaagaagc atttaaatcg ctgcagcttc ctatgttcat 1200tctctcccgc acacaacata ctgagacttc accagggaaa agcaagcacc tgggagccac 1260ccgaccaggt gcgttgccca cacggggtaa ggagatggac gcgtttcctc gcggtctcta 1320ttgcccgcgg aaggaacacg ggaaagcatg actaattcat gtgtgtgatc ccagagtagg 1380ctgacctggg gcggagaaca gttggcctaa cttttaggtg gttgccgaag ggtgaggggg 1440tggaagagag ctgggagtct gaaacttgat tcctcgttgc ctctactttc ctcaatctag 1500ggacaacgtg tagatttaaa aatgtgtacc agtacaaatt aatatcctag tatataaaaa 1560gggcagcccc aaaagccagt aattgtaatg tttgcgggct taaccccccc cccccccaaa 1620gctatgtgcc tttctactaa gatactggtt gttccaaccc cttccctgat ctgcacggcc 1680tgttgtacag aaaacacaaa ccctccaggg tctaaataca tagctttgtt tgtaattcaa 1740atccctgccc aaagtgcagg cttgtggact gctgtgagtc tgtgatcttt gccctctgca 1800gttcttcagg gtctctgaca ggtgggcagt ggagtaaggt acagaaattt ctcttttata 1860cagctctcct cacacccaac attgtatggg tgacagtgcc acctctggca gctcccagaa 1920cactaaaacc acaaagtgtt taggttggac atttatttat tgtaacagaa cagtaatatt 1980tcaatatgag atactggctg aagctttaca gaacaatcta gcagagaata ttagatactg 2040caaagaaaaa acatatataa tattgataaa ttgtaactac tttaaagcta caacaaagca 2100catttcctaa gcccaagtag ctgcaaaatg aagacacaga agacatgaga aacactcaaa 2160gaaacatgtt atcaaacttg taggaaggta aagtttaaaa aaatgtgtat tagaggtatc 2220ttgattttta atttaaaact atcctattaa tctctttacc 226031268PRTMus musculus 31Met Ser Ala Ala Leu Phe Ser Leu Asp Ser Pro Val Arg Gly Thr Pro 1 5 10 15 Trp Pro Thr Glu Pro Ala Ala Phe Tyr Glu Pro

Gly Arg Val Asp Lys 20 25 30 Pro Gly Arg Gly Pro Glu Pro Gly Asp Leu Gly Glu Leu Gly Ser Thr 35 40 45 Thr Pro Ala Met Tyr Asp Asp Glu Ser Ala Ile Asp Phe Ser Ala Tyr 50 55 60 Ile Asp Ser Met Ala Ala Val Pro Thr Leu Glu Leu Cys His Asp Glu 65 70 75 80 Leu Phe Ala Asp Leu Phe Asn Ser Asn His Lys Ala Ala Gly Ala Gly 85 90 95 Gly Leu Glu Leu Leu Gln Gly Gly Pro Thr Arg Pro Pro Gly Val Gly 100 105 110 Ser Val Ala Arg Gly Pro Leu Lys Arg Glu Pro Asp Trp Gly Asp Gly 115 120 125 Asp Ala Pro Gly Ser Leu Leu Pro Ala Gln Val Ala Val Cys Ala Gln 130 135 140 Thr Val Val Ser Leu Ala Ala Ala Ala Gln Pro Thr Pro Pro Thr Ser 145 150 155 160 Pro Glu Pro Pro Arg Gly Ser Pro Gly Pro Ser Leu Ala Pro Gly Thr 165 170 175 Val Arg Glu Lys Gly Ala Gly Lys Arg Gly Pro Asp Arg Gly Ser Pro 180 185 190 Glu Tyr Arg Gln Arg Arg Glu Arg Asn Asn Ile Ala Val Arg Lys Ser 195 200 205 Arg Asp Lys Ala Lys Arg Arg Asn Gln Glu Met Gln Gln Lys Leu Val 210 215 220 Glu Leu Ser Ala Glu Asn Glu Lys Leu His Gln Arg Val Glu Gln Leu 225 230 235 240 Thr Arg Asp Leu Ala Gly Leu Arg Gln Phe Phe Lys Lys Leu Pro Ser 245 250 255 Pro Pro Phe Leu Pro Pro Thr Gly Ala Asp Cys Arg 260 265 326464DNAMus musculus 32gtcatgtgac tggggactgt agtaagacag gtgccttcag ttcactctca gtaaggggct 60ggttgcctgc atgagtgtgt gctgtgtgtc agagtggatt ggagttgaaa aagcttgact 120ggcgtcattc gggagctgga tggcttggga catgtgcagc caagactctg tatggagtga 180catagagtgt gctgctctgg ttggtgagga ccagcctctt tgcccagatc ttcctgaact 240tgacctttct gaacttgatg tgaatgactt ggatacagac agctttctgg gtggattgaa 300gtggtgtagc gaccaatcgg aaatcatatc caaccagtac aacaatgagc ctgcgaacat 360atttgagaag atagatgaag agaatgaggc aaacttgcta gcggttctca cagagacact 420ggacagtctc cccgtggatg aagacggatt gccctcattt gatgcactga cagatggagc 480cgtgaccact gacaacgagg ccagtccttc ctccatgcct gacggcaccc ctccccctca 540ggaggcagaa gagccgtctc tacttaagaa gctcttactg gcaccagcca acactcagct 600cagctacaat gaatgcagcg gtcttagcac tcagaaccat gcagcaaacc acacccacag 660gatcagaaca aaccctgcca ttgttaagac cgagaattca tggagcaata aagcgaagag 720catttgtcaa cagcaaaagc cacaaagacg tccctgctca gagcttctca agtatctgac 780cacaaacgat gaccctcctc acaccaaacc cacagaaaac aggaacagca gcagagacaa 840atgtgcttcg aaaaagaagt cccatacaca accgcagtcg caacatgctc aagccaaacc 900aacaacttta tctcttcctc tgaccccaga gtcaccaaat gaccccaagg gttccccatt 960tgagaacaag actattgagc gaaccttaag tgtggaactc tctggaactg caggcctaac 1020tcctcccaca actcctcctc ataaagccaa ccaagataac cctttcaagg cttcgccaaa 1080gctgaagccc tcttgcaaga ccgtggtgcc accgccaacc aagagggccc ggtacagtga 1140gtgttctggt acccaaggca gccactccac caagaaaggg cccgagcaat ctgagttgta 1200cgcacaactc agcaagtcct cagggctcag ccgaggacac gaggaaagga agactaaacg 1260gcccagtctc cggctgtttg gtgaccatga ctactgtcag tcactcaatt ccaaaacgga 1320tatactcatt aacatatcac aggagctcca agactctaga caactagact tcaaagatgc 1380ctcctgtgac tggcaggggc acatctgttc ttccacagat tcaggccagt gctacctgag 1440agagactttg gaggccagca agcaggtctc tccttgcagc accagaaaac agctccaaga 1500ccaggaaatc cgagcggagc tgaacaagca cttcggtcat ccctgtcaag ctgtgtttga 1560cgacaaatca gacaagacca gtgaactaag ggatggcgac ttcagtaatg aacaattctc 1620caaactacct gtgtttataa attcaggact agccatggat ggcctatttg atgacagtga 1680agatgaaagt gataaactga gctacccttg ggatggcacg cagccctatt cattgttcga 1740tgtgtcgcct tcttgctctt cctttaactc tccgtgtcga gactcagtgt caccaccgaa 1800atccttattt tctcaaagac cccaaaggat gcgctctcgt tcaagatcct tttctcgaca 1860caggtcgtgt tcccgatcac catattccag gtcaagatca aggtccccag gcagtagatc 1920ctcttcaaga tcctgttact actatgaatc aagccactac agacaccgca cacaccgcaa 1980ttctcccttg tatgtgagat cacgttcaag gtcaccctac agccgtaggc ccaggtacga 2040cagctatgaa gcctatgagc acgaaaggct caagagggat gaataccgca aagagcacga 2100gaagcgggag tctgaaaggg ccaaacagag agagaggcag aagcagaaag caattgaaga 2160gcgccgtgtg atttacgttg gtaaaatcag acctgacaca acgcggacag aattgagaga 2220ccgctttgaa gtttttggtg aaattgagga atgcaccgta aatctgcggg atgatggaga 2280cagctatggt ttcatcacct accgttacac ctgtgacgct ttcgctgctc ttgagaatgg 2340atatacttta cgcaggtcga acgaaactga cttcgagctg tacttttgtg gacggaagca 2400atttttcaag tctaactatg cagacctaga taccaactca gacgattttg accctgcttc 2460caccaagagc aagtatgact ctctggattt tgatagttta ctgaaggaag ctcagagaag 2520cttgcgcagg taacgtgttc ccaggctgag gaatgacaga gagatggtca atacctcatg 2580ggacagcgtg tcctttccca agactcttgc aagtcatact taggaatttc tcctacttta 2640cactctctgt acaaaaataa aacaaaacaa aacaacaata acaacaacaa caacaacaat 2700aacaacaaca accataccag aacaagaaca acggtttaca tgaacacagc tgctgaagag 2760gcaagagaca gaatgataat ccagtaagca cacgtttatt cacgggtgtc agctttgctt 2820tccctggagg ctcttggtga cagtgtgtgt gcgtgtgtgt gtgtgggtgt gcgtgtgtgt 2880atgtgtgtgt gtgtacttgt ttggaaagta catatgtaca catgtgagga cttgggggca 2940cctgaacaga acgaacaagg gcgacccctt caaatggcag catttccatg aagacacact 3000taaaacctac aacttcaaaa tgttcgtatt ctatacaaaa ggaaaataaa taaatataaa 3060ttaaaaggaa agaaaactca caaaccaccc taaaatgaca ctgctgatgc ctgttgtcag 3120cctccggtac cgtcttttca gaaagtgcaa aacccagaaa gtgcaaaacc aacctgcagc 3180aagctctctc tctctcttaa tgtaatcatt acgtgacaat cccgaagaca ctacaggttc 3240catagaactc atatccacct ctctctctct ctctctctct ctctctctct ctctctctct 3300cctctctcct ctctcctctc tccctccctt ctttgccatt gaatctgggt gggagaggat 3360actgcaggca ccagatgcta aactttccta acattttgaa gtttctgtag tttgtccttt 3420gtcctgacac ctatgtatat gttcaaaatg ttgatcttcc actgcagatt ttgaaaagcc 3480ttgttattgg tcaagcgggg agtgtgttca gtggctcctt ctgaggagca gacgcggtgt 3540tacatgagta ctgagagttg agtagaactc tctggatgtg ttcagatagt gtaattgcta 3600cattctctga tgtagttaag tatttacaga tgttaaatgg agtattttta ttttatgtac 3660atactctaca actatgttct tttttgttac agctatgcac tgtaaatgca gccttctttt 3720caaaactgct aaatttttct taatcaagaa tattcaaatg taattatgag gtgaaacaat 3780tattgtacac taacatattt agaagctaaa cttactgctt atatatattt gattgtaaaa 3840aaaaaaaaaa acaaaaccaa caaaacaaaa gacagtgtgt gtgtgtgtgt ccgttgagtg 3900caagtccaac aaaatggcgc ttcacgcaca tccatccctt cttaggtgag cttcaatcta 3960agcatcttgt caacaacaac aaaaatccta ggcccctcaa ggtattaacc acttctgcaa 4020tatttttcca cattttcttg ttgcttgttt ttctttgaag ttttatacac tggatttgtt 4080aggggaatga aattttctca tctaaaattt ttctagacaa tatcatgatt ttatgtaaag 4140tctctcaatg gggaaccatt aagaaatgtt tttattttct ctatcaacag tagatttgaa 4200actagaggtc aaaaaaaatc tttttaaaat gctgttttgt tttaattttt gtgattttaa 4260tttgatacaa aatgctgagg taataattac agtatgattt ttacaatagt caatgtgtgt 4320ctgaagacta tctttgaagc cagtatctct ttcccttggc agagtatgat gatggtattt 4380aatctgtatt ttttacagtt atacatcctg taaaatactg atatttcatt cctttgttta 4440ctaaagagac atatttatca gttgcagata gcctatttat tataaattaa gagatgatga 4500aaataataag gtcagtggag actttctacc cagggtgcat ggcagttgtc aggctggagt 4560gtaccttctt cgtttgggaa actcagctct cgcagaagca gtgttccatc tttcactagc 4620atggcctctg atacgaccat ggtgttgttc ttggtgacat tgcttctgct aaatttaata 4680ttaataataa taaatgtcag aaaaaaaacc ctccattttg agcatcagga tttcatctga 4740gtatggagtc gctgccatgg gagtcactaa actttggagt atgtatttca tttccaaatt 4800gagatgcatt tactgtttgg ctgacatgaa ttttctggaa gatatgatag acctactact 4860taaccgtttt tgtttgtttt tttttctttg ttgttgttgt tttgtttttt gtttttttgt 4920ttttctctct cacccaacac tatcttacaa aatgggtttc acccccaggc caatgcagct 4980aattttgaca gctgcattca tttatcacca gcatattgtg ttctgagtga atccactgtc 5040tgtcctgtcg aatgcttgct caagtgtttg gcttattatt tctaagtaga tagaaagcaa 5100taaataacta tgaaataaaa aagaattgtg ttcacaggtt ctgcgttaca acagtaacac 5160atctttaatc cgcctaattc ttgttctgta ggataaatgc aggtatttta actctttgtg 5220aacgccaaac taaagtttac agtctttctt tctgaatttt gagtatcttc tgttgtagaa 5280taataataaa aagactatta agagcaataa attattttta agaaatcaat atttagtaaa 5340tcctgttatg tgtttaagga ccagatgcgt tctctatttt gcctttaaat ttttgtgatc 5400caactttaaa aacatacgtt gtcttgtttg ccctggatca tggacatgac taaaattttg 5460tggtttcttt tcttacttat caaaagacaa cactacagat ttcatgttga ggattcattg 5520agctctcacc ctctggcctg acaaatcttg ttaccatgaa gatagttttc ctccgtggac 5580ttcaaattgc atctaaaatt agtgaagctt gtgtatctta tgcagacact gtgggtagcc 5640catcaaaata taagctgtaa gctttgttcc tttcattttt ttttttttac ttcttttggg 5700agagaatatt tccaacaaac acatgcaccc caccaacagg ggaggcaaat ttcagcatag 5760atctataaga ctttcagatg accatgggcc attgccttca tgctgtggta agtactacat 5820ctacaatttt ggtacccgaa ctggtgcttt agaaatgcgg ggtttttatt aaaaaaaaaa 5880aaaagaaatg tagcagaata attcttttag tgcagcaact cagtttttgt aaaggactct 5940gagaacactt gggctgtgaa cattcaaagc agcagagagg gaacctggca ctattggggt 6000aaagtgtttg ggtcagttga aaaaaaggaa accttttcat gcctttagat gtgagctaac 6060agtaggtaat gatcatgtgt ccctttttga tggctgtacg aagaacttca atcactgtag 6120tctaagatct gatctataga tgacctagaa tagccatgta atataatgtg atgattctaa 6180atttgtacct atgtgacaga cattttcaat aatgtgaaaa ctgcagattt gatggagcta 6240ctttaagatt tgtaggtgaa agtgtgctac tgttggttga actatgctga agagggaaag 6300tgagtgatta gtttgagccc ttgctggctc ttttccacct gccaattcta catgtattgt 6360tgtggtttta ttcattgtat gaaaattcct gtgatttttt tttaaatgtg cagtacacat 6420cagcctcact gagctaataa agggaaaaga atgtttcaaa tcta 646433797PRTMus musculus 33Met Ala Trp Asp Met Cys Ser Gln Asp Ser Val Trp Ser Asp Ile Glu 1 5 10 15 Cys Ala Ala Leu Val Gly Glu Asp Gln Pro Leu Cys Pro Asp Leu Pro 20 25 30 Glu Leu Asp Leu Ser Glu Leu Asp Val Asn Asp Leu Asp Thr Asp Ser 35 40 45 Phe Leu Gly Gly Leu Lys Trp Cys Ser Asp Gln Ser Glu Ile Ile Ser 50 55 60 Asn Gln Tyr Asn Asn Glu Pro Ala Asn Ile Phe Glu Lys Ile Asp Glu 65 70 75 80 Glu Asn Glu Ala Asn Leu Leu Ala Val Leu Thr Glu Thr Leu Asp Ser 85 90 95 Leu Pro Val Asp Glu Asp Gly Leu Pro Ser Phe Asp Ala Leu Thr Asp 100 105 110 Gly Ala Val Thr Thr Asp Asn Glu Ala Ser Pro Ser Ser Met Pro Asp 115 120 125 Gly Thr Pro Pro Pro Gln Glu Ala Glu Glu Pro Ser Leu Leu Lys Lys 130 135 140 Leu Leu Leu Ala Pro Ala Asn Thr Gln Leu Ser Tyr Asn Glu Cys Ser 145 150 155 160 Gly Leu Ser Thr Gln Asn His Ala Ala Asn His Thr His Arg Ile Arg 165 170 175 Thr Asn Pro Ala Ile Val Lys Thr Glu Asn Ser Trp Ser Asn Lys Ala 180 185 190 Lys Ser Ile Cys Gln Gln Gln Lys Pro Gln Arg Arg Pro Cys Ser Glu 195 200 205 Leu Leu Lys Tyr Leu Thr Thr Asn Asp Asp Pro Pro His Thr Lys Pro 210 215 220 Thr Glu Asn Arg Asn Ser Ser Arg Asp Lys Cys Ala Ser Lys Lys Lys 225 230 235 240 Ser His Thr Gln Pro Gln Ser Gln His Ala Gln Ala Lys Pro Thr Thr 245 250 255 Leu Ser Leu Pro Leu Thr Pro Glu Ser Pro Asn Asp Pro Lys Gly Ser 260 265 270 Pro Phe Glu Asn Lys Thr Ile Glu Arg Thr Leu Ser Val Glu Leu Ser 275 280 285 Gly Thr Ala Gly Leu Thr Pro Pro Thr Thr Pro Pro His Lys Ala Asn 290 295 300 Gln Asp Asn Pro Phe Lys Ala Ser Pro Lys Leu Lys Pro Ser Cys Lys 305 310 315 320 Thr Val Val Pro Pro Pro Thr Lys Arg Ala Arg Tyr Ser Glu Cys Ser 325 330 335 Gly Thr Gln Gly Ser His Ser Thr Lys Lys Gly Pro Glu Gln Ser Glu 340 345 350 Leu Tyr Ala Gln Leu Ser Lys Ser Ser Gly Leu Ser Arg Gly His Glu 355 360 365 Glu Arg Lys Thr Lys Arg Pro Ser Leu Arg Leu Phe Gly Asp His Asp 370 375 380 Tyr Cys Gln Ser Leu Asn Ser Lys Thr Asp Ile Leu Ile Asn Ile Ser 385 390 395 400 Gln Glu Leu Gln Asp Ser Arg Gln Leu Asp Phe Lys Asp Ala Ser Cys 405 410 415 Asp Trp Gln Gly His Ile Cys Ser Ser Thr Asp Ser Gly Gln Cys Tyr 420 425 430 Leu Arg Glu Thr Leu Glu Ala Ser Lys Gln Val Ser Pro Cys Ser Thr 435 440 445 Arg Lys Gln Leu Gln Asp Gln Glu Ile Arg Ala Glu Leu Asn Lys His 450 455 460 Phe Gly His Pro Cys Gln Ala Val Phe Asp Asp Lys Ser Asp Lys Thr 465 470 475 480 Ser Glu Leu Arg Asp Gly Asp Phe Ser Asn Glu Gln Phe Ser Lys Leu 485 490 495 Pro Val Phe Ile Asn Ser Gly Leu Ala Met Asp Gly Leu Phe Asp Asp 500 505 510 Ser Glu Asp Glu Ser Asp Lys Leu Ser Tyr Pro Trp Asp Gly Thr Gln 515 520 525 Pro Tyr Ser Leu Phe Asp Val Ser Pro Ser Cys Ser Ser Phe Asn Ser 530 535 540 Pro Cys Arg Asp Ser Val Ser Pro Pro Lys Ser Leu Phe Ser Gln Arg 545 550 555 560 Pro Gln Arg Met Arg Ser Arg Ser Arg Ser Phe Ser Arg His Arg Ser 565 570 575 Cys Ser Arg Ser Pro Tyr Ser Arg Ser Arg Ser Arg Ser Pro Gly Ser 580 585 590 Arg Ser Ser Ser Arg Ser Cys Tyr Tyr Tyr Glu Ser Ser His Tyr Arg 595 600 605 His Arg Thr His Arg Asn Ser Pro Leu Tyr Val Arg Ser Arg Ser Arg 610 615 620 Ser Pro Tyr Ser Arg Arg Pro Arg Tyr Asp Ser Tyr Glu Ala Tyr Glu 625 630 635 640 His Glu Arg Leu Lys Arg Asp Glu Tyr Arg Lys Glu His Glu Lys Arg 645 650 655 Glu Ser Glu Arg Ala Lys Gln Arg Glu Arg Gln Lys Gln Lys Ala Ile 660 665 670 Glu Glu Arg Arg Val Ile Tyr Val Gly Lys Ile Arg Pro Asp Thr Thr 675 680 685 Arg Thr Glu Leu Arg Asp Arg Phe Glu Val Phe Gly Glu Ile Glu Glu 690 695 700 Cys Thr Val Asn Leu Arg Asp Asp Gly Asp Ser Tyr Gly Phe Ile Thr 705 710 715 720 Tyr Arg Tyr Thr Cys Asp Ala Phe Ala Ala Leu Glu Asn Gly Tyr Thr 725 730 735 Leu Arg Arg Ser Asn Glu Thr Asp Phe Glu Leu Tyr Phe Cys Gly Arg 740 745 750 Lys Gln Phe Phe Lys Ser Asn Tyr Ala Asp Leu Asp Thr Asn Ser Asp 755 760 765 Asp Phe Asp Pro Ala Ser Thr Lys Ser Lys Tyr Asp Ser Leu Asp Phe 770 775 780 Asp Ser Leu Leu Lys Glu Ala Gln Arg Ser Leu Arg Arg 785 790 795 347404DNAUnknownsource/note="Description of Unknown PGC-1 alpha promoter 2kb luciferase plasmid sequence" 34ggtacccctg tgctctctct agcttcacat accccttttt atgaaggtag aggacagagt 60ggctgtttta agcagatcat tagcttcatg gatgtgctgg gttagtttct tttcttttct 120tttcttttct ttttttaaag tagaattagg tggcaaaaaa agaaagaaag aaagaaagaa 180agaaagaaat tatcttttca aagcaaagaa aaagaaaatc ctgccacaat tcagtgtgag 240caagttaaga tatcaaaaca ataatgcaga gttatctagt gcaagcaacc atctgtaaga 300agaggtgtgc atgaggtttg aaaaagtatg gtggatgcct aatggatgac aaagagaaac 360caaagtatca gttaccatca ggatgccagg attgcttgat aatgatgaat aaacatcgac 420tctttatagc taagtggcca gcattatttc acagttgact gtgtggaaag tagagcccat 480gacctttgtc ctgaatttta atagtttact gaagttttac attaagtaaa aatctaattg 540gcaagaaaaa ttagtgtttc tttgtgggag tacagagtaa ggctactaat tgcatgtaga 600aaatgatccc agggttgtct ctctctctct ctgaattaga aaattaaatg aataatacct 660ttctgatgtt tggaagagga cagttgtagc agtgaagtat attcacctaa tttagaatgt 720caaagctgtt ggccatcttg ttctgttcac ttattgacaa actgtcgatt tcattaccat 780attccctgtc tactagttgt ggacatccaa gcaaacagac cccttatacg gggtctgaag 840caaacagcaa gcttgtgggc tttgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 900tgtgtgtgtg tgcacgctcg ctgcatttct ttctttcact ttactgtatt agtaactgac 960tatataaagt cagactgaga gaagtcacca atgttttcct tctaagggga gagaaaataa 1020agagcacatt aaattaacct cagtggaata ggagtttaaa tgaatggtgc tttataaatt 1080atatttagat gcatagggac ttttttcttc ttctaaatta ctgtcaagta aaggaaatga 1140gctggttttt gagtctgtgg ggtttgaggt attgtgaaaa tatgttttca aaggtcccct 1200gtgcatttct cactgggcct ggaagggtta agtctgagca cccaagtgtt atggaaagtg 1260ctgagagttg gttatgtcct ctgtctgtaa tgtcacagga aaaacagtgg cacctgcatt 1320acccctcatt gactcaggaa cgacaaaaaa gtattagtaa gcaaagctca agaaatgagt 1380atctctgctg ataccatttc agtgtttttc cttcattccc tggacattct tgatttcaaa 1440aacaaactgt acagcccaag gcactagggt tggagtccaa tgtttattca aaaaggcacc 1500ctgaagccat gaggaagact gtgctacata tgagaaaaga aataaggggt gggggcaggt 1560gagtagctaa gctgtttcag ggatggcagc agcaattgta ttttctagca tttgttttct 1620gggagcctat

gagatccacg gaaagaatca tgagggggaa cccaagagtc tagggtgttg 1680tggcttgctt gctttacaag gagcaaggca aactgcagta acagtttagg agactgcatt 1740ctctactgcc aaggagacag ctgatttggg gtagagaaat ttgtttagac ctaaacaaat 1800gtggcggttt tgttgactaa acatggaaag aaagaaagaa agaaagaaag aaagaaagaa 1860agaaagaaag aaagaaagaa agaaacaaag aaagaaagaa agaaaggaag gaaggaagga 1920agaaaggaag aaaggaagga aggaaggaag gaaagaaagg agagagagaa agaaaatcgg 1980gggtgttgcc ttcaaacact cctctaatag ggagggaaaa aaaagaatct catgaaaatg 2040tatcacatga ggagcgcttg cttcagttcc aagctgagtc tggggctact tggaaaccat 2100ttcttaaagc acacacattt taggcaaggg tgtagttact gtgtcagtaa caggggatct 2160ttgctatttg cctgttttgg atggaaaata aatttaaaaa aaaaagattg caggagattt 2220gagttattat gtgagcaggg ctccggttta gagttggtgg cattcaaagc tggcttcagt 2280cacagtgtga tgcttgaagc ctcccaaagg ccaagtgttt ccttttcttt cttctatttt 2340ttttttcctc tctctctaag cgttacttca ctgaggcaga gggctgcctt ggagtgacgt 2400caggagtttg tgcagcaagc ttgcacagga gaagggaggc tgggtgagtg acagcccagc 2460ctacttttta atagctttgt catgtgactg gggactgtag taagacaggt gccttcagtt 2520cactctcagt aaggggctgg ttgcctgcat gagtgtgtgc tgtgtgtcag agtggattgg 2580agttgaaaaa gcttgactgg cgtcattcgg gagctggctc gagatctgcg atctaagtaa 2640gcttggcatt ccggtactgt tggtaaagcc accatggaag acgccaaaaa cataaagaaa 2700ggcccggcgc cattctatcc gctggaagat ggaaccgctg gagagcaact gcataaggct 2760atgaagagat acgccctggt tcctggaaca attgctttta cagatgcaca tatcgaggtg 2820gacatcactt acgctgagta cttcgaaatg tccgttcggt tggcagaagc tatgaaacga 2880tatgggctga atacaaatca cagaatcgtc gtatgcagtg aaaactctct tcaattcttt 2940atgccggtgt tgggcgcgtt atttatcgga gttgcagttg cgcccgcgaa cgacatttat 3000aatgaacgtg aattgctcaa cagtatgggc atttcgcagc ctaccgtggt gttcgtttcc 3060aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc tcccaatcat ccaaaaaatt 3120attatcatgg attctaaaac ggattaccag ggatttcagt cgatgtacac gttcgtcaca 3180tctcatctac ctcccggttt taatgaatac gattttgtgc cagagtcctt cgatagggac 3240aagacaattg cactgatcat gaactcctct ggatctactg gtctgcctaa aggtgtcgct 3300ctgcctcata gaactgcctg cgtgagattc tcgcatgcca gagatcctat ttttggcaat 3360caaatcattc cggatactgc gattttaagt gttgttccat tccatcacgg ttttggaatg 3420tttactacac tcggatattt gatatgtgga tttcgagtcg tcttaatgta tagatttgaa 3480gaagagctgt ttctgaggag ccttcaggat tacaagattc aaagtgcgct gctggtgcca 3540accctattct ccttcttcgc caaaagcact ctgattgaca aatacgattt atctaattta 3600cacgaaattg cttctggtgg cgctcccctc tctaaggaag tcggggaagc ggttgccaag 3660aggttccatc tgccaggtat caggcaagga tatgggctca ctgagactac atcagctatt 3720ctgattacac ccgaggggga tgataaaccg ggcgcggtcg gtaaagttgt tccatttttt 3780gaagcgaagg ttgtggatct ggataccggg aaaacgctgg gcgttaatca aagaggcgaa 3840ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa acaatccgga agcgaccaac 3900gccttgattg acaaggatgg atggctacat tctggagaca tagcttactg ggacgaagac 3960gaacacttct tcatcgttga ccgcctgaag tctctgatta agtacaaagg ctatcaggtg 4020gctcccgctg aattggaatc catcttgctc caacacccca acatcttcga cgcaggtgtc 4080gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg ccgttgttgt tttggagcac 4140ggaaagacga tgacggaaaa agagatcgtg gattacgtcg ccagtcaagt aacaaccgcg 4200aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac cgaaaggtct taccggaaaa 4260ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca agaagggcgg aaagatcgcc 4320gtgtaattct agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat 4380gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 4440gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat 4500tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa 4560aacctctaca aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt 4620caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac 4680tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc 4740tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4800cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4860gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4920gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4980gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5040ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5100aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5160tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5220ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5280gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5340ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5400ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5460agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5520ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5580aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 5640tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 5700cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5760tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 5820cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 5880ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 5940gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 6000gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 6060gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6120gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6180tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6240aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6300cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6360caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6420cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6480ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6540aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6600tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 6660cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 6720cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 6780tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 6840ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 6900cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 6960tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 7020ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 7080cgaattttaa caaaatatta acgtttacaa tttcccattc gccattcagg ctgcgcaact 7140gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat 7200aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt 7260ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca 7320tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg 7380tgccagaaca tttctctatc gata 7404351857DNAMus musculus 35gtgtgacgac aaggtgaccg ggctgagggg acgggctgag gagaagtcac actctgacag 60gagcctgtga gaccaacagc ctgacggggt ctcggttgag gggacgcggg ctgagaagtc 120acgttctgac aggactgtgt gacagacaag atttgaaaga agcggtgaac cactgatatt 180caggacattt ttaaaaacaa gactaccctt tactgaaatt accatggttg acacagagat 240gccattctgg cccaccaact tcggaatcag ctctgtggac ctctccgtga tggaagacca 300ctcgcattcc tttgacatca agccctttac cacagttgat ttctccagca tttctgctcc 360acactatgaa gacattccat tcacaagagc tgacccaatg gttgctgatt acaaatatga 420cctgaagctc caagaatacc aaagtgcgat caaagtagaa cctgcatctc caccttatta 480ttctgaaaag acccagctct acaacaggcc tcatgaagaa ccttctaact ccctcatggc 540cattgagtgc cgagtctgtg gggataaagc atcaggcttc cactatggag ttcatgcttg 600tgaaggatgc aagggttttt tccgaagaac catccgattg aagcttattt atgataggtg 660tgatcttaac tgccggatcc acaaaaaaag tagaaataaa tgtcagtact gtcggtttca 720gaagtgcctt gctgtgggga tgtctcacaa tgccatcagg tttgggcgga tgccacaggc 780cgagaaggag aagctgttgg cggagatctc cagtgatatc gaccagctga acccagagtc 840tgctgatctg cgagccctgg caaagcattt gtatgactca tacataaagt ccttcccgct 900gaccaaagcc aaggcgaggg cgatcttgac aggaaagaca acggacaaat caccatttgt 960catctacgac atgaattcct taatgatggg agaagataaa atcaagttca aacatatcac 1020ccccctgcag gagcagagca aagaggtggc catccgaatt tttcaagggt gccagtttcg 1080atccgtagaa gccgtgcaag agatcacaga gtatgccaaa aatatccctg gtttcattaa 1140ccttgatttg aatgaccaag tgactctgct caagtatggt gtccatgaga tcatctacac 1200gatgctggcc tccctgatga ataaagatgg agtcctcatc tcagagggcc aaggattcat 1260gaccagggag ttcctcaaaa gcctgcggaa gccctttggt gactttatgg agcctaagtt 1320tgagtttgct gtgaagttca atgcactgga attagatgac agtgacttgg ctatatttat 1380agctgtcatt attctcagtg gagaccgccc aggcttgctg aacgtgaagc ccatcgagga 1440catccaagac aacctgctgc aggccctgga actgcagctc aagctgaatc acccagagtc 1500ctctcagctg ttcgccaagg tgctccagaa gatgacagac ctcaggcaga tcgtcacaga 1560gcacgtgcag ctactgcatg tgatcaagaa gacagagaca gacatgagcc ttcaccccct 1620gctccaggag atctacaagg acttgtatta gcaggaaagt cccacccgct gacaacgtgt 1680tccttctatt gattgcacta ttattttgag ggaaaaaaat ctgacaccta agaaatttac 1740tgtgaaaaag catttaaaaa caaaaagttt tagaacatga tctattttat gcatattgtt 1800tataaagata catttacaat ttacttttaa tattaaaaat taccacatta taaaatt 185736475PRTMus musculus 36Met Val Asp Thr Glu Met Pro Phe Trp Pro Thr Asn Phe Gly Ile Ser 1 5 10 15 Ser Val Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe Asp Ile 20 25 30 Lys Pro Phe Thr Thr Val Asp Phe Ser Ser Ile Ser Ala Pro His Tyr 35 40 45 Glu Asp Ile Pro Phe Thr Arg Ala Asp Pro Met Val Ala Asp Tyr Lys 50 55 60 Tyr Asp Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro 65 70 75 80 Ala Ser Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Arg Pro 85 90 95 His Glu Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys 100 105 110 Gly Asp Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly 115 120 125 Cys Lys Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile Tyr Asp 130 135 140 Arg Cys Asp Leu Asn Cys Arg Ile His Lys Lys Ser Arg Asn Lys Cys 145 150 155 160 Gln Tyr Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn 165 170 175 Ala Ile Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu 180 185 190 Ala Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp 195 200 205 Leu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe 210 215 220 Pro Leu Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr 225 230 235 240 Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly 245 250 255 Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser 260 265 270 Lys Glu Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val 275 280 285 Glu Ala Val Gln Glu Ile Thr Glu Tyr Ala Lys Asn Ile Pro Gly Phe 290 295 300 Ile Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val 305 310 315 320 His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly 325 330 335 Val Leu Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys 340 345 350 Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe 355 360 365 Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile 370 375 380 Phe Ile Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn 385 390 395 400 Val Lys Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu 405 410 415 Leu Gln Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys 420 425 430 Val Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val 435 440 445 Gln Leu Leu His Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His 450 455 460 Pro Leu Leu Gln Glu Ile Tyr Lys Asp Leu Tyr 465 470 475 371769DNAMus musculus 37caaaacacca gtgtgaatta cagcaaatct ctgttttatg ctgttatggg tgaaactctg 60ggagattctc ctgttgaccc agagcatggt gccttcgctg atgcactgcc tatgagcact 120tcacaagaaa ttaccatggt tgacacagag atgccattct ggcccaccaa cttcggaatc 180agctctgtgg acctctccgt gatggaagac cactcgcatt cctttgacat caagcccttt 240accacagttg atttctccag catttctgct ccacactatg aagacattcc attcacaaga 300gctgacccaa tggttgctga ttacaaatat gacctgaagc tccaagaata ccaaagtgcg 360atcaaagtag aacctgcatc tccaccttat tattctgaaa agacccagct ctacaacagg 420cctcatgaag aaccttctaa ctccctcatg gccattgagt gccgagtctg tggggataaa 480gcatcaggct tccactatgg agttcatgct tgtgaaggat gcaagggttt tttccgaaga 540accatccgat tgaagcttat ttatgatagg tgtgatctta actgccggat ccacaaaaaa 600agtagaaata aatgtcagta ctgtcggttt cagaagtgcc ttgctgtggg gatgtctcac 660aatgccatca ggtttgggcg gatgccacag gccgagaagg agaagctgtt ggcggagatc 720tccagtgata tcgaccagct gaacccagag tctgctgatc tgcgagccct ggcaaagcat 780ttgtatgact catacataaa gtccttcccg ctgaccaaag ccaaggcgag ggcgatcttg 840acaggaaaga caacggacaa atcaccattt gtcatctacg acatgaattc cttaatgatg 900ggagaagata aaatcaagtt caaacatatc acccccctgc aggagcagag caaagaggtg 960gccatccgaa tttttcaagg gtgccagttt cgatccgtag aagccgtgca agagatcaca 1020gagtatgcca aaaatatccc tggtttcatt aaccttgatt tgaatgacca agtgactctg 1080ctcaagtatg gtgtccatga gatcatctac acgatgctgg cctccctgat gaataaagat 1140ggagtcctca tctcagaggg ccaaggattc atgaccaggg agttcctcaa aagcctgcgg 1200aagccctttg gtgactttat ggagcctaag tttgagtttg ctgtgaagtt caatgcactg 1260gaattagatg acagtgactt ggctatattt atagctgtca ttattctcag tggagaccgc 1320ccaggcttgc tgaacgtgaa gcccatcgag gacatccaag acaacctgct gcaggccctg 1380gaactgcagc tcaagctgaa tcacccagag tcctctcagc tgttcgccaa ggtgctccag 1440aagatgacag acctcaggca gatcgtcaca gagcacgtgc agctactgca tgtgatcaag 1500aagacagaga cagacatgag ccttcacccc ctgctccagg agatctacaa ggacttgtat 1560tagcaggaaa gtcccacccg ctgacaacgt gttccttcta ttgattgcac tattattttg 1620agggaaaaaa atctgacacc taagaaattt actgtgaaaa agcatttaaa aacaaaaagt 1680tttagaacat gatctatttt atgcatattg tttataaaga tacatttaca atttactttt 1740aatattaaaa attaccacat tataaaatt 176938505PRTMus musculus 38Met Gly Glu Thr Leu Gly Asp Ser Pro Val Asp Pro Glu His Gly Ala 1 5 10 15 Phe Ala Asp Ala Leu Pro Met Ser Thr Ser Gln Glu Ile Thr Met Val 20 25 30 Asp Thr Glu Met Pro Phe Trp Pro Thr Asn Phe Gly Ile Ser Ser Val 35 40 45 Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe Asp Ile Lys Pro 50 55 60 Phe Thr Thr Val Asp Phe Ser Ser Ile Ser Ala Pro His Tyr Glu Asp 65 70 75 80 Ile Pro Phe Thr Arg Ala Asp Pro Met Val Ala Asp Tyr Lys Tyr Asp 85 90 95 Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser 100 105 110 Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Arg Pro His Glu 115 120 125 Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly Asp 130 135 140 Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys 145 150 155 160 Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile Tyr Asp Arg Cys 165 170 175 Asp Leu Asn Cys Arg Ile His Lys Lys Ser Arg Asn Lys Cys Gln Tyr 180 185 190 Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn Ala Ile 195 200 205 Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu 210 215 220 Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg 225 230 235 240 Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu 245 250 255 Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys 260 265 270 Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp 275 280 285 Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu 290 295 300 Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala 305 310 315 320 Val Gln Glu Ile Thr Glu Tyr Ala Lys Asn Ile Pro Gly Phe Ile Asn 325 330 335 Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu 340 345 350 Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu 355 360 365 Ile

Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu 370 375 380 Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val 385 390 395 400 Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile 405 410 415 Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys 420 425 430 Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln 435 440 445 Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Val Leu 450 455 460 Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln Leu 465 470 475 480 Leu His Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu 485 490 495 Leu Gln Glu Ile Tyr Lys Asp Leu Tyr 500 505 391644DNAMus musculus 39aagtgccggg caatctgggc ttaacgggtc ctccctgccc gagcaagagg aagggacgct 60cacctttgag ctgctccaca gcgccgcctc tgcactggca ctacctagcc caggtggctc 120tgcaggagtc cgaagtcgcg ggtttcgtgc ccgcatcagg caacagtgcc actgttgtct 180tcagggctga gtccttttgt tcttgcactc acgcctctct gccctccaag ccaggatggt 240gaacccgaca acttccgaag tgcaacccac catgggggtc aagatcttct cagccggagt 300ttcagcttgc ctggcagata tcatcacctt cccgctggac actgccaaag tccgccttca 360gatccaaggt gaaggccagg cttccagtac cattaggtat aaaggtgtcc tagggaccat 420caccaccctg gcaaaaacag aaggattgcc gaaactgtac agcggtctgc ctgcgggcat 480tcagaggcaa atcagctttg cctcactcag gattggcctc tacgactcag tccaagagta 540cttctcttca gggagagaaa cacctgcctc tctcggaaac aagatctcag ccggcttaat 600gactggaggt gtggcagtgt tcattgggca gcctacagag gtcgtgaagg tcagaatgca 660agcccagagc catctgcatg ggatcaaacc ccgctacacg gggacctaca atgcttacag 720agttatagcc accacagaaa gcttgtcaac actttggaaa gggacgaccc ctaatctaat 780gagaaatgtc atcatcaatt gtacagagct ggtaacatat gacctcatga agggggccct 840tgtaaacaac aaaatactgg cagatgacgt cccctgccat ttactgtcag ctcttgttgc 900cgggttttgc accacactcc tggcctctcc agtggatgtg gtaaaaacaa gattcatcaa 960ctctctgcca ggacagtacc caagcgtacc aagctgtgcg atgtccatgt acaccaagga 1020aggaccgacg gcctttttca aagggtttgt ggcttctttt ctgcgactcg ggtcctggaa 1080cgtcatcatg tttgtgtgct ttgaacagct gaaaaaagag ctgatgaagt ccagacagac 1140agtggattgt accacataag caacttggag gaagagatac tgaacatcat tgggcttcta 1200tgctgggaga ccacgaataa aaccaaccaa agaaatcaaa tgaacagctc cgttgacttt 1260atttacatta caagatcatt tccagtagag agttttgaaa cctcttttaa ttttttttaa 1320agggaaaact aacacataca catagttttt attcttactg tcttaaagac agaagagcat 1380agcattcact aatattttga gaaaataata cctatataaa gtcctgtatt taactggtct 1440ttggggagag gtgggagtgt atgactgggt ataaagaatt ctgattacag ctcaaactag 1500tgggaaggaa aaattagtcc aaaacccttt acatcgataa acactttaaa aaagaaagct 1560atcaaaaaaa tattgccatt tcatcttatt tattgaccac agttcacagc taatatactc 1620aataaagtat tgctaattcc atct 164440307PRTMus musculus 40Met Val Asn Pro Thr Thr Ser Glu Val Gln Pro Thr Met Gly Val Lys 1 5 10 15 Ile Phe Ser Ala Gly Val Ser Ala Cys Leu Ala Asp Ile Ile Thr Phe 20 25 30 Pro Leu Asp Thr Ala Lys Val Arg Leu Gln Ile Gln Gly Glu Gly Gln 35 40 45 Ala Ser Ser Thr Ile Arg Tyr Lys Gly Val Leu Gly Thr Ile Thr Thr 50 55 60 Leu Ala Lys Thr Glu Gly Leu Pro Lys Leu Tyr Ser Gly Leu Pro Ala 65 70 75 80 Gly Ile Gln Arg Gln Ile Ser Phe Ala Ser Leu Arg Ile Gly Leu Tyr 85 90 95 Asp Ser Val Gln Glu Tyr Phe Ser Ser Gly Arg Glu Thr Pro Ala Ser 100 105 110 Leu Gly Asn Lys Ile Ser Ala Gly Leu Met Thr Gly Gly Val Ala Val 115 120 125 Phe Ile Gly Gln Pro Thr Glu Val Val Lys Val Arg Met Gln Ala Gln 130 135 140 Ser His Leu His Gly Ile Lys Pro Arg Tyr Thr Gly Thr Tyr Asn Ala 145 150 155 160 Tyr Arg Val Ile Ala Thr Thr Glu Ser Leu Ser Thr Leu Trp Lys Gly 165 170 175 Thr Thr Pro Asn Leu Met Arg Asn Val Ile Ile Asn Cys Thr Glu Leu 180 185 190 Val Thr Tyr Asp Leu Met Lys Gly Ala Leu Val Asn Asn Lys Ile Leu 195 200 205 Ala Asp Asp Val Pro Cys His Leu Leu Ser Ala Leu Val Ala Gly Phe 210 215 220 Cys Thr Thr Leu Leu Ala Ser Pro Val Asp Val Val Lys Thr Arg Phe 225 230 235 240 Ile Asn Ser Leu Pro Gly Gln Tyr Pro Ser Val Pro Ser Cys Ala Met 245 250 255 Ser Met Tyr Thr Lys Glu Gly Pro Thr Ala Phe Phe Lys Gly Phe Val 260 265 270 Ala Ser Phe Leu Arg Leu Gly Ser Trp Asn Val Ile Met Phe Val Cys 275 280 285 Phe Glu Gln Leu Lys Lys Glu Leu Met Lys Ser Arg Gln Thr Val Asp 290 295 300 Cys Thr Thr 305 411879DNAMus musculus 41gagcgggctg gttagcagcg cacgtgccag gctccggggc ccttctgctt atacacaatt 60tctttctgtc ctgggtttct tcgtccctga gacccactcc atcttctact tctttggctc 120tcgcccagct ccctacccca agctctgtaa ctcgtcgtct gcaaaatcga aatggacaca 180tccatgaatt tctcacgcgg gttaaaaatg gacctgatgc aaccctatga cttcgagacg 240tttcaggact taaggccctt tttggaggag tactgggtaa gctcatttct catagtggtc 300gtctatctgt tgctcatcgt tgttggccag acctacatga gaacgcggaa gagcttcagc 360ttgcagaggc ctctcatcct ctggtccttc ttcctggcaa tattcagtat cctgggtact 420ctgaggatgt ggaagtttat ggcaacagtg atgtttacag tgggcctcaa gcaaaccgtg 480tgctttgcca tctacacgga tgacgccgta gtcagattct ggtcctttct ctttcttctc 540agcaaggttg ttgaactggg agacacggcc ttcatcatcc tgcgtaagcg tccactcatc 600tttgtccact ggtaccacca cagcacagtg ctactgttca caagctttgg atacaagaac 660aaagtgcctt cgggtggctg gttcatgacc atgaactttg gcgtccattc tgtcatgtac 720acttactaca ctatgaaggc tgccaaactg aagcatccta atcttctccc catggtcatc 780accagcctgc agattctgca gatggttctg ggcaccatct ttggcatact gaattacatc 840tggaggcagg agaaaggatg ccacacaaca acggaacact tcttctggtc ttttatgcta 900tatgggacct atttcatcct attcgctcac ttcttccacc gagcctacct caggcccaag 960ggcaaagttg catccaagag ccaatgagag taggaaagaa agatggagcc tcagccgttc 1020ctccgtggca ctaagggtat gggagaatga ttagggtacc tccctgtatg gtttccccca 1080tgggatatgt accctcaaag ttgcaggaag ctatgacaac caagaaatgt cacccttggg 1140gatagggggt gtgtggtttg gtactttgat gtttctgtct ttaatgtgaa ggaaaaccaa 1200gccctaggaa ggagatagga ctgaggtcct taaaatggag ttatttatat ttatatttag 1260aaatctttct cttcttgctc tatttttaaa agaggtcaac atgatcttga ggatttgtgg 1320acttggaggg gaggggagag tggactgact ctgtggtagg aggaggctga ctctggggag 1380tgagtgatct gcaggggggg agcctgaggg tgtgtggaag gacagaggca cacacaaaca 1440ctcaataaga attctaggcc tggtaggcgc ttaataaatg tcttttacag actagaagtt 1500tattgctgtt agagacccaa gcctctgaaa ggaacagtga gaaacaagtc ctaaaatcat 1560aggctaaact gcagaagaga tcgctgcgtg gaagcacggg tgaccagacc taccggctgt 1620cctcccagag accaccaggt ggcgtcgcag caccagcaga gaacgatccc tgtcagctag 1680ctggtcacac caagcttcat actcctttta ctccaggaga ccctgcttcc ctatcctgga 1740ggaaaggagt gagacggcaa ccttagaagc ctggacgctc actcggttcc tgtacatcca 1800gctgtccttt tggacaatgg gaaggatgaa gggaaggagg gctcaaccgc aagtctgaat 1860catcaggcct ttgacagtc 187942271PRTMus musculus 42Met Asp Thr Ser Met Asn Phe Ser Arg Gly Leu Lys Met Asp Leu Met 1 5 10 15 Gln Pro Tyr Asp Phe Glu Thr Phe Gln Asp Leu Arg Pro Phe Leu Glu 20 25 30 Glu Tyr Trp Val Ser Ser Phe Leu Ile Val Val Val Tyr Leu Leu Leu 35 40 45 Ile Val Val Gly Gln Thr Tyr Met Arg Thr Arg Lys Ser Phe Ser Leu 50 55 60 Gln Arg Pro Leu Ile Leu Trp Ser Phe Phe Leu Ala Ile Phe Ser Ile 65 70 75 80 Leu Gly Thr Leu Arg Met Trp Lys Phe Met Ala Thr Val Met Phe Thr 85 90 95 Val Gly Leu Lys Gln Thr Val Cys Phe Ala Ile Tyr Thr Asp Asp Ala 100 105 110 Val Val Arg Phe Trp Ser Phe Leu Phe Leu Leu Ser Lys Val Val Glu 115 120 125 Leu Gly Asp Thr Ala Phe Ile Ile Leu Arg Lys Arg Pro Leu Ile Phe 130 135 140 Val His Trp Tyr His His Ser Thr Val Leu Leu Phe Thr Ser Phe Gly 145 150 155 160 Tyr Lys Asn Lys Val Pro Ser Gly Gly Trp Phe Met Thr Met Asn Phe 165 170 175 Gly Val His Ser Val Met Tyr Thr Tyr Tyr Thr Met Lys Ala Ala Lys 180 185 190 Leu Lys His Pro Asn Leu Leu Pro Met Val Ile Thr Ser Leu Gln Ile 195 200 205 Leu Gln Met Val Leu Gly Thr Ile Phe Gly Ile Leu Asn Tyr Ile Trp 210 215 220 Arg Gln Glu Lys Gly Cys His Thr Thr Thr Glu His Phe Phe Trp Ser 225 230 235 240 Phe Met Leu Tyr Gly Thr Tyr Phe Ile Leu Phe Ala His Phe Phe His 245 250 255 Arg Ala Tyr Leu Arg Pro Lys Gly Lys Val Ala Ser Lys Ser Gln 260 265 270 43393DNAMus musculus 43aaagtgctgc acgtccttgg gcgaagaggg gaggtgactc ggagctgctg aggacgcaaa 60atgagggccc tacgggtctc ccaggctctg gtccggtctt ttagctcatc taccagaagc 120cacttagaaa accgtgtggc agagaagcag aagctcttcc aggccgacaa tgacctccca 180gtacacttga aaggcggggg aatggacaac gtcctgtaca gactgaccat gacgctgact 240ctggggggca ctgcctactg cttatactgc ttgggctggg cctccttccc ccacaagaag 300tgacaccaag aagcttggag gacttggaca aacatcaata aatatgctaa tctcttgaga 360acccaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 3934480PRTMus musculus 44Met Arg Ala Leu Arg Val Ser Gln Ala Leu Val Arg Ser Phe Ser Ser 1 5 10 15 Ser Thr Arg Ser His Leu Glu Asn Arg Val Ala Glu Lys Gln Lys Leu 20 25 30 Phe Gln Ala Asp Asn Asp Leu Pro Val His Leu Lys Gly Gly Gly Met 35 40 45 Asp Asn Val Leu Tyr Arg Leu Thr Met Thr Leu Thr Leu Gly Gly Thr 50 55 60 Ala Tyr Cys Leu Tyr Cys Leu Gly Trp Ala Ser Phe Pro His Lys Lys 65 70 75 80 45383DNAMus musculus 45cagctataaa aagccccagc tggctaccta agtggtcaga ggacgtgcag cggacattca 60gggtgcctct ttggggccaa ggaaggagtg cgaccccgag aatcatgcca aggctccccc 120ctatcctgcg gctgctccaa gcgcctgcga agttcacagt ggttcccaaa gcccatgtct 180ctgccaagcc agccaaaact cccacttccg ccgtggagca ggctgtgggg atctcagcca 240tagtcgttgg cttcatggtt ccagcaggat gggtcttagc ccacttggag agctataaaa 300agagctccgc agcatgaagt tgcacatccc tgagagattt cattaaacat gtccatctgc 360ctaaaaaaaa aaaaaaaaaa aaa 3834670PRTMus musculus 46Met Pro Arg Leu Pro Pro Ile Leu Arg Leu Leu Gln Ala Pro Ala Lys 1 5 10 15 Phe Thr Val Val Pro Lys Ala His Val Ser Ala Lys Pro Ala Lys Thr 20 25 30 Pro Thr Ser Ala Val Glu Gln Ala Val Gly Ile Ser Ala Ile Val Val 35 40 45 Gly Phe Met Val Pro Ala Gly Trp Val Leu Ala His Leu Glu Ser Tyr 50 55 60 Lys Lys Ser Ser Ala Ala 65 70 471164DNAMus musculus 47ccagaagaga aaaacaaact gacaagagcc accaacatca ccaaatcctg gggttttggg 60ccctcggtac cgtttgcgca cgaaggggcg tggccccggg acccaggcca gggagccaga 120actattcgct gctcgcagga gcgcagcctg tcgccaaggt cgggtcaagt cgtcgcgggg 180cgtggctgat agggcagtga tttaagagac gcggctttgg gacaggagga cccgcaccaa 240tggagaccgc cagggactac gcgggagccc tcatcaggcc cctgacattc atgggattgc 300agactaagaa ggtcctactg acccccctca tacatccagc tcgccctttt cgagtttcaa 360accatgaccg aagtagccgg cgtggggtga tggccagcag cctgcaggaa cttatcagca 420agactctgga tgtcttagtc atcacaactg gcctggttac gctggtgctg gaggaggacg 480gcaccgtggt ggacacagag gagttctttc agaccttaag ggacaacacg catttcatga 540tcttggaaaa gggacagaaa tggacaccgg gtagtaagta tgtcccagtc tgcaagcaac 600caaagaaatc gggaatagcc agagtcacct tcgacctata caggctgaac cccaaggact 660tcctcggctg tctcaatgtc aaagccacga tgtacgagat gtactcggtg tcctacgaca 720tccgatgcac aagcttcaag gccgtgttaa ggaatctgct gaggtttatg tcctatgctg 780cacagatgac gggacagttc ctggtctatg cgggcacata catgctccga gtactgggcg 840atacagaaga gcagccatcc cccaagccta gcaccaaagg ctggttcatg taaccagggc 900acagctacag aggcccaggg accctgctct ctgttatagg ctgtgggatg ccaggggaag 960gaatgggggc ttgggggtgg tacccagtgc agggctgagt agcaggattc ctgcaaagga 1020aaggcggcag aggggccttt caagcgcttt aggaagggat caacagcgga gtgtgtggga 1080actgcgtgga tacgaatcag tttctttgga tccttacata ctgtaataaa ccagtcacat 1140gagtcgtctt tgatacttcc ttgc 116448217PRTMus musculus 48Met Glu Thr Ala Arg Asp Tyr Ala Gly Ala Leu Ile Arg Pro Leu Thr 1 5 10 15 Phe Met Gly Leu Gln Thr Lys Lys Val Leu Leu Thr Pro Leu Ile His 20 25 30 Pro Ala Arg Pro Phe Arg Val Ser Asn His Asp Arg Ser Ser Arg Arg 35 40 45 Gly Val Met Ala Ser Ser Leu Gln Glu Leu Ile Ser Lys Thr Leu Asp 50 55 60 Val Leu Val Ile Thr Thr Gly Leu Val Thr Leu Val Leu Glu Glu Asp 65 70 75 80 Gly Thr Val Val Asp Thr Glu Glu Phe Phe Gln Thr Leu Arg Asp Asn 85 90 95 Thr His Phe Met Ile Leu Glu Lys Gly Gln Lys Trp Thr Pro Gly Ser 100 105 110 Lys Tyr Val Pro Val Cys Lys Gln Pro Lys Lys Ser Gly Ile Ala Arg 115 120 125 Val Thr Phe Asp Leu Tyr Arg Leu Asn Pro Lys Asp Phe Leu Gly Cys 130 135 140 Leu Asn Val Lys Ala Thr Met Tyr Glu Met Tyr Ser Val Ser Tyr Asp 145 150 155 160 Ile Arg Cys Thr Ser Phe Lys Ala Val Leu Arg Asn Leu Leu Arg Phe 165 170 175 Met Ser Tyr Ala Ala Gln Met Thr Gly Gln Phe Leu Val Tyr Ala Gly 180 185 190 Thr Tyr Met Leu Arg Val Leu Gly Asp Thr Glu Glu Gln Pro Ser Pro 195 200 205 Lys Pro Ser Thr Lys Gly Trp Phe Met 210 215 492083DNAMus musculus 49cttcccatcc cccaggaata tataaagagc cccaacctca gccactgacc gaccctggcc 60aacaggcatc tgtccttgtt aattacagag agacagtccc aaactccggg agctccgcct 120ggatttgctg gcctgcagca gccagggact ggcgtgtctc cctctctgct gaatccaggt 180attcccacct gcttctctga aggatggaca tgacggacgg ctgccagttc tccccttctg 240agtacttcta tgaaggctcc tgtatcccct caccagagga tgagtttggg gaccagtttg 300agccaagagt agcagccttc ggagcacaca aagctgagct gcagggctca gacgatgagg 360agcacgtgcg tgcacctacc ggccaccacc aggctggcca ctgcctcatg tgggcctgca 420aagcttgcaa gaggaagtcc actaccatgg atcggcgcaa ggccgccacc atgcgcgagc 480gtagacgcct gaagaaggtc aaccaagctt tcgagacgct caagaggtgc accaccacca 540accctaacca gagactcccc aaggtggaga tcctcaggaa tgccatccgc tacattgaga 600gcctccagga gctgctgagg gaacaggtgg agaactatta cagcctgccg ggacagagct 660gctctgagcc caccagcccc acctccaact gctctgacgg catgcctgaa tgtaacagcc 720ctgtctggtc ccgaaagaac agcagctttg acagcatcta ctgtcctgat gtatcaaatg 780catgtgctgc agataaaagc tccgtgtcca gcttggattg cttgtccagc attgtggatc 840ggatcacgtc tacagagcca tccgagctgg ctcttcagga cacagcttcc ctctctccag 900cgaccagcgc caactcacag cctgctaccc cgggaccctc cagctccaga cttatctatc 960acgtattatg aactctctcc cgatgatcac tcctgctagg agggcgtcct tcatggagga 1020aaagaagccc tgaagctgaa ggaaagacaa gctgggcaga atacgtgctt ttcggttgta 1080aatactgtct tgccacttta tgagaaaata gatttaactg aaagtcacat ttgcaataat 1140ggattctcct ctgcctgttc tttttgcttt cggttttttt tttttttttt tttttagctt 1200ccaattgctt tagatacatg attccagaaa tatttttctg ttggaggcaa ttaattgaca 1260gttacttaga gtaattctta acttatacat atatattgta aatattgcac atcaaaataa 1320ctttggtatt tagagctcta tatttttctt caaaataaca ttttaacagc ttggaatcca 1380ttacagggaa ttaaaaatat atttaacttt tgcttttctc tttaatcttt tgttaatagt 1440gtatcatcaa atgaaaatat aacagttgtg cctaatggta tatactttct taaaatcttt 1500taatcgtata atcttacatc ttttcttata agaaatactt ctttcaatgt aagctataaa 1560taatacattg agggcaattt caaactatta aaaatgtaaa tttccccata aataacattg 1620aaataactaa tttgtttctt ggcctttaaa aataacatcc ccaatgaaat tagcaaacca 1680tgaacacgaa acatttaaga atgggttaaa tatgatcaca cagttagcct tgtagatatg 1740tattgaaata atttatgaat ttcttttaga tttgttgatg tcacttgtaa aaatattaca 1800tttccattgt aagcacattt caagaatgcc tggtaaatga agcccccttt tctttgttgt 1860tatttcatac aatgtccagt tgtatataaa aaaaaaggat tgtaaaattt tataggataa 1920tatcatttgt ttaagcaaaa aagcttaaaa agtattatgt cattttacta tatacagtac 1980tttgccaatc atgagccagg ttttattaac tatttgtata tgccttaaaa taacttgata 2040aataaatgta ctattattat caataaaata tttaaaggag gtg 208350255PRTMus musculus

50Met Asp Met Thr Asp Gly Cys Gln Phe Ser Pro Ser Glu Tyr Phe Tyr 1 5 10 15 Glu Gly Ser Cys Ile Pro Ser Pro Glu Asp Glu Phe Gly Asp Gln Phe 20 25 30 Glu Pro Arg Val Ala Ala Phe Gly Ala His Lys Ala Glu Leu Gln Gly 35 40 45 Ser Asp Asp Glu Glu His Val Arg Ala Pro Thr Gly His His Gln Ala 50 55 60 Gly His Cys Leu Met Trp Ala Cys Lys Ala Cys Lys Arg Lys Ser Thr 65 70 75 80 Thr Met Asp Arg Arg Lys Ala Ala Thr Met Arg Glu Arg Arg Arg Leu 85 90 95 Lys Lys Val Asn Gln Ala Phe Glu Thr Leu Lys Arg Cys Thr Thr Thr 100 105 110 Asn Pro Asn Gln Arg Leu Pro Lys Val Glu Ile Leu Arg Asn Ala Ile 115 120 125 Arg Tyr Ile Glu Ser Leu Gln Glu Leu Leu Arg Glu Gln Val Glu Asn 130 135 140 Tyr Tyr Ser Leu Pro Gly Gln Ser Cys Ser Glu Pro Thr Ser Pro Thr 145 150 155 160 Ser Asn Cys Ser Asp Gly Met Pro Glu Cys Asn Ser Pro Val Trp Ser 165 170 175 Arg Lys Asn Ser Ser Phe Asp Ser Ile Tyr Cys Pro Asp Val Ser Asn 180 185 190 Ala Cys Ala Ala Asp Lys Ser Ser Val Ser Ser Leu Asp Cys Leu Ser 195 200 205 Ser Ile Val Asp Arg Ile Thr Ser Thr Glu Pro Ser Glu Leu Ala Leu 210 215 220 Gln Asp Thr Ala Ser Leu Ser Pro Ala Thr Ser Ala Asn Ser Gln Pro 225 230 235 240 Ala Thr Pro Gly Pro Ser Ser Ser Arg Leu Ile Tyr His Val Leu 245 250 255 511854DNAMus musculus 51aggggccagg acgccccagg acacgactgc tttcttcacc actcctctga caggacagga 60cagggaggag gggtagagga cagccggtgt gcattccaac ccacagaacc tttgtcattg 120tactgttggg gttccggagt ggcagaaagt taagacgact ctcacggctt gggttgaggc 180tggacccagg aactgggata tggagcttct atcgccgcca ctccgggaca tagacttgac 240aggccccgac ggctctctct gctcctttga gacagcagac gacttctatg atgacccgtg 300tttcgactca ccagacctgc gcttttttga ggacctggac ccgcgcctgg tgcacatggg 360agccctcctg aaaccggagg agcacgcaca cttccctact gcggtgcacc caggcccagg 420cgctcgtgag gatgagcatg tgcgcgcgcc cagcgggcac caccaggcgg gtcgctgctt 480gctgtgggcc tgcaaggcgt gcaagcgcaa gaccaccaac gctgatcgcc gcaaggccgc 540caccatgcgc gagcgccgcc gcctgagcaa agtgaatgag gccttcgaga cgctcaagcg 600ctgcacgtcc agcaacccga accagcggct acccaaggtg gagatcctgc gcaacgccat 660ccgctacatc gaaggtctgc aggctctgct gcgcgaccag gacgccgcgc cccctggcgc 720cgctgccttc tacgcacctg gaccgctgcc cccaggccgt ggcagcgagc actacagtgg 780cgactcagat gcatccagcc cgcgctccaa ctgctctgat ggcatgatgg attacagcgg 840ccccccaagc ggcccccggc ggcagaatgg ctacgacacc gcctactaca gtgaggcggc 900gcgcgagtcc aggccaggga agagtgcggc tgtgtcgagc ctcgactgcc tgtccagcat 960agtggagcgc atctccacag acagccccgc tgcgcctgcg ctgcttttgg cagatgcacc 1020accagagtcg cctccgggtc cgccagaggg ggcatcccta agcgacacag aacagggaac 1080ccagaccccg tctcccgacg ccgcccctca gtgtcctgca ggctcaaacc ccaatgcgat 1140ttatcaggtg ctttgagaga tcgactgcag cagcagaggg cgcaccaccg taggcactcc 1200tggggatggt gtccctggtt cttcacgccc aaaagatgaa gcttaaatga cactcttccc 1260aactgtcctt tcgaagccgt tcttccagag ggaagggaag agcagaagtc tgtcctagat 1320ccagccccaa agaaaggaca tagtcctttt tgttgttgtt gttgtagtcc ttcagttgtt 1380tgtttgtttt ttcatgcggc tcacagcgaa ggccacttgc actctggctg cacctcactg 1440ggccagagct gatccttgag tggccaggcg ctcttccttt cctcatagca caggggtgag 1500ccttgcacac ctaagccctg ccctccacat ccttttgttt gtcactttct ggagccctcc 1560tggcacccac ttttccccac agcttgcgga ggccactcag gtctcaggtg taacaggtgt 1620aaccataccc cactctcccc cttcccgcgg ttcaggacca cttatttttt tatataagac 1680ttttgtaatc tattcgtgta aataagagtt gcttggccag agcgggagcc ccttgggcta 1740tatttatctc ccaggcatgc tgtgtagtgc aacaaaaact ttgtatgttt attcctcaag 1800cgggcgagcc tcgaggctcg ctcgctcagg tgttggaaat aaagacgcta attt 185452318PRTMus musculus 52Met Glu Leu Leu Ser Pro Pro Leu Arg Asp Ile Asp Leu Thr Gly Pro 1 5 10 15 Asp Gly Ser Leu Cys Ser Phe Glu Thr Ala Asp Asp Phe Tyr Asp Asp 20 25 30 Pro Cys Phe Asp Ser Pro Asp Leu Arg Phe Phe Glu Asp Leu Asp Pro 35 40 45 Arg Leu Val His Met Gly Ala Leu Leu Lys Pro Glu Glu His Ala His 50 55 60 Phe Pro Thr Ala Val His Pro Gly Pro Gly Ala Arg Glu Asp Glu His 65 70 75 80 Val Arg Ala Pro Ser Gly His His Gln Ala Gly Arg Cys Leu Leu Trp 85 90 95 Ala Cys Lys Ala Cys Lys Arg Lys Thr Thr Asn Ala Asp Arg Arg Lys 100 105 110 Ala Ala Thr Met Arg Glu Arg Arg Arg Leu Ser Lys Val Asn Glu Ala 115 120 125 Phe Glu Thr Leu Lys Arg Cys Thr Ser Ser Asn Pro Asn Gln Arg Leu 130 135 140 Pro Lys Val Glu Ile Leu Arg Asn Ala Ile Arg Tyr Ile Glu Gly Leu 145 150 155 160 Gln Ala Leu Leu Arg Asp Gln Asp Ala Ala Pro Pro Gly Ala Ala Ala 165 170 175 Phe Tyr Ala Pro Gly Pro Leu Pro Pro Gly Arg Gly Ser Glu His Tyr 180 185 190 Ser Gly Asp Ser Asp Ala Ser Ser Pro Arg Ser Asn Cys Ser Asp Gly 195 200 205 Met Met Asp Tyr Ser Gly Pro Pro Ser Gly Pro Arg Arg Gln Asn Gly 210 215 220 Tyr Asp Thr Ala Tyr Tyr Ser Glu Ala Ala Arg Glu Ser Arg Pro Gly 225 230 235 240 Lys Ser Ala Ala Val Ser Ser Leu Asp Cys Leu Ser Ser Ile Val Glu 245 250 255 Arg Ile Ser Thr Asp Ser Pro Ala Ala Pro Ala Leu Leu Leu Ala Asp 260 265 270 Ala Pro Pro Glu Ser Pro Pro Gly Pro Pro Glu Gly Ala Ser Leu Ser 275 280 285 Asp Thr Glu Gln Gly Thr Gln Thr Pro Ser Pro Asp Ala Ala Pro Gln 290 295 300 Cys Pro Ala Gly Ser Asn Pro Asn Ala Ile Tyr Gln Val Leu 305 310 315 531518DNAMus musculus 53ggggctgcgg gagcttgggg gccagtggca ggaacaagcc ttttccgacc tgatggagct 60gtatgagaca tccccctatt tctaccagga gccccacttc tatgatgggg aaaactacct 120tcctgtccac cttcagggct tcgagccccc gggctatgag cggactgagc tcagcttaag 180cccggaagcc cgagggcccc tggaagaaaa gggactgggg acccctgagc attgtccagg 240ccagtgcctg ccgtgggcat gtaaggtgtg taagaggaag tctgtgtcgg tggaccggag 300gagggcagcc acactgaggg agaagcgcag gctcaagaaa gtgaatgagg ccttcgaggc 360cctgaagagg agcaccctgc tcaaccccaa ccagcggctg cctaaagtgg agatcctgcg 420cagcgccatc cagtacattg agcgcctaca ggccttgctc agctccctca accaggagga 480gcgcgatctc cgctacagag gcgggggcgg gccccagccc atggtgccca gtgaatgcaa 540ctcccacagc gcctcctgca gtccggagtg gggcaatgca ctggagttcg gtcccaaccc 600aggagatcat ttgctcgcgg ctgaccctac agacgcccac aatctgcact cccttacgtc 660catcgtggac agcatcacgg tggaggatat gtctgttgcc ttcccagacg aaaccatgcc 720caactgagat tgtctgtcag gctgggtgtg catgtgagcc cccaagttgg tgtcaaaagc 780catcacttct gtagcagggg gcttttaagt ggggctgtcc tgatgtccag aaaacagccc 840tgggctgcca caagccagac tccccactcc ccattcacat aaggctaaca cccagcccag 900cgagggaatt tagctgactc cttaaagcag agagcatcct cttctgagga gagaaagatg 960gagtccagag agcccccttg ttaatgtccc tcagtggggc aaactcagga gcttcttttt 1020tgtttatcat aatatgcctc gaattccacc ccccaccccc aaaatgaaac cgtttgagag 1080acatgagtgc cctgacctgg acaagtgtgc acatctgttc tagtctcttc ctgaagccag 1140tggctgggct gggcctgccc tgagttgaga gagaaggggg aggagctatc cggttccaaa 1200gcctctgggg gccaagcatt tgcagtggat cttgggaacc ttccagtgct ttgtgtattg 1260tttattgttt tgtgtgttgt ttgtaaagct gccgtctgac caaggtctcc tgtgctgatg 1320ataccgggaa caggcaggga agggggtggg ggctcttggg gtgacttctt ttgttaacta 1380agcattgtgt ggttttgcca attttttttc ttttgtaatt cttttgctaa cttatttgga 1440tttccttttt taaaaaatga ataaagactg gttgctatca aaaaaaaaaa aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaa 151854224PRTMus musculus 54Met Glu Leu Tyr Glu Thr Ser Pro Tyr Phe Tyr Gln Glu Pro His Phe 1 5 10 15 Tyr Asp Gly Glu Asn Tyr Leu Pro Val His Leu Gln Gly Phe Glu Pro 20 25 30 Pro Gly Tyr Glu Arg Thr Glu Leu Ser Leu Ser Pro Glu Ala Arg Gly 35 40 45 Pro Leu Glu Glu Lys Gly Leu Gly Thr Pro Glu His Cys Pro Gly Gln 50 55 60 Cys Leu Pro Trp Ala Cys Lys Val Cys Lys Arg Lys Ser Val Ser Val 65 70 75 80 Asp Arg Arg Arg Ala Ala Thr Leu Arg Glu Lys Arg Arg Leu Lys Lys 85 90 95 Val Asn Glu Ala Phe Glu Ala Leu Lys Arg Ser Thr Leu Leu Asn Pro 100 105 110 Asn Gln Arg Leu Pro Lys Val Glu Ile Leu Arg Ser Ala Ile Gln Tyr 115 120 125 Ile Glu Arg Leu Gln Ala Leu Leu Ser Ser Leu Asn Gln Glu Glu Arg 130 135 140 Asp Leu Arg Tyr Arg Gly Gly Gly Gly Pro Gln Pro Met Val Pro Ser 145 150 155 160 Glu Cys Asn Ser His Ser Ala Ser Cys Ser Pro Glu Trp Gly Asn Ala 165 170 175 Leu Glu Phe Gly Pro Asn Pro Gly Asp His Leu Leu Ala Ala Asp Pro 180 185 190 Thr Asp Ala His Asn Leu His Ser Leu Thr Ser Ile Val Asp Ser Ile 195 200 205 Thr Val Glu Asp Met Ser Val Ala Phe Pro Asp Glu Thr Met Pro Asn 210 215 220 551233DNAMus musculus 55taagctgggg tctgcctgtc cccatgagta ccagactaat gagacctggc cactttctcc 60tcatttctgt ctgtacgatt gtcagtggat ctgacgacac caaaagggct caggatgcta 120ctgttgcaag ctctcctgtt cctcttaatc ctgcccagtc atgccgaaga tgacgttact 180acaactgaag agctagctcc tgctttggtc cctccaccca agggaacttg tgcaggttgg 240atggcaggca tcccaggaca tcctggccac aatggcacac caggccgtga tggcagagat 300ggcactcctg gagagaaggg agagaaagga gatgcaggtc ttcttggtcc taagggtgag 360acaggagatg ttggaatgac aggagctgaa gggccacggg gcttccccgg aacccctggc 420aggaaaggag agcctggaga agccgcttat gtgtatcgct cagcgttcag tgtggggctg 480gagacccgcg tcactgttcc caatgtaccc attcgcttta ctaagatctt ctacaaccaa 540cagaatcatt atgacggcag cactggcaag ttctactgca acattccggg actctactac 600ttctcttacc acatcacggt gtacatgaaa gatgtgaagg tgagcctctt caagaaggac 660aaggccgttc tcttcaccta cgaccagtat caggaaaaga atgtggacca ggcctctggc 720tctgtgctcc tccatctgga ggtgggagac caagtctggc tccaggtgta tggggatggg 780gaccacaatg gactctatgc agataacgtc aacgactcta catttactgg ctttcttctc 840taccatgata ccaactgact gcaactaccc atagcccata caccaggaga atcatggaac 900agtcgacaca ctttcagctt agtttgagag attgatttta ttgcttagtt tgagagtcct 960gagtattatc cacacgtgta ctcacttgtt cattaaacga ctttataaaa aataatttgt 1020gttcctagtc cagaaaaaaa ggcactccct ggtctccacg actcttacat ggtagcaata 1080acagaatgaa aatcacattt ggtatggggg cttcacaata ttcgcatgac tgtctggaag 1140tagaccatgc tatttttctg ctcactgtac acaaatattg ttcacataaa ccctataatg 1200taaatatgaa atacagtgat tactcttctc act 123356247PRTMus musculus 56Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His 1 5 10 15 Ala Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 20 25 30 Pro Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 40 45 His Pro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 50 55 60 Pro Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys 65 70 75 80 Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 90 95 Phe Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr 100 105 110 Val Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val 115 120 125 Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn 130 135 140 His Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu 145 150 155 160 Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val 165 170 175 Ser Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr 180 185 190 Gln Glu Lys Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu 195 200 205 Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His 210 215 220 Asn Gly Leu Tyr Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe 225 230 235 240 Leu Leu Tyr His Asp Thr Asn 245 57926DNAMus musculus 57aggggcagga ggtaagaggc aggagtccat aaaacagccc tgagagcctg ctgggtcagt 60gcctgctgtc agaatgcaca gctccgtgta cttcgtggct ctggtgatcc tgggagcggc 120tgtatgtgca gcacagcccc gaggccggat tctgggtggc caggaggccg cagcccatgc 180tcggccctac atggcttccg tgcaagtgaa cggcacacac gtgtgcggtg gcaccctgct 240ggacgagcag tgggtgctca gtgctgcaca ctgcatggat ggagtgacgg atgacgactc 300tgtgcaggtg ctcctgggtg cccactccct gtccgcccct gaaccctaca agcgatggta 360tgatgtgcag agtgtagtgc ctcacccggg cagccgacct gacagccttg aggacgacct 420cattcttttt aagctatccc agaatgcctc gttgggtccc cacgtgagac ccctaccctt 480gcaatacgag gacaaagaag tggaacccgg cacgctctgc gacgtggctg gttggggtgt 540ggtcacccat gcaggacgca ggcctgatgt cctgcatcaa ctcagagtgt caatcatgaa 600ccggacaacc tgcaatctgc gcacgtacca tgacggggta gtcaccatta acatgatgtg 660tgcagagagc aaccgcaggg acacttgcag gggagactcc ggcagccctc tagtgtgcgg 720ggatgcagtc gaaggtgtgg ttacgtgggg ctctcgcgtc tgtggcaatg gcaaaaagcc 780gggcgtctat acccgagtgt catcctaccg gatgtggatc gaaaacatca caaatggtaa 840catgacatcc tgaggggaca ccagagacac gtggctcagg gaaacaagag acacgtggct 900cacaataaat gcatgcatct gagaaa 92658259PRTMus musculus 58Met His Ser Ser Val Tyr Phe Val Ala Leu Val Ile Leu Gly Ala Ala 1 5 10 15 Val Cys Ala Ala Gln Pro Arg Gly Arg Ile Leu Gly Gly Gln Glu Ala 20 25 30 Ala Ala His Ala Arg Pro Tyr Met Ala Ser Val Gln Val Asn Gly Thr 35 40 45 His Val Cys Gly Gly Thr Leu Leu Asp Glu Gln Trp Val Leu Ser Ala 50 55 60 Ala His Cys Met Asp Gly Val Thr Asp Asp Asp Ser Val Gln Val Leu 65 70 75 80 Leu Gly Ala His Ser Leu Ser Ala Pro Glu Pro Tyr Lys Arg Trp Tyr 85 90 95 Asp Val Gln Ser Val Val Pro His Pro Gly Ser Arg Pro Asp Ser Leu 100 105 110 Glu Asp Asp Leu Ile Leu Phe Lys Leu Ser Gln Asn Ala Ser Leu Gly 115 120 125 Pro His Val Arg Pro Leu Pro Leu Gln Tyr Glu Asp Lys Glu Val Glu 130 135 140 Pro Gly Thr Leu Cys Asp Val Ala Gly Trp Gly Val Val Thr His Ala 145 150 155 160 Gly Arg Arg Pro Asp Val Leu His Gln Leu Arg Val Ser Ile Met Asn 165 170 175 Arg Thr Thr Cys Asn Leu Arg Thr Tyr His Asp Gly Val Val Thr Ile 180 185 190 Asn Met Met Cys Ala Glu Ser Asn Arg Arg Asp Thr Cys Arg Gly Asp 195 200 205 Ser Gly Ser Pro Leu Val Cys Gly Asp Ala Val Glu Gly Val Val Thr 210 215 220 Trp Gly Ser Arg Val Cys Gly Asn Gly Lys Lys Pro Gly Val Tyr Thr 225 230 235 240 Arg Val Ser Ser Tyr Arg Met Trp Ile Glu Asn Ile Thr Asn Gly Asn 245 250 255 Met Thr Ser 593171DNAMus musculus 59gccgcccggg gaccagactg gaagatgcct gggggcccgg gcgcgccctc ttctcccgca 60gcatcctcag gctcctcgcg tgccgcgccg tcggggatcg cggcatgccc tctgtcccca 120cctcccctag cccgagggtc tccacaagct tccggtcccc ggcgcggcgc cagcgtcccg 180cagaagctgg cggagactct gagcagccag tatggactga acgtgttcgt ggcggggctg 240ctgttcctgc tggcctgggc ggtgcacgcg acgggtgtgg gcaagagcga cctgctgtgc 300gttctaaccg cgcttatgct gctgcagctg ctctggatgc tgtggtacgt gggtcgcagc 360tacatgcagc gccgcctcat ccgccccaag gacacgcacg cgggtgcgcg ctggcttcgc 420ggaagcatca cgttgttcgc gtttatcact gtcgtcctgg gatgcttgaa agtcgcatac 480ttcattggat tctcggagtg cttgtcagcc accgagggag ttttcccagt cacccatgca 540gtgcataccc tattgcaggt gtatttcctc tggggccatg ctaaggatat catcatgtct 600ttcaaaacac tggaaaggtt tggggtgatc cattcagtgt tcacgaacct cctactgtgg 660gccaacagcg tcctgaatga atcaaagcac cagctgaatg agcacaagga acggctgatc 720actctgggct ttggcaacat

caccatcgtt ttggatgacc acacaccaca gtgtaactgc 780acaccacccg ccctctgctc tgccctctcc catgggattt actatctgta ccccttcaac 840attgagtacc agatcctggc ctcgaccatg ctctacgtgc tgtggaagaa catcgggcgc 900agagtggaca gctcccagca ccagaagatg cagtgcagat tcgacggggt cctagtgggc 960tccgtgctgg gcttgacagt gctggctgcc accatcgccg tggttgtggt gtacatgatc 1020cacatcgggc gctccaaatc caagagcgag tcggctctca tcatgttcta tttgtacgct 1080atcacggtgc tgctgcttat gggggccgca gggctagtcg gaagctggat ttacagggtg 1140gatgagaagt ctctggatga gtcaaagaac ccagcgcgca agctggatgt tgacctgttg 1200gtggccaccg gctccggctc ctggctcctc tcctggggct ccattctggc catcgcctgt 1260gctgagactc gcccaccgta cacctggtac aacctgccct actcggtcct ggtgatcgtg 1320gagaagtatg tccaaaacat tttcatcatc gagtccgtgc acctcgagcc tgagggggtc 1380ccggaggatg tgcgcactct gcgtgtggtc accgtctgca gcagcgaggc tgccgcactg 1440gctgcatcca ctctcgggag ccaggggatg gcccaggatg ggtcacctgc tgtcaatgga 1500aatctgtgtc tgcagcagag gtgtgggaaa gaggaccagg agtctggctg ggaaggagct 1560acggggacaa cccgatgtct ggacttcctt cagggcggca tgaagaggag gcttctcaga 1620aacatcacgg cctttctgtt tctttgcaac atctcgcttt ggattccccc tgcctttggc 1680tgccgtcccg agtatgacaa cggattggag gaaattgtct ttggctttga accctggata 1740attgtggtca acctggccat gcccttttcc attttctacc ggatgcacgc agctgctgcc 1800ctctttgagg tctattgtaa gatctagcct gagtcctcat gaaaggagaa gggacggggg 1860agcaaggggg ttctgcagcc acctgcgaag ggcggggatg agcaaacact gtctgacaaa 1920ggcgggaagg atgccttttg ttctgactgt ctggagttgc cctcgacctg ggggagagcg 1980agcagttaaa tcaactgcac aagatagctg gagcctgtcc ttagccaatt ccaatcagat 2040ccaaaatgag ttgagcattt tctggctgag accctgtgtt ttagaagaag gtgatccagg 2100tttggatgaa aatgatggag tttttcataa caagcatttc ctgtctgtaa tttgtatgac 2160tgtgtacaac ttttttctgg ccatctgtgt atcaatccat ttacattttt tttttttcct 2220gctcagggtt actgggtttg gaaacaaatg cattttagtt tgtagtttaa tatttgagct 2280ggtttaattt tggataaaaa ttagttttga tcaactgcat gagagtctga ttctagactt 2340gttctacaaa cacaagacct tccctaaatt tttggtccat ttttatttgt gttcattatg 2400aaatattaaa agaaaataaa cattgatgtt ctcttttttg ctacatctat atccagttcc 2460tgacgtagaa aatctgaata tttttgggag gagagacagg aaaaatggtc catgttgcac 2520tgctaacagt tactctgaac accatggtgt agctttcagc tgtggtgccg tgcagacttt 2580tctatattgt tgtacttgct agtgataacc tgacatttct aatgttctcc tcatgctttc 2640ctatttgaca gtctgatgac acctttctct acactgtcat aaattcctgg caaacatcat 2700tttactgctg tcagaatctc aaactaatgt accacaatta acctcctttg cctttttttg 2760attttagaag gctcacaatg ttgctttcct aagtggctct gcgaaagcca agcgtgtttc 2820taaaacattt ctcttattta aaaattgcat tttaaaagag acgattggcc ctacatatcc 2880attgatccca catctacatt cagccaattt tgcatagaaa atgttttgta agagttgggt 2940cttggggttg gagagatggc ctgctgctct caaaaaggac cagagtttgg tttttagtat 3000ggcatcagtt ggtttacaac tgcctgcaat tctagctcca gaagatctga tattcttctc 3060aggtttccag ggacaccagc actcacatgc aagagtgtgc tacccccatg aataataaaa 3120aataataata ataaagcttt caaaaaggaa aaaaaaaaaa aaaaaaaaaa a 317160600PRTMus musculus 60Met Pro Gly Gly Pro Gly Ala Pro Ser Ser Pro Ala Ala Ser Ser Gly 1 5 10 15 Ser Ser Arg Ala Ala Pro Ser Gly Ile Ala Ala Cys Pro Leu Ser Pro 20 25 30 Pro Pro Leu Ala Arg Gly Ser Pro Gln Ala Ser Gly Pro Arg Arg Gly 35 40 45 Ala Ser Val Pro Gln Lys Leu Ala Glu Thr Leu Ser Ser Gln Tyr Gly 50 55 60 Leu Asn Val Phe Val Ala Gly Leu Leu Phe Leu Leu Ala Trp Ala Val 65 70 75 80 His Ala Thr Gly Val Gly Lys Ser Asp Leu Leu Cys Val Leu Thr Ala 85 90 95 Leu Met Leu Leu Gln Leu Leu Trp Met Leu Trp Tyr Val Gly Arg Ser 100 105 110 Tyr Met Gln Arg Arg Leu Ile Arg Pro Lys Asp Thr His Ala Gly Ala 115 120 125 Arg Trp Leu Arg Gly Ser Ile Thr Leu Phe Ala Phe Ile Thr Val Val 130 135 140 Leu Gly Cys Leu Lys Val Ala Tyr Phe Ile Gly Phe Ser Glu Cys Leu 145 150 155 160 Ser Ala Thr Glu Gly Val Phe Pro Val Thr His Ala Val His Thr Leu 165 170 175 Leu Gln Val Tyr Phe Leu Trp Gly His Ala Lys Asp Ile Ile Met Ser 180 185 190 Phe Lys Thr Leu Glu Arg Phe Gly Val Ile His Ser Val Phe Thr Asn 195 200 205 Leu Leu Leu Trp Ala Asn Ser Val Leu Asn Glu Ser Lys His Gln Leu 210 215 220 Asn Glu His Lys Glu Arg Leu Ile Thr Leu Gly Phe Gly Asn Ile Thr 225 230 235 240 Ile Val Leu Asp Asp His Thr Pro Gln Cys Asn Cys Thr Pro Pro Ala 245 250 255 Leu Cys Ser Ala Leu Ser His Gly Ile Tyr Tyr Leu Tyr Pro Phe Asn 260 265 270 Ile Glu Tyr Gln Ile Leu Ala Ser Thr Met Leu Tyr Val Leu Trp Lys 275 280 285 Asn Ile Gly Arg Arg Val Asp Ser Ser Gln His Gln Lys Met Gln Cys 290 295 300 Arg Phe Asp Gly Val Leu Val Gly Ser Val Leu Gly Leu Thr Val Leu 305 310 315 320 Ala Ala Thr Ile Ala Val Val Val Val Tyr Met Ile His Ile Gly Arg 325 330 335 Ser Lys Ser Lys Ser Glu Ser Ala Leu Ile Met Phe Tyr Leu Tyr Ala 340 345 350 Ile Thr Val Leu Leu Leu Met Gly Ala Ala Gly Leu Val Gly Ser Trp 355 360 365 Ile Tyr Arg Val Asp Glu Lys Ser Leu Asp Glu Ser Lys Asn Pro Ala 370 375 380 Arg Lys Leu Asp Val Asp Leu Leu Val Ala Thr Gly Ser Gly Ser Trp 385 390 395 400 Leu Leu Ser Trp Gly Ser Ile Leu Ala Ile Ala Cys Ala Glu Thr Arg 405 410 415 Pro Pro Tyr Thr Trp Tyr Asn Leu Pro Tyr Ser Val Leu Val Ile Val 420 425 430 Glu Lys Tyr Val Gln Asn Ile Phe Ile Ile Glu Ser Val His Leu Glu 435 440 445 Pro Glu Gly Val Pro Glu Asp Val Arg Thr Leu Arg Val Val Thr Val 450 455 460 Cys Ser Ser Glu Ala Ala Ala Leu Ala Ala Ser Thr Leu Gly Ser Gln 465 470 475 480 Gly Met Ala Gln Asp Gly Ser Pro Ala Val Asn Gly Asn Leu Cys Leu 485 490 495 Gln Gln Arg Cys Gly Lys Glu Asp Gln Glu Ser Gly Trp Glu Gly Ala 500 505 510 Thr Gly Thr Thr Arg Cys Leu Asp Phe Leu Gln Gly Gly Met Lys Arg 515 520 525 Arg Leu Leu Arg Asn Ile Thr Ala Phe Leu Phe Leu Cys Asn Ile Ser 530 535 540 Leu Trp Ile Pro Pro Ala Phe Gly Cys Arg Pro Glu Tyr Asp Asn Gly 545 550 555 560 Leu Glu Glu Ile Val Phe Gly Phe Glu Pro Trp Ile Ile Val Val Asn 565 570 575 Leu Ala Met Pro Phe Ser Ile Phe Tyr Arg Met His Ala Ala Ala Ala 580 585 590 Leu Phe Glu Val Tyr Cys Lys Ile 595 600 612822DNAMus musculus 61gcatctttcc ccctcaagcg ggtctcacta gatcccggag agccttggtg ctctccggtt 60ccgtgggttg tggcagtgag tcccaccaga cccgcccttt gcacacggct tccgaacgcc 120ggggtctcgt gccggccagg cccggaccct ataccctatt catttttttc ttattgcagc 180gcctgagtct tttcttcttt taaaacaaga tgccgtcggg tttccagcag atcggctctg 240acgatgggga accccctcgg cagcgagtga ctggaacact ggtcctagct gtattctcag 300ctgtgcttgg ctcccttcag tttggctata acattggggt tatcaatgcc ccacagaagg 360tgattgaaca gagctacaat gcaacgtggc tgggtaggca aggtcctggg ggaccggatt 420ccatcccaca aggcaccctc actacgctct gggctctctc cgtggccatc ttctctgtgg 480gtggcatgat ctcttccttt ctcattggca tcatttctca atggttggga aggaaaaggg 540ctatgctggc caacaatgtc ttggccgtgt tggggggcgc cctcatgggc ctagccaatg 600ccgcggcctc ctatgagata ctcattcttg gacggttcct cattggcgcc tactcagggc 660taacatcagg gctggtgccc atgtatgtgg gagaaatcgc ccccactcat cttcggggtg 720ccttgggaac actcaaccaa ctggccatcg tcattggcat tctggttgcc caggtgctgg 780gcttggagtc tatgctgggc acagctaccc tgtggccact gcttctggct ctcacagtac 840tccctgctct cctgcagctg attctgctgc ccttctgtcc tgagagcccc agatacctct 900acatcatccg gaacctggag gggcctgccc gaaagagtct aaagcgcctg accggctggg 960ctgatgtgtc tgacgcacta gctgagctga aggatgagaa acggaagttg gagagagagc 1020gtccaatgtc cttgctccag ctcctgggca gccgcaccca ccggcagcct ctgatcatcg 1080cagtggtgct gcagctgagc caacagctct caggcatcaa tgctgttttc tactattcaa 1140ccagcatctt cgagtcggct ggggtgggac agccagccta cgccaccata ggagctggtg 1200tggtcaatac ggtcttcacg ttggtctcgg tgctcttagt agaacgagct ggacgacgga 1260cactccatct gttgggcctg gccggcatgt gtggctgtgc catcttgatg accgtggctc 1320tgctgctgct ggaacgggtt ccagccatga gctatgtctc catcgtggcc atatttggct 1380ttgtggcctt ctttgagatt ggccctggcc ccattccctg gttcattgtg gcagagctct 1440tcagccaggg cccccgccca gccgccatgg ctgtcgctgg tttctccaac tggacctgta 1500acttcattgt cggcatgggt ttccagtatg ttgcggatgc tatgggtcct tacgtcttcc 1560ttctatttgc cgtcctcctg cttggcttct tcatcttcac cttcctaaaa gtgcctgaaa 1620ccagaggccg gacgtttgac cagatctcag ctgccttccg acggacacct tcccttttag 1680agcaggaggt gaaacccagt acagaacttg aatacttagg gccagatgag aatgactgag 1740gggcaaaaca gggtgggaga gccaccctct ccacccagac tccctccttt cctctacagc 1800actttagccc tctcttccct gttacctcca ggttgaagga acagcagcct ggggaactgg 1860gaagctgaag ggaggggtgg tccatgtacc cctcattccc cctgtgtgat tcttttggat 1920tatttatgtg tgtggctagg ctgtggccac ctagatgggc tttctccgtc ctgccttcct 1980cctgccccta cccagactca gctctagact actttcctcc cctttcgaga aggggtctgc 2040aggagggtgg ggtggccctg aattcatcag gataaacagc aggggtgggt gtgtgagcga 2100gtgctttcct cccacaaact ggcacttcca ctgaactctt gccacacagg ctctgggtga 2160agggggttgt cttgacccct ccagggcaaa ggatacaccc tcccaaaatc tagccctgcc 2220tccccacagg ctccaccctc ccgggcaaag gaacacaata gtacatacct gacagggcaa 2280ggacggttag agcgcatcag tctccatttg gggccctagg ttgttcccag ggctgcaaag 2340cgtaggtacc aacactttct tgttcccctc caggaagggt gctaaacccg aaagcttctg 2400accaactaag ggcgggaggg gatttgaaag gctgcctata aacactggtt gggagggagc 2460ctttggtatt tttgtatgtt ttgaagaacg gatagggagc agaaacccaa gggctgctgt 2520attaaatgtg tatatagaga ttcgtccata aagtcactgt ttgaagatga gtgtcctgtg 2580ctggaggaac tggagggtgt gcaaaaagaa aatgcctcac tcaccctttg cacttcatcc 2640ttcctgggct cagggcaccg aggcctctag attctgtcac ctttttttta ggggggggcg 2700ggtttcgaga cagggtttct ctgtcctgga actcactttg tagaccaggc tggccttgaa 2760ctcaagaaat ccgcctgcct ctgcctcccg attgctggga ttaaaggtgt gcgtgtgcca 2820cc 282262509PRTMus musculus 62Met Pro Ser Gly Phe Gln Gln Ile Gly Ser Asp Asp Gly Glu Pro Pro 1 5 10 15 Arg Gln Arg Val Thr Gly Thr Leu Val Leu Ala Val Phe Ser Ala Val 20 25 30 Leu Gly Ser Leu Gln Phe Gly Tyr Asn Ile Gly Val Ile Asn Ala Pro 35 40 45 Gln Lys Val Ile Glu Gln Ser Tyr Asn Ala Thr Trp Leu Gly Arg Gln 50 55 60 Gly Pro Gly Gly Pro Asp Ser Ile Pro Gln Gly Thr Leu Thr Thr Leu 65 70 75 80 Trp Ala Leu Ser Val Ala Ile Phe Ser Val Gly Gly Met Ile Ser Ser 85 90 95 Phe Leu Ile Gly Ile Ile Ser Gln Trp Leu Gly Arg Lys Arg Ala Met 100 105 110 Leu Ala Asn Asn Val Leu Ala Val Leu Gly Gly Ala Leu Met Gly Leu 115 120 125 Ala Asn Ala Ala Ala Ser Tyr Glu Ile Leu Ile Leu Gly Arg Phe Leu 130 135 140 Ile Gly Ala Tyr Ser Gly Leu Thr Ser Gly Leu Val Pro Met Tyr Val 145 150 155 160 Gly Glu Ile Ala Pro Thr His Leu Arg Gly Ala Leu Gly Thr Leu Asn 165 170 175 Gln Leu Ala Ile Val Ile Gly Ile Leu Val Ala Gln Val Leu Gly Leu 180 185 190 Glu Ser Met Leu Gly Thr Ala Thr Leu Trp Pro Leu Leu Leu Ala Leu 195 200 205 Thr Val Leu Pro Ala Leu Leu Gln Leu Ile Leu Leu Pro Phe Cys Pro 210 215 220 Glu Ser Pro Arg Tyr Leu Tyr Ile Ile Arg Asn Leu Glu Gly Pro Ala 225 230 235 240 Arg Lys Ser Leu Lys Arg Leu Thr Gly Trp Ala Asp Val Ser Asp Ala 245 250 255 Leu Ala Glu Leu Lys Asp Glu Lys Arg Lys Leu Glu Arg Glu Arg Pro 260 265 270 Met Ser Leu Leu Gln Leu Leu Gly Ser Arg Thr His Arg Gln Pro Leu 275 280 285 Ile Ile Ala Val Val Leu Gln Leu Ser Gln Gln Leu Ser Gly Ile Asn 290 295 300 Ala Val Phe Tyr Tyr Ser Thr Ser Ile Phe Glu Ser Ala Gly Val Gly 305 310 315 320 Gln Pro Ala Tyr Ala Thr Ile Gly Ala Gly Val Val Asn Thr Val Phe 325 330 335 Thr Leu Val Ser Val Leu Leu Val Glu Arg Ala Gly Arg Arg Thr Leu 340 345 350 His Leu Leu Gly Leu Ala Gly Met Cys Gly Cys Ala Ile Leu Met Thr 355 360 365 Val Ala Leu Leu Leu Leu Glu Arg Val Pro Ala Met Ser Tyr Val Ser 370 375 380 Ile Val Ala Ile Phe Gly Phe Val Ala Phe Phe Glu Ile Gly Pro Gly 385 390 395 400 Pro Ile Pro Trp Phe Ile Val Ala Glu Leu Phe Ser Gln Gly Pro Arg 405 410 415 Pro Ala Ala Met Ala Val Ala Gly Phe Ser Asn Trp Thr Cys Asn Phe 420 425 430 Ile Val Gly Met Gly Phe Gln Tyr Val Ala Asp Ala Met Gly Pro Tyr 435 440 445 Val Phe Leu Leu Phe Ala Val Leu Leu Leu Gly Phe Phe Ile Phe Thr 450 455 460 Phe Leu Lys Val Pro Glu Thr Arg Gly Arg Thr Phe Asp Gln Ile Ser 465 470 475 480 Ala Ala Phe Arg Arg Thr Pro Ser Leu Leu Glu Gln Glu Val Lys Pro 485 490 495 Ser Thr Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp 500 505 6321DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 63gccctgagta atcacttaaa g 216419DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 64ccgggccctg agtaatcac 196522DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 65ccttgcaata cgaggacaaa ga 226618DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 66cacaccccaa ccagccac 186722DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 67gcactggcaa gttctactgc aa 226823DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 68gtaggtgaag agaacggcct tgt 236922DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 69caccctgatg ttattgggtt ca 227021DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 70ttagcgccca cttattccac t 217121DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 71catcctggga cgagatgaac t 217220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 72tgacaagcgt taccacaggc 207320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 73ctgaccttcg gcacgacatt 207423DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 74ccaccataag ccgtgtaaaa gac 237521DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 75caagaacagc aacgagtacc g 217621DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 76gtcactggtc aactccagca c 217721DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 77acgacttcct ctccgacctc t 217821DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 78cgaggctcac gtaaccgtag t 217921DNAArtificial Sequencesource/note="Description of Artificial

Sequence Synthetic primer" 79cgacttcagc gcctacattg a 218019DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 80ctagcgacag accccacac 198121DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 81atcacaactg gcctggttac g 218221DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 82tactacccgg tgtccatttc t 218320DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 83cagcgtcatg gtcagtctgt 208420DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 84agaaaaccgt gtggcagaga 208520DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 85gaaccatgaa gccaacgact 208620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 86gcgaagttca cagtggttcc 208721DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 87gagtgggaac tggtagtgtt g 218819DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 88cgcacagagc gatgaaggt 198922DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 89gtaggactcc cggcttcttt ct 229022DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 90agtctggtcc aagaatccga ag 229121DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 91gatggttctg ggcaccatct t 219220DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 92cgttgttgtg tggcatcctt 209321DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 93ccaggaaaca tcagtgagtc c 219422DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 94ggatggaact tggaatcggt ca 229519DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 95ccctgcgacg agaaagctc 199622DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 96gctcttttcg ttgaggcaaa cc 229722DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 97ctgctgactt taacacaggg ag 229821DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 98ggttgccact ttttacccca g 219920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 99actaggaccc cgtcgaatct 2010020DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 100accatgctct acgtgctgtg 2010119DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 101ccctgccatt gttaagacc 1910219DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 102tgctgctgtt cctgttttc 1910322DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 103tgaaagaagc ggtgaaccac tg 2210422DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 104tggcatctct gtgtcaacca tg 2210518DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 105gcatggtgcc ttcgctga 1810622DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 106tggcatctct gtgtcaacca tg 2210719DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 107cagcacggtg aagccattc 1910817DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 108gcgtgcatcc gcttgtg 1710920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 109gtcacagcac atgacggagg 2011021DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 110tcttccagat gctcgggata c 2111123DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 111cccttgcagt tctaagttca aca 2311221DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 112acctttgaca agcggactct c 2111322DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 113cagacctgcc ttacgactat gg 2211421DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 114ctcggtggcg ttgagattgt t 2111521DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 115actgccacac ctccagtcat t 2111621DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 116ctttgcctca ctcaggattg g 2111721DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 117cagccgtcca gtgttatgtt g 2111822DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 118tgccccaatt tatcaaggca aa 2211920DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 119ggcaagcagt gcagctagaa 2012023DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 120cataactgtc tttcgaggtg tgg 2312121DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 121cagcctttgg agaaccagtc t 2112221DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 122tcccgctcag agattccatc a 2112321DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 123agaaagtagc cagatggagc c 2112421DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 124atgtgagcct agtagctgtc a 2112521DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 125gataggccgt gtggtgaaat c 2112621DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 126accaggaggg aaaccatact c 2112723DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 127ccctgacatt tatgggatca cag 2312822DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 128catccagagt cttgctgatg ag 2212919DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 129cttcgcgggg tagtgttgg 1913019DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 130ggccagactt cgacgacaa 1913121DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 131gtattgggca acctcattcc c 2113221DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 132gcccccagga tactgaagat t 2113325DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 133gccaaaccaa caactttatc tcttc 2513425DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 134cacacttaag gtgcgttcaa tagtc 2513519DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 135ctggaatagc ggcgtgctt 1913620DNAArtificial Sequencesource/note="Description of Artificial Sequence Synthetic primer" 136aataacactg gacgtcgggc 20

Claims

1. A method for preventing or treating obesity or an obesity-related disorder in a subject comprising inducing both Prdm16 and C/EBPβ expression and/or activity sufficient to activate brown fat cell differentiation, wherein the differentiated brown fat cells increase energy expenditure to thereby prevent or treat obesity or an obesity-related disorder in the subject.

2. The method of claim 1, where in the obesity-related disorder is selected from the group consisting of: insulin resistance, type II diabetes, hypertension, hyperuricemia, fatty liver, non-alcoholic fatty liver disease, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia.

3. The method of claim 1, wherein the obesity-related disorder is Type II diabetes.

4. The method of claim 1, wherein the subject is human.

5. A method for increasing energy expenditure in a mammal comprising inducing both Prdm16 and C/EBPβ expression and/or activity sufficient to activate brown fat cell differentiation in the mammal, wherein the differentiated brown fat cells promote energy expenditure thereby increasing energy expenditure in the mammal.

6. The method of claim 5, wherein the mammal is a human.

7. A method for treating obesity or an obesity-related disorder in a subject comprising administering to the subject an agent that induces expression and/or activity of both Prdm16 and C/EBPβ, wherein Prdm16 and C/EBPβ expression and/or activity increases respiration and energy expenditure to thereby treat obesity or an obesity-related disorder in the subject.

8. The method of claim 7, wherein the respiration is total respiration or uncoupled respiration.

9. The method of claim 7, wherein the respiration is assessed using positron emission tomography.

10. The method of claim 7, where in the obesity-related disorder is selected from the group consisting of: insulin resistance, type II diabetes, hypertension, hyperuricemia, fatty liver, non-alcoholic fatty liver disease, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, Prader-Labhart-Willi syndrome, anorexia, and cachexia.

11. The method of claim 7, wherein the obesity-related disorder in Type II diabetes.

12. The method of claim 7, wherein the subject is a human.

13-50. (canceled)

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