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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) Internationa] Patent Classification ^ : 

C07K 13/00, 15/28, A61K 37/36, C12N 
15/18, 15/10, 15/66 



Al 



(11) International PubUcatioii Number: WO 94/21681 

(43) International Publication Date: 29 September 1994 (29.09.94) 



(21) International ApplicatioD Number: PCTAJS94A)3019 

(22) International Filing Date: 18 March 1994 (18.03.94) 



(30) Priority Data: 

08/033^ 



19 March 1993 (19.03.93) 



US 



(60) Parent AppBcation or Grant 
(63) Related by Continuation 
US 

Filed on 



08/033^ (OP) 
19 March 1993 (19.03.93) 



(71) Applicant (for all designated States except US): JOHNS HOP- 

KINS UNIVERSITY SC:H00L OF MEDICINE [US/US]; 
720 Rutland Avenue, Baltimore, MD 21205 (US). 

(72) Inventors; and 

(75) Inventors/AppUcants (for US only): I FF Se-Tm [US/US]; 
6711 Chokebeiry Road, Baltimore, MD 21209 (US). 
McPHERRON, Alexandra, C [US/US]; 3905 Keswick 
Road, Baltimore, MD 21211 (US). 

(74) Agaits: WETHERELL, John, R. et al.; Spensley Horn Jubas 
& Lubitz, 1880 Ontury Park East, 5th Floor, Los Angeles, 
CA 90067 (US). 



(81) Designated States: CA, JP, US, European patent (AT, BE, CH, 
DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, FT. SE). 



Published 

With imerruxtional search report. 



CO 

m 

CO 



01 

m 
o 



(54) Title: GROWTH DIFFERENTIATION FACIOR-8 

(57) Abstract 

(jTOWth differentiation factor-8 (GDF-8) 
is disclosed along with its polynucleotide se- 
quence and amino add sequence. Also dis- 
closed arc diagnostic and therapeutic me&ods 
of using the GDF*8 polypeptide and polynu- 
cleotide sequences. 



I 



HEART 

LUNG 

THYMUS 

BRAIN 

KIDNEY 

SEMINAL VESICLE 

PANCREAS 

INTESTINE 

SPLEEN 

TESTIS 

MUSCLE 

LIVER 

OVARY 

FAT 

UTERUS 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


GB 


United Kingdom 


MR 


MauritinU 


AU 


Australia 


GE 


Georgift 


MW 


Malawi 


BB 


Barbados 


GN 


Guinea 


NE 


Niger 


BE 


Belgium 


GR 


Greece 


^^L 


Netherlaofb 


BF 


Burtana Faao 


HU 


Hungary 


NO 


Norway 


BG 


Bulgaria 


IE 


Ireland 


NZ 


New Zealand 


BJ 


Beoio 


IT 


Italy 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


FT 


Portugal 


BY 


Belans 


KE 


Kenya 


RO 


Romania 


CA 


Canada 


KG 


Kyxgysion 


RD 


Russian Federation 


CF 


Central African Republic 


KP 


Detnocntic People's Republic 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzalaod 


KR 


Republic of Korea 


SI 


Slovenia 


a 


Cflte d'lvoire 


KZ 


Kazakhstan 


SK 


Slovakia 


CM 


CatDOooo 


U 


Ltectttenstem 


SN 


Senegal 


CN 




LK 


Sri Lanka 


TD 


Chad 


CS 


Czechoslovakia 


hV 


Luxembourg 


TG 


Togo 


CZ 


Czech Republic 


LV 


Latvia 


TJ 


Tajikistan 


DE 


Gcnnany 


MC 


Monaco 


TT 


Thmdad and Tobago 


DK 


Detnnaik 


MD 


Republic of Moldova 


UA 


Uknine , 


ES 


Spain 


MG 


Madagascar 


US 


United States of America 


n 


Finland 


ML 


Mali 


UZ 


Uzfaelcistan 


FR 


France 


MN 


Mongolia 


VN 


Viet Nam 


GA 


Gabon 











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-1- 

GROWTH DIFFERENTIATION FACTOR-8 

This application is a continuation-in-part application of the U.S. Application 
Serial No. 08/033.923 filed on 3/19/93. 

BACKGROUND OF THE INVENTION 

5 1. Field of the Invention 

The invention relates generally to growth factors and specifically to a new 
member of the transforming growth factor beta (TGF-^) superfamily, which is 
denoted, growth differentiation factor-8 (GDF-8). 

2. Description of Related Art 

10 The transforming growth factor fi (TGF-^) superfamily encompasses a group 
of structurally-related proteins which affect a wide range of differentiation 
processes during embryonic development. The family includes. Mulierian 
inhibiting substance (MIS), which is required for normal male sex development 
(Behringer, et al., Nature, 345:167, 1990). Drosophila decapentaplegic {DPP) 

15 gene product, which is required for dorsal-ventral axis formation and 
morphogenesis of the imaginal disks (Padgett, et al.. Nature. 325:81-84, 1987), 
the Xenopus Vg-1 gene product, which localizes to the vegetal pole of eggs 
((Weeks, et al., Cell, 51:861-867. 1987), the activins (Mason, et al.. Biochem. 
Biophys. Res. Gommun.. 135:957-964, 1986), which can induce the formation 

20 of mesoderm and anterior structures in Xenopus embryos (Thomsen. et al.. 
Cell, 63:485. 1990). and the bone morphogenetic proteins (BMPs, osteogenin. 
OP-1) which can induce de novo cartilage and bone formation (Sampath. et 
al., J. Biol. Chem., 265:13198, 1990). The IGF-ps can influence a variety of 
differentiation processes, including adipogenesis. myogenesis. chor>drogenesis, 



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-2- 

hematopoiesis, and epithelial cell differentiation (for review, see Massague, Cell 
43:437. -'987). 

The proteins of the TGF-^ family are initially synthesized as a large precursor 
protein which subsequently undergoes proteolytic cleavage at a cluster of basic 

5 residues approximately 110-140 amino acids from the C-terminus. The C- 
terminal regions, or mature regions, of the proteins are all structurally related 
and the different family members can be classified into distinct subgroups 
based on the extent of their homology. Although the homologies within 
particular subgroups range from 70% to 90% amino acid sequence Identity, the 

10 homologies between subgroups are significantly lower, generally ranging from 
only 20% to 50%. In each case, the active species appears to be a disulfide- 
linked dimer of C-terminal fragments. Studies have shown that when the pro- 
region of a member of the TGF-^ family is coexpressed with a mature region 
of another member of the TGF-^ family, intracellular dimerization and secretion 

15 of biologically active homodimers occur (Gray, A., and Maston, A., Science, 
247:1328. 1990). Additional studies by Hammonds, et al., (Molec. Endocrin. 
5:149, 1991) showed that the use of the BMP-2 pro-region combined with the 
BMP-4 mature region led to dramatically improved expression of mature BMP- 
4. For most of the family men^bers that have been studied, the .homodimeric 

20 species has been found to be biologically active, -but for other family members, 
like the inhibins (Ung, et al.. Nature, 321:779, 1986) and the TGF-^s <Cheifet2. 
et al„ Cell, 4g:409, 1987), heterodimers have also been detected, and these 
appear to have different biobgical properties than the respective homodimers. 

Identification of new factors that are tissue-specific in their expression pattern 
25 will provide a greater understanding of that tissue's devebpment and function. 



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-3- 

SUMMARY OF THE INVENTION 

The present invention provides a cell growth and differentiation factor, GDF-8, 
a polynucleotide sequence which encodes the factor, and antibodies which are 
immunoreactive with the factor. This factor appears to relate to various cell 
5 proliferative disorders, especially those involving those involving muscle, nerve, 
and adipose tissue. 

Thus, in one embodiment, the invention provides a method for detecting a cell 
proliferative disorder of muscle, nerve, or fat origin and which is associated 
with GDF-8. In another embodiment, the invention provides a method for 
1 0 treating a cell proliferative disorder by suppressing or enhancing GDF-8 activity. 



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-4- 

BRIEF DESCRIPTION OF THE DRAWINGS 

FIGURE 1 is a Northern blot showing expression of GDF-8 mRNA in adutt 
tissues. The probe was a partial murine GDF-8 clone, 

FIGURE 2 shows nucleotide and predicted amino acid sequences of murine 
5 GDF-8 (FIGURE 2a) and human GDF-8 (FIGURE 2b). The putative dibasic 
processing sites in the murine sequence are boxed, 

FIGURE 3 shows the alignment of the C-terminal sequences of GDF-8 with 
other members of the TGF-^ superfamily. The conserved cysteine residues are 
boxed. Dashes denote gaps introduced in order to maximize alignment, 

1 0 FIGURE 4 shows amino acid homologies among different members of the TGF- 
p superfamily. Numbers represent percent amino acid identities between each 
pair calculated from the first conserved cysteine to the G-terminus. Boxes 
repreisent homologies among highly-related members within particular 
subgroups. 

15 FIGURE 5 shows the sequence of GDF-8. Nucleotide and amino acid 
sequences of murine (FIGURE 5a) and human (FIGURE 5b) GDF-8 cDNA 
clones are shown. Numbers indicate nucleotide position relative to the 5* end. 
Consensus N-linked glycosylation signals are shaded. The putative RXXR 
proteolytic cleavage sites are boxed. 

20 FIGURE 6 shows a hydropathicity profile of GDF-8. Average hydrophobicity 
values for murine (FIGURE 6a) and human (FIGURE 6b) <3DF-8 were calculated 
using the method of Kyte and Doolittle (J, Mol. Biol., 157:105-132. 1982). 
Positive numbers indicate increasing hydrophobicity. 



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-5- 

FIGURE 7 shows a comparison of murine and human GDF-8 amino acid 
sequences. The predicted murine sequence is shown in the top lines and the 
predicted human sequence is shown in the bottom lines. Numbers indicate 
amino acid position relative to the N-termlnus. Identities between the two 
5 sequences are denoted by a vertical line. 

FIGURE 8 shows the expression of GDF-8 in bacteria. BL21 (DE3) (pLysS) 
cells carrying a pRSET/GDF-8 expression plasmid were induced with 
isopropylthio-^-galactoside, and the GDF-8 fusion protein was purified by metal 
chelate chromatography. Lanes: total=total cell lysate; soluble = soluble protein 

10 fraction; insoluble= insoluble protein fraction (resuspended in 10 mM Tris pH 
8.0, 50 mM sodium phosphate, 8 M urea, and 10 mM ^-mercaptoethanol 
[buffer B]) loaded onto the column; pellet= insoluble protein fraction discarded 
before loading the column; flowthrough= proteins not bound by the column; 
washes = washes carried out in buffer B at the indicated pH*s. Positions of 

15 molecular weight standards are shown at the right. Arrow indicates the 
position of the GDF-8 fusion protein. 

FIGURE 9 shows the expression of GDF-8 in mammalian cells. Chinese 
hamster ovary cells were transfected with pMSXND/GDF-8 expression plasmids 
and selected in G418. Conditioned media from G418-resistant cells (prepared 
20 from cells transfected with constructs in which GDF-8 was cloned in either the 
antisense or sense orientation) were concentrated, electrophoresed under 
reducing conditions, blotted, and probed with anti-GDF-8 antibodies and 
[^^^IjiodoproteinA. Arrow indicates the position of the processed GDF-8 
protein. 



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-6- 

FIGURE 10 shows the expression of GDF-8 mRNA. Poly A-selected RNA {5 
;ig each) prepared from adult tissues (FIGURE 10a) or placentas and embryos 
(FIGURE 10b) at the indicated days of gestation was electrophoresed on 
formaldehyde gels, blotted, and probed with full length murine GDF-8. 

5 FIGURE 1 1 shows chromosomal mapping of human GDF-8. DNA samples 
prepared from human/rodent somatic cell hybrid lines were subjected to PGR, 
electrophoresed on agarose gels, blotted, and probed. The human 
chromosome contained in each of the hybrid cell lines is identified at the top 
of each of the first 24 lanes (1-22. X, and Y). In the lanes designated M, OHO. 
10 and H. the starting DNA template was total genomic DNA from mouse, 
hamster, and human sources, respectively. In the lane marked B1 . no template 
DNA was used. Numbers at left indicate the mobilities of DNA standards. 



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-7- 

DETAILED DESCRIPTION OF THE INVENTION 

The present invention provides a growth and differentiation factor. GDF-8 and 
a polynucleotide sequence encoding GDF-8. GDF-8 is expressed at highest 
levels in muscle and at lower levels in adipose tissue. In one embodiment, the 
5 invention provides a method for detection of a cell proliferative disorder of 
muscle, nerve, or fat origin which is associated with GDF-8 expression. In 
another embodiment, the invention provides a method for treating a cell 
proliferative disorder by using an agent which suppresses or enhances GDF-8 
activity. 

The TGF-^ superfamily consists of multifunctional polypeptides that control 
proliferation, differentiation, and other functions in many cell types. Many of the 
peptides have regulatory, both positive and negative, effects on other peptide 
growth factors. The structural homology between the GDF-8 protein of this 
invention and the members of the TGF-^ family, indicates that GDF-8 is a new 
member of the family of growth and differentiation factors. Based on the 
known activities of many of the other members, it can be expected that GDF-8 
will also possess biological activities that will make it useful as a diagnostic and 
therapeutic reagent. 

In particular, certain members of this superfamily have expression patterns or 
20 possess activities that relate to the function of the nervous system. For 
example, the inhibins and activins have been shown to be expressed in the 
brain (Meunier, et al., Proc. Natl. Acad. Sci.. USA, 85:247, 1988; Sawchenko, 
et al.. Nature, 334:615, 1988). and activin has been shown to be capable of 
functioning as a nerve cell survival molecule {Schubert, et al., Nature, 344:868. 
25 1990). Another family member, namely, GDF-1. is nervous system-specific in 
its expression pattern (Lee, S.J., Proc. Natl. Acad. Sci., USA, ^:4250, 1991), 
and certain other family members, such as Vgr-1 (Lyons, et al., Proc. Natl 



10 



15 



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-8- 

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 known to be expressed in the 
nervous system. Because it is known that skeletal muscle produces a factor 
5 or factors that promote the survival of motor neurons (Brown. Trends 
Neurosci., 7:10, 1984). the expression of GDF-8 in muscle suggests that one 
activity of GDF-8 may be as a trophic factor for neurons. In this regard, GDF-8 
may have applications in the treatment of neurodegenerative diseases, such 
as amyotrophic lateral sclerosis, or in maintaining cells or tissues in culture 
10 prior to transplantation. 

GDF-8 may also have applications in treating disease processes involving 
muscle, such as in musculodegenerative diseases or in tissue repair due to 
trauma. In this regard, many other members of the TGF-^ family are also 
important mediators of tissue repair. TGF-^ has been shown to have marked 
effects on the formation of collagen and to cause a striking angiogenic 
response in the newborn mouse (Roberts, et al.. Proc. Natl. Acad. Sci.. USA 
83:4167. 1986). TGF-^ has also been shown to inhibit the differentiation of 
myoblasts in culture (Massague, et al., Proc. Natl. Acad. Sci., USA 83:8206, 
1986). Moreover, because myoblast cells may be used as a vehicle for 
delivering genes to muscle for gene therapy, the properties of GDF-8 could be 
exploited for maintaining cells prior to transplantatton or for enhancing the 
efficiency of the fusion process. 

The expression of GDF-8 in adipose tissue also raises the possibility of 
applications for GDF-8 in the treatment of obesity or of disorders related to 
25 abnormal proliferation of adipocytes. In this regard. TGF-^ has been shown to 
be a potent inhibitor of adipocyte differentiation in vitro (Ignotz and Massague, 
Proc. Natl. Acad. Sci.. USA 82:8530, 1985). 



15 



20 



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-9- 

The term "substantially pure" as used herein refers to GDF-8 which is 
. substantially free of other proteins, lipids, carbohydrates or other materials with 
which it is naturally associated. One skilled in the art can purify GDF-8 using 
standard techniques for protein purification. The substantially pure polypeptide 
5 will yield a single major band on a non-reducing polyacrylamide gel. The purity 
of the GDF-8 polypeptide can also be determined by amino-terminal amino 
acid sequence analysis. GDF-8 polypeptide includes functional fragments of 
the polypeptide, as long as the activity of GDF-8 remains. Smaller peptides 
containing the biological activity of GDF-8 are included in the invention. 

The invention provides polynucleotides encoding the GDF-8 protein. These 
polynucleotides include DNA. cDNA and RNA sequences which encode GDF-8. 
It is understood that all polynucleotides encoding all or a portion of GDF-8 are 
also included herein, as long as they encode a polypeptide with GDF-8 activity. 
Such polynucleotides include naturally occurring, synthetic, and intentionally 
manipulated polynucleotides. For example. GDF-8 polynucleotide may be 
subjected to site-directed mutagenesis. The polynucleotide sequence for GDF- 
8 also includes antisense sequences. The polynucleotides of the invention 
include sequences that are degenerate as a result of the genetic code. There 
are 20 natural amino acids, most of which are specified by more than one 
codon. Therefore, all degenerate nucleotide sequences are Included in the 
invention as long as the amino acid sequence of GDF-8 polypeptide encoded 
by the nucleotide sequence is functionally unchanged. 

Specifically disclosed herein is a genomic DNA sequence containing a portion 
of the GDF-8 gene. The sequence contains an open reading frame 
25 corresponding to the predicted C-terminal region of the GDF-8 precursor 
protein. The encoded polypeptide is predicted to contain two potential 
proteolytic processing sites (KR and RR). Cleavage of the precursor at the 
downstream site would generate a mature biologically active C^terminal 



15 



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-10- 

fragment of 109 amino acids with a predicted molecular weight of 
approximately 12.400. Also, disclosed are full length murine and human GDF-8 
cDNA sequences. The murine pre-pro-GDF-8 protein is 376 amino acids in 
length, which is encoded by a 2676 base pair nucleotide sequence, beginning 
5 at nucleotide 104 and extending to a TGA stop codon at nucleotide 1232. The 
human GDF-8 protein is 375 amino acids and is encoded by a 2743 base pair 
sequence, with the open reading frame beginning at nucleotide 59 and 
extending to nucleotide 1184. 

The C-terminal region of GDF-8 following the putative proteolytic processing 
site shows significant homology to the known members of the TGF-^s 
superfamily. The GDF-8 sequence contains most of the residues that are 
highly consen/ed in other family members (see FIGURE 3). Like the IGF-fis 
and inhibin ^s, GDF-8 contains an extra pair of cysteine residues in addition to 
the 7 cysteines found in virtually all other family members. Among the known 
family members, GDF-8 is most homologous to Vgr-1 (45% sequence identity) 
(see FIGURE 4). 

Minor modifications of the recombinant GDF-8 primary amino acid sequence 
may result in proteins which have substantially equivalent activity, as compared 
to the GDF-8 polypeptide described herein. Such modifications may be 
20 deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the 
polypeptides produced by these modifications are included herein as long as 
the biological activity of GDF-8 still exists. Further, deletion of one or more 
amino acids can also result in a modification of the structure of the resultant 
molecule without significantly altering its biological activity. This can lead to the 
25 development of a smaller active molecule which would have broader utility. For 
example, one can remove amino or oarboxy terminal amino acids whrch are 
not required for GDF-8 biological activity. 



10 



15 



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The nucleotide sequence encoding the GDF-8 polypeptide of the invention 
includes the disclosed sequence and conservative variations thereof. The term 
"conservative variation" as used herein denotes the replacement of an amino 
acid residue by another, biologically similar residue. Examples of conservative 
5 variations include the substitution of one hydrophobic residue such as 
isoleucine, valine, leucine or methionine for another, or the substitution of one 
polar residue for another, such as the substitution of arginine for lysine, 
glutamic for aspartic acid, or glutamine for asparagine, and the like. The term 
"conservative variation" also includes the use of a substituted amino acid in 
1 0 place of an unsubstituted parent amino acid provided that antibodies raised to 
the substituted polypeptide also immunoreact with the unsubstituted polypep- 
tide. 

DNA sequences of the invention can be obtained by several methods. For 
example, the DNA can be isolated using hybridization techniques which are 

15 well known in the art. These include, but are not limited to: 1) hybridization of 
genomic or cDNA libraries with probes to detect homologous nucleotide 
sequences, 2) polymerase chain reaction (PGR) on genomic DNA or cDNA 
using primers capable of annealing to the DNA sequence of interest, and 3) 
antibody screening of expression libraries to detect cloned DNA fragments with 

20 shared structural features. 

Preferably the GDF-8 polynucleotide of the invention is derived from a 
mammalian organism, and most preferably from a mouse, rat, or human. 
Screening procedures which rely on nucleic acid hybridization make it possible 
to isolate any gene sequence from any organism, provided the appropriate 
25 probe is available. Oligonucleotide probes, which correspond to apart of the 
sequence encoding the protein in question, can be synthesized chemteally. 
This requires that short, oligopeptide stretches of amino acid sequence must 
be known. The DNA sequence encoding the protein can be deduced from the 



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genetic code, however, the degeneracy of the code must be taken into 
account. It Is possible to perform a mixed addition reaction when the 
sequence is degenerate. This includes a heterogeneous mixture of denatured 
double-stranded DNA. For such screening, hybridization is preferably 
5 performed on either single-stranded DNA or denatured double-stranded DNA. 
Hybridization is particularly useful in the detection of cDNA clones derived from 
sources where an extremely low amount of mRNA sequences relating to the 
polypeptide of interest are present. In other words, by using stringent 
hybridization conditions directed to avoid non-specific binding, it is possible. 
10 for example, to allow the autoradiographic visualization of a specific cDNA 
clone by the hybridization of the target DNA to that single probe in the mixture 
which is its complete complement (Wallace, et al., Nucl. Acid Res.. 9:879, 
1981). 

The development of specific DNA sequences encoding GDF-8 can also be 
1 5 obtained by: 1) isolation of double-stranded DNA sequences from the genomic 
DNA; 2) chemical manufacture of a DNA sequence to provide the necessary 
codons for the polypeptide of interest; and 3) in vitro synthesis of a double- 
stranded DNA sequence by reverse transcription of mRNA isolated from a 
eukaryotic donor cell. In the latter case, a double-stranded DNA complement 
20 of mRNA is eventually formed which is generally referred to as cDNA. 

Of the three above-noted methods for developing specific DNA sequences for 
use in recombinant procedures, the isolation of genomic DNA isolates is the 
least common. This is especially true when it is desirable to obtain the 
microbial expression of mammalian polypeptides due to the presence of 
25 introns. 



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The synthesis of DNA sequences is frequently the method of choice when the 
entire sequence of amino acid residues of the desired polypeptide product is 
known. When the entire sequence of amino acid residues of the desired 
polypeptide is not known, the direct synthesis of DNA sequences is not 
5 possible and the method of choice is the synthesis of cDNA sequences. 
Among the standard procedures for isolating cDNA sequences of interest is the 
formation of plasmid- or phage-carrying cDNA libraries which are derived from 
reverse transcription of mRNA which is abundant in donor cells that have a 
high level of genetic expression. When used in combination with polymerase 

10 chain reaction technology, even rare expression products can be cloned. In 
those cases where significant portions of the amino acid sequence of the 
polypeptide are known, the production of labeled single or double-stranded 
DNA or RNA probe sequences duplicating a sequence putatively present in the 
target cDNA may be employed in DNA/DNA hybridization procedures which are 

15 carried out on cloned copies of the cDNA which have been denatured into a 
single-stranded form (Jay, et al.. Nucl. Acid Res., 11:2325, 1983). 

A cDNA expression library, such as lambda gtll, can be screened irKiirectly 
for GDF-8 peptides having at least one epitope, using antibodies specific for 
GDF-8. Such antibodies can be either polyclonally or monoclonally derived 
20 and used to detect expression product indk^ative of the presence of GDF-S 
cDNA. 

DNA sequences encoding GDF-8 can be expressed in vitro by DNA transfer 
into a suitable host cell. "Host cells" are cells in which a vector can be 
propagated and its DNA expressed. The term also includes any progeny of 
25 the subject host cell. It is understood that all progeny may not be identteal to 
the parental cell since there may be mutations that occur during replrcation. 
However, such progeny are included when the term "host cell" is used. 



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-14- 

Methods of stable transfer, meaning that the foreign DNA is continuously 
maintained in the host, are known in the art. 

In the present invention, the GDF-8 polynucleotide sequences may be inserted 
into a recombinant expression vector. The term "recombinant expression 
5 vector*' refers to a plasmid. virus or other vehicle known in the art that has 
been manipulated by insertion or incorporation of the GDF-8 genetic 
sequences. Such expression vectors contain a promoter sequence which 
facilitates the efficient transcription of the inserted genetic sequence of the host. 
The expression vector typically contains an origin of replication, a promoter, as 

10 well as specific genes which allow 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 
expression in mammalian cells (Lee and Nathans, J. Biol, Chem., 263:3521, 

15 1988) and baculovirus-derived vectors for expression in insect cells. The DNA 
segment can be present in the vector operably linked to regulatory elements, 
for example, a promoter (e.g.. T7. metallothionein I, or polyhedrin promoters). 

Polynucleotide sequences encoding GDF-8 can be expressed in either 
prokaryotes or eukaryotes. Hosts can include mterobial, yeast, insect and 

20 mammalian organisms. Methods of expressing DNA sequences having 
eukaryotic or viral sequences in prokaryotes are well known in the art. 
Biologically functional viral and plasmid DNA vectors capable of expression and 
replication in a host are known in the art. Such vectors are used to incorp- 
orate DNA sequences of the invention. Preferably, the mature C-terminal 

25 region of GDF-8 is expressed from a cDNA clone containing the entire coding 
sequence of GDF-8. Alternatively, the C-terminal portion of GDF-8 can be 
expressed as a fusion protein with the pro- region of another member of the 
JGF'fi family or co-expressed with another pro- region (see for example. 



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-15- 

Hammonds, et al., Molec. Endocrin. 5:149. 1991; Gray, A., and Mason. A., 
Science, 247:1328. 1990). 

Transformation of a host cell with recombinant DNA may be carried out by 
conventional techniques as are well known to those skilled in the art. Where 
5 the host is prokaryotic. such as E. coii. competent cells which are capable of 
DNA uptake can be prepared from cells harvested after exponential growth 
phase and subsequently treated by the CaCl2 method using procedures well 
known in the art. Alternatively. MgCl2 or RbCI can be used. Transformation 
can also be performed after forming a protoplast of the host cell if desired. 

10 When the host is a eukaryote. such methods of transfection of DNA as calcium 
phosphate co-precipitates, conventional mechanical procedures such as 
microinjection, electroporation, insertion of a plasmid encased in liposomes, or 
virus vectors may be used. Eukaryotic cells can also be cotransformed with 
DNA sequences encoding the GDF-8 of the invention, and a second foreign 

15 DNA molecule encoding a selectable phenotype, such as the herpes simplex 
thymidine kinase gene. 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, (see. for example. 
Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Gluzman ed.. 1982). 

20 Isolation and purification of microbial expressed polypeptide, or fragments 
thereof, provided by the invention, may be carried out by conventional means 
including preparative chromatography and immunological separations involving 
monoclonal or polyclonal antibodies. 

The invention includes antibodies immunoreactive with GDF-8 polypeptide or 
25 functional fragments thereof. Antibody which consists essentially of pooled 
monoclonal antibodies with different epitopic specificities, as well as distinct 



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monoclonal antibody preparations are provided. Monoclonal antibodies are 
made from antigen containing fragments of the protein by methods well known 
to those skilled in the art (Kohler, et al., Nature. 256:495, 1975). The term 
antibody as used in this invention is meant to include intact molecules as well 
5 as fragments thereof, such as Fab and F(ab')2. which are capable of binding 
an epitopic determinant on GDF-8. 

The term "cell-proliferative disorder" denotes malignant as well as non-malignant 
cell populations which often appear to differ from the surrounding tissue both 
morphologically and genotypicaliy. Malignant cells (i.e. cancer) develop as a 

10 result of a multistep process. The GDF-8 polynucleotide that is an antisense 
molecule is useful in treating malignancies of the various organ systems, 
particularly, for example, cells in muscle or adipose tissue. Essentially, any 
disorder which is etiologically linked to altered expression of GDF-8 could be 
considered susceptible to treatment with a GDF-8 suppressing reagent. One 

15 such disorder is a malignant cell proliferative disorder, for example. 

The invention provides a method for detecting a cell prolifarative disorder of 
muscle or adipose tissue which comprises contacting an anti-GDF-8 antibody 
with a cell suspected of having a GDF-8 associated disorder and detecting 
binding to the antibody. The antibody reactive with GDF-8 is labeled with a 

20 compound which allows detection of binding to GDF-8. For purposes of the 
invention, an antibody specific for GDF-8 polypeptide may be used to detect 
the level of GDF-8 in biological fluids and tissues. Any specimen containing a 
detectable amount of antigen can be used. A preferred sample of this 
invention is muscle tissue. The level of GDF-8 in the suspect cell can be 

25 compared with the level In a normal cell to determine whether the subject has 
a GDF-8-associated cell proliferative disorder. Preferably the subject is human. 



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The antibodies of the invention can be used in any subject in which it is 
desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy. 
The antibodies of the invention are suited for use. for example, in immuno- 
assays in which they can be utilized in liquid phase or bound to a solid phase 
5 carrier. In addition, the antibodies in these immunoassays can be detectably 
labeled in various ways. Examples of types of immunoassays which can utilize 
antibodies of the invention are competitive and non-competitive immunoassays 
in either a direct or indirect format. Examples of such immunoassays are the 
radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection 
10 of the antigens using the antibodies of the invention can be done utilizing 
immunoassays which are run in either the forward, reverse, or simultaneous 
modes, including immunohistochemical assays on physiological samples. 
Those of skill in the art will know, or can readily discern, other immunoassay 
formats without undue experimentation. 

1 5 The antibodies of the invention can be bound to many different carriers and 
used to detect the presence of an antigen comprising the polypeptide of the 
invention. Examples of well-known carriers include glass, polystyrene, 
polypropylene, polyethylene, dextran, nylon, amylases, natural and modified 
celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier 

20 can be either soluble or insoluble for purposes of the invention. Those skilled 
in the art will know of other suitable carriers for binding antibodies, or will be 
able to ascertain such, using routine experimentation. 

There are many different labels and methods of labeling known to those of 
ordinary skill in the art. Examples of the types of labels which can be used in 
25 the present invention include enzymes, radioisotopes, fluorescent compounds, 
colloidal metals, chemiluminescent compounds, phosphorescent compounds, 
and bioluminescent compounds. Those of ordinary skill in the art will know of 



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other suitable labels for binding to the antibody, or will be able to ascertain 
such, using routine experimentation. 

Another technique which may also result in greater sensitivity consists of 
coupling the antibodies to low molecular weight haptens. These haptens can 
5 then be specifically detected by means of a second reaction. For example, it 
is common to use such haptens as biotin, which reacts with avidin, or 
dinitrophenyl, puridoxal, and fluorescein, which can react with specific anti- 
hapten antibodies. 

In using the monoclonal antibodies of the invention for the in vivo detection of 
10 antigen, the detectably labeled antibody is given a dose which is diagnostically 
effective. The term "diagnostically effective" means that the amount of 
detectably labeled monoclonal antibody is administered in sufficient quantity to 
enable detection of the site having the antigen comprising a polypeptide of the 
invention for which the monoclonal antibodies are specific. 

15 The concentration of detectably labeled monoclonal antibody which is 
administered should be sufficient such that the binding to those cells having 
the polypeptide is detectable compared to the background. . Further, it is 
desirable that the detectably labeled monoclonal antibody be rapidly cleared 
from the circulatory system in order to give the best target-to-backgrourKl 

20 signal ratio. 

As a rule, the dosage of detectably labeled monoclonal antibody for in vivo 
diagnosis will vary depending on such factors as age, sex, and extent of 
disease of the individual. Such dosages may vary, for example, depending on 
whether multiple injections are given, antigenic burden, and other factors 
25 known to those of skill in the art. 



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For in vivo diagnostic imaging, the type of detection instrument available is a 
major factor in selecting a given radioisotope. The radioisotope chosen must 
have a type of decay which is detectable for a given type of instrument Still 
another important factor in selecting a radioisotope for in vivo diagnosis is that 
5 deleterious radiation with respect to the host is minimized. Ideally, a radio- 
isotope used for in vivo imaging will lack a particle emission, but produce a 
large number of photons in the 140-250 keV range, which may readily be 
detected by conventional gamma cameras. 

For in vivo diagnosis radioisotopes may be bound to immunoglobulin either 
10 directly or indirectly by using an intermediate functional group. Intermediate 
functional groups which often are used to bind radioisotopes which exist as 
metallic ions to immunoglobulins are the bifunctional chelating agents such as 
diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid 
(EDTA) and similar molecules. Typical examples of metallic ions which can be 

111 Q7 R7 fifi 

1 5 bound to the monoclonal antibodies of the invention are In, Ru. Ga, Ga, 
72as 89zr.and ^Olj,. 

The monoclonal antibodies of the invention can also be labeled with a 
paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic 
resonance imaging (MR!) or electron spin resonance (ESR). In general, any 
20 conventional method for Nnsualizing diagnostic imaging can be utilized. Usually 
gamma and positron emitting radioisotopes are used for camera imagir^g and 
paramagnetic isotopes for MRI. Elements which are particularly useful in such 
techniques include ^ ^^Gd.^^Mn.^ ^^Dy.^^Cr.and ^®Fe. 

The monoclonal antibodies of the invention can be used in vitro and in vivo to 
25 monitor the course of amelioration of a GDF-8-associated disease in a subject. 
Thus, for example, by measuring the increase or decrease in the number of 
cells expressing antigen comprising a polypeptide of the invention or changes 



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in the concentration of such antigen present in various body fluids, it would be 
possible to determine whether a particular therapeutic regimen aimed at 
ameliorating the GDF-8-associated disease is effective. The term "ameliorate" 
denotes a lessening of the detrimental effect of the GDF-8-associated disease 
5 in the subject receiving therapy. 

The. present invention identifies a nucleotide sequence that can be expressed 
in an altered manner as compared to expression in a normal cell, therefore it 
is. possible to design appropriate therapeutic or diagnostic techniques directed 
to this sequence. Thus, where a cell-proliferative disorder is associated with 

10 the expression of GDF-8. nucleic acid sequences that interfere with GDF-8 
expression at the translational level can be used. This approach utilizes, for 
example, antisense nucleic acid and ribozymes to block translation of a specific 
GDF-8 mRNA, either by masking that mRNA with an antisense nucleic acid or 
by cleaving it with a ribozyme. Such disorders include neurodegenerative 

15 diseases, for example. 

Antisense nucleic acids are DNA or RNA molecules that are complementary to 
at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 
2g2:40, 1990). In the cell, the antisense nucleic acids hybridize to the 
corresponding mRNA, forming a double-stranded molecule. The antisense 

20 nucleic acids interfere with the translation of the mRNA. since the cell will not 
translate a mRNA that is double-stranded. Antisense oligomers of about 15 
nucleotides are preferred, since they are easily synthesized and are less likely 
to cause problems than larger molecules when introduced into the target GDF- 
8-producing cell. The use of antisense methods to inhibit the in vitro 

25 translation of genes is well known in the art {Marcus-Sakura, Anal.Biochem., 
172:289, 1988). 



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Ribozymes are RNA molecules possessing the ability to specifically cleave 
other single-stranded RNA in a manner analogous to DNA restriction 
endonucleases. Through the modification of nucleotide sequences which 
encode these RNAs, it is possible to engineer molecules that recognize specific 
5 nucleotide sequences in an RNA molecule and cleave it (Cech, J.Amer.Med. 
Assn., 260:3030. 1988). A major advantage of this approach is that, because 
they are sequence-specific, only mRNAs with particular sequences are 
inactivated. 

There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, 
10 Nature, 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes 
recognize sequences which are four bases in length, while "hammerhead"-type 
ribozymes recognize base sequences 11-18 bases in length. The longer the 
recognition sequence, the greater the likelihood that the sequence will occur 
exclusively in the target mRNA species. Consequently, hammerhead-type 
15 ribozymes are preferable to tetrahymena-type ribozymes for inactivating a 
specific mRNA species and 18-based recognition sequences are preferable to 
shorter recognition sequences. 

The present invention also provides gene therapy for the treatment of cell 
proliferative or immunologic disorders which are mediated toy GDF-8 protein. 

20 Such therapy would achieve its therapeutic effect by introduction of the GDF-8 
antisense polynucleotide into cells having the proliferative disorder. Delivery of 
antisense GDF-8 polynucleotide can be achieved using a recombinant expres- 
sion vector such as a chimeric virus or a colloidal dispersion system. 
Especially preferred for therapeutic delivery of antisense sequences is the use 

25 of targeted liposomes. 



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Various viral vectors which can be utilized for gene therapy as taught herein 
include adenovirus, herpes virus, vaccinia, or. preferably, an RNA virus such 
as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or 
avian retrovirus. Examples of retroviral vectors in which a single foreign gene 
5 can be inserted include, but are not limited to: Moloney murine leukemia virus 
(MoMuLV). Harvey murine sarcoma virus (HaMuSV), murine mammary tumor 
virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional 
retroviral vectors can incorporate multiple genes. All of these vectors can 
transfer or incorporate a gene for a selectable marker so that transduced cells 

10 can be identified and generated. By inserting a GOF-8 sequence of interest _ 
into the viral vector, along with another gene which encodes the ligand for a 
receptor on a specific target cell, for example, the vector is now target specific. 
Retroviral vectors can be made target specific by attaching, for example, a 
sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using 

15 an antibody to target the retroviral vector. Those of skill in the art will know of, 
or can readily ascertain without undue experimentation, specific polynucleotide 
sequences which can be inserted into the retroviral genome or attached to a 
viral envelope to allow target specific delivery of the retroviral vector containing 
the GDF-8 antisense polynucleotide. 

Since recombinant retroviruses are defective, they require assistance in order 
to produce infectious vector particles. This assistance can be provided, for 
example, by using helper cell lines that contain plasmkis encoding all of the 
structural genes of the retrovirus under the control of regulatory sequences 
within the LTR. These plasmids are missing a nucleotide sequence which . 
enables the packaging mechanism to recognize an RNA transcript for 
encapsidation. Helper cell lines which have deletions of the packaging signal 
include, but are not limited to ^2, PA317 and PA12. for example. These cell 
lines produce empty virions, since no genome is packaged. If a retroviral 
vector is introduced into such cells in which the packaging signal is intact, but 



20 



25 



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the structural genes are replaced by other genes of interest, the vector can be 
packaged and vector virion produced. 

Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected 
with plasmids encoding the retroviral structural genes gag. pol and env, by 
5 conventional calcium phosphate transfection. These cells are then transfected 
with the vector plasmid containing the genes of interest. The resulting cells 
release the retroviral vector into the culture medium. 

Another targeted delivery system for GDF-8 antisense polynucleotides is a 
colloidal dispersion system. Colloidal dispersion systems include macromole- 

10 cule complexes, nanocapsules, microspheres, beads, and lipid-based systems 
including oii-in-water emulsions, micelles, mixed micelles, and liposomes. The 
preferred colloidal system of this invention is a liposome. Liposomes are 
artificial membrane vesicles which are useful as delivery vehicles In vitro and 
in vivo. It has been shown that large unilamellar vesicles (LUV), which range 

15 in size from 0.2-4.0 /im can encapsulate a substantial percentage of an 
aqueous buffer containing large macromolecules. RNA, DNA and intact virions 
can be encapsulated within the aqueous interior and be delivered to cells in a 
biologically active form (Fraley, et al.. Trends Biochem, Sci., 6:77, 1981). In 
addition to mammalian cells, liposomes have been used for delivery of 

20 polynucleotides in plant, yeast and bacterial cells. In order for a liposome to 
be an efficient gene transfer vehicle, the following characteristics should be 
present: (1) encapsulation of the genes of interest at high efficiency while not 
compromising their biological activity; <2) preferential and substantial binding 
to a target cell in comparison to non-target cells; (3) delivery of the aqueous 

25 contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) 
accurate and effective expression of genetic infomnation (Mannino. et al., 
Biotechniques, 6:682, 1988). 



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The composition of the liposome is usually a combination of phospholipids, 
particularly high-phase-transition-temperature phospholipids, usually in 
combination with steroids, especially cholesterol. Other phospholipids or other 
lipids may also be used. The physical characteristics of liposomes depend on 
5 pH, ionic strength, and the presence of divalent cations. 

Examples of lipids useful in liposome production include phosphatidyl 
compounds, such as phosphatidylglycerol, phosphatidylcholine, 
phosphatidylserine,phosphatidylethanolamine.sphingolipids.cerebrosides,and 
gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid 
10 moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon 
atoms, and is saturated. Illustrative phospholipids include egg phosphatidyl- 
choline, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. 

The targeting of liposomes can be classified based on anatomical and 
mechanistic factors. Anatomical classification is based on the level of 

15 selectivity, for example, organ-specific, cell-specific, and organelle-specific. 
Mechanistic targeting can be distinguished based upon whether It is passive 
or active. Passive targeting utilizes the natural tendency of liposomes to 
distribute to cells of the reticulo-endothelial system (RES) in organs which 
contain sinusoidal capillaries. Active targeting, on the other hand, involves 

20 alteration of the liposome by coupling the liposome to a specific ligand such 
as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the 
composition or size of the liposome in order to achieve targeting to organs and 
cell types other than the naturally occurring sites of localization. 

The surface of the targeted delivery system may be modified in a variety of 
25 ways. In the case of a liposomal targeted delivery system, lipid groups can be 
incorporated into the lipid bilayer of the liposome in order to maintain the 



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•25- 

targeting ligand in stable association with the liposomal bilayer. Various linking 
groups can be used for joining the lipid chains to the targeting ligand. 

Due to the expression of GDF-8 in muscle and adipose tissue, there are a 
variety of applications using the polypeptide, polynucleotide, and antibodies of 
5 the invention, related to these tissues. Such applications include treatment of 
cell proliferative disorders involving these and other tissues, such as neural 
tissue. In addition, GDF-8 may be useful in various gene therapy procedures." 

The data in Example 6 shows that the human GDF-8 gene is located on 
chromosome 2. By comparing the chromosomal location of GDF-8 with the 

10 map positions of various human disorders, it should be possible to determine 
whether mutations in the GDF-8 gene are involved in the etiology of human 
diseases. For example, an autosomal recessive form of juvenile amyotrophic 
lateral sclerosis has been shown to map to chromosome 2 {Hentati. et al.. 
Neurology, 42 [Suppl.3]:201, 1992). More precise mapping of GDF-8 and 

15 analysis of DNA from these patients may indicate that GDF-8 is, in fact, the 
gene affected in this disease. In addition, GDF-8 is useful for distinguishing 
chromosome 2 from other chromosomes. 

The following examples are intended to illustrate but not limit the invention. 
While they are typical of those that might be used, other procedures known to 
20 those skilled in the art may alternatively be used. 



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EXAMPLE 1 

IDENTIFICATION AND ISOLATION OF A NOVEL 
TGF-B FAMILY MEMBER 

To identify a new member of the TGF-^ superfamily. degenerate 
5 oligonucleotides were designed which corresponded to two conserved regions 
among the known family members: ope region spanning the two tryptophan 
residues conserved in all family members except MIS and the other region 
spanning the invariant cysteine residues near the C-terminus, These primers 
were used for polymerase chain reactions on mouse genomic DNA followed 
1 0 by subcloning the PGR products using restriction sites placed at the 5' ends 
of the primers, picking individual E. coli colonies carrying these subcloned 
inserts, and using a combination of random sequencing and hybridization 
analysis to eliminate known members of the superfamily. 

GDF-8 was identified from a mixture of PGR products obtained with the primers 
1 5 SJL1 41 : 5'-GGGGAATTC<3GITGG(G/C/A)A(G/A/T/G)(A/G) A(T/G)TGG(A/G)TI 

(A/G)TI{T/G)GICG-3' <SEQ ID N0:1) 
SJL1 47: 5'-GGGGAATTG(G/A)GAI(G/G)C(G/A)GA{G/A)GT{G/A/T/G) 

TGIAGI(G/A)(T/G)GAT-3' (SEC ID N0:2) 

PGR using these primers was carried out with 2 ^g mouse genomic DNA at 
20 94**G for 1 min, 50^0 for 2 min. and 72**G for 2 min for 40 cycles. 

PGR products of approximately 280 bp were gel-purified, digested with Eco fll. 
gel"purrfied again, and subcloned in the Bluescript vector (Stratagene, San 
Diego, GA). Bacterial colonies carrying individual subclones were picked into 
96 well microtiter plates, and multiple replicas were prepared by plating the 
25 cells onto nitrocellulose. The replicate fitters were hybridized to probes 



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representing known members of the family, and DNA was prepared from non- 
hybridizing colonies for sequence analysis. 

The primer combination of SJL141 and SJL147, encoding the amino acid 
sequences GW(H/Q/N/K/D/E)(D/N)W(V/I/M)(V/I/M)(A/S)P (SEQ ID N0:9) and 
5 M(V/I/M/T/A)V(D/E)SC(G/A)C (SEQ ID NO:10). respectively, yielded four 
previously identified sequences (BMP-4, inhibin ^B, GDF-3 and GDF-5) and one 
novel sequence, which was designated GDF-8, among 110 subclones 
analyzed. 

Human GDF-8 was isolated using the primers: 

1 0 ACM1 3: 5'-CGCGGATCCAG AAGTCAAGGTGACAGACACAC-3' (SEQ ID N0:3) ; 
and 

ACM14: 5'-CGCGGATCCTCCTCATGAGCACCCACAGCGGTC-3' (SEQ ID N0;4) 

PGR using these primers was carried out with one ^g human genomic DNA at 
94"C for 1 min, 58*^0 for 2 min, and 72*'C for 2 min for 30 cycles. The PGR 
15 product was digested with Bam HI. gel-purified, and subcloned in the 
Bluescript vector (Stratagene, San Francisco. CA). 

EXAMPLE 2 

EXPRESSION PATTERN AND SEQUENCE OF GDF^ 

To determine the expression pattern of GDF-8. RNA samples prepared from 
20 a variety of adult tissues were screened by Northern analysis. RNA isolation 
and Northern analysis were carried out as described previously (Lee. S.-J.. 
Mol. Endocrinol.. 4:1034, 1990) except that hybridization was carried out in 5X 
SSPE. 10% dextran sulfate. 50% formamide. 1% SDS, 200 ^g/m\ salmon DNA, 
and 0.1% each of bovine serum albumin, ficoll. and polyvinylpyrrolidone. Five 



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micrograms of twice poly A-selected RNA prepared from each tissue {except 
for muscle, for which only 2 ^9 RNA was used) were electrophoresed on 
formaldehyde gels, blotted, and probed with GDF-8. As shown in FIGURE 1 , 
the GDF-8 probe detected a single mRNA species expressed at highest levels 
5 in muscle and at significantly lower levels in adipose tissue. 

To obtain a larger segment of the GDF-8 gene, a mouse genomic library was 
screened with a probe derived from the GDF-8 PGR product. The partial 
sequence of a GDF-8 genomic clone is shown in FIGURE 2a. The sequence 
contains an open reading frame corresponding to the predicted C-terminal 
region of the GDF-8 precursor protein. The predicted GDF-8 sequence 
contains two potential proteolytic processing sites, which are boxed. Cleavage 
of the precursor at the second of these sites would generate a mature 0- 
terminal fragment 109 amino acids in length with a predicted molecular weight 
of 12,400. The partial sequence of human GDF-8 is shown in FIGURE 2b. 
Assuming no PCR-induced errors during the isolation of the human clone, the 
human and mouse amino acid sequences in this region are 100% identical. 

The C-terminal region of GDF-8 following the putative proteolytic processing 
site shows significant homology to the known members of the TGF-jS 
superfamily (FIGURE 3). FIGURE 3 shows the alignment of the C-terminal 
20 sequences of GDF-8 with the corresponding regions of human GDF-1 (Lee, 
Proc. Natl. Acad. Sci. USA. 88:4250-4254, 1991), human BMP-2 and 4 
(Wozney, et al.. Science, 242:1528-1534. 1988), human Vgr-1 (Celeste, et al., 
Proc. Natl. Acad. Sci. USA, 82:9843-9847. 1990), human OP-1 (Ozkaynak, et 
a!.. EMBO J., 9:2085-2093. 1990), human BMP-5 (Celeste, et al., Proc. Natl. 
25 Acad. Sci. USA. 87:9843-9847, 1990), human BMP-3 (Wozney, et al.. Science, 
242:1528-1534, 1988), human MIS (Gate, et al.. Cell, 45:685-698, 1986), human 
inhibin alpha, and pB (Mason, et al., Biochem, Biophys. Res. Commun.. 
135:957-964. 1986), human TGf-pl (Derynck. et al.. Nature. 316:701-705. 



10 



15 



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1985). humanTGF-/92 (deMartin. et al., EMBO J., 6:3673-3677. 1987), and 
human TGF-/93 (ten Dijke, et a!.. Proc. Natl. Acad. Sci. USA. 85:4715-4719, 
1988). The conserved cysteine residues are boxed. Dashes denote gaps 
introduced in order to maximize the alignment 

5 GDF-8 contains most of the residues that are highly conserved in other family 
members, including the seven cysteine residues with their characteristic 
spacing. Like the TGF-^s and inhibin ^s, GDF-8 also contains two additional 
cysteine residues. In the case of TGF-^2, these two additional cysteine 
residues are known to form an intramolecular disulfide bond (Daopin, et al., 
10 Science. 25Z:369, 1992; Schlunegger and Grutter, Nature, 358:430, 1992). 

FIGURE 4 shows the amino acid homologies among the different members of 
the TGF-^ superfamily. Numbers represent percent amino acid identities 
between each pair calculated from the first conserved cysteine to the C- 
terminus. Boxes represent homologies among highly-related members within 
15 particular subgroups. In this region, GDF-8 is most honr>ologous to Vgr-1 (45% 
sequence identity). 

EXAMPLE 3 

ISOLATION OF cDNA CLONES ENCODING MURINE AND HUMAN GDF-8 

In order to isolate full-length cDNA clones encoding murine and human GDF-8, 
20 cDNA libraries were prepared in the lambda ZAP II vector (Stratagene) using 
RNA prepared from skeletal muscle. From 5 /ig of twice poly A-selected RNA 
prepared from murine and human muscle, cDNA libraries consisting of 4.4 
million and 1.9 million recombinant phage, respectively, were constructed 
according to the instructions provided by Stratagene. These libraries were 
25 screened without amplification. Library screening and characterizatton of cDNA 



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inserts were carried out as described previously (Lee, Mol. Endocrirx)!, 4:1034- 
1040). 

From 2,4 x 1 0^ recombinant phage screened from the murine muscle cDNA 
library, greater than 280 positive phage were identified using a murine GDF-8 

5 probe derived from a genomic clone, as described in Example 1 . The entire 
nucleotide sequence of the longest cDNA insert analyzed is shown in FIGURE 
5a and SEQ ID N0:11. The 2676 base pair sequence contains a single long 
open reading frame beginning with a methionine codon at nucleotide 104 and 
extending to a TGA stop codon at nucleotide 1232. Upstream of the putative 

10 initiating methionine codon is an in-frame stop codon at nucleotide 23. The 
predicted pre-pro-GDF-8 protein is 376 amino acids in length. The sequence 
contains a core of hydrophobic amino acids at the N-terminus suggestive of 
a signal peptide for secretion (FIGURE 6a), one potential N-glycosylation site 
at asparagine 72, a putative RXXR proteolytic cleavage site at amino acids 264- 

15 267, and a C-terminal region showing significant homology to the known 
members of the TGF-^ superfamily. Cleavage of the precursor protein at the 
putative RXXR site would generate a mature C-terminal GDF-8 fragment 109 
amino acids in length with a predicted molecular weight of approximately 
12,400. 

20 From 1.9 x 10^ recombinant phage screened from the human muscle cDNA 
library, 4 positive phage were identified using a human GDF-8 probe derived 
by polymerase chain reaction on human genomic DNA, The entire nucleotide 
sequence of the longest cDNA insert is shown in FIGURE 5b and SEQ ID 
N0:13. The 2743 base pair sequence contains a single long open reading 

25 frame beginning with a methionine codon at nucleotide 59 and extending to a 
TGA stop codon at nucleotide 1 184. The predicted pre-pro-GDF-8 protein is 
375 amino acids in length. The sequence contains a core of hydrophobic 
amino acids at the N-terminus suggestive of a signal peptide for secretion 



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(FIGURE 6b). one potential N-glycosylation site at asparagine 71, and a 
putative RXXR proteolytic cleavage site at amino acids 263-266. FIGURE 7 
shows a comparison of the predicted murine (top) and human (bottom) GDF-8 
amino acid sequences. Numbers indicate amino acid position relative to the 
5 N-terminus. Identities between the two sequences are denoted by a vertical 
line. Murine and human GDF-8 are approximately 94% identical in the 
predicted pro-regions and 100% identical following the predicted RXXR 
cleavage sites. 



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EXAMPLE 4 

PREPARATION OF AK'TIBODIES AGAINST GDF-8 AND 
EXPRESSION OF GDF-8 IN MAMMALIAN CELLS 

In order to prepare antibodies against GDF-8, GDF-8 antigen was expressed 
5 as a fusion protein in bacteria. A portion of murine GDF-8 cDNA spanning 
amino acids 268-376 (mature region) was inserted into the pRSET vector 
(Invitrogen) such that the GDF-8 coding sequence was placed in frame with the 
initiating methionine codon present in the vector; the resulting construct 
created an open reading frame encoding a fusion protein with a molecular 

10 weight of approximately 16.600. The fusion construct was transformed into 
BL21 {DE3) (pLysS) cells, and expression of the fusion protein was induced by 
treatment with isopropylthio-^-galactoside as described (Rosenberg, et al.. 
Gene, 56:125-135), The fusion protein was then purified by metal chelate 
chromatography according to the instructions provided by Invitrogen. A 

15 Coomassie blue-stained gel of unpurified and purified fusion proteins is shown 
in FIGURE 8. 

The purified fusion protein was used to immunize both rabbits and chickens. 
Immunization of rabbits was carried out by Spring Valley Labs (Sykesvilie, MD), 
and immunization of chickens was carried out by HRP, Inc. (Denver, PA). 
20 Western analysis of sera both from immunized rabbits and from immunized 
chickens demonstrated the presence of antibodies directed against the fusion 
protein. 

To express GDF-8 in mammalian cells, the murine GDF-8 cDNA sequence from 
nucleotides 48-1303 was cloned in both orientations downstream of the 
25 metallothionein I promoter in the pMSXND expression vector; this vector 
contains processing signals derived from SV40, a dihydrofolate reductase 
gene, and a gene conferring resistance to the antibiotic G418 <Lee and 



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Nathans, J. Biol. Chem., 263:3521-3527). The resulting constructs were 
transfected into Chinese hamster ovary cells, and stable tranfectants were 
selected in the presence of G418. Two milliliters of conditioned rriedia 
prepared from the G41 8-resistant cells were dialyzed, lyophilized, 
5 electrophoresed under denaturing, reducing conditions, transferred to 
nitrocellulose, and incubated with anti-GDF-8 antibodies (described above) and 
[^^^l]iodoprotein^. 

As shown in FIGURE 9, the rabbit GDF-8 antibodies (at a 1:500 dilution) 
detected a protein of approximately the predicted molecular weight for the 

10 mature G-terminal fragment of GDF-8 in the conditioned media of cells 
transfected with a construct in which GDF-8 had been cloned in the correct 
(sense) orientation with respect to the metallothionein promoter (lane 2); this 
band was not detected in a similar sample prepared from cells transfected with 
a control antisense construct (lane 1). Similar results were obtained using 

15 antibodies prepared in chickens. Hence, GDF-8 is secreted and proteolytically 
processed by these transfected mammalian cells. 

EXAMPLE 5 
EXPRESSION PATTERN OF GDF-8 

To determine the pattern of GDF-8, 5 m9 of twice poly A-selected RNA 
20 prepared from a variety of murine tissue sources were subjected to Northern 
analysis. As shown in FIGURE 10a (and as shown previously in Example 2), 
the GDF-8 probe detected a single mRNA species present almost exclusively 
in skeletal muscle among a large number of adult tissues surveyed. On longer 
exposures of the same blot, significantly lower -but detectable levels of GDF-8 
25 mRNA were seen in fat, brain, thymus, heart, and lung. Hence, these results 
confirm the high degree of specificity of <aDF-8 expression in skeletal muscle. 
GDF-8 mRNA was also detected in mouse embryos at both gestational ages 



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(day 12.5 and day 18.5 post-coital) examined but not in placentas at various 
stages of development (FIGURE 10b). 

EXAMPLE 6 
CHROMOSOMAL LOCALIZATION OF GDF-8 

5 In order to map the chromosomal location of GDF-8, DNA samples from 
human/rodent somatic cell hybrids (DnA^inga. et al., Genomics, 16:311-413. 
1993; Dubois and Naylor, Genomics, 16:315-319. 1993) were analyzed by 
polymerase chain reaction followed by Southern blotting. Polymerase chain 
reaction was carried out using primer #83, 5'- 

10 CGCGGATCCGTGGATCTAAATGAGAACAGTGAGC-3' (SEQ ID N0:15) and 
primer #84, 5'-GGCGAATTCTCAGGTAATGATTGTTTCCGTTGTAGCG-3'{SEQ 
ID N0:16) for 40 cycles at 94*0 for 2 minutes, 60"C for 1 minute, and 72'*C 
for 2 minutes. These primers correspond to nucleotides 119 to 143 (flanked 
by a Bam H1 recognition sequence), and nucleotides 394 to 418 (flanked by 

15 an Eco R1 recognition sequence), respectively, in the human GDF-8 cDNA 
sequence. PGR products were electrophoresed on agarose gels, blotted, and 
probed with oligonucleotide #100, 5'-ACACTAAATCTTCAAGAATA-3' (SEQ ID 
N0:17), which corresponds to a sequence internal to the region flanked by 
primer #83 and #84. Filters were hybridized in 6 X SSC. 1 X Denhardt's 

20 solution. 100Mg/ml yeast transfer RNA. and 0.05% sodium pyrophosphate at 
50^C. 

As shown in FIGURE 1 1 . the human-specific probe detected a band of the 
predicted size (approximately 320 base pairs) in the positive control sample 
(total human genomic DNA) and in a single DNA sample from the 
25 human/rodent hybrid panel. This positive signal corresponds to human 
chromosome 2. The human chromosome contained in each of the hybrid cell 
lines is identified at the top of each of the first 24 lanes (1-22. X, and Y). In the 



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lanes designated M, CHO. and H, the starting DNA template was total genomic 
DNA from mouse, hamster, and human sources, respectively. In the lane 
marked B1 . no template DNA was used. Numbers at left indicate the mobilities 
of DNA standards. These data show that the human GDF-8 gene is located 
5 on chromosome 2. 

Although the invention has been described with reference to the presently 
preferred embodiment, it should be understood that various modifications can 
be made without departing from the spirit of the invention. Accordingly, the 
invention is limited only by the following claims. 



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SUMMARY OF SEQUENCES 

SEQ ID NO: 1 is the nucleic acid sequence for clone SJL141. 

SEQ ID NO: 2 is the nucleic acid sequence for clone SJL147. 

SEQ ID NO: 3 is the nucleic acid sequence for clone ACM13. 

5 SEQ ID NO: 4 is the nucleic acid sequence for clone ACM14. 

SEQ ID NO: 5 is the partial nucleotide sequence and deduced amino acid 
sequence for murine GDF-8. 

SEQ ID NO: 6 is the deduced partial amino acid sequence for murine GDF-8. 

SEQ ID NO: 7 is the partial nucleotide sequence and deduced amino add 
10 sequence for human GDF-8. 

SEQ ID NO: 8 is the deduced partial amino acid sequence for human GDF-8. 

SEQ ID NO: 9 is the amino acid sequence for primer SJL141. 

SEQ ID NO: 10 is the amino acid sequence for primer SJL147. 

SEQ ID NO: 1 1 is the nucleotide and deduced amino acid sequence for murine . 
15 GDF-S. 

SEQ ID NO: 12 is the deduced amino acid sequence for murine GDF-8. 



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SEQ ID NO: 13 is the nucleotide and deduced amino acid sequence for human 
GDF-8. 

SEQ ID NO: 14 is the deduced amino acid sequence for human GDF-8. 

SEQ ID NO*s: 15 and 16 are nucleotide sequences for primer #83 and #84, 
5 respectively, which were used to map human GDF-8 in human/rodent somatic 
cell hybrids. 

SEQ ID N0:17 is the nucleotide sequence of oligonucleotide #100 which 
corresponds to a sequence internal to the region flanked by primer #83 and 
#84. 



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SEQUENCE LISTING 

(1) GENERAL INFORMATION: 

(i) APPLICANT: THE JOHNS HOPKINS UNIVERSITY 

6 (ii) TITLE OF INVENTION: GROWTH DIFFERENTIATION FACTOR-8 

(iii) NUMBER OF SEQUENCES: 17 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: Spensley Horn Jubas & Lubitz 
10 (B) STREET: 1880 Century Park East - Suite 500 

(C) CITY: Los Angeles 

(D) STATE: California 

(E) COUNTRY: USA 

(F) ZIP: 90067 

15 (v) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

(C) OPERATING SYSTEM: PC- DOS/MS • DOS 

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 

20 (vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: PCT 

(B) FILING DATE: 18-MAR-1994 

(C) CLASSIFICATION: 

(viii) ATTORNEY/AGENT INFORMATION: 
25 (A) NAME: Wetherell. Jr., Ph.D., John R. . 

(B) REGISTRATION NUMBER: 31,678 

(C) REFERENCE/DOCKET NUMBER: FD-3A13 CIP PCT 

(ix) TELECOMMUNICATION INFORMATION: 
(A) TELEPHONE: (619) 455-5100 
30 (B) TELEFAX: (619) 455-5110 

(2) INFORMATION FOR SEQ ID N0:1: 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 



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(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 
(I>) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



5 (vii) IMMEDIATE SOURCE: 

(B) CLONE: SJL141 

(ix) FEATURE: 

(A) NAME/KEY: modif ied^base 

(B) LOCATION: 1..35 

10 (D) OTHER INFORMATION: /mod_base" i 

/note= ""B" is defined as "I" (inosine)' 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 

CCGGAATTCG GBTGGVANRA YTGGRTBRTB KCBCC 
35 

15 (2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 33 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 
20 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: SJLU7 

(ix) FEATURE: 
25 (A) NAME/KEY: CDS 

(B) LOCATION: 1. .33 

(ix) FEATURE: 

(A) NAME/KEY: modif ied^base 

(B) LOCATION: 1..33 

30 (D) OTHER INFORMATION: /mod^base- i 

/note- ""B" is defined as "I" (inosine)" 



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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

CCGGAATTCR CABSCRCARC TNTCBACBRY CAT 

.33 

(2) INFORMATION FOR SEQ ID NO: 3: 

5 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 32 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

10 (ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: ACM13 

(ix) FEATURE: 

(A) NAME/KEY: CDS 
15 (B) LOCATION: 1..32 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

CGCGGATCCA GAAGTCAAGG TGACAGACAC AC 
32 

(2) INFORMATION FOR SEQ ID NO: 4: 

20 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 33 base pairs 

(B) TYPE: nucleic acid 

(C) iSTRANDEDNESS : single 

(D) TOPOLOGY: linear 

25 (ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: ACM14 



(ix) FEATURE: 



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(A) NAME/KEY: CDS 

(B) LOCATION: 1..33 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: 

CGCGGATCCT CCTCATGAGC ACCCACAGCG GTC 
5 33 

(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 550 base pairs 

(B) TYPE: nucleic acid 
10 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: mouse GDF-8 

15 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 59.. 436 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

TTAAGGTAGG AAGGATTTCA GGCTCTATTT ACATAATTGT TCTTTCCTTT TCACACAG 
20 58 

AAT CCC TTT TTA GAA GTC AAG GTG ACA GAC ACA CCC AAG AGG TCC CGG 
106 

Asn Pro Phe Leu Glu Val Lys Val Thr Asp Thr Pro Lys Arg Ser Arg 
1 5 10 15 

25 AGA GAC TTT GGG CTT GAC TGC GAT GAG CAC TCC ACG GAA TCC CGG TGC 

154 

Arg Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys 
20 25 30 

TGC CGC TAC CCC CTC ACG GTC GAT TTT GAA GCC TTT GGA TGG GAC TCG 
30 202 



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Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp 
35 40 45 

ATT ATC GCA CCC AAA AGA TAT AAG GCC Akl TAG TGC TCA GGA GAG TGT 
250 

5 He He Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys 
50 55 60 

GAA TTT GTG TTT TTA CAA AAA TAT CCG CAT ACT CAT CTT GTG CAC CAA 
298 

Glu Phe Val Phe Leu Gin Lys Tyr Pro His Thr His Leu Val His Gin 
10 65 70 75 80 

GCA AAC CCC AGA GGC TCA GCA GGC CCT TGC TGC ACT CCG ACA AAA ATG 
346 

Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met 
85 90 95 

15 TCT CCG ATT AAT ATG CTA TAT TTT AAT GGC AAA GAA CAA ATA ATA TAT 

394 

Ser Pro He Asn Met Leu Tyr Phe Asn Gly Lys Glu Gin He He Tyr 
100 105 110 

GGG AAA ATT CCA GCC ATG GTA GTA GAC CGC TGT GGG TGC TCA 
20 436 

Gly Lys He Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 
115 120 125 

TGAGCTTTGC ATTAGGTTAG AAACTTCCCA AGTCATGGAA GGTCTTCCCC TCAATTTGGA 
496 

25 AACTGTGAAT TCCTGCAGCC CGGGGGATCC ACTAGTTCTA GAGCGGCCGC CACC 

550 



(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 126 amino acids 
30 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 



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-43- 

Asn Pro Phe Leu Glu Val Lys Val Thr Asp Thr Pro Lys Arg Ser Arg 

1 5 ' 10 15 

Arg Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys 

20 25 30 

5 Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp 

35 40 45 

lie lie Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys 

50 55 60 



Glu Phe Val Phe Leu Gin Lys Tyr Pro His Thr His Leu Val His Gin 
10 65 70 75 80 

Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met 
85 90 95 

Ser Pro lie Asn Met Leu Tyr Phe Asn Gly Lys Glu Gin lie lie Tyr 
100 105 110 

15 Gly Lys He Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 
115 120 125 

(2) INFORMATION FOR SEQ ID N0:7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 326 base pairs 
20 (B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
25 (B) CLONE: human GDF-8 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 3. .326 



(xi) 



SEQUENCE DESCRIPTION: SEQ ID NO: 7: 



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CA AAA AGA TCG AGA AGG GAT TTT GGT CTT GAG TGT GAT GAG CAC TCA 
A7 

Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys Asp Glu His Ser 
15 10 15 

5 ACA GAA TCA CGA TGC TGT CGT TAG CCT CTA ACT GTG GAT TTT GAA GCT 
95 

Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala 
20 25 30 

TTT GGA TGG GAT TGG ATT ATC GCT CCT AAA AGA TAT AAG GCC AAT TAG 
10 143 

Phe Gly Trp Asp Trp lie lie Ala Pro Lys Arg Tyr Lys Ala Asn Tyr 
35 40 45 

TGC TGT GGA GAG TGT GAA TTT GTA TTT TTA CAA AAA TAT CCT CAT ACT 
191 

15 Cys Ser Gly Clu Cys Glu Phe Val Phe Leu Gin Lys Tyr Pro His Thr 
50 55 60 

CAT CTG GTA CAC CAA GGA AAC CCC AGA CCT TCA GGA GGC CCT TGC TGT 
239 

His Leu Val His Gin Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys 
20 65 70 75 

ACT CCC ACA AAG ATG TCT CCA ATT AAT ATG CTA TAT TTT AAT GGC AAA 
287 

Thr Pro Thr Lys Met Ser Pro lie Asn Met Leu Tyr Phe Asn Gly Lys 
80 85 90 95 

25 GAA CAA ATA ATA TAT GGG AAA ATT CCA <;CG ATG GTA GTA 

326 

Glu Gin He He Tyr Gly Lys He Pro Ala Met Val Val 
100 105 



(2) INFORMATION FOR SEQ ID NO: 8: 

30 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 108 amino acids 

(B) TYPE: amino acid 
<D) TOPOLOGY: linear 



<ii) MOLECULE TYPE: protein 



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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr 
1 5 10 15 

Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe 
5 20 25 30 

Gly Trp Asp Trp lie lie Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys 
35 ^0 ^ A5 

Ser Gly Glu Cys Glu Phe Val Phe Leu Gin Lys Tyr Pro His Thr His 
50 55 60 

10 Leu Val His Gin Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr 
65 70 75 80 

Pro Thr Lys Met Ser Pro lie Asn Met Leu Tyr Phe Asn Gly Lys Glu 
85 90 95 

Gin He He Tyr Gly Lys He Pro Ala Met Val Val 
15 100 105 



(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 9 amino acids 

(B) TYPE: amino acid 

20 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: peptide 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: SJL141 



25 (ix) FEATURE: 

(A) NAME/KEY: Peptide 

(B) LOCATION: 1. .9 

(D) OTHER INFORMATION: /note« "His - His. Asn. Lys, Asp or 
Glu; Asp «= Asp or Asn; Val = Val, He or Met; Ala 
30 - Ala or Ser." 



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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Gly Trp His Asp Trp Val Val Ala Pro 
1 5 

(2) INFORMATION FOR SEQ ID NO: 10: 

5 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 8 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

10 (ii) MOLECULE TYPE: peptide 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: SJL147 

(ix) FEATURE: 

(A) NAME/KEY: Peptide 
15 (B) LOCATION: 1..8 

(D) OTHER INFORMATION: /not€= "He - He, Val. Met, Thr or 
Ala; Asp = Asp or Glu; Gly « Gly or Ala." 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Met He Val Asp Ser Cys Gly Cys 
20 1 5 

(2) INFORMATION FOR SEQ ID NO: 11: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 2676 base pairs 

(B) TYPE: nucleic acid 
25 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) 



IMMEDIATE SOURCE: 
(B) CLONE: Murine GDF-8 



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(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 104.. 1231 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 

5 GTCTCTCGGA CGGTACATGC ACTAATATTT CACTTGGCAT TACTCAAAAG CAAAAAGAAG 

60 

AAATAAGAAC AAGGGAAAAA AAAAGATTGT GCTGATTTTT AAA ATG ATG CAA AAA 
115 

Met Met Gin Lys 
10 1 

CTG CAA ATG TAT GTT /TAT ATT TAC CTG TTC ATG CTG ATT GCT GCT GGC 
163 

Leu Gin Met Tyr Val Tyr lie Tyr Leu Phe Met Leu lie Ala Ala Gly 
5 10 15 20 

15 CCA GTG GAT CTA AAT GAG GGC AGT GAG AGA GAA GAA AAT GTG GAA AAA 

211 

Pro Val Asp Leu Asn Glu Gly Ser Glu Arg Glu Glu Asn Val Glu Lys 
25 30 35 

GAG GGG CTG TGT AAT GCA TGT GCG TGG AGA CAA AAC ACG A<;G TAC TCC 
20 259 

Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gin Asn Thr Arg Tyr Ser 
40 45 50 

AGA ATA GAA GCC ATA AAA ATT CAA ATC CTC AGT AAG CTG GGC CTG GAA 
307 

25 Arg lie Glu Ala He Lys He Gin He Leu Ser Lys Leu Arg Leu Glu 
55 60 65 

ACA GCT CCT AAC ATC AGC AAA GAT GCT ATA AGA CAA CTT CTG CCA AGA 
355 

Thr Ala Pro Asn He Ser Lys Asp Ala He Arg Gin Leu Leu Pro Arg 
30 70 75 80 

GCG CCT CCA CTC CGG GAA CTG ATC GAT CAG TAC GAC GTG CAG AGG -GAT 
403 

Ala Pro Pro Leu Arg Glu Leu He Asp Gin Tyr Asp Val Gin Arg Asp 
85 90 95 100 



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GAC AGC ACT GAT GGC TCT TTG GAA GAT GAC GAT TAT CAC GCT ACC ACG 
451 

Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His Ala Thr Thr 
105 110 115 

5 GAA ACA ATC ATT ACC ATG CCT ACA GAG TCT GAC TTT CTA ATG CAA GCG 

499 

Glu Thr lie lie Thr Met Pro Thr Glu Ser Asp Phe Leu Met Gin Ala 
120 125 130 

GAT GGC AAG CCC AAA TGT TGC TTT TTT AAA TTT AGC TCT AAA ATA CAG 
10 547 

Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser Lys lie Gin 
135 140 145 

TAG AAC AAA GTA GTA AAA GCC CAA CTG TGG ATA TAT CTC AGA CCC GTC 
595 

15 Tyr Asn Lys Val Val Lys Ala Gin Leu Trp lie Tyr Leu Arg Pro Val 

150 155 160 

AAG ACT CCT ACA ACA GTG TTT GTG CAA ATC CTG AGA CTC ATC AAA CCC 
643 

Lys Thr Pro Thr Thr Val Phe Val Gin lie Leu Arg Leu lie Lys Pro 
20 165 170 175 180 

ATG AAA GAC GGT ACA AGG TAT ACT GGA ATC CGA TCT CTG AAA CTT GAC 
691 

Met Lys Asp Gly Thr Arg Tyr Thr Gly lie Arg Ser Leu Lys Leu Asp 
185 190 195 

25 ATG AGC CCA GGC ACT GGT ATT TGG CAG AGT ATT GAT GTG AAG ACA CTG 

739 

Met Ser Pro Gly Thr Gly He Trp Gin Ser He Asp Val Lys Thr Val 
200 205 210 

TTG CAA AAT TGG CTC AAA CAG CCT GAA TCC AAC TTA GGC ATT GAA ATC 
30 787 

Leu Gin Asn Trp Leu Lys Gin Pro Glu Ser Asn Leu Gly He Glu He 
215 220 225 

AAA GCT TTG GAT GAG AAT GGC CAT GAT CTT GCT GTA ACC TTC CCA GGA 
835 

35 Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr Phe Pro Gly 
230 235 240 



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CCA GGA GAA GAT GGG CTG AAT CCC TTT TTA GAA GTC AAG GTG ACA GAC 
883 

Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys Val Thr Asp 
245 250 255 260 

5 ACA CCC AAG AGG TCC GGG AGA GAC TTT GGG CTT GAC TGC GAT GAG CAC 

931 

Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys Asp Glu His 
265 270 275 

TCC ACG GAA TCC CGG TGC TGC CGC TAG CCC CTC ACG GTC CAT TTT GAA 
10 979 

Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu 
280 285 290 

GCC TTT GGA TGG GAC TGG ATT ATC GCA CCC AAA AGA TAT AAG GCC AAT 
1027 

15 Ala Phe Gly Trp Asp Trp He He Ala Pro Lys Arg Tyr Lys Ala Asn 
295 300 305 

TAG TGC TCA GGA GAG TGT GAA TTT GTG TTT TTA CAA AAA TAT CGG CAT 
1075 

Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gin Lys Tyr Pro His 
20 310 315 320 

ACT CAT CTT GTG CAC CAA GCA AAC CCC AGA GGG TCA GCA GGG CCT TGC 
1123 

Thr His Leu Val His Gin Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys 
325 330 335 340 

25 TGC ACT CCG ACA AAA ATG TCT CCC ATT AAT ATG CTA TAT TTT AAT GGC 

1171 

Cys Thr Pro Thr Lys Met Ser Pro He Asn Met Leu Tyr Phe Asn -Gly 
345 350 355 

AAA GAA CAA ATA ATA TAT GGG AAA ATT CCA GCC ATG GTA GTA GAC C-GC 
30 1219 

Lys Glu Gin He He Tyr Gly Lys He Pro Ala Met Val Val Asp Arg 
360 365 370 

TGT GGG TGC TCA TGAGCTTTGC ATTAGGTTAG AAACTTCCCA ACTCATGGAA 
1271 

35 Cys Gly Cys Ser 
375 



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GGTCTTCCCC TCAATTTCGA AACTGTGAAT TCAAGCACCA CAGGCTGTAG CGCTTGAGTA 
1331 

TGCTCTAGTA ACGTAAGCAC AAGCTACAGT GTATGAACTA AAAGAGAGAA TAGATGCAAT 
1391 

5 GGTTGGCATT CAACCACCAA AATAAACCAT ACTATAGGAT GTTGTATGAT TTCCAGAGTT 

1451 

TTTGAAATAG ATGGAGATCA AATTACATTT ATGTCCATAT ATGTATATTA CAACTACAAT 
1511 

CTAGGCAAGG AAGTGAGAGC ACATCTTGTG GTCTGCTGAG TTAGGAGGGT ATGATTAAAA 
10 1571 

GGTAAAGTCT TATTTCCTAA CAGTTTCACT TAATATTTAC AGAAGAATCT ATATGTAGCC 
1631 

TTTGTAAAGT GTAGGATTGT TATCATTTAA AAACATCATG TACACTTATA TTTGTATTGT 
1691 

15 ATACTTGGTA AGATAAAATT CCACAAAGTA GGAATGGGGC CTCACATACA CATTGCCATT 

1751 

CCTATTATAA TTGGACAATC CACCACGGTG CTAATGCAGT GCTGAATCGC TCCTACTGGA 
1811 

CCTCTCGATA GAACACTCTA CAAAGTACGA GTCTCTCTCT CCCTTCCAGG TGCATCTCCA 
20 1871 

CACACACAGC ACTAAGTGTT CAATGCATTT TCTTTAAGGA AAGAAGAATC TTTTTTTCTA 
1931 

GAGGTCAACT TTCAGTCAAC TCTAGCACAG CGGGAGTGAC TGCTGCATCT TAAAAGGGAG 
1991 

25 CCAAACAGTA TTCATTTTTT AATCTAAATT TCAAAATCAC TGTCTGCCTT TATCACAT€G 

2051 

CAATTTTGTG GTAAAATAAT GGAAATGACT GGTTCTATCA ATATTGTATA AAAGACTCTG 
2111 

AAACAATTAC ATTTATATAA TATGTATACA ATATT<3TTTT GTAAATAAGT GTCTCCTTTT 
30 2171 



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PCT/US94/03019 



ATATTTACTT TGGTATATTT TTACACTAAT GAAATTTCAA ATCATTAAAG TACAAAGACA 
2231 

TGTCATGTAT CACAAAAAAG GTGACTGCTT CTATTTCAGA GTGAATTAGC AGATTCAATA 
2291 

5 GTGGTCTTAA AACTCTGTAT GTTAAGATTA GAAGGTTATA TTACAATCAA TTTATGTATT 

2351 

TTTTACATTA TCAACTTATG GTTTCATGGT GGCTGTATCT ATGAATGTGG CTCCCAGTCA 
2411 

AATTTCAATG CCCCACCATT TTAAAAATTA CAAGCATTAC TAAACATACC AACATGTATC 
10 2471 

TAAAGAAATA CAAATATGGT ATCTCAATAA CAGCTACTTT TTTATTTTAT AATTTGACAA 
2531 

TGAATACATT TCTTTTATTT ACTTCAGTTT TATAAATTGG AACTTTGTTT ATCAAATGTA 
2591 

15 TTGTACTCAT AGCTAAATGA AATTATTTCT TACATAAAAA TGTGTAGAAA CTATAAATTA 

2651 

AAGTGTTTTC ACATTTTTGA AAGGC 
2676 



(2) INFORMATION FOR SEQ ID NO: 12: 

20 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 376 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 

Met Met Gin Lys Leu Gin Met Tyr Val Tyr He Tyr Leu Phe Met Leu 
1 5 10 15 



He Ala Ala Gly Pro Val Asp Leu Asn Glu Gly Ser Glu Arg Glu Glu 
20 25 30 



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Asn Val Glu Lys Glu Gly Leu Cys 
35 40 

Thr Arg Tyr Ser Arg He Glu Ala 
50 55 

5 Leu Arg Leu Glu Thr Ala Pro Asn 
65 70 

Leu Leu Pro Arg Ala Pro Pro Leu 
85 



-52- 

Asn Ala Cys Ala Trp Arg Gin Asn 
45 

He Lys lie Gin He Leu Ser Lys 
60 

He Ser Lys Asp Ala He Arg Gin 
75 80 

Arg Glu Leu He Asp Gin Tyr Asp 
90 95 



Val Gin Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr 
10 100 105 110 

His Ala Thr Thr Glu Thr He He Thr Met Pro Thr Glu Ser Asp Phe 
115 120 125 

Leu Met Gin Ala Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser 
130 135 140 

15 Ser Lys He Gin Tyr Asn Lys Val Val Lys Ala Gin Leu Trp He Tyr 

145 150 155 160 

Leu Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gin He Leu Arg 
165 170 175 



Leu He Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly He Arg Ser 
20 180 185 190 

Leu Lys Leu Asp Met Ser Pro Gly Thr Gly He Trp Gin Ser He Asp 
195 200 205 



Val Lys Thr Val Leu Gin Asn Trp Leu Lys Gin Pro Glu Ser Asn Leu 
210 215 220 

25 Gly He Glu He Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val 

225 230 235 240 



Thr Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val 
245 250 255 



Lys Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp 
30 260 265 270 



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Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 
275 280 285 

Val Asp Phe Glu Ala Phe Gly Trp Asp Trp lie He Ala Pro Lys Arg 
290 295 300 

5 Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gin 
305 310 315 320 

Lys Tyr Pro His Thr His Leu Val His Gin Ala Asn Pro Arg Gly Ser 
325 330 335 

Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro He Asn Met Leu 
10 3A0 345 350 

Tyr Phe Asn Gly Lys Glu Gin He He Tyr Gly Lys He Pro Ala Met 
355 360 365 

Val Val Asp Arg Cys Gly Cys Ser 
370 375 

15 (2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 2743 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 
20 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 

(B) CLONE: Human GDF-8 

(ix) FEATURE: 
25 (A) NAME/KEY: CDS 

(B) LOCATION: 59.. 1183 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 

AAGAAAAGTA AAAGGAAGAA ACAAGAACAA GAAAAAAGAT TATATTGATT TTAAAATC 
58 



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-54- 



ATG CAA AAA CTG CAA CTC TGT GTT TAT ATT TAG CTG TTT ATG CTG ATT 
106 

Met Gin Lys Leu Gin Leu Cys Val Tyr He Tyr Leu Phe Met Leu He 
1 3 10 15 

5 GTT GCT GGT CCA GTG GAT CTA AAT GAG AAC ACT GAG CAA AAA GAA AAT 

154 

Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gin Lys Glu Asn 
20 25 30 

GTG GAA AAA GAG GGG CTG TGT AAT GCA TGT ACT TGG AGA CAA AAC ACT 
10 202 

Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gin Asn Thr 
35 40 45 

AAA TCT TCA AGA ATA GAA GCC ATT AAG ATA CAA ATC CTC ACT AAA CTT 
250 

15 Lys Ser Ser Arg He Glu Ala He Lys He Gin He Leu Ser Lys Leu 

50 55 60 

CGT CTG GAA ACA GCT CCT AAC ATC AGC AAA GAT GTT ATA AGA CAA CTT 
298 

Arg Leu Glu Thr Ala Pro Asn He Ser Lys Asp Val He Arg Gin Leu 
20 65 70 75 80 

TTA CCC AAA GCT CCT CCA CTC CGG GAA CTG ATT GAT CAG TAT GAT GTC 
346 

Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu He Asp Gin Tyr Asp Val 
85 90 95 

25 CAG AGG GAT GAC AGC AGC GAT GGC TCT TTG GAA GAT GAG GAT TAT CAC 

394 

Gin Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 
100 105 110 

GCT ACA ACG GAA ACA ATC ATT ACC ATG CCT ACA GAG TCT GAT TTT CTA 
30 442 

Ala Thr Thr Glu Thr He He Thr Met Pro Thr Glu Ser Asp Phe Leu 
115 120 125 

ATG CAA GTG CAT GGA AAA CCC AAA TCT TGC TTC TTT AAA TTT AGC TCT 
490 

35 Met Gin Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 
130 135 140 



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-55- 



AAA ATA CAA TAC AAT AAA GTA GTA AAG GCC CAA CTA TGG ATA TAT TTG 
538 

Lys lie Gin Tyr Asn Lys Val Val Lys Ala Gin Leu Trp lie Tyr Leu 
145 150 155 160 

5 AGA CCC GTC GAG ACT COT ACA ACA GTG TTT GTG CAA ATC CTG AGA CTC 

586 

Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gin He Leu Arg Leu 
165 170 175 

ATC AAA CCT ATG AAA GAC GGT ACA AGG TAT ACT GGA ATC CGA TCT CTG 
10 634 

He Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly He Arg Ser Leu 
180 185 190 

AAA CTT GAC ATG AAC CCA GGC ACT GGT ATT TGG GAG AGC ATT GAT GTG 
682 

15 Lys Leu Asp Met Asn Pro Gly Thr Gly He Trp Gin Ser He Asp Val 

195 200 205 

AAG ACA GTG TTG CAA AAT TGG CTC AAA CAA CCT GAA TCC AAC TTA GGC 
730 

Lys Thr Val Leu Gin Asn Trp Leu Lys Gin Pro Glu Ser Asn Leu Gly 
20 210 215 220 

ATT GAA ATA AAA GCT TTA GAT GAG AAT GGT CAT GAT CTT GCT GTA ACC 
778 

He Glu He Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 
225 230 235 240 

25 TTC CCA GGA CCA GGA GAA GAT GGG CTG AAT CCG TTT TTA GAG GTC AAG 

826 

Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys 
245 250 255 

GTA ACA GAC ACA CCA AAA AGA TCC AGA A^G GAT TTT ^T CTT GAC T<;T 
30 874 

Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 
260 265 270 

GAT GAG CAC TCA ACA GAA TCA CGA TGC TGT CGT TAC CCT CTA ACT GTG 
922 

35 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 
275 280 285 



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-56- 



GAT TTT GAA GCT TTT GGA TGG GAT TGG ATT ATC GOT CCT AAA A€A TAT 
970 

Asp Phe Glu Ala Phe Gly Trp Asp Trp He He Ala Pro Lys Arg Tyr 
290 295 300 

5 AAG GCC AAT TAG TGC TCT GGA GAG TGT GAA TTT GTA TTT TTA GAA AAA 

1018 

Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gin Lys 
305 310 315 320 

TAT CCT CAT ACT CAT CTG GTA CAC CAA GCA AAG CCC AGA GCT TCA GCA 
10 1066 

Tyr Pro His Thr His Leu Val His Gin Ala Asn Pro Arg Gly Ser Ala 
325 330 335 

GGC CCT TGC TGT ACT CCC ACA AAG ATG TCT CCA ATT AAT ATG GTA TAT 
1114 

15 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro He Asn Met Leu Tyr 

340 345 350 

TTT AAT GGC AAA GAA CAA ATA ATA TAT GGG AAA ATT CCA GCG ATG GTA 
1162 

Phe Asn Gly Lys Glu Gin He He Tyr Gly Lys He Pro Ala Met Val 
20 355 360 365 

GTA GAC CGC TGT GGG TGC TCA TGAGATTTAT ATTAAGGGTT CATAACTTCC 
1213 

Val Asp Arg Cys Gly Cys Ser 
370 375 

25 TAAAACATGG AAGGTTTTCC CCTCAACAAT TTTGAAGCTG TGAAATTAAG TACCACAGGC 

1273 

TATAGGCCTA GAGTATGCTA CAGTCACTTA AGCATAAGCT ACAGTATGTA AACTAAAAGG 
1333 

GGGAATATAT GCAATGGTTG GCATTTAACC ATCCAAACAA ATCATACAAG AAAGTTTTAT 
30 1393 

GATTTCCAGA GTTTTTGAGC TAGAAGGAGA TCAAATTACA TTTATCTTCC TATATATTAC 
1453 

AACATCGGCG AGGAAATGAA AGCGATTCTC CTTGACTTCT GATGAATTAA AGGAGTATGC 
1513 



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-57- 



TTTAAAGTCT ATTTCTTTAA AGTTTTGTTT AATATTTACA GAAAAATCCA CATACAGTAT 
1573 

TGGTAAAATG CAGGATTGTT ATATACCATC ATTCGAATCA TCCTTAAACA CTTGAATTTA 
1633 

5 TATTGTATGG TAGTATACTT GGTAAGATAA AATTCCACAA AAATAGGGAT GGTGCAGCAT 

1693 

ATGCAATTTC CATTCCTATT ATAATTGACA CAGTACATTA ACAATCCATG CCAACGGTGC 
1753 

TAATACGATA GGCTGAATGT CTGAGGCTAC CAGGTTTATC ACATAAAAAA CATTCAGTAA 
10 1813 

AATAGTAAGT TTCTCTTTTC TTCAGGTGCA TTTTCCTACA CCTCCAAATG AGGAATGGAT 
1873 

TTTCTTTAAT GTAAGAAGAA TCATTTTTCT AGAGGTTGGC TTTCAATTCT GTAGCATACT 
1933 

15 TGGAGAAACT GCATTATCTT AAAAGGCAGT CAAATGGTGT TTGTTTTTAT CAAAATGTCA 

1993 

AAATAACATA CTTGGAGAAG TATGTAATTT TGTCTTTGGA AAATTACAAC ACTGCCTTTG 
2053 

CAACACTGCA GTTTTTATGG TAAAATAATA GAAATGATCG ACTCTATCAA TATTGTATAA 
20 2113 

AAAGACTGAA ACAATGCATT TATATAATAT GTATACAATA TTGTTTTGTA AATAAGTGTC 
2173 

TCCTTTTTTA TTTACTTTGG TATATTTTTA CACTAAGGAC ATTTCAAATT AAGTACTAAC 
2233 

25 GCACAAAGAC ATGTCATGCA TCACAGAAAA GCAACTACTT ATATTTCAGA <5CAAATTAGC 

2293 

AGATTAAATA GTGGTCTTAA AACTCCATAT GTTAATGATT AGATGGTTAT ATTACAATCA 
2353 

TTTTATATTT TTTTACATGA TTAACATTCA CTTATG^ATT GATGATGGCT GTATAAAGTG 
30 2413 



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-58- 



AATTTGAAAT TTCAATGGTT TACTGTCATT GTGTTTAAAT CTCAACGTTC CATTATTTTA 
2473 

ATACTTGCAA AAACATTACT AAGTATACCA AAATAATTGA CTCTATTATC TGAAATGAAG 
2533 

5 AATAAACTGA TGCTATCTCA ACAATAACTG TTACTTTTAT TTTATAATTT GATAATGAAT 

2593 

ATATTTCTGC ATTTATTTAC TTCTGTTTTG TAAATTGGGA TTTTGTTAAT CAAATTTATT 
2653 

GTACTATGAC TAAATGAAAT TATTTCTTAC ATCTAATTTG TAGAAACAGT ATAAGTTATA 
10 2713 

TTAAAGTGTT TTCACATTTT TTTGAAAGAC 
2743 



(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 
15 (A) LENGTH: 375 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 

20 Met Gin Lys Leu Gin Leu Cys Val Tyr He Tyr Leu Phe Met Leu He 
15 10 15 

Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gin Lys Glu Asn 
20 25 30 

Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gin Asn Thr 
25 35 40 45 

Lys Ser Ser Arg He Glu Ala He Lys He Gin lie Leu Ser Lys Leu 
50 55 60 

Arg Leu Glu Thr Ala Pro Asn He Ser Lys Asp Val He Arg Gin Leu 
65 70 75 80 



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-59- 



Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu lie Asp Gin Tyr Asp Val 
85 90 95 



Gin Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 
100 105 110 

5 Ala Thr Thr Glu Thr He He Thr Met Pro Thr Glu Ser Asp Phe Leu 
115 120 125 

Met Gin Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 
130 135 lAO 

Lys He Gin Tyr Asn Lys Val Val Lys Ala Gin Leu Trp He Tyr Leu 
10 145 150 155 160 



Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gin He Leu Arg Leu 
165 170 175 

He Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly He Arg Ser Leu 
180 185 190 



15 Lys Leu Asp Met Asn Pro Gly Thr Gly He Trp Gin Ser He Asp Val 

195 200 205 

Lys Thr Val Leu Gin Asn Trp Leu Lys Gin Pro Glu Ser Asn Leu Gly 
210 215 220 

He Glu He Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 
20 225 230 235 240 

Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys 
245 250 255 

Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 
260 265 270 

25 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 
275 280 285 

Asp Phe Glu Ala Phe Gly Trp Asp Trp He He Ala Pro Lys Arg Tyr 
290 295 300 



30 



Lys Ala Asn Tyr Cys Ser Gly Glu Cys 
305 310 



Glu Phe Val Phe Leu Gin Lys 
315 320 



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-60- 

Tyr Pro His Thr His Leu Val His Gin Ala Asn Pro Arg Gly Ser Ala 
325 330 335 

Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro lie Asn Met Leu Tyr 
340 345 350 

5 Phe Asn Gly Lys Glu Gin He He Tyr Gly Lys He Pro Ala Met Val 
355 360 365 

Val Asp Arg Cys Gly Cys Ser 
370 375 



(2) INFORMATION FOR SEQ ID NO: 15; 

10 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 34 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

15 (ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: #83 

(ix) FEATURE: 

(A) NAME/KEY: CDS 
20 (B) LOCATION: 1..34 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 

CGCGGATCCG TGGATCTAAA T-GAGAACACT GAGC 
34 

(2) INFORMATION FOR SEQ ID NO: 16: 

25 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 37 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



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-61- 



(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: #84 



(ix) FEATURE: 
5 (A) NAME/KEY: CDS 

(B) LOCATION: 1. .37 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 



CGCGAATTCT CAGGTAATGA TTGTTTCCGT TGTAGCG 
37 



10 (2) INFORMATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 
15 (D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: DNA (genomic) 



(vii) IMMEDIATE SOURCE: 
(B) CLONE: #100 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 



20 ACACTAAATC TTCAAGAATA 

20 



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-62- 



CLftlMS 

1. Substantially pure growth differentiation factor-8 {GDF-8) and functional 
fragments thereof. 

2. An isolated polynucleotide sequence encoding the GDF-8 polypeptide 
of clainn 1 . 

3. The polynucleotide of claim 2. wherein the GDF-8 nucleotide sequence 
is selected from the group consisting of the nucleic acid sequence of 

a. FIGURE 5a, wherein T can also be U; 

b. FIGURE 5b. wherein T can also be U; 

5 c, nucleic acid sequences complenr^entary to FIGURE 5a; 

d. nucleic acid sequences complementary to FIGURE 5b; 

e. fragments of a. or c. that are at least 15 bases in length and that 
will selectively hybridize to genomic DNA which encodes the 
GDF-8 protein of FIGURE 5a; and 

10 f. fragments of b. or d. that are at least 15 bases In length and that 

will selectively hybridize to genomic DNA which encodes the 
GDF-8 protein of FIGURE 5b. 

4. The polynucleotide sequence of claim 2, wherein the polynucleotide is 
isolated from a mammalian cell. 

5. The polynucleotide of claim 4, wherein the mammalian cell is selected 
from the group consisting of mouse, fat. and human cell. 

6. An expression vector including the polynucleotide of claim 2. 



7. 



The vector of claim 6. wherein the vector is a plasmid. 



wo 94/21681 PCTAJS94/03019 

-63- 



8. The vector of claim 6. wherein the vector is a virus. 

9. A host cell stably transformed with the vector of claim 6. 

10. The host cell of claim 9, wherein the cell is prokaryotic. 

11. The host cell of claim 9. wherein the cell is eukaryotic. 

12. Antibodies reactive with the polypeptide of claim 1 or fragments thereof. 

13. The antibodies of claim 12, wherein the antibodies are polyclonal. 

14. The antibodies of claim 12. wherein the antibodies are monoclonal. 

1 5. A method of detecting a cell proliferative disorder comprising 
contacting the antibody of claim 12 with a specimen of a subject 
suspected of having a GDF-8 associated disorder and detecting binding 
of the antibody. 

16. The method of claim 15, wherein the cell is a muscle cell.. 

17. The method of claim 15, wherein the detecting is in vivo. 

18. The method of claim 17, wherein the antibody is detectably labeled. 

19. The method of claim 18, wherein the detectable label is selected from 
the group consisting of a radioisotope, a fluorescent compound, a 
bioluminescent compound and a chemiluminescent compound. 

20. The method of claim 15, wherein the detection is in vitro. 



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-64- 

21 . The method of claim 20, wherein the antibody is detectably labeled. 

22. The method of claim 21 . wherein the label is selected from the group 
consisting of a radioisotope, a fluorescent compound, a bioluminescent 
compound, a chemoluminescent compound and an enzyme. 

23. A method of treating a cell proliferative disorder associated with 
expression of GDF-8, comprising contacting the cells with a reagent 
which suppresses the GDF-8 activity. 

24. The method of claim 23. wherein the reagent is an anti-GDF-8 antibody. 

25. The method of claim 23, wherein the reagent is a GDF-8 antisense 
sequence. 

26. The method of claim 23, wherein the cell is a muscle cell. 

27. The method of claim 23, wherein the reagent which suppresses GDF-8 
activity is introduced to a cell using a vector. 

28. The method of claim 27, wherein the vector is a colloidal dispersbn 
system. 

29. The method of claim 28, wherein the colloidal dispersion system is a 
liposome. 

30. The method of claim 29, wherein the liposome is essentially target 
specific. 

31. The method of claim 30. wherein the liposome is anatomicaHy targeted. 



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-65- 

32. The method of claim 31, wherein the liposome is mechanistically 
targeted. 

33. The method of claim 32. wherein the mechanistic targeting is passive. 

34. The method of claim 32, wherein the mechanistic targeting is active. 

35. The method of claim 34. wherein the liposome is actively targeted by 
coupling with a moiety selected from the group consisting of a sugar, 
a glycolipid, and a protein. 

36. The method of claim 35. wherein the protein moiety is an antibody. 

37. The method of claim 36. wherein the vector Is a virus. 

38. The method of claim 37, wherein the virus is an RNA virus, 

39. The method of claim 38, wherein the RNA virus is a retrovirus. 

40. The method of claim 39, wherein the retrovirus is essentially target 
specific. 

The method of claim 40, wherein a moiety for target specificity is 
encoded by a polynucleotide inserted into the retrovirat genome. 

The method of claim 40, wherein a moiety for target specificity is 
selected from the group consisting of a sugar, a glycolipid, and a 
protein. 



41. 



42. 



43. 



The method of claim 42, wherein the protein is an antibody. 



wo 94/21681 



PCrAJS94/03019 



1/15 



c/5 

I— r> 
^ o s 
< :s >- 
UJ z> x 
X -J 



>- 



QC = 



—J 

o 

CO 
LU 
> 



CO 



CO 

<t 

LU 
QC 
O 

<t 

a. 



00 
LU 



LU 

Q. 
CO 



CO 
LU 



cr 

LU 



cr 



or 

O -J 



O 
00 

=> 
S 




-2.9 kb 



FIG. 1 

SUBSTITUTE SHEET (RULE 26) 



wo 94/21681 



PCT/US94/03019 



2/15 



1 TTAAGGTAGGAAGGATTTCAGGCTCTATTTACATAATTGTTCTTTCCTTTTCACACAGAA 60 

N 

61 TCCCTTTTTAGAAGTCAAGGTGACAGACACACC CAAGAG GTC CCGGAG AGACTTTGGGCT 120 

P F L E V K V T D T P flTR] S fR^ 0 F G L 
121 TGACTGCGATGAGCACTCCACGGAATCCCGGTGCTGCCGCTACCCCCTCACGGTCGATTT 180 

DCDEHSTESRCCRYPLTVOF 
181 7GAAGCCTTTGGATGGGACTGGATTATCGCACCCAAAAGATATAAGGCCAATTACTGCTC 240 

EAFGWDWI lAPKRYKANYCS 
241 AGGAGAGTGTGAATTTGTGTTTTTACAAAAATATCCGCATACTCATCTTGTGCACCAAGC 300 

GECEFVFLQKYPHTHLVHOA 
301 AAACCCCAGAGGCTCAGCAGGCCCTTGCTGCACTCCGACAAAAATGTCTCCCATTAATAT 360 

NPRGSAGPCCTPTKMSPINM 
361 GCTATATTTTAATGGCAAAGAACAAATAATATATGGGAAAATTCCAGCCATOGTAGTAGA 420 

LYFNGKEQllYGKIPAMVVD 
421 CCGCTGTGGGTGCTCATGAGCTTTGCATTAGGTTAGAAACTTCCCAAGTCATGGAAGGTC 480 

R C G C S » 

481 TTCCCCTCAATTTCGAAACTGTGAATTCCTGCAGCCCGGGGGATCCACTAGTTGTAGAGC 540 
541 GGCCGCCACC 550 _ 

FIG.2a 



1 CAAAAAGATCCAGAAGGGATTTTGGTCTTGACTGTGATCAGCACTCAACAGAATCACGAT 60 

fK~R] S EU DFGLDCDEHSTES«C 
61 GCTGTCGTTACCCTCTAACTGTGGATTTTGAAGCTTTIGGATGGGATTCGATTATCGCTC 120 

CRYPLTVDFEAFGWDWI lAP 
121 CTAAAAGATATAAGGCCAATTACTGCTCTGGAGAGTGTGAATTTGTATTTTTACAAAAAT 180 

KRYKANYCSGECEFVFLQKY 
181 ATCCTCATACTCATCTGGTACACCAAGCAAACCCCAGAGGTTCAGCAGGCCCTTGCTCTA 240 

PHTHLVHQANPRGSAGPCCT 
241 CTCCCACAAAGATGTCTCCAATTAATATCCTATATTTTAATGGCAAAGAACAAATAATAT 300 

PTKMSPINMLYFNGKEOIIY 
301 ATGGGAAAATTCCAGCGATGGTAGTA 326 

G K I P A M V V 

FIG.2b 



SUBSTITUTE SHEET (RULE 26) 



wo 94/21681 



PCT/US94/03019 



GDr-8 

GDF-1 

BMP-2 

BMP-4 

Vgr-1 

OP-1 

BMP-5 

BMP-3 

MIS 

Inhibina 
Inhibin^A 
Inhibin^B 
TGF- pi 
TGF- fi2 
TGF- pi 



3/15 

srrdfgldcdehstesrScrypltvdf-eafgwd-wiiapkryk, 
rprrdaepvlgggpgga^arrlyvsf-revgwhrwviaprgfl 
rekroakhkqrkrlkssa<rhplyvdf-s0vgwndwivappgyhaf 
krspkhhsqrarkimncrrhslyvdf-sdvgwndwivappgyqaf 
srgsgssdyngselktackkhelyvsf-qdlgwqdwiiapkgy; 

lrmanvaensssdqroa|c <KHEL YVSF-RDLGWQDWI I APEGYAA' 
KKHELYVSF-RDLGWQDWl lAPEGYAAF 
RYLKVDF-ADIGWSEWI ISPKSFOA' 
RELSVOL- — RAERSVL IPETYQANMOX' 



SRMSSVGDYNTSEQKO; 
EQTLKKARRKOWIEPRI 
GPGRAQRSAGATAADG! 

alrllqrppeep, 
hrrrrrglecdgkv-ni 
hrirkrglecogrt-nli 
hrraldtnycfssteki 

KKRALOAAYCFRNVQDI 



IRVALNISF-QELGWERWIVYPPSFIFI 
KKQFFVSF-KDlGWNOWilAPSG' 
RQQFFIDF-^LIGWNOWIIAPTGYYGI 
VRQLYIOFRKDLGWK-WI HEPKGYHANFl 
LRPLYIOFKRDLGWK-Wl HEPKGYNANF| 
KKRALDTNYCFRNLEENCCVRPLYIOFRQOLGWK-WVHEPKGYYANFi 




FVFLQKYP 

iPVALSGSGGPP 
fPLADHLNS— 
fPLADHLNS— 
IFPLNAHMNA— 
fPLNSYMNA— 
IFPLNAHMNA— 
iFPMPKSLKPS- 
;WPQSORNPRY- 
LHIPPNLSLPV- 
HIAGTSGSSL- 
:GaPAYLAGVPGSAS- 

CflCiPYIWSLD 

^YLWSSO 

^YLRSAD 



GOF-8 

GDF-1 

BMP-2 

BlyP-4 

Vgr-1 

OP-1 

BMP-5 

BMP-3 

MIS 

Inhibina 
inhibin/iA 
Inhibin^B 
TGF- ^1 
TGF- fi2 
TGF- /I3 



-HTHLVHQANPRG- 



SAGRCQT-PTKMSPINMLYF-NGKEOI lYGKIPAMWDR 



ALNHAVLRALMHA-AAPGAAOL 
-TNHAIVQTLVNS— VNSKIPI 
-TNHAIVOTLVNS— VNSSIPK; 
-TNHAIVOTLVHL-WNPEYVPKI 
-TNHAIVOTLVHF-INPETVPKI 




'-f ARLSP I SVLFF-ONSONWLRQYEOMWDEI 
'-PTELSA1SMLYL-0ENEKWLKNYQDMWE- 
'-PTELSAlSMLYL-OEYDKWLKNYQEMWEi 
.-PTKLNAISVLYF-OONSNVI LKKYRI 

...... '(jA-PTQLNAISVLYF-ODSSNVILKKYRl _ 

-TNHAIVOTLM-*fPDHWKrcc!A--PTKLNAISVLYF-O0SSNVlLKKYRNMWR^ 
-NHAT 1 OS I VRA-VGWPG I PETO-PEKMSSLS I LFF-OENKNVVLKVYPI*ITVESf|lb? 
-CNHWLLLKMQA-RGAALARPPfcQV-PTAYAGKLL ISLSEER- ISAHHVPMylVATr'"'"^ 



-PGAPPTPAQPYS LLPGAQP 



LPGTMRPLHVRTTSOGGYSFKYETVPNLLT^ 
i^-PTKLRPMSMLYY-€OGQNl IKKOI(M«IVEEl 




-SFHSTVINHYRMRGHSPFANLK« „- 

-SFHTAVVI^YRMRGLNPGT-VN9Cdl--PTKLSTMSMLYF-00EYNIVKR0VPrAilVEE|q^ 
-TQYSI<VLALYI«)--HNPGASAAPW\^--PQALEPLPim-VGRKPKV-EQLSr«<IVRS^^ 
-TQHSRVLSLYNT--INPEASASPpW--S(X)LEPLTILYY-IGKTPKI-EOLSNMIWSP<; 
-TTHSTVLGLYNT--LNPEASASf]gV--PQDLEPLTILYY-VGRTPKV-£QLSNMVVKSCk 



FIG.3 



SUBSTITUTE SHEET {RULE 26) 



wo 94/21681 



PCT/US94/03019 



4/15 



cNitoioior^ooo^cNi 
I I I I I I I ' ' 



CD O C_D 





ro 


^5 
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ro 


ro 


OO 
ro 


oo 

ro 


ro 


tn 
CM 


tiD 
ro 


1 

ro 


ro 


OO 
ro 


to 

ro 


CM 
ro 


CM 


CM 


to 

ro 


P^ 
ro 




CNI 

ro 


CX3 
C>J 


ro 


ro 


to 

ro 


m 

ro 


ro 


*o 

CM 


ro 


ro 
ro 


ro 


OO 
ro 


to 

ro 


CM 

ro 


ro 

CM 


CM 
CM 


ro 


<o 




ro 
ro 


(O 

CNI 


to 
ro 


ro 
ro 


lO 
ro 


to 

ro 


ro 


ro 
eg 


to 

ro 


ro 


to 

ro 


ro 


ro 


CM 

ro 


oo 


ro 
CM 




to 

ro 






iO 
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ro 


ro 


to 

ro 


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CM 


CM 




CM 


P^ 

to 


P^ 
ro 


lO 
CM 


lO 
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ro;0 
to O 






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to 


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JDUiqjqui 


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to 

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to 

CM 


Csl 


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sm 


ro 


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to 

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CM 


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CM 


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OO O 



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P^ O . 



O 
O 



C5 

c c c 
lo lo lo 



-S-Pk ^ ^ - 

D> O C3D GD S 



— CM ro 
QQ, Oa_ QQ_ 



CD CD 



SUBSrmJTE SHEET (RULE 26) 



wo 94/2K81 



PCT/US94/03019 



5/15 

1 GTCTCTCGGACGGTACATGCACTAATATTTCACTTGGCATTACTCAAAAGCAAAAAGAAG 60 
61 AAATAAGAACAAGGGAAAAAAAAAGATTGTGCTGATTTTTAAAATGATGCAAAAACTGCA 120 

M M Q K L Q 

121 AATGTATGTTTATATTTACCTGTTCATGCTGATTGCTGCTGGCCCAGTGGATCTAAATCA 180 
MYVYiYLFMLIAAGPVDLNE 

181 GGGCAGTGAGAGAGAAGAAAATGTGGAAAAAGAGGGGCTGTGTAATGCATGTGCGTGGAG 240 
GSEREENVEKEGLCNACAWR 

241 ACAAAACACGAGGTACTCCAGAATAGAAGCCATAAAAATTCAAATCCTCAGTAAGCTGCG 300 
QNTRYSRIEAIKIOILSKLR 

301 CCTGGAAACAGCTCCTAACATCAGCAAAGATGCTATAAGACAACTTCTGCCAAGAGCGCC 360 



L E T A P [N:V1::.SJ KDAIRQLLPRAP 
361 TCCACTCCGGGAACTGATCGATCAGTACGACGTCCAGAGGGATGACAGCAGTGArcGCTC 420 

PLRELIDOYOVORDDSSDOS 
421 TTTGGAAGATGACGATTATCACGCTACCACGGAAACAATCATTACCATGCCTACAGAGTC 480 

LEDDDYHATTETIITMPTES 
481 TGACTTTCTAATGCAAGCGGATGGCAAGCCCAAATGTTGCTTTTTTAAAT7TAGCTCTAA 540 

DFLMOADGKPKCCFFKFSSK 
541 AATACAGTACAACAAAGTAGTAAAAGCCCAACTGTGGATATATCTCAGACCCGTCAAGAC 600 

lOYNKVVKAQLWlYLRPVKT 
601 TCCTACAACAGTGTTTGTGCAAATCCTGAGACTCATCAAACCCATGAAAGACGGTACAAG 660 

PTTVFVQILRLIKPMKDGTR 
661 GTATACTGGAATCGGATCTCTGAAACTTGACATCAGCCCAGGCACTGGTATTTGGCAGAG 720 

YTGIRSLKLOMSPGTGIWQS 
721 TATTGATGTGAAGACAGTGTTGCAAAATTGGCTCAAACAGCCTGAATCCAACTTAGGCAT 780 

IDVKTVLQNWLKQPESNLGI 
781 TGAAATCAAAGCTTTGGAT<;aGAATGGCCATGATCTT€CTGTAACCTTCCCAGGACCAGG 840 

EIKALDENGHOLAVTFPGPG 
841 AGAAGAmCTGAATCCCTTTTTAGAAGTCAAGGTGACAGACACACCCAAGAGGTCCCG 900 



EDGLNPFLEVKVTDTPK |R S R 
901 GAGAGACTTT€GGCTTGACTGCGATGAGCACTCCAGGGAATGG{X5GTGCTGC(X;CTACCC 960 

T|DFGLDCDEHSTESRCCRYP 
961 CCTCACGGTCGATTTTGAAGCCTTTGGATGGGACTOGATTAmAGCCAAAAGATATAA 1020 

LTVDFEAFGWDWI lAPKRYK 
1021 GGCGAATTACTGCTGAGGAGAGTGTGAATTTGTGTTTTTACAAAAATATCOJGATACTCA 1080 

ANYCSGECEfVFLQKYPHTH 
1081 TGTTGTGGACGAAGGAAACCGGAGAGGCTCAGGAGGCGCTTGGTGCAGTGOIAGAAAAAT 1140 

LVHOANPRGSAGPCGTPTKM 
1141 GTCTCGGATTAATATGGTATATTTTAATGGCAAAGAACAAATAATATATGGGAAAATTGC 1200 

SPINMLYFNGKEQIIYGKIP 
1201 AGCGATGGTAGTAGAGCGCTGTGGGTGCTGATGAGCTTTGCATTAGGTTAGAAAGTTGGG 1260 

AMVVDRGGCS* 



FIG.Sa 

SUBSTITUTE SHEET (RULE 26) 



wo 94/21681 PCTAJS94/03019 

6/15 



1261 AAGTCATGGAAGGTCTTCCCCTCAATTTCGAAACTGTCAATTCAAGCACCACAGGCTGTA 1320 

1321 GGCCTTGAGTATGCTCTAGTAACGTAAGCACAAGC7ACAGTGTATGAACTAAAAGAGAGA 1380 

1381 ATAGATGCAATGGTTGGCATTCAACCACCAAAATAAACCATACTATAGGATGTTGTATGA 1440 

1441 TTTCCAGAGTTTTTGAAAIAGATGGAGATCAAATTACATTTATGTCCATATATGTATATT 1500 

1501 ACAACTACAATCTAGGCAAGGAAGTGAGAGCACATCTTGTGGTCTGCTGAGTTAGGAGGG 1560 

1561 TATGATTAAAA(X;TAAAGTCTTATTtCCTAACAGTTTCACTTAATATTTACAGAAGAATC 1620 

1621 TATATGTAGCCTTTGTAAAGTGTAGGATTGTTATCATTTAAAAACATCATGTACACTTAT 1680 

1681 ATTTGTATTGTATACTTGGTAAGATAAAATTCCACAAAGTAGGAATGGGGCCTCACATAC 1740 

1741 ACATTGCCATTCCTATTATAATTGGACAATCCACCACGGTGCTAATCCAGTGCTGAATGG 1800 

1801 CTCCTACTGGACCTCTGGATAGAACACTCTACAAAGTAOGAGTCTCTCTCTCCCTTCCAG 1860 

1861 GTGCATCTCCACACACACAGCACTAAGTGTTCAATGCATTTTCTTTAAGGAAAGAAGAAT 1920 

1921 CTTTTTTTCTAGAGGTCAACTTTCAGTCAACTCTAGCACAGCGGGAGTGACTGCTGCATC 1980 

1981 TTAAAAGGCAGCCAAACAGTATTCATTTTTTAATCTAAATTTCAAAATCACTGTCTGCCT 2040 

2041 TTATCACATGGCAATTTTGTGGTAAAATAATGGAAATGACTGGTTCTATCAATATTGTAT 2100 

2101 AAAAGACTCTGAAACAATTACATTTATATAATATGTATACAATATTGTTTTGTAAATAAG 2160 

2161 TGTCTCCTTTTATATTTACTTrcGTATATTTTTACACTAATGAAATTTCAAATCATTAAA 2220 

2221 GTACAAAGACATGTCATGTATCACAAAAAAGGTGACTGCTTCTATTTCAGAGTGAATTAG 2280 

2281 CAGATTCAATAGTGGTCTTAAAACTCTGTATGTTAAGATTAGAAGGTTATATTAGAATCA 2340 

234 1 ATTTATGTATTTTTTACATTATCAACTTATGGTTTCATGGmTGTATCTATGAATGTG 2400 

2401 GCTCCCAGTCAAATTTCAATGCCCCACCATTTTAAAAATTACAAGCATTACTAAACATAC 2460 

2461 CAACATGTATCTAAAGAAATACAAATATGGTATCTCAATAACAGCTACTTTTTTATTTTA 2520 

2521 TAATTTGACAATGAATACATTTCTTTTATTTACTTCAGTTTTATAAATTGGAACTTTGTT 2580 

2581 TATCAAATGTATTGTACTCATAGCTAAATOAAATTATTTCTTACATAAAAATGTGTAGAA 2640 

2641 ACTATAAATTAAAGTCTTTTCACATTTTTGAAAGGC 2676 

FIG.Sb 



SUBSTITUTE SHEET (fiULE 26) 



wo 94/21681 



PCTAJS94/03019 



7/15 

1 AAGAAAAGTAAAAGGAAGAAACAAGAACAAGAAAAAAGATTATATTGATTTTAAAATCAT 60 

M 

61 GCAAAAACTGCAACTCTGTGTTTATATTTACCTGTTTATGCTGATTGTTGCTGGTCCAGT 120 
QKLOLCVY.IYLFMLIVAGPV 

121 GGATCTAAATGAGAACAGTGAGCAAAAAGAAAATGTGGAAAAAGAGGGGCTGTGTAATGC 180 
DLNENSEOKENVEKEGLCNA 

181 ATGTACTTGGAGACAAAACACTAAATCTTCAAGAATAGAAGCCATTAAGATACAAATCCT 240 
C T W R Q N T K S S R I E A I K 1 0 i L 

241 CAGTAAACTTCGTCTGGAAACAGCTCCTAACATCAGCAAAGATGTTATAAGACAACTTTT 300 



S K ,L R L E T A P :N::.I::S- K. 0 V I R 0 L L 



301 ACCCAAAGCTCCTCCACTCCGGGAACTGATTGATCAGTATGATGTCCAGAGGGATGACAG 350 

PKAPPLRELIDQYDVORDDS 
361 CAGCGATGGCTCTTTGGAAGATGACGATTATCACGCTACAACGGAAACAATCATTACCAT 420 

SDGSLEDOOYHATTETIITM 
421 GCCTACAGAGTCTGATTTTCTAATGCAAGTGGATGGAAAACCCAAATGTTCCTTCTTTAA 480 

PTESDFLMOVDGKPKCCFFK 
481 ATTTAGCTCTAAAATACAATACAATAAAGTAGTAAAGCCCCAACTATGGA7ATATTTGAG 540 

FSSKIQYNKVVKAOLWIYLR 
541 ACCCGTCGAGACTCCTACAACAGTGTTTGTGCAAATCCTGAGACTCATCAAACCTATGAA 600 

PVETPTTVFVOILRLIKPMK 
601 AGACGGTACAAGGTATACTGGAATCCGATCTCTGAAACTTGACATGAACCCAGGCACTGG 660 

DGTRYTGIRSLKLDMNPGTG 
661 TATTTGGCAGAGCATTGATGTGAAGACAGTGTTGCAAAATTGGCTCAAACAACCTGAATC 720 

IWOSIDVKTVLQNWLKOPES 
721 CAACTTAGGCATTGAAATAAAAGCTTTAGATGAGAATCGTCATGATCTTGCTGTAACCTT 780 

NLGIEIKALDENGHOLAVTF 
781 CCCAGGACCAGGAGAAGATGGGCTGAATCCGTTTTTAGAGGTCAAGGTAACAGACACACC 840 

PGPGEDGLNPFLEVKVTDTP 
841 AAAAAGATCCAGAAGGGATTTTGGTCTTGACTGTGATCAGCACTCAACAGAATCACGATG 900 



K |R S R R| DF. GLDCDEHSTESRC 
901 CTGTCGTTACCCTCTAACTGTGGATTTTGAAGCTTTTGGATCGGATTGGATTATCOCTCC 960 

CRYPLTVDFEAFGWDWIIAP 
961 TAAAAGATATAAGGCCAATTACTGCTCTGGAGAGTGTGAATTTGTATTTTTACAAAAATA 1020 

KRYKANYCSGECEFVFLQKY 
1021 TCCTCATACTCATCTGGTACACCAAGCAAACCCCAGAGGTTCAGCAGGCCCTTGCTGTAC 1080 

PHTHLVHOANPRGSAGPCCT 
1081 TCCCACAAAGATGTCTCCAATTAATATGCTATATTTTAATGGOAAAGAACAAATAATATA 1140 

PTKMS'PINMLYFNGKEOI lY 
1141 TGGGAAAATTCCAGCGATGGTAGTAGACCGCTGTGGGTGCTCATGAGATTTATATTAAGC 1200 

GKIPAMVVDRCGCS* 



FIG.5C 



SUBSTITUTE SHEET (fiULE 26) 



wo 94/21681 



8/15 



PCT/US94/03019 



1201 GTTCATAACTTCCTAAAACATGGAAGGTTTTCCCCTCAACAATTTTGAAGCTGTGAAATT 1260 

1261 AAGTACCACAGGCTATAGGCCTAGAGTATGCTACAGTCACTTAAGCATAAGCTACAGTAT 1320 

1321 GTAAACTAAAAGGGGGAATATATGCAATGGTTGGCATTTAACCATCCAAACAAATCATAC 1380 

1381 AAGAAAGTTTTATGATTTCCAGAGTTTTTGAGCTAGAAGGAGATCAAATTACATTTATGT 1440 

1441 TCCTATATATTACAACATCGGCGAGGAAATGAAAGCGATTCTCC7TGAGITCTGATGAAT 1500 

1501 TAAAGGAGTATGCTTTAAAGTCTATTTCTTTAAAGTTTTGTTTAATATTTACAGAAAAAT 1560 

1561 CCACATACAGTATTGGTAAAATGCAGGATTGTTATATACCATCATTCGAATCATCCTTAA 1620 

1621 ACACTTGAATTTATATTGTATGGTAGTATACTTGGTAAGATAAAATTCCACAAAAATAGG 1680 

1 681 GATGGTGCAGCATATGCAATTTCCATTCCTATTATAATTGACACAGTACATTAACAATCC 1 740 

1741 ATGCCAACGGTGCTAATACGATAGGCTGAATGTCTGAGGCTACCAGGTTTATCACATAAA 1800 

1801 AAACATTCAGTAAAATAGTAAGTTTCTCTTTTCTTCAGGTGCATTTTCCTACACCTCCAA 1860 

1861 ATGAGGAATGGATTTTCTTTAATGTAAGAAGAATCATTTTTCTAGAGGTTGGCTTTCAAT 1920 

1921 TCTGTAGCATACTTGGAGAAACTGCATTATCTTAAAAGGCAGTCAAATGGTGTTTGTTTT 1980 

1981 TATCAAAATGTCAAAATAACATACTTGGAGAAGTATGTAATTTTGTCTTTGGAAAATTAC 2040 

2041 AACACTGCCTTTCCAACACTGCAGTTTTTATGGTAAAATAATAGAAATGATCGACTCTAT 2100 

2101 CAATATTGTATAAAAAGACTGAAACAATGCATTTATATAATATGTATACAATATTGTTTT 2160 

2161 GTAAATAAGTGTCTCCTTTTTTATTTACTTTGGTATATTTTTACACTAAGGACATTTCAA 2220 

2221 ATTAAGTACTAAGGCACAAAGACATGTCATGCATCACAGAAAAGCAACTACTTATATTTC 2280 

2281 AGAGCAAATTAGCAGATTAAATAGTGGTCTTAAAACTCCATATGTTAATGATTAGATGGT 2340 

2341 TATATTACAATCATTTTATATTTTTTTACATGATTAACATTCACTTATGGATTCATGATG 2400 

2401 GCTGTATAAAGTGAATTTGAAATTTCAATGGTTTACTGTCATTGTCTTTAAATCTCAACG 2460 

2461 TTCCATTATTTTAATACTTGCAAAAACATTACTAAGTATACCAAAATAATTGACTCTATT 2520 

2521 ATCTGAAATGAAGAATAAACTGATGCTATCTCAACAATAACTGTTACTTTTATTTTATAA 2580 

2581 TTTGATAATGAATATATTTCTGCATTTATTTACTTCTGTTTTGTAAATTGGGATTTTGTT 2640 

2641 AATCAAATTTATTGTACTATGACTAAATGAAATTATTTCTTACATCTAATTTGTAGAAAC 2700 

2701 AGTATAAGTTATATTAAAGTGTTTTCACATTTTTTTGAAAGAC 2743 



FIG.Sd 



SUBSTITUTE SHE€T (RULE 26) 



wo 94/21681 



PCTAJS94/03019 




SUBSTfTUTE SHEET (RULE 26) 



wo 94/21681 



PCT/US94/03019 



10/15 



1 hliOKLQMYVYIYLFMLiAAGPVDLNEGSEREENVEKEGLCNACAWRONTR 50 

illll lllllllll llllllli II lllllllllill lllll 
1 MOKLOLCVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACTWRQNTK 49 

51 YSRIEAIKIQILSKLRLETAPNISKOAIRQLLPRAPPLRELIDQYDVQRD 100 

lllllllllllllllllllllllll llllll lllllllillllllll 
50 SSRIEAIKIQILSKLRLETAPNISKOVIRaiPKAPPLRELlOOYDVORD 99 

101 OSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIOYN 150 

lllllllllllllllllllllllllllllll llllllllllllllllll 
100 DSSDGSLEODDYHATTETIIIMPTESDFLIylQVDGKPKCCFFKFSSKlOYN 149 

151 KWKAQLWIYLRPVKTPTTVFVOILRLIKPMKOGTRYTGIRSLKLOMSPG 200 

llllllllllllll lllllllllilllllllillllllllllilll II 
150 KWKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLOMNPG 199 

201 TGIWQSIDVKTVLQNWLKQPESNLGIEIKALOENGHDLAVTFPGPGEOGL 250 

llllllllllllillllllllllllllllllllllllllllllllllill 
200 TGIWQSIDVKTVLQNWLKQPESNLGIEIKALOENGHOLAVTFPGPGEDGL 249 

251 NPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWII 300 

llllllllllllllllllllllilllllllllllllllllllllllllll 
250 NPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIl 299 

301 APKRYKANYCSGECEFVFIOKYPHTHLVHQANPRGSAGPCCTPTKMSPIN 350 

llllliilllllllllllllllllllllllllllllllllllllllllll 
300 APKRYKANYCSGECEFVFLOKYPHTHLVHOANPRGSAGPCCTPTKMSPIN 349 

351 MLYFNGKEQIIYGKIPAMWDRCGCS 376 

llllllllllllllllllllllllil 
350 MLYFNGKEOIIYGKIPAMWDRCGCS 375 



FIG.7 



SUBSTITW SHEET (BULE 26) 



wo 94/21681 



PCT/US94/03019 



11/15 



X 



9 NOIiDVaJ 

t7 NOIiOVMd 
2 NOIiDVMd 
Z NOIiDVaj 

I Noiiovaj 

£*9H<J HSVM 
9Hd HSVM 

HonoaHi-MOid 

(QVOl) 

3iamosNi 
3i8mos 

"IViOi 



I 



OJ f*- CD 

ro cvi 

f I t 





00 
CD 



SUBSnTUTt SHEET (RULE 26) 



wo 94/21681 



PCTAJS94/03019 



12/15 



ANTISENSE SENSE 




FIG. 9 



SUBSniUTE SHEET (RULE 26) 



wo 94/21681 



PCT/US94/03019 



13/15 



on 
cvi 
I 



sna3in 

AdVAO 

«3An 
3iosnw 

SliS3i 
N331dS 
3NliS31NI 
SV3aDNVd 
31DIS3A 1VNIW3S 
ASNQIM 
NIVdG 
SnWAHi 
9Nm 
XMV3H 



SUBSTITUTE SHEET (RULE 26) 



wo 94/21681 



PCr/XiS94/03019 



14/15 

<l <l <t 

H- I— 

z s: 2 o o 

lu uj >: >: 

o o o ^ oc: 

< ^ < CO CD 

-J — I 2 s 

C3l CL tU UJ 



CL 



^ -O -O TO 

in m in 

CM CD cJ CO 




-2.9 kb 



FI6. 10b 



SUBSTITUTE SHEET <fiULE 26) 



wo 94/216S1 



PCT/US94/03019 



15/15 



00 



o 
o 




X 

CM 
CM 

CD 
CD 

ro 

CM 



o 
m 

GO 
CD 

in 
ro 

CM 



^ mrorocM 

CD 

o 

ID 



SUBSTITUTE SHEET <RULE 2^ 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US94/03019 



A. CLASSmCATlON OF SUBJECT MATTER 

IPC(5) :C07K 13/00, 15/28: A61K 37/36; C12N 15/18, 15/10, 15/66 
US CL :530/399. 387.1: 536/23.5; 514/12; 435/69.1, 320.1. 252.3 
According to Inicmational Patent Classification (IPC) or to both national classification and IPC 


B. FIELDS SEARCHED 


Minimum documentation searched (classification system followed by classification symbols) 


Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 


Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) 
Please See Extra Sheet. 


C. DOCUMENTS CONSIDERED TO BE RELEVANT 


Category* 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


A 


Molecular Endocrinology, VOLUME 6, NUMBER 11, issued 
1992, "Isolation of Vgr-2, a Novel Member of the 
Transforming Growth Factor-beta-Related Gene Family", 
pages 1961-1968. 


1-43 


A 


Proceedings of the National Academy of Sciences, VOLUME 
88, issued May 1 991 , "Expression of growth/differentiation 
factor 1 in the nervous system: Conservation of a bicistronic 
structure", pages 4250-4254, 


1-43 


A 


Molecular Endocrinology, VOLUME 4, NUMBER 7, issued 
1990, "Identification of a Novel Member (GDF-1) of the 
Transforming Growth Factor-beta Superfamily", pages 1034- 
1040. 


1-43 


fx] Further documents arc listed in the continuation of Box C. Q Sec patent family annex. 


* Speci&l calegonet of cited documeati: 

'A* documciitdefmiiit the geaenl tttte of the art which is not couidered 
to be p«n of paiticiilir relcvuicc 

earlier document publisbed oo or aAcr tltt intenutioiuU filing dite 

*L* document whidi may throw doubu on priority cbuin(t) or which i» 
cited to establiah the publintioo d«te of another citation or other 
special reason (as specified) 

*0* doctiment referring to an oral disclosure, use. exhibition or other 

*p' document publbhed prior to the tntemaiiooal filing dale out later than 
tbc priority date dahued 


'T* btcr docimieni published after the intematioQal filing date or priority 
date and not in conflict with the appUcatitm but cited to understand the 
principle or tfaeoiy underlying the invention 

'X* document of particular relevance; the claimed invention cannot be 
considered novel or cannot be coosiderod to involve an inventive step 
when the document is taken alone 

'Y* document of particular relevance: the claimed invention cannot be 
considered to involve an inventive step when the document is 
combined with one or more other such documents, such combination 
being obvious to a person skilled in the an 

document member of the same patem family 


Date of the actual completion of the international search 
29 APRIL 1994 


Date of mailij^^^e^^^i^tional search repon 


Name and mailing address of the ISA/US 
Commissioner of Patenia and Tr&deniarics 
Box PCT 

Washington. D.C. 20231 
FacsimUe No. (703) 305-3230 


Authorized officer >^ o •» / / 
Telephone No . (703) 308-0 1 96 



Form PCT/ISA/210 (second shect)(July 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 

PCT/US94/03019 



C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


A 


The Journal of Biological Chemistry, VOLUME 268, NUMBER 
5, issued 15 February 1993, "GDF-3 and GDF-9: Two New 
Members of the Transforming Growth Factor-beta Superfamily 
Containing a Novel Pattern of Cysteines", pages 3444-3449. 


1-43 



Form PCT/ISA/210 (continuation of second shect)(July 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 

PCT/US94/03019 



B. FIELDS SEARCHED 

Electronic data bases consulted (Name of data base and where practicable terms used): 

APS, Dialog: File Biochem. Medicine 

search terms: Growth differentiation factor-8. GDF-8 

Sequence Data: PIR, SwissPro, GenBank 



Form PCT/ISA/210 (extra sheet)(July 1992)* 



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

Defective images within this document are accurate representations of the original 
documents submitted by the applicant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□TfADED text or DRAWING 

2^ BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

□ REFERENCE(S) OR EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.