MXPA03009768A - Use of follistatin to increase muscle mass - Google Patents

Abstract

The present invention provides a substantially purified growth differentiation factor (GDF) receptor, including a GDF-8 (myostatin) receptor, as well as functional peptide portions thereof. In addition, the invention provides a virtual representation of a GDF receptor or a functional peptide portion thereof. The present invention also provides a method of modulating an effect of myostatin on a cell by contacting the cell with an agent that affects myostatin signal transduction in the cell. In addition, the invention provides a method of ameliorating the severity of a pathologic condition, which is characterized, at least in part, by an abnormal amount, development or metabolic activity of muscle or adipose tissue in a subject, by modulating myostatin signal transduction in a muscle cell or an adipose tissue cell in the subject. The invention also provides a method of modulating the growth of muscle tissue or adipose tissue in a eukaryotic organism by administering an agent that affects myostatin signal transduction to the organism.

Description

USE OF FOLISTATINE TO INCREASE THE MUSCLE MASS Field of the Invention This invention relates generally to receptors of growth differentiation factor (GDF), and more specifically to GDF-8 receptors (myostatin), to compositions that affect the myostatin signal transduction in a cell, and methods of using such compositions to modulate the myostatin signal transduction in a cell. Background The amount of time, effort, and money spent in the United States each year by people trying to lose weight is increasing. For many of these people, the goal is not merely to look better, but more important, to avoid the inevitable medical problems associated with being overweight. More than half of the adult population in the United States is considered overweight. In addition, 20 to 30 percent of adult men, and 30 to 40 percent of adult women in the United States, are considered obese, with the highest rates among the poor and minorities. Obesity, which is defined as at least approximately 20 percent above the mean level of adiposity, has increased dramatically in prevalence during the past decades, and is becoming a major problem among the pediatric population. percent of all children are now considered overweight, a number that represents a doubling over the past five years. Obesity and the medical problems that can be directly attributed to it, are a leading cause of pathology and mortality throughout the world. Obesity is an important risk factor for the development of different pathological conditions, including atherosclerosis, hypertension, heart attack, type II diabetes, gallbladder disease, and certain cancers, and contributes to premature death. Heart disease is the leading cause of death in the United States, and type II diabetes afflicts more than 16 million people in the United States, and is one of the leading causes of death by disease. More than 80 percent of type II diabetes occurs in obese people. Although type II diabetes affects all races, it is particularly prevalent among Native Americans, African Americans, and Hispanics. Significantly, type II diabetes, which used to occur almost exclusively in adults over 40 years of age, now occurs in children, having almost tripled the cases reported during the last five years. Type II diabetes, also called non-insulin-dependent diabetes, is characterized by reduced insulin secretion in response to glucose, and by the body's resistance to the action of insulin, even when the levels of insulin in the circulation are generally normal or elevated. Type II diabetes affects the function of a variety of different tissues and organs, and can lead to vascular disease, kidney failure, retinopathy, and neuropathy. In contrast to the medical problems associated with obesity, the severe weight loss that commonly occurs in patients with certain chronic diseases also presents a challenge to medical intervention. The molecular basis for this weight loss, referred to as cachexia, is not well understood. However, it is clear that cachexia complicates the management of these diseases, and is associated with a poor prognosis for patients. The effects of cachexia are evident in the waste syndrome that occurs in cancer and AIDS patients. Although great efforts have been made to try to elucidate the biological processes involved in the regulation of body weight,. Results have provided more fantasies than real value. For example, the discovery of leptin has been proclaimed as a discovery in the understanding of the molecular basis for the accumulation of fat in humans, and with it, the promise of a cure for obesity. Studies in animals indicated that leptin is involved in the transmission of internal signals that regulate appetite, and suggested that leptin could be useful in treating humans. humans who suffer from obesity. However, progress in the use of leptin to treat obesity has been slow, and so far, leptin has not met initial expectations. The treatment of the pathological obese is currently limited to surgery to remove portions of the intestine, thereby reducing the amount of food (and calories) absorbed. For the moderate obese, the only "treatment" is to eat a healthy diet and exercise regularly, a method that has proven modest success at best. Accordingly, there is a need to identify the biological factors involved in the regulation of body weight, including the development of muscle and the accumulation of fat, so that methods for the treatment of disorders such as obesity and cachexia can be developed. The present invention satisfies this need, and provides additional advantages. SUMMARY OF THE INVENTION The present invention relates to a substantially purified growth differentiation factor receptor. A growth differentiation factor receptor of the invention can be, for example, a myostatin receptor, a GDF-11 receptor, or another growth differentiation factor receptor. A myostatin receptor, for example, interacts specifically with at least myostatin, and may also specifically interact with one or a few growth factor peptides. additional mature. Also provided are polynucleotides that encode a growth differentiation factor receptor, anti-bodies that specifically interact with a growth differentiation factor receptor, and the like. The present invention also relates to a method for modulating the effect of a growth differentiation factor, by affecting the signal transduction effected by the growth differentiation factor. For example, a method is provided for modulating an effect of myostatin on a cell by contacting the cell with an agent that affects the transduction of the myostatin signal in the cell. In one embodiment, the agent alters a specific interaction of myostatin with a myostatin receptor expressed by the cell, thereby modulating the transduction of the myostatin signal in the cell. The myostatin receptor can be an activin receptor, or it can be any other receptor that can make contact with a mature myostatin or a functional peptide portion thereof, such that the transduction of the myostatin signal is activated. In another embodiment, the agent binds to a myostatin receptor, thereby improving the binding of myostatin to the receptor, or competing with myostatin for the receptor. As such, the agent can increase the transduction of the myostatin signal, or it can reduce or inhibit the transduction of the myostatin signal. In yet another modality, the agent acts intracellularly to alter the transduction of the myostatin signal in the cell. A useful agent for modulating the signal transduction of the growth differentiation factor in a cell can be a peptide, a mimetic peptide, a polynucleotide, a small organic molecule, or any other agent, and can act as a transduction agonist. of the signal of the growth differentiation factor, or as an antagonist of the signal transduction of the growth differentiation factor. In one embodiment, the peptide agent alters a specific interaction of myostatin with a myostatin receptor. This peptide agent can be, for example, a peptide that binds or otherwise sequesters myostatin, thereby affecting the ability of myostatin to interact specifically with its receptor. These agents are exemplified by a mutant myostatin receptor, for example, a soluble extracellular domain of a myostatin receptor, which can interact specifically with myostatin.; by a prodrug of myostatin, which can interact specifically with myostatin; and by a mutant myostatin polypeptide that is resistant to proteolytic cleavage in a mature prodrug or myostatin, and that can interact specifically with myostatin, and are useful as myostatin signal transduction antagonists, which reduce or inhibit the transduction of the myostatin signal in a cell.

In another embodiment, the peptide agent can interact specifically with a myostatin receptor expressed by a cell, thereby competing with myostatin for the receptor. This peptide agent is employed by an anti-myostatin receptor antibody, or by an anti-idiotypic anti-body anti-myostatin anti-body anti-idiotype. This peptide agent provides the additional advantage that it can be selected not only for its ability to specifically interact with a myostatin receptor, thereby competing with myostatin for the receptor, but can also be selected to have the ability to activate or Do not activate the transduction of the myostatin signal. Accordingly, a peptide agent which specifically interacts with a myostatin receptor expressed by a cell, and which activates the transduction of the myostatin-dependent signal, such as a myostatin agonist, can be used to increase the transduction of the myostatin signal in the cell, whereas a peptide agent can be used that specifically interacts with a myostatin receptor expressed by a cell, but does not activate the transduction of the myostatin signal, such as a myostatin antagonist, to reduce or inhibit the transduction of the myostatin signal in the cell. A useful agent in a method of the invention may also be a polynucleotide. In general, but not necessarily, the polynucleotide is introduced into the cell, where it performs its function either directly, or immediately after the transcription or translation, or both. For example, the polynucleotide agent can encode a peptide, which is expressed in the cell and modulates the activity of myostatin. This expressed peptide can be, for example, a mutant myostatin receptor, such as an extracellular domain of soluble myostatin receptor; an extracellular domain of myostatin receptor operatively associated with a membrane anchoring domain; or a mutant myostatin receptor that lacks protein kinase activity. A peptide expressed from a polynucleotide agent can also be a peptide that affects the level or activity of an intracellular polypeptide component of a signal transduction pathway of the growth differentiation factor. The intracellular polypeptide can be, for example, a Smad polypeptide, such as a dominant negative Smad, which, as disclosed herein, can affect the transduction of the myostatin signal in a cell. Accordingly, a polynucleotide agent can encode a dominant Smad 2, Smad 3, or Smad 4 polypeptide which, upon expression in the cell, reduces or inhibits the transduction of the myostatin signal in the cell; or it can encode a Smad 6 or Smad 7 polypeptide which, on its expression, reduces the transduction of the myostatin signal in the cell. A polynucleotide agent can also encode an intracellular c-ski polypeptide, whose Expression may reduce or inhibit the transduction of the myostatin signal. A polynucleotide agent useful in a method of the invention may also be, or may encode, an anti-sense molecule, a ribosome, or a triplexing agent. For example, the polynucleotide can be (or can encode) an anti-sense nucleotide sequence, such as an anti-sense c-ski nucleotide sequence, which can increase the transduction of the myostatin signal in a cell; or an anti-sense Smad nucleotide sequence, which can increase the transduction of the myostatin signal, or which can reduce or inhibit the transduction of the myostatin signal, depending on the particular Smad anti-sense nucleotide sequence. The present invention also relates to a method for reducing the severity of a pathological condition, which is characterized, at least in part, by an amount, development, or abnormal metabolic activity of muscle or adipose tissue in a subject. This method involves modulating the transduction of the growth differentiation factor signal in a cell associated with the pathological condition, for example, modulating the transduction of the myostatin signal in a muscle cell or in an adipose tissue cell in the subject. Different pathological conditions are susceptible to be alleviated using a method of the invention, including, for example, waste disorders such as cachexia, anorexia, muscular dystrophies, neuromuscular diseases; and metabolic disorders such as obesity and type II diabetes. The present invention further relates to a method for modulating the growth of muscle tissue or adipose tissue in a eukaryotic organism, by administering to the organism an agent that affects signal transduction mediated by a growth factor differentiation receptor. . In one embodiment, a method is carried out to modulate the growth of muscle tissue or adipose tissue by administering an agent that affects the transduction of the myostatin signal. In another embodiment, the agent affects the transduction of the GDF-11 signal, or the transduction of the myostatin and GDF-11 signal. The agent can be, for example, an agent that alters the specific interaction of myostatin with a myostatin receptor, an agent that reduces or inhibits the specific interaction of myostatin with a myostatin receptor, or any other agent as given to know in the present. The eukaryotic organism can be a vertebrate, for example a mammal, a bird, or a pool organism, or it can be an invertebrate, for example, a mollusk such as a shrimp, a shell, a squid, an octopus, a snake, or a slug. The present invention also relates to a method for identifying an agent that specifically interacts with a growth differentiation factor (GD.F) receptor. East The screening assay of the invention can be carried out, for example, by contacting a growth differentiation factor receptor with a test agent, and determining that the test agent interacts specifically with the growth differentiation factor receptor. , thereby identifying an agent that specifically interacts with a growth differentiation factor receptor. The growth differentiation factor receptor can be any growth differentiation factor receptor, in particular a myostatin receptor, and the agent can be an agonist of the growth differentiation factor receptor, which increases the signal transduction of the growth factor. growth differentiation factor, or an antagonist of the growth differentiation factor receptor, that reduces or inhibits the signal transduction of the growth differentiation factor. This method of the invention is useful for screening a library of test agents, in particular a combination library of test agents. The present invention also provides a virtual representation of a growth differentiation factor receptor, or a functional peptide portion of a growth differentiation factor receptor, eg, a virtual representation of the GDF-8 receptor or the GDF- receptor. eleven. In one embodiment, the virtual representation includes an agent that interacts specifically with the factor receptor. growth differentiation. As such, the invention further provides a method for identifying an agent that specifically interacts with a growth differentiation factor (GDF) receptor, or a functional peptide portion of a growth differentiation factor receptor, by utilizing a computer system. For example, the method can be performed by testing a virtual test agent to determine the ability to interact specifically with a receptor for the virtual growth differentiation factor or the functional peptide portion thereof; and detecting a specific interaction of the virtual test agent with the recipient of the virtual growth differentiation factor or the functional peptide portion thereof, thereby identifying an agent that specifically interacts with a growth differentiation factor receptor or a portion thereof. of functional peptide thereof. Brief Description of the Figures Figure 1 shows the amino acid sequences of murine promyostatin (SEQ ID NO: 4); rat promyostatin (SEQ ID NO: 6); human promyostatin (SEQ ID NO: 2); mandrel promyostatin (SEQ ID NO: 10); bovine promyostatin (SEQ ID NO: 12); swine promyostatin (SEQ ID NO: 14); ovine promyostatin (SEQ ID NO: 16); chicken promyostatin (SEQ ID NO: 8); turkey promyostatin (SEQ ID NO: 18); and zebrafish promiostatin (SEQ ID NO: 20). The amino acids are numbered in relation to the human promyostatin (SEQ ID NO: 2). The dotted lines indicate the holes introduced to maximize homology. The identical residues between the sequences are shaded. Figure 2 shows the amino acid sequences of murine promyostatin (SEQ ID NO: 4) and zebrafish promyostatin (SEQ ID NO: 20), and portions of the amino acid sequences of salmon allele promyostatin 1 (SEQ ID NO: 20). NO: 27, "salmon 1"), and salmon allele promyostatin 2 (SEQ ID NO: 29, "salmon 2"). The amino acid position is indicated in relation to the human promyostatin to the left of each row (compare Figure 1, first amino acid of salmon 1 corresponds to human promyostatin 218, first amino acid of salmon 2 corresponds to human promyostatin 239). The dotted lines indicate the holes introduced to maximize homology. The positions of relative amino acids, including voids, are indicated along the top of each row. The identical residues between the sequences are shaded. Detailed Description of the Invention The present invention provides a substantially purified peptide portion of a promyostatin polypeptide. Promiostatin, which has been previously referred to as the growth differentiation factor-8 (GDF-8), comprises an amino-terminal prodomain and a mature C-terminal myostatin peptide (see US Patent 5, 827, 733). The activity of myostatin is effected by the mature myostatin peptide in followed by its dissociation of promiostatin. Accordingly, promyostatin is a precursor polypeptide that proteolitically dissociates to produce active myostatin. As disclosed herein, the prodrug of myostatin can inhibit myostatin activity, GDF-11 activity, or both. The present invention also provides a substantially purified peptide portion of a pro-GDF-11 polypeptide. Pro-GDF-11, which has previously been referred to in general as GDF-11, comprises an amino-terminal prodomain and a mature C-terminal GDF-11 peptide (see International Publication WO 98/35019, which is incorporated herein by reference). the present as a reference). The activity of GDF-11 is effected by the mature GDF peptide following its dissociation from pro-GDF-11. Accordingly, pro-GDF-11, such as promyostatin, is a precursor polypeptide that proteolytically dissociates to produce the active GDF-11. As disclosed herein, the prodomain of GDF-11 can inhibit GDF-11 activity, myostatin activity, or both. Promiostatin and pro-GDF-11 are members of the transimorphic growth factor-ß superfamily (TGF-ß), which consists of multifunctional polypeptides that control proliferation, differentiation, and other functions in different cell types. The TGF-β superfamily, which encompasses a group of structurally related proteins that affect a broad range of differentiation processes during embryonic development, includes, for example, the Mullerian inhibitory substance (MIS), which is required for the normal development of the male sex (Behringer et al., Nature 345: 167, 1990), the genetic product decapentaplégico de Drosophila (DPP), which is required for the formation of the dorsal-ventral axis and the morphogenesis of imaginal discs (Padgett et al., Nature 325: 81-84, 1987), the genetic product Vg-1 of Xenopus, which it is located in the vegetable pole of eggs (Weeks et al., Cell 51: 861-867, 1987), activins (Mason et al., Biochem. Biophys. Res. Comm. 135: 957-964, 1986), which can induce the formation of mesoderm and anterior structures in Xenophus embryos (Thomsen et al., Cell 63: 485, 1990), and bone morphogenic proteins (MBPs, osteogenin, OP -1), which can induce the. formation of cartilage and de novo bone (Sampath et al., J. Biol. Chem. 265: 13198, 1990). Members of the TGF-β family can influence a variety of differentiation processes, including adipogenesis, myogenesis, cronogenesis, hematopoiesis, and epithelial cell differentiation (Massague, Cell 49: 437, 1987; Massague, Ann. Rev. Biochem. 67: 753-791, 1998; each of which is incorporated herein by reference). Many of the members of the TGF-β family have regulatory effects (positive or negative) on other peptide growth factors. In particular, certain members of the TGF-β superfamily have expression patterns or possess activities that are related to the function of the nervous system. For example, inhibins and activins are expressed in the brain (Meunier et al., Proc. Nati, Acad. Sci. USA 85: 247, 1988; Sawchenko et al., Nature 334: 615, 1988), and activin can function as a survival molecule of nerve cells (Schubert et al., Nature 344: 868, 1990) . Another member of the family, the growth differentiation factor-1 (GDF-1), is specific to the nervous system in its expression pattern (Lee, Proc. Nati, Acad. Sci., USA 88: 4250, 1991), and other members of the family, such as Vgr-1 (Lyons et al., Proc. Nati, Acad. Sci., USA 86: 4554, 1989), Jones et al., Development 111: 531, 1991), OP -1 (Ozkaynak et al., J. Biol. Chem. 267: 25220, 1992), and BMP-4 (Jones et al., Development 111: 531, 1991), are also expressed in the nervous system. Because skeletal muscle produces a factor or factors that promote the survival of motor neurons (Brown, Trends Neurosci, 7:10, 1984), the expression of myostatin (GDF-8) and GDF-11 in muscle suggests that myostatin and GDF-11 may be trophic factors for neurons. As such, methods for modulating the activity of myostatin, of GDF-11, or both, may be useful for the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis or muscular dystrophy, or to maintain cells or tissues in culture. before the transplant The TGF-β family proteins are synthesized as large precursor proteins, which subsequently undergo proteolytic dissociation in a cluster of basic residues of approximately 110 to 140 amino acids from the C terminus, resulting in the formation of a prodomain peptide and a peptide mature C-terminal. The C-terminal mature peptides of the members of this protein family are structurally related, and the different members of the family can be classified into different subgroups, based on the extent of their homology. Although the homologies within the particular subgroups are from 70 percent to 80 percent amino acid sequence identity, the homologies between the subgroups are significantly lower, being generally 20 percent to 50 percent. In each case, the active species appears to be a disulfide-linked dimer of C-terminal peptide fragments. Promiostatin and pro-GDF-11 polypeptides have been identified in mammal, bird, and pool species, and myostatin is active in several other species, including vertebrates and invertebrates. During embryonic development and in adult animals, for example, myostatin is expressed specifically by cells of the myogenic lineage (McPherron et al., Nature 387: 83-90, 1997, which is incorporated herein by reference). During early embryogenesis, the Myostatin is expressed by cells in the myotome compartment of developing somites. In later embryonic stages and adult animals, myostatin is widely expressed in skeletal muscle tissue, although expression levels vary considerably from muscle to muscle. Myostatin expression is also detected in adipose tissue, although at lower levels than in muscle. In a similar way, GDF-11 is expressed in skeletal muscle and adipose tissue, as well as in the adult thymus, in the spleen, and in the uterus, and is also expressed in the brain at different stages of development. Promiostatin polypeptides of different species share a substantial sequence identity, and the amino acid sequences of the C-terminal sequence of mature human, murine, rat, and chicken miostatin are 100 percent identical (see Figure 1) . Promiostatin polypeptides are exemplified herein (see Figure 1) by human promyostatin (SEQ ID NO: 2); murine promyostatin (SEQ ID NO: 4); rat promyostatin (SEQ ID NO: 6), mandrel promyostatin (SEQ ID NO: 10); bovine promiostatin (SEQ ID NO: 12); the porcine promiostatin (SEQ ID NO: 14); ovine promyostatin (SEQ ID NO: 16); chicken promyostatin (SEQ ID NO: 8); turkey promiostatin (SEQ ID NO: 18); and the zebrafish promiostatin (SEQ ID NO: 20). Promiostatin polypeptides are also exemplified herein by a polypeptide comprising the portions of the salmon allele 1 (SEQ ID NO: 27; "salmon 1") and of the salmon allele 2 (SEQ ID NO: 29; "salmon 2", see Figure 2). The nucleic acid molecules encoding these promyostatin polypeptides are disclosed herein as SEQ ID NOs: 1, 3, 5, 9, 11, 13, 15, 7, 17, 19, 26, and 28, respectively (see also McPherron and Lee, Proc. Nati, Acad. Sci., USA 94: 12457, 1997, which is incorporated herein by reference). A pro-GDF-11 polypeptide is exemplified herein by human pro-GDF-11 (SEQ ID NO: 25), which is encoded by SEQ ID NO: 24. In view of the extensive conservation between the polypeptides of promyostatin, particularly among species as diverse as humans and fish, it would be a routine matter to obtain polynucleotides that encode myostatin from any species, including the remains of the salmon 1 and salmon 2 sequences, and to identify the expression of Promiostatin or myostatin in any species. In particular, the mature myostatin sequence shares significant homology with other members of the TGF-β superfamily, and myostatin contains most of the residues that are highly conserved among the other members of the family and in other species. In addition, myostatin, such as TGF-β? and inhibins, contains an extra pair of cysteine residues in addition to the seven cysteine residues present in virtually all other members of the family. Myostatin is more homologous to VG -1 (sequence identity of 45 percent). Like other members of the TGF-β superfactor, myostatin is synthesized as a larger precursor promiostatin polypeptide that proteolytically dissociates into an active myostatin peptide. Polynucleotides encoding promiostatin polypeptides from different organisms can be identified using well-known methods and algorithms, based on identity (or homology) with the disclosed sequences. Homology or identity is often measured using sequence analysis software, such as the Sequence Analysis Software Package from the Genetics Computer Group (University of Wisconsin, Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705, United States) . This software matches similar sequences by assigning degrees of homology to different deletions, substitutions, and other modifications. The terms "homology" and "identity", when used herein in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or that have a specified percentage of residues of amino acids or nucleotides that are equal, when compared and aligned for maximum correspondence over a comparison window or designated region, measured using any number of sequence comparison algorithms, or by manual alignment and visual inspection.

For sequence comparison, normally one sequence acts as a reference sequence, with which the test sequences are compared. When a sequence comparison algorithm is used, the test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and the program parameters of the sequence algorithm are designated. You can use the default program parameters, or you can designate alternative parameters. The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequences relative to the reference sequence, based on the parameters of the program. The term "comparison window" is widely used herein to include reference to a segment of any of the number of contiguous positions, for example from about 20 to 600 positions, eg, amino acid or nucleotide positions, usually from about 50 to about 200 positions, more usually from about 100 to about 150 positions, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after optimally aligning the two sequences. Methods of sequence alignment for comparison are well known in this field. The optimal alignment of sequences for comparison can be driving, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2: 482, 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443 , 1970), using the similarity search method of Person and Lipman (Proc. Nati. Acad. Sci. USA 85: 2444, 1988), each of which is incorporated herein by reference; through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin, United States); or by manual alignment and visual inspection. Other algorithms to determine homology or identity include, for example, in addition to the BLAST program (Basic Local Alignment Search Tool) at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Multiple Protein Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS , BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals and Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS , WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global Alignment Program) (Global Alignment Program)), GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local Content Program) )), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA (Pattern-Induced Multi- sequence Alignment (Alignment of Multiple Sequences Induced by the Pattern)), SAGA (Sequence Alignment by Genetic Algorithm (Alignment of Sequences Using the Algorithm Ge phonetic)), and WHAT-IF. These alignment programs can also be used to screen genome databases, in order to identify polynucleotide sequences having substantially identical sequences. A number of genome databases are available for comparison, including, for example, a substantial portion of the human genome that is available as part of the Human Genome Sequencing Pro ect (Human Genome Sequencing Project) (J. Roach, http: // / Weber, U.Washington.edu / Droach / human genome progress 2.html). In In addition, at least 21 genomes have been sequenced in their entirety, including, for example, M. genitalium, M. jannaschii, H. influenzae, E. coli, yeast (5. cerevisiae), and D. melanogas-ter. Significant progress has been made in the sequencing of the genomes of model organisms, such as mouse, C. elegans, and Arabldopsis sp .. Several databases that contain genomic information annotated with some functional information, are maintained by different organizations, and they are accessible through the Internet, for example htt: // wwwtigr. org / tdb; http: // genetics.wisc.edu; htt: // genome-www. Stanford edu / Dball; htt: // hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov; http: / / ebi. ac.uk; http://Pasteur.fr/other/biology; as well as htt: // www. genome wi. -mit. edu An example of a useful algorithm is that of the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al.

(Nucleic Acids Res. 25: 3389-3402, 1977; J. Mol. Biol. 215: 403-410, 1990, each of which is incorporated herein by reference). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). This algorithm involves first identifying the pairs of high-score sequences (HSPs) by identifying short words of a length W in the requested sequence, matching or satisfying some threshold score of positive value T by aligning with a word of the same length in the base sequence of data. T is referred to as the score threshold of. the word neighbor (Altschul and | co-workers, supra, 1977, 1990). These initial neighbor word impacts act as sowings to initiate searches in order to find longer HSPs that contain them. The word hits extend in both directions along each sequence so that the cumulative alignment score can be increased. The cumulative scores are calculated using, for the nucleotide sequences, the M parameters (reward score for a pair of paired residues, always> 0). For the amino acid sequences, a score matrix is used to calculate the cumulative score. The extent of word hits in each direction stops when: the cumulative alignment score falls out by the amount X from its maximum reached value; the cumulative score goes to zero or less, due to the accumulation of one or more negative scoring residue alignments; or the end of any sequence is reached. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, as omission, a word length (W) of 11, an expectation (E) of 10, M = 5, N = 4, and a comparison of both chains. For the amino acid sequences, the BLASTP program uses, as a default, a word length of 3, and expectations (C) of 10, and the BLOSUM62 score matrix (see Henikof and Henikof, Proc. Nati. Acad. Sci. USA 89: 10915, 1989), alignments (?) Of 50, expectation (E) of 10, M = 5, N = -4, and a comparison of both chains. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Nati, Acad. Sci., USA 90: 5873, 1993, which is incorporated herein by reference). A measure of the similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and very preferably less than about 0.001. In one embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST"). In particular, five specific BLAST programs are used to carry out the following task: (1) BLASTP and BLAST3 compare a requested sequence of amino acids against a database of protein sequences; (2) BLASTN compares a requested sequence of nucleotides against a database of nucleotide sequences; (3) BLASTX compares the conceptual translation products of six frames of a ordered nucleotide sequence (both chains) against a database of protein sequences; (4) TBLASTN compares a sequence of ordered protein against a database of translated nucleotide sequences in all six reading frames (both chains); and (5) TBLASTX compares translations of six frames of a requested sequence of nucleotides against translations of six frames from a database of nucleotide sequences. BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs", between an ordered amino acid or nucleic acid sequence, and a test sequence that is preferably obtained from a database of protein or nucleic acid sequences. Highly scoring segment pairs of preference are identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256: 1443-1445, 1992, Henikoff and Henikoff, Proteins 17: 49-61, 1993, each of which is incorporated herein by reference). reference). Less preferable, matrices can also be used PAM or PAM250 (Schwartz and Dayhoff, editors, "Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure" (Washington, National Biomedical Research Foundation 1978)). BLAST programs are accessible through the U.S. National Library of Medicine, for example at www.ncbi.nlm.gov. The parameters used with the previous algorithms can be adapted depending on the length of the sequence and the degree of homology studied. In some embodiments, the parameters may be the default parameters used by the algorithms in the absence of user instructions. A polynucleotide encoding a promyostatin can be derived from any organism, including, for example, mouse, rat, cow, pig, human, chicken, turkey, zebrafish, salmon, fin fish, other aquatic organisms, and other species . Examples of aquatic organisms include those belonging to the Pool class, such as trout, small scale trout, ayu, carp, crossed carp, golden fish, goby, whitebait, eel, conger, sardine, flying fish, sea bass, bream sea, parrotfish, biting fish, mackerel, sarda, tuna, bonito, yellow tail, perch, flounder, sole, turbot, puffer fish, triggerfish; those belonging to the class of Cephalopods, such as squid, cuttlefish, octopus; those belonging to the Pelecipod class, such as clams (for example, hard shell, manila, Quahog, Surf, soft shell); coquinas, mussels, bigarros; shells (for example, sea, bay, cayo); snail, slugs, sea cucumbers; shell of ark; oysters (eg, C. virginica, Gulf, New Zealand, Pacific); those belonging to the class of Gastropods, such as turban shell, abalone (for example, green, pink, red); and those belonging to the Crustacean class, such as lobster, including, but not limited to, Spiny, Rock, and American; shrimp; shrimp, including, but not limited to, M. rosenbergii, P. styllrolls, P. indicus r P. jeponious r P. onodon, P. vannemel r M. ensis, S. melantho, N. norvegious, cold water shrimp; crab, including, but not limited to, Blue, jackdaw, stone, king, queen, snow, chestnut, no debris, Jonah, Mangrove, soft shell; shearing, krill, prawns; River crayfish, including, but not limited to, Blue, Brown, Red-breasted, Red-breasted, Soft-breasted, White; Annelida; Cordados, including, but not limited to, reptiles, such as lizards and turtles; Amphibians, including frogs; and Echinoderms, including, but not limited to, sea urchins. The present invention provides substantially purified peptide portions of a promyostatin polypeptide, and substantially purified peptide portions of a pro-GDF-11 polypeptide. As used herein, the reference to a "pro-GDF", eg, promyostatin or pro-GDF-11, means the full-length polypeptide, including the amino-terminal prodomain and the biologically active carboxy-terminal GDF peptide . In addition, the prodomain includes a peptide of signal (leader sequence), comprising approximately the first 15 to 30 amino acids in the amino terminus of the prodomain. The signal peptide can be dissociated from the full-length pro-GDF polypeptide, which can also be dissociated at a proteolytic cleavage site Arg-Xaa-Xaa-Arg (SEQ ID NO: 21). The reference herein to amino acid residues is made with respect to the full-length pro-GDF polypeptides, as shown in Figures 1 and 2 (see also Sequence Listing). It should also be recognized that reference is made herein to particular peptides that begin or end at "about" a particular amino acid residue. The term "about" is used in this context because it is recognized that a particular protease can dissociate a pro-GDF polypeptide in or immediately adjacent to a proteolytic cleavage recognition site., or one or a few amino acids from the recognition site. As such, reference, for example, to a myostatin prodomain having a sequence of approximately amino acid residues 1 to 263 of SEQ ID NO: 4, would include a portion of the amino-terminal peptide of promyostatin including the peptide of signal, and has a carboxyl terminus terminating at amino acid residue 257 to amino acid residue 269, preferably at amino acid residue 260 to amino acid residue 266. In a similar manner, the signal peptide can be dissociated at any position from about the residue amino acid 15 to 30 of a pro-GDF polypeptide, for example, at residue 15, 20, 25, or 30, without affecting the function, for example, of a remaining prodomain. Accordingly, for convenience, reference is generally made herein to a peptide portion of a pro-GDF polypeptide, from which the signal peptide has been cleaved, starting at approximately 20 amino acid residue. However, it will be recognized that the dissociation of the signal peptide can be at any amino acid position within about the first 15 to 30 amino-terminal amino acids of a pro-GDF polypeptide. As such, reference, for example, to a myostatin prodomain. having a sequence of approximately amino acid residues 20 to 263 of SEQ ID NO: 4, would include a portion of promyostatin peptide lacking approximately the first 15 to 30 amino acids of promyostatin, comprising the signal peptide, and have a carboxyl terminus terminating at amino acid residue 267 to amino acid residue 269, preferably at amino acid residue 260 to amino acid residue 266. In general, a pro-GDF polypeptide or a GDF prodomain is referred to herein. , starting approximately at the amino acid 1. In view of the above disclosure, however, it will be recognized that these pro-GDF polypeptides or prodrominies of the growth differentiation factor lacking the signal peptide, are also encompassed within the present invention. In addition, in this aspect, it must be recognized that the presence or absence of a signal peptide in a peptide of the invention, may have influence, for example, on the compartments of a cell through which a peptide will pass, for example, a prodomain of myostatin, and in which will be finely located the peptide, including if the peptide will be secreted from the cell. Accordingly, the present invention further provides a substantially purified signal peptide portion of a pro-GDF polypeptide. As disclosed herein, this signal peptide can be used to direct an agent, in particular a peptide agent, to the same cell compartments as the naturally occurring growth differentiation factor from which the peptide was derived. signal peptide. The term "peptide" or "peptide portion" is used broadly herein to mean two or more amino acids linked by a peptide bond. The term "fragment" or "proteolytic fragment" is also used herein to refer to a product that can be produced by a proteolytic reaction on a polypeptide, i.e., a peptide produced after dissociation of a peptide bond in the polypeptide . Although the term "proteolytic fragment" is generally used herein to refer to a peptide that can be produced by a proteolytic reaction, it must be recognized that the fragment does not necessarily have to be produced by a proteolytic reaction, but also that it is it can produce using chemical synthesis methods or recombinant DNA technology methods, as described in more detail below, to produce a synthetic peptide that is equivalent to a proteolytic fragment. In view of the disclosed homology of promeostatin with other members of the TGF-β superfamily, it will be recognized that a peptide of the invention is characterized, in part, because it is not present in the previously disclosed members of this superfamily. Whether a peptide portion of a promyostatin or pro-GDF-11 polypeptide is present in a previously disclosed member of the TGF-β superfamily can be easily determined using the computation algorithms described above. Generally speaking, a peptide of the invention contains at least about 6 amino acids, usually containing about 10 amino acids, and may contain 15 or more amino acids, in particular 20 or more amino acids. It should be recognized that the term "peptide" is not used herein to suggest a particular size or number of amino acids that comprise the molecule, and that a peptide of the invention can contain up to several or more amino acid residues. For example, a full-length mature C-terminal myostatin peptide contains more than 100 amino acids, and a full-length prodomain peptide can contain more than 260 amino acids. As used herein, the term "substantially purified" or "substantially pure" or "isolated" means that the molecule to which it refers, for example, a peptide or a polynucleotide, is in a form that is relatively free of proteins, nucleic acids, lipids, carbohydrates, or other materials with which it is naturally associated. In general, a substantially pure peptide, polynucleotide, or other molecule, constitute at least 20 percent of a sample, generally constituting at least about 50 percent of a sample, usually constitute at least about 80 percent of a sample. shows, and in particular constitute approximately 90 percent or 95 percent or more of a sample. A determination can be made that a peptide or a polynucleotide of the invention is substantially pure using well known methods, for example, by performing electrophoresis and identifying the particular molecule as a relatively separate band. For example, a substantially pure polynucleotide can be obtained by cloning the polynucleotide, or by chemical or enzymatic synthesis. A substantially pure peptide can be obtained, for example, by a chemical synthesis method, or by employing protein purification methods, followed by proteolysis, and if desired, further purification by chromatographic or electrophoretic methods. A peptide of the invention can be identified by comparison with a promyostatin or pro-GDF-11 sequence, and determining that the amino acid sequence of the The peptide is contained within the promyostatin or pro-GDF-11 polypeptide sequence, respectively. However, it must be recognized that a peptide of the invention need not be identical to a corresponding amino acid sequence of promiostatin or pro-GDF-11. Thus, a peptide of the invention may correspond to an amino acid sequence of a promyostatin polypeptide, for example, but may vary from a naturally occurring sequence, for example, containing one or more D-amino acids instead of a Corresponding L-amino acid; or containing one or more amino acid analogues, for example, an amino acid that has been derivatized or otherwise modified in its reactive side chain. In a similar manner, one or more peptide bonds in the peptide can be modified. In addition, a reactive group may be modified at the amino terminus or at the carboxyl terminus, or both. These peptides can be modified, for example, to have a better stability to a protease, an oxidizing agent, or other reactive material that can find the peptide in a biological environment, and therefore, can be particularly useful in the realization of a method. of the invention. Of course, the peptides can be modified to have reduced stability in a biological environment, such that the period of time in which the peptide is active in the environment is reduced. The sequence of a peptide of the invention can also be modified in comparison with the corresponding sequence in a promyostatin or pro-GDF-11 polypeptide, by incorporating a conservative amino acid substitution with one or a few amino acids in the peptide. Conservative amino acid substitutions include the replacement of an amino acid residue with another amino acid residue having relatively the same chemical characteristics, for example, the substitution of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine, on the other, or the substitution of one polar residue for another, for example the replacement of arginine with lysine; or of glutamic acid by aspartic acid; or from glutamine to asparagine; or similar. Examples of the positions of a promyostatin polypeptide that can be modified are apparent from an examination of Figure 1, which shows several amino acid differences in the myostatin prodomain and in the mature myostatin peptide., which do not substantially affect the activity of promyostatin or myostatin. The present invention also provides a substantially purified proteolytic fragment of a growth differentiation factor (GDF) polypeptide (a pro-GDF polypeptide), or a functional peptide portion thereof. The proteolytic fragments of a pro-GDF polypeptide are exemplified herein by the proteolytic fragments of a promyostatin polypeptide, and the proteolytic fragments of a pro-GDF-11 polypeptide. As it is known in thepresent, a peptide portion of a pro-GDF polypeptide that is equivalent to a proteolytic fragment of a pro-GDF can be produced, either by a chemical method or by a recombinant DNA method. In view of the present disclosure, proteolytic fragments of other polypeptides of the growth differentiation factor can be made and used easily. In general, peptides corresponding to the proteolytic fragments of a pro-GDF polypeptide are exemplified by a mature carboxy-terminal (C-terminal) growth differentiation factor fragment, which can specifically interact with a differentiation factor receptor. of growth, and affect the signal transduction of the growth differentiation factor, and an amino-terminal prodomain fragment, which may include a signal peptide, and, as disclosed herein, may interact specifically with a pro-GDF polypeptide or with a mature growth differentiation factor peptide, and affect its ability to effect transduction of the growth differentiation factor signal. For example, proteolytic fragments of a promyostatin polypeptide include a mature C-terminal myostatin peptide, which can specifically interact with a myostatin receptor, and induce transduction of the myostatin signal; and an amino-terminal prodomain fragment, which can interact specifically with myostatin, thereby reducing or inhibiting the ability of myostatin to induce myostatin signal transduction. A proteolytic fragment of a pro-GDF polypeptide, or a functional peptide portion thereof, is characterized, in part, by having or affecting an activity associated with the stimulation or inhibition of signal transduction of the differentiation factor of the increase. For example, a promyostatin polypeptide or a functional peptide portion thereof,. may have a myostatin receptor binding activity, a myostatin signal transducing or stimulating activity, a myostatin binding activity, a promyostatin binding activity, or a combination thereof. Accordingly, the term "functional peptide moiety", when used herein with reference to a pro-GDF polypeptide, means a peptide portion of the pro-GDF polypeptide that can specifically interact with its receptor, and stimulate or inhibit the signal transduction of the growth differentiation factor; which can interact specifically with a mature growth differentiation factor or with a pro-GDF; or that exhibits cellular localization activity, that is, the activity of a signal peptide. It should be recognized that a functional peptide portion of the mature full-length myostatin peptide, for example, does not need to have the same activity of mature myostatin, Including the ability to stimulate the transduction of the myostatin signal, because the functional peptide portions of the mature peptide may have, for example, an ability to interact specifically with a myostatin receptor without also having the ability to activate the path of signal transduction. Methods for identifying this functional peptide portion of a pro-GDF polypeptide, which may be useful as a myostatin antagonist, are disclosed herein, or are otherwise known in the art. Accordingly, in one embodiment, a functional peptide portion of a promyostatin polypeptide can specifically interact with a myostatin receptor, and can act as an agonist to stimulate myostatin signal transduction, or as an antagonist to reduce or inhibit the transduction of the myostatin signal. In another embodiment, a functional peptide portion of a promyostatin polypeptide can specifically interact with a promyostatin polypeptide, or with a mature myostatin peptide, thereby blocking transduction of the myostatin signal. This portion of the functional peptide of promyostatin can act, for example, by preventing dissociation of a promyostatin polypeptide to mature myostatin.; forming a complex with the mature myostatin peptide; or through some other mechanism. When a peptide-myostatin complex is formed, the complex can block the transduction of the myostatin signal, for example, by reducing or inhibiting the ability of myostatin to interact specifically with its receptor, or by binding to the receptor in a manner that lacks the ability to induce myostatin signal transduction . Proteolytic fragments of a pro-GDF polypeptide can be produced by dissociating the polypeptide at a proteolytic cleavage site having a consensus amino acid sequence Arg-Xaa-Xaa-Arg (SEQ ID NO: 21). These proteolytic recognition sites are exemplified by the sequence Arg-Ser-Arg-Arg (SEQ ID NO: 22) shown as amino acid residues 263 to 266 in SEQ ID NO: 1 (promiosta-tine), or the residues of amino acids 295 to 298 of SEQ ID NO: 25 (human pro-GDF-11, see also relative positions 267 to 270 of Figure 2), and for the Arg-Ile-Arg-Arg sequence (SEQ ID NO: 23) shown as amino acid residues 263 to 266 in SEQ ID NO: 20. In addition to the proteolytic cleavage site for the signal peptide, the promyostatin polypeptides, for example, contain two additional potential proteolytic processing sites (Lys. -Arg- and Arg-Arg). The dissociation of a promyostatin polypeptide at or near the last proteolytic processing site, which is contained within the consensus proteolytic dissociation site Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) (see, for example, the amino acid residues 263 to 266 of SEQ ID NO: 2), generates a fragment of mature human biotically active C-terminal myostatin. The full-length mature myostatin peptides exemplified contain from about 103 to about 109 amino acids, and have a predicted molecular weight of approximately 12,400 Daltons (Da). In addition, myostatin can form dimers, which have an expected molecular weight of approximately 23 to 30 kilodalons (kDa). The dimers may be myostatin homodimers, or they may be heterodimers, for example, with GDF-11 or another member of the TGF-β family or the growth differentiation factor. A proteolytic fragment of the invention is exemplified by a prodomain of the growth differentiation factor, for example, a myostatin prodomain, which includes approximately amino acid residues 20 to 262 of a promyostatin polypeptide, or a functional peptide portion of the same, or a GDF-11 prodomain, which includes approximately amino acid residues 20 to 295 of a pro-GDF-11 polypeptide, or a functional peptide portion thereof, each of which may additionally contain the signal peptide comprising approximately amino acids 1 to 20 of the respective pro-GDF polypeptide. The prodrugs of myostatin are further exemplified by approximately amino acid residues 20 to 263, as stipulated in SEQ ID NO: 4 and SEQ ID NO: 6; as well as by approximately amino acid residues 20 to 262, as stipulated in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO : 20, which can be produced by proteolytic cleavage of a corresponding promyostatin polypeptide, and can be chemically synthesized, or can be expressed from a recombinant polynucleotide encoding the proteolytic fragment. A functional peptide portion of a myostatin prodomain is employed by a peptide portion of a myostatin prodomain that can interact specifically with myostatin or with promyostatin. A GDF-11 prodomain is exemplified by approximately amino acid residues 20 to 295 of SEQ ID NO: 25, which may further include the signal peptide comprising approximately amino acid residues 1 to 20 of SEQ ID NO: 25 , and a functional peptide portion of a GDF-11 prodomain is exemplified by a peptide portion of a GDF-11 prodomain that can interact specifically with the mature GDF-11 polypeptide or with a pro-GDF-11 polypeptide. Preferably, the functional peptide portion of a prodomain of the growth differentiation factor inhibits the ability of the corresponding growth differentiation factor, or a related growth differentiation factor, to stimulate signal transduction, for example, by reducing or the inhibition of the ability of the growth differentiation factor to interact specifically with its receptor, or by linking to the receiver as an inactive complex. In one embodiment, the present invention provides a functional fragment of a pro-GDF polypeptide, in particular a functional fragment of a prodomain of the growth differentiation factor, operatively linked to a signal peptide of the growth differentiation factor, preferably a myostatin signal peptide or a GDF-11 signal peptide comprising approximately the first 15 to 30 amino-terminal amino acids of promyostatin or of pro-GDF-11, respectively. As disclosed herein, a prodrug of myostatin or a prodomain of GDF-11 may interact with mature myostatin, GDF-11, or both, thus reducing or inhibiting the ability of mature growth differentiation factor to interact specifically with your receiver (see Examples 7 and 8). Accordingly, a functional peptide portion of a myostatin prodomain can be obtained, for example, by examining peptide portions of a myostatin prodomain, using the methods provided herein, and by identifying the peptide portions. functional proteins that can interact specifically with myostatin or with promyostatin, and can reduce or inhibit the ability of myostatin to interact specifically with a myostatin receptor, or to stimulate the transduction of the myostatin signal.

A functional peptide portion of a myostatin prodomain that can interact specifically with myostatin, or a functional peptide portion of another prodomain of the growth differentiation factor, can also be identified using any of several known assays useful for identifying interactions. of protein-specific proteins. These assays include, for example, gel electrophoresis methods, affinity chromatography, the Fields and Song two-hybrid system (Nature 340: 245-246, 1989; see also US Patent 5,283,173; Fearon et al., Proc. Nati. Acad Sci. USA 89: 7958-7962; Chien et al., Proc. Nati, Acad. Sci. USA 88: 9578-9582, 1991; Young, Biol. Reprod. 58: 302-311 (1998), each of which is incorporated herein by reference), the two-hybrid reverse assay (Leana and Hannink, Nucí Acids Res. 24: 3341-3347, 1996, which is incorporated herein by reference), the repressed transactivator system (US Patent 5,885,779, which is incorporated herein by reference), the phage display system (Lowman, Ann.Rev. Biophys., Biomol. Struct. 26: 401-424, 1997, which is incorporated herein by reference). reference), the GST-HIS extraction assays, mutant operators (international publication O 98/01879, which is incorporated herein by reference). erencia), the protein recruitment system (US Patent 5,776,689, which is incorporated herein by reference), and the like (see, for example, Mathis, Clin. Chem. 41: 139-147, 1995 Lam, Anticancer Drug Res. 12: 145-167, 1997; Phizicky et al., Microbiol. Rev. 59: 94-123, 1995; each of which is incorporated herein by reference). A functional peptide portion of a prodomain of the growth differentiation factor can also be identified using molecular modeling methods. For example, an amino acid sequence of a mature myostatin peptide can be introduced into a computer system having appropriate modeling software, and a three-dimensional representation of myostatin ("virtual myostatin") can be produced. A promyostatin amino acid sequence can also be introduced into the computer system, such that the modeling software can simulate portions of the promiostatin sequence, e.g., portions of the prodomain, and can identify the peptide portions of the prodomain that can interact specifically with the virtual myostatin. A baseline can be previously defined for a specific interaction by modeling virtual myostatin and a full-length myostatin prodomain, and by identifying the amino acid residues in the virtual myostatin that are "contacted" by the prodomain, because it is known that this interaction inhibits the activity of myostatin. It should be recognized that these methods, including two-hybrid assays and molecular modeling methods, can also be used to identify other molecules of specific interaction encompassed within the present invention. Accordingly, methods such as the two hybrid assay can be used to identify a growth differentiation factor receptor, such as a myostatin receptor, using, for example, a myostatin peptide or a peptide portion thereof. that specifically interacts with an Act RIIA or Act RIIB receptor as a binding component of the assay, and identifying a growth differentiation factor receptor, that specifically interacts with the myostatin peptide. In a similar manner, molecular modeling methods can be used to identify an agent that specifically interacts with a peptide of the mature growth differentiation factor, such as mature myostatin, or with a growth factor differentiation receptor, and, for Consequently, it may be useful as an agonist or an antagonist of signal transduction mediated by the growth differentiation factor or by the growth differentiation factor receptor. This agent can be, for example, a functional peptide portion of a prodrug of myostatin or a prodomain of GDF-11, or a chemical agent that mimics the action of the prodomain of the growth differentiation factor. Modeling systems useful for the purposes disclosed herein may be based on the structural information obtained, for example, by crystallographic analysis or nuclear magnetic resonance analysis, or on the primary sequence information (see, for example, Dunbrack et al., "Meeting review: the Second meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2)" (Asilomar, California, 13-16 December 1996) Fold Des 2 (2): R27-42, (1997), Fischer and Eisenberg, Protein Sci. 5: 947-55, 1996; (see also, US Patent 5,436,850); Havel, Prog. Biophys., Mol. Biol. 56: 43-78, 1991, Lichtarge et al., J. Mol. Biol. 274: 325-37, 1997, Matsumoto et al., J. Biol. Chem. 270: 19524-31, 1995; Sali et al. J. Biol. Chem. 268: 9023-34, 1993; Sali, Molec., Med. Today 1: 270-7, 1995; Sali, Curr. Opin. Biotechnol., 6: 437-51, 1995b; Sali et al., Proteins. 23: 318-26, 1995c; Sali, Nature Struct., Biol. 5: 1029-1032, 1998; US Patent 5,933,819; US Patent 5,265,030, each of which is incorporated herein by reference). The coordinates of the crystal structure of a promyostatin polypeptide or a growth differentiation factor receptor can be used to design compounds that bind to the protein and alter its physical or physiological properties in a variety of ways. The coordinates of the structure of the protein can also be used to computationally search for databases of peptide molecules to determine agents that bind to the polypeptide to develop modulating or binding agents, which can act as agonists or antagonists of the Transduction of signal of the growth differentiation factor. These agents can be identified by kinetic computer fitting data using standard equations (see, for example, Segel, "Enzyme Kinetics" (J. Wiley &Sons 1975), which is incorporated herein by reference. crystal structure data for designing inhibitors or binding agents are known in this field For example, the coordinates of the growth differentiation factor receptor can be superimposed on other available coordinates of similar receptors, including recipients having a linked inhibitor, to provide an approximation of the way in which the inhibitor interacts with the receptor, and computer programs used in the practice of rational drug design can also be used, in order to identify compounds that reproduce similar interaction characteristics to the found, for example, between a mature myostatin and a co-crystallized myostatin prodomain. Detailed knowledge of the nature of the specific interactions allows modification of the compounds to alter or improve solubility, pharmacokinetics, and the like, without affecting the binding activity. The computer programs to carry out the activities necessary to design agents using the information of the structure of the crystal are well known. The Examples of these programs include Catalyst DatabaseTM - an information retrieval program that accesses chemical databases, such as the BioByte Master File, Derwent WDI and ACD; Catalyst / HYPOMR - generates model compounds and hypotheses to explain the activity variations with the structure of the candidate drugs; LudiMR - adjusts the molecules in the active site of a protein, by identifying and pairing the complementary polar and hydrophobic groups; and LeapfrogMR - "grows" new ligands using a genetic algorithm with parameters under user control. Different machines can be used for general purposes with these programs, or it may be more convenient to build a more specialized apparatus to carry out the operations. In general, the mode is implemented in one or more computer programs that run on programmable systems, each comprising at least one processor, at least one data storage system (including volatile and non-volatile memory, and / or hardware elements). storage), at least one input device, and at least one output device. The program runs on the processor to perform the functions described herein. Each program can be implemented in any desired computer language, including, for example, machine, assembly, high-level procedure, or object-oriented programming languages, to communicate It is with a computer system. In any case, the language can be a compiled or interpreted language. The computer program will normally be stored in a storage medium or device, for example, a ROM, CD-ROM, magnetic or optical medium, or the like, which is readable by a programmable computer for general or special purposes, to configure and operating the computer when the storage medium or device is read by the computer to carry out the procedures described herein. The system can also be considered as implemented as a computer readable storage medium, configured with a computer program, where the storage medium so configured makes a computer operate in a specific and previously defined manner, to carry out the functions described herein. The embodiments of the invention include systems, for example, Internet-based systems, in particular computer systems that store and manipulate coordinate information obtained by crystallographic or nuclear magnetic resonance analysis, or information of amino acid or nucleotide sequences, such as It is made known in the present. As used herein, the term "computer system" refers to hardware components, software components, and data storage components used to analyze coordinates or sequences, as stipulated. at the moment. The computer system usually includes a processor to process, access, and manipulate the sequence data. The processor can be any well-known type of central processing unit, for example, a Pentium II or Pentium III processor from Intel Corporation, or a similar processor from Sun, Motorola, Compaq, Advanced MicroDevices or International Business Machines. Typically, the computer system is a general-purpose system comprising the processor and one or more internal data storage components for storing data, and one or more data recovery devices for retrieving the data stored in the storage components of the computer. data. A person skilled in the art can readily appreciate that any of the computer systems currently available is suitable. In one embodiment, the computer system includes a processor connected to a busbar, which is connected to a main memory, preferably implemented as RAM, and one or more internal data storage devices, such as a hard disk or other computer readable medium having data recorded therein. In some embodiments, the computer system further includes one or more data recovery devices for reading the data stored in the internal data storage devices. The data recovery device can represent for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or a modem capable of being connected to a remote data storage system (for example, via the Internet). In some embodiments, the internal data storage device is a removable computer-readable medium, such as a floppy disk, a compact disc, a magnetic tape, etc., which contains the control logic and / or the data recorded therein. . The computer system may conveniently include, or may be programmed by appropriate software, to read the control logic and / or the data from the data storage component, once inserted in the data recovery device. The computer system generally includes a visual display, which is used to display the output of a computer user. It should also be noted that the computer system can be linked to other computer systems in a network, or in a wide area network, to provide centralized access to the computer system. When it is desired to identify a chemical entity that specifically interacts with myostatin or with a growth differentiation factor receptor, any of several methods can be used to track chemical entities or fragments, by their ability to interact specifically with the molecule. This process can beby inspection visual, for example, of myostatin and a prodomain of myostatin, on the computer screen. Peptide portions selected from the prodomain, or chemical entities that can act as mimics, can then be placed in a variety of orientations, or in platform, within an individual binding site of myostatin. Platforming can be carried out using software such as Quanta and Sybyl, followed by a minimization of energy and molecular dynamics with conventional molecular mechanical force fields, such as CHARMM and AMBER. The specialized computer programs may be particularly useful for selecting portions of a prodomain peptide, or useful chemical entities, for example, as an agonist or antagonist of the growth differentiation receptor. These programs include, for example, GRID (Goodford, J. Med. Chem., 28: 849-857, 1985, available from the University of Oxford, Oxford, United Kingdom); MCSS (Miranker and Karplus, Proteins: Structure, Function and Genetics 11: 29-34, 1991, available from Molecular Simulations, Burlington, MA); AUTODOCK (Goodsell and Olsen, Proteins: Structure. Function, and Genetics 8: 195-202, 1990, available from Scripps Research Institute, La Jolla, California, United States); DOCK (Kuntz et al., J. Mol. Biol. 161: 269-288, 1982, available from the University of California, San Francisco, California, United States), each of which is incorporated herein by reference. reference. Suitable peptides or agents that have been selected can be assembled into a single compound or linking agent. The assembly can be carried out by visual inspection of the relationship of the fragments with each other on the three-dimensional image displayed on a computer screen, followed by the manual construction of the model using software such as Quanta or Sybyl. Useful programs to assist a person skilled in the art in connecting individual chemical entities or fragments include, for example, CAVEAT (Bartlett et al., Special Pub., Royal Chem. Soc. 78: 182-196, 1989, available in University of California, Berkeley, California, United States); three-dimensional database systems, such as MACCS-3D (MDL Information Systems, San Leandro, California, United States, for a review, see Martin, J. Med. Chem. 35: 2145-2154, 1992); HOOK (available from Molecular Simulations, Burlington, Mass.), Each of which is incorporated herein by reference. In addition to the method of constructing or identifying these specific interaction agents in a stepwise fashion, a fragment or chemical entity at a time, as described above, the agents can be designed as a whole or de novo, using either a site active, or optionally, including some portions of a known agent that specifically interacts, for example, a prodrug of myostatin from full length, which interacts specifically with myostatin. These methods include, for example, LUDI (Bohm, J. Comp.Aid.Olec.Design 6: 61-78, 1992, available from Biosym Technologies, San Diego California, United States); LEGEND (Nishibata and Itai, Tetrahedron 47: 8985, 1991, available in Molecular Simulations, Burlington MA); LeapFrog (available from Tripos Associates, St. Louis MO), and those described by Cohen et al. (J. Med. Chem. 33: 883-894, 1990) and by Navia and Murcko, Curr. Opin. Struct. Biol. 2: 202-210, 1992, each of which is incorporated herein by reference). In the matter, specific computer software is available to evaluate the energy of deformation of compounds and the electrostatic interaction. Examples of programs designed for these uses include Gaussian 92, revision C (Frisen, Gaussian, Inc., Pittsburgh PA, 1992); AMBER, version 4.0 (Kollman, University of California, San Francisco, 1994); GUANTA / CHARMM (Molecular Simulations, Inc., Burlington MA, 1994); and Insight II / Discover (Biosysm Technologies Inc., San Diego, California, United States, 1994). These programs can be implemented using, for example, a Silicon Graphics workstation, IRIS 4D / 35 or an IBM RISC / 6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art, of which speed and capacity are continually modified.

A molecular modeling process for identifying an agent that specifically interacts with a molecule of interest, for example, with a mature growth differentiation factor peptide, such as mature myostatin, or with a growth differentiation factor receptor, can be carry out as is disclosed in the present. In a first step, a virtual representation of an objective molecule is performed, for example, myostatin. Accordingly, in one embodiment, the present invention provides a virtual representation of an objective molecule, wherein the objective molecule is selected from a pro-GDF polypeptide, for example, promyostatin; a peptide portion of a pro-GDF polypeptide; a growth differentiation factor receptor, and a relevant domain of a growth differentiation factor receptor, e.g., a growth factor differentiation binding domain. The virtual representation of the objective molecule can be displayed or kept in the memory of a computer system. The process begins in a start state, which comprises the virtual objective molecule, then moves to a state where a database containing one or more virtual test molecules is stored in a memory of a computer system. As described above, the memory can be any type of memory, including RAM, or an internal storage device. Then the process moves to a state where it determines the ability of a first virtual test molecule to interact specifically with the virtual objective molecule, where the database containing the virtual test molecule is opened, which may be one of a population of test molecules, for the analysis of an interaction of the virtual objective molecule and the virtual test molecule, and the analysis is made. The determination of a specific interaction can be made based on the calculations made by the software maintained in the computer system, or by a comparison with a previously determined specific interaction, which can be stored in a memory of the computer system and can be access as appropriate. Then the process moves to a state where, when a specific interaction is detected, the virtual test molecule is displayed, or stored in a second computer database. If appropriate, the process is repeated for the virtual objective molecule and a second virtual test molecule, a third virtual test molecule, and so on, as desired. If a determination is made that a virtual test molecule interacts specifically with the virtual target molecule, the identified virtual test molecule is moved from the database and can be displayed to the user. This state notifies the user that the molecule with the name or structure exhibited, interacts specifically with the objective molecule within the limitations that were introduced. Once the name of the test molecule identified for the user is displayed, the process moves to a decision state, where a determination is made as to whether there are more virtual test molecules in the database, or if they are going to examine. If there are no more molecules in the database, then the process ends in an end state. However, if there are more test molecules in the database, then the process moves to a state where a pointer moves to the next test molecule in the database, so that it can be examined to determine the specific link activity. In this way, the new molecule is examined to determine its ability to interact specifically with the virtual objective molecule. The methods described above can be used in different aspects encompassed within the claimed invention. Accordingly, the methods can be used to identify a peptide portion of a promiostatin prodrug that can interact specifically with myostatin, and reduce or inhibit the ability of myostatin to interact with its receptor, or otherwise affect the ability of myostatin to effect signal transduction. In a similar way, the methods can be used to identify small organic molecules that mimic the action of a prodi- of the growth differentiation factor, thus reducing or inhibiting the myostatin or GDF-11 signal transduction. The methods can also be used to identify agents that specifically interact with a growth differentiation factor receptor, eg, an Act RIIA receptor, Act RIIB, or other growth differentiation factor receptor, these agents being useful as agonists. or growth-differentiating factor receptor antagonists, which can modulate the signal transduction of growth differentiation factor in a cell. In addition, the methods provide a means to identify previously unknown pro-GDF polypeptides or growth-differentiation factor receptors, for example, by identifying the conserved structural features of the particular polypeptides. In a manner similar to other members of the TGF-β superfamily, the active growth differentiation factor peptides are expressed as precursor polypeptides, which dissociate into a mature, biologically active form. In accordance with the foregoing, in yet another embodiment, the proteolytic fragment of a pro-GDF polypeptide is a mature growth differentiation factor peptide, or a functional peptide portion of a mature growth differentiation factor peptide, wherein , as described above, the functional peptide portion can have the activity of a agonist or antagonist of the growth differentiation factor. The proteolytic fragment can be a mature C-terminal myostatin peptide, which includes approximately amino acid residues 268 to 374 of a promiostatin polypeptide (see Figure 1, see also Figure 2), or a GDF- peptide. 11 C-terminal mature, which includes approximately amino acid residues 299 to 407 of a pro-GDF-11 polypeptide. The full-length mature myostatin peptides are exemplified by approximately amino acid residues 268 to 375, as stipulated in SEQ ID NO: 4 and SEQ ID NO: 6; by about amino acid residues 267 to 374, as set forth in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 14 , SEQ ID NO: 16, and SEQ ID NO: 20, and by approximately amino acid residues 49 to 157 of SEQ ID NO: 27 and approximately amino acid residues 28 to 136 of SEQ ID NO: 29. A full length mature GDF-11 peptide is exemplified by approximately amino acid residues 299 to 407 of SEQ ID NO: 25. The portions peptide functionalities of mature growth differentiation factor peptides are exemplified by portions of mature myostatin peptide or mature GDF-11 having agonist or antagonist activity with respect to the activity of a mature growth differentiation factor peptide. . Preferably, the activity of the mature growth differentiation factor peptide is a capacity for interact specifically with your receiver. As disclosed herein, a mature myostatin pep (referred to herein in general as "myostatin") can induce myostatin signal transduction activity, by specific interaction with a myostatin receptor expressed on the surface of a cell (see Example 7). Accordingly, a portion of myostatin functional pep can be obtained by examining pep portions of a mature myostatin pep, using a method as described herein (Example 7) or as otherwise known in the art. , and identifying the functional pep portions of myostatin that interact specifically with a myostatin receptor, for example, an activin type IIA receptor (Act RILA) or an Act RIIB receptor expressed in a cell (Act RILA, Cell 65: 973- 982, 1991; Act RIIB Cell 68: 97-108, 1992, both incorporated herein by reference in its entirety). A prodrug of myostatin can reduce or inhibit the activity of myostatin signal transduction. In one embodiment, the myostatin prodomain can interact specifically with myostatin, thereby reducing or inhibiting the ability of the myostatin pep to interact specifically with its receptor. As disclosed herein, a precursor promiostatin also lacks the ability to interact specifically with a receptor Myostatin, and therefore, mutations in promyostatin that reduce or inhibit the ability of promyostatin to dissociate in mature myostatin, provide a means to reduce or inhibit myostatin signal transduction. In accordance with the foregoing, in another embodiment, the present invention provides a mutant pro-GDF polypep, which contains one or more amino acid mutations that interrupt the proteolytic cleavage of the mutant pro-GDF to a mature growth factor-like pep. active. A mutant pro-GDF polypep of the invention may have a mutation that affects dissociation at a proteolytic cleavage site, such as the consensus proteolitic cleavage recognition site Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) , which is present in the pro-GDF polypeps. Accordingly, the mutation can be a mutation of an Arg residue of SEQ ID NO: 21, such that a mutant promiostatin, for example, can not be cleaved into a prodomain of myostatin and a mature myostatin pep. However, the mutation may also be at a different site from the proteolytic cleavage site, and may alter the ability of the protease to bind to the pro-GDF polypep, to effect proteolysis at the cleavage site. A mutant pro-GDF polypep of the invention, for example, a mutant promyostatin or a mutant pro-GDF-11, may have a dominant negative activity with respect to myostatin or GDF-11, and Consequently, it may be useful to reduce or inhibit the transduction of the myostatin or GDF-11 signal in a cell. The present invention also provides a substantially purified polynucleo, which encodes a pep portion of a promyostatin polypep or an imitating promyostatin, or a pep portion of a pro-GDF-11 or mutant pro-GDF-11 polypep, as described above. As discussed in more detail below, the invention also provides useful polynucleos as agents for modulating the effect of myostatin on a cell, and further provides a polynucleo that encodes a growth differentiation factor receptor, or a functional pep portion. of the same. Examples of these polynucleos are provided in the following disclosure. As such, it should be recognized that the following disclosure is relevant to the different embodiments of the invention as disclosed herein. The term "polynucleo" is widely used herein to mean a sequence of two or more deoxyri-bonucleos or ribonucleos that are linked together by a phosphodiester linkage. As such, the term "polynucleotide" includes RNA and DNA, which may be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and may be single-stranded or double-stranded, as well as a DNA / RNA hybrid. In addition, the term "polynucleotide", as used herein, includes the molecules of naturally occurring nucleic acids, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by chemical synthesis methods or by enzymatic methods, such as the chain reaction of the polymerase (PCR). In different embodiments, a polynucleotide of the invention may contain nucleoside or nucleotide analogs, or a base structure linkage other than a phosphodiester linkage (see above). In general, nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine, or thymine, linked with 2'-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine, or uracil, linked with ribose . However, a polynucleotide can also contain nucleotide analogs, including synthetic nucleotides that do not occur naturally, or naturally occurring, modified nucleotides. These nucleotide analogs are well known in the art and are commercially available, as well as polynucleotides containing these nucleotide analogs (Lin et al., Nucí Acids Res. 22: 5220-5234 (1994); Jellinek et al., Biochemistry 34 : 11363-11372 (1995); Pagratis et al., Nature Biotechnol., 15: 68-73 (1997), each of which is incorporated herein by reference). The covalent bond linking the nucleotides of a polynucleotide is generally a phosphodiester linkage. Without However, the covalent bond may also be any of numerous other linkages, including a thiodiéster linkage, a phosphorothioate linkage, a peptide bond, or any other link known to those skilled in the art as useful for linking nucleotides to the of producing synthetic polynucleotides (see, for example, Tam et al., Nucí Acids Res. 22: 977-986 (1994); Ecker and Crooke, BioTechnology 13: 351360 (1995), each of which is incorporated herein) as reference) . Incorporation of nucleotide analogs that do not occur naturally, or of linkages that bind to nucleotides or analogs, may be particularly useful where the polynucleotide is to be exposed to an environment that may contain a nucleolytic activity, including, for example, , a tissue culture medium, or after its administration to a living subject, because the modified polynucleotides may be less susceptible to degradation. A polynucleotide comprising naturally occurring nucleotides and phosphodiester linkages can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogues or covalent bonds other than phosphodiester linkages, will generally be synthesized chemically, although an enzyme, such as T7 polymerase, can incorporate certain types of nucleotide analogs in a polynucleotide, and, therefore, it can be used to produce this polynucleotide in a recombinant manner from an appropriate template (Jellinek et al., supra, 1995). When a polynucleotide encodes a peptide, for example, a portion of the promyostatin peptide or a peptide agent, the coding sequence is generally contained in a vector, and is operably linked to the appropriate regulatory elements, including, if desired, a promoter. specific tissue or enhancer. The encoded peptide may be further operably linked, for example, with a peptide tag, such as a His-6 tag or the like, which may facilitate identification of the expression of the agent in the target cell. A poly-histidine tag peptide, such as His-6, can be detected using a divalent cation, such as nickel ion, cobalt ion, or the like. Additional peptide tags include, for example, a FLAG epitope, which can be detected using an anti-FLAG anti-body (see, for example, Hopp et al., BioTechology 6: 1204 (1988)).; US Patent 5,011,912, each of which is incorporated herein by reference); a c-myc epitope, which can be detected using a specific anti-body for the epitope; biotin, which can be detected using streptavidin or avidin; and glutathione S-transferase, which can be detected using glutathione. These signs can provide the additional advantage that they can facilitate the isolation of the operably linked peptide or the peptide agent, for example, when it is desired to obtain a substantially purified peptide corresponding to a proteolytic fragment of a myostatin polypeptide. As used herein, the term "operably linked" or "operatively associated" means that two or more molecules are placed one with respect to the other, such that they act as a single unit, and perform a function that is It can be attributed to one or both molecules, or a combination thereof. For example, a polynucleotide sequence encoding a peptide of the invention can be operably linked to a regulatory element, in which case, the regulatory element confers its regulatory effect on the polynucleotide in a manner similar to the way in which the regulatory element would affect a polynucleotide sequence with which it is normally associated in a cell. A first polynucleotide coding sequence can also be operably linked to a second (or more) coding sequence, such that a chimeric polypeptide can be expressed from the operably linked coding sequences. The chimeric polypeptide may be a fusion polypeptide, wherein the two (or more) encoded peptides are translated into a single polypeptide, i.e., they are linked in a covalent manner via a peptide bond; or they can be translated as two separate peptides that, after their translation, can associate operatively with one another to form a stable complex. A chimeric polypeptide generally demonstrates some or all of the characteristics of each of its peptide components. As such, a chimeric polypeptide may be particularly useful for carrying out the methods of the invention, as disclosed herein. For example, in one embodiment, a method of the invention can modulate the transduction of the myostatin signal in a cell. Accordingly, when a peptide component of a chimeric polypeptide encodes a localization domain in a cellular compartment, and a second peptide component encodes a dominant Smad negative polypeptide, the functional chimeric polypeptide can be translocated to the cellular compartment designated by the localization domain. in a cellular compartment, and may have the dominant negative activity of the Smad polypeptide, thereby dominating the transduction of the myostatin signal in the cell. Cell compartmentalization domains are well known and include, for example, a localization domain in the plasma membrane, a nuclear localization signal, a mitochondrial membrane localization signal, a localization signal in the endoplasmic reticulum, or the like (see, for example, Hancock et al., EMBO J. 10: 4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8: 3960-3963, 1988; US 5,776,689, each of which is incorporated herein by reference). This domain can be useful for directing an agent to a particular compartment in the cell, or for directing the agent to be secreted from a cell. For example, a kinase domain of a myostatin receptor, such as Act RIIB, is generally associated with the inner surface of the plasma membrane. Accordingly, a chimeric polypeptide comprising a dominant negative myostatin receptor kinase domain, eg, a dominant Act RIIB negative receptor, lacking kinase activity, may further comprise a localization domain on the plasma membrane, localizing in this way the kinase domain of Act RIIB negative dominant in the inner cell membrane. As disclosed herein, a pro-GDF signal peptide has cell localization activity. As used herein, the term "cell localization activity" refers to the ability of a signal peptide to direct the translocation of a peptide operably linked thereto., to one or more specific intracellular compartments, or to direct the secretion of the molecule from the cell. As such, a pro-GDF signal peptide may be particularly useful for directing the translocation of a peptide or other agent operably linked to the signal peptide, to the same intracellular compartments as a naturally expressed growth differentiation factor having substantially the same signal peptide. In addition, the signal peptide, for example, a promyostatin signal peptide, comprising approximately the first 15 to 30 amino acids of promiostatin, can direct the secretion of an agent operatively linked from the cell, through the same path as the naturally occurring pro-GDF comprising the signal peptide. Accordingly, agents particularly useful for carrying out a method of the invention include a prodomain of growth differentiation factor or a functional peptide portion thereof, which is operably linked to a signal peptide of growth differentiation factor, preferably a promyostatin or pro-GDF-11 signal peptide. A polynucleotide of the invention, including a polynucleotide agent useful in carrying out a method of the invention, can be contacted directly with an objective cell. For example, oligonucleotides useful as anti-sense molecules, ribosims, or triplexing agents, can be contacted directly with an objective cell, after which they enter the cell and perform their function. A polynucleotide agent can also specifically interact with a polypeptide, for example, a myostatin receptor (or a myostatin), thereby altering the ability of myostatin to specifically interact with the receptor. These polynucleotides, as well as the methods for making and identifying these polynucleotides, are disclosed in present, or otherwise known in the art (see, for example, O'Connell et al., Proc. Nati, Acad. Sci., USA 93: 5883-5887, 1996; Tuerk and Gold, Science 249: 505- 510, 1990; Gold et al., Ann. Rev. Biochem. 64: 763-797, 1995, each of which is incorporated herein by reference). A polynucleotide of the invention, which can encode a peptide portion of a pro-GDF polypeptide, such as promyostatin, or which can encode a mutant promiostatin polypeptide, or which can encode a growth factor differentiation receptor or a functional peptide portion thereof, or which may be a polynucleotide agent useful in carrying out a method of the invention, may be contained in a vector, which may facilitate manipulation of the polynucleotide, including introduction of the polynucleotide into a cell objective The vector can be a cloning vector, which is useful for maintaining the polynucleotide, or it can be an expression vector, which contains, in addition to the polynucleotide, regulatory elements useful for expressing the polynucleotide, and, when the polynucleotide encodes a peptide, to express the encoded peptide in a particular cell. An expression vector can contain the expression elements necessary to achieve, for example, a sustained transcription of the coding polynucleotide, or the regulatory elements can be operably linked to the polynucleotide before being cloned into the vector.

An expression vector (or polynucleotide) generally contains or encodes a promoter sequence, which can provide constitutive, or if desired, inducible or tissue-specific expression or specificity of the developmental stage of the coding polynucleotide, a sequence of recognition of poly-A, and a ribosome recognition site or an entry site to the internal ribosome, or other regulatory elements, such as an enhancer, which may be tissue-specific. The vector may also contain the elements required for replication in a prokaryotic or eukaryotic host system, or both, as desired. These vectors, which include vectors of plasmids and viral vectors, such as bacteriophages, baculoviruses, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Semliki rainforest viruses, and adeno-associated virus vectors, are well known and can be obtained from a commercial source (Promega, Madison WI; Stratagene, La Jolla California, United States; GIBCO / BRL, Gaithersburg MD), or can be constructed by a technician in this field (see, for example, Meth. Enzymol., Volume 185, Goeddel, editor (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther 1: 51-64, 1994, Flotte, J. Bioenerg, Biomemb 25: 37-42, 1993, Kirshenbaum et al, J. Clin Invest 92: 381-387, 1993, each of which incorporated herein by reference). A tetracycline-inducible (tet) promoter may be particularly useful for boosting the expression of a polynucleotide. of the invention, for example, a polynucleotide encoding a dominant negative form of myostatin, wherein the proteolytic processing site has been mutated, or which codes for a prodomain of myostatin, which can form a complex with a myostatin peptide. mature, or that encodes a dominant negative form of a growth factor receptor. After administration of the tetracycline, or a tetracycline analog, to a subject containing a polynucleotide operably linked to a tetracycline-inducible promoter, expression of the encoded peptide is induced, whereby, the peptide can affect its activity, for example, wherein a peptide agent can reduce or inhibit the transduction of the myostatin signal. This method can be used, for example, to induce muscle hypertrophy in an adult organism. The polynucleotide can also be operably linked to a tissue-specific regulatory element, eg, a muscle cell-specific regulatory element, such that the expression of a coded peptide is restricted to an individual's muscle cells, or to muscle cells. of a mixed population of cells in culture, for example, the culture of an organ. The specific regulatory elements of muscle cells are well known in the art, including, for example, the muscle creatine kinase promoter (Stern-Berg et al., Mol. Cell. Biol. 8: 2896-2909, 1988, which incorporated herein by reference), and the myosin light chain enhancer / promoter (Donoghue et al, Proc. Nati, ACAD, Sci., USA 88: 5847-5851, 1991, which is incorporated in the present as reference). Viral expression vectors can be particularly useful for introducing a polynucleotide into a cell, in particular a cell of a subject. Viral vectors provide the advantage that they can infect host cells with relatively high efficiency, and can infect specific cell types. For example, a polynucleotide encoding a myostatin prodomain or a functional peptide portion thereof, can be cloned into a baculovirus vector, which can then be used to infect an insect host cell, thereby providing a means for produce large quantities of the encoded prodomain. The viral vector can also be derived from a virus that infects the cells of an organism of interest, for example, to the cells of a vertebrate host, such as the cells of a mammalian, bird, or pool host. Viral vectors can be particularly useful for introducing a polynucleotide useful in carrying out a method of the invention in an objective cell. Viral vectors have been developed for use in particular host systems, in particular mammalian systems, and include, for example, retroviral vectors, other lentivirus vectors such as those based on the human immunodeficiency virus (HIV), adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccine virus vectors, and the like (see Miller and Rosman, BioTechniques 7: 980-990, 1992, Anderson et al, Nature 392: 25-30 Supplement, 1998, Verma and Somia, Nature 389: 239-242, 1997, Wilson, New Engl. J. Med. 334: 1185-1187 (1996), each of which is incorporated herein by reference). When retroviruses are used, for example, for gene transfer, replicating competent retroviruses can theoretically develop due to recombination of the retroviral vector and viral genetic sequences in the packaging cell line used to produce the retroviral vector. Cellular packaging lines where replication competent virus production has been reduced or eliminated by recombination can be used to minimize the possibility of replication competent retrovirus. All supernatants of the retroviral vector used to infect cells are screened for replicating competent viruses by conventional assays, such as polymerase chain reaction and reverse transcriptase assays. Retroviral vectors allow the integration of a heterologous gene into a genome of a host cell, which allows the gene to be passed to daughter cells following cell division.

A polynucleotide, which may be contained in a vector, may be introduced into a cell by any of a variety of methods known in the art (Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); and collaborators, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1987, and supplements until 1995), each of which is incorporated herein by reference). These methods include, for example, transfection, lipofection, microinjection, electroporation, and with viral vectors, infection; and may include the use of liposomes, microemulsions, or the like, which may facilitate the introduction of the polynucleotide into the cell, and may protect the polynucleotide from degradation prior to its introduction into the cell. The selection of a particular method will depend, for example, on the cell in which the polynucleotide is to be introduced, as well as on whether the cell is isolated in culture, or is in a tissue or organ in culture, or in sltu. The introduction of a polynucleotide into a cell by infection with a viral vector is particularly convenient, because it can efficiently introduce the nucleic acid molecule into a cell ex vivo or in vivo (see, for example, US Patent 5,399,346, which is incorporated herein by reference). Moreover, viruses are very specialized, and can be selected as vectors based on the ability to infect and spread in one or a few specific cell types. Accordingly, its natural specificity can be used to direct the nucleic acid molecule contained in the vector to specific cell types. As such, an HIV-based vector can be used to infect T cells, a vector based on an adenovirus can be used, for example, to infect respiratory epithelial cells, a vector based on a herpes virus can be used to infect cells neuronal, and similar. Other vectors, such as adeno-associated viruses, can have a greater range of host cells, and therefore, can be used to infect different cell types, although viral or non-viral vectors can also be modified with specific receptors or ligands. to alter objective specificity through events mediated by the receiver. The present invention also provides anti-bodies that specifically bind to a peptide portion of a promyostatin polypeptide or an imitating promiostatin polypeptide. Particularly useful anti-bodies of the invention include anti-bodies that specifically bind to a myostatin prodomain, or a functional peptide portion thereof, and anti-bodies that bind to a promyostatin polypeptide, and reduce inhibit the proteolytic dissociation of promyostatin to a mature myostatin peptide. In addition, an anti-body of the invention can be an anti-body which is specifically linked to a growth differentiation factor receptor, or a functional peptide portion thereof, as described below. The methods for preparing and isolating an anti-body of the invention are described in more detail below, the disclosure of which is incorporated herein by reference. Myostatin is essential for proper regulation of skeletal muscle mass. Compared to wild-type mice, mice with myostatin clearance, lacking myostatin, have 2 to 3 times the amount of muscle due to a combination of hyperplasia and hypertrophy. As disclosed herein, mice with myostatin removal also have a dramatic reduction in fat accumulation, due, at least in part, to an increased anabolic rate of skeletal muscle tissue throughout the body. Inversely, overexpression of myostatin in hairless mice, induced a waste syndrome, which resembles the cachectic state observed in human patients suffering from chronic diseases such as cancer or AIDS. As is further disclosed herein, the activity of myostatin can be mediated through signal transduction having the characteristics of the Smad signal transduction pathway. In accordance with the above, the invention provides methods for modulating an effect of myostatin on a cell, by contacting the cell with an agent that affects the transduction of the myostatin signal in the cell. As used herein, the term "modular", when used with reference to an effect of myostatin on a cell, means that the transduction of the myostatin signal in the cell is increased or reduced or inhibited. The terms "increase" and "reduce or inhibit" are used with reference to a baseline level of myostatin signal transduction activity, which may be the activity level of the signal transduction pathway in the absence of myostatin, or the level of activity in a normal cell in the presence of myostatin. For example, the myostatin signal transduction pathway exhibits a particular activity in a muscle cell contacted with myostatin, and after further contact of the muscle cell with a myostatin prodomain, the transduction activity of the myostatin can be reduced or inhibited. the myostatin signal. As such, a myostatin prodomain is a useful agent for reducing or inhibiting the transduction of the myostatin signal. In a similar manner, a prodomain of another member of the growth differentiation factor family, such as a prodomain of GDF-11, or of another member of the TGF-β family, such as an activin prodomain, a MIS prodomain , or similar, may be useful to reduce the transduction of the myostatin signal. The terms "reduce" or "inhibit" are used together in the present, because it is recognized that, in some cases, the level of myostatin signal transduction is it can reduce below a level that can be detected by a particular test. As such, it can not be determined using this assay if there remains a low level of transduction of the myostatin signal, or if signal transduction is completely inhibited. As used herein, the term "myostatin signal transduction" refers to the series of events, in general a series of protein-protein interactions, that occur in a cell due to the specific interaction of myostatin. with a myostatin receptor expressed on the surface of the cell As such, the transduction of the myostatin signal can be detected, for example, by detecting a specific interaction of myostatin with its receptor on a cell, by detection of the phosphorylation of one or more polypeptides involved in a pathway of myostatin signal transduction in the cell, by detecting the expression of one or more genes that are specifically induced due to myostatin signal transduction, or by detection of a phenotypic change that occurs in response to myostatin signal transduction (see examples). known herein, a useful agent in a method of the invention can act as an agonist to stimulate the transduction of the myostatin signal, or as an antagonist to reduce or inhibit myostatin signal transduction. The methods of the present invention are exemplified in general in the present with respect to myostatin. However, it should be recognized that the methods of the invention can more broadly encompass the modulation of an effect of other peptides of the growth differentiation factor, for example GDF-11, on a cell, by contacting the cell with an agent that affects the signal transduction due to the growth differentiation factor in the cell. Methods for practicing the full scope of the invention will be readily known in view of the present disclosure, which includes, for example, methods for identifying growth differentiation factor receptors, methods for identifying agents that modulate signal transduction due to a specific interaction of the growth differentiation factor with its receptor, and the like. A pathway of transduction of the myostatin signal is exemplified in the present by the Smad path, which is initiated when myostatin interacts specifically with the extracellular domain of an activin type II receptor, and is propagated through interactions of intracellular polypeptides, including Smad proteins, in the cell. In general, myostatin signal transduction is associated with phosphorylation or dephosphorylation of specific intracellular polypeptides, such as Smad polypeptides. Accordingly, the transduction of the myostatin signal in a cell can be detected by detecting a higher level of phosphorylation of one or more Smad polypeptides in the presence of myostatin, compared to the level of phosphorylation of the polypeptides in the absence of myostatin. A method of the invention provides a means to increase or decrease the transduction of the myostatin signal, and consequently, the level of phosphorylation of a Smad polypeptide involved in a myostatin signal transduction path will increase above a normal level, or will be reduced below an expected level in the presence of myostatin, respectively. A method of the invention can be carried out, for example, by contacting, under suitable conditions, an objective cell and an agent that affects the transduction of the myostatin signal in the cell. Suitable conditions can be provided by placing the cell, which can be an isolated cell or can be a component of a tissue or organ, in an appropriate culture medium, or by contacting the cell in situ in an organism. For example, a medium containing the cell can be contacted with an agent that affects the ability of myostatin to interact specifically with a myostatin receptor expressed in the cell, or with an agent that affects the transduction pathway of the cell. the myostatin signal in the cell. In general, the cell is a component of a tissue or organ in a subject, in which case, the contact of the cell may comprise administering the agent to the subject. However, the cell can also be manipulated in culture, it can then be maintained in culture, administered to a subject, or used to produce a non-human transgenic animal. A useful agent in a method of the invention can be any type of molecule, for example, a polynucleotide, a peptide, a mimetic peptide, peptoids such as vinylogous peptoids, a small organic molecule, or the like, and can act on any of different ways to affect myostatin signal transduction. The agent can act extracellularly by binding myostatin or a myostatin receptor, such as an activin receptor, thereby altering the ability of myostatin to interact specifically with its receptor, or it can act intracellularly to alter the transduction of the myostatin signal in the cell. In addition, the agent may be an agonist, which mimics or enhances the effect of myostatin in a cell, for example, the ability of myostatin to interact specifically with its receptor, thereby increasing the transduction of the myostatin signal in the cell; or it may be an antagonist, which reduces or inhibits the effect of myostatin on a cell, thereby reducing or inhibiting the transduction of the myostatin signal in the cell. As used herein, the term "specific interaction" or "specific link", or the like, means that two molecules form a complex that is relatively stable under physiological conditions. The term is used in the present with reference to different interactions, including, for example, the interaction of myostatin and a myostatin receptor, the interaction of the intracellular components of a myostatin signal translation pathway, the interaction of an anti-body and its antigen , and the interaction of a prodomain of myostatin with myostatin. A specific interaction can be characterized by a dissociation constant of at least about lxl Cr6 M, in general of at least about lxl O "7 M, usually at least about lxlO" 8 M, and in particular at least about lxlCr5 M or lxl 0 ~ 10 M or more. A specific interaction is generally stable under physiological conditions, including, for example, conditions that occur in a living individual, such as a human or other vertebrate or invertebrate, as well as conditions that occur in a cell culture, such as those used to maintain mammalian cells or cells of another vertebrate organism or of an invertebrate organism. In addition, a specific interaction such as the extracellular interaction of a myostatin and myostatin prodomain, is generally stable under conditions such as those used for aquaculture of a commercially valuable marine organism. Methods for determining whether two molecules interact specifically are well known and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

An agent that alters a specific interaction of myostatin with its receptor can act, for example, by binding to myostatin, so that it can not interact specifically with its cellular receptor, by competing with myostatin for the linkage. with its receptor, or otherwise deviating the requirement that myostatin interact specifically with its receptor, in order to induce the transduction of the myostatin signal. A truncated myostatin receptor, such as a soluble extracellular domain of a myostatin receptor, is an example of an agent that can bind to myostatin, thereby sequestering myostatin, and reducing or inhibiting its ability to specifically interact with a myostatin. Myostatin receptor cell surface. A myostatin prodomain or a functional peptide portion thereof, is another example of an agent that can bind to myostatin, thereby reducing or inhibiting the ability of myostatin to interact specifically with a cell surface myostatin receptor. These myostatin antagonists are useful in the practice of a method of the invention, in particular to reduce or inhibit the transduction of the myostatin signal in a cell. Follistatin is another example of an agent that can bind to myostatin, thereby reducing or inhibiting the ability of myostatin to interact specifically with its receptor. Follistatin can bind to, and inhibit, the activity of different members of the TGF-β family, including myostatin (GDF-8, US patent 6,004,937), and GDF-11 (Gamer et al., Devel. Biol. 208: 222-232, 1999), and consequently, You can use it to carry out a method as disclosed. Although the use of follistatin to modulate the effects of myostatin had previously been described (US Pat. No. 6,004,937), it was not known, prior to the present disclosure, that follistatin reduces or inhibits the ability of myostatin to specifically interact with a receptor. of myostatin, such as Act RIIB. A useful agent in a method of the invention can also interact with a cellular myostatin receptor, thereby competing with myostatin for the receptor. This agent can be, for example, an anti-body that specifically binds with a cell surface myostatin receptor, including all or a portion of the myostatin binding domain, thereby preventing myostatin from interacting specifically with the receiver. This myostatin anti-receptor anti-body can be selected for its ability to specifically bind to the receptor without activating the myostatin signal transduction, and therefore, may be useful as a myostatin antagonist to reduce or inhibit signal transduction of myostatin; or it can be selected for its ability to bind specifically to the receptor and activate the transduction of the myostatin signal, acting as a this way as a myostatin agonist. The anti-body can be reproduced using a myostatin receptor, or the extracellular domain of the receptor, as an immunogen, or it can be an anti-idiotype anti-body, which reproduces against an anti-myostatin anti-body, and mimics to myostatin. The anti-growth factor receptor antibodies are described in more detail below. A useful agent in a method of the invention may also be an agent that reduces or inhibits the proteolytic cleavage of a pro-GDF polypeptide to an active mature growth differentiation factor peptide, thereby reducing or inhibiting signal transduction. of the growth differentiation factor. This agent can be a protease inhibitor, in particular one that inhibits the activity of a protease that recognizes and dissociates a proteolytic recognition site Arg-Xaa-Xaa-Arg (SEQ ID NO: 21). When the pro-GDF is promiostatin, an anti-myostatin anti-body that reduces or inhibits the specific binding of a protease with the proteolytic cleavage site Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) in myostatin can also be used to reduce or inhibit the proteolysis of promyostatin, thereby reducing the amount of mature myostatin produced. This anti-body can be linked to the proteolytic cleavage site, or it can be linked to some other site on the pro-GDF polypeptide, in such a way that the link of, and the dissociation by, protease. In addition, an agent useful in a method of the invention may be a mutant myostatin receptor that, for example, lacks the myostatin signal transduction activity, in response to the myostatin binding, or that has a transduction activity of the constitutive myostatin signal. For example, a mutant myostatin receptor may have a point mutation, a deletion, or the like, in its kinase domain, such that the receptor lacks kinase activity. This dominant mutant negative myostatin receptor lacks the ability to transmit the myostatin signal transduction, despite the fact that it can specifically bind to myostatin. A useful agent in a method of the invention can also modulate the level or activity of an intracellular polypeptide involved in a myostatin signal transduction pathway. As disclosed herein, the regulation of muscle growth by myostatin may involve components of a signal transduction pathway that is activated by activin type II receptors (see Examples 7 and 9; Example 14). Myostatin interacts specifically with activin type IIB receptors (Act RIIB) expressed in COS cells in culture (Example 7). The low binding affinity indicates that the binding of myostatin with Act RIIB in vivo may involve other factors, similar to TGF-β, which has a significantly higher affinity for the type 11 receptor, when the type I receptor is also present (Attisano et al., Cell 75: 671-680, 1993), or to other systems that require other molecules to present the ligand to the signaling receptor (Massague, s pra, 1998; Wang et al., Cell 67: 795-805, 1991). The specific interaction of myostatin with Act RIIB indicates that the transduction of the myostatin signal may involve the components of the Sitiad signal transduction pathway. Accordingly, the Smad signal transduction path provides an objective to modulate the effect of myostatin in a cell, and agents that affect the Smad pathway may also be useful for modulating the transduction of the myostatin signal in a cell. Agents useful for modulating the level or activity of the intracellular components of the polypeptide of a growth factor differentiation signal transduction include agonists, which can increase the activity of signal transduction, and antagonists, which can reduce or inhibit the activity of signal transduction. With respect to myostatin, for example, agents that can increase the activity of myostatin signal transduction are exemplified by phosphatase inhibitors, which can reduce or inhibit the dephosphorylation of Smad polypeptides, thus prolonging the activity of transduction Smad signal. Dominant dominant Smad 6 or Smad 7 polypeptides, which may negate the inhibitory effect of Smad 6 and Smad 7 on myostatin signal transduction, are additional examples of agents that can increase the activity of myostatin signal transduction through the increase in the transduction of the Smad signal. Antagonists that can reduce or inhibit myostatin signal transduction activity are exemplified by dominant Smad negative polypeptides, such as dominant Smad 2, Smad 3, or Smad 4, where the phosphorylation sites have been mutated. C-terminals. Smad inhibitory polypeptides, such as Smad 6 and Smad 7, which inhibit the activation of Smad 2 and Smad 3; and a c-ski polypeptide, which binds to the Smad polypeptides and inhibits signal transduction, are additional examples of antagonists useful for reducing or inhibiting myostatin signal transduction by reducing the transduction of the Smad signal. When the agent that acts intracellularly is a peptide, can be contacted with the cell directly, or a polynucleotide encoding the peptide (or polypeptide) can be introduced into the cell, and the peptide can be expressed in the cell. It is recognized that some of the peptides useful in a method of the invention are relatively large, and therefore, may not easily traverse a cell membrane. However, different methods are known to introduce a peptide in a cell. The selection of a method for introducing this peptide into a cell will depend, in part, on the characteristics of the objective cell, to which the polypeptide is to be provided. For example, when the target cells, or a few types of cells that include the target cells, express a receptor that, when linked to a particular ligand, is internalized in the cell, the peptide agent can be operatively associated with the ligand. Upon binding to the receptor, the peptide is translocated to the cell by endocytosis mediated by the receptor. The peptide agent can also be encapsulated in a liposome, or it can be formulated into a lipid complex, which can facilitate entry of the peptide into the cell, and can be further modified to express a receptor (or ligand) as above. The peptide agent can also be introduced into a cell by designing the peptide to contain a protein transduction domain, such as the TAT protein transduction domain of the human immunodeficiency virus, which facilitates the translocation of the peptide into the cell (see Schwarce et al., Science 285: 1569-1572 (1999), which is incorporated herein by reference, see also Derossi et al., J. Biol. Chem. 271: 18188 (1996)). The objective cell can also be contacted with a polynucleotide encoding the peptide agent, which can be expressed in the cell. The expressed peptide agent it can be a mutant growth differentiation factor receptor, or a peptide portion thereof. An example of a mutant growth differentiation factor receptor includes a kinase deficient form of a myostatin receptor, such as Act RIIA or Act RIIB negative dominant, which may, but does not need, to have the ability to specifically bind to a ligand (for example, myostatin); and a myostatin receptor or other truncated growth differentiation factor, such as a soluble form of a myostatin receptor, which binds to myostatin, thereby sequestering it so that it does not interact specifically with a cellular myostatin receptor; a dominant negative form of a Smad polypeptide, such as a dominant negative Smad 3, wherein the C-terminal phosphorylation sites have been mutated (Liu et al., Proc. Nati, Acad. Sci. USA 94: 10669-10674, 1997); a Smad 7 polypeptide, which inhibits the activation of Smad 2 and Smad 3 (Heldin et al., Nature 390: 465-471, 1997); or a c-ski polypeptide, which can bind to a Smad polypeptide and inhibit signal transduction by Smad (Sutrave et al., Genes Devel., 4: 1462-1472, 1990). The expression of a c-ski peptide agent in a cell can be particularly useful for modulating the transduction of the myostatin signal. Mice lacking c-ski show a severe reduction in skeletal muscle mass (Berk et al., Genes Devel., 11: 2029-2039, 1997), whereas transgenic mice that overexpress c-ski in muscle, show dramatic muscle hypertrophy (Sutrave et al., supra, 1990). c-ski interacts with, and blocks, the activity of certain Smad proteins, including Smad 2, Smad 3, and Smad 4, which mediate TGF-β signaling and activin type II receptors (Luo et al., Genes Devel. 13: 2196-1106, 1999; Stroschein et al., Science 286: 771-774, 1999; Sun et al., Mol. Cell 4: 499-509, 1999a; Sun et al., Proc. Nati. Acad. Sci. USA 96 : 112442-12447, 1999b; Akiyoshi et al., J. Biol. Chem. 274: 35269, 1999). Accordingly, in view of the present disclosure, in which the activity of myostatin can be mediated through the Act RIIB binding, it will be recognized that the activity of myostatin, or any growth differentiation factor that is used, can be modulated. a Smad path, by increasing or reducing the expression of c-ski in an objective cell. A useful agent in a method of the invention can be a polynucleotide, which can be contacted with, or introduced into, a cell as described above. In general, but not necessarily, the polynucleotide is introduced into the cell, where it performs its function either directly, or immediately after transcription or translation, or both. For example, as described above, the polynucleotide can encode a peptide agent, which is expressed in the cell and modulates the activity of myostatin. This peptide expressed it may be, for example, an immisinase promyostatin polypeptide, which can not be dissociated in active myostatin; or it may be a mutant myostatin receptor, for example, an extracellular domain of the truncated myostatin receptor; an extracellular domain of the myostatin receptor operatively associated with a membrane anchoring domain; or a mutant myostatin receptor that lacks the protein kinase activity. The methods for introducing a polynucleotide into a cell are exemplified below, or are otherwise known in the art. A polynucleotide agent useful in a method of the invention may also be, or may encode, an anti-sense molecule, a ribosome, or a triplexing agent. For example, the polynucleotide can be (or can encode) an anti-sense nucleotide sequence, such as a c-ski anti-sense nucleotide sequence, which can act as an agonist to increase the transduction of the myostatin signal in a cell; or an anti-sense Sitiad nucleotide sequence, which can act as either an agonist to increase the transduction of the myostatin signal, or as an antagonist to reduce or inhibit the myostatin signal transduction, depending on the sequence of nucleotides anti-sense Smad particular. These polynucleotides can be contacted directly with an objective cell, and when recovered by the cell, they can carry out their anti-sense, ribosomal or triplexante; or they can be encoded by a polynucleotide that is introduced into a cell, after which the polynucleotide is expressed to produce, for example, an anti-sense RNA or ribosome molecule, which performs its activity. A polynucleotide, ribosime, or antisense triplexant agent, is complementary to an objective sequence, which may be a DNA or RNA sequence, for example, messenger RNA, and may be a coding sequence, a nucleotide sequence comprising a binding of intron-exon, a regulatory sequence such as a Shine-Delgarno sequence, or the like. The degree of complementarity is such that the polynucleotide, for example, an anti-sense polynucleotide, can interact specifically with the target sequence in a cell. Depending on the total length of the anti-sense polynucleotide or other polynucleotide, one or a few bad matches can be tolerated with respect to the target sequence without losing the specificity of the polynucleotide for its objective sequence. Accordingly, few, if any, mismatches would be tolerated in an anti-sense molecule consisting, for example, of 20 nucleotides, while several mismatches would not affect the hybridization efficiency of an anti-sense molecule that is complementary, for example, to the full length of an objective mRNA encoding a cellular polypeptide. The number of bad pairings that can be tolerated can be estimated, for example, using formulas well known for determining the kinetics of hybridization (see Sambrook et al., supra, 1989), or it can be determined empirically using the methods disclosed herein or otherwise known in the art, in particular by determining that the presence of the polynucleotide, ribosime, or triplexant anti-sense agent in a cell reduces the level of the target sequence or the expression of a polypeptide encoded by the target sequence in the cell. A polynucleotide useful as an anti-sense molecule, a ribosome, or a triplexing agent, can inhibit translation or dissociate the nucleic acid molecule, thereby modulating the transduction of the myostatin signal in a cell. For example, an anti-sense molecule can be linked to an mRNA to form a double-stranded molecule that can not be translated into a cell. Antisense oligonucleotides of at least about 15 to 25 nucleotides are preferred, because they are easily synthesized, and can hybridize specifically with an objective sequence, alth longer antisense molecules can be expressed from a polynucleotide introduced into the target cell. Specific nucleotide sequences useful as anti-sense molecules can be identified using well known methods, for example, gene advancement methods (see, for example, Seimiya et al., J. Biol. Chem. 272: 4631-4636 (1997). ), which is incorporated herein by reference). When the anti-sense molecule comes into contact directly with an objective cell, it can be operatively associated with a chemically reactive group, such as iron-bound EDTA, which dissociates a target DNA at the site of hybridization. In comparison, a triplexing agent can stop transcription (Maher et al, Antisense Res. Devel. 1: 227 (1991); Helene, Anticancer Drug Design 6: 569 (1991)). Accordingly, a triplexing agent can be designed to recognize, for example, a sequence of a Smad gene regulatory element, thereby reducing or inhibiting the expression of a Smad polypeptide in the cell, thereby modulating signal transduction of myostatin in an objective cell. The present invention also provides a method for identifying an agent that can alter the effect of a growth differentiation factor, such as myostatin, in a cell, in particular agents that can alter the ability of the growth differentiation factor to interact specifically with your cellular receiver These agents can act by increasing or reducing the ability of the growth differentiation factor to specifically interact with its receptor, and therefore, are useful to increase or decrease the transduction of the growth differentiation factor signal, respectively. A screening method of the invention is exemplified herein using a myostatin receptor, for example, an activin type II receptor, such as Act RITA or Act RIIB.

A screening method of the invention can be carried out, for example, by contact, under suitable conditions, of myostatin, or of a functional peptide portion thereof, a myostatin receptor such as Act RIIA or Act RIIB, and an agent to be tested. The myostatin, the receptor, and the agent can be contacted in any order, as desired. As such, the screening method can be used to identify agents that can competitively or non-competitively inhibit the binding of myostatin to the receptor, agents that can mediate or enhance the binding of myostatin to the receptor, agents that can induce dissociation of the specifically bound myostatin of the receptor, and agents that otherwise affect the ability of myostatin to induce signal transduction, these agents having an agonist or antagonist activity. Appropriate control reactions are performed to confirm that the action of the agent is specific with respect to myostatin or another growth factor receptor. Suitable conditions for carrying out a screening method of the invention can be any conditions that allow the myostatin to interact specifically with its receptor, including the methods disclosed herein (see Examples 7 and 9), or known from another way in this field. Accordingly, suitable conditions for carrying out the screening test can be, for example, conditions in vitro using a myostatin receptor substantially purified; cell culture conditions, using a cell that normally expresses a myostatin receptor, for example, an adipocyte or a muscle cell, or a cell that has been genetically modified to express a functional myostatin receptor on its surface; or conditions in situ, as they appear in an organism. A screening method of the invention can also be carried out using molecular modeling methods, as described above. The use of a molecular modeling method provides a convenient and cost-effective means of identifying these agents, among a large population, such as a combination library of potential agents, that are more likely to interact specifically with a factor receptor. growth differentiation, thus reducing the number of potential agents that need to be traced using a biological assay. By identifying agents that interact specifically with a growth factor differentiation receptor, such as Act RIB, using a molecular modeling method, the selected agents can be examined for their ability to modulate an effect of a growth differentiation factor, such as myostatin, on a cell, using the methods disclosed in the present. The ability of a test agent to modulate a effect of myostatin, can be detected using the methods disclosed herein (see Examples 7 and 9), or otherwise known in the art. The term "test agent" or "test molecule" is widely used herein to mean any agent that is being examined to determine its agonist or antagonist activity in a method of the invention. Although the method is generally used as a screening assay to identify previously unknown molecules that can act as agonist or antagonist agents, as described herein, the methods can also be employed to confirm that an agent known to be have a particular activity, in fact have the activity, for example, in the standardization of the activity of the agent. A method of the invention can be carried out, for example, by contacting myostatin with a cell that has been genetically modified to express an Act RIIB receptor, and by determining the effect of an agent, for example, an Act RIIB dominant negative, by examining the phosphorylation of a Smad polypeptide involved in the pathway of myostatin signal translation. If desired, the cell can also be genetically modified to contain a reporter nucleotide sequence, whose expression depends on the pathway of myostatin signal transduction, for example, after activation of the Smad path, and the effect of the test agent by comparing the expression of the sequence of reporter nucleotides in the presence and absence of the agent, myostatin, or both. The expression of the reporter nucleotide sequence can be detected, for example, by detecting a transcript of RNA from the reporter nucleotide sequence, or by detecting a polypeptide encoded by the reporter nucleotide sequence. A reporter polypeptide can be, for example, a β-lactamase, chloramphenicol acetyltransferase, adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin-B phosphotransferase, thymidine kinase, β-galactosidase, luciferase, or a polypeptide of Xanthine-guanine phosphoribosyl transferase, or the like, and can be detected, for example, by the detection of radioactivity, luminescence, chemiluminescence, fluorescence, enzymatic activity, or specific binding due to the reporter polypeptide. A screening method of the invention provides the advantage that it can be adapted to high production analysis, and therefore, can be used to screen combination libraries of test agents in order to identify agents that can modulate an effect. of myostatin on a cell, including agents that can alter a specific interaction of myostatin and a myostatin receptor. Methods for preparing a combination library of molecules that can be tested to determine a desired activity are well known in the art, and include, for example, methods for making a library of phage display peptides, which may be limited peptides (see, for example, US Patent 5,622,699, US Patent 5,206,347, Scott and Smith, Science 249: 386-390, 1990; collaborators, Gene 109: 13-19, 1991, each of which is incorporated herein by reference); a library of peptides (US Patent 5,264,563, which is incorporated herein by reference); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14: 83-92, 1995); a library of nucleic acids (O'Connell et al., supra, 1996; Tuerk and Gold, supra, 1990; Gold et al., Supra, 1995; each of which is incorporated herein by reference); a library of oligosaccharides (York et al., Carb. Res., 285: 99-128, 1996; Liang et al., Science, 274: 1520-1522, 1996; Ding et al., Adv. Expt. Med. Biol., 376 : 261-269, 1995; each of which is incorporated herein by reference); a lipoprotein library (de Kruif et al., FEBS Lett., 399: 232-236, 1996, which is incorporated herein by reference); a library of glycoproteins or glycolipids (Araoglu et al., J. Cell Biol., 130: 567-577, 1995, which is incorporated herein by reference); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem., 37: 1385-1401, 1994; Ecker and Crooke, Bio / Technology, 13: 351-360, 1995 each of which is incorporated herein by reference Inc) . Polynucleotides can be particularly useful as agents that can modulate a specific interaction of myostatin and its receptor, because nucleic acid molecules that have binding specificity for cellular targets, including cellular polypeptides, exist naturally, and because synthetic molecules having this specificity, can be easily prepared and identified (see, for example, US patent 5,750,342, which is incorporated herein by reference). In view of the present disclosure, it will be recognized that different animal model systems can be used as search tools to identify the agents useful for practicing a method of the invention. For example, transgenic mice or other experimental animals can be prepared using the different constructions of myostatin inhibitors disclosed herein, and the transgenic non-human organism can be examined directly to determine the effect produced by the expression of different levels of a protein. particular agent in the body. In addition, the transgenic organism, for example, a transgenic mouse, can be crossed with other mice, for example with ob / ob, db / db, or agouti lethal yellow mutant mice, to determine the optimal levels of expression of an inhibitor of Myostatin useful for the treatment or prevention of a disorder such as obesity, type II diabetes, or the like. As such, the present invention provides transgenic non-human organisms, in particular transgenic organisms that contain a polynucleotide encoding a myostatin prodomain, which may include the myostatin signal peptide, a pro-peptide (see examples), or a polynucleotide that encodes a promyostatin polypeptide mutant In addition, the invention provides transgenic non-human organisms that express high levels of follistatin, or that express a dominant Act RIIB receptor negative (see examples). These organisms exhibit dramatic increases in muscle mass, similar to those seen in mice with myostatin clearance (see, for example, US Pat. No. 5,994,618, incorporated herein by reference). As described herein, these animal models are important for identifying agents to improve muscle growth for therapeutic purposes and for agricultural applications. Methods for producing transgenic non-human animals are known in the art (see, for example, US 6, 140, 552, 5,998,698, 6,218,596, all of which are incorporated herein by reference). A myostatin polynucleotide of the invention is derived from any organism, including mouse, rat, cow, pig, human, chicken, sheep, turkey, fin fish, and other aquatic organisms and other species. These polynucleotides can be used to make transgenic animals as described herein. The examples of aquatic animals for that can be made transgenic (and from which the myostatin polynucleotide can be derived) include those belonging to the Pool class, such as salmon, trout, small-scale trout, ayu, carp, crossed carp, golden fish, goby, whitebait, eel, conger, sardine, zebrafish, flying fish, sea bass, sea bream, parrot fish, biting fish, mackerel, sarda, tuna, bonito, yellow tail, perch, flounder, flounder, turbot, puffer fish, triggerfish; those belonging to the class of Cephalopods, such as squid, cuttlefish, octopus; those belonging to the Pelecipod class, such as clams (for example, hard shell, Manila, Quahog, Surf, concha Blanda); coquinas, mussels, bigarros; shells (for example, sea, bay, cayo); snail, slugs, sea cucumbers; shell of ark; oysters (eg, C. virginica, Gulf, New Zealand, Pacific); those belonging to the Gastropod class, such as turban shell, abalone (for example, green, pink, red); and those belonging to the Crustacean class, such as lobster, including, but not limited to, Spiny, Rock, and American; shrimp; shrimp, including, but not limited to, M. rosenbergií r P. styllrolls, P. indicus, P. jeponious, P. monodon, P. vannemel, M. ensis, S. melantho, N. norvegious, cold water shrimp; crab, including, but not limited to, Blue, jackdaw, stone, king, queen, snow, chestnut, no debris, Jonah, Mangrove, soft shell; shearing, krill, prawns; crayfish, including, but not limited to, Blue, Brown, Red Pincer, Red Swamp, Soft Shell, white; Annelida; Cordados, including, but not limited to, reptiles, such as lizards and turtles; Amphibians, including frogs; and Echinoderms, including, but not limited to, sea urchins. Different methods for producing a transgenic animal are known. In one method, an embryo is harvested in the pronuclear stage (an "embryo of a cell") from a female, and the transgene is microinjected into the embryo, in which case, the transgene will be chromosomally integrated into the germ cells and in the somatic cells of the resulting mature animal. In another method, totipotent embryonic cells are isolated, and the transgene is incorporated into the totipotent cells by electroporation, plasmid transfection, or microinjection; the totipotent cells are then reintroduced into the embryo, where they colonize and contribute to the germ line. Methods for microinjection of polynucleotides in mammalian species are described, for example, in US Pat. No. 4,873,191, which is incorporated herein by reference. In still another method, embryonic cells are infected with a retrovirus containing the transgene, whereby the germ cells of the embryo have the transgene chromosomally integrated therein. When the animals that are going to be transgenic are birds, the microinjection in the pronucleus of the fertilized egg is problematic, because the fertilized eggs of birds they usually pass through cell division during the first 20 hours in the oviduct, and consequently, the pronucleus is inaccessible. Accordingly, the method of infection with retroviruses to make transgenic bird species is preferred (see US Pat. No. 5,162,215, which is incorporated herein by reference). If microinjection is to be used with bird species, however, the embryo can be obtained from a hen slaughtered approximately 2.5 hours after the previous egg has been laid, the transgene is microinjected into the cytoplasm of the germinal disc, and it is grown the embryo in a host cover until maturing (Love et al., Biotechnology 12, 1994). When the animals to be transgenic are bovine or porcine, the microinjection can be hindered by the opacity of the eggs, thus making the nuclei difficult to identify using a traditional differential interference contrast microscope. To overcome this problem, the eggs can first be centrifuged to segregate the pronuclei for better visualization. The non-human transgenic animals of the invention can be bovine, swine, sheep, pool, poultry, or other animals. The transgene can be introduced into the embryonic target cells at different stages of development, and different methods are selected, depending on the stage of development of the objective embryonic cell. The zygote is the best objective for microinjection. The use of zygotes as a target for genetic transfer has a great advantage, because the injected DNA can be incorporated into the host gene before the first dissociation (Brinster et al., Proc. Nati. Acad. Sci., USA 82: 4438- 4442, 1985). As a consequence, all cells of the non-human transgenic animal carry the incorporated transgene, thereby contributing to an efficient transmission of the transgene to the progeny of the founder, because 50% of the germ cells will house the transgene. A transgenic animal can be produced by crossing two chimeric animals, each of which includes exogenous genetic material inside the cells used in reproduction. 25 percent of the resulting progeny will be from transgenic animals that are homozygous for the exogenous genetic material, 50 percent of the resulting animals will be heterozygous, and the remaining 25 percent will lack the exogenous genetic material and will have a wild-type phenotype . In the microinjection method, the transgene is digested and purified to be free from any vector DNA, for example, by gel electrophoresis. The transgene may include an operably associated promoter, which interacts with cellular proteins involved in transcription, and provides for constitutive expression, tissue specific expression, specific expression of the developmental stage, or Similary. These promoters include those of cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus, as well as those from the genes encoding metallothionein, skeletal actin, fosphenol pyruvate carboxylase (PEPCK), phosphoglycerate (PGK) ), dihydrofolate reductase (DHFR), and thymidine kinase (TK). Promoters can also be employed from viral long terminal repeats (LTRs), such as Rous LTR sarcoma virus. When the animals to be made transgenic are birds, the preferred promoters include those for the chicken globin gene, chicken lysozyme gene, and bird leukosis gene. Constructs useful in the transfection of plasmids from totipotent embryonic cells will employ additional regulatory elements, including, for example, enhancer elements to stimulate transcription, splice acceptors, termination and polyadenylation signals, ribosome binding sites to allow translation, and similar. In the retroviral infection method, the developing non-human embryo can be cultured in vitro up to the blastocyst stage. During this time, blastomeres may be targets for retroviral infection (Jaenich, Proc. Nati, Acad. Sci., USA 73: 1260-1264, 1976). An efficient infection of blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al., Manipula-ting the Mouse Enbryo (Cold Spring Harbor Laboratory Press, 1986). The system of the viral vector used to introduce the transgene is usually a defective retrovirus in replica carrying the transgene (Jahner et al., Proc. Nati, Acad. Sci., USA 82: 6927-6931, 1985, Van der Putten et al. Proc. Nati, Acad. Sci. USA 82: 6148-6152, 1985). Transfection is obtained easily and efficiently by culturing blastomeres on a monolayer of virus producing cells (Van der Putten et al., Supra, 1985, Stewart et al., EMBO J. 6: 383-388, 1987) . In an alternative way, the infection can be carried out at a later stage. Virus or virus-producing cells can be injected into the blastocell (Jahner et al., Nature 298: 623-628, 1982). Most of the founders will be the mosaic for the transgene, because the incorporation occurs only in a subset of the cells that formed the non-human transgenic animal. In addition, the founder may contain different retroviral insertions of the transgene in different positions of the genome, which will generally be segregated in the progeny. In addition, it is also possible to introduce transgenes in the germ line, albeit with an efficiency, by retroviral intrauterine infection of the embryo at mid pregnancy (Jahner et al., Supra, 1982). The embryonic totipotent cell (ES) can also be directed for the introduction of the transgene. The totipotent embryonic cells are obtained from embryos of pre-implantation cultured in vitro and fused with the embryos (Evans et al, Nature 292: 154-156, 1981; Bradley et al., Nature 309: 255-258, 1984; Gossler et al., Proc. Nati. Acad. Sci. , USA 83: 9065-9069, 1986; Robertson et al., Nature 322: 445-448, 1986). Transgenes can be introduced efficiently into totipotent embryonic cells by DNA transection or by retrovirus-mediated transduction. These transformed embryonic totipotent cells can then be combined with the blasts of a non-human animal. Subsequently, embryonic totipotent cells colonize the embryo and contribute to the germline of the resulting chimeric animal (see Jaenisch, Science 240: 1468-1474, 1988). As disclosed herein, myostatin may exert its activity, at least in part, through the Smad signal transduction pathway, and the expression of myostatin may be associated with different pathological conditions. As such, the present invention provides new targets for the treatment of different pathological conditions associated with myostatin, including metabolic conditions such as obesity and type II diabetes. In accordance with the foregoing, the present invention provides methods for reducing the severity of a pathological condition in a subject, wherein the pathological condition is characterized at least in part by an abnormal amount, development, or metabolic activity of a patient. muscle or adipose tissue, by modulating the transduction of the myostatin signal in a muscle cell or in an adipose tissue cell in the subject. Myostatin functions as a negative regulator of muscle growth (McPherron et al., Supra, 1997). The mice with myostatin clearance weighed approximately 25 percent to 30 percent more than the wild-type litters, and this increase in body weight in the mice examined resulted entirely from a dramatic increase in skeletal muscle tissue weight. In mice lacking myostatin, the skeletal muscles weighed approximately 2 to 3 times more than the corresponding muscles of the wild-type baits. This increase in muscle weight in mice with homozygous elimination resulted from a combination of hyperplasia and hypertrophy. As disclosed herein, mice with heterozygous myostatin removal also had an increase in skeletal muscle mass, although to a lesser degree than that observed in homozygous mutant mice, thus demonstrating that myostatin acts in a dose dependent manner in vivo (see Example 1). In addition, overexpression of myostatin in animals had the opposite effect with respect to muscle growth. For example, hairless mice that carried tumors that expressed myostatin, developed a waste syndrome characterized by a dramatic loss of muscle and fat weight (see Exercise 8). This syndrome in hairless mice resembled the cachectic state that occurs in patients with chronic diseases such as cancer or AIDS. The serum levels of the material immunoreactive with myostatin have been correlated with the condition of the patients with respect to muscle waste (Gonzalez-Kadavid et al., Proc. Nati, Acad. Sci., USA 95: 14938-14943, 1998, which it is incorporated herein by reference). Therefore, patients with AIDS, who also showed signs of cachexia, measured by the loss of total body weight, had slightly increased levels of serum immunosorbent material with myostatin in serum, compared with normal males without AIDS, or with AIDS patients that they did not have weight loss. However, the interpretation of these results was complicated, because the myostatin immunoreactive material detected in the serum samples did not have the mobility on SDS gels that was expected for the authentic processed myostatin. As is disclosed herein, myostatin not only affects muscle mass, but also affects the overall metabolism of an organism. For example, myostatin is expressed in adipose tissue, and mice deficient in myostatin have a dramatic reduction in fat accumulation as animals age (see Examples II and III). Although this mechanism is not proposed for the action of myostatin, the effect of myostatin may be the direct effect of myostatin on adipose tissue, or it may be an indirect effect caused by the lack of myostatin activity on skeletal muscle tissue. Regardless of the mechanism, the overall anabolic effect on muscle tissue that results in the reduction of myostatin activity can alter the body's overall metabolism and affect energy storage in the form of fat, as demonstrated by the introduction of a mutation of myostatin in an obese mouse strain (lethal yellow mice agouti (Ay)), which suppressed fat accumulation by five times (see Example 5). Abnormal glucose metabolism was also partially suppressed in agouti mice that contained the myostatin mutation. These results demonstrate that methods that inhibit myostatin can be used to treat or prevent metabolic diseases such as obesity and type II diabetes. The methods of the invention are useful, for example, to lessen the severity of different pathological conditions, including, for example, cachexia associated with chronic diseases such as cancer (see Norton et al., Crit. Rev. Oncol. Hematol., 7: 289-327, 1987), as well as conditions such as type II diabetes, obesity, and other metabolic disorders. As used herein, the term "pathological condition" refers to a disorder that is characterized, at least in part, by an abnormal amount, development, or metabolic activity of the muscle or adipose tissue. These pathological conditions, which include, for example, obesity; conditions associated with obesity, for example atherosclerosis, hypertension, and myocardial infarction; muscular waste disorders such as muscular dystrophy, neuromuscular diseases, cachexia, and anorexia; and metabolic disorders such as type II diabetes, which in general, but not necessarily, is associated with obesity, are particularly susceptible to treatment using a method of the invention. As used herein, the term "abnormal", when used with reference to the amount, development, or metabolic activity of the muscle or adipose tissue, is used in a relative sense as compared to an amount, development, or activity metabolic that an expert clinician or other relevant professional would recognize as normal or ideal. These normal or ideal values are known by the clinician, and are based on average values generally observed or desired in a healthy individual of a corresponding population. For example, the clinician would know that obesity is associated with a body weight that is approximately 20 percent above an "ideal" weight range for a person of a particular height and body type. However, the clinician would recognize that a weightlifter is not necessarily obese simply by virtue of having a body weight that is 20 percent or more greater than the expected weight for a person of the same height and body type in a correspondingly different population. In a similar manner, the technician would know that a patient presenting what appears to be abnormally reduced muscle activity could be identified as having abnormal muscle development, for example, subjecting the patient to different strength tests, and comparing the results with the expected for an average healthy individual in a corresponding population. A method of the invention can lessen the severity of a pathological condition that is characterized, at least in part, by an abnormal amount, development, or metabolic activity in muscle or adipose tissue, by modulating signal transduction of myostatin in a muscle or adipose tissue cell associated with the etiology of the condition. As used herein, the term "lessen", when used with reference to the severity of a pathological condition, means that the signs or symptoms associated with the condition are reduced. The signs or symptoms that will be monitored will be characteristic of a particular pathological condition, and will be known by the expert clinician, as well as the methods to monitor the signs and conditions. For example, when the pathological condition is type II diabetes, the skilled clinician can monitor glucose levels, glucose elimination rates, and the like in the subject.

When the pathological condition is obesity or cachexia, the clinician can simply monitor the subject's body weight. The agent to be administered to the subject is administered under conditions that facilitate the contact of the agent with the objective cell, and if appropriate, the entry of the cell. The entry of a polynucleotide agent into a cell, for example, can be facilitated by incorporating the polynucleotide into a viral vector that can infect the cells. If a viral vector specific for the cell type is not available, the vector can be modified to express a specific receptor (or ligand) for a ligand (or receptor) expressed in the target cell, or it can be encapsulated within a liposome, which can also be modified to include this ligand (or receptor). A peptide agent can be introduced into a cell by different methods, including, for example, by designing the peptide to contain a protein transduction domain, such as the TAT protein transduction domain of the human immunodeficiency virus, which can facilitate the translocation of the peptide to the cell (see Schwarce et al., supra, 1999; Derossi et al., Supra, 1996). The presence of the agent in the target cell can be directly identified, for example, by the operative linkage of a detectable label with the agent, by using a specific anti-body for the agent, in in particular a peptide agent, or by detecting a downstream effect due to the agent, for example a reduced phosphorylation of a Smad polypeptide in the cell. An agent can be labeled to be detectable using methods well known in the art (Hermanson, "Bioconjugate Techiques" (Academic Press 1996), which is incorporated herein by reference, see also Harlow and Lane, supra, 1988). For example, a peptide or a polynucleotide agent can be labeled with different detectable moieties, including a radiolabel, an enzyme such as alkaline phosphatase, biotin, a fluorophor, and the like. When the agent is contained in a kit, the reagents for labeling the agent can also be included in the kit, or the reagents can be purchased separately from a commercial source. A useful agent in a method of the invention can be administered to the site of the pathological condition, or it can be administered by any method that provides the target cells with the polynucleotide or the peptide. As used herein, the term "objective cells" means the muscle cells or adipocytes that are to be contacted with the agent. To be administered to a living subject, the agent is generally formulated into a suitable pharmaceutical composition to be administered to the subject. Therefore, the invention provides pharmaceutical compositions containing an agent, which is useful for modulating the signal transduction of Myostatin in a cell, in a pharmaceutically acceptable vehicle. As such, the agents are useful as medicaments for the treatment of a subject suffering from a pathological condition as defined herein. Pharmaceutically acceptable carriers are well known in the art, and include, for example, aqueous solutions, such as water or physiologically regulated whey, or other solvents or vehicles, such as glycols, glycerol, oils such as olive oil, or organic esters injectables. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds which act, for example, to stabilize or increase the absorption of the conjugate. These physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. A person skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physicochemical characteristics of the therapeutic agent and on the route of administration of the composition, which may be, for example. , orally or parenterally, such as intravenously, and by injection, intubation, or other method known in the art. The pharmaceutical composition may also contain a second reagent, such as a diagnostic reagent, a nutritional substance, a toxin, or a therapeutic agent, for example, a chemotherapeutic agent for cancer. The agent can be incorporated into an encapsulating material, such as in an oil-in-water emulsion, a microemulsion, miscelium, mixed miscelium, liposome, microsphere, or other polymeric matrix (see, for example, Gregoriadis, Liposome Technology, Volume 1 (CRC Press, Boca Raton, FL 1984), Fraley et al, Trends Biochem, Sci., 6:77 (1981), each of which is incorporated herein by reference). For example, liposomes, which consist of phospholipids or other lipids, are non-toxic, physiologically acceptable and metabolizable vehicles, which are relatively simple to make and administer. "Stealth" liposomes (see, for example, patents US 5,882,679; 5,395,619; and 5,225,212, each of which is incorporated herein by reference) are an example of these encapsulating materials particularly useful for preparing a pharmaceutical composition useful for practice of a method of the invention, and similarly, other "masked" liposomes may be used, these liposomes prolonging the time that the therapeutic agent remains in the circulation. For example, cationic liposomes can also be modified with specific receptors or ligands (Morishita et al., J. Clin. Invest., 91: 2580-2585 (1993), which is incorporated herein by reference). In addition, a polynucleotide agent can be introduced into a cell using, for example, adenovirus-polylysine DNA complexes (see, for example, Michael et al., J. Biol. Chem. 268: 6866-6869 (1993), which is incorporated herein by reference). The route of administration of a pharmaceutical composition containing an agent that alters the transduction of the myostatin signal will depend, in part, on the chemical structure of the molecule. For example, polypeptides and polynucleotides are not particularly useful when administered orally, because they can be degraded in the digestive tract. However, methods for chemically modifying polypeptides are known, for example, to make them less susceptible to degradation by endogenous proteases, or more absorbable through the alimentary tract (see, for example, Blondelle et al., Supra, 1995). Ecker and Crook, supra, 1995). In addition, a peptide agent can be prepared using D-amino acids, or it can contain one or more domains based on peptidomimetics, which are organic molecules that mimic the structure of the peptide domain; or based on a peptoid, such as a vinylogo peptoide. A pharmaceutical composition, as disclosed herein, can be administered to an individual by different routes, including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally. , intrarectally, intracisternally, or by absorption passive or facilitated through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. In addition, the pharmaceutical composition can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment, or active, for example, using a nasal spray or inhalant, in which case, a component of the composition is an appropriate propellant. A pharmaceutical composition can also be administered to the site of a pathological condition, for example, intravenously or intra-arterially in a blood vessel supplying a tumor. The total amount of an agent to be administered in the practice of a method of the invention, can be administered to a subject as a single dose, either as a bolus, or by infusion over a relatively short period of time, or it can be administered using a fractionated treatment protocol, wherein multiple doses are administered over a prolonged period of time. A person skilled in the art would know that the amount of the pharmaceutical composition for treating a pathological condition in a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments that are administered. go to administer In view of these factors, the technician would adjust the particular dose as necessary. In general, the formulation of the pharmaceutical composition and the routes and frequency of administration, are determined, initially, using clinical trials in Phase I and Phase II. The pharmaceutical composition can be formulated for an oral formulation, such as a tablet, or a form of solution or suspension; or may comprise a mixture with an organic or inorganic carrier or excipient suitable for oral or parenteral applications, and may be mixed, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, granules, capsules, suppositories, solutions, emulsions, suspensions, or other suitable form to be used. The vehicles, in addition to those disclosed above, may include glucose, lactose, mannose, acacia gum, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch. , urea, triglycerides of medium chain length, dextrans, and other vehicles suitable for use in the preparation of preparations, in a solid, semi-solid, or liquid form. In addition, auxiliary agents, stabilizers, thickeners, or colorants, and perfumes may be used, for example, a stabilizing dry agent such as triulose (see, for example, US Pat. No. 5,314,695). The present invention also provides a method for modulating the growth of muscle tissue or adipose tissue in a subject. As disclosed herein, the growth differentiation factor receptors, such as as Act RIIA and Act RIIB are involved in mediating the effects of a growth differentiation factor, such as myostatin, which is involved in the formation of muscle tissue and adipose tissue. Accordingly, in one embodiment, a method for modulating the growth of muscle tissue or adipose tissue includes affecting the signal transduction from a growth differentiation factor receptor, such as an activin receptor, for example Act RIIA or Act. RIIB. This method can be carried out by contacting the cells of the tissue with, or expressing in the cells, a mutant growth differentiation factor receptor, having a dominant negative activity, a constitutive activity, or the like. In another embodiment, a method for modulating the growth of muscle tissue or adipose tissue in an organism is carried out by administering to the organism an agent that affects the transduction of the myostatin signal. Preferably, the agent is, or encodes, a myostatin prodomain or a mutant promyostatin polypeptide, any of which may include a myostatin signal peptide. As used herein, the term "growth" is used in a relative sense when referring to the muscle tissue mass or adipose tissue mass in an organism, which has been subjected to a method of the invention, compared to a corresponding organism that has not been subjected to a method of the invention. By Consequently, when carrying out a method of the invention, in such a way that the transduction of the myostatin signal has been reduced or inhibited, it will be recognized that the growth of muscle tissue in the organism would result in an increase in muscle mass. in the organism, comparing with the muscle mass of a corresponding organism (or population of organisms) in which the transduction of the myostatin signal has not been affected in this way. A method of the invention may be useful for increasing muscle mass or reducing the fat content of an organism, or both. For example, when this method is carried out on an organism that is useful as a food source, the protein content of the food can be increased, the cholesterol level can be reduced, and the quality of the food material can be improved. A method of the invention may also be useful for reducing the growth of muscle tissue in an organism, for example, an organism that is detrimental to an environment, such that the organism is less able to compete in the environment. Accordingly, a method of the invention can be carried out on any eukaryotic organism expressing myostatin, including a vertebrate organism, for example a mammalian organism, bird, or pool, or it can be an invertebrate organism, for example a mollusc, echinoderm , gastropod, or cephalopod. The agent can be any agent that alters the myostatin signal transduction, as disclosed herein, and can be administered to the organism in any convenient manner. For example, when the organism to be treated is fish, shrimp, shells, or the like, which are grown in aquaculture, the agent can be added to the water in which the organisms are kept, or it can be included in their food , in particular where the agent is a soluble peptide or a small organic molecule. When the agent used in a method of the invention is a polynucleotide encoding a peptide agent, an anti-sense people, or the like, germ cells of a non-human organism containing the polynucleotide can be selected, and transgenic organisms can be produced that express the agent. Preferably, the polynucleotide is under the control of an inducible regulatory element, such that the agent encoded by the polynucleotide can be expressed at a desired time and for a duration. In accordance with the above, the present invention provides non-human transgenic organisms, as well as food products produced by these organisms. These food products have a higher nutritional value, due to the increase in muscle tissue. Transgenic non-human animals can be of any species, as disclosed herein, including vertebrate organisms, such as cattle, pigs, sheep, chickens, turkeys and fish, and invertebrate species, such as shrimp, lobsters, Crabs, squid, oysters, and abalone. The regulation of TGF-β family members and their specific interactions with cell surface receptors is beginning to be elucidated. Therefore, co-expression of the prodomain of a member of the TGF-β family with a mature region of another member of the TGF-β family is associated with intracellular dimerization, and secretion of biologically active homodimers occurs (Gray et al. , Science 247: 1328, 1990). For example, the use of the BMP-2 prodomain with the mature BMP-4 region led to a dramatically improved expression of mature BMP-4 (Hammonds et al., (Mol Endocrinol 5: 149, 1991)). For most family members that have been studied, homodimeric species are biologically active, while for other family members, such as inhibins (Ling et al., Nature 321: 779, 1986), and TGFs -βe (Cheifetz et al., Cell 48: 409, 1987), heterodimers have also been detected, and appear to have different biological properties than the respective homodimers. The receptor-ligand interaction studies have revealed a great deal of information about how cells respond to external stimuli, and have led to the development of therapeutically important compounds, such as erythropoietin, colony-stimulating factors, and PDGF. Consequently, continuous efforts have been made to identify Carry the receptors that mediate the action of members of the TGF-β family. As disclosed herein, myostatin interacts specifically with an activin type II receptor. The identification of this interaction provides targets for identifying antagonists and agonists useful for agricultural and human therapeutic purposes, for example, for the treatment of different pathological conditions, such as obesity, type II diabetes, and cachexia. The identification of this specific interaction also provides a means to identify other myostatin receptors, as well as the specific receptors of other growth differentiation factors. In accordance with the above, the present invention provides growth differentiation factor receptors, which interact specifically with a growth differentiation factor, or with a combination of growth differentiation factors, for example, with myostatin, GDF-11, or both A growth differentiation factor receptor of the invention is exemplified herein by a myostatin receptor, in particular an activin type II receptor, which interacts specifically with myostatin and with GDF-11. However, myostatin receptors that specifically interact with myostatin, but not with GDF-11, are also encompassed within the present invention, as well as GDF-11 receptors that interact specifically with GDF-11 but not with myostatin. , and similar. For greater convenience of In the discussion, the receptors of the invention are referred to herein in general as a "growth differentiation factor receptor", and are employed by a myostatin receptor, which is a receptor that interacts specifically with at least myostatin . As such, although reference is generally made to a specific interaction of myostatin with a myostatin receptor, it will be recognized that the present disclosure covers more broadly any growth differentiation factor receptor, including a GDF-11 receptor, that specifically interacts with at least GDF-11. Also provided is a recombinant cell line expressing a growth-differentiating factor receptor polypeptide, as well as anti-bodies that specifically bind to the receptor, substantially purified polynucleotides encoding the receptor, and growth-differentiation factor receptor polypeptides. substantially purified. Peptide portions of a growth differentiation factor receptor are also provided, including, for example, soluble extracellular domains of a growth differentiation factor receptor, such as a myostatin receptor which, as disclosed herein can alter the specific interaction of myostatin with a cellular myostatin receptor; a constitutively active intracellular kinase domain of a growth differentiation factor receptor, which can induce, stimulate, or otherwise maintain the transduction of the growth differentiation factor signal in a cell; or another truncated portion of a growth differentiation factor receptor that has the ability to modulate myostatin or other signal transduction of the growth differentiation factor. The invention also provides methods for identifying a growth factor differentiation receptor polypeptide, including methods for screening genomic or cDNA libraries, which can be expression libraries, using nucleotide probes or antibody probes; methods for screening cells that respond to, and therefore, express the receptor, using, for example, a growth differentiation factor such as myostatin or a functional peptide portion thereof; two-hybrid assays, as described above, using, for example, the peptide of growth differentiation factor as a component of a hybrid, and peptides expressed from a cDNA library, which is prepared from cells that express a receptor for the growth differentiation factor, as components of second hybrids, and the like. As described above, agents that specifically interact with a growth differentiation factor receptor, eg, a myostatin receptor, such as Act RIIB, can be identified using the receiver to track these agents. Conversely, an agent that has been identified as having the ability to interact specifically with a myostatin receptor, such as the Act RIIB receptor, can be used to screen additional myostatin receptors or other receptors for growth differentiation factor. This method may include incubating components such as the agent (or myostatin or other growth differentiation factor), and a cell that expresses a growth differentiation factor receptor, which may be a truncated membrane bound receptor or a soluble receptor. , under conditions sufficient to allow the agent (or the growth differentiation factor) to interact specifically with the recipient; measure the agent (or the growth differentiation factor) bound to the recipient; and isolate the receiver. A method of molecular modeling as described above, may also be useful as a screening method to identify a growth differentiation factor receptor, or a functional peptide portion thereof. Also provided are non-human transgenic animals that have a phenotype characterized by the expression of a growth factor differentiation receptor, the phenotype being conferred by a transgene contained in the somatic and germ cells of the animal. The transgene comprises a polynucleotide that encodes the differential factor receptor. growth, for example, the myostatin receptor, or the polypeptide. The methods for producing these transgenic animals are disclosed herein or are otherwise known in the art. The present invention provides a substantially purified polynucleotide that encodes all or a portion of the peptide of a growth factor differentiation receptor. Although a growth differentiation factor receptor is exemplified herein as an activin type II receptor, polynucleotides encoding type II activin receptors (US Pat. No. 5,885,794) have been described above. Accordingly, it should be recognized that these activin type II receptors are not encompassed within the present invention (Massague, supra, 1998, Heldin et al., Supra, 1997). In a similar manner, activin type I receptors, including Act RIB; TGF-β receptors, including TGF-βRI and TGF-βRII; and the BMP receptors, including BMP RIA, BMP RIB, and BMP RII, have already been described and are well known in the field (Massague, supra, 1998; Heldin et al., Supra, 1997), and therefore, are not encompassed within the receptors of growth differentiation factor of the invention. A polynucleotide of the invention can encode a polypeptide having a myostatin receptor activity, for example, a myostatin binding activity, or it can encode a mutant myostatin receptor, for example, a mutant myostatin receptor having a mutation in a kinase domain, such that the mutant acts as a dominant negative myostatin receptor (see above). Therefore, a polynucleotide of the invention can be a polynucleotide that occurs naturally, synthetically, or intentionally manipulated. For example, portions of the mRNA sequence may be altered due to alternating RNA splicing patterns, or to the use of alternating promoters for RNA transcription. As another example, the polynucleotide can be subjected to site-directed mutagenesis. The polynucleotide can also be an anti-sense nucleotide sequence. Polynucleotides of the growth differentiation factor receptor of the invention include sequences that are degenerated as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Accordingly, all degenerate nucleotide sequences are included within the invention, provided that the amino acid sequence of the growth differentiation factor receptor polypeptide encoded by the polynucleotide is functionally unchanged. Nucleotide sequences encoding myostatin receptor polypeptides are also included. Oligonucleotide portions of a polynucleotide encoding a growth differentiation factor receptor of the invention are also encompassed within the scope of the invention. present invention. These oligonucleotides are generally at least about 15 bases in length, which is sufficient to allow the oligonucleotide to selectively hybridize to a polynucleotide encoding the receptor, and can be at least about 18 nucleotides or 21 nucleotides or more in length. As used herein, the term "selective hybridization" or "selectively hybridizing" refers to hybridization under moderately restrictive or highly restrictive physiological conditions, which can distinguish related nucleotide sequences from unrelated nucleotide sequences. In nucleic acid hybridization reactions, the conditions used to achieve a particular level of restriction will vary, depending on the nature of the nucleic acids that are hybridizing. For example, in the selection of hybridization conditions, the length, the degree of complementarity, the composition of the nucleotide sequence (e.g., the relative content of GC: AT), and the type of nucleic acid, can be considered. that is, if the oligonucleotide or the objective nucleic acid sequence is DNA or RNA. A further consideration is whether one of the nucleic acids is immobilized, for example, on a filter. The methods for selecting the appropriate restriction conditions can be determined empirically, or they can be estimated using different formulas, as is well known in the art (see, for example, example, Sambrook et al., supra, 1989). An example of progressively higher restriction conditions is as follows: 2X SSC / 0.1% SDS at about room temperature (hybridization conditions); 0.2X SSC / 0.1 percent SDS at approximately room temperature (conditions of low restraint); 0.2X SSC / 0.1% SDS at approximately 42 ° C (moderate restraint conditions); and 0. IX SSC at approximately 68 ° C (conditions of high restriction). Washing can be carried out using only one of these conditions, for example, conditions of high restriction, or each of the conditions may be used, for example, for 10 to 15 minutes each, in the order listed above, repeating any or all of the steps listed. A polynucleotide encoding the growth differentiation factor receptor of the invention can be obtained by any of several methods. For example, the polynucleotide can be isolated using hybridization or computer-based techniques, as is well known in the art. These methods include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) screening of anti-bodies in expression libraries to detect cloned DNA fragments with shared structural characteristics; 3) polymerase chain reaction (PCR) on genomic DNA or CDNA, using primers capable of annealing to the DNA sequence of interest; 4) searches of sequences databases in computer to look for similar sequences (see above); 5) differential screening of a stolen DNA library; and 6) two-hybrid assays, using, for example, a peptide of mature growth differentiation factor in one of the hybrids. In view of the present disclosure that an activin receptor interacts specifically with myostatin, oligonucleotide probes can be designed based on the sequence encoding an activin receptor, for example, a sequence encoding the extracellular domain, which binds to Myostatin, and is used to screen a library prepared from cells such as muscle cells or adipocytes, which respond to myostatin, thereby facilitating the identification of a polynucleotide that encodes a myostatin receptor. The selected clones can be further screened, for example, by subcloning the inserts into an expression vector, and following the expression of the cloned sequences, the screening of the polypeptides expressed using myostatin. A polynucleotide of the invention, for example, a polynucleotide that encodes a myostatin receptor, can be derived from a vertebrate species, including a mammalian, avian, or pool species, or from a species of invertebrate. Tracing procedures that rely on nucleic acid hybridization allow the isolation of any genetic sequence from any organism, provided that the appropriate probe is available. Oligonucleotide probes that correspond to a part of the sequence encoding the protein in question can be chemically synthesized. This requires that short stretches of oligopeptides of the amino acid sequence be known. A polynucleotide sequence encoding the receptor can be deduced from the genetic code, taking into account the degeneracy of the genetic code. Accordingly, mixed addition reactions can be carried out when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For this screening, preferential hybridization is carried out on single-stranded DNA or on denatured double-stranded DNA. Hybridization is particularly useful in the detection of derived cDNA clones from sources where an extremely low amount of mRNA sequences related to the polypeptide of interest is present. Therefore, by using constraining hybridization conditions directed to avoid non-specific binding, autoradiographic visualization can be used to identify a specific cDNA clone, by hybridizing the target DNA to an oligonucleotide probe in the mixture, which is the complete nucleic acid complement objective (Wallace et al., Nucí, Acid Res., 9: 879, 1981, which is incorporated herein by reference). Alternatively, a subtractive library can be used, thereby eliminating non-specific cDNA clones. When the entire amino acid sequence of a desired polypeptide is not known, direct synthesis of DNA sequences is not possible, and the method of choice is the synthesis of cDNA sequences. Among the conventional methods for isolating cDNA sequences of interest, is the formation of cDNA libraries prepared in plasmids or phages, wherein the libraries are derived from the reverse transcription of the mRNA which is abundant in donor cells having a high level of genetic expression. When used in combination with the polymerase chain reaction technology, even rare expression products can be cloned. When significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA or double-stranded RNA probe sequences that duplicate a sequence presumably present in the target cDNA can be employed in the hybridization procedures carried out on cloned copies of the cDNA, which have denatured in a single chain form (Jay et al., Nucí, Acid Res., 11: 2325, 1983, which is incorporated herein by reference). You can trace a cDNA expression library, such as the lambda gtll library, to determine the peptides receptors of the growth differentiation factor, using an anti-body specific for a growth differentiation factor receptor / for example, an anti-body anti-Act RIIB. The anti-body can be polyclonal or monoclonal, and can be used to detect the expression product that indicates the presence of a cDNA of the growth factor differentiation receptor. This expression library can also be screened with a growth differentiation factor peptide, for example, with myostatin, or a functional peptide portion thereof, to identify a clone that encodes at least a portion of a binding domain. myostatin of a myostatin receptor. Polynucleotides that encode mutant growth differentiation factor receptor and mutant growth factor receptor polynucleotide receptors are also encompassed within the invention. An alteration in a polynucleotide that encodes a growth differentiation factor receptor may be an intragenic mutation, such as point mutation, nonsense mutation (STOP), missense mutation, splice site mutation, or frame change , or it can be a heterozygous or homozygous deletion, and it can be a mutation that occurs naturally, or it can be designed using recombinant DNA methods, for example. These alterations can be detected using methods conventional ones known to those skilled in the art, including, but not limited to, nucleotide sequence analysis, Southern blot analysis, an analysis based on polymerase chain reaction, such as a multiplex polymerase chain reaction, or analysis of labeled sites of the sequence (STS), or analysis of in situ hybridization. The receptor polypeptides. of growth differentiation factor can be analyzed by standard SDS-PAGE, immunoprecipitation analysis, Western blot analysis, or the like. The mutant growth differentiation factor receptors are exemplified by the truncated growth differentiation factor receptors, including a soluble extracellular domain, which may have the ability to bind specifically to its cognate growth differentiation factor, but lack a kinase domain; a kinase domain of intracellular growth differentiation factor receptor, which may exhibit a constitutive kinase activity; as well as by growth differentiation factor receptors containing a point mutation, which alters the receptor kinase activity or the ligand binding capacity of the receptor; and similar. These mutants of growth differentiation factor receptors are useful for modulating the signal transduction of the growth differentiation factor, and, therefore, for practicing different methods of the invention.

A polynucleotide that encodes a growth differentiation factor receptor can be expressed in vitro by introducing the polynucleotide into a suitable host cell. The "host cells" can be any cells in which the particular vector can be propagated, and, where appropriate, where a polynucleotide contained in the vector can be expressed.The term "host cells" includes any progeny of an original host cell It is understood that all progeny of the host cell may not be identical to the progenitor cell, due, for example, to mutations that occur during replication, however, this progeny is included when the term "host cell" is used. Methods for obtaining a host cell that contains in a transient or stable manner an introduced polynucleotide of the invention are well known in the art A growth differentiation factor receptor polynucleotide of the invention can be inserted into a vector, which may be a cloning vector or a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus, or other vehicle known in the art that has been manipulated by the insertion or incorporation of a polynucleotide, in particular with respect to the present invention, a polynucleotide that encode all or a portion of the peptide of a growth factor differentiation receptor. These expression vectors contain a promoter sequence, which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector generally contains a replication origin, a promoter, as well as specific genes that allow the phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to, the T7-based expression vector, for expression in bacteria (Rosenberg et al., Gene 56: 125, 1987), the pMSXND expression vector for its expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263: 3521, 1988), and baculovirus derived vectors for expression in insect cells. The DNA segment may be present in the vector operably linked to the regulatory elements, for example, a promoter, which may be a T7 promoter, a metallothionein I promoter, a polyhedrin promoter, or other desired promoter, in particular promoters. tissue-specific or inducible promoters. A polynucleotide sequence that encodes a growth differentiation factor receptor can be expressed either in prokaryotes or in eukaryotes. Hosts may include microbial, yeast, insect, and mammalian organisms. Methods for expressing polynucleotides having eukaryotic or viral sequences in prokaryotes are well known in the art, as well as biologically functional viral and plasmid DNA vectors. able to express themselves and replicate in a host. The methods for constructing an expression vector containing a polynucleotide of the invention are well known, as well as the factors that must be considered in the selection of transcriptional or translational control signals, including, for example, if the The polynucleotide will preferably be expressed in a particular cell type or under particular conditions (see, for example, Sambrook et al., supra, 1989). A variety of host cells / expression vector systems can be used to express a growth factor differentiation receptor coding sequence, including, but not limited to, microorganisms, such as bacteria transformed with DNA expression vectors. recombinant bacteriophage, plasmid DNA, cosmid DNA; yeast cells transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors, such as a cauliflower mosaic virus or a tobacco mosaic virus, or transformed with recombinant plasmid expression vectors such as a Ti plasmid; insect cells infected with recombinant virus expression vectors, such as baculoviruses; animal cell systems infected with recombinant virus expression vectors, such as a retrovirus, adenovirus, or a vaccine virus vector; and systems of transformed animal cells, genetics- mind designed for stable expression. When the expressed growth differentiation factor receptor is modified after translation, for example, by glycosylation, it may be particularly convenient to select a host cell / expression vector system that can effect the desired modification, for example, a host system. host cell / mammalian expression vector. Depending on the host cell / vector system used, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like, can be used in the expression vector (Bitter et al., Meth. Enzymol., 153: 516-544, 1987). For example, when cloning into bacterial systems, inducible promoters such as pL of the bacteriophage?, Plac can be used., ptrp, ptac (ptrp-lac hybrid promoter) and the like. When cloning into mammalian cell systems, promoters derived from the genome of mammalian cells, for example a promoter of human or mouse metallothionein, or from mammalian virus, for example a long terminal repeat of retroviruses, can be used. , a delayed adenovirus promoter, or a 7.5 K vaccine virus promoter. Promoters produced by recombinant or synthetic DNA techniques can also be used to provide transcription of the sequence of coding of the growth differentiation factor receptors inserted. In yeast cells, a number of vectors containing constitutive or inducible promoters can be used (see Ausubel et al., Supra, 1987, see chapter 13, Grant et al., Meth. Enzymol., 153: 516-544, 1987; Glover, DNA Cloning Volume II (IRL Press, 1986), see Chapter 3, Bitter, Meth. Enzymol 152: 673-684, 1987, see also The Molecular Biology of the Yeast Saccharomyces (eds., Strathern et al., Cold Spring Harbor Laboratory Press, 1982), Volumes I and II). A constitutive yeast promoter, such as ADH or LEU2, or an inducible promoter such as GAL (Rothstein, DNA Cloning Volume II. (Supra, 1986), chapter 3) can be used. Alternatively, vectors that promote the integration of foreign DNA sequences into the yeast chromosome can be used. Eukaryotic systems, in particular mammalian expression systems, allow for appropriate post-translational modifications of the expressed mammalian proteins. Eukaryotic cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and conveniently insertion into the plasma membrane of the gene product, can be used as host cells for the expression of a factor receptor polypeptide. difference- growth, or a functional peptide portion thereof. Mammalian cellular systems using recombinant viruses or viral elements can be designed to direct expression. For example, when adenovirus expression vectors are used, the coding sequence of the growth differentiation factor receptors can be ligated to an adenovirus transcription / translation control complex, for example the late promoter and the tripartite leader sequence. . Alternatively, the 7.5 K vaccine virus promoter can be used (Mackett et al., Proc. Nati, Acad. Sci., USA 79: 7415-7419, 1982, Mackett et al., J. Virol. 49: 857 -864, 1984; Panicali et al., Proc. Nati, Acad. Sci. USA 79: 4927-4931, 1982). Particularly useful are bovine papilloma virus vectors, which can be replicated as extrachromosomal elements (Sarver et al., Mol.Cell. Biol. 1: 486, 1981). Shortly after the entry of this DNA into mouse cells, the plasmid replicates up to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require the integration of the plasmid into the chromosome of the host cell, thus producing a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene. In an alternative way, the retroviral genome can be modified to used as a vector capable of introducing and directing the expression of the gene of growth factor differentiation receptors in host cells (Cone and Mulligan, Proc Nati, Acad. Sci., USA 81: 6349-6353, 1984). High level expression can also be achieved using inducible promoters, including, but not limited to, the metallothionein II promoter and the heat shock promoters. For the long term, stable expression of the high yield production of recombinant proteins is preferred. Instead of using expression vectors containing viral replication origins, the host cells can be transformed with the cDNA of the growth differentiation factor receptor controlled by appropriate expression control elements, such as promoter or enhancer sequences, transcription terminators. , and polyadenylation sites, and a selectable marker .. The selectable marker in the recombinant plasmid can confer resistance to selection, and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. For example, following the introduction of the foreign DNA, the designed cells can be allowed to grow for 1 to 2 days in an enriched medium, and then they are changed to a selective medium. A number of selection systems can be used, including, but not limited to, the thymidine kinase genes of the herpes simplex virus (Wigler et al., Cell 11: 223, 1977), hypoxanthine-guanine phosphoribosyl transferase (Szybalska and Szybalski, Proc. Nati, Acad. Sci., USA 48: 2026, 1982), and adenosine phosphoribosyltransferasetrase (Lowy et al., Cell 22: 817, 1980) in tk-, hgprt-, or aprt cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Nati, Acad. Sci., USA 77: 3567, 1980; O'Hare et al. Proc. Nati, Acad. Sci., USA 78: 1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Nati, Acad. Sci., USA 78: 2072, 1981); neo, which confers resistance to aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150: 1, 1981), and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147, 1984). ). Additional selectable genes have also been described, including trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to use histinol in place of histidine (Hartman and Mulligan, Proc. Nati, Acad. Sci. USA 85: 8047, 1988); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoromethyl) -DL-ornithine, DFMO (McConlogue, Curr. Comm. Mol. Biol. (Cold Spring Harbor Laboratory Press, 1987)). When the host is a eukaryote, methods of DNA transfection such as coprecipitates can be employed in calcium phosphate, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid housed in liposomes, or virus vectors. Eukaryotic cells can also be co-transformed with DNA sequences encoding the growth differentiation factor receptors of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes thymidine kinase gene. simplex Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40), or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (Gluzman, Eukariotic Viral Vectors (Cold Spring Harbor Laboratory Press, 1982). )). The invention also provides stable recombinant cell lines, which cells express growth differentiation factor receptor polypeptides, and contain DNA encoding growth factor differentiation receptors. Suitable cell types include, but are not limited to, NIH 3T3 (murine) cells, C2C12 cells, L6 cells, and P19 cells. The myoblasts C2C12 and L6 spontaneously differentiate in culture, and form myotubes depending on the particular growth conditions (Yaffe and Saxel, Nature 270: 725-727, 1977; Yaffe, Proc. Nati. Acad. Sci., USA 61: 77 -483, 1968). P19 is a line of embryonic carcinoma cells. These cells are described, for example, in the Cell Line Catalog of the American Type Culture Collection (ATCC). These cells can be stably transformed using well-known methods (see, for example, Ausubel et al., Supra, 1995, see sections 9.5.1-9.5.6). A growth differentiation factor receptor can be expressed from a recombinant polynucleotide of the invention using inducible or constitutive regulatory elements, as described herein. The desired protein coding sequence and an operably linked promoter can be introduced into a recipient cell, either as a DNA molecule (or AR) without replication, which can be a linear molecule or a covalently closed circular molecule. Expression of the desired molecule can occur due to transient expression of the introduced sequence, or the polynucleotide can be stably maintained in the cell, for example, by its integration into a chromosome of the host cell, thus allowing a more permanent expression. In accordance with the above, the cells may be stably or transiently transformed (transfected) cells. An example of a vector that can be employed is one that is capable of integrating the desired genetic sequences into the chromosome of the host cell. Cells that have stably integrated the DNA introduced into their chromosomes can also be selected by entering one or more markers that allow the selection of the host cells contain the expression vector. The marker can complement an auxotrophy in the host, such as leu2 or ura3, which are common auxotrophic yeast markers; they can confer resistance to biocides, for example to an antibiotic or to heavy metal ions, such as copper, or the like. The selectable marker gene can be linked directly to the DNA genetic sequences to be expressed, or it can be introduced into the same cell by cotransfection. The introduced sequence can be incorporated into a plasmid or viral vector capable of having autonomous replication in the recipient host. Any of a variety of vectors can be used for this purpose. Factors of importance in the selection of a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from cells not containing the vector; the number of copies of the vector desired in a particular host cell; and if it is desirable to be able to "throw" the vector between the host cells of different species. For a mammalian host, several vector systems are available for expression. One class of vectors utilizes DNA elements that provide for the autonomous replication of extrachromosomal plasmids derived from animal viruses, for example, a bovine papilloma virus, bovine papilloma virus, polyoma, adenovirus, or SV40 virus. A second class of vectors includes the expression vectors of the vaccine virus. A third class of vectors relies on the integration of the desired genetic sequences in the host chromosome. Cells that have stably integrated the DNA introduced into their chromosomes can also be selected by introducing one or more marker genes (as described above), which allow the selection of host cells containing the expression vector. The selectable marker gene can be linked directly to the DNA sequences to be expressed, or it can be introduced into the same cell by cotransfection. Additional elements may be included to provide optimal synthesis of an encoded mRNA or peptide, including, for example, splicing signals, promoters or transcription enhancers, and transcription or translation termination signals. CDNA expression vectors incorporating appropriate regulatory elements are well known in the art (see, for example, Okayama, Mol, Cell, Biol. 3: 280, 1983). Once the vector or DNA sequence containing the construct has been prepared, for expression, the DNA construct can be introduced into an appropriate host. Different methods can be used to introduce the polynucleotide into a cell, including, for example, transfection or transformation methods, such as protoplast fusion, precipitation with calcium phosphate, and electroporation or other conventional techniques, for example, infection, wherein the vector is a viral vector. The invention also provides transgenic animals, which have cells that express a recombinant growth differentiation factor receptor. These transgenic animals may be selected to have a reduced fat content or an increased muscle mass, or both, and therefore, may be useful as a source of foodstuffs with high muscle and protein content, and a reduced fat content and cholesterol. The animals have been chromosomally altered in their germ cells and somatic cells, so that the production of a growth differentiation factor, in particular myostatin, is maintained at a "normal" level, but the myostatin receptor is produced in amounts reduced, or is completely interrupted, resulting in the cells of the animals having a reduced ability to bind to myostatin, and consequently, have higher than normal levels of muscle tissue, preferably without increasing the levels of fat or choleste-rol. In accordance with the foregoing, the present invention also includes food products provided by the animals. These food products have a higher nutritional value, due to the increase in muscle tissue. The non-human transgenic animals of the invention include bovine, porcine, ovine, and bird animals, as well as other vertebrates, and also include transgenic invertebrates. The invention also provides a method for producing animal food products having a higher muscle content. This method can include the modification of the genetic formation of the germ cells of a pronuclear embryo of the animal, implanting the embryo in the oviduct of a pseudo-pregnant female, thus allowing the embryo to mature to the full-term progeny. the progeny to determine the presence of the transgene in order to identify the positive progeny for the transgene, to cross the progeny positive for the transgene in order to obtain positive progeny for the additional transgene, and to process the progeny to obtain a food material. Modification of the germ cell comprises altering the genetic composition to reduce or inhibit the expression of the naturally occurring gene encoding the production of a myostatin receptor protein. For example, the transgene may comprise an anti-sense molecule that is specific for a polynucleotide that encodes a myostatin receptor; may comprise a non-functional sequence that replaces or intervenes in the endogenous myostatin receptor gene or the transgene; or can encode a myostatin receptor antagonist, for example, a dominant negative myostatin receptor, such as a dominant RIIB negative act.

As used herein, the term "animal" refers to any bird, fish, or mammal, except a human being, and includes any stage of development, including embryonic and fetal stages. Within the meaning of the term "animal", farm animals, such as pigs, goats, sheep, cows, horses, rabbits, and the like, are included; rodents such as mice; and domestic pets, such as cats and dogs. In addition, the term "organism" is used herein to include animals as described above, as well as other eukaryotes, including, for example, other vertebrates such as reptiles and amphibians, as well as invertebrates as described above. As used herein, the term "transgene", when used with reference to an animal or an organism, means that the cells of the animal or organism have been genetically engineered to contain an exogenous polynucleotide sequence that is stably maintained with the cells. The manipulation can be, for example, microinjection of a polynucleotide or infection with a recombinant virus containing the polynucleotide. Accordingly, the term "transgenic" is used herein to refer to animals (organisms) in which one or more cells receive a recombinant polynucleotide, which can be integrated into a chromosome of the cell, or can be maintained as a extrachromosomal replication polynucleotide, as it could be designed on a chromosome artificial yeast The term "transgenic animal" also includes a transgenic animal of the "germ cell line". A transgenic animal of the germ cell line is a transgenic animal in which genetic information has been taken and incorporated into a germline cell, conferring therefore the ability to transfer information to the progeny. If in fact this progeny owns some or all that information, the progeny are also considered as of transgenic animals. The invention also encompasses transgenic organisms. A transgenic organism can be any organism whose genome has been altered by in vitro manipulation of an early stage embryo or a fertilized egg, or by any transgenic technology to induce a specific genetic elimination. The term "genetic elimination" refers to the targeted disruption of a gene in a cell or in vivo, which results in a complete loss of function. An objective gene in a transgenic animal can be rendered non-functional by an insertion directed to the gene to become non-functional, for example, by homologous recombination, or by any other method to interrupt the function of a gene in a cell. The transgene to be used in the practice of the present invention may be a DNA sequence comprising a coding sequence of factor receptor receptors. differentiation of modified growth. Preferably, the gene of the modified growth differentiation factor receptor is one that is altered by the homologous direction towards the embryonic totipotent cells. For example, the entire mature C-terminal region of the growth differentiation factor receptor gene can be deleted (see Example 13). Optionally, the alteration (or deletion) may be accompanied by the insertion of, or replacement with, another polynucleotide, for example a non-functional growth differentiation factor receptor sequence. An "elimination" phenotype can also be conferred by the introduction or expression of an anti-sense growth differentiation factor receptor polynucleotide in a cell of the organism, or by the expression of an anti-body or a factor receptor. of differentiation of the dominant negative growth in the cells. Where appropriate, polynucleotides encoding proteins that have growth factor-factor receptor activity, but which differ in the nucleotide sequence of a genetic sequence of growth differentiation factor that occurs naturally due to the degeneration of the genetic code, as well as truncated forms, allelic variants, and inter-species homologs. The present invention also provides anti-bodies that specifically bind to a factor receptor. differentiation of growth, and that block the link of the growth differentiation factor with the recipient. These anti-bodies may be useful, for example, to ameliorate a pathological condition, such as a cell proliferative disorder associated with muscle tissue. A monoclonal anti-body that specifically binds with a growth factor differentiation receptor, in particular with a myostatin receptor, can increase the development of skeletal muscles. In the preferred embodiments of the claimed methods, a monoclonal antibody, polypeptide, or polynucleotide of the growth differentiation factor receptor is administered to a patient suffering from a pathological condition, such as a muscle wasting disease, a neuromuscular disorder, atrophy muscular, aging or similar. The anti-body of the growth differentiation factor receptor, in particular an anti-body anti-myostatin receptor, can also be administered to a patient suffering from a pathological condition, such as muscular dystrophy, spinal cord injury. , traumatic injury, congestive obstructive pulmonary disease (COPD), AIDS, or cachexia. In a preferred embodiment, the myostatin anti-receptor anti-body is administered to a patient with muscle wasting disease or disorder, by intravenous, intramuscular, or subcutaneous injection; preferably, a monoclonal anti-body within a dose range of between about 0.1 micrograms / kg and about 100 mg / kg; more preferably between about 1 microgram / kg and 75 mg / kg; most preferably from about 10 mg / kg to 50 mg / kg. The anti-body can be administered, for example, by bolus injection or by slow infusion. Slow infusion is preferred over a period of 30 minutes to 2 hours. The anti-body anti-myostatin receptor, or other anti-body anti-receptor growth factor, can be formulated into a suitable formulation for administration to a patient. These formulations are known in the art. The dosage regimen will be determined by the physician attending, considering different factors that modify the action of the myostatin receptor protein, for example, the amount of tissue that it is desired to form, the site of tissue damage, the condition of the tissue damaged, the size of a wound, the type of tissue damaged, the age, sex, and diet of the patient, the severity of any infection, the time of administration, and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and the types of agents, such as anti-myostatin receptor anti-bodies, which are to be used in the composition. In general, systemic or injectable administration is given, such as intravenous, intramuscular, or subcutaneous injection. Administration generally starts at a dose that is minimally effective, and the dose is increased during a previously selected time course, until a positive effect is observed. Subsequently, increasing increases in dosage are made, limiting these increasing increases to levels such that they produce a corresponding increase in the effect, while taking into account any adverse effects that may arise. The addition of other known growth factors, such as Igf I (insulin-like growth factor I), human growth hormone, bovine, or chicken, which can help increase muscle mass, to the final composition, can also affect to the dosage. In the embodiment wherein an anti-myostatin receptor anti-body is administered, the anti-body is generally administered within a dose range of about 0.1 micrograms / kg to about 100 mg / kg; more preferably between about 100 mg / kg and 50 mg / kg. As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of those anti-bodies. An anti-body useful in a method of the invention, or an antigen binding fragment thereof, is characterized, for example, by having specific binding activity for an epitope of a growth factor differentiation receptor, for example, a myostatin receptor. In addition, as described above, an anti- The body of the invention can be an anti-body that specifically binds to a peptide portion of a promyostatin polypeptide, in particular a myostatin prodomain or a functional peptide portion thereof. It will be recognized that the following methods, which exemplify the preparation and characterization of the growth differentiation factor receptor anti-bodies, are further applicable to the preparation and characterization of additional anti-bodies of the invention, including anti-bodies that specifically bind to a prodrug of myostatin, anti-bodies that specifically bind to a promyostatin polypeptide and reduce or inhibit the proteolytic cleavage of promyostatin to myostatin, and the like. The terms "specifically binds" or "specific binding activity", when used with reference to an anti-body, mean that an interaction of the anti-body and a particular epitope, has a dissociation constant of at least about lxlO ". s, generally at least about lxlO "7, usually at least about lxlO" 8, and in particular at least about lxlO "9 or lxlO" 10 or less As such, within the definition of an anti-body, include the Fab, F (ab ') 2 / Fd, and Fv fragments of an antibody, which retain the specific binding activity for an epitope of a growth differentiation factor receptor For the purposes of the present invention, for example , HE considers that an anti-body that specifically reacts with an epitope of a myostatin receptor substantially does not react with a TGF-β receptor or with a BMP receptor if the anti-body has at least a two-fold binding affinity or more, in general at least one binding affinity of five times or more, and in particular at least one binding affinity of 10 times or more for the myostatin receptor, compared to the TGF-β or BMP receptor. The term "anti-body", as used herein, includes anti-bodies that occur naturally, as well as anti-bodies that do not occur naturally, including, for example, single-chain anti-bodies, chimeric, bifunctional and humanized antibodies, as well as antigen binding fragments thereof. These antibodies that do not occur naturally, can be constructed using solid phase peptide synthesis, can be produced in a recombinant manner, or can be obtained, for example, by screening combination libraries consisting of variable heavy chains and variable light chains (see Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference). ). These and other methods for making, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional anti-bodies are well known to those skilled in the art (Winter and Harris, Immunol., Today 14: 243- 246, 1993; Wardet al., Nature 341: 544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, Second Edition (Oxford University Press 1995); each of which is incorporated herein by reference). Anti-bodies that specifically bind to a growth differentiation factor receptor can be reproduced using the receptor as an immunogen, and removing anti-bodies that cross-react, for example, with a TGF-β type I receptor or type II, with an activin receptor such as Act RIB, Act RIIA, or Act RIIB, or with a BMP receptor, such as BMP RII, BMP RIA, and BMP RIB (see Massague, supra, 1998). An anti-body of the invention can be conveniently reproduced using a peptide portion of a myostatin receptor that is not present in a TGF-β, activin, or BMP receptor. In a similar manner, an anti-body that specifically binds to a myostatin prodomain, can be reproduced using the prodomain, or a functional peptide portion thereof, as the immunogen. When this peptide is non-immunogenic, it can be made immunogenic by coupling the hapten with a carrier molecule, such as bovine serum albumin (BSA) or bore 1-hole hemocyanin (KLH), or by expressing the peptide portion as a fusion protein. In this field, other different carrier molecules and methods for coupling a hapten with a carrier molecule (see, for example, Harlow and Lane, supra, 1988). If desired, a kit incorporating an anti-body or other useful agent can be prepared in a method of the invention. This kit may contain, in addition to the agent, a pharmaceutical composition in which the agent can be reconstituted for administration to a subject. The kit may also contain, for example, reagents to detect the anti-body, or to detect the specific binding of the anti-body with a growth factor differentiation receptor. These detectable reagents useful for labeling or otherwise identifying the anti-body are described herein and are known in the art. Methods for reproducing polyclonal anti-bodies, for example in a rabbit, goat, mouse, or other mammal, are well known in the art (see, for example, Green et al., "Production of Polyclonal Antisera", in Immunochemical Protocols ( Manson, editor, Humana Press 1992), pages 1-5, Coligan et al, "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters," in Curr. Protocols Immunol. (1992), Section 2.4.1; of which is incorporated herein by reference). In addition, monoclonal antibodies can be obtained using methods that are well known and routine in this field (Harlow and Lane, supra, 1988). For example, the spleen cells of a mouse immunized with a receptor myostatin, or an epitopic fragment thereof, can be fused with an appropriate myeloma cell line, such as SP / 02 myeloma cells, to produce hybridoma cells. The cloned hybridoma cell lines can be screened using the labeled antigen to identify clones that secrete monoclonal antibodies having the appropriate specificity, and the hybridomas can be isolated that express antibodies having a desirable specificity and affinity, and are used as a continuous source of anti-bodies. The antibodies can be further screened for their inability to specifically bind to the myostatin receptor. These anti-bodies are useful, for example, to prepare standardized kits for clinical use. A recombinant phage that expresses, for example, an anti-body myostatin antireceptor of a single chain, also provides an anti-body that can be used for the preparation of standardized kits. Methods for preparing monoclonal anti bodies are well known (see, for example, Kohler and Milstein, Nature 256: 495, 1975, which is incorporated herein by reference).; see also Coligan et al., supra, 1992, see sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988). Said in a brief manner, monoclonal anti-bodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of the production of anti-bodies by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting the positive clones that produce antibodies to the antigen, and Isolate the anti-bodies from the hybridoma cultures. Monoclonal anti-bodies can be isolated and purified from the hybridoma cultures by a variety of well-established techniques, including, for example, affinity chromatography with SEPHAROSE protein-A, size exclusion chromatography, and exchange chromatography. ions (Coligan et al., supra, 1992, see sections 2.7.1.-2.7.12, and sections 2.9.1-2.9.3, see also Barnes et al., "Purification of Immunoglobulin G (IgG)", in Meth .: Molec. Biol. 10: 79-104 (Humana Press 1992), which are incorporated herein by reference). The methods of in vitro and in vivo multiplication of the monoclonal antibodies are well known to those skilled in the art. The in vitro multiplication can be carried out in a suitable culture medium, such as an Eagle Medium Modified by Dulbecco or an RPMI 1640 medium, optionally filled with mammalian serum, such as fetal calf serum, or trace elements and supplements that support growth, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. In vitro production provides preparations of relatively pure anti-bodies, and allows to scale to produce large quantities of the desired anti-bodies. The large-scale hybridoma culture can be carried out by homogeneous suspension culture in an air-lift reactor, in a continuous agitator reactor, or in immobilized or trapped cell culture. In vivo multiplication can be carried out by injection of cellular clones in mammals histocompatible with the progenitor cells, for example, syngeneic mice, to cause the growth of anti-body producing tumors. Optionally, the animals are prepared with a hydrocarbon, especially oils, such as pristane (tetramethylpentadecane) before injection. After 1 to 3 weeks, the desired monoclonal anti body is recovered from the body fluid of the animal. Therapeutic applications for the anti-bodies disclosed herein are also a part of the present invention. For example, the anti-bodies of the present invention can also be derived from a sub-human primate anti-body. General techniques for reproducing therapeutically useful antibodies in mandrels can be found, for example, in Goldenberg et al., International publication WO 91/11465 (1991); and in Losman et al., Int. J. Cancer 46: 310, 1990, each of which is incorporated herein by reference). An anti-body anti-differential factor receptor- Therapeutically useful growth can also be derived from a "humanized" monoclonal anti-body. The humanized monoclonal anti-bodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin to a human variable domain, and then the human residues are replaced in the structure regions of the mouse. the counterparts of the murine. The use of anti-body components derived from humanized monoclonal anti-bodies obviates the potential problems associated with the immunogenicity of the murine constant regions. The general techniques for cloning murine immunoglobulin variable domains are known (see, for example, Orlandi et al, Proc. Nati, Acad. Sci. USA 86: 3833, 1989, which is incorporated herein by reference in its entirety) . Techniques for producing humanized monoclonal anti bodies are also known (see, for example, Jones et al., Nature 321: 522, 1986; Riechmann et al., Nature 332: 323, 1988; Verhoeyen et al., Science 239: 1534, 1988; Carter and collaborators, Proc. Nati Acad. Sci., USA 89: 4285, 1992; Sandhu, Crit. Rev. Biotechnol. 12: 437, 1992; and Singer et al., J. Immunol. 150: 2844, 1993; each of which is incorporated herein by reference). The anti-bodies of the invention can also be derived from fragments of isolated human anti-bodies from a combination immunoglobulin library (see, for example, Barbas et al., METHODS: A Companion to Methods in Immunology 2: 119, 1991; inter et al., Ann. Rev. Immuno., 12: 433, 1994; one of which is incorporated herein by reference). Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from Stratagene Cloning Systems (La Jolla, California, United States). An anti-body of the invention can also be derived from a human monoclonal anti-body. These anti-bodies are obtained from transgenic mice that have been "engineered" to produce specific human anti-bodies in response to the stimulus. antigenic In this matter, elements of the human heavy and light chain sites are introduced into the strains of mice derived from totipotent embryonic cell lines containing targeted alterations of the endogenous heavy and light chain sites. Transgenic mice can synthesize human anti-bodies specific for human antigens, and mice can be used to produce anti-human body secretion hybridomas. Methods for obtaining human anti-bodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368: 856, 1994; and Taylor et al., Int. Immuno1. 6: 579, 1994; each of which is incorporated herein by reference. The anti-body fragments of the present invention can be prepared by the proteolytic hydrolysis of the antibody, or by the expression of E. coli of the DNA encoding the fragment. Antibody fragments can be obtained by digestion with pepsin or papain of the whole anti-bodies by conventional methods. For example, fragments of anti-bodies can be produced by enzymatic cleavage of the anti-bodies with pepsin to provide a 5S fragment denoted as F (ab ').?. This fragment can be further dissociated using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from the dissociation of the disulfide bonds, to produce monovalent 3.5S Fab 'fragments. Alternatively, an enzymatic dissociation using pepsin yields two monovalent Fab 'fragments and a Fe fragment directly (see, for example, Goldenberg, US Pat. 4, 036, 945 and US Pat. 4, 331, 647, each of which it is incorporated by reference, and the references contained therein, Nison off et al., Arch. Biochem. Biophys., 89: 230, 1960; Porter, Biochem. J. 73: 119, 1959; Edelman et al., Meth. Enzymol. , 1: 422 (Academic Press 1967), each of which is incorporated herein by reference, see also Coligan et al., Supra, 1992, see sections 2.8.1-2.8.10, and 2.10.1-2.10 .4).

Other methods for dissociating anti-bodies, such as the separation of heavy chains to form monovalent light / heavy chain fragments, additional fragment dissociation, or other enzymatic, chemical, or genetic techniques, may also be used, in the understanding of that the fragments bind specifically with the antigen that is recognized by the intact anti-body. For example, the Fv fragments comprise an association of VH and VL chains. This association can be non-covalent (Inbar et al., Proc. Nati, Acad. Sci., USA 69: 2659, 1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond, or they can be crosslinked by chemicals such as glutaraldehyde (Sandhu, supra, 1992). Preferably, the Fv fragments comprise the VH and VL chains connected by a peptide linker. These single chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide by bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97, 1991; Bird and collaborators, Science 242: 423-426, 1988; Ladner et al., US Patent 4, 946, 778; Pack et al., Bio / Technology 11: 1271-1277, 1993; each of which is incorporated herein by reference; see also Sandhu, supra, 1992. Another form of an anti-body fragment is a peptide encoding a single complementarity determining region (CDR). Peptides of complementarity determining region ("minimal recognition units") can be obtained by constructing genes that encode the complementarity determining region of an anti-body of interest. These genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region of the RNA of the anti-body producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106, 1991, which is incorporated herein by reference). The invention also provides a method for identifying a growth differentiation factor receptor polypeptide. This method can be carried out, for example, by the incubation of components comprising the growth differentiation factor polypeptide, and a cell expressing a full-length receptor or a truncated receptor under conditions sufficient to allow the factor of Growth differentiation is linked to the receptor; measure the binding of the differentiation factor polypeptide of the growth with the receiver; and isolate the receiver. The growth differentiation factor can be any of the kngrowth differentiation factors (eg, GDF-1-16), and is preferably GDF-8 (myostatin) or GDF-11. The methods for isolating the receivers are described in more detail in the examples section below. In accordance with the foregoing, the invention also provides a substantially purified growth differentiation factor receptor, as well as peptides and peptide derivatives of a growth differentiation factor receptor having fewer amino acid residues than a factor receptor. of differentiation of growth that occurs naturally. These peptides and peptide derivatives are useful as research and diagnostic tools in the study of muscular waste diseases and the development of more effective therapies. The invention further provides variants of the growth differentiation factor receptor. As used herein, the term "growth differentiation factor receptor variant" means a molecule that stimulates at least part of the structure of the growth differentiation factor receptors. Growth differentiation factor receptor variants may be useful to reduce or inhibit the binding of the growth differentiation factor, thereby lessening a condition pathological, as is disclosed in the present. Examples of growth differentiation factor receptor variants include, but are not limited to, truncated growth differentiation factor receptors, such as a soluble extracellular domain of a growth differentiation factor receptor; a dominant negative growth differentiation factor receptor, such as a dominant Act RIIB negative receptor, that lacks kinase activity; or other truncated or mutant growth differentiation factor receptors. The invention relates not only to peptides and peptide derivatives of a naturally occurring growth differentiation factor receptor, but also to variants of the growth differentiation factor receptor, including mutant growth differentiation factor receptors, and chemically synthesized derivatives of growth differentiation factor receptors that specifically bind with a growth differentiation factor, for example myostatin. For example, changes in the amino acid sequence of a growth factor differentiation receptor are contemplated in the present invention. The growth differentiation factor receptors can be altered by changing the DNA encoding the protein. Preferably, only alterations of conservative amino acids are undertaken, using amino acids that have properties same or similar. Illustrative amino acid substitutions include changes from alanine to serine; Arginine to Usina; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine. The variants useful for the present invention comprise analogs, homologues, muteins and mimetics of a growth differentiation factor receptor, which retain the ability to specifically bind to their respective growth differentiation factors. In another embodiment, variant growth differentiation factor receptors having dominant negative activity are also contemplated, regardless of whether the variant also interacts specifically with its growth differentiation factor. Peptides of the growth differentiation factor receptors refer to portions of the amino acid sequence of the growth differentiation factor receptors having these capacities. Variants can be generated directly from the growth differentiation factor receptors themselves by chemical modification, by digestion with proteolytic enzymes, or by combinations thereof. Additionally, genetic engineering techniques can be employed, as well as methods for synthesizing polypeptides directly from amino acid residues. Peptides can be synthesized by commonly used methods, such as protection with t-BOC or FMOC from alpha-amino groups. Both methods involve stepwise synthesis where a single amino acid is added at each step, starting from the C-terminus of the peptide (Coligan et al., Current Protocols in Immunology (Wiley Interscience, 1991), Unit 9, which is incorporated herein by reference). reference). The peptides of the invention can also be synthesized by well-known solid-phase peptide synthesis methods (Merrifield, J. Am. Chem. Soc. 85: 2149, 1962; Stewart and Young, Solid Phase Peptides Synthesis (Freeman, San. Francisco, 1969), see pages 27-62, each of which is incorporated herein by reference), using a copoly (styrene-divinylbenzene) containing 0.1-1.0 mMol amines / g of polymer. Upon completion of the chemical synthesis, the peptides can be deprotected and dissociated from the polymer by treatment with liquid HF-anisole at 10 percent for approximately 1 / 4-1 hours at 0 ° C. After the evaporation of the reagents, the peptides are extracted from the polymer with a 1 percent acetic acid solution, which is then lyophilized to provide the raw material. This can usually be purified by techniques such as gel filtration on Sephadex G-15, using 5 percent acetic acid as a solvent. Lyophilization of the appropriate fractions from the column will produce the homogeneous peptide or peptide derivatives, which can then be characterized by conventional techniques such as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantification by Edman degradation in solid phase. Non-peptidic compounds that mimic the binding and function of growth-differentiating factor receptors ("mimetics") can be produced by the approach illustrated by Saragovi et al. (Science 253: 792-95, 1991, which is incorporated in the present as a reference). Mimetics are molecules that mimic the elements of the secondary structure of the protein (Johnson et al., "Peptide Turn imetics", in Biotechnology and Pharmacy (Pezzuto et al., Eds.; Chapman and Hall, New York 1993), which is incorporated herein by reference). The underlying reason behind the use of peptide mimetics is that the base structure of the peptide of the proteins exists mainly to orient the side chains of amino acids, in such a way that molecular interactions are facilitated. For the purposes of In the present invention, an appropriate mimetic can be considered as the equivalent of a growth differentiation factor receptor. Longer peptides can be produced by the "native chemical" ligation technique, which binds the peptides together (Dawson et al., Science 266: 776, 1994, which is incorporated herein by reference). Variants can be created by recombinant techniques, using genomic or cDNA cloning methods. Site-specific and site-directed mutagenesis techniques can be employed (Ausubel et al., Supra, 1989 and 1990 until 1993), see volume 1, chapter 8; Protein Engineering (Oxender and Fox, editors, A. Liss, Inc., 1987)). In addition, linker scanning techniques can be employed and mediated by polymerase chain reaction for mutagenesis (Stockton Press 1989); Ausubel et al., Supra, 1989 to 1993). In the references cited above, the sequencing, structure, and protein modeling approaches for use with any of the above techniques are disclosed. The present invention also provides binding agents of growth differentiation factor receptors that block the specific binding of a growth differentiation factor to its receptor. These agents are useful, for example, as research and diagnostic tools in the study of muscle waste disorder, as described above, and as effective therapeutics, and can be identified using the methods disclosed herein, for example, a method of molecular modeling. In addition, pharmaceutical compositions comprising growth factor receptor receptor binding agents may represent effective therapeutics. In the context of the invention, the phrase "growth differentiation factor receptor binding agent" denotes a ligand that occurs naturally of a growth differentiation factor receptor, eg, GDF-1 through GDF-16; a synthetic ligand of growth differentiation factor receptors, or an appropriate derivative of natural or synthetic ligands. The determination and isolation of the ligands are well known in the art (Lerner, Trends Neurosci, 17: 142-146, 1994, which is incorporated herein by reference). In still another embodiment, the present invention relates to binding agents of growth factor differentiation receptors that interfere with the binding between a growth differentiation factor receptor and a growth differentiation factor. These binding agents can interfere by competitive inhibition, by non-competitive inhibition, or by uncompetitive inhibition. Interference with the normal link between the growth differentiation factor receptors and one or more growth differentiation factors may result in an effect pharmacological useful. The invention also provides a method for identifying a composition that binds to a growth differentiation factor receptor. The method includes incubating components comprising the composition and a growth differentiation factor receptor under conditions sufficient to allow the components to specifically interact, and measuring the binding of the composition to the growth differentiation factor receptors. Compositions that bind to growth differentiation factor receptors include peptides, peptidomyruetics, polypeptides, chemical compounds, and biological agents, as described above. Incubation includes exposing the reagents to conditions that allow contact between the test composition and the growth differentiation factor receptors, and provide the conditions suitable for a specific interaction, as it would happen in vivo. The contact can be in solution or in solid phase. The test / composition ligand may optionally be a combination library for screening a plurality of compositions, as described above. The compositions identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a DNA sequence. specific, such as polymerase chain reaction, oligomer restriction (Saiki et al., Bio / Technology 3: 1008-1012, 1985, which is incorporated herein by reference), allele-specific oligonucleotide probe analysis (ASO ) (Conner et al., Proc. Nati, Acad. Sci., USA 80: 278, 1983, which is incorporated herein by reference), oligonucleotide ligation assays (OLAs) (Landegren et al., Science 241: 1077, 1988, which is incorporated herein by reference), and the like (see Landegren et al., Science 242: 229-237, 1988, which is incorporated herein by reference). To determine whether a composition can be complexed with the receptor protein, the induction of an exogenous gene can be monitored by monitoring changes at the protein level, a protein encoded by the exogenous gene, or any other given method. to know in the present. When a composition that can induce transcription of the exogenous gene is identified, it is concluded that this composition can specifically bind to the receptor protein encoded by the nucleic acid encoding the initial sample test composition. The expression of the exogenous gene can be monitored by a functional assay or by an assay to determine a protein product, for example. Accordingly, the exogenous gene is a gene that provides an expression product assayable / measurable, in order to allow the detection of exogenous gene expression. These exogenous genes include, but are not limited to, reporter genes, such as the chloramphenicol acetyl transferase gene, an alkaline phosphatase gene, β-galactosidase, a luciferase gene, a green fluorescent protein gene, guanine phosphoribosyltransferase -xanthin, alkaline phosphatase, and antibiotic resistance genes, such as neomycin phosphotransferase (see above). The expression of the exogenous gene indicates a specific interaction of a composition and a growth differentiation factor receptor; therefore, the binding or blocking composition can be identified and isolated. The compositions of the present invention can be extracted and purified from the culture medium or from a cell by the use of commonly known protein purification techniques employed, including, for example, extraction, precipitation, ion exchange chromatography, chromatography. by affinity, gel filtration, and the like. The compositions can be isolated by affinity chromatography using the extracellular domain of the modified receptor protein linked to a column matrix, or by heparin chromatography. In the screening method of the invention, combination chemistry methods are also included to identify the chemical compounds that bind to the receptor Growth differentiation factor, as described above. Accordingly, the screening method is also useful for identifying variants, binding or blocking agents, etc., which functionally, if not physically (e.g., spherically) act as antagonists or agonists, as desired. The following examples are intended to illustrate, but not limit, the invention. EXAMPLE 1 Myostatin Acts in a Way Dependent on Dosage This example demonstrates that the activity of myostatin to inhibit muscle growth depends on the level of myostatin expression in vivo. Myostatin is a negative regulator of skeletal muscle mass (McPherron et al., Supra, 1997, McPherron and Lee, supra, 1997). The mice with myostatin elimination that were homozygous for a suppression of the myostatin gene, had an increase of 25 to 30 percent in the total body mass. An examination of the mice with homozygous clearance revealed that the increased muscle mass was due to an increase of approximately 100 to 200 percent in the skeletal muscle mass throughout the body. Mice that were heterozygous for the myostatin mutation, also had an increase in total body mass. However, the increase in mass of the heterozygotes was less than that of the homozygotes, and it was statistically significant only in one age group and sex among the many examined. In order to determine whether the heterozygous mice have an intermediate phenotype between that of the wild-type mice and the homozygous myostatin-knockout mice, the analysis of muscle weights was extended to the heterozygous mice. The individual muscles sampled from the heterozygous mice weighed approximately 25 to 50 percent more than those from the wild-type mice. These results demonstrate that mice that are heterozygous for the suppression of a myostatin gene, have a phenotype that is intermediate between that of the wild type mice and that of the homozygous myostatin knockout mice, and show that myostatin produces a dose-dependent effect in vivo. These results indicate that manipulation of myostatin activity may be useful in the treatment of muscle wasting diseases and other metabolic disorders associated with myostatin activity. In addition, the dose dependent effect of myostatin indicates that a therapeutic effect can be obtained without achieving complete inhibition of myostatin activity, thereby allowing adjustment of myostatin activity, for example, if a certain level of activity produces undesirable effects in a subject. Example 2 THE EFFECT OF MIOSTATIN REDUCES AGE IN MICE WITH GENETIC REMOVAL This example demonstrates that a reduced difference in body weight between wild type mice and mice with homozygous myostatin removal is associated with a decline in weight muscle of the mutant mice. The mice with myostatin clearance weighed approximately 25 to 30 percent more than the wild-type mice at five months of age (McPherron et al., Supra, 1997). However, this difference in total body weights became significantly smaller or completely disappeared as the animals aged. In order to determine whether this effect was due to a relative weight loss in the mice with genetic deletion due, for example, to muscle degeneration, or a relatively greater weight gain of the wild-type mice, a detailed analysis was made of muscle weights as a function of age. In all ages examined from two months to 17 months, the pectoralis muscle weighed significantly more in homozygous mutant mice than in wild-type litters. The most dramatic difference was observed at 5 months, at which time, the weight of the pectoralis was approximately 200 percent greater in the mutant mice. Although the weight of pectoralis declined slightly at older ages, the weight of this muscle in the mutant mice was still more than twice that of the wild-type mice. This same basic trend was observed in all other muscles examined, including triceps brachii, quacrceps, gastrocnemius and plantaris, and tibialis anteior. Similar trends were observed in both male and female mice. These results demonstrate that the reduced difference in total body weights between the mutant and wild-type mice observed with aging is due to a slight decline in muscle weights in the mutant mice. Example 3 MIOSTATIN AFFECTS FAT ACCUMULATION IN A WAY DOSE DEPENDENT This example demonstrates that mice with myostatin elimination fail to accumulate fat, and that the reduction in fat accumulation is associated with the level of myostatin expression in vivo. Because the decline in muscle weights in the myostatin mutants, as demonstrated in Example 2, did not fully account for the observation that the wild type animals eventually weighed approximately the same as the mutant mice, the amount of accumulation of fat in wild-type and mutant mice. Inguinal, epididymal, and retroperitoneal fat pads were examined in male mice. There was no difference in the weights of any of these fat cushions between the wild-type and mutant mice at two months of age. For 5 to 6 months of age, the wild type and heterozygous elimination mice both exhibited a large weight range of the fat pad, and on average, the fat pad weights increased by approximately 3 times to 5 times for the time when the animals reached 9 to 10 months of age. Due to the large weight range of the fat cushions observed in these animals, some animals showed a much greater increase (approximately 10 times) than others. In contrast to the wild-type and heterozygously deleted mice, the weights of the fat cushions of the homozygous mutant mice with myostatin were in a relatively narrow range, and were virtually identical in the two-month-old mice and in the mice. from 9 to 10 months of age. Accordingly, the increase in fat accumulation that occurred with aging in the wild-type mice was not observed in mice with homozygous myostatin clearance. This difference in fat accumulation, together with the slight decline in muscle weights, as a function of age in the homozygous mutant mice, was fully accounted for by the observation that the wild-type animals eventually had the same total body weight as the mutants. The weights of the average fat co ines of the mice groups of mice had a normal response in a glucose tolerance test. The results demonstrate that homozygous myostatin knockout mice can maintain normal serum glucose levels, even when their serum insulin level is lower than that of the wild-type animals. Table 1. PARAMETERS IN SERUM + / + indicates the wild-type mice; - / - indicates the mice with homozygous elimination. In order to determine whether differences in metabolic rates could explain the lack of fat accumulation in the mutant mice, the oxygen consumption rate of the wild-type and mutant mice was compared using a calorimeter. The mutant mice had a lower basal metabolic rate and a lower overall metabolic rate than the wild-type control mice. These results indicate that the lack of fat accumulation in myostatin mutant mice is not due to a higher speed of metabolic activity. with heterozygous elimination at 9 to 10 months of age, they were intermediate between those of wild-type mice and homozygous mutant mice. Although this difference was not statistically significant, due to the wide weight range of the fat pads in these and in the wild-type mice, however, these results indicate that the myostatin has a dose-dependent effect on the accumulation of fat, similar to its effect on muscle growth. Example 4 EFFECT OF. THE MYTHATATIN ON METABOLISM This example demonstrates that insulin and glucose levels in serum, as well as metabolic activity, are affected by the level of myostatin expression. In order to determine whether skeletal muscle hypertrophy and lack of fat accumulation in myostatin mutant mice is due to an effect on the overall metabolism, the metabolic profile of the mutant mice was examined. The levels of serum triglycerides and serum cholesterol were significantly lower in the myostatin mutant mice, compared to the wild-type control mice (Table 1). Serum insulin levels also appeared to be lower in myostatin mutant mice. However, fasting and fasting glucose levels were both indistinguishable between the homozygous mutant mice and the wild-type mice (Table 1), and both Example 5 MIOSTATIN AFFECTS FAT ACCUMULATION IN GENETICALLY OBESE MICE This example demonstrates that a lack of myostatin expression suppresses fat accumulation in mice that are a genetic model for obesity. In order to determine whether the loss of myostatin activity could suppress fat accumulation, not only in normal mice but also in obese mice, the effect of the myostatin mutation on lethal yellow mice agouti (Ay), which they represent a genetic model of obesity (Yen et al., FASEB J. 8: 479-488, 1994). Mice were generated that were doubly heterozygous for the lethal yellow and myostatin mutations, and the progeny of the crosses of these doubly heterozygous mice were examined. The total body weight of the Ay / a mice, myostatin - / - double mutant, was dramatically reduced (approximately 9 g), compared with that of the mouse and / or, myostatin + / +. This reduction in total body weight was even more dramatic, considering that the Ay / a double mutant, myostatin - / -, had approximately 2 to 3 times more skeletal muscle than the Ay / a mouse, myostatin + / +. The double mutant had approximately 10 g more muscle than the mouse Ay / a, myostatin + / +, and consequently, the reduction of total weight in the rest of the tissues It was approximately 19 g. The reduction in total body weight resulted from a reduction in the overall fat content. As shown in Table 2, the weights of the parametrial and retroperitoneal fat cushions were reduced from 5 times to 6 times in the double mutant Ay / a, myostatin - / -, compared with the mouse Ay / a, myostatin + / +. These results indicate that the presence of the myostatin mutation dramatically suppresses fat accumulation in obesity. The presence of the myostatin mutation also dramatically affected glucose metabolism. Agouti lethal mice lacking the myostatin mutation, had very abnormal glucose tolerance test results, frequently reaching serum glucose levels of 450 to 600 mg / dl, and recovering only slowly up to the levels of the baseline during a period of 4 hours. Female agouti lethal mice were less affected than male mice, and some females responded almost normally in this test, as described previously (see Yen et al., Supra, 1994). In contrast, although the Ay / a mice, myostatin - / - had slightly abnormal glucose tolerance tests, none of these animals had the large abnormalities observed in the Ay / a mice and myostatin + / + mice. These results indicate that the mutation of myostatin suppressed at least partially the development of metabolism abnormal glucose in lethal agouti mice. Significantly, mice that were heterozygous for the myostatin mutation, had an intermediate response, comparing with myostatin + / + and myostatin - / - mice, thus confirming the dose-dependent effect of myostatin . Example 6 PURIFICATION OF RECOMBINANT MIOSTATIN This example provides a method for preparing and isolating recombinant myostatin. In order to elucidate the biological activity of myostatin, large quantities of myostatin protein were purified for the bioassays. Stable Chinese hamster ovary (CHO) cell lines producing high levels of myostatin protein were generated by co-amplifying a myostatin expression cassette with a dihydrofolate reductase cassette, using a methotre-xato selection scheme (McPherron et al., Supra, 1997). Myostatin was purified from the conditioned medium of the highest production line by successive fractionation on hydroxyapatite, lentil-lentin SEPHAROSE, DEAE agarose, and SEPHAROSE heparin. The analysis of silver staining revealed that the purified protein obtained after these four steps of column chromatography (referred to as "heparin eluate") consisted of two species with approximate molecular masses. kilodáltones (kDa) and 12 kilodáltones. The purified protein preparation was determined by different criteria to represent a two-peptide complex of the myostatin prodomain and a disulfide-linked dimer of the mature C-terminal myostatin peptides. First, Western blot analysis, using anti-bodies reproduced against specific portions of the promyostatin sequence, identified the 35-kb band as the prodomain, and the 12-kb band as the mature C-terminal peptide. Second, under non-reducing conditions, the species that reacted with the anti-bodies directed against the mature C-terminal peptide, had an electrophoretic mobility consistent with a disulfide-linked dimer. Third, the molar ratio of the prodomain to the mature C-terminal peptide was about 1: 1. Fourth, the prodomain and the mature C-terminal peptide were co-purified through the four steps of column chromatography. Finally, the mature C-terminal peptide was bound to the lentil-lectin column, even when the C-terminal region does not contain N-linked glycosylation signals in consensus, indicating that the mature C-terminal peptide was bound to the column due to its interaction with the prodomain peptide, which contains a potential N-linked glycosylation site. These results indicate that myostatin produced by genetically modified CHO cells is secreted in a Proteolytically processed form, and that the resulting prodomain and the mature C-terminal region are associated in a non-covalent manner to form a complex containing two prodomain peptides and a disulfide-linked dimer of the C-terminal proteolytic fragments, in a manner similar to that described for TGF-β. In the TGF-β complex, the C-terminal dimer exists in an inactive latent form (Miyazono-et al., J. Biol. Chem. 263: 6407-6415, 1988), and the active species can be released from this complex latent by its treatment with acid, chaotropic agents, reactive oxygen species, or plasmin, or by interactions with other proteins, including thrombospondin and αβ6 integrin (Lawrence et al, Biochem. Biophys. Res. Comm. 133: 1026-1034 , 1985, Lyons et al, J. Cell Biol. 106: 1659-1665, 1988, Schultz-Cherry and Murphy-Ullrich, J. Cell Biol. 122: 923-932, 1993, Barcellos-Hoff and Dix, Mol. Endocrinol. 10: 1077-1083, 1996; Munger et al., Cell 96: 319-328, 1999). In addition, the addition of the purified prodomain peptide (also known as the peptide associated with latency or LAP) to the TGF-β complex, inhibits the biological activity of the purified C-terminal dimer in vitro and in vivo (Gentry and Nash, Biochemistry 29 : 6851-6857, 1990; Bottinger et al., Proc. Nati, Acad. Sci., USA 93: 5877-5882, 1996). The heparin eluate, which consisted of a prodomain complex and the mature C-terminal peptide, was further purified using a C4 HPLC reverse phase column. The dimer C-terminal was eluted from the HPLC column before the prodomain, thus allowing the isolation of the C-terminal dimer to be released from the prodomain. Fractions containing mostly prodomain were also obtained, although these fractions contained small amounts of the C-terminal dimer. Some of the protein was also present as higher molecular weight complexes. The nature of the higher molecular weight complexes is unknown, but, based on Western blot analysis in the presence or absence of reducing agents, these complexes may contain at least one prodomain peptide and a mature myostatin peptide C- terminal linked by one or more disulfide bonds. In fact, most of the mature C-terminal peptide present in the HPLC fraction enriched for the propeptide (HPLC fractions 35-37) was present in these high molecular weight complexes. These higher molecular weight complexes possibly represent inappropriately folded proteins that are secreted by genetically modified CHO cells. Example 7 MIOSTATINES INTERACT SPECIFICALLY WITH A RECEIVER ACTIVINE This example demonstrates that myostatin binds specifically to an activin type II receptor expressed in cultured cells, and that this specific binding is inhibited by a prodrug of myostatin.

The receptors have been identified for some members of the TGF-β family, and the majority are individual membrane extension serine / threonine kinases (Massague and Weis-Garcia, Cancer Surveys 27: 41-64, 1996). For example, it is known that activin type II receptors (Act RIIA and / or Act RIIB) bind to the members of the TGF-β superfamily. The phenotype of mice lacking the Act RIIB receptor showed anterior / posterior axial pattern defects and renal abnormalities that were very similar to those seen in mice with GDF-11 clearance (McPherron et al., Nat. Genet, 22: 260 -264, 1999; Oh and Li, Genes Devel., 11: 1812-1826, 1997). Because the amino acid sequences of GDF-11 and myostatin (GDF-8) are 90 percent identical in the mature C-terminal region, the ability of myostatin to interact specifically with an activin type II receptor was examined. Myostatin was labeled by radioiodination, and binding studies were performed using transfected COS cells with an expression construct of Act RIIB. Myostatin interacted specifically with transfected COS cells. The myostatin binding was competed in a dose-dependent manner by the excess of unlabeled myostatin, and was significantly lower in the control COS cells, which were transfected with an empty vector. No significant binding was present with the cells transfected with an expression construct of BMP RII or TGF-βRII. The myostatin binding with the cells transfected with Act RIIB was saturable, and the binding affinity was about 5 nM, determined by Scatchard analysis. The receptor binding assay was also used to examine the ability of the myostatin prodomain to inhibit the ability of the mature C-terminal dimer to interact specifically with Act RIIB in this system. The addition of the purified prodomain peptide blocked the ability of the C-terminal dimer to bind to COS cells transfected with Act RIIB in a dose-dependent manner. These results indicate that the myostatin prodomain is a natural inhibitor of myostatin. EXAMPLE 8 INCREASED LEVELS OF MIOSTATIN INDUCE LOSS OF PE50 This example demonstrates that high levels of myostatin can lead to substantial weight loss in vivo. In a set of experiments, CHO cells expressing myostatin were injected into hairless mice. Hairless mice that had tumors of CHO cells expressing myostatin showed a severe waste during the course of approximately 12 to 16 days following the injection of the cells. This waste syndrome was not observed in the hairless mice injected with any of a variety of control CHO lines that had undergone a selection process similar but that did not express myostatin. In addition, the myostatin coding sequence in the construct used to transfect the CHO cells was under the control of a metallothionein promoter, and the waste syndrome was exacerbated when the mice that had the tumors expressing myostatin were maintained with water containing sulfate of zinc. Western blot analysis revealed high levels of myostatin protein in the serum of hairless mice that had CHO cells expressing myostatin. These results indicate that the waste syndrome was induced in response to the high level of myostatin in the hairless mice, and as described below, this result was confirmed by the observation of similar effects in the mice injected with purified myostatin. The dramatic weight loss observed in hairless mice that had CHO cells expressing myostatin was primarily due to a disproportionate loss of both fat and muscle weight. The weights of the white fat cushions (white intrascapular fat, uterine, and retroperitoneal) were reduced by more than 90 percent, compared to mice that had control CHO cell tumors. The muscle weights were also severely reduced, weighing the individual muscles about half as much as those of the mice expressing myostatin, as well as the control mice, by day 16. This loss in muscle weight was. reflected by a corresponding reduction in fiber sizes and protein content. Mice that had tumors of CHO cells that expressed myostatin, also became severely hypoglycemic. However, the weight loss and hypoglycemia were not due to a difference in feed intake, because all the mice consumed equivalent amounts of food in each time interval examined during the course of 16 days of the study. These results indicate that overexpression of myostatin induces a dramatic loss of weight, which resembles the cachectic waste syndrome that occurs in patients suffering from chronic diseases such as cancer or AIDS. With more chronic administrations using lower doses of myostatin, changes in fat weight were observed. For example, twice daily injections of 1 microgram of protein myostatin for 7 days resulted in a reduction of approximately 50 percent in the weights of a number of different white fat cushions (white, uterine, intrascapular fat cushions). , and retroperitoneal), without a significant effect on brown fat (intrascapular coffee). These results confirm that myostatin can induce weight loss, and in extreme cases, an in vivo waste syndrome. Example 9 CHARACTERIZATION OF THE MIOSTATIN LINK WITH A RECEIVER ACTIVINE This example describes a method to characterize the relationship of myostatin binding to an activin receptor, with the biological effects produced by myostatin in vivo. Mice with clearance of Act RIIA or Act RIIB can be used to confirm that Act RIIA or Act RIIB is a receptor for myostatin in vivo. A detailed muscle analysis of these mice can determine whether the removal of an activin receptor is associated with a change in the number or size of muscle fibers. Because the homozygous double mutant Act RIIA / Act RIIB dies early during embryogenesis (Song et al., Devel. Biol. 213: 157-169, 1999), only the different homozygous / heterozygous combinations can be examined. However, mice can be generated with tissue-specific or conditional elimination, in such a way that both genes can be "suppressed" only in the muscle tissue, thus allowing the post-natal examination of the mice with homozygous double elimination. The effect on the adipose tissue can be examined with the aging of the mice to determine whether the number of adipocytes or the accumulation of lipid by these adipocytes in the elimination mice is altered. The number and size of adipocytes are determined by preparing cell suspensions from tissue treated with collagenase (Rodbell, J. Biol. Chem. 239: 375-380, 1964, Hirsc? A and Gallian, J. Lipid Res. 9: 110-119 , 1968). The total lipid content in the animals is determined by measuring the dry carcass weights, and then the residual weights of the dried carcass after lipid extraction (Folch et al., J. Biol. Chem. 226: 497-509, 1957). A variety of serum parameters can also be examined, including glucose and insulin with food and fasting, triglycerides, cholesterol, and leptin. As disclosed above, serum triglycerides and serum insulin are reduced in mutant myostatin animals. The ability of mice with clearance of the activin receptor to respond to an exogenous glucose load using glucose tolerance tests can also be examined. As disclosed above, the response to a glucose load was essentially identical in the wild-type mice and in the myostatin mutants at five months of age. This observation can be extended by measuring these parameters in the mice as they age. You can also measure serum insulin levels at different times during glucose tolerance tests. Basal metabolic rates can also be monitored using a calorimeter (Columbus Instruments). As disclosed above, myostatin mutant mice have a lower metabolic rate at three months of age than their wild-type counterparts. This analysis can be extended to older mice, and can also be measured the respiratory quotient in these animals. The ability to maintain normal thermogenesis can be determined by measuring the basal body temperatures, as well as its ability to maintain body temperature when placed at 4 ° C. The brown fat weights and the levels of expression of UCP1, UCP2, and UCP3 can also be examined in brown fat, white fat, muscle, and other tissues (Schrauwen et al., 1999). The ingestion of food can be monitored in relation to the weight gain, and the efficiency of the food can be calculated. In addition, the weight gain of animals placed on high fat diets can be monitored. Wild-type mice maintained on a high-fat diet accumulate fat rapidly, whereas the results disclosed herein indicate that mutant activin receptor animals will remain relatively thin. The results of these studies can provide a more complete profile of the effect of myostatin in mice, in particular with respect to their overall metabolic status, thus providing an overview on whether the ability of mice with myostatin removal to suppress Fat accumulation is an anabolic effect of the myostatin mutation in the muscle that leads to a change in energy utilization, so that little energy is available to be stored in the form of fat. For example, reduced fat accumulation may be due to a higher thermogenesis index. These results will also provide a baseline for comparing the effect of myostatin activity in the context of different genetic models of obesity and type II diabetes. Example 10 CHARACTERIZATION OF THE EFFECTS OF. THE MIOSTATIN IN GENETIC MODELS OF OBESITY AND DIABETES TYPE II This example describes the methods to determine the effect of myostatin in the treatment of obesity or type II diabetes. The dramatic reduction in overall fat accumulation in myostatin mutant mice, compared to wild-type mice, indicates that the activity of myostatin can be manipulated to treat or prevent obesity or type II diabetes. The effect of the myostatin mutation can be examined in the context of several well-characterized mouse models of these metabolic diseases, including, for example, "obese" (ob / ob) mice, "diabetic" (db / db) mice, and lethal yellow mutant strains agouti (AY). Each of these strains is abnormal for virtually every parameter and test described above (see, for example, Yen et al., Supra, 1994, Friedman and Halaas, Nature 395: 763-770, 1998). You can examine the ability of the myostatin mutation to slow down or suppress the development of these abnormalities in the mice carrying these other mutations, by constructing doubly mutant, then subjecting the mutant double animals, together with the appropriate control baits carrying only the ob / ob mutations, db / db, or lethal yellow agouti, to the different tests. As disclosed above, the mutation of myostatin in the A mice was associated with an approximately five-fold suppression of fat accumulation in myostatin mutant A mice, and with a partial suppression of the abnormal glucose metabolism development, as assessed by glucose tolerance tests. These results can be extended to include additional animals of different ages, and similar studies can be carried out with the ob / ob and db / db mutants. Because both mutations are recessive, mice can be generated that are doubly homozygous for the myostatin mutation, and either the ob or db mutation. In order to examine the effects of partial loss of myostatin function in these genetic model systems, mice that are homozygous for the ob or db mutation and heterozygous for the myostatin mutation are also examined. Mice that are doubly heterozygous for myostatin and ob mutations have been generated, and the progeny of pairs of these doubly heterozygous mice can be examined, in particular with respect to the accumulation of fat and glucose metabolism. The partial suppression of either or both abnormalities in obese mutants may indicate that myostatin is a target for the treatment of obesity and type II diabetes. EXAMPLE 11 CHARACTERIZATION OF TRANSGENIC MICE EXPRESSING NEGATIVE NEGATIVE POLYPEPTIDES THAT MAY AFFECT MIOSTATIN ACTIVITY This example describes methods for characterizing the effect of myostatin post-natally, by expression of dominant negative polypeptides that can block the expression of myostatin or the transduction of the myostatin signal. Myostatin Inhibitors Post-natal modulation of myostatin activity can be used to determine the effect of myostatin on the number of muscle fibers (hyperplasia) and the size of muscle fibers (hypertrophy). For these studies, mice with conditional myostatin elimination may be used, in which the myostatin gene is deleted at defined times during the life of the animal. The PET regulator in combination with the recombinase ere provides a system for generating these mice. In this system, erectile expression is induced by the administration of doxycycline. Transgenic mice can also be generated expressing a myostatin inhibitor from an inducible promoter, such that myostatin activity can be reduced or inhibited at defined times during the life of the animal. The tetracycline regulators are useful for generating these transgenic mice, in which the expression of myostatin is induced by doxycycline. A modification of the test system, which uses the co-expression of a hybrid inverse transactivator-tet (fusion protein of the activation domain of VP16 with the mutant inverse tet repressor) and a hybrid trans-repressor-tet (repressor domain fusion protein) KRAB of mammalian Koxl with the native tet repressor) may be particularly useful for producing the transgenic mice (Rossi et al., Nat. Genet 20: 389-393, 1998, Forster et al., Nucí Acids Res. 27: 708 -710, 1999). In this system, the hybrid inverse transactivator-tet is linked to tet operator sequences, and activates transcription only in the presence of tetracycline; the hybrid trans-repressor-tet is linked to the tet operator sequences, and represses transcription only in the absence of tetracycline. By coexpression of these two fusion proteins, the basal activity of the target promoter is silenced by the trans-repressor-tet in the absence of tetracycline, and is activated by the reverse transactivator-tet on the administration of tetracycline. Two types of transgenic lines can be generated. In the first type, the transgene encodes a myostatin inhibitor polypeptide under the control of a muscle-specific promoter, eg, the muscle creatine kinase promoter (Stern-berg et al., supra, 1988), or the enhancer / promoter of myosin light chain (Donoghue et al., supra, 1991.) Individual transgenic lines are screened for the specific expression of tet regulators in skeletal muscle, and several independent lines are selected and examined for each of the two promoters , in order to confirm that any effects observed are not due, for example, to the specific effects of the integration site A construction containing both tet regulators has been constructed under the control of the myosin light chain promoter / enhancer, and can be used for pronuclear injections In the second type of line, the transgene contains a myostatin inhibitor polypeptide under the control of a minimal CMV promoter that also contains tet operator sequences. The myostatin inhibitor can be a dominant negative form of myostatin, or a prodrug of myostatin which, as disclosed herein, can inhibit myostatin activity. Dominant negative forms of members of the TGF-β family have been described (see, for example, Lopez et al., Mol Cell Biol. 12: 1674-1679, 1992; Wittbrodt and Rosa, Genes Devel. 8: 1448-1462 , 1994), and contain, for example, a mutant proteolytic cleavage site, preventing this so that the protein is processed in biologically active species. When co-expressed in a cell with the endogenous wild-type gene, the mutant protein forms non-functional heterodimers with the wild type protein, thus acting as a dominant negative. A mutant raiostatin polypeptide containing a mutation at the promiostatin dissociation site has been constructed and can be examined for a dominant negative effect by co-expressing the mutant with wild-type myostatin in varying proportions in 293 cells. The conditioned medium can be examined from 293 cells transiently transfected with the constructs by Western blot analysis, and the ability of the mutant to block the formation of mature C-terminal dimers can be examined. An expression construct encoding only the myostatin prodomain can also be used. As disclosed above, the prodomain forms a tight complex with the mature C-terminal dimer, and blocks the ability of the mature C-terminal myostatin dimer to bind to Act RIIB in the cells expressing the recipient in culture. By analogy to TGF-β, the prodrug of myostatin can also maintain the mature C-terminal dimer in an inactive latent complex in vivo. These transgenic animals can be bred with those that express the tet regulators to generate lines dually transgenic that contain both the tet regulators and the objective inhibitory construction. These double transgenic lines can be traced to determine those in which all the different components are properly expressed. Northern blot analysis can be used using the RNA obtained from different muscles and control tissues of representative mice in each line, before and after the administration of doxycycline in drinking water, in order to identify these transgenic lines. Transgenic lines that do not express the transgene in any tissue in the absence of doxycycline, and that express the transgene only in the muscle in the presence of doxycycline, will be selected. Doxycycline is administered to the selected transgenic animals, and the effect on muscle mass is examined. Doxycycline can be administered to pregnant mothers to induce inhibitor expression during embryogenesis. The effect of the blocking of myostatin activity during the development of the transgenic animals can be compared with the effects observed in the mice with myostatin elimination. Because the promoters to boost the expression of tet regulators can be induced at a later time during development, other than the time in which myostatin is first expressed, the effect on muscle mass in mice can be compared transgenic with the effect that occurs in mice with myostatin elimination.

The effect of inhibiting myostatin activity post-natally can be examined by administering doxycycline to dually transgenic mice at different times after birth. The treatment with doxycycline can begin, for example, at three weeks of age, and the animals can be analyzed at five months of age, which is the age at which the difference in muscle weights in the Myostatin elimination against wild-type mice. The animals are examined to determine the effects of the inhibitor on muscle mass. The muscles can also be examined histologically to determine the effects on the number of fibers and the size of the fibers. In addition, an analysis of fiber types of different muscles in the transgenic mice can be carried out to determine if there is a selective effect on the type I or type II fibers. Doxycycline can be administered in different doses and at different times to characterize the effect of myostatin inhibitors. You can also keep dually transgenic mice chronically with doxycycline, and then examined to determine the effects on the weights of the fat pads and other relevant metabolic parameters, as described above. The results of these studies can confirm that modulation of myostatin activity post-natally, 1 can increase muscle mass or reduce the accumulation of fat, thus indicating that the direction of myostatin may be useful for the treatment of a variety of muscular and metabolic waste diseases clinically. Myostatin Transgenic mice containing a myostatin transgene can also be examined, and the effects produced on the expression of myostatin can be compared with those observed in hairless mice containing CHO cells expressing myostatin. In a manner similar to that described above, myostatin can be placed under the control of conditional (tet) and tissue-specific regulatory elements, and the expression of myostatin in the transgenic mice can be examined to determine whether a syndrome of waste similar to that observed in hairless mice. The myostatin transgene can include, for example, processing signals derived from SV40, such that the transgene of the endogenous myostatin gene can be distinguished. Serum samples of the myostatin transgenic mice can be isolated at different times following the administration of doxycycline, and the level of the transgenic myostatin product in the serum can be determined. The total body weights of the animals are monitored over time to determine if the animals show significant weight loss. In addition, the individual muscles and fat pads are isolated and weighed, and the number, size, and type of muscle fibers in selected muscle samples. The level of expression of the myostatin transgene can be varied by varying the dose of doxycycline administered to the animals. The expression of the transgene can be monitored using, for example, Northern blot analysis of muscle transgene RNA levels, or serum myostatin protein levels. The identification of specific expression levels of the myostatin transgene allows a correlation of the degree of waste induced by myostatin. The transgenic lines can also be crossed with mice with myostatin elimination to generate mice in which the only source of myostatin is expression from the transgene. The expression of myostatin can be examined at different times during development, and the effect of myostatin on the number of fibers, the size of the fibers, and the type of fibers can be determined. The availability of mice in which the expression of myostatin can be controlled in a precise and rapid manner, provides a powerful tool to further characterize the myostatin signal transduction pathway, and to examine the effects of different agents that can potentially be useful for modulating myostatin signal transduction. Effects of myostatin signal transduction can generate transgenic mice containing dominant negative forms of a pathway of myostatin signal transduction, which may include components of a TGF-β signal transduction pathway, which is expressed specifically in skeletal muscle. As disclosed herein, Smad proteins, which mediate signal transduction through a pathway induced by type II activin receptors, may be involved in the transduction of the myostatin signal. Act RIIB can be linked to GDF-11, which is highly related to myostatin (McPherron et al, supra, 1997, Gamer et al, supra, 1999, Nakashima et al, Mech.Devel.80: 185-189, 1999) , and the expression of c-ski, which can be linked to a Smad 2 inhibitor, Smad 3, and Smad 4, dramatically affects muscle growth (Sutrave et al., supra, 1990; Berk et al., supra, 1997; see also Luo and collaborators, supra, 1999, Stroschein et al, supra, 1999, Sun et al, supra, 1999a and b, Akiyoshi et al., supra, 1999). As disclosed herein, myostatin interacts specifically with the Act RIIB, and therefore, may exert its biological effect, at least in part, by binding to type II activin receptors in vivo, and activating the signaling path Smad. The role of the Smad signaling pathway in the regulation of muscle growth can be examined using lines of transgenic mice that are blocked, or that can be blocked, at specific points in the signal transduction path Smad / Act RIIB. The muscle creatine kinase promoter or the myosin light chain enhancer / promoter can be used to drive the expression of different inhibitors of the Smad signal transduction pathway. A useful inhibitor in this system may include, for example, follistatin; a dominant negative RIIB Act receptor; a dominant Smad negative polypeptide, such as Smad 3; c-ski; or a Smad inhibitor polypeptide, such as Smad 7. Follistatin can bind and inhibit the activity of certain members of the TGF-β family, including GDF-11 (Gamer et al., supra, 1999). Dominant negative forms of a type II activin receptor can be obtained by expression of a truncated growth differentiation factor receptor, for example, by expression of the extracellular domain, in particular a soluble form of an extracellular domain of Act RIIB, or by expression of a truncated Act RIIB receptor that lacks the kinase domain, or that contains a mutation such that the mutant receptor lacks kinase activity. Smad 7 functions as a Smad inhibitor that can block the signaling pathway induced by activin, TGF-β and BMP. For example, a dominant negative form of Smad 3 can be constructed by mutation of the C-terminal phosphorylation sites of Smad 3, thereby blocking the function of Smad 3 (Liu et al., Supra, 1997). The expression of c-ski has been correlated with muscle hypertrophy in transgenic mice (Sutrave et al., Supra, 1990). Transgenic mice can be prepared, and each founder line can be examined to determine the specific expression of the appropriate muscle of the transgene. The selected mice are examined to determine the total body weights, the weights of the individual muscles, and the sizes, numbers and types of muscle fibers. Lines demonstrating a clear effect on muscle mass can be further examined to determine fat accumulation and other relevant metabolic parameters, as described above. The use of these different agents to direct specific steps in the signal transduction pathway Smad / activin receptor, is particularly informative, because the signaling pathways for the different agents overlap in different steps. For example, follistatin binds to and inhibits the activity of activin and GDF-11, but not of TGF-β, whereas a dominant Smad 3 negative can block signaling through both activin receptors and of TGF-β. Smad 7 may have even more pleotropic, because it blocks the signaling through the BMP receptors as well. The studies can allow the identification of specific objectives to modulate the activity of myostatin, thus providing different strategies for develop drugs or other agents that modulate the myostatin signal transduction, and consequently, the activity of myostatin. In particular, the transgene lines described herein can be used to determine the effect of blocking the myostatin function or the Smad signaling pathway post-natally on the development of obesity or type II diabetes. For example, inhibitory transgenes can be crossed in the mutant mice ob / ob, db / db, and Ay. In the absence of doxycycline, an inhibitory transgene is not expressed, and therefore, the animals are indistinguishable from each of the parent mutant mice. In the presence of doxycycline, the inhibitor is expressed, and may block myostatin activity. The effect of blocking myostatin activity on the development of metabolic abnormalities in these mutant animals can be examined. The expression of the inhibitor can be induced at an early age, for example, at 3 weeks of age, to maximize the effect. In addition, myostatin activity can be blocked before the time when the metabolic abnormalities become so severe that they are irreversible. The animals can be maintained with doxycycline, and can be evaluated at different ages using the tests described above, including those related to fat accumulation and glucose metabolism. Any delay in age can be identified to which one or more test results become abnormal in the mutant animals ob / ob, db / db, and Ay. Similar studies can be carried out using older animals, which have developed some of the signs of obesity or type II diabetes, and the effect of blocking myostatin activity on different parameters, including fat weight and metabolism can be determined of glucose. The results of these studies can also identify specific objectives that can be manipulated in an effort to prevent or treat obesity or type II diabetes. Example 12 CHARACTERIZATION OF THE EFFECT OF THE MIOSTATIN ON THE INDUCTION OF CAQUEXIA This example describes methods to determine the role of myostatin signal transduction in the development and progression of cachexia. The activin receptor and the Smad pathway may constitute at least part of the signal transduction pathway involved in the mediation of myostatin activity in normal individuals, and therefore, may be involved in mediating the effects that occur in a individual due to excessive levels of myostatin. As disclosed herein, cachexia, for example, can be mediated, at least in part, by abnormally high levels of myostatin. As such, the methods to manipulate transduction Signaling through the Smad pathway can provide a new strategy to develop drugs for the treatment of muscular waste in general and cachexia in particular. The role of the Smad signaling pathway in cachexia can be examined by examining the susceptibility of the different transgenic lines described above to cachexia, which can be induced, for example, with interleukin-6 (IL-6; contributors, Endocrinology 128: 2657-2659, 1991, which is incorporated herein by reference), tumor necrosis factor- (TNF-a, Oliff et al., Cell 50: 555-563, 1987, which is incorporated herein) as a reference), or certain tumor cells. In the case of IL-6 and TNF-a, the inhibitory transgenes can be crossed in a hairless mouse background, and then the animals can be stimulated with CHO cells that produce IL-6 or TNF-α, which induce the waste in hairless mice when overexpressed in this way. CHO cells that overproduce IL-6 or TNF-α can be prepared using the methods described above to generate myostatin overproducing cells. For example, the TNF-α cDNA can be cloned into the expression vector pMSXND (Lee and Nathans, J. Biol. Chem. 263: 3521-3527, 1988), and then the cells bearing the amplified copies of the construct of expression can be selected stepwise in increasing concentrations of methotrexate. For these studies, cells can also be used tumor cells, such as Lewis lung carcinoma cells (Matthys et al., Eur. J. Cancer 27: 182-187, 1991, which is incorporated herein by reference), or colon adenocarcinoma cells 26 (Tanaka et al., J. Cancer Res. 50: 2290-2295, 1990, which is incorporated herein by reference), which can induce cachexia in mice. These cell lines cause severe waste when they grow as tumors in mice. Accordingly, the effect of these tumors on the different transgenic mice described herein can be examined. It is recognized that different tumor cells will only grow in certain genetic backgrounds. For example, Lewis lung carcinoma cells are cultured routinely in C57 BL / 6 mice, and colon carcinoma cells 26 are cultured routinely in BALB / c mice. Therefore, transgenes can be backcrossed in these or other genetic backgrounds to allow the growth of tumor cells. Different parameters can be monitored, including total body weight, individual muscle weight, size and number of muscle fibers, food intake, and serum parameters, including glucose levels. In addition, serum myostatin levels and myostatin RNA levels in muscle can be examined to confirm that the highest expression of myostatin is correlated with cachexia. The results of these studies can confirm that the action of myostatin is downstream from the agents Cachexia inducers in these experimental models. The results can also confirm that the Smad signaling pathway is essential for the development of cachexia in these models, and can demonstrate that a therapeutic benefit can be obtained in the treatment of cachexia by modulation of Smad signaling. Example 13 IDENTIFICATION AND CHARACTERIZATION OF GROWTH DIFFERENTIATION FACTOR RECEIVERS - 8 (GDF-8) AND GDF-11 This example describes methods for identifying and characterizing cell surface receptors for GDF-8 (myostatin) and GDF-11 . The purified GDF-8 and GDF-11 proteins will be used primarily for testing in order to determine biological activity. In order to identify the potential target cells for GDF-8 and GDF-11, the action cells that express their receptors will be searched. For this purpose, the purified protein will be radioiodinated using the chloramine T method, which has been used successfully to mark other members of this superfamily, such as TGF-β (Cheifetz et al., Supra, 1987), activins (Sugino and collaborators, J. Biol. Chem. 263: 15249-15252, 1988), and BMPs (Paralkar et al., Proc. Nati, Acad. Sci., USA 88: 3397-3401, 1991), for receptor binding studies. The mature processed forms of GDF-8 and GDF-11 each contain multiple residues of tyrosine. Two different approaches will be taken to identify the receptors for these proteins. An approach will determine the er, affinity, and distribution of the receptors. Any of the whole cells grown in culture, frozen sections of embryos or adult tissues, or whole membrane fractions prepared from cultured tissues or cells, will be incubated with the labeled protein, and the amount or distribution of the protein will be determined. linked For experiments involving cell lines or membranes, the amount of binding will be determined by measuring either the amount of radioactivity bound to the cells in the dish after several washes, or, in the case of membranes, the amount of radioactivity deposited with the cells. the membranes after centrifugation, or retained with the membranes on a filter. For experiments involving primary cultures, where the er of cells may be more limited, link sites will be directly visualized overlapping with the photographic emulsion. For experiments involving frozen sections, ligand binding sites will be visualized by exposing these sections to high resolution beta-max hyper-film; If a finer location is required, the sections will be immersed in a photographic emulsion. For all these experiments, the specific binding will be determined by adding an excess of unlabeled protein as a competitor (for example, see Lee and Nathans, supra, 1988). A second approach will be to characterize the receptor biochemically. The membrane preparations or potential objective cells grown in culture will be incubated with the labeled ligand and cross-linked. covalent receptor / ligand complexes using disuccinimidyl suberate, which has been commonly used to identify receptors for a variety of ligands, including members of the TGF-β superfamily (Massague and Like, J. Biol. Chem. 260: 2636 -2645, 1985). The crosslinked complexes are separated by electrophoresis on SDS polyacrylamide gels to look for the bands marked in the absence, but not in the presence, of excess unlabelled protein. The molecular weight of the assumed receptor will be estimated by subtracting the molecular weight of the ligand. An important issue that these experiments will solve is whether the signal of GDF-8 and GDF-11 through type I and type II receptors is like many other members of the TGF-β superfamily (Massague and Weis-Garcia, supra, 1996). ). Once a method has been performed to detect receptors for these molecules, a more detailed analysis will be carried out to determine the binding affinities and the specificities. A Scatchard analysis will be used to determine the er of binding sites and the dissociation constants. By conducting cross-competition analysis between GDF-8 and GDF-11, it will be possible to determine if they are capable of link to the same receiver and their relative affinities. These studies will give an indication on whether the molecules signal through the same receptors or through different receptors. Competence experiments will be conducted using other members of the TGF-β family to determine specificity. Some of these ligands will be available commercially, and some others are available from Genetics Institute, Inc. For these experiments, a variety of embryonic and adult tissues and cell lines will be tested. Based on the specific expression of GDF-8 in the skeletal muscle, and the phenotype of the mice with GDF-8 elimination, the initial studies focus on embryonic and adult muscle tissue for membrane preparation and for the studies of Receivers using frozen sections. In addition, the myoblasts will be isolated and cultured from the embryos on different days of gestation, or satellite cells of adult muscles, as described (Vivarelli and Cossu, Devel. Biol. 117: 319-325, 1986; Cossu et al. collaborators, Cell Diff., 9: 357-368, 1980). The link studies on these primary cells will be carried out after several days in culture, and the binding sites will be located by autoradiography, in such a way that the binding sites can be co-localized with different myogenic markers, such as myocin. muscle (Vivarelli et al., J. Cell Biol. 107: 2191-2197, 1988), and the linkage will be correlated with the differentiation state of the cells, such as the formation of multinucleated myotubes. In addition to using primary cells, cell lines will be used to search for receptors. In particular, the initial focus will be on three cell lines, C2C12, L6, and P19. The myoblasts C2C12 and L6 spontaneously differentiate in culture and form myotubes, depending on the particular culture conditions (Yaffe and Saxel, supra, 1977, Yaffe, supra, 1968). P19 embryonic carcinoma cells can be induced to differentiate into different cell types, including skeletal muscle cells, in the presence of dimethyl sulfoxide (Rudnicki and McBurney, Teratocarcinomas and Embryonic Stem Cells: A practical approach (EJ Robertson, IRL Press, Cambridge 1987)). Receptor binding studies on these cell lines will be carried out under different culture conditions and in different stages of differentiation. Although initial studies will focus on muscle cells, other tissues and cell types will be examined for the presence of GDF-8 and GDF-11 receptors. In these binding studies, the homodimer of recombinant human GDF-8 (rhGDF-8) will be used. RhGDF-8 was expressed using CHO cells, and purified to a purity of about 90 percent. RhGDF-8 had the expected molecular weight of 25 kDa at 27 kDa, and after reduction, it was reduced to the 12 kDa monomer. Using GDF-8 labeled with 1-125 in a receptor-ligand binding assay, two myoblast cell lines, L6 and G-8, were linked with GDF-8. The link was specific, because unlabeled GDF-8 competed effectively for the ligand-labeled linkage. The dissociation constant (Kd) was 370 pM, and the L6 myoblasts have a high number (5.00 receptors / cell) of cell surface binding proteins. GDF-11 (BMP-11) is highly homologous (> 90 percent) to GDF-8. The receptor binding studies revealed that GDF-8 and GDF-11 bound to the same binding proteins on the L6 myoblasts. It is important to establish whether or not GDF-8 binds to the known TGF-β receptor. TGF-β did not compete for the GDF-8 binding, indicating that the GDF-8 receptor is distinct from the TGF-β receptor. The GDF-8 receptor was not expressed in all myoblast cell lines, including four myoblast cell lines, C2C12, G7, MLB13MYC cl4, and BC3H1, which do not bind to GDF-8. The gene or genes that encode receptors for GDF-8 and GDF-11 can be obtained. As a first step towards understanding the mechanism by which GDF-8 and GDF-11 exert their biological effects, it is important to clone the genes that encode their receptors. From the previous experiments, it will become clearer if GDF-8 and GDF-11 bind to the same receptor or to different receptors. There will also be considerable information regarding the tissue distribution and cell type of these receptors. Using this information, two different approaches will be taken to clone the receptor genes. The first approach will be to use an expression cloning strategy. In fact, this was the strategy that was originally used by Mathews and Vale (Cell 65: 973-982, 1991), and by Lin et al. (Cell 68: 775-785, 1992) to clone the first activin receptors and TGF-β. Selected RNA with poly-A will be obtained from the tissue or cell type that expresses the highest relative number of high affinity binding sites, and will be used to prepare a cDNA library in the mammalian expression vector pcDNA- 1, which contains a CMV promoter and an SV40 replication origin. The library will be applied, and the cells of each plate will be grouped in a broth, and they will freeze. Aliquots of each group will be grown for the preparation of the DNA. Each individual group will be transiently transfected into COS cells on camera slides, and the transfected cells will be incubated with iodinated GDF-8 or GDF-11. After washing the unbound protein, the ligand binding sites will be visualized by autoradiography. Once a positive group is indicated, the cells of that group will be reapplied at a lower density, and the process will be repeated. Then, the positive groups will be applied, and the individual colonies will be collected in grids, and they will be analyzed again as described (Wong et al., Science 228: 810-815, 1985). Initially, group sizes of 1,500 will be tracked colonies In order to make sure to identify a positive clone in a mixture of this complexity, a control experiment will be carried out using TGF-β and a cloned type II receptor. The coding sequence for the TGF-β type II receptor will be cloned into the vector pcDNA-1, and bacteria transformed with this construct will be mixed with bacteria from our library in different proportions, including 1: 1,500. Then the DNA prepared from this mixture will be transfected, in COS cells, incubated with iodinated TGF-ß, and visualized by autoradiography. If positive signs are observed in a ratio of 1: 1,500, the groups of 1,500 clones will be tracked. Otherwise, smaller group sizes corresponding to the proportions in which the method is sensitive enough to identify a positive signal in the control experiments will be used. A second parallel strategy will also be used to try to clone GDF-8 and GDF-11 receptors, taking advantage of the fact that most of the receptors for members of the TGF-β superfamily that have been identified belong to the family of membrane-extending serine / threonine kinase (Massague and Weis-Garcia, supra, 1996). Because the cytoplasmic domains of these receptors are related in sequence, degenerate polymerase chain reaction probes will be used to clone members of this family of receptors that are expressed in tissues containing sites of link for GDF-8 and GDF-11. In fact, this is the approach that has been used to identify the majority of the members of this family of recipients. The general strategy will be to design degenerate primers corresponding to the conserved regions of the known receptors, use these primers for the polymerase chain reaction on cDNA prepared from the appropriate RNA samples (more possibly from skeletal muscle), subclone the products of the polymerase chain reaction, and finally sequencing the individual subclones. As the sequences are identified, they will be used as hybridization probes to eliminate duplicate clones from the additional analysis. The receptors that are identified will then be tested for their ability to bind to purified GDF-8 and GDF-11. Because this screening will produce only small polymerase chain reaction products, full-length cDNA clones will be obtained for each receptor from the cDNA libraries prepared from the appropriate tissue, inserted into the pcDNA-1 vector, transfected to the COS cells, and the transfected cells tested for their capacity to link to GDF-8 or GDF-11 iodized. Ideally, each receptor that is identified in this screening will be tested to determine its ability to bind to these ligands. However, the number of receptors that are identified may be large, and the isolation of all full-length cDNAs and their proof They may require considerable effort. Almost certainly, some of the receptors that are identified will correspond to known receptors, and for these, the obtaining of clones of full-length cDNAs from other researchers, or the amplification of the coding sequences by polymerase chain reaction based on the published sequences, must be direct. For novel sequences the tissue distribution will be determined by Northern blot analysis, and the highest priority will be directed to the receptors whose expression pattern most closely resembles the distribution of the GDF-8 and / or GDF binding sites. -11, as determined previously. In particular, it is known that these receptors fall into two classes, type I and type II, which can be distinguished based on the sequence, and which are required, both for a complete activity. Certain ligands can not bind to type I receptors in the absence of type II receptors, while others are able to bind to both types of receptor (Massague and Weis-Garcia, supra, 1996). The crosslinking experiments illustrated above should give some indication as to whether both type I and type II receptors are also involved in the signaling of GDF-8 GDF-11. If so, it will be important to clone both receptor subtypes in order to fully understand the way in which GDF-8 and GDF-11 transmit their signals. Because it can not be predicted if the type I receptor is able or not to interact with GDF-8 and GDF-11 in the absence of the type II receptor, first type II receptors will be cloned. Only after at least one type II receptor for these ligands has been identified will an attempt be made to identify type I receptors for GDF-8 and GDF-11. The general strategy will be to co-transfect the type II receptor with each of the type I receptors that are identified in the polymerase chain reaction screening, and then assay the transfected cells by cross-linking. If the type I receptor is part of the receptor complex for GDF-8 or GDF-11, two species of reticulated receptors must be detected in the transfected cells, one corresponding to the type I receptor, and the other corresponding to the type II receptor. The search for GDF-8 and GDF-11 receptors is further complicated by the fact that at least one member of the TGF-β superfamily, i.e., GDNF, is able to signal through a completely different type of receptor complex which involves a GPI-linked component (GDNFR-alpha) and a receptor tyrosine kinase (c-ret, Trupp et al., Nature 381: 785-789, 1996; Durbec et al., Nature 381: 789-793, 1996; Treanor et al, Nature 382: 80-83, 1996; Jing et al., Cell 85: 1113-1124, 1996). Although the DNF gene is the most distantly related member of the TGF-β superfamily, it is certainly possible that other members of the TGF-β family may also signal through a receptor system analogous. If GDF-8 and GDF-11 signal through a similar receptor complex, the expression screening approach should be able to identify at least the GPI-linked component (the GDNFR-alpha was actually identified using a tracing approach). expression) of this complex. In the case of GDNF, similar phenotypes of mice deficient in GDNF and c-ret suggested c-ret as a potential receptor for GDNF. EXAMPLE 14 PREPARATION AND CHARACTERIZATION OF MICE WITH ELIMINATION OF GDF-11 The phenotype of mice with GDF-11 deletion in several respects resembles the phenotype of mice that carry a deletion of a receptor for some members of the TGF-β superfamily, including the activin type IIB receptor (Act RIB). To determine the biological function of GDF-11, the GDF-11 gene was disrupted by homologous targeting in embryonic totipotent cells. A genomic library of murine 129 SvJ in lambda FIXII was prepared according to the instructions provided by Stratagene (La Jolla, California, United States). The structure of the GDF-11 gene was deduced from the restriction mapping and partial sequencing of phage clones isolated from the library. The vectors to prepare the steering construction were kindly provided by Philip Soriano and irk Thomas. To ensure that the resulting mice were null for the GDF-11 function, the entire mature C-terminal region was deleted and replaced by a neo cassette. Rl ES cells were transfected with the targeting construct, selected with ganciclovir (2 μ?) And G418 (250 micrograms / ml), and analyzed by Southern blot analysis. The homologous direction of the GDF-11 gene was observed in 8/155 clones of ES cells doubly resistant to ganciclo-vir / G418. Following the injection of several directed clones into the C57BL / 6J blasts, chimeras were obtained from an ES clone that produced heterozygous pups when they crossed with both females C57BL / 6J and 129 / SvJ. Crosses of the C57BL / 6J / 129SvJ heterozygous Fl heterozygotes produced 49 wild-type (34 percent), 94 heterozygous (66 percent), and no homozygous mutant adult progeny. In a similar manner, there were no adult homozygous null animals in the 129 / SvJ background (32 wild-type animals (36 percent) and 56 heterozygous mutants (64 percent)). To determine the age at which the homozygous mutants were stained, isolated embryo baits were genotyped at different gestation ages from heterozygous females that had crossed with heterozygous males. In all the embryonic stages examined, the homozygous mutant embryos were present at approximately the predicted frequency of 25 percent. Among the newborn mice born hybrids, the different genotypes were also represented in the Mendilian ratio of 1: 2: 1 (34 + / + (28%), 61 +/- (50%), and 28 - / - (23%)). The homozygous mutant mice were born alive and were able to breathe and nourish themselves. However, all homozygous mutants died, within the first 24 hours after birth. The precise cause of death is unknown, but the lethality may have been related to the fact that the kidneys in the homozygous mutants were severely hypoplastic, or were completely absent. The homozygous mutant animals were easily recognizable by their severely shortened or absent tails. To further characterize the defects of the tail in these homozygous mutant animals, their skeletons were examined to determine the degree of alteration of the caudal vertebrae. However, a comparison of wild-type and mutant skeletal preparations of late-stage embryos and newborn mice revealed differences not only in the caudal region of the animals, but in many other regions as well. In almost every case in which differences were observed, the abnormalities appeared to represent homeotic transformations of vertebral segments in which some particular segments seemed to have a typical morphology of earlier segments. These transformations were evident throughout the axial skeleton that extended from the cervical region to the caudal region. Except for the defects seen in the axial skeleton, the rest of the skeleton, such as the skull and limb bones, appeared normal. Previous transformations of the vertebrae in the mutant newborn animals were more readily apparent in the thoracic region, where there was a dramatic increase in the number of thoracic segments (T). All the wild-type mice examined showed the typical pattern of 13 thoracic vertebrae, each with its associated pair of ribs. In contrast, the homozygous mutant mice showed a surprising increase in the number of thoracic vertebrae. All the homozygous mutants examined had 4 to 5 extra pairs of ribs for a total of 17 to 18, although in more than 1/3 of these animals, the 18th rib appeared rudimentary. Therefore, the segments that would normally correspond to the lumbar segments (L) Ll to L4 or L5, appeared to have been transformed into thoracic segments in the mutant animals. Moreover, the transformations within the thoracic region where one thoracic vertebra had a morphological characteristic of another thoracic vertebra were also evident. For example, in wild-type mice, the first seven pairs of ribs were attached to the sternum, and the remaining six were not attached or were free. In the homozygous mutants, there was an increase in the number of both bound and rib free pairs to 10-11 and 7-8, respectively. By Consequently, the thoracic segments T8, T9, TIO, and in some cases even Til, which have all floating ribs in the wild-type animals, were transformed into imitating animals to have a typical characteristic of more anterior thoracic segments, that is, the presence of ribs attached to the sternum. In a manner consistent with this finding, the spinous process of transition and the transitional joint processes normally found in TIO in wild-type animals were found in place in T13 in the homozygous mutants. Additional transformations within the thoracic region were also observed in certain mutant animals. For example, in wild type mice, the ribs derived from IT usually touched the upper part of the sternum. However, in 2/23 hybrids and 2/3 homozygous 129 / SvJ mutant mice examined, T2 appeared to have transformed to have a morphology that resembled that of TI; that is, in these animals, the ribs derived from T2 extended to touch the upper part of the sternum. In these cases, the ribs derived from IT seemed to merge into the second pair of ribs. Finally, in 82 percent of the homozygous mutants, the long spinous process normally present in T2 was changed to the position of T3. In other homozygous mutants, the asymmetric fusion of a pair of vertebroesternales ribs in other thoracic levels was seen. The previous transformations were not restricted to the thoracic region. Most of the previous transformation we observed was at the level of the sixth cervical vertebra (C6). In wild-type mice, C6 is easily identifiable by the presence of two anterior tubers on the ventral side. In several homozygous mutant mice, although one of these two previous tubers was present in C6, the other was present in the C7 position instead. Therefore, in these mice, C7 seemed to have partially transformed to have a morphology that resembled that of C6. Another homozygous mutant had two anterior tubers on C7, but retained one on C6, for a complete transformation of C7 to C6, but a partial transformation of C6 to C5. Transformations of the axial skeleton also extended to the lumbar region. While wild type animals normally have only six lumbar vertebrae, the homozygous mutants had from 8 to 9. At least six of the lumbar vertebrae in the mutants must have been derived from segments that would normally have resulted in sacral vertebrae and caudal, that the data described above suggest that four to five lumbar segments were transformed into the thoracic segments. Accordingly, the homozygous mutant mice had a total of 33-34 presacral vertebrae, compared to 26 presacral vertebrae normally present in the wild-type mice. The most common presacral vertebral patterns were C7 / T18 / L8 and C7 / T18 / L9 for the mutant mice, comparing with C7 / T13 / L6 for the wild-type mice. The presence of additional presacral vertebrae in the mutant animals was obvious, even without a detailed examination of the skeletons, because the position of the hind limbs in relation to the front limbs was subsequently displaced by 7 to 8 segments. Although the sacral and caudal vertebrae were also affected in the homozygous mutant mice, the exact nature of each transformation was not so readily identifiable. In wild-type mice, the sacral segments SI and S2 normally have extensive cross-sectional processes, compared to S3 and S4. In the mutants, there did not appear to be an identifiable SI or S2 vertebra. Instead, the mutant animals had several vertebrae that appeared to have a morphology similar to S3. In addition, the transverse processes of the four sacral vertebrae were usually fused with each other, although in newborns, often only fusions of the first three vertebrae are seen. However, in the homozygous mutants, the transverse processes of the sacral vertebrae were usually not fused. In the most caudal region, all the mutant animals also had severely malformed vertebrae, with extensive cartilage fusions. Although the severity of the fusions made it difficult to count the total number of vertebrae in the caudal region, up to 15 transverse processes were counted in several animals. It could not be determined if these represented sacral or caudal vertebrae in the mutants, because no criteria could be established to distinguish S4 from caudal vertebrae, even in wild-type newborn animals. Regardless of their identities, the total number of vertebrae in this region was significantly reduced from the normal number of approximately 30. Thus, although the mutants had significantly more thoracic and lumbar vertebrae than the wild-type mice, the total number of segments in the mutants, due to the truncation of the tails. Heterozygous mice also showed abnormalities in the axial skeleton, although the phenotype was much lighter than in homozygous mice. The most obvious abnormality in the heterozygous mice was the presence of an additional thoracic segment with a pair of associated ribs. This transformation was present in each heterozygous animal examined, and in each case, the additional pair of ribs was attached to the sternum. Therefore, T8, whose associated rib usually does not touch the sternum, appeared to have morphed to a characteristic morphology of a more anterior thoracic vertebra, and Ll appeared to have morphed to a characteristic morphology of a posterior thoracic vertebra. Other abnormalities that indicated previous transformations to different degrees in heterozygous mice were also seen. These included a change in the long prickly process characteristic of T2 by a segment up to T3, a change of the articular and spinous processes from TIO to Til, a change of the anterior tubercle on C6 to C7, and the transformation of T2 to TI where the rib associated with T2 touched the upper part of the sternum. In order to understand the basis for the abnormalities in the axial pattern seen in the GDF-11 mutant mice, mutant embryos isolated at different stages of development were examined and compared with the wild-type embryos. Through a morphological examination, homozygous mutant embryos isolated up to day 9.5 of gestation were not easily distinguishable from the corresponding wild-type embryos. In particular, the number of somites present at any given developmental age was identical between the mutant and wild type embryos, suggesting that the rate of somite formation was not altered in the mutants. For day 10.5 to 11.5 p.c., mutant embryos could be easily distinguished from wild-type embryos by posterior displacement of the hind limb by 7-8 somites. Abnormalities in the development of the tail at this stage were also easily apparent. Taken together, these data suggest that the abnormalities observed in the mutant skeletons represented true transformations of segment identities, rather than the insertion of additional segments, for example, by a higher rate of somitogenesis.

It is known that alterations in the expression of homeocuadro-containing genes cause transformations in Drosophila and in vertebrates. To see if the expression patterns of the Hox genes (genes that contain homeocomponent of the vertebrae) were altered in null mutants in GDF-11, the expression pattern of three representative Hox genes, Hoxc-6, Hoxc-8, was determined , and Hoxc-11, on day 12.5 pc of wild-type embryos, and heterozygous and homozygous mutants, by the total amount of in situ hybridization. The expression pattern of Hoxc-6 in the wild-type embryos was extended in the vertebrae 8-15, corresponding to the thoracic segments T1-T8. However, in the homozygous mutants, the expression pattern of Hoxc-6 subsequently changed, and expanded to the pre-vertebrae 9-18 (T2-T11). A similar change is seen with the Hoxc-8 probe. In wild-type embryos, Hoxc-8 was expressed in the pre-vertebrae 13-18 (T6-T11), but in the homozygous mutant embryos, Hoxc-8 was expressed in the vertebrae 14-22 (T7-T15). Finally, the expression of Hoxc-11 also changed later, in that the previous limit of expression changed from the vertebrae 28 of the wild-type embryos to the fortertebrae 36 in the mutant embryos. (Note that since the position of the hind limb in the mutant embryos also changed subsequently, the expression patterns of Hoxc-11 in the wild type and mutant appeared similar in relation to the hind limbs).

These data provide additional evidence that the skeletal abnormalities seen in mutant animals represent homeotic transformations. The phenotype of GDF-11 mice suggested that GDF-11 acts early during embryogenesis as a global regulator of axial pattern. To begin to examine the mechanism by which GDF-11 exerts its effects, the pattern of expression of GDF-11 in early mouse embryos was examined by the total amount of in situ hybridization. In these stages, the primary expression sites of GDF-11 correlated precisely with the known sites in which mesodermal cells are generated. The expression of GDF-11 was detected first on day 8.25-8.5 p.c. (8-10 somites) in the primitive striped region, which is the site where the input cells form the mesoderm of the developing embryo. The expression remained in the primitive streak on day 8.75, but for day 9.5 pc, when the tail button replaced the primitive streak as the source of new mesodermal cells, the expression of GDF-11 was moved to the button from the tail. Therefore, in these early stages, GDF-11 appears to be synthesized in the developing embryo region where new mesodermal cells are present, and presumably acquire their position identity. The phenotype of the mice with elimination of GDF-11 in several aspects resembled the phenotype of the mice that they carried a suppression of a receptor for some members of the TGF-β superfamily, the activin receptor type IIB (Act RIIB). As in the case of mice with elimination of GDF-11, mice with Act RIIB removal have extra pairs of ribs and a spectrum of renal defects ranging from hypoplastic kidneys to complete absence of kidneys. The similarity in the phenotypes of these mice presents the possibility that Act RIIB may be a receptor for GDF-11. However, Act RIIB can not be the only receptor for GDF-11, because the phenotype of mice with elimination of GDF-11 is more severe than the phenotype of mice with Act-RIIB. For example, while animals with elimination of GDF-11 have 4 to 5 extra pairs of ribs, and show homeotic transformations through the axial skeleton, animals with Act RIIB removal have only three extra pairs of ribs, and do not show transformations in other axial levels. In addition, the data indicate that kidney defects in mice with GDF-11 clearance are also more severe than those in mice with Act RIIB removal. Mice with Act RIIB removal show defects in left / right axis formation, such as pulmonary isomerism, and a range of cardiac defects that we have not yet observed in mice with GDF-11 clearance. Act RIIB can be linked to activins and certain BMPs, although none of the knockout mice generated for these ligands show defects in the formation of the left / right axis. If GDF-11 acts directly on mesodermal cells to establish position identity, the data presented here would be consistent either with a short range or with morphogenic models for the action of GDF-11. That is, GDF-11 can act on mesodermal precursors to establish patterns of Hox gene expression as these cells are being generated at the site of GDF-11 expression, or alternatively, GDF-11 produced at the posterior end of the embryo can diffuse to form a morphogenic gradient. Whatever the mechanism of action of GDF-11, the fact that the gross anterior / posterior pattern is still present in the animals with elimination of GDF-11, suggests that GDF-11 may not be the only regulator of the specification before, after. However, it is clear that the GDF-11 has an important role as the global regulator of the axial pattern, and that an additional study of this molecule will lead to new important perspectives on the way in which identity of position is established along the anterior / posterior axis in the vertebrate embryo. Similar phenotypes are expected in animals with elimination of GDF-8. For example, animals with elimination of GDF-8 are expected to have a greater number of ribs, kidney defects, and anatomical differences when compared to the wild type.

EXAMPLE 15 PRODUCTION OF TRANSGENIC MICE EXPRESSING THE PROPÉTIDE OF MYIOSTATIN, FOLISTATIN, OR A KINNER NEGATIVE RIIB ACT Purification of myostatin. A Chinese hamster ovary cell line (CHO) carrying amplified copies of a myostatin expression construct was transfected with an expression construct for the PACE furin protease (kindly provided by Monique Davies) in order to improve the processing of the precursor protein. A conditioned medium (prepared by Cell Trends, Middletown, MD) was successively passed over hydroxylapatite (eluted with 200 mM sodium phosphate, pH 7.2), lentil-lectin Sepharose (eluted with 50 mM Tris, pH 7.4, 500 mM NaCl , 500 mM methyl material), DEAE agarose (collected material that flowed through the column in 50 mM Tris, pH 7.4, 50 mM NaCl), and heparin Sepharose (eluted with 50 mM Tris, pH 7.4, 200 NaCl mM). The eluate of the heparin column was then ligated to a reverse phase C4 HPLC column, and eluted with a gradient of acetonitrile in 0.1 percent trifluoroacetic acid. Anti-bodies directed against the mature C-terminal protein were described above (see US patent 5,827,733, incorporated herein by reference). In order to reproduce anti-bodies against the propeptide, the portion of the human myostatin protein that extended into amino acids 122-261 was expressed in bacteria using the RSET vector (Invitrogen, San Diego, California, United States), purified by chromatography on nickel chelate, and injected into rabbits. Immunizations were carried out by Spring Valley Labs (Woodbine, MD). Link of the receiver. The purified myostatin was radioiodinated using the chloramine T method (Frolik, C.A., Wake-field, L., Smith, D.M. and Sporn, M.B. (1984) J. Biol. Chem. 259, 10995-1100). COS-7 cells cultured in six- or twelve-well plates were transfected with 1-2 micrograms of pCMV5 or a pCMV5 / receptor construct, using lipofectamine (Gibco, Rockville, MD). The crosslinking experiments were carried out two days after the transfection, as described (Franzén, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schultz, P., Heldin, CH and Miyazono , K. (1993) Cell 75, 681-692). For quantitative receptor binding assays, the cell monolayers were washed twice with phosphate-buffered serum containing 1 mg / ml bovine serum albumin, and incubated with labeled myostatin in the presence or absence of different concentrations of non-myostatin. labeled, propeptide, or follistatin, at 4 ° C. The cells were then washed three times with the same regulator, lysed in 0.5 N NaOH, and counted in a gamma counter. The specific binding was calculated as the difference in the bound myostatin between the cells transfected with Act RIIB, and the cells transfected with the vector. This method of calculating the specific link was especially important in the evaluation of the effect of the propeptide, because the addition of the propeptide also reduced the non-specific binding in a concentration-dependent manner. Transgenic mice. The DNAs encoding a truncated form of amino acids 1-174 which extended into Act RIIB of murine, amino acids 1-267 which extended into the murine myostatin propeptide, and the short form of human folistatin, were cloned into the MDAF2 vector containing the myosin light chain promoter and the 1/3 enhancer (McPherron, AC and Lee, SJ (1993) J. Biol. Chem. 268, 3444-3449). Purified transgenes were used, including the myosin light chain regulatory sequences and the SV40 processing sites for the microinjections. All microinjections and embryo transfers were carried out by the Johns Hopkins School of Medicine Transgenic Core Facility. Transgenic founders in a hybrid background of SJL / C57BL / 6 were crossed with wild type C57BL / 6 mice, and all the studies were carried out using the progeny Fl. For the analysis of muscle weights, the individual muscles of both sides of almost all animals were dissected, and the average left and right muscle weights were used. The analysis of the numbers and sizes of the fibers was carried out as described (McPherron, A.C., Lawler, A.M. and Lee, S.J. (1997) Nature 387, 83-90). RNA isolation and Northern analysis were carried out as described (McPherron, A.C. and Lee, S.J. (1993) J. Biol. Chem. 268, 3444-3449). In order to overproduce the myostatin protein, a line of CHO cells was produced which carried amplified copies of the myostatin expression construct (McPherron, A.C., Lawler, A.M. 6 Lee, S.J. (1997) Nature 387, 83-90). Myostatin was purified from the conditioned medium of this cell line, by successive fractionation on hydroxylapatite, lentil-lectin Sepharose, DEAE agarose, and heparin Sepharose. Silver-stained analysis of the purified protein preparation revealed the presence of two protein species of 29 kd and 12.5 kd. A variety of data suggested that this purified protein consisted of a non-covalent complex of two propeptide molecules linked to a disulfide-linked C-terminal dimer. First, by Western analysis, the 29 kd and 12.5 kd species were immunoreactive with the anti-bodies reproduced against the bacterially expressed fragments of myostatin extending to the propeptide and the mature C-terminal region, respectively. Second, in the absence of reducing agents, the C-terminal region had an electrophoretic mobility consistent with that of a dimer. Third, the two species were present in a molar ratio of approximately 1: 1. And fourth, the C-terminal dimer was retained on the lectin column, and could be eluted with methyl agar, even when this portion of the protein does not contain potential N-linked glycosylation sites; the simplest interpretation of these data is that the C-terminal region was linked to the lectin indirectly by being present in a tight complex with the propeptide, which has a glycosylation signal. Because it is known that the C-terminal dimer is a biologically active molecule for other members of the TGF-β family, the C-terminal dimer of myostatin was purified from its propeptide by reverse phase HPLC. The fractions containing the purified C-terminal dimer (32-34) appeared to be homogeneous. However, the most enriched fractions for the propeptide (35-37) were contaminated with small amounts of C-terminal dimer, and with high molecular weight complexes that most likely represented misfolded proteins. Most members of the TGF-β superfamily have been shown to signal via the binding of serine / threonine kinase receptors, followed by the activation of Smad proteins (Heldin, CH Miyazono, K. and ten Dijke, P. ( 1997) Nature 390, 465-471, Massagué, J., Blain, SW and Lo, RS (2000) Cell 103, 295-309). The initial event when the signaling path is triggered is the link of the ligand with a type II receptor. In order to determine if myostatin is capable of binding to any of the known type II receptors for related ligands, cross-linking studies were performed with the radioiodinated radioiodinated myostatin dimer C-terminal.

COS-7 cells transfected with expression constructs for TGF-β, BMP, or activin type II receptors. Cross-linked complexes of predicted size (full length receptor bound to myostatin) were detected for cells expressing Act RIIA or Act RIIB. Higher levels of binding to Act RIIB were observed than to Act RIIA in both cross-linking and standard receptor binding assays, and therefore, receptor binding studies were focused on Act RIIB. The binding of myostatin with Act RIIB was specific (the binding could be competed for an excess of unlabeled myostatin) and saturable, and assuming that all the myostatin protein was bioactive, we estimated the dissociation constant by Scatchard analysis as approximately 10 nM. In the case of TGF-β, it is known that the affinity for the type II receptor is significantly higher in the presence of the appropriate type I receptor, and that other molecules are involved in the presentation of the ligand to the receptor. In order to determine if activin type II receptors may be involved in myostatin signaling in vivo, we investigated the effect of expressing a dominant negative form of Act RIIB in mice. For this purpose, we generated a construct in which a truncated form of Act RIIB lacking the kinase domain downstream of a skeletal muscle specific myosin light chain promoter / enhancer was placed. From pronuclear injections of In this construction, a total of seven positive founder animals were identified for the transgene. Analysis of these founding animals at seven months of age revealed that the seven had significant increases in skeletal muscle mass, weighing the individual muscles of these founder animals up to 125 percent more than those of non-transgenic control animals derived from injections similar (Table 2). Three lines of evidence suggested that the increases in muscle weights in these founding animals resulted from the expression of the transgene. First, the progeny analysis derived from the crosses of three founder animals (the other four founder animals did not generate sufficient numbers of progeny for the analysis) with wild-type C57BL / 6 mice, showed that increases in muscle weights correlated with the presence of transgenes (Table 3). Second, although the muscle weights varied among the different transgenic lines, the magnitude of the increase was highly consistent between the animals of any given line for all the muscles examined and both for males and for females (Table 3). For example, all the muscles of both male and female mice of the C5 line weighed approximately 30 to 60 percent more than those of the control animals, while all the muscles of the Cll mice weighed approximately 110 to 180 percent. plus. Third, the Northern analysis of the RNA samples prepared from the transgenic animals, showed that the expression of the transgene was restricted to the skeletal muscle, and that the relative levels of expression of the transgene correlated with the relative magnitude of the increase in muscle weights (Table 3) . For example, animals of the Cll line, which had the greatest increases in muscle weights, also had the highest expression levels of the transgene. These data showed that the expression of a dominant negative form of Act RIIB can cause increases in muscle mass similar to those seen in mice with myostatin clearance. In mice with myostatin clearance, it has been shown that the increase in muscle mass results from increases in both the number of fibers and the size of the fibers. In order to determine if the expression of Act RIIB negative dominant also causes both hyperplasia and hypertrophy, sections of the gastrocnemius and plantaris muscles of animals of line C27 were analyzed. Compared to the control muscles, the muscles of the C27 animals showed a clear increase in the overall cross-sectional area. This increase in area resulted partially from an increase in the number of fibers. At the widest point, the gastrocnemius and plantaris muscles had a total of 10015 + 1143 fibers in the animals of the C27 line (n = 3), compared with 7871 + 364 fibers in the control animals (n = 3). Without However, muscle fiber hypertrophy also contributed to the increase in the total area. The average diameter of the fiber was 51 microns in the animals of the C27 line, compared to 43 microns in the control animals. Consequently, the increase in muscle mass appeared to result from an increase of approximately 27 percent in the number of fibers, and from a 19 percent increase in fiber diameter (assuming the fibers were regularly cylindrical, this increase in diameter results in an increase of approximately 40 percent in the cross-sectional area). However, except for the increase in the number and size of the fibers, the muscles of the transgenic animals seemed regularly normal. In particular, there were no obvious signs of degeneration, such as widely variable fiber sizes (standard deviation of fiber sizes was similar between control and transgenic animals), or extensive fibrosis or fat infiltration. These approaches were used to explore other possible strategies to inhibit myostatin. First, we investigated the effect of the myostatin propeptide. In the case of TGF-β, it is known that the C-terminal dimer is maintained in a dormant complex inactive with other proteins, including its propeptide, and that the propeptide of TGF-β can have an inhibitory effect on the activity of TGF-β. ß, both in vitro and in vivo (Miyazono, K., Hellman, U., Wernstedt, C. and Heldin, CH (1988) J. Biol. Chem. 263, 6407-6415; Gentry, L.E. and Nash, B.W. (1990) Biochem. 29, 6851-6857; Bdttinger, E.P., Factor, V.M., Tsang, M.L.S., Weatherbee, J.A., Kopp, J.B., Qian, S.W., Wakefield, L, M., Roberts, A.B., Thorgeirsson, S.S. and Sporn, M.B. (1996) Proc. Nati Acad. Sel. USA 93, 5877-5882). The observation that the C-terminal dimer of myostatin and the co-purified propeptide presented the possibility that myostatin may normally exist in a similar latent complex, and that the myostatin propeptide may have an inhibitory activity. Second, we examined the effect of follistatin, which has been shown to be able to bind and inhibit the activity of several members of the TGF-β family. In particular, follistatin can block the activity of GDF-11, which is highly related to myostatin, and it has been shown that mice with elimination of follistatin have a reduced muscle mass at birth, which would be consistent with the overactivity of the myostatin (Gamer, L., Wolfman, N., Celeste, A., Hattersley, G., Hewick, R. and Rosen, V. (1999) Dev. Biol. 208, 222-232; Matzuk, MM, Lu, N., Vogel, H., Selheyer, K., Roop, DR and Braley, A. (1995) Nature 374, 360-363). The effect of propeptide and follistatin in vitro was then studied. Both the myostatin propeptide and follistatin were able to block the binding of the C-terminal dimer with Act RIIB. Ki of follistatin was estimated at approximately 470 pM, and that of propeptide at least 50 more times. The calculation of the Ki for the propeptide, however, assumes that the entire protein in the final preparation - represented the biologically active propeptide, and therefore, is probably an overestimation. As described above, the propeptide preparation was contaminated with both small amounts of C-terminal dimer and misfolded high molecular weight species. In order to determine if these molecules are also capable of blocking the activity of myostatin in vivo, transgenic mice were generated in which the promoter / enhancer of the light chain of myosin was used to boost the expression of either the propeptide of myostatin or folistati-na. From pronuclear injections of the propeptide construction, three transgenic mouse lines were identified (two of these, B32A and B32B, represented insertion sites of the independent segregating transgene in an original founder animal), which showed greater muscle formation. . As shown in Table 3, the muscle weights of the animals of each line increased by approximately 20 to 110 percent, compared with those of the non-transgenic control animals. Northern analysis of the RNA samples prepared from representative animals from each of these lines showed that the expression levels of the transgene correlated with the magnitude of the increase in muscle weights. In a specific way, the animals of the line B32A, which had only an increase of approximately 20 to 40 percent in muscle mass, had the lowest levels of expression of the transgene, and animals of lines B32B and B53, which had an increase of approximately 70 to 110 percent in muscle mass, had the highest levels of expression of the transgene. Perhaps in a significant way, the muscle weights in the animals that were dually transgenic for the insertion sites B32A and B32B were similar to those observed in the transgenic animals only for the B32B insertion site (Table 3), despite the fact that double-transgenic animals seemed to have higher levels of expression of the transgene. These findings suggest that the effects seen in the B32B line (and in the B53 line) were the maximum achievable by overexpression of the propeptide. As in the case of animals that expressed the dominant negative form of Act RIIB, the animals that expressed the propeptide showed increases both in the number and in the size of the muscle fibers. The analysis of the gastrocnemius and plantaris muscles of two animals that were doubly transgenic for insertion sites B32A and B32B, showed that fiber numbers increased by approximately 40 percent (both animals had 11940 and 10420 fibers), and fiber diameters increased by approximately 21 percent (up to 52 microns), compared to the control animals. The most dramatic effects on skeletal muscle were obtained using the construction of follistatin. Two founding animals (F3 and F66) showed an increase in muscle formation (Table 2). In one of these animals (F3), muscle weights increased by 194 to 327 percent in relation to the control animals, resulting from a combination of hyperplasia (66 percent increase in the number of fibers up to 13051 in the gastrocnemius / plantaris) and hypertrophy (increase of 28 percent in the diameter of the fibers to 55 microns). Although we did not analyze the muscle weights of the mice with myostatin removal in a SJL / C57BL / 6 hybrid pool, the increases in muscle mass observed in the founding animal F3 were significantly greater than the increases we have seen in the null animals in myostatin from other genetic backgrounds. These results suggest that at least part of the effect of follistatin may result from the inhibition of another ligand in addition to myostatin. Clearly, an analysis of additional follistatin transgenic lines will be essential to determine if other ligands may also be involved in the down regulation of muscle growth. Following proteolytic processing, the C-terminal dimer of myostatin is likely to remain in a latent complex with its propeptide, and perhaps also other proteins. Myostatin is also negatively regulated by follistatin, which binds to the C-terminal dimer, and inhibits its ability to link with the receivers. The release of the C-terminal dimer from these inhibitory proteins by unknown mechanisms allows the myostatin to bind to the activin type II receptors. By analogy with other members of the family, we presume that the activation of these receptors then leads to the activation of a type II receptor and Smad proteins. This global model of regulation and signaling of myostatin is consistent not only with the data presented here, but also with other genetic data. As described above, it has been shown that mice with elimination of follistatin have a reduced muscle mass at birth, which is what one would expect for the non-inhibited myostatin activity. A similar muscle phenotype has been reported for mice lacking ski, which has been shown to inhibit the activity of Smad 2 and 3, and the opposite phenotype, that is, an excess of skeletal muscle, has been observed in mice overexpress ski. Based on the present findings, one hypothesis is that these observed phenotypes reflect the overactivity and subactivity, respectively, of myostatin in these mice. Although all in vitro and genetic data are consistent with the global model we have presented here, these data would also be consistent with alternative models involving other receptors and ligands. For example, no We know the mechanism by which the truncated form of the Act RIIB improves muscle growth in our transgenic mice. It is possible that the truncated receptor is not acting to block signaling in the target cell, but rather is merely acting as a sink to deplete the extracellular concentrations of myostatin. It is also possible that the truncated receptor is blocking the signaling of other ligands in addition to myostatin. In this regard, it has been shown that the dominant negative forms of activin type II receptors can block the signaling of a variety of different ligands related to TGF-β in other species. In a similar way, our data does not definitively show that follistatin is blocking myostatin activity in vivo to promote muscle growth. In this regard, the extraordinary degree of muscle formation seen in one of the founder animals expressing follistatin suggests that other ligands sensitive to follistatin may be involved in the regulation of muscle growth. Nevertheless, to date, myostatin is the only secreted protein that has been shown to have a negative role in the regulation of muscle mass in vivo. Although additional experiments will be required to test aspects of this global model, and to identify the other signaling components, our data suggest that myostatin antagonists, such as follistatin and myostatin propeptide, or antagonists Table 2. Muscle weights (rrtg) All animals (including controls) represent F0 hybrid SJL / C57BL / 6 mice born from injected embryos.

Table 3 P l M l p? Propeptide B32A (n = 9) 78. í 3 + 2.9 *** 100 1 + 3.7 *** 206 0 + 2.7 *** 138 9 + 3.1 *** B32B (n = 2) 131 0 + 18.4 151 5+ 23.3 315 5 + 58.7 199 5 + 2 .7 B32 + B (n = 4) 109 3 + 9.5 * 132 8 + 6.0 ** 270 8 + 6.9 *** 177 0 + 2.4 *** B53 (n = 6) 134 7 + 7.7 *** 148 2 + 12.1 *** 303 8 + 18.5 *** 212 8 + 12.9 *** * p < 0.05, ** p < 0.01, *** p < 0.001. All (including controls) represent the four-month-old progeny of the transgenic founders (SJL / C57BL / 6) crossed with wild-type C57BL / 6 mice.

Although the invention has been described with reference to the previous examples, it will be understood that the modifications and variations are encompassed within the spirit and scope of the invention. According to the foregoing, the invention is limited only by the following claims.

Claims (42)

1. CLAIMS 1. A transgenic non-human animal, whose genome contains a nucleic acid sequence comprising an activin type II receptor gene, truncated, and a muscle-specific promoter operably linked and integrated into the animal's genome, where the acid sequence nucleic acid is expressed so as to result in high levels of truncated type II activin receptor and increased muscle mass in the animal compared to a corresponding non-transgenic animal.
2. 2. The transgenic animal of claim 1, wherein the muscle-specific promoter is a myosin light chain promoter / enhancer.
3. 3. The transgenic animal of claim 1, wherein the activin type II receptor is RILA, or RIIB.
4. 4. The transgenic animal of claim 3, wherein the truncated activin RIIB receptor lacks kinase activity.
5. 5. The transgenic animal of claim 1, wherein the truncated activin RIIB receptor comprises the amino acid residues 1-174 of activin RIIB.
6. 6. A non-human transgenic animal whose genome contains a nucleic acid sequence comprising a prodomain of myostatin and a muscle-specific promoter operably linked and integrated into the animal's genome, where the nucleic acid sequence is expressed so as to give as a result, high levels of myostatin prodomain and increased muscle mass in the animal compared to a corresponding non-transgenic animal.
7. 7. The transgenic animal of claim 6, wherein the myostatin prodomain comprises amino acid residues 1 to 262 of a promyostatin polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12 , 14, 16, 18 or 20, or a functional peptide portion of said myostatin prodomain.
8. The transgenic animal of claim 6, wherein the myostatin prodomain comprises an amino acid sequence selected from the group consisting of: amino acid residues of from about 20 to 263, as set forth in SEQ ID NO: 4; the amino acid residues of about 20 to 262, as indicated in SEQ ID NO: 2; the amino acid residues of about 20 to 262, as indicated in SEQ ID NO: 10; the amino acid residues of about 20 to 262, as indicated in SEQ ID NO: 12; the amino acid residues of around 20 to 262, as indicated in SEQ ID NO: 8; the amino acid residues of around 20 to 263, as indicated in SEQ ID NO: 6; the amino acid residues of around 20 to 262, as indicated in SEQ ID NO: 18; the amino acid residues of around 20 to 262, as indicated in SEQ ID NO: 14; the amino acid residues of around 20 to 262, as indicated in SEQ ID NO: 16; the amino acid residues of around 20 to 262, as indicated in SEQ ID NO: 20; and a functional peptide portion thereof.
9. 9. The transgenic animal of claim 6, wherein the myostatin prodomain further comprises a myostatin signal peptide.
10. 10. The transgenic animal of claim 6, wherein the muscle-specific promoter is a myosin light chain promoter / speaker.
11. 11. A non-human transgenic animal, the genome of which contains a nucleic acid sequence comprising a gene of follistatin and a muscle-specific promoter operably linked and integrated into the animal's genome, wherein the nucleic acid sequence is expressed so as to give as a result elevated levels of folistatin and increased muscle mass in the animal compared to a corresponding non-transgenic animal.
12. 12. The transgenic animal of claim 11, wherein the muscle-specific promoter is a myosin light chain promoter / enhancer.
13. 13. An expression cassette, comprising a DNA segment encoding a truncated activin RIIB receptor gene, operably linked to a muscle-specific control sequence.
14. 14. The expression cassette of claim 13, where the muscle-specific promoter is a myosin light chain promoter / enhancer.
15. 15. An expression cassette, comprising a DNA segment encoding a myostatin prodomain gene operably linked to a muscle-specific control sequence.
16. 16. The expression cassette of claim 15, wherein the muscle-specific promoter is a myosin light chain promoter / speaker.
17. 17. An expression cassette, comprising a DNA segment encoding a follistatin gene operably linked to a muscle-specific control sequence.
18. 18. The expression cassette of claim 17, wherein the muscle-specific promoter is a myosin light chain promoter / speaker.
19. 19. A method for tissue-specific folistatin expression in a transgenic animal, comprising expressing an expression cassette of claim 17 in the cells of a transgenic animal, wherein the expression cassette is integrated into the animal's genome, where the cassette is expressed so as to result in high levels of folistatin in the animal, thereby resulting in increased muscle mass in the transgenic animal relative to a corresponding non-transgenic animal.
20. 20. A cell or cell line isolated from the animal of any of claims 1, 6 or 11, wherein said cell expresses the truncated type II activin receptor, the myostatin prodomain, or follistatin, respectively.
21. 21. A method of inhibiting the binding of myostatin to an activin type II receptor, which comprises contacting myostatin with follistatin, thereby inhibiting binding to the receptor.
22. 22. The method of claim 21, wherein the inhibition of ligation is by the C-terminus of myostatin.
23. 23. The method of claim 21, wherein the activin receptor is Act RIIA or Act RIIB.
24. 24. A non-human transgenic animal, whose genome comprises a DNA construct comprising a DNA segment encoding a follistatin protein operably linked to a promoter heterologous to the endogenous folistatin gene, effective for expression in muscle cells, where the expression of said construction of DNA in muscle cells results in an increase in the muscle mass of said animal.
25. 25. The non-human animal according to claim 24, wherein said DNA construct has been introduced into an ancestor of said animal.
26. 26. The non-human animal according to claim 24, wherein said DNA construct is introduced to said animal or ancestor of said animal in an embryonic stage.
27. 27. The non-human animal according to claim 24, wherein said follistatin protein is a truncated, mutant or other variant form of follistatin protein as compared to the wild type.
28. 28. The non-human animal according to claim 24, wherein said DNA construct is an MDAF2 expression plasmid containing said DNA segment encoding a follistatin protein.
29. 29. The non-human animal according to claim 24, wherein the animal is a mammal.
30. 30. The non-human animal according to claim 29, wherein said mammal is a mouse.
31. 31. The non-human animal according to claim 29, wherein said mammal is a porcine.
32. 32. The non-human animal according to claim 29, wherein said mammal is a bovine.
33. 33. The non-human animal of claim 24, wherein said animal is a species of bird.
34. 34. The non-human animal of claim 33, wherein the bird species is a chicken or a turkey.
35. 35. The non-human animal of claim 24, wherein said animal is an aquatic species.
36. 36. The non-human animal of claim 35, wherein the aquatic species is a finfish.
37. 37. The non-human animal of claim 36, wherein the finfish is a salmon, trout, small-scale trout, ayu, carp, crossed carp, golden fish, goby, whitebait, eel, conger eel, sardine, flying fish, sea bass, sea bream, parrotfish, biting fish, mackerel, sarda, tuna, bonito, yellow tail, perch, flounder, sole, turbot, puffer fish, triggerfish.
38. 38. The non-human animal of claim 35, wherein the aquatic species is a clam, cochin, mussel, bigarro, shell, snail, slug, sea cucumber, ark shell, oyster, turban shell, abalone, lobster, shrimp , shrimp, cold water shrimp, crab, shearing, krill, prawn, river crab, Annelida, lizard, turtle, frog, or sea urchins.
39. 39. The non-human animal of claim 24, wherein the animal is a sheep.
40. 40. A method of producing a chimeric, non-human animal, the method comprising: obtaining an ovum from animal ovaries; ripen the ovum in vitro; fertilize the mature ovule in vitro to form a zygote; introducing to the zygote in vitro a nucleic acid construct comprising in operative association a DNA sequence encoding a truncated activin type II receptor, a myostatin pro-peptide, or follistatin, and a regulatory sequence that promotes the expression of the sequence of DNA encoding the polypeptide; to mature the zygote to an embryo of the pre-implantation stage in vitro; and transplanting the embryo to a female receiving animal, where the female animal breeds the embryo to produce a chimeric animal.
41. 41. A method of producing animal feed products having increased muscle mass, comprising: a) introducing a transgene encoding folistatin, myostatin pro-peptide or a truncated activin type II receptor, into germ cells of a pro-nuclear embryo of the animal; b) implanting the embryo in the oviduct of a pseudo-pregnant female, thereby allowing the embryo to mature into full-term progeny; c) testing progeny for the presence of the transgene to identify positive progeny to the transgene; d) crossing the positive progeny in the transgene to obtain more positive progeny to the transgene; Y e) process the progeny to obtain food products.
42. 42. A method of producing food products from poultry, swine, piscine or bovines having increased muscle mass, comprising: a) introducing a transgene encoding folistatin, myostatin pro-peptide or a truncated type II activin receptor to an embryo of an animal bird, pig, Piscine or bovine; b) cultivate the embryo under conditions through which progeny are bred; c) having the progeny tested for the presence of the transgene to identify positive progeny to the transgene; d) crossing the positive progeny to the transgene; and e) process the progeny to obtain food products.