U.S. patent application number 09/859211 was filed with the patent office on 2002-10-24 for growth differentiation factor-8.
This patent application is currently assigned to Johns Hopkins University School of Medicine. Invention is credited to Lee, Se-Jin, McPherron, Alexandra C..
Application Number | 20020157125 09/859211 |
Document ID | / |
Family ID | 27739431 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020157125 |
Kind Code |
A1 |
Lee, Se-Jin ; et
al. |
October 24, 2002 |
Growth differentiation factor-8
Abstract
A transgenic non-human animal of the species selected from the
group consisting of avian, bovine, ovine and porcine having a
transgene which results in disrupting the production of and/or
activity of growth differentiation factor-8 (GDF-8) chromosomally
integrated into the germ cells of the animal is disclosed. Also
disclosed are methods for making such animals, and methods of
treating animals, including humans, with antibodies or antisense
directed to GDF-8. The animals so treated are characterized by
increased muscle tissue.
Inventors: |
Lee, Se-Jin; (Baltimore,
MD) ; McPherron, Alexandra C.; (Baltimore,
MD) |
Correspondence
Address: |
Lisa A. Haile, Ph.D.
Gray Cary Ware & Freidenrich LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121-2189
US
|
Assignee: |
Johns Hopkins University School of
Medicine
|
Family ID: |
27739431 |
Appl. No.: |
09/859211 |
Filed: |
May 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09859211 |
May 15, 2001 |
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09019070 |
Feb 5, 1998 |
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09019070 |
Feb 5, 1998 |
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08862445 |
May 23, 1997 |
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08862445 |
May 23, 1997 |
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08847910 |
Apr 28, 1997 |
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08847910 |
Apr 28, 1997 |
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08795071 |
Feb 5, 1997 |
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5994618 |
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08795071 |
Feb 5, 1997 |
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08525596 |
Oct 26, 1995 |
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5827733 |
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08525596 |
Oct 26, 1995 |
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PCT/US94/03019 |
Mar 18, 1994 |
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PCT/US94/03019 |
Mar 18, 1994 |
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08033923 |
Mar 19, 1993 |
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Current U.S.
Class: |
800/15 ; 800/16;
800/17; 800/18; 800/19; 800/20 |
Current CPC
Class: |
A01K 2227/10 20130101;
A61K 38/00 20130101; A01K 2267/02 20130101; A01K 67/0276 20130101;
A61K 48/00 20130101; A01K 2227/105 20130101; C12N 15/8509 20130101;
A01K 2217/00 20130101; A01K 2267/03 20130101; A01K 2267/0362
20130101; C07K 2319/00 20130101; C07K 16/22 20130101; C07K 14/475
20130101; A01K 2217/075 20130101; A01K 2207/15 20130101; A01K
2227/30 20130101; C07K 14/495 20130101 |
Class at
Publication: |
800/15 ; 800/16;
800/17; 800/19; 800/20; 800/18 |
International
Class: |
A01K 067/027 |
Claims
1. A transgenic non-human animal having a transgene disrupting or
interfering with expression of growth differentiation factor-8
(GDF-8) chromosomally integrated into the germ cells of the
animal.
2. The transgenic animal of claim 1, wherein the animal is selected
from the group of species consisting of avian, bovine, ovine,
piscine, murine, and porcine.
3. The transgenic animal of claim 1 where the species is avian.
4. The transgenic animal of claim 1 where the species is
bovine.
5. The transgenic animal of claim 1 where the species is
porcine.
6. The transgenic animal of claim 1 where the species is ovine.
7. The transgenic animal of claim 1 where the species is
piscine.
8. The transgenic animal of claim 1, wherein the transgene
comprises GDF-8 antisense polynucleotide(s).
9. The transgenic animal of claim 1, wherein the transgene
comprises a gene encoding a dominant negative GDF-8
polypeptide.
10. The transgenic animal of claim 1, wherein the animal is
homozygous or heterozygous for GDF-8 polynucleotide.
11. A chicken or turkey egg produced by the transgenic animal of
claim 3.
12. Beef obtained from the transgenic animal of claim 4.
13. Milk obtained from the transgenic animal of claim 4.
14. Pork obtained from the transgenic animal of claim 5.
15. Lamb obtained from the transgenic animal of claim 6.
16. Chicken or turkey meat produced by the transgenic animal of
claim 3.
17. A method of producing animal food products having increased
muscle mass comprising: a) introducing a transgene disrupting or
interfering with expression of growth differentiation factor-8
(GDF-8) into an embryo into germ cells of a pronuclear embryo of
the animal; b) implanting the embryo into the oviduct of a
pseudopregnant female thereby allowing the embryo to mature to full
term progeny; c) testing the progeny for presence of the transgene
to identify transgene-positive progeny; d) cross-breeding
transgene-positive progeny to obtain further transgene-positive
progeny; and e) processing the progeny to obtain foodstuff.
18. The method of claim 17, wherein the transgene comprises GDF-8
antisense polynucleotides.
19. The method of claim 17, wherein the transgene comprises a gene
encoding a dominant negative GDF-8 polypeptide.
20. A method of producing avian food products having reduced
cholesterol levels comprising: a) introducing a transgene
disrupting or interfering with expression of growth differentiation
factor-8 (GDF-8) into an embryo of an avian animal; b) culturing
the embryo under conditions whereby progeny are hatched; c) testing
the progeny for presence of the transgene to identify
transgene-positive progeny; d) cross-breeding transgene-positive
progeny; and e) processing the progeny to obtain foodstuff.
21. The method of claim 20, wherein the transgene comprises GDF-8
antisense polynucleotides.
22. The method of claim 20, wherein the transgene comprises a gene
encoding a dominant negative GDF-8 polypeptide.
23. The transgenic animal of claim 20, wherein the transgene
comprises a polynucleotide encoding a truncated GDF-8
polypeptide.
24. A method for increasing the muscle mass in an animal comprising
administering to the animal an antibody, or fragment thereof, which
binds to GDF-8 polypeptide.
25. The method of claim 24, wherein anti-GDF-8 antibody is
administered to a domesticated animal.
26. The method of claim 24, wherein the antibody is a monoclonal
antibody or a polyclonal antibody.
27. The method of claim 24, wherein the anti-GDF-8 antibody is
administered by intravenous, intramuscular, multiple bolus, or
subcutaneous injections.
28. The method of claim 27, wherein the anti-GDF-8 antibody is
administered within a dose range of 0.1 ug/kg to 100 mg/kg.
29. The method of claim 27, wherein the antibody is formulated in a
formulation suitable for administration by injection into an
animal.
30. A method of inhibiting the growth regulating actions of GDF-8
comprising contacting a GDF-8 agent with fetal or adult muscle
cells or progenitor cells.
31. The method of claim 30, wherein the agent is selected from the
group consisting of a monoclonal antibody, an antisense nucleic
acid and a dominant negative encoding nucleic acid sequence or
polypeptide.
32. The method of claim 31, wherein the antibody is a humanized
monoclonal antibody or a chimeric monoclonal antibody or fragment
thereof.
33. The method of claim 30, wherein the agent is administered to a
patient suffering from a disorder selected from the group
consisting of muscle wasting disease, neuromuscular disorder,
muscle atrophy and aging.
34. The method of claim 30, wherein the agent is administered to a
patient suffering from a disorder selected from the group
consisting of muscular dystrophy, spinal cord injury, traumatic
injury, congestive obstructive pulmonary disease (COPD), AIDS and
cachechia.
35. The method of claim 30, wherein the agent is administered to a
patient with muscle wasting disease or disorder by intravenous,
intramuscular or subcutaneous injection.
36. The method of claim 31, wherein the monoclonal antibody is
administered within a dose range between about 0.1/kg to about 100
mg/kg.
37. The method of claim 31, wherein the monoclonal antibody is
formulated in a formulation suitable for administration to a
patient.
38. A method for treating a muscle or adipose tissue disorder in a
subject, comprising administering a therapeutically effective
amount of a GDF-8 agent to the subject, thereby inhibiting abnormal
growth of muscle or adipose tissue.
39. The method as in claim 38, wherein the GDF-8 agent is selected
from the group consisting of an antisense polynucleotide, a
polynucleotide encoding a dominant negative GDF-8 polypeptide, a
GDF-8 antibody and a polynucleotide encoding a truncated GDF-8
polypeptide.
40. The method of claim 38, wherein the disorder is a cancer
selected from the group consisting of muscle, connective tissue, or
bone.
41. The method of claim 38, wherein the subject has an obesity
disorder.
42. A method of inhibiting the growth regulating actions of GDF-8
in a subject comprising administering to the subject, a GDF-8 agent
that inhibits the action of GDF-8 in the subject.
43. The method of claim 42, wherein the GDF-8 agent is selected
from the group consisting of an antisense polynucleotide, a
polynucleotide encoding a dominant negative GDF-8 polypeptide, a
GDF-8 antibody and a polynucleotide encoding a truncated GDF-8
polypeptide.
44. A method for identifying a compound that affects GDF-8 activity
or gene expression comprising: a) incubating the compound with
GDF-8 polypeptide, or with a recombinant cell expressing GDF-8
under conditions sufficient to allow the components to interact;
and b) determining the effect of the compound on GDF-8 activity or
expression.
45. The method of claim 44, wherein the effect is inhibition of
GDF-8 activity or expression.
46. The method of claim 44, wherein the effect is stimulation of
GDF-8 activity or expression.
47. An isolated polynucleotide encoding a truncated GDF-8
polypeptide wherein the truncation is a loss of the C-terminal
active fragment of GDF-8.
48. The isolated polynucleotide of claim 47, wherein the
polynucleotide is as shown in FIG. 12a.
Description
[0001] This application is a continuation-in-part application of
U.S. application Ser. No. 08/862,445, filed May 23, 1997, which is
a continuation-in-part application of U.S. application Ser. No.
08/847,910, filed Apr. 28, 1997, which is a continuation-in-part of
U.S. application Ser. No. 08/795,071, filed Feb. 5, 1997, which is
a continuation-in-part application of U.S. application Ser. No.
08/525,596, filed Oct. 25, 1995, which is a 371 application of
PCT/US94/03019 filed on Mar. 18, 1994, which is a
continuation-in-part of U.S. application Ser. No. 08/033,923 filed
on Mar. 19, 1993, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates generally to growth factors and
specifically to a new member of the transforming growth factor beta
(TGF-.beta.) superfamily, which is denoted, growth differentiation
factor-8 (GDF-8) and methods of use for modulating muscle cell and
adipose tissue growth.
[0004] 2. Description of Related Art
[0005] The transforming growth factor .beta. (TGF-.beta.)
superfamily encompasses a group of structurally-related proteins
which affect a wide range of differentiation processes during
embryonic development. The family includes, Mullerian inhibiting
substance (MIS), which is required for normal male sex development
(Behringer, et al., Nature, 345:167, 1990), Drosophila
decapentaplegic (DPP) 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. Commun., 135:957-964, 1986), which can
induce the formation 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 TGF-.beta.s can influence a variety of
differentiation processes, including adipogenesis, myogenesis,
chondrogenesis, hematopolesis, and epithelial cell differentiation
(for review, see Massague, Cell 49:437, 1987).
[0006] The proteins of the TGF-.beta. family are initially
synthesized as a large precursor protein which subsequently
undergoes proteolytic cleavage at a cluster of basic 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 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-.beta. family is
coexpressed with a mature region of another member of the
TGF-.beta. family, intracellular dimerization and secretion of
biologically active homodimers occur (Gray, A. et al., 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 members that
have been studied, the homodimeric species has been found to be
biologically active, but for other family members, like the
inhibins (Ling, et al., Nature, 321 :779, 1986) and the TGF-.beta.s
(Cheifetz, et al., Cell, 48:409, 1987), heterodimers have also been
detected, and these appear to have different biological properties
than the respective homodimers.
[0007] In addition it is desirable to produce livestock and game
animals, such as cows, sheep, pigs, chicken and turkey, fish which
are relatively high in musculature and protein, and low in fat
content. Many drug and diet regimens exist which may help increase
muscle and protein content and lower undesirably high fat and/or
cholesterol levels, but such treatment is generally administered
after the fact, and is begun only after significant damage has
occurred to the vasculature. Accordingly, it would be desirable to
produce animals which are genetically predisposed to having higher
muscle content, without any ancillary increase in fat levels.
[0008] The food industry has put much effort into increasing the
amount of muscle and protein in foodstuffs. This quest is
relatively simple in the manufacture of synthetic foodstuffs, but
has been met with limited success in the preparation of animal
foodstuffs. Attempts have been made, for example, to lower
cholesterol levels in beef and poultry products by including
cholesterol-lowering drugs in animal feed (see e.g. Elkin and
Rogler, J. Agric. Food Chem. 1990, 38, 1635-1641). However, there
remains a need for more effective methods of increasing muscle and
reducing fat and cholesterol levels in animal food products.
SUMMARY OF THE INVENTION
[0009] 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
proliferative disorders, especially those involving muscle, nerve,
and adipose tissue.
[0010] 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 treating a cell proliferative
disorder by suppressing or enhancing GDF-8 activity.
[0011] In another embodiment, the subject invention provides
non-human transgenic animals which are useful as a source of food
products with high muscle and protein content, and reduced fat and
cholesterol content. The animals have been altered chromosomally in
their germ cells and somatic cells so that the production of GDF-8
is produced in reduced amounts, or is completely disrupted,
resulting in animals with decreased levels of GDF-8 in their system
and higher than normal levels of muscle tissue, preferably without
increased fat and/or cholesterol levels. Accordingly, the present
invention also includes food products provided by the animals. Such
food products have increased nutritional value because of the
increase in muscle tissue. The transgenic non-human animals of the
invention include bovine, porcine, ovine and avian animals, for
example.
[0012] The subject invention also provides a method of producing
animal food products having increased muscle content. The method
includes modifying the genetic makeup of the germ cells of a
pronuclear embryo of the animal, implanting the embryo into the
oviduct of a pseudopregnant female thereby allowing the embryo to
mature to full term progeny, testing the progeny for presence of
the transgene to identify transgene-positive progeny,
cross-breeding transgene-positive progeny to obtain further
transgene-positive progeny and processing the progeny to obtain
foodstuff. The modification of the germ cell comprises altering the
genetic composition so as to disrupt or reduce the expression of
the naturally occurring gene encoding for production of GDF-8
protein. In a particular embodiment, the transgene comprises
antisense polynucleotide sequences to the GDF-8 protein.
Alternatively, the transgene may comprise a non-functional sequence
which replaces or intervenes in the native GDF-8 gene.
[0013] The subject invention also provides a method of producing
avian food products having improved muscle content. The method
includes modifying the genetic makeup of the germ cells of a
pronuclear embryo of the avian animal, implanting the embryo into
the oviduct of a pseudopregnant female into an embryo of a chicken,
culturing the embryo under conditions whereby progeny are hatched,
testing the progeny for presence of the genetic alteration to
identify transgene-positive progeny, cross-breeding
transgene-positive progeny and processing the progeny to obtain
foodstuff.
[0014] The invention also provides a method for treating a muscle
or adipose tissue disorder in a subject. The method includes
administering a therapeutically effective amount of a GDF-8 agent
to the subject, thereby inhibiting abnormal growth of muscle or
adipose tissue. The GDF-8 agent may include an antibody, a GDF-8
antisense molecule or a dominant negative polypeptide, for example.
In one aspect, a method for inhibiting the growth regulating
actions of GDF-8 by contacting an anti-GDF-8 monoclonal antibody, a
GDF-8 antisense molecule or a dominant negative polypeptide (or
polynucleotide encoding a dominant negative polypeptide) with fetal
or adult muscle cells or progenitor cells is included. These agents
can be administered to a patient suffering from a disorder such as
muscle wasting disease, neuromuscular disorder, muscle atrophy,
obesity or other adipocyte cell disorders, and aging, for
example.
[0015] The invention also provides a method for a method for
identifying a compound that affects GDF-8 activity or gene
expression includng incubating the compound with GDF-8 polypeptide,
or with a recombinant cell expressing GDF-8 under conditions
sufficient to allow the compounds to interact and determining the
effect of the compound on GDF-8 activity or expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A is a Northern blot showing expression of GDF-8 mRNA
in adult tissues. The probe was a partial murine GDF-8 clone.
[0017] FIG. 1B is a Southern blot showing GDF-8 genomic sequences
identified in mouse, rat, human, monkey, rabbit, cow, pig, dog and
chicken.
[0018] FIGS. 2A to 2D shows partial nucleotide and predicted amino
acid sequences of murine GDF-8 (FIG. 2a; SEQ ID NOS: 5 and 6,
respectively), human GDF-8 (FIG. 2b; SEQ ID NOS: 7 and 8,
respectively), rat GDF-8 (FIG. 2c; SEQ ID NOS: 32 and 33,
respectively) and chicken GDF-8 (FIG. 2d; SEQ ID NOS: 34 and 35).
The putative dibasic processing sites in the murine sequence are
boxed.
[0019] FIG. 3a shows the alignment of the C-terminal sequences of
GDF-8 with other members of the TGF-.beta. superfamily (SEQ ID NOS:
12 and 36-49, respectively). The conserved cysteine residues are
boxed. Dashes denote gaps introduced in order to maximize
alignment.
[0020] FIG. 3b shows the alignment of the C-terminal sequences of
GDF-8 from human (SEQ ID NO: 14), murine (SEQ ID NO: 12), rat (SEQ
ID NO:33), and chicken (SEQ ID NO: 35) sequences.
[0021] FIG. 4 shows amino acid homologies among 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 particular subgroups.
[0022] FIGS. 5a to 5d show the sequence of GDF-8. Nucleotide and
amino acid sequences of murine (FIG. 5a and 5b)(GenBank accession
number U84005; SEQ ID NO:11 and 12, respectively) and human (FIG.
5c and 5d; SEQ ID NO:13 and 14, respectively) 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 (SEQ ID NO: 50) proteolytic cleavage sites are boxed.
[0023] FIG. 6 shows a hydropathicity profile of GDF-8. Average
hydrophobicity values for murine (FIG. 6a) and human (FIG. 6b)
GDF-8 were calculated using the method of Kyte and Doolittle (J.
Mol. Biol, 157:105-132, 1982). Positive numbers indicate increasing
hydrophobicity.
[0024] FIGS. 7a and 7b show a comparison of murine and human GDF-8
amino acid sequences (SEQ ID NOS: 12 and 14, respectively). 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-terminus. Identities
between the two sequences are denoted by a vertical line.
[0025] FIG. 8 shows the expression of GDF-8 in bacteria. BL21 (DE3)
(pLysS) cells carrying a pRSET/GDF-8 expression plasmid were
induced with isopropylthio-.beta.-galactoside, and the GDF-8 fusion
protein was purified by metal chelate chromatography. Lanes:
total=total cell lysate; soluble=soluble protein fraction;
insoluble-insoluble protein fraction (resuspended in 10 Mm Tris pH
8.0, 50 mM sodium phosphate, 8 M urea, and 10 mM P-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 molecular weight standards are
shown at the right. Arrow indicates the position of the GDF-8
fusion protein.
[0026] FIG. 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 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 [.sup.125I]iodoproteina. Arrow indicates the position of the
processed GDF-8 protein.
[0027] FIGS. 10a and 10b show the expression of GDF-8 mRNA. Poly
A-selected RNA (5.mu.g each) prepared from adult tissues (FIG. 10a)
or placentas end embryos (FIG. 10b) at the indicated days of
gestation was electrophoresed on formaldehyde gels, blotted, and
probed with full length murine GDF-8.
[0028] FIG. 11 shows chromosomal mapping of human GDF-8. DNA
samples prepared from human/rodent somatic cell hybrid lines were
subjected to PCR, 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, CHO, and H, the starting DNA
template was total genomic DNA from mouse, hamster, and human
sources, respectively. In the lane marked B 1, no template DNA was
used. Numbers at left indicate the mobilities of DNA standards.
[0029] FIG. 12a shows a map of the GDF-8 locus (top line) and
targeting construct (second line). The black and stippled boxes
represent coding sequences for the pro- and C-terminal regions,
respectively. The white boxes represent 5 and 3 untranslated
sequences. A probe derived from the region downstream of the 3
homology fragment and upstream of the most distal HindIII site
shown hybridizes to an 11.2 kb HindIII fragment in the GDF-8 gene
and a 10.4 kb fragment in an homologously targeted gene.
Abbreviations: H, HindIII; X, Xba I.
[0030] FIG. 12b shows a Southern blot analysis of offspring derived
from a mating of heterozygous mutant mice. The lanes are as
follows: DNA prepared from wild type 129 SV/J mice (lane 1),
targeted embryonic stem cells (lane 2), F1 heterozygous mice (lanes
3 and 4), and offspring derived from a mating of these mice (lanes
5-13).
[0031] FIGS. 13a and 13b show the muscle fiber size distribution in
mutant and wild type littermates.
[0032] FIG. 13a shows the smallest cross-sectional fiber widths
measured for wild type (n=1761) and mutant (n=1052) tibialis
cranial.
[0033] FIG. 13b shows wild type (n=900) and mutant (n=900)
gastrocnemius muscles, and fiber sizes were plotted as a percent of
total fiber number. Standard deviations were 9 and 10 .mu.m,
respectively, for wild type and mutant tibialis cranial is and 11
and 9 .mu.m, respectively, for wild type and mutant gastrocnemius
muscles. Legend: o-o, wild type; _-_, mutant.
[0034] FIG. 14a shows the nucleotide and deduced amino acid
sequence for baboon GDF-8 (SEQ ID NO:18 and 19, respectively).
[0035] FIG. 14b shows the nucleotide and deduced amino acid
sequence for bovine GDF-8 (SEQ ID NO: 20 and 21, respectively).
[0036] FIG. 14c shows the nucleotide and deduced amino acid
sequence for chicken GDF-8 (SEQ ID NO:22 and 23, respectively).
[0037] FIG. 14d shows the nucleotide and deduced amino acid
sequence for rat GDF-8 (SEQ ID NO:24 and 25, respectively).
[0038] FIG. 14e shows the nucleotide and deduced amino acid
sequence for turkey GDF-8 (SEQ ID NO:26 and 27, respectively).
[0039] FIG. 14f shows the nucleotide and deduced amino acid
sequence for porcine GDF-8 (SEQ ID NO:28 and 29, respectively).
[0040] FIG. 14g shows the nucleotide and deduced amino acid
sequence for ovine GDF-8 (SEQ ID NO:30 and 31, respectively).
[0041] FIGS. 15a and 15b show an alignment between murine, rat,
human, porcine, ovine, baboon, bovine, chicken, and turkey GDF-8
amino acid sequences (SEQ ID NO:12, 25, 14, 29, 31, 19, 21, 23 and
27, respectively).
DETAILED DESCRIPTION OF THE INVENTION
[0042] 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.
[0043] The animals contemplated for use in the practice of the
subject invention are those animals generally regarded as useful
for the processing of food stuffs, i.e. avian such as meat bred and
egg laying chicken and turkey, ovine such as lamb, bovine such as
beef cattle and milk cows, piscine and porcine. For purposes of the
subject invention, these animals are referred to as "transgenic"
when such animal has had a heterologous DNA sequence, or one or
more additional DNA sequences normally endogenous to the animal
(collectively referred to herein as "transgenes") chromosomally
integrated into the germ cells of the animal. The transgenic animal
(including its progeny) will also have the transgene integrated
into the chromosomes of somatic cells.
[0044] The TGF-.beta. 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-.beta. 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.
[0045] In particular, certain members of this superfamily have
expression patterns or 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, 1990). Another family member, namely, GDF-1, is
nervous system-specific in its expression pattern (Lee, S. J.,
Proc. Natl. Acad. Sci., USA, 88:4250, 1991), and certain other
family members, such as Vgr-1 (Lyons, et al., Proc. Natl. 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 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 muscular
dystrophy, or in maintaining cells or tissues in culture prior to
transplantation.
[0046] 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-.beta. family are also important mediators of
tissue repair. TGF-.beta. 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-.beta. 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 transplantation or for enhancing the
efficiency of the fusion.
[0047] 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 abnormal proliferation of adipocytes. In
this regard, TGF-.beta. has been shown to be a potent inhibitor of
adipocyte differentiation in vitro (Ignotz and Massague, Proc.
Natl. Acad. Sci., USA 82:8530, 1985).
[0048] Polypeptides, Polynucleotides, Vectors and Host Cells
[0049] The invention provides substantially pure GDF-8 polypeptide
and isolated polynucleotides that encode GDF-8. 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 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.
[0050] 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 GDF8 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.
[0051] Specifically disclosed herein is a genomic DNA sequence
containing a portion of the GDF-8 gene. The sequence contains an
open reading frame 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 fragment of 109
and 103 amino acids for murine and human species, respectively,
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
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. GDF-8 is also
capable of forming dimers, or heterodimers, with an expected
molecular weight of approximately 23-30KD (see Example 4). For
example, GDF-8 may form heterodimers with other family members,
such as GDF-11.
[0052] Also provided herein are the biologically active C-terminal
fragments of chicken (FIG. 2c) and rat (FIG. 2d) GDF-8. The full
length nucleotide and deduced amino acid sequences for baboon,
bovine, chicken, rat, ovine, porcine, and turkey are shown in FIGS.
14a-g and human and murine are shown in FIG. 5. As shown in FIG.
3b, alignment of the amino acid sequences of human, murine, rat and
chicken GDF-8 indicate that the sequences are 100% identical in the
C-terminal biologically active fragment. FIG. 15 a and 15b also
show the alignment of GDF-8 amino acid sequences for murine, rat,
human, baboon, porcine, ovine, bovine, chicken and turkey. Given
the extensive conservation of amino acid sequences between species,
it would now be routine for one of skill in the art to obtain the
GDF-8 nucleic acid and amino acid sequence for GDF-8 from any
species, including those provided herein, as well as piscine, for
example.
[0053] The C-terminal region of GDF-8 following the putative
proteolytic processing site shows significant homology to the known
members of the TGF-.beta. superfamily. The GDF-8 sequence contains
most of the residues that are highly conserved in other family
members and in other species(see FIGS. 3a and 3b and 15a and 15b).
Like the TGF-.beta.s and inhibin .beta.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 FIG.
4).
[0054] 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 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 development of a
smaller active molecule which would have broader utility. For
example, one can remove amino or carboxy terminal amino acids which
are not required for GDF-8 biological activity.
[0055] 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 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
place of an unsubstituted parent amino acid provided that
antibodies raised to the substituted polypeptide also immunoreact
with the unsubstituted polypeptide.
[0056] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
techniques which are 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 (PCR) 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 shared structural features.
[0057] Preferably the GDF-8 polynucleotide of the invention is
derived from a mammalian organism, and most preferably from mouse,
rat, cow, pig, or human. GDF-8 polynucleotides from chicken,
turkey, fish and other species are also included herein. Screening
procedures which rely on nucleic acid hybridization make it
possible to isolate any gene sequence from any organism, provided
the appropriate probe is available. Given the extensive nucleotide
and amino acid homology between species, it would be routine for
one of skill in the art to obtain polynucleotides encoding GDF-8
from any species. Oligonucleotide probes, which correspond to a
part of the sequence encoding the protein in question, can be
synthesized chemically. This requires that short, oligopeptide
stretches of amino acid sequence must be known. The DNA sequence
encoding the protein can be deduced from the 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 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, 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).
[0058] The development of specific DNA sequences encoding GDF-8 can
also be 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 of mRNA is eventually
formed which is generally referred to as cDNA.
[0059] 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 introns.
[0060] 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 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 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 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).
[0061] A cDNA expression library, such as lambda gtl 1, can be
screened indirectly for GDF-8 peptides having at least one epitope,
using antibodies specific for GDF-8. Such antibodies can be either
polyclonally or monoclonally derived and used to detect expression
product indicative of the presence of GDF-8 cDNA.
[0062] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0063] An example of progressively higher stringency conditions is
as follows: 2.times. SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times. SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times. SSC at about 68 C (high stringency conditions). Washing
can be carried out using only one of these conditions, e.g., high
stringency conditions, or each of the conditions can be used, e.g.,
for 10-15 minutes each, in the order listed above, repeating any or
all of the steps listed. However, as mentioned above, optimal
conditions will vary, depending on the particular hybridization
reaction involved, and can be determined empirically.
[0064] 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 the subject host cell. It is
understood that all progeny may not be identical to the parental
cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is
used. Methods of stable transfer, meaning that the foreign DNA is
continuously maintained in the host, are known in the art.
[0065] In the present invention, the GDF-8 polynucleotide sequences
may be inserted into a recombinant expression vector. The term
"recombinant expression vector" refers to a plasmid, virus or other
vehicle known in the art that has been manipulated by 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 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, 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 1, or polyhedrin promoters).
[0066] Polynucleotide sequences encoding GDF-8 can be expressed in
either prokaryotes or eukaryotes. Hosts can include microbial,
yeast, insect and mammalian organisms. Methods of expressing DNA
sequences having eukaryotic or viral sequences in prokaryotes are
well known in the art. Biologically finctional viral and plasmid
DNA vectors capable of expression and replication in a host are
known in the art. Such vectors are used to incorporate DNA
sequences of the invention. Preferably, the mature C-terminal
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 TGF-.beta. family or
co-expressed with another pro-region (see for example, Hammonds, et
al., Molec. Endocrin., 5:149, 1991; Gray, A., and Mason, A.,
Science, 247:1328, 1990).
[0067] 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 the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired.
[0068] 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
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).
[0069] 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.
[0070] GDF-8 Antibodies and Methods of Use
[0071] The invention includes antibodies immunoreactive with GDF-8
polypeptide or functional fragments thereof. Antibody which
consists essentially of pooled monoclonal antibodies with different
epitopic specificities, as well as distinct 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 as fragments thereof, such as Fab
and F(ab').sub.2, Fv and SCA fragments which are capable of binding
an epitopic determinant on GDF-8.
[0072] (1) An Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain.
[0073] (2) An Fab' fragment of an antibody molecule can be obtained
by treating a whole antibody molecule with pepsin, followed by
reduction, to yield a molecule consisting of an intact light chain
and a portion of a heavy chain. Two Fab' fragments are obtained per
antibody molecule treated in this manner.
[0074] (3) An (Fab').sub.2 fragment of an antibody can be obtained
by treating a whole antibody molecule with the enzyme pepsin,
without subsequent reduction. A (Fab').sub.2 fragment is a dimer of
two Fab' fragments, held together by two disulfide bonds.
[0075] (4) An Fv fragment is defined as a genetically engineered
fragment containing the variable region of a light chain and the
variable region of a heavy chain expressed as two chains.
[0076] (5) A single chain antibody ("SCA") is a genetically
engineered single chain molecule containing the variable region of
a light chain and the variable region of a heavy chain, linked by a
suitable, flexible polypeptide linker.
[0077] As used in this invention, the term "epitope" refers to an
antigenic determinant on an antigen, such as a GDF-8 polypeptide,
to which the paratope of an antibody, such as an GDF-8-specific
antibody, binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics.
[0078] As is mentioned above, antigens that can be used in
producing GDF-8-specific antibodies include GDF-8 polypeptides or
GDF-8 polypeptide fragments. The polypeptide or peptide used to
immunize an animal can be obtained by standard recombinant,
chemical synthetic, or purification methods. As is well known in
the art, in order to increase immunogenicity, an antigen can be
conjugated to a carrier protein. Commonly used carriers include
keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum
albumin (BSA), and tetanus toxoid. The coupled peptide is then used
to immunize the animal (e.g., a mouse, a rat, or a rabbit). In
addition to such carriers, well known adjuvants can be administered
with the antigen to facilitate induction of a strong immune
response.
[0079] 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 genotypically.
Malignant cells (i.e. cancer) develop as a result of a multistep
process. The GDF-8 polynucleotide that is an antisense molecule or
that encodes a dominant negative GDF-8 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
agent (e.g., a suppressing or enhancing agent). One such disorder
is a malignant cell proliferative disorder, for example.
[0080] The invention provides a method for detecting a cell
proliferative 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 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 compared with the level in a
normal cell to determine whether the subject has a GDF-8-associated
cell proliferative disorder. Such methods of detection are also
useful using nucleic acid hybridization to detect the level of
GDF-8 mRNA in a sample or to detect an altered GDF-8 gene.
Preferably the subject is human.
[0081] 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 immunoassays in which they can
be utilized in liquid phase or bound to a solid phase 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 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.
[0082] 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
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.
[0083] 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 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 other suitable labels for binding to the antibody, or will
be able to ascertain such, using routine experimentation.
[0084] Another technique which may also result in greater
sensitivity consists of coupling the antibodies to low molecular
weight haptens. These haptens can 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 dinitrophenyi,
puridoxal, and fluorescein, which can react with specific
antihapten antibodies.
[0085] In using the monoclonal antibodies of the invention for the
in vivo detection of 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.
[0086] 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-background signal ratio.
[0087] 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 known to those of skill
in the art.
[0088] 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 deleterious radiation with respect to the host is
minimized. Ideally, a radioisotope 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.
[0089] For in vivo diagnosis radioisotopes may be bound to
immunoglobulin either 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 bound to the
monoclonal antibodies of the invention are .sup.111In, .sup.97Ru,
.sup.67Ga, .sup.68Ga, 72As, 89Zr and 201Tl.
[0090] 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 (MRI) or electron spin
resonance (ESR). In general, any conventional method for
visualizing diagnostic imaging can be utilized. Usually gamma and
positron emitting radioisotopes are used for camera imaging and
paramagnetic isotopes for MRI. Elements which are particularly
useful in such techniques include .sup.157Gd, .sup.55Mn,
.sup.162Dy, .sup.52Cr, and .sup.56Fe.
[0091] The monoclonal antibodies of the invention can be used in
vitro and in vivo to 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 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 in the
subject receiving therapy.
[0092] Additional Methods of Treatment and Diagnosis
[0093] 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.
Treatment includes administration of a reagent which modulates
activity. The term "modulate" envisions the suppression or
expression of GDF-8 when it is over-expressed, or augmentation of
GDF-8 expression when it is underexpressed. When a
muscle-associated disorder is associated with GDF-8 overexpression,
such suppressive reagents as antisense GDF-8 polynucleotide
sequence, dominant negative sequences or GDF-8 binding antibody can
be introduced into a cell. In addition, an anti-idiotype antibody
which binds to a monoclonal antibody which binds GDF-8 of the
invention, or an epitope thereof, may also be used in the
therapeutic method of the invention. Alternatively, when a cell
proliferative disorder is associated with underexpression or
expression of a mutant GDF-8 polypeptide, a sense polynucleotide
sequence (the DNA coding strand) or GDF-8 polypeptide can be
introduced into the cell. Such muscle-associated disorders include
cancer, muscular dystrophy, spinal cord injury, traumatic injury,
congestive obstructive pulmonary disease (COPD), AIDS or cachecia.
In addition, the method of the invention can be used in the
treatment of obesity or of disorders related to abnormal
proliferation of adipocytes. One of skill in the art can determine
whether or not a particular therapeutic course of treatment is
successful by several methods described herein (e.g., muscle fiber
analysis or biopsy; determination of fat content). The present
examples demonstrate that the methods of the invention are useful
for decreasing fat content, and therefore would be useful in the
treatment of obesity and related disorders (e.g., diabetes).
Neurodegenerative disorders are also envisioned as treated by the
method of the invention.
[0094] Thus, where a cell-proliferative disorder is associated with
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 diseases,
for example. In addition, dominant-negative GDF-8 mutants would be
useful to actively interfere with function of normal GDF-8.
[0095] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262:40, 1990). In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will not
translate a mRNA that is double-stranded.
[0096] 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 translation of genes is well known in the art
(Marcus-Sakura, Anal. Biochem., 172:289, 1988).
[0097] 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 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.
[0098] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, 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 ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0099] In another embodiment of the present invention, a nucleotide
sequence encoding a GDF-8 dominant negative protein is provided.
For example, a genetic construct that contain such a dominant
negative encoding gene may be operably linked to a promoter, such
as a tissue-specific promoter. For example, a skeletal muscle
specific promoter (e.g., human skeletal muscle a-actin promoter) or
developmentally specific promoter (e.g., MyHC 3, which is
restricted in skeletal muscle to the embryonic period of
development, or an inducible promoter (e.g., the orphan nuclear
receptor TIS1).
[0100] Such constructs are useful in methods of modulating a
subject s skeletal mass. For example, a method include transforming
an organism, tissue, organ or cell with a genetic construct
encoding a dominant negative GDF-8 protein and suitable promoter in
operable linkage and expressing the dominant negative encoding
GDF-8 gene, thereby modulating muscle mass by interfering with
wild-type GDF-8 activity.
[0101] GDF-8 most likely forms dimers, homodimers or heterodimers
and may even form heterodimers with other GDF family members, such
as GDF-11 (see Example 4). Hence, while not wanting to be bound by
a particular theory, the dominant negative effect described herein
may involve the formation of non-functional homodimers or
heterodimers of dominant negative and wild-type GDF-8 monomers.
More specifically, it is possible that any non-functional homodimer
or any heterodimer formed by the dimerization of wild-type and/or
dominant negative GDF-8 monomers produces a dominant effect by: 1)
being synthesized but not processed or secreted; 2) inhibiting the
secretion of wild type GDF-8; 3) preventing normal proteolytic
cleavage of the preprotein thereby producing a nonfunctional GDF-8
molecule; 4) altering the affinity of the non-functional dimer
(e.g., homodimeric or heterodimeric GDF-8) to a receptor or
generating an antagonistic form of GDF-8 that binds a receptor
without activating it; or 5) inhibiting the intracellular
processing or secretion of GDF-8 related or TGF-B family
proteins.
[0102] Non-functional GDF-8 can function to inhibit the growth
regulating actions of GDF-8 on muscle cells that include a dominant
negative GDF-8 gene. Deletion or missense dominant negative forms
of GDF-8 that retain the ability to form dimers with wild-type
GDF-8 protein but do not function as wild-type GDF-8 proteins may
be used to inhibit the biological activity of endogenous wild-type
GDF-8. For example, in one embodiment, the proteolytic processing
site of GDF-8 may be altered (e.g., deleted) resulting in a GDF-8
molecule able to undergo subsequent dimerization with endogenous
wild-type GDF-8 but unable to undergo further processing into a
mature GDF-8 form. Alternatively, a non-functional GDF-8 can
function as a monomeric species to inhibit the growth regulating
actions of GDF-8 on muscle cells.
[0103] Any genetic recombinant method in the art may be used, for
example, recombinant viruses may be engineered to express a
dominant negative form of GDF-8 which may be used to inhibit the
activity of wild-type GDF-8. Such viruses may be used
therapeutically for treatment of diseases resulting from aberrant
over-expression or activity of GDF-8 protein, such as in
denervation hypertrophy or as a means of controlling GDF-8
expression when treating disease conditions involving muscle, such
as in musculodegenerative diseases or in tissue repair due to
trauma or in modulating GDF-8 expression in animal husbandry (e.g.,
transgenic animals for agricultural purposes).
[0104] The invention provides a method for treating a muscle or
adipose tissue disorder in a subject. The method includes
administering a therapeutically effective amount of a GDF-8 agent
to the subject, thereby inhibiting abnormal growth of muscle or
adipose tissue. The GDF-8 agent may include a GDF-8 antisense
molecule or a dominant negative polypeptide, for example. A
"therapeutically effective amount" of a GDF-8 agent is that amount
that ameliorates symptoms of the disorder or inhibits GDF-8 induced
growth of muscle, for example, as compared with a normal
subject.
[0105] Gene Therapy
[0106] The present invention also provides gene therapy for the
treatment of cell proliferative or immunologic disorders which are
mediated by GDF-8 protein. Such therapy would achieve its
therapeutic effect by introduction of the GDF-8 antisense or
dominant negative encoding polynucleotide into cells having the
proliferative disorder. Delivery of antisense or dominant negative
GDF-8 polynucleotide can be achieved using a recombinant expression
vector such as a chimeric virus or a colloidal dispersion system.
Especially preferred for therapeutic delivery of antisense or
dominant negative sequences is the use of targeted liposomes. In
contrast, when it is desirable to enhance GDF-8 production, a
"sense" GDF-8 polynucleotide or functional equivalent (e.g., the
C-term active region) is introduced into the appropriate
cell(s).
[0107] 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 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 can be identified and
generated. By inserting a GDF-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 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.
[0108] 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 plasmids 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
encapsulation. Helper cell lines which have deletions of the
packaging signal include, but are not limited to .psi.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 the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced.
[0109] Alternatively, NIH 3T3 or other tissue culture cells can be
directly transfected with plasmids encoding the retroviral
structural genes gag, pol and env, by 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.
[0110] Another targeted delivery system for GDF-8 polynucleotides
is a colloidal dispersion system. Colloidal dispersion systems
include macromolecule complexes, nanocapsules, microspheres, beads,
and lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a 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 in size from 0.2-4.0 .mu.m 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 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 contents of the vesicle to the
target cell cytoplasm at high efficiency; and (4) accurate and
effective expression of genetic information (Manning, et al.,
Biotechniques, 6:682, 1988).
[0111] 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 pH, ionic
strength, and the presence of divalent cations.
[0112] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidyiglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0113] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0114] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand.
[0115] 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 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. In embodiments where GDF-8 polypeptide is administered
to a subject, the dosage range is about 0.1 ug/kg to 100 mg/kg;
more preferably from about 1 ug/kg to 75 mg/kg and most preferably
from about 10 mg/kg to 50 mg/kg.
[0116] Chromosomal Location of GDF-8
[0117] 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 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 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.
[0118] Transgenic Animals and Methods of Making the same
[0119] Various methods to make the transgenic animals of the
subject invention can be employed. Generally speaking, three such
methods may be employed. In one such method, an embryo at the
pronuclear stage (a "one cell embryo") is harvested from a female
and the transgene is microinjected into the embryo, in which case
the transgene will be chromosomally integrated into both the germ
cells and somatic cells of the resulting mature animal. In another
such method, embryonic stem cells are isolated and the transgene
incorporated therein by electroporation, plasmid transfection or
microinjection, followed by reintroduction of the stem cells into
the embryo where they colonize and contribute to the germ line.
Methods for microinjection of mammalian species is described in
U.S. Pat. No. 4,873,191. In yet another such method, embryonic
cells are infected with a retrovirus containing the transgene
whereby the germ cells of the embryo have the transgene
chromosomally integrated therein. When the animals to be made
transgenic are avian, because avian fertilized ova generally go
through cell division for the first twenty hours in the oviduct,
microinjection into the pronucleus of the fertilized egg is
problematic due to the inaccessibility of the pronucleus.
Therefore, of the methods to make transgenic animals described
generally above, retrovirus infection is preferred for avian
species, for example as described in U.S. Pat. No. 5,162,215. If
microinjection is to be used with avian species, however, a
recently published procedure by Love et al., (Biotechnology, Jan.
12, 1994) can be utilized whereby the embryo is obtained from a
sacrificed hen approximately two and one-half hours after the
laying of the previous laid egg, the transgene is microinjected
into the cytoplasm of the germinal disc and the embryo is cultured
in a host shell until maturity. When the animals to be made
transgenic are bovine or porcine, microinjection can be hampered by
the opacity of the ova thereby making the nuclei difficult to
identify by traditional differential interference-contrast
microscopy. To overcome this problem, the ova can first be
centrifuged to segregate the pronuclei for better
visualization.
[0120] The "non-human animals" of the invention bovine, porcine,
ovine and avian animals (e.g., cow, pig, sheep, chicken, turkey).
The "transgenic non-human animals" of the invention are produced by
introducing "transgenes" into the germline of the non-human animal.
Embryonal target cells at various developmental stages can be used
to introduce transgenes. Different methods are used depending on
the stage of development of the embryonal target cell. The zygote
is the best target for micro-injection. The use of zygotes as a
target for gene transfer has a major advantage in that in most
cases the injected DNA will be incorporated into the host gene
before the first cleavage (Brinster et al., Proc. Natl. Acad. Sci.
USA 82:4438-4442, 1985). As a consequence, all cells of the
transgenic non-human animal will carry the incorporated transgene.
This will in general also be reflected in the efficient
transmission of the transgene to offspring of the founder since 50%
of the germ cells will harbor the transgene.
[0121] The term "transgenic" is used to describe an animal which
includes exogenous genetic material within all of its cells. A
"transgenic" animal can be produced by cross-breeding two chimeric
animals which include exogenous genetic material within cells used
in reproduction. Twenty-five percent of the resulting offspring
will be transgenic i.e., animals which include the exogenous
genetic material within all of their cells in both alleles. 50% of
the resulting animals will include the exogenous genetic material
within one allele and 25% will include no exogenous genetic
material.
[0122] In the microinjection method useful in the practice of the
subject invention, the transgene is digested and purified free from
any vector DNA e.g. by gel electrophoresis. It is preferred that
the transgene include an operatively associated promoter which
interacts with cellular proteins involved in transcription,
ultimately resulting in constitutive expression. Promoters useful
in this regard include those from cytomegalovirus (CMV), Moloney
leukemia virus (MLV), and herpes virus, as well as those from the
genes encoding metallothionin, skeletal actin, P-enolpyruvate
carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine
kinase. Promoters for viral long terminal repeats (LTRs) such as
Rous Sarcoma Virus can also be employed. When the animals to be
made transgenic are avian, preferred promoters include those for
the chicken .beta.-globin gene, chicken lysozyme gene, and avian
leukosis virus. Constructs useful in plasmid transfection of
embryonic stem cells will employ additional regulatory elements
well known in the art such as enhancer elements to stimulate
transcription, splice acceptors, termination and polyadenylation
signals, and ribosome binding sites to permit translation.
[0123] Retroviral infection can also be used to introduce transgene
into a non-human animal, as described above. The developing
non-human embryo can be cultured in vitro to the blastocyst stage.
During this time, the blastomeres can be targets for retro viral
infection (Jaenich, R., Proc. Natl. Acad. Sci USA 73:1260-1264,
1976). Efficient infection of the blastomeres is obtained by
enzymatic treatment to remove the zona pellucida (Hogan, et al.
(1986) in Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector
system used to introduce the transgene is typically a
replication-defective retro virus carrying the transgene (Jahner,
et al., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der
Putten, et al., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985).
Transfection is easily and efficiently obtained by culturing the
blastomeres on a monolayer of virus-producing cells (Van der
Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele (D.
Jahner et al., Nature 2-98:623-628, 1982). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic nonhuman animal.
Further, the founder may contain various retro viral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line, albeit with low
efficiency, by intrauterine retroviral infection of the
midgestation embryo (D. Jahner et al., supra).
[0124] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(M. J. Evans et al. Nature 292:154-156, 1981; M. O. Bradley et al.,
Nature 309: 255-258, 1984; Gossler, et al., Proc. Natl. Acad. Sci
USA 83: 9065-9069, 1986; and Robertson et al., Nature 322:445-448,
1986). Transgenes can be efficiently introduced into the ES cells
by DNA transfection or by retro virus-mediated transduction. Such
transformed ES cells can thereafter be combined with blastocysts
from a nonhuman animal. The ES cells thereafter colonize the embryo
and contribute to the germ line of the resulting chimeric animal.
(For review see Jaenisch, R., Science 240: 1468-1474, 1988).
[0125] "Transformed" means a cell into which (or into an ancestor
of which) has been introduced, by means of recombinant nucleic acid
techniques, a heterologous nucleic acid molecule. "Heterologous"
refers to a nucleic acid sequence that either originates from
another species or is modified from either its original form or the
form primarily expressed in the cell.
[0126] "Transgene" means any piece of DNA which is inserted by
artifice into a cell, and becomes part of the genome of the
organism (i.e., either stably integrated or as a stable
extrachromosomal element) which develops from that cell. Such a
transgene may include a gene which is partly or entirely
heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism.
Included within this definition is a transgene created by the
providing of an RNA sequence which is transcribed into DNA and then
incorporated into the genome. The transgenes of the invention
include DNA sequences which encode GDF-8, and include GDF-sense,
antisense, dominant negative encoding polynucleotides, which may be
expressed in a transgenic non-human animal. The term "transgenic"
as used herein additionally includes any organism whose genome has
been altered by in vitro manipulation of the early embryo or
fertilized egg or by any transgenic technology to induce a specific
gene knockout. The term gene knockout as used herein, refers to the
targeted disruption of a gene in vivo with complete loss of
function that has been achieved by any transgenic technology
familiar to those in the art. In one embodiment, transgenic animals
having gene knockouts are those in which the target gene has been
rendered nonfunctional by an insertion targeted to the gene to be
rendered non-functional by homologous recombination. As used
herein, the term "transgenic" includes any transgenic technology
familiar to those in the art which can produce an organism carrying
an introduced transgene or one in which an endogenous gene has been
rendered non-functional or "knocked out." An example of a transgene
used to "knockout" GDF-8 function in the present Examples is
described in Example 8 and FIG. 12a. Thus, in another embodiment,
the invention provides a transgene wherein the entire mature
C-terminal region of GDF-8 is deleted.
[0127] The transgene to be used in the practice of the subject
invention is a DNA sequence comprising a modified GDF-8 coding
sequence. In a preferred embodiment, the GDF-8 gene is disrupted by
homologous targeting in embryonic stem cells. For example, the
entire mature C-terminal region of the GDF-8 gene may be deleted as
described in the examples below. Optionally, the GDF-8 disruption
or deletion may be accompanied by insertion of or replacement with
other DNA sequences, such as a non-functional GDF-8 sequence. In
other embodiments, the transgene comprises DNA antisense to the
coding sequence for GDF-8. In another embodiment, the transgene
comprises DNA encoding an antibody or receptor peptide sequence
which is able to bind to GDF-8. The DNA and peptide sequences of
GDF-8 are known in the art, the sequences, localization and
activity disclosed in WO94/21681 and pending U.S. patent
application Ser. No. 08/033,923, filed on Mar. 19, 1993,
incorporated by reference in its entirety. The disclosure of both
of these applications are hereby incorporated herein by reference.
Where appropriate, DNA sequences that encode proteins having GDF-8
activity but differ in nucleic acid sequence due to the degeneracy
of the genetic code may also be used herein, as may truncated
forms, allelic variants and interspecies homologues.
[0128] The invention also includes animals having heterozygous
mutations in GDF-8 or partial inhibition of GDF-8 function or
expression. A heterozygote would exhibit an intermediate increase
in muscle mass as compared to the homozygote as shown in Table 4
below. In other words, partial loss of function leads to a partial
increase in muscle mass. One of skill in the art would readily be
able to determine if a particular mutation or if an antisense
molecule was able to partially inhibit GDF-8. For example, in vitro
testing may be desirable initially by comparison with wild-type or
untreated GDF-8 (e.g., comparison of northern blots to examine a
decrease in expression).
[0129] After an embryo has been microinjected, colonized with
transfected embryonic stem cells or infected with a retrovirus
containing the transgene (except for practice of the subject
invention in avian species which is addressed elsewhere herein) the
embryo is implanted into the oviduct of a pseudopregnant female.
The consequent progeny are tested for incorporation of the
transgene by Southern blot analysis of blood samples using
transgene specific probes. PCR is particularly useful in this
regard. Positive progeny (G0) are crossbred to produce offspring
(G1) which are analyzed for transgene expression by Northern blot
analysis of tissue samples. To be able to distinguish expression of
like-species transgenes from expression of the animals endogenous
GDF-8 gene(s), a marker gene fragment can be included in the
construct in the 3' untranslated region of the transgene and the
Northern probe designed to probe for the marker gene fragment. The
serum levels of GDF-8 can also be measured in the transgenic animal
to establish appropriate expression. Expression of the GDF-8
transgenes, thereby decreasing the GDF-8 in the tissue and serum
levels of the transgenic animals and consequently increasing the
muscle tissue content results in the foodstuffs from these animals
(i.e. eggs, beef, pork, poultry meat, milk, etc.) having markedly
increased muscle content, and preferably without increased, and
more preferably, reduced levels of fat and cholesterol. By practice
of the subject invention, a statistically significant increase in
muscle content, preferably at least a 2% increase in muscle content
(e.g., in chickens), more preferably a 25% increase in muscle
content as a percentage of body weight, more preferably greater
than 40% increase in muscle content in these foodstuffs can be
obtained.
[0130] Additional Methods of Use
[0131] Thus, the present invention includes methods for increasing
muscle mass in domesticated animals, characterized by inactivation
or deletion of the gene encoding growth and differentiation
factor-8 (GDF-8). The domesticated animal is preferably selected
from the group consisting of ovine, bovine, porcine, piscine and
avian. The animal may be treated with an isolated polynucleotide
sequence encoding growth and differentiation factor-8 which
polynucleotide sequence is also from a domesticated animal selected
from the group consisting of ovine, bovine, porcine, piscine and
avian. The present invention includes methods for increasing the
muscle mass in domesticated animals characterized by administering
to a domesticated animal monoclonal antibodies directed to the
GDF-8 polypeptide. The antibody may be an anti-GDF-8, and may be
either a monoclonal antibody or a polyclonal antibody.
[0132] The invention includes methods comprising using an
anti-GDF-8 monoclonal antibody, antisense, or dominant negative
mutants as a therapeutic agent to inhibit the growth regulating
actions of GDF-8 on muscle cells. Muscle cells are defined to
include fetal or adult muscle cells, as well as progenitor cells
which are capable of differentiation into muscle. The monoclonal
antibody may be a humanized (e.g., either fully or a chimeric)
monoclonal antibody, of any species origin, such as murine, ovine,
bovine, porcine or avian. Methods of producing antibody molecules
with various combinations of "humanized" antibodies are well known
in the art and include combining murine variable regions with human
constant regions (Cabily, et al. Proc.Natl.Acad.Sci. USA, 81:3273,
1984), or by grafting the murine-antibody complementary determining
regions (CDRs) onto the human framework (Richmann, et al., Nature
332:323, 1988). Other general references which teach methods for
creating humanized antibodies include Morrison, et al., Science,
229:1202, 1985; Jones, et al., Nature, 321:522, 1986; Monroe, et
al., Nature 312:779, 1985; Oi, et al., BioTechniques, 4:214, 1986;
European Patent Application No. 302,620; and U.S. Pat. No.
5,024,834. Therefore, by humanizing the monoclonal antibodies of
the invention for in vivo use, an immune response to the antibodies
would be greatly reduced.
[0133] The monoclonal antibody, GDF-8 polypeptide, or GDF-8
polynucleotide (all "GDF-8 agents") may have the effect of
increasing the development of skeletal muscles. In preferred
embodiments of the claimed methods, the GDF-8 monoclonal antibody,
polypeptide, or polynucleotide is administered to a patient
suffering from a disorder selected from the group consisting of
muscle wasting disease, neuromuscular disorder, muscle atrophy or
aging. The GDF-8 agent may also be administered to a patient
suffering from a disorder selected from the group consisting of
muscular dystrophy, spinal cord injury, traumatic injury,
congestive obstructive pulmonary disease (COPD), AIDS or cachechia.
In a preferred embodiment, the GDF-8 agent is administered to a
patient with muscle wasting disease or disorder by intravenous,
intramuscular or subcutaneous injection; preferably, a monoclonal
antibody is administered within a dose range between about 0.1
mg/kg to about 100 mg/kg; more preferably between about 1 ug/kg to
75 mg/kg; most preferably from about 10 mg/kg to 50 mg/kg. The
antibody may be administered, for example, by bolus injunction or
by slow infusion. Slow infusion over a period of 30 minutes to 2
hours is preferred. The GDF-8 agent may be formulated in a
formulation suitable for administration to a patient. Such
formulations are known in the art.
[0134] The dosage regimen will be determined by the attending
physician considering various factors which modify the action of
the GDF-8 protein, e.g. amount of tissue desired to be formed, the
site of tissue damage, the condition of the damaged tissue, the
size of a wound, type of damaged tissue, the patient's age, sex,
and diet, the severity of any infection, time of administration and
other clinical factors. The dosage may vary with the type of matrix
used in the reconstitution and the types of agent, such as
anti-GDF-8 antibodies, to be used in the composition. Generally,
systemic or injectable administration, such as intravenous (IV),
intramuscular (IM) or subcutaneous (Sub-Q) injection.
Administration will generally be initiated at a dose which is
minimally effective, and the dose will be increased over a
preselected time course until a positive effect is observed.
Subsequently, incremental increases in dosage will be made limiting
such incremental increases to such levels that produce a
corresponding increase in effect, while taking into account any
adverse affects that may appear. The addition of other known growth
factors, such as IGF I (insulin like growth factor I), human,
bovine, or chicken growth hormone which may aid in increasing
muscle mass, to the final composition, may also affect the dosage.
In the embodiment where an anti-GDF-8 antibody is administered, the
anti-GDF-8 antibody is generally administered within a dose range
of about 0.1 ug/kg to about 100 mg/kg.; more preferably between
about 10 mg/kg to 50 mg/kg.
[0135] Progress can be monitored by periodic assessment of tissue
growth and/or repair. The progress can be monitored, for example,
x-rays, histomorphometric determinations and tetracycline
labeling.
[0136] Screening for GDF-8 Modulating Compounds
[0137] In another embodiment, the invention provides a method for
identifying a compound or molecule that modulates GDF-8 protein
activity or gene expression. The method includes incubating
components comprising the compound, GDF-8 polypeptide or with a
recombinant cell expressing GDF-8 polypeptide, under conditions
sufficient to allow the components to interact and determining the
effect of the compound on GDF-8 activity or expression. The effect
of the compound on GDF-8 activity can be measured by a number of
assays, and may include measurements before and after incubating in
the presence of the compound. Compounds that affect GDF-8 activity
or gene expression include peptides, peptidomimetics, polypeptides,
chemical compounds and biologic agents. Assays include Northern
blot analysis of GDF-8 mRNA (for gene expression), Western blot
analysis (for protein level) and muscle fiber analysis (for protein
activity).
[0138] The above screening assays may be used for detecting the
compounds or molecules that bind to the GDF-8 receptor or GDF-8
polypeptide, in isolating molecules that bind to the GDF-8 gene,
for measuring the amount of GDF-8 in a sample, either polypeptide
or RNA (mRNA), for identifying molecules that may act as agonists
or antagonists, and the like. For example, GDF-8 antagonists are
useful for treatment of muscular and adipose tissue disorders
(e.g., obesity).
[0139] Incubating includes conditions which allow contact between
the test compound and GDF-8 polypeptide or with a recombinant cell
expressing GDF-8 polypeptide. Contacting includes in solution and
in solid phase, or in a cell. The test compound may optionally be a
combinatorial library for screening a plurality of compounds.
Compounds identified in the method of the invention can be further
evaluated, detected, cloned, sequenced, and the like, either in
solution or after binding to a solid support, by any method usually
applied to the detection of a specific DNA sequence such as PCR,
oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012,
1985), allele-specific oligonucleotide (ASO) probe analysis
(Conner, et al., Proc. Natl. Acad. Sci. USA, 80:278, 1983),
oligonucleotide Landegren, et al., Science, 241:1077, 1988), and
the like. Molecular techniques for DNA analysis have been reviewed
(Landegren, et al., Science, 242:229-237, 1988).
[0140] All references cited herein are hereby incorporated by
reference in their entirety.
[0141] 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 those skilled in the art may
alternatively be used.
EXAMPLE 1
Identification and Isolation of a Novel TGF-.beta. Family
Member
[0142] To identify a new member of the TGF-.beta. superfamily,
degenerate oligonucleotides were designed which corresponded to two
conserved regions among the known family members: one 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 by
subcloning the PCR 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.
[0143] GDF-8 was identified from a mixture of PCR products obtained
with the primers
1 SJL141: 5'-CCGGAATTCGGITGG(G/C/A)A(G/A/T/C)(A/G)A(T/C)TG-
G(A/G)TI (SEQ ID NO: 1) (A/G)TI(T/G)CICC-3' SJL147:
5'-CCGGAATTC(G/A)CAI(G/C)C(G/A)CA(G/A)CT(GIA/T/C) (SEQ ID NO: 2)
TCIACI(G/A)(T/C)CAT-3'
[0144] PCR using these primers was carried out with 2 .mu.g mouse
genomic DNA at 94.degree. C. for 1 min, 50.degree. C. for 2 min,
and 72.degree. C. for 2 min for 40 cycles. PCR products of
approximately 280 bp were gel-purified, digested with Eco Rl,
gel-purified again, and subcloned in the Bluescript vector
(Stratagene, San Diego, Calif.). Bacterial colonies carrying
individual subcclones were picked into 96 well microtiter plates,
and multiple replicas were prepared by plating the cells onto
nitrocellulose. The replicate filters were hybridized to probes
representing known members of the family, and DNA was prepared from
nonhybridizing colonies for sequence analysis.
[0145] The primer combination of SJL141 and SJL147, encoding the
amino acid sequences GW(HIQ/N/KID/E)(DIN)W(V/I/M)(V/IIM)(A/S)P (SEQ
ID NO:9) and 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,PB, GDF-3 and GDF-5) and one novel sequence, which was
designated GDF-8, among 110 subclones analyzed.
[0146] Human GDF-8 was isolated using the primers:
2 ACM13: 5'-CGCGGATCCAGAGTCAAGGTGACAGACACAC-3' (SEQ ID NO: 3); and
ACM14: 5'-CGCGGATCCTCCTCATGAGCACCCACAGCGGTC-3' (SEQ ID NO: 4)
[0147] PCR using these primers was carried out with one .mu.g human
genomic DNA at 94.degree. C. for 1 min, 58.degree. C. for 2 min,
and 72.degree. C. for 2 min for 30 cycles. The PCR product was
digested with Bam Hl, gel-purified, and subcloned in the Bluescript
vector (Stratagene, San Francisco, Calif.).
EXAMPLE 2
Expression Pattern and Sequence of GDF-8
[0148] To determine the expression pattern of GDF-8, RNA samples
prepared from 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 5.times. SSPE, 10%
dextran sulfate, 50% formamide, 1% SDS, 200 .mu.g/ml salmon DNA,
and 0.1% each of bovine serum albumin, ficoll, and
polyvinylpyrrolidone. Five micrograms of twice poly A-selected RNA
prepared from each tissue (except for muscle, for which only 2
.mu.g RNA was used) were electrophoresed on formaldehyde gels,
blotted, and probed with GDF-8. As shown in FIG. 1, the GDF-8 probe
detected a single mRNA species expressed at highest levels in
muscle and at significantly lower levels in adipose tissue.
[0149] To obtain a larger segment of the GDF-8 gene, a mouse
genomic library was screened with a probe derived from the GDF-8
PCR product. The partial sequence of a GDF-8 genomic clone is shown
in FIG. 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 C 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 FIG. 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.
[0150] The C-terminal region of GDF-8 following the putative
proteolytic processing site shows significant homology to the known
members of the TGF-P; superfamily (FIG. 3). FIG. 3 shows the
alignment of the C-terminal 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, 87:9843-9847, 1990), human OP-1 (Ozkaynak, et
al., EMBO J., 9:2085-2093, 1990), human BMP-5 (Celeste, et al.,
Proc. Natl. Acad. Sci. USA, 87:9843-9847, 1990), human BMP-3
(Wozney, et al., Science, 242:1528-1534, 1988), human MiS (Cate, et
al. Cell, 45:685-698,1986), human inhibin alpha, .beta.A, and
.beta.B (Mason, et al., Biochem, Biophys. Res. Commun.,
135:957-964, 1986), human TGF-.beta.1 (Derynck, et al., Nature,
316:701 -705, 1985), humanTGF-R2 (deMartin, et al., EMBO J.,
6:3673-3677, 1987), and human TGF-.beta.3 (ten Dijke, et al., 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.
[0151] 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-.beta.s
and inhibin .beta.s, GDF-8 also contains two additional cysteine
residues. In the case of TGF-.beta.2, these two additional cysteine
residues are known to form an intramolecular disulfide bond
(Daopin, et al., Science, 257:369, 1992; Schlunegger and Grutter,
Nature, 358:430, 1992).
[0152] FIG. 4 shows the amino acid homologies among the different
members of the TGF-.beta. 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 particular subgroups. In this
region, GDF-8 is most homologous to Vgr-1 (45% sequence
identity).
EXAMPLE 3
Isolation of cDNA Clones Encoding Murine and Human GDF-8
[0153] In order to isolate full-length cDNA clones encoding murine
and human GDF-8, cDNA libraries were prepared in the lambda ZAP II
vector (Stratagene) using RNA prepared from skeletal muscle. From 5
.mu.g 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 screened
without amplification. Library screening and characterization of
cDNA inserts were carried out as described previously (Lee, Mol.
Endocrinol., 4:1034-1040).
[0154] From 2.4.times.10.sup.6 recombinant phage screened from the
murine muscle cDNA library, greater than 280 positive phage were
identified using a murine GDF-8 probe derived from a genomic clone,
as described in Example 1. The entire nucleotide sequence of the
longest cDNA insert analyzed is shown in FIG. 5a and 5b and SEQ ID
NO: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 initiating methionine codon is an in-frame stop codon
at nucleotide 23. The predicted pre-pro-GDF-8 protein is 76 amino
acids in length. The sequence contains a core of hydrophobic amino
acids at the N-terminus suggestive of a signal peptide for
secretion (FIG. 6a), one potential N-glycosylation site at
asparagine 72, a putative RXXR (SEQ ID NO: 50) proteolytic cleavage
site at amino acids 264-267, and a C-terminal region showing
significant homology to the known members of the TGF-.beta.
superfamily. Cleavage of the precursor protein at the putative RXXR
(SEQ ID NO: 50) site would generate a mature C-terminal GDF-8
fragment 109 amino acids in length with a predicted molecular
weight of approximately 12,400.
[0155] From 1.9.times.10.sup.6 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 FIG. 5c and 5d and SEQ ID NO:13. The 2743 base
pair sequence contains a single long open reading frame beginning
with a methionine codon at nucleotide 59 and extending to a TGA
stop codon at nucleotide 1184. 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 (FIG. 6b), one potential N-glycosylation site
at asparagine 71, and a putative RX)(R proteolytic cleavage site at
amino acids 263-266. FIG. 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 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 (SEQ ID
NO: 50) cleavage sites.
EXAMPLE 4
Dimerization of GDF-8
[0156] To determine whether the processing signals in the GDF-8
sequence are functional and whether GDF-8 forms dimers like other
members of the TGF-.beta. superfamily, the GDF-8 cDNA was stably
expressed in CHO cells. The GDF-8 coding sequence was cloned into
the pMSXND expression vector (Lee and Nathans, J. Biol. Chem.,
263:3521,(1988) and transfected into CHO cells. Following G418
selection, the cells were selected in 0.2 .mu.M methotrexate, and
conditioned medium from resistant cells was concentrated and
electrophoresed on SDS gels. Conditioned medium was prepared by
Cell Trends, Inc. (Middletown, Md.). For preparation of anti-GDF-8
serum, the C-terminal region of GDF-8 (amino acids 268 to 376) was
expressed in bacteria using the RSET vector (Invitrogen, San Diego,
Calif.), purified using a nickle chelate column, and injected into
rabbits. All immunizations were carried out by Spring Valley Labs
(Woodbine, Md.). Western analysis using [.sup.125I]iodoprotein A
was carried out as described (Burnette, W. N., Anal. Biochem.,
112:195, 1981). Western analysis of conditioned medium prepared
from these cells using an antiserum raised against a
bacterially-expressed C-terminal fragment of GDF-8 detected two
protein species with apparent molecular weights of approximately
52K and 15K under reducing conditions, consistent with unprocessed
and processed forms of GDF-8, respectively. No bands were obtained
either with preimmune serum or with conditioned medium from CHO
cells transfected with an antisense construct. Under non-reducing
conditions, the GDF-8 antiserum detected two predominant protein
species with apparent molecular weights of approximately 101K and
25K, consistent with dimeric forms of unprocessed and processed
GDF-8, respectively. Hence, like other TGF-.beta. family members,
GDF-8 appears to be secreted and proteolytically processed, and the
C-terminal region appears to be capable of forming a
disulfide-linked dimer.
EXAMPLE 5
Preparation of Antibodies Against GDF-8 and Expression of GDF-8 in
Mammalian Cells
[0157] In order to prepare antibodies against GDF-8, GDF-8 antigen
was expressed 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
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-.beta.-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 Coomassie blue-stained gel of unpurified and purified
fusion proteins is shown in FIG. 8.
[0158] The purified fusion protein was used to immunize both
rabbits and chickens. Immunization of rabbits was carried out by
Spring Valley Labs (Sykesville, Md.), and immunization of chickens
was carried out by HRP, Inc. (Denver, Pa.). Western analysis of
sera both from immunized rabbits and from immunized chickens
demonstrated the presence of antibodies directed against the fusion
protein.
[0159] 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 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 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 media
prepared from the G418-resistant cells were dialyzed, lyophilized,
electrophoresed under denaturing, reducing conditions, transferred
to nitrocellulose, and incubated with anti-GDF-8 antibodies
(described above) and [.sup.125I]iodoproteinA.
[0160] As shown in FIG. 9, the rabbit GDF-8 antibodies (at a 1:500
dilution) detected a protein of approximately the predicted
molecular weight for the mature C-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 antibodies prepared in chickens. Hence, GDF-8 is secreted and
proteolytically processed by these transfected mammalian cells.
EXAMPLE 6
Expression Pattern of GDF-8
[0161] To determine the pattern of GDF-8, 5 .mu.g of twice poly
A-selected RNA prepared from a variety of murine tissue sources
were subjected to Northern analysis. As shown in FIG. 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 mRNA
were seen in fat, brain, thymus, heart, and lung. Hence, these
results confirm the high degree of specificity of GDF-8 expression
in skeletal muscle. GDF-8 MRNA was also detected in mouse embryos
at both gestational ages (day 12.5 and day 18.5 post-coital)
examined but not in placentas at various stages of development
(FIG. 10b). To further analyze the expression pattern of GDF-8, in
situ hybridization was performed on mouse embryos isolated at
various stages of development.
[0162] For all in situ hybridization experiments, probes
corresponding to the C-terminal region of GDF-8 were excluded in
order to avoid possible cross-reactivity with other members of the
superfamily. Whole mount in situ hybridization analysis was carried
out as described (Wilkinson, D. G., In Situ Hybridization, A
Practical Approach, pp. 75-83, IRL. Press, Oxford, 1992) except
that blocking and antibody incubation steps were carried out as in
Knecht et al. (Knecht, et al., Development, 121:1927, 1955).
Alkaline phosphatase reactions were carried out for 3 hours for day
10.5 embryos and overnight for day 9.5 embryos. Hybridization was
carried out using digoxigenin-labelled probes spanning nucleotides
8-811 and 1298-2676, which correspond to the pro-region and 3'
untranslated regions, respectively. In situ hybridization to
sections was carried out as described (Wilkinson, et al., Cell,
50:79, 1987) using .sup.35S-labelled probes ranging from
approximately 100-650 bases in length and spanning nucleotides
8-793 and 1566-2595. Following hybridization and washing, slides
were dipped in NTB-3 photographic emulsion, exposed for 16-19 days,
developed and stained with either hematoxylin and eosin or
toluidine blue. RNA isolation, poly A selection, and Northern
analysis were carried out as described previously (McPherron and
Lee, J. Biol. Chem., 268:3444, 1993).
[0163] At all stages examined, the expression of GDF-8 mRNA
appeared to be restricted to developing skeletal muscle. At early
stages, GDF-8 expression was restricted to developing somites. By
whole mount in situ hybridization analysis, GDF-8 mRNA could first
be detected as early as day 9.5 post coitum in approximately
one-third of the somites. At this stage of development,
hybridization appeared to be restricted to the most mature (9 out
of 21 in this example), rostral somites. By day 10.5 p.c., GDF-8
expression was clearly evident in almost every somite (28 out of 33
in this example shown). Based on in situ hybridization analysis of
sections prepared from day 10.5 p.c. embryos, the expression of
GDF-8 in somites appeared to be localized to the myotome
compartment. At later stages of development, GDF-8 expression was
detected in a wide range of developing muscles.
[0164] GDF-8 continues to be expressed in adult animals as well. By
Northern analysis, GDF-8 mRNA expression was seen almost
exclusively in skeletal muscle among the different adult tissues
examined. A significantly lower though clearly detectable signal
was also seen in adipose tissue. Based on Northern analysis of RNA
prepared from a large number of different adult skeletal muscles,
GDF-8 expression appeared to be widespread although the expression
levels varied among individual muscles.
EXAMPLE 7
Chromosomal Localization of GDF-8
[0165] In order to map the chromosomal location of GDF-8, DNA
samples from human/rodent somatic cell hybrids (Drwinga, 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
3 primer #83, 5'- CGCGGATCCGTGGATCTAAATGAGAACAGTGAGC-3'(SEQ ID NO:
15) and primer #84, 5'-CGCGAATTCTCAGGTAATGATTGTTTC- CGTTGTAGCG-3'
(SEQ ID NO: 16)
[0166] for 40 cycles at 94.degree. C. for 2 minutes, 60.degree. C.
for 1 minute, and 72.degree. C. for 2 minutes. These primers
correspond to nucleotides 119 to 143 (flanked by a Bam Hl
recognition sequence), and nucleotides 394 to 418 (flanked by an
Eco Rl recognition sequence), respectively, in the human GDF-8 cDNA
sequence. PCR products were electrophoresed on agarose gels,
blotted, and probed with oligonucleotide #100,
5'-ACACTAAATCTTCAAGAATA-3' (SEQ ID NO:17), which corresponds to a
sequence internal to the region flanked by primer #83 and #84.
Filters were hybridized in 6.times. SSC, 1.times. Denhardt's
solution, 100.mu.g/ml yeast transfer RNA, and 0.05% sodium
pyrophosphate at 50.degree. C.
[0167] As shown in FIG. 11, 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 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 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 on chromosome 2.
EXAMPLE 8
GDF-8 Transgenic Knockout Mice
[0168] The GDF-8, we disrupted the GDF-8 gene was disrupted by
homologous targeting in embryonic stem cells. To ensure that the
resulting mice would be null for GDF-8 function, the entire mature
C-terminal region was deleted and replaced by a neo cassette (FIG.
12a). A murine 129 SV/J genomic library was prepared in lambda FIX
II according to the instructions provided by Stratagene (La Jolla,
Calif.). The structure of the GDF-8 gene was deduced from
restriction mapping and partial sequencing of phage clones isolated
from this library. Vectors for preparing the targeting construct
were kindly provided by Philip Soriano and Kirk Thomas University.
R1 ES cells were transfected with the targeting construct, selected
with gancyclovir (2 .mu.M) and G418 (250 .mu.g/ml), and analyzed by
Southern analysis. Homologously targeted clones were injected into
C57BL/6 blastocysts and transferred into pseudopregnant females.
Germline transmission of the targeted allele was obtained in a
total of 9 male chimeras from 5 independently-derived ES clones.
Genomic Southern blots were hybridized at 42/C as described above
and washed in 0.2.times. SSC, 0.1% SDS at 42/C.
[0169] For whole leg analysis, legs of 14 week old mice were
skinned, treated with 0.2 M EDTA in PBS at 4/C for 4 weeks followed
by 0.5 M sucrose in PBS at 4/C. For fiber number and size analysis,
samples were directly mounted and frozen in isopentane as described
(Brumback and Leech, Color Atlas of Muscle Histochemistry, pp.
9-33, PSG Publishing Company, Littleton, Mass., 1984). Ten to 30
.mu.m sections were prepared using a cryostat and stained with
hematoxylin and eosin. Muscle fiber numbers were determined from
sections taken from the widest part of the tibialis cranialis
muscle. Muscle fiber sizes were measured from photographs of
sections of tibialis cranialis and gastrocnemius muscles. Fiber
type analysis was carried out using the mysosin ATPase assay after
pretreatment at pH 4.35 as described (Cumming, et al., Color Atlas
of Muscle Pathology, pp. 184-185, 1994) and by immunohistochemistry
using an antibody directed against type I myosin (MY32, Sigma) and
the Vectastain method (Vector Labs); in the immunohistochemical
experiments, no staining was seen when the primary antibodies were
left out. Carcasses were prepared from shaved mice by removing the
all of the internal organs and associated fat and connective
tissue. Fat content of carcasses from 4 month old males was
determined as described (Leshner, et al., Physiol. Behavior, 9:281,
1972).
[0170] For protein and DNA analysis, tissue was homogenized in 150
mM NaCl, 100 mM EDTA. Protein concentrations were determined using
the Biorad protein assay. DNA was isolated by adding SDS to 1%,
treating with 1 mg/ml proteinase K overnight at 55/C, extracting 3
times with phenol and twice with chloroform, and precipitating with
ammonium acetate and EtOH. DNA was digested with 2 mg/ml RNase for
1 hour at 37/C, and following proteinase K digestion and phenol and
chloroform extractions, the DNA was precipitated twice with
ammonium acetate and EtOH.
[0171] Homologous targeting of the GDF-8 gene was seen in 13/131
gancyclovir/G418 doubly-resistant ES cell clones. Following
injection of these targeted clones into blastocysts, we obtained
chimeras from 5 independently-derived ES clones that produced
heterozygous pups when crossed to C57BL/6 females (FIG. 12b).
Genotypic analysis of 678 offspring derived from crosses of F1
heterozygotes showed 170 +/+(25%), 380 +/- (56%), and 128 -/-
(19%). Although the ratio of genotypes was close to the expected
ratio of 1:2: 1, the smaller than expected number of homozygous
mutants appeared to be statistically significant (p<0.001).
[0172] Homozygous mutants were viable and fertile when crossed to
C57BL/6 mice and to each other. Homozygous mutant animals, however,
were approximately 30% larger than their heterozygous and wild type
littermates (Table 1). The difference between mutant and wild type
body weights appeared to be relatively constant irrespective of age
and sex in adult animals. Adult mutants also displayed an abnormal
body shape, with pronounced shoulders and hips. When the skin was
removed from animals that had been sacrificed, it was apparent that
the muscles of the mutants were much larger than those of wild type
animals. The increase in skeletal muscle mass appeared to be
widespread throughout the body. Individual muscles isolated from
homozygous mutant animals weighed approximately 2-3 times more than
those isolated from wild type littermates (Table 2). Although the
magnitude of the weight increase appeared to roughly correlate with
the level of GDF-8 expression in the muscles examined. To determine
whether the increased muscle mass could account for the entire
difference in total body weights between wild type and mutant
animals or whether many tissues were generally larger in the
mutants, we compared the total body weights to carcass weights. As
shown in Table 3, the difference in carcass weights between wild
type and mutant animals was comparable to the difference in total
body weights. Moreover, because the fat content of mutant and wild
type animals was similar, these data are consistent with all of the
total body weight difference resulting from an increase in skeletal
muscle mass, although we have not formally ruled out the
possibility that differences in bone mass might also contribute to
the differences in total body mass.
[0173] To determine whether the increase in skeletal muscle mass
resulted from hyperplasia or from hypertrophy, histologic analysis
of several different muscle groups was performed. The mutant muscle
appeared grossly normal. No excess connective tissue or fat was
seen nor were there any obvious signs of degeneration, such as
widely varying fiber sizes (see below) or centrally-placed nuclei.
Quantitation of the number of muscle fibers showed that at the
widest portion of the tibialis cranialis muscle, the total cell
number was 86% higher in mutant animals compared to wild type
littermates [mutant=5470+/-121 (n=3), wild type =2936+/-288 (n=3);
p<0.01]. Consistent with this result was the finding that the
amount of DNA extracted from mutant muscle was roughly 50% higher
than from wild type muscle [mutant=350 .mu.g (n=4), wild type=233
.mu.g (n=3) from pooled gastrocnemius, plantaris, triceps brachii,
tibialis cranialis, and pectoralis muscles; p=0.05]. Hence, a large
part of the increase in skeletal muscle mass resulted from muscle
cell hyperplasia. However, muscle fiber hypertrophy also appeared
to contribute to the overall increase in muscle mass. As shown in
FIG. 13, the mean fiber diameter of the tibialis cranialis muscle
and gastrocnemius muscle was 7% and 22% larger, respectively, in
mutant animals compared to wild type littermates, suggesting that
the cross-sectional area of the fibers was increased by
approximately 14% and 49%, respectively. Notably, although the mean
fiber diameter was larger in the mutants, the standard deviation in
fiber sizes was similar between mutant and wild type muscle,
consistent with the absence of muscle degeneration in mutant
animals. The increase in fiber size was also consistent with the
finding that the protein to DNA ratio (w/w) was slightly increased
in mutant compared to wild type muscle [mutant=871+/-111 (n=4),
wildtype=624+/-85 (n=3); p<0.05].
[0174] Table 4 shows a comparison between muscle weight (in grams)
from wild-type (+/+), heterozyous (+/-) and a homozygous knock-out
mice (-/-). The muscle mass is increased in heterozyogous as
compared to wild-type animals. Finally, fiber type analysis of
various muscles was carried out to determine whether the number of
both type I (slow) and type II (fast) fibers was increased in the
mutant animals. In most of the muscles examined, including the
tibialis cranialis muscle, the vast majority of muscle fibers were
type II in both mutant and wild type animals. Hence, based on the
cell counts discussed above, the absolute number of type II fibers
were increased in the tibialis cranialis muscle. In the soleus
muscle, where the number of type I fibers was sufficiently high
that we could attempt to quantitate the ratio of fiber types could
be quantiated, the percent of type I fibers was decreased by
approximately 33% in mutant compared to wild type muscle [wild
type=39.2+/-8.1 (n=3), mutant=26.4+/-9.3 (n=4)]; however, the
variability in this ratio for both wild type and mutant animals was
too high to support any firm conclusions regarding the relative
number of fiber types.
EXAMPLE 9
Isolation of Rat and Chicken GDF-8
[0175] In order to isolate rat and chicken GDF-8 cDNA clones,
skeletal muscle cDNA libraries prepared from these species were
obtained from Stratagene and screened with a murine GDF-8 probe.
Library screening was carried out as described previously (Lee,
Mol. Endocrinol., 4:1034-1040) except that final washes were
carried out in 2.times. SSC at 65 C. Partial sequence analysis of
hybridizing clones revealed the presence of open reading frames
highly related to murine and human GDF-8. Partial sequences of rat
and chicken GDF-8 are shown in FIGS. 2c and 2d, respectively, and
an alignment of the predicated rat and chicken GDF-8 amino acid
sequences with those of murine and human GDF-8 are shown in FIG.
3b. Full length rat and chicken GDF-8 is shown in FIGS. 14d and
14c, respectively and sequence alignment between murine, rat,
human, baboon, porcine, ovine, bovine, chicken, and turkey
sequences is shown in FIGS. 15a and 15b. All sequences contain an
RSRR (SEQ ID NO: 51) sequence that is likely to represent the
proteolytic processing site. Following this RSRR (SEQ ID NO: 51)
sequence, the sequences contain a C-terminal region that is 100%
conserved among all four species. The absolute conservation of the
C-terminal region between species as evolutionarily far apart as
humans and chickens, and baboons and turkeys, suggests that this
region will be highly conserved in many other species as well.
[0176] Similar methodology was used to obtain the nucleotide and
amino acid sequences for baboon (SEQ ID NO:18 and 19, respectively;
FIG. 14a); bovine (SEQ ID NO:20 and 21, respectively; FIG. 14b);
turkey (SEQ ID NO:26 and 27, respectively; FIG. 14e); porcine (SEQ
ID NO:28 and 29, respectively; FIG. 14f); and ovine (SEQ ID NO:30
and 31, respectively; FIG. 14g).
[0177] 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.
Sequence CWU 1
1
51 1 35 DNA Artificial Sequence oligonucleotide for PCR 1
ccggaattcg gntggvanra ytggrtnrtn kcncc 35 2 33 DNA Artificial
Sequence oligonucleotide for PCR 2 ccggaattcr canscrcarc tntcnacnry
cat 33 3 31 DNA Artificial Sequence oligonucleotide for PCR 3
cgcggatcca gagtcaaggt gacagacaca c 31 4 33 DNA Artificial Sequence
oligonucleotide for PCR 4 cgcggatcct cctcatgagc acccacagcg gtc 33 5
550 DNA Mus musculus CDS (59)...(436) 5 ttaaggtagg aaggatttca
ggctctattt acataattgt tctttccttt tcacacag 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 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 tgg 202 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 aat tac tgc tca gga gag tgt
250 Ile Ile 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 Gln Lys Tyr Pro His Thr His Leu Val
His Gln 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 tct ccc att aat atg cta tat ttt aat ggc
aaa gaa caa ata ata tat 394 Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly
Lys Glu Gln Ile Ile Tyr 100 105 110 ggg aaa att cca gcc atg gta gta
gac cgc tgt ggg tgc tca 436 Gly Lys Ile Pro Ala Met Val Val Asp Arg
Cys Gly Cys Ser 115 120 125 tgagctttgc attaggttag aaacttccca
agtcatggaa ggtcttcccc tcaatttcga 496 aactgtgaat tcctgcagcc
cgggggatcc actagttcta gagcggccgc cacc 550 6 126 PRT Mus musculus 6
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 Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly
Trp Asp Trp 35 40 45 Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr
Cys Ser Gly Glu Cys 50 55 60 Glu Phe Val Phe Leu Gln Lys Tyr Pro
His Thr His Leu Val His Gln 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 Ile Asn Met
Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr 100 105 110 Gly Lys Ile
Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 115 120 125 7 326 DNA
Homo sapiens CDS (3)...(326) 7 ca aaa aga tcc aga agg gat ttt ggt
ctt gac tgt gat gag cac tca 47 Lys Arg Ser Arg Arg Asp Phe Gly Leu
Asp Cys Asp Glu His Ser 1 5 10 15 aca gaa tca cga tgc tgt cgt tac
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 tac 143 Phe Gly Trp Asp Trp Ile
Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr 35 40 45 tgc tct gga gag
tgt gaa ttt gta ttt tta caa aaa tat cct cat act 191 Cys Ser Gly Glu
Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr 50 55 60 cat ctg
gta cac caa gca aac ccc aga ggt tca gca ggc cct tgc tgt 239 His Leu
Val His Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys 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 Ile Asn Met Leu Tyr Phe Asn Gly Lys 80
85 90 95 gaa caa ata ata tat ggg aaa att cca gcg atg gta gta 326
Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val Val 100 105 8 108
PRT Homo sapiens 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 20 25 30 Gly Trp Asp Trp Ile Ile Ala Pro
Lys Arg Tyr Lys Ala Asn Tyr Cys 35 40 45 Ser Gly Glu Cys Glu Phe
Val Phe Leu Gln Lys Tyr Pro His Thr His 50 55 60 Leu Val His Gln
Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr 65 70 75 80 Pro Thr
Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu 85 90 95
Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val Val 100 105 9 9 PRT
Artificial Sequence amino acid encoded by oligonucleotide for PCR 9
Gly Trp Xaa Xaa Trp Xaa Xaa Xaa Pro 1 5 10 8 PRT Artificial
Sequence amino acid encoded by oligonucleotide for PCR 10 Met Xaa
Val Xaa Ser Cys Xaa Cys 1 5 11 2676 DNA Mus musculus CDS
(104)...(1231) 11 gtctctcgga cggtacatgc actaatattt cacttggcat
tactcaaaag caaaaagaag 60 aaataagaac aagggaaaaa aaaagattgt
gctgattttt aaa atg atg caa aaa 115 Met Met Gln Lys 1 ctg caa atg
tat gtt tat att tac ctg ttc atg ctg att gct gct ggc 163 Leu Gln Met
Tyr Val Tyr Ile Tyr Leu Phe Met Leu Ile Ala Ala Gly 5 10 15 20 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 agg tac tcc
259 Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln Asn Thr Arg Tyr Ser
40 45 50 aga ata gaa gcc ata aaa att caa atc ctc agt aag ctg cgc
ctg gaa 307 Arg Ile Glu Ala Ile Lys Ile Gln Ile 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 Ile Ser Lys Asp Ala Ile Arg Gln
Leu Leu Pro Arg 70 75 80 gcg cct cca ctc cgg gaa ctg atc gat cag
tac gac gtc cag agg gat 403 Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln
Tyr Asp Val Gln Arg Asp 85 90 95 100 gac agc agt 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 gaa aca atc att acc
atg cct aca gag tct gac ttt cta atg caa gcg 499 Glu Thr Ile Ile Thr
Met Pro Thr Glu Ser Asp Phe Leu Met Gln Ala 120 125 130 gat ggc aag
ccc aaa tgt tgc ttt ttt aaa ttt agc tct aaa ata cag 547 Asp Gly Lys
Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser Lys Ile Gln 135 140 145 tac
aac aaa gta gta aaa gcc caa ctg tgg ata tat ctc aga ccc gtc 595 Tyr
Asn Lys Val Val Lys Ala Gln Leu Trp Ile 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 Gln Ile Leu Arg Leu Ile Lys Pro
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 Ile Arg Ser Leu
Lys Leu Asp 185 190 195 atg agc cca ggc act ggt att tgg cag agt att
gat gtg aag aca gtg 739 Met Ser Pro Gly Thr Gly Ile Trp Gln Ser Ile
Asp Val Lys Thr Val 200 205 210 ttg caa aat tgg ctc aaa cag cct gaa
tcc aac tta ggc att gaa atc 787 Leu Gln Asn Trp Leu Lys Gln Pro Glu
Ser Asn Leu Gly Ile Glu Ile 215 220 225 aaa gct ttg gat gag aat ggc
cat gat ctt gct gta acc ttc cca gga 835 Lys Ala Leu Asp Glu Asn Gly
His Asp Leu Ala Val Thr Phe Pro Gly 230 235 240 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 aca ccc
aag agg tcc cgg 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 tac ccc ctc acg gtc gat ttt gaa 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 Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys
Ala Asn 295 300 305 tac tgc tca gga gag tgt gaa ttt gtg ttt tta caa
aaa tat ccg cat 1075 Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu
Gln Lys Tyr Pro His 310 315 320 act cat ctt gtg cac caa gca aac ccc
aga ggc tca gca ggc cct tgc 1123 Thr His Leu Val His Gln Ala Asn
Pro Arg Gly Ser Ala Gly Pro Cys 325 330 335 340 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 Ile 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 cgc 1219 Lys
Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg 360 365
370 tgt ggg tgc tca tgagctttgc attaggttag aaacttccca agtcatggaa
1271 Cys Gly Cys Ser 375 ggtcttcccc tcaatttcga aactgtgaat
tcaagcacca caggctgtag gccttgagta 1331 tgctctagta acgtaagcac
aagctacagt gtatgaacta aaagagagaa tagatgcaat 1391 ggttggcatt
caaccaccaa aataaaccat actataggat gttgtatgat ttccagagtt 1451
tttgaaatag atggagatca aattacattt atgtccatat atgtatatta caactacaat
1511 ctaggcaagg aagtgagagc acatcttgtg gtctgctgag ttaggagggt
atgattaaaa 1571 ggtaaagtct tatttcctaa cagtttcact taatatttac
agaagaatct atatgtagcc 1631 tttgtaaagt gtaggattgt tatcatttaa
aaacatcatg tacacttata tttgtattgt 1691 atacttggta agataaaatt
ccacaaagta ggaatggggc ctcacataca cattgccatt 1751 cctattataa
ttggacaatc caccacggtg ctaatgcagt gctgaatggc tcctactgga 1811
cctctcgata gaacactcta caaagtacga gtctctctct cccttccagg tgcatctcca
1871 cacacacagc actaagtgtt caatgcattt tctttaagga aagaagaatc
tttttttcta 1931 gaggtcaact ttcagtcaac tctagcacag cgggagtgac
tgctgcatct taaaaggcag 1991 ccaaacagta ttcatttttt aatctaaatt
tcaaaatcac tgtctgcctt tatcacatgg 2051 caattttgtg gtaaaataat
ggaaatgact ggttctatca atattgtata aaagactctg 2111 aaacaattac
atttatataa tatgtataca atattgtttt gtaaataagt gtctcctttt 2171
atatttactt tggtatattt ttacactaat gaaatttcaa atcattaaag tacaaagaca
2231 tgtcatgtat cacaaaaaag gtgactgctt ctatttcaga gtgaattagc
agattcaata 2291 gtggtcttaa aactctgtat gttaagatta gaaggttata
ttacaatcaa tttatgtatt 2351 ttttacatta tcaacttatg gtttcatggt
ggctgtatct atgaatgtgg ctcccagtca 2411 aatttcaatg ccccaccatt
ttaaaaatta caagcattac taaacatacc aacatgtatc 2471 taaagaaata
caaatatggt atctcaataa cagctacttt tttattttat aatttgacaa 2531
tgaatacatt tcttttattt acttcagttt tataaattgg aactttgttt atcaaatgta
2591 ttgtactcat agctaaatga aattatttct tacataaaaa tgtgtagaaa
ctataaatta 2651 aagtgttttc acatttttga aaggc 2676 12 376 PRT Mus
musculus 12 Met Met Gln Lys Leu Gln Met Tyr Val Tyr Ile Tyr Leu Phe
Met Leu 1 5 10 15 Ile Ala Ala Gly Pro Val Asp Leu Asn Glu Gly Ser
Glu Arg Glu Glu 20 25 30 Asn Val Glu Lys Glu Gly Leu Cys Asn Ala
Cys Ala Trp Arg Gln Asn 35 40 45 Thr Arg Tyr Ser Arg Ile Glu Ala
Ile Lys Ile Gln Ile Leu Ser Lys 50 55 60 Leu Arg Leu Glu Thr Ala
Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln 65 70 75 80 Leu Leu Pro Arg
Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp 85 90 95 Val Gln
Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr 100 105 110
His Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe 115
120 125 Leu Met Gln Ala Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe
Ser 130 135 140 Ser Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu
Trp Ile Tyr 145 150 155 160 Leu Arg Pro Val Lys Thr Pro Thr Thr Val
Phe Val Gln Ile Leu Arg 165 170 175 Leu Ile Lys Pro Met Lys Asp Gly
Thr Arg Tyr Thr Gly Ile Arg Ser 180 185 190 Leu Lys Leu Asp Met Ser
Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp 195 200 205 Val Lys Thr Val
Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu 210 215 220 Gly Ile
Glu Ile 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 260 265 270 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
Ile Ile Ala Pro Lys Arg 290 295 300 Tyr Lys Ala Asn Tyr Cys Ser Gly
Glu Cys Glu Phe Val Phe Leu Gln 305 310 315 320 Lys Tyr Pro His Thr
His Leu Val His Gln Ala Asn Pro Arg Gly Ser 325 330 335 Ala Gly Pro
Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu 340 345 350 Tyr
Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met 355 360
365 Val Val Asp Arg Cys Gly Cys Ser 370 375 13 2743 DNA Homo
sapiens CDS (59)...(1183) 13 aagaaaagta aaaggaagaa acaagaacaa
gaaaaaagat tatattgatt ttaaaatc 58 atg caa aaa ctg caa ctc tgt gtt
tat att tac ctg ttt atg ctg att 106 Met Gln Lys Leu Gln Leu Cys Val
Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10 15 gtt gct ggt cca gtg gat
cta aat gag aac agt gag caa aaa gaa aat 154 Val Ala Gly Pro Val Asp
Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30 gtg gaa aaa gag
ggg ctg tgt aat gca tgt act tgg aga caa aac act 202 Val Glu Lys Glu
Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40 45 aaa tct
tca aga ata gaa gcc att aag ata caa atc ctc agt aaa ctt 250 Lys Ser
Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile 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 Ile Ser Lys Asp Val Ile Arg Gln Leu 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 Ile Asp Gln Tyr
Asp Val 85 90 95 cag agg gat gac agc agc gat ggc tct ttg gaa gat
gac gat tat cac 394 Gln 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 442 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro
Thr Glu Ser Asp Phe Leu 115 120 125 atg caa gtg gat gga aaa ccc aaa
tgt tgc ttc ttt aaa ttt agc tct 490 Met Gln Val Asp Gly Lys Pro Lys
Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 aaa ata caa tac aat aaa
gta gta aag gcc caa cta tgg ata tat ttg 538 Lys Ile Gln Tyr Asn Lys
Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 aga ccc gtc
gag act cct aca aca gtg ttt gtg caa atc ctg aga ctc 586 Arg Pro Val
Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 atc
aaa cct atg aaa gac ggt aca agg tat act gga atc cga tct ctg 634 Ile
Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185
190 aaa ctt gac atg aac cca ggc act ggt att tgg cag agc att gat gtg
682 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile 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 Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn
Leu Gly 210 215 220 att gaa ata aaa gct tta gat gag aat ggt cat gat
ctt gct gta acc 778 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp
Leu Ala Val Thr 225 230 235 240 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 agg gat ttt ggt ctt
gac tgt 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 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr
Pro Leu Thr Val 275 280 285 gat ttt gaa gct ttt gga tgg gat tgg att
atc gct cct aaa aga tat 970 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile
Ile Ala Pro Lys Arg Tyr 290 295 300 aag gcc aat tac tgc tct gga gag
tgt gaa ttt gta ttt tta caa aaa 1018 Lys Ala Asn Tyr Cys Ser Gly
Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 tat cct cat act
cat ctg gta cac caa gca aac ccc aga ggt tca gca 1066 Tyr Pro His
Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 ggc
cct tgc tgt act ccc aca aag atg tct cca att aat atg cta tat 1114
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile 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 Gln Ile Ile Tyr Gly Lys Ile Pro Ala
Met Val 355 360 365 gta gac cgc tgt ggg tgc tca tgagatttat
attaagcgtt cataacttcc 1213 Val Asp Arg Cys Gly Cys Ser 370 375
taaaacatgg aaggttttcc cctcaacaat tttgaagctg tgaaattaag taccacaggc
1273 tataggccta gagtatgcta cagtcactta agcataagct acagtatgta
aactaaaagg 1333 gggaatatat gcaatggttg gcatttaacc atccaaacaa
atcatacaag aaagttttat 1393 gatttccaga gtttttgagc tagaaggaga
tcaaattaca tttatgttcc tatatattac 1453 aacatcggcg aggaaatgaa
agcgattctc cttgagttct gatgaattaa aggagtatgc 1513 tttaaagtct
atttctttaa agttttgttt aatatttaca gaaaaatcca catacagtat 1573
tggtaaaatg caggattgtt atataccatc attcgaatca tccttaaaca cttgaattta
1633 tattgtatgg tagtatactt ggtaagataa aattccacaa aaatagggat
ggtgcagcat 1693 atgcaatttc cattcctatt ataattgaca cagtacatta
acaatccatg ccaacggtgc 1753 taatacgata ggctgaatgt ctgaggctac
caggtttatc acataaaaaa cattcagtaa 1813 aatagtaagt ttctcttttc
ttcaggtgca ttttcctaca cctccaaatg aggaatggat 1873 tttctttaat
gtaagaagaa tcatttttct agaggttggc tttcaattct gtagcatact 1933
tggagaaact gcattatctt aaaaggcagt caaatggtgt ttgtttttat caaaatgtca
1993 aaataacata cttggagaag tatgtaattt tgtctttgga aaattacaac
actgcctttg 2053 caacactgca gtttttatgg taaaataata gaaatgatcg
actctatcaa tattgtataa 2113 aaagactgaa acaatgcatt tatataatat
gtatacaata ttgttttgta aataagtgtc 2173 tcctttttta tttactttgg
tatattttta cactaaggac atttcaaatt aagtactaag 2233 gcacaaagac
atgtcatgca tcacagaaaa gcaactactt atatttcaga gcaaattagc 2293
agattaaata gtggtcttaa aactccatat gttaatgatt agatggttat attacaatca
2353 ttttatattt ttttacatga ttaacattca cttatggatt catgatggct
gtataaagtg 2413 aatttgaaat ttcaatggtt tactgtcatt gtgtttaaat
ctcaacgttc cattatttta 2473 atacttgcaa aaacattact aagtatacca
aaataattga ctctattatc tgaaatgaag 2533 aataaactga tgctatctca
acaataactg ttacttttat tttataattt gataatgaat 2593 atatttctgc
atttatttac ttctgttttg taaattggga ttttgttaat caaatttatt 2653
gtactatgac taaatgaaat tatttcttac atctaatttg tagaaacagt ataagttata
2713 ttaaagtgtt ttcacatttt tttgaaagac 2743 14 375 PRT Homo sapiens
14 Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile
1 5 10 15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys
Glu Asn 20 25 30 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp
Arg Gln Asn Thr 35 40 45 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile
Gln Ile Leu Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile
Ser Lys Asp Val Ile Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro
Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp
Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Thr
Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu 115 120 125
Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130
135 140 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr
Leu 145 150 155 160 Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln
Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr
Thr Gly Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr
Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn
Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys
Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 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 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 Ile Ile Ala
Pro Lys Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu
Phe Val Phe Leu Gln Lys 305 310 315 320 Tyr Pro His Thr His Leu Val
His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr
Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly
Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 Val
Asp Arg Cys Gly Cys Ser 370 375 15 34 DNA Artificial Sequence
oligonucleotide for PCR 15 cgcggatccg tggatctaaa tgagaacagt gagc 34
16 37 DNA Artificial Sequence oligonucleotide for PCR 16 cgcgaattct
caggtaatga ttgtttccgt tgtagcg 37 17 20 DNA Artificial Sequence
oligonucleotide for PCR 17 acactaaatc ttcaagaata 20 18 1128 DNA
Baboon CDS (1)...(1125) 18 atg caa aaa ctg caa ctc tgt gtt tat att
tac ctg ttt atg ctg att 48 Met Gln Lys Leu Gln Leu Cys Val Tyr Ile
Tyr Leu Phe Met Leu Ile 1 5 10 15 gtt gct ggt cca gtg gat cta aat
gag aac agt gag caa aaa gaa aat 96 Val Ala Gly Pro Val Asp Leu Asn
Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30 gtg gaa aaa gag ggg ctg
tgt aat gca tgt act tgg aga caa aac act 144 Val Glu Lys Glu Gly Leu
Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40 45 aaa tct tca aga
ata gaa gcc att aaa ata caa atc ctc agt aaa ctt 192 Lys Ser Ser Arg
Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 cgt ctg
gaa aca gct cct aac atc agc aaa gat gct ata aga caa ctt 240 Arg Leu
Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln Leu 65 70 75 80
tta ccc aaa gcg cct cca ctc cgg gaa ctg att gat cag tat gat gtc 288
Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85
90 95 cag agg gat gac agc agc gat ggc tct ttg gaa gat gac gat tat
cac 336 Gln 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 tta 384 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser
Asp Phe Leu 115 120 125 atg caa gtg gat gga aaa ccc aaa tgt tgc ttc
ttt aaa ttt agc tct 432 Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe
Phe Lys Phe Ser Ser 130 135 140 aaa ata caa tac aat aaa gtg gta aag
gcc caa cta tgg ata tat ttg 480 Lys Ile Gln Tyr Asn Lys Val Val Lys
Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 aga ccc gtc gag act cct
aca aca gtg ttt gtg caa atc ctg aga ctc 528 Arg Pro Val Glu Thr Pro
Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 atc aaa cct atg
aaa gac ggt aca agg tat act gga atc cga tct ctg 576 Ile Lys Pro Met
Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 aaa ctt
gac atg aac cca ggc act ggt att tgg cag agc att gat gtg 624 Lys Leu
Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205
aag aca gtg ttg caa aat tgg ctc aaa caa cct gaa tcc aac tta ggc 672
Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210
215 220 att gaa ata aaa gct tta gat gag aat ggt cat gat ctt gct gta
acc 720 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val
Thr 225 230 235 240 ttc cca gga cca gga gaa gat ggg ctg aat ccc ttt
tta gag gtc aag 768 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe
Leu Glu Val Lys 245 250 255 gta aca gac aca ccc aaa aga tcc aga agg
gat ttt ggt ctt gac tgt 816 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 tcg cga
tgc tgt cgt tac cct cta act gtg 864 Asp Glu His Ser Thr Glu Ser Arg
Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 gat ttt gaa gct ctt gga
tgg gat tgg att atc gct cct aaa aga tat 912 Asp Phe Glu Ala Leu Gly
Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 aag gcc aat tac
tgc tct gga gag tgt gaa ttt gta ttt tta caa aaa 960 Lys Ala Asn Tyr
Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 tat
cct cat act cat ctg gta cac caa gca aac ccc aga ggt tca gca 1008
Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325
330 335 ggc cct tgc tgt act ccc aca aag atg tct cca att aat atg cta
tat 1056 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met
Leu Tyr 340 345 350 ttt aat ggc aaa gaa caa ata ata tat ggg aaa att
cca gcc atg gta 1104 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys
Ile Pro Ala Met Val 355 360 365 gta gac cgc tgc ggg tgc tca tga
1128 Val Asp Arg Cys Gly Cys Ser 370 375 19 375 PRT Baboon 19 Met
Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10
15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn
20 25 30 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln
Asn Thr 35 40 45 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile
Leu Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys
Asp Ala Ile Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg
Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser
Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu
Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 Met Gln
Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140
Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145
150 155 160 Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu
Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly
Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile
Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu
Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu
Asp Glu Asn Gly His Asp Leu Ala Val Thr 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 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
275 280 285 Asp Phe Glu Ala Leu Gly Trp Asp Trp Ile Ile Ala Pro Lys
Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val
Phe Leu Gln Lys 305 310 315 320 Tyr Pro His Thr His Leu Val His Gln
Ala Asn Pro Arg Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr
Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu
Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 Val Asp Arg
Cys Gly Cys Ser 370 375 20 1128 DNA Bovine CDS (1)...(1125) 20 atg
caa aaa ctg caa atc tct gtt tat att tac cta ttt atg ctg att 48 Met
Gln Lys Leu Gln Ile Ser Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10
15 gtt gct ggc cca gtg gat ctg aat gag aac agc gag cag aag gaa aat
96 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn
20 25 30 gtg gaa aaa gag ggg ctg tgt aat gca tgt ttg tgg agg gaa
aac act 144 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Leu Trp Arg Glu
Asn Thr 35 40 45 aca tcg tca aga cta gaa gcc ata aaa atc caa atc
ctc agt aaa ctt 192 Thr Ser Ser Arg Leu Glu Ala Ile Lys Ile Gln Ile
Leu Ser Lys Leu 50 55 60 cgc ctg gaa aca gct cct aac atc agc aaa
gat gct atc aga caa ctt 240 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys
Asp Ala Ile Arg Gln Leu 65 70 75 80 ttg ccc aag gct cct cca ctc ctg
gaa ctg att gat cag ttc gat gtc 288 Leu Pro Lys Ala Pro Pro Leu Leu
Glu Leu Ile Asp Gln Phe Asp Val 85 90 95 cag aga gat gcc agc agt
gac ggc tcc ttg gaa gac gat gac tac cac 336 Gln Arg Asp Ala Ser Ser
Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 gcc agg acg gaa
acg gtc att acc atg ccc acg gag tct gat ctt cta 384 Ala Arg Thr Glu
Thr Val Ile Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125 acg caa
gtg gaa gga aaa ccc aaa tgt tgc ttc ttt aaa ttt agc tct 432 Thr Gln
Val Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140
aag ata caa tac aat aaa cta gta aag gcc caa ctg tgg ata tat ctg 480
Lys Ile Gln Tyr Asn Lys Leu Val Lys Ala Gln Leu Trp Ile Tyr Leu 145
150 155 160 agg cct gtc aag act cct gcg aca gtg ttt gtg caa atc ctg
aga ctc 528 Arg Pro Val Lys Thr Pro Ala Thr Val Phe Val Gln Ile Leu
Arg Leu 165 170 175 atc aaa ccc atg aaa gac ggt aca agg tat act gga
atc cga tct ctg 576 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly
Ile Arg Ser Leu 180 185 190 aaa ctt gac atg aac cca ggc act ggt att
tgg cag agc att gat gtg 624 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile
Trp Gln Ser Ile Asp Val 195 200 205 aag aca gtg ttg cag aac tgg ctc
aaa caa cct gaa tcc aac tta ggc 672 Lys Thr Val Leu Gln Asn Trp Leu
Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 att gaa atc aaa gct tta
gat gag aat ggc cat gat ctt gct gta acc 720 Ile Glu Ile Lys Ala Leu
Asp Glu Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 ttc cca gaa
cca gga gaa gat gga ctg act ccc ttt tta gaa gtc aag 768 Phe Pro Glu
Pro Gly Glu Asp Gly Leu Thr Pro Phe Leu Glu Val Lys 245 250 255 gta
aca gac aca cca aaa aga tct agg aga gat ttt ggg ctt gat tgt 816 Val
Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265
270 gat gaa cac tcc aca gaa tct cga tgc tgt cgt tac cct cta act gtg
864 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
275 280 285 gat ttt gaa gct ttt gga tgg gat tgg att att gca cct aaa
aga tat 912 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys
Arg Tyr 290 295 300 aag gcc aat tac tgc tct gga gaa tgt gaa ttt gta
ttt ttg caa aag 960 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val
Phe Leu Gln Lys 305 310 315 320 tat cct cat acc cat ctt gtg cac caa
gca aac ccc aga ggt tca gcc 1008 Tyr Pro His Thr His Leu Val His
Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 ggc ccc tgc tgt act cct
aca aag atg tct cca att aat atg cta tat
1056 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu
Tyr 340 345 350 ttt aat ggc gaa gga caa ata ata tac ggg aag att cca
gcc atg gta 1104 Phe Asn Gly Glu Gly Gln Ile Ile Tyr Gly Lys Ile
Pro Ala Met Val 355 360 365 gta gat cgc tgt ggg tgt tca tga 1128
Val Asp Arg Cys Gly Cys Ser 370 375 21 375 PRT Bovine 21 Met Gln
Lys Leu Gln Ile Ser Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10 15
Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20
25 30 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Leu Trp Arg Glu Asn
Thr 35 40 45 Thr Ser Ser Arg Leu Glu Ala Ile Lys Ile Gln Ile Leu
Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp
Ala Ile Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Leu Glu
Leu Ile Asp Gln Phe Asp Val 85 90 95 Gln Arg Asp Ala Ser Ser Asp
Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Arg Thr Glu Thr
Val Ile Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125 Thr Gln Val
Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 Lys
Ile Gln Tyr Asn Lys Leu Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150
155 160 Arg Pro Val Lys Thr Pro Ala Thr Val Phe Val Gln Ile Leu Arg
Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile
Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp
Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu Lys
Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu Asp
Glu Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 Phe Pro Glu Pro
Gly Glu Asp Gly Leu Thr 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
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 Ile Ile Ala Pro Lys Arg
Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe
Leu Gln Lys 305 310 315 320 Tyr Pro His Thr His Leu Val His Gln Ala
Asn Pro Arg Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr Lys
Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly Glu Gly Gln
Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 Val Asp Arg Cys
Gly Cys Ser 370 375 22 1128 DNA Gallus gallus CDS (1)...(1125) 22
atg caa aag ctg gca gtc tat gtt tat att tac ctg ttc atg cag atc 48
Met Gln Lys Leu Ala Val Tyr Val Tyr Ile Tyr Leu Phe Met Gln Ile 1 5
10 15 gcg gtt gat ccg gtg gct ctg gat ggc agt agt cag ccc aca gag
aac 96 Ala Val Asp Pro Val Ala Leu Asp Gly Ser Ser Gln Pro Thr Glu
Asn 20 25 30 gct gaa aaa gac gga ctg tgc aat gct tgt acg tgg aga
cag aat aca 144 Ala Glu Lys Asp Gly Leu Cys Asn Ala Cys Thr Trp Arg
Gln Asn Thr 35 40 45 aaa tcc tcc aga ata gaa gcc ata aaa att caa
atc ctc agc aaa ctg 192 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln
Ile Leu Ser Lys Leu 50 55 60 cgc ctg gaa caa gca cct aac att agc
agg gac gtt att aag cag ctt 240 Arg Leu Glu Gln Ala Pro Asn Ile Ser
Arg Asp Val Ile Lys Gln Leu 65 70 75 80 tta ccc aaa gct cct cca ctg
cag gaa ctg att gat cag tat gat gtc 288 Leu Pro Lys Ala Pro Pro Leu
Gln Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 cag agg gac gac agt
agc gat ggc tct ttg gaa gac gat gac tat cat 336 Gln Arg Asp Asp Ser
Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 gcc aca acc
gag acg att atc aca atg cct acg gag tct gat ttt ctt 384 Ala Thr Thr
Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 gta
caa atg gag gga aaa cca aaa tgt tgc ttc ttt aag ttt agc tct 432 Val
Gln Met Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135
140 aaa ata caa tat aac aaa gta gta aag gca caa tta tgg ata tac ttg
480 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu
145 150 155 160 agg caa gtc caa aaa cct aca acg gtg ttt gtg cag atc
ctg aga ctc 528 Arg Gln Val Gln Lys Pro Thr Thr Val Phe Val Gln Ile
Leu Arg Leu 165 170 175 att aag ccc atg aaa gac ggt aca aga tat act
gga att cga tct ttg 576 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr
Gly Ile Arg Ser Leu 180 185 190 aaa ctt gac atg aac cca ggc act ggt
atc tgg cag agt att gat gtg 624 Lys Leu Asp Met Asn Pro Gly Thr Gly
Ile Trp Gln Ser Ile Asp Val 195 200 205 aag aca gtg ctg caa aat tgg
ctc aaa cag cct gaa tcc aat tta ggc 672 Lys Thr Val Leu Gln Asn Trp
Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 atc gaa ata aaa gct
ttt gat gag act gga cga gat ctt gct gtc aca 720 Ile Glu Ile Lys Ala
Phe Asp Glu Thr Gly Arg Asp Leu Ala Val Thr 225 230 235 240 ttc cca
gga cca gga gaa gat gga ttg aac cca ttt tta gag gtc aga 768 Phe Pro
Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Arg 245 250 255
gtt aca gac aca ccg aaa cgg tcc cgc aga gat ttt ggc ctt gac tgt 816
Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260
265 270 gat gag cac tca acg gaa tcc cga tgt tgt cgc tac ccg ctg aca
gtg 864 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr
Val 275 280 285 gat ttc gaa gct ttt gga tgg gac tgg att ata gca cct
aaa aga tac 912 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro
Lys Arg Tyr 290 295 300 aaa gcc aat tac tgc tcc gga gaa tgc gaa ttt
gtg ttt cta cag aaa 960 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe
Val Phe Leu Gln Lys 305 310 315 320 tac ccg cac act cac ctg gta cac
caa gca aat ccc aga ggc tca gca 1008 Tyr Pro His Thr His Leu Val
His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 ggc cct tgc tgc aca
ccc acc aag atg tcc cct ata aac atg ctg tat 1056 Gly Pro Cys Cys
Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 ttc aat
gga aaa gaa caa ata ata tat gga aag ata cca gcc atg gtt 1104 Phe
Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360
365 gta gat cgt tgc ggg tgc tca tga 1128 Val Asp Arg Cys Gly Cys
Ser 370 375 23 375 PRT Gallus gallus 23 Met Gln Lys Leu Ala Val Tyr
Val Tyr Ile Tyr Leu Phe Met Gln Ile 1 5 10 15 Ala Val Asp Pro Val
Ala Leu Asp Gly Ser Ser Gln Pro Thr Glu Asn 20 25 30 Ala Glu Lys
Asp Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40 45 Lys
Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55
60 Arg Leu Glu Gln Ala Pro Asn Ile Ser Arg Asp Val Ile Lys Gln Leu
65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Gln Glu Leu Ile Asp Gln Tyr
Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp
Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro
Thr Glu Ser Asp Phe Leu 115 120 125 Val Gln Met Glu Gly Lys Pro Lys
Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 Lys Ile Gln Tyr Asn Lys
Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 Arg Gln Val
Gln Lys Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 Ile
Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185
190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val
195 200 205 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn
Leu Gly 210 215 220 Ile Glu Ile Lys Ala Phe Asp Glu Thr Gly Arg Asp
Leu Ala Val Thr 225 230 235 240 Phe Pro Gly Pro Gly Glu Asp Gly Leu
Asn Pro Phe Leu Glu Val Arg 245 250 255 Val Thr Asp Thr Pro Lys Arg
Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 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 Ile Ile Ala Pro Lys Arg Tyr 290 295 300 Lys
Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310
315 320 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser
Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn
Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys
Ile Pro Ala Met Val 355 360 365 Val Asp Arg Cys Gly Cys Ser 370 375
24 1131 DNA Rattus norvegicus CDS (1)...(1128) 24 atg att caa aaa
ccg caa atg tat gtt tat att tac ctg ttt gtg ctg 48 Met Ile Gln Lys
Pro Gln Met Tyr Val Tyr Ile Tyr Leu Phe Val Leu 1 5 10 15 att gct
gct ggc cca gtg gat cta aat gag gac agt gag aga gag gcg 96 Ile Ala
Ala Gly Pro Val Asp Leu Asn Glu Asp Ser Glu Arg Glu Ala 20 25 30
aat gtg gaa aaa gag ggg ctg tgt aat gcg tgt gcg tgg aga caa aac 144
Asn Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln Asn 35
40 45 aca agg tac tcc aga ata gaa gcc ata aaa att caa atc ctc agt
aaa 192 Thr Arg Tyr Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser
Lys 50 55 60 ctc cgc ctg gaa aca gcg cct aac atc agc aaa gat gct
ata aga caa 240 Leu Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala
Ile Arg Gln 65 70 75 80 ctt ctg ccc aga gcg cct cca ctc cgg gaa ctg
atc gat cag tac gac 288 Leu Leu Pro Arg Ala Pro Pro Leu Arg Glu Leu
Ile Asp Gln Tyr Asp 85 90 95 gtc cag agg gat gac agc agt gac ggc
tct ttg gaa gat gac gat tat 336 Val Gln Arg Asp Asp Ser Ser Asp Gly
Ser Leu Glu Asp Asp Asp Tyr 100 105 110 cac gct acc acg gaa aca atc
att acc atg cct acc gag tct gac ttt 384 His Ala Thr Thr Glu Thr Ile
Ile Thr Met Pro Thr Glu Ser Asp Phe 115 120 125 cta atg caa gcg gat
gga aag ccc aaa tgt tgc ttt ttt aaa ttt agc 432 Leu Met Gln Ala Asp
Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser 130 135 140 tct aaa ata
cag tac aac aaa gtg gta aag gcc cag ctg tgg ata tat 480 Ser Lys Ile
Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr 145 150 155 160
ctg aga gcc gtc aag act cct aca aca gtg ttt gtg caa atc ctg aga 528
Leu Arg Ala Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg 165
170 175 ctc atc aaa ccc atg aaa gac ggt aca agg tat acc gga atc cga
tct 576 Leu Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg
Ser 180 185 190 ctg aaa ctt gac atg agc cca ggc act ggt att tgg cag
agt att gat 624 Leu Lys Leu Asp Met Ser Pro Gly Thr Gly Ile Trp Gln
Ser Ile Asp 195 200 205 gtg aag aca gtg ttg caa aat tgg ctc aaa cag
cct gaa tcc aac tta 672 Val Lys Thr Val Leu Gln Asn Trp Leu Lys Gln
Pro Glu Ser Asn Leu 210 215 220 ggc att gaa atc aaa gct ttg gat gag
aat ggg cat gat ctt gct gta 720 Gly Ile Glu Ile Lys Ala Leu Asp Glu
Asn Gly His Asp Leu Ala Val 225 230 235 240 acc ttc cca gga cca gga
gaa gat ggg ctg aat ccc ttt tta gaa gtc 768 Thr Phe Pro Gly Pro Gly
Glu Asp Gly Leu Asn Pro Phe Leu Glu Val 245 250 255 aaa gta aca gac
aca ccc aag agg tcc cgg aga gac ttt ggg ctt gac 816 Lys Val Thr Asp
Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp 260 265 270 tgc gat
gaa cac tcc acg gaa tcg cgg tgc tgt cgc tac ccc ctc acg 864 Cys Asp
Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 275 280 285
gtc gat ttc gaa gcc ttt gga tgg gac tgg att att gca ccc aaa aga 912
Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg 290
295 300 tat aag gct aat tac tgc tct gga gag tgt gaa ttt gtg ttc tta
caa 960 Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu
Gln 305 310 315 320 aaa tat ccg cat act cat ctt gtg cac caa gca aac
ccc aga ggc tcg 1008 Lys Tyr Pro His Thr His Leu Val His Gln Ala
Asn Pro Arg Gly Ser 325 330 335 gca ggc cct tgc tgc acg cca aca aaa
atg tct ccc att aat atg cta 1056 Ala Gly Pro Cys Cys Thr Pro Thr
Lys Met Ser Pro Ile Asn Met Leu 340 345 350 tat ttt aat ggc aaa gaa
caa ata ata tat ggg aaa att cca gcc atg 1104 Tyr Phe Asn Gly Lys
Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met 355 360 365 gta gta gac
cgg tgt ggg tgc tcg tga 1131 Val Val Asp Arg Cys Gly Cys Ser 370
375 25 376 PRT Rattus norvegicus 25 Met Ile Gln Lys Pro Gln Met Tyr
Val Tyr Ile Tyr Leu Phe Val Leu 1 5 10 15 Ile Ala Ala Gly Pro Val
Asp Leu Asn Glu Asp Ser Glu Arg Glu Ala 20 25 30 Asn Val Glu Lys
Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln Asn 35 40 45 Thr Arg
Tyr Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys 50 55 60
Leu Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln 65
70 75 80 Leu Leu Pro Arg Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln
Tyr Asp 85 90 95 Val Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu
Asp Asp Asp Tyr 100 105 110 His Ala Thr Thr Glu Thr Ile Ile Thr Met
Pro Thr Glu Ser Asp Phe 115 120 125 Leu Met Gln Ala Asp Gly Lys Pro
Lys Cys Cys Phe Phe Lys Phe Ser 130 135 140 Ser Lys Ile Gln Tyr Asn
Lys Val Val Lys Ala Gln Leu Trp Ile Tyr 145 150 155 160 Leu Arg Ala
Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg 165 170 175 Leu
Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser 180 185
190 Leu Lys Leu Asp Met Ser Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp
195 200 205 Val Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser
Asn Leu 210 215 220 Gly Ile Glu Ile 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 260 265 270 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 Ile Ile Ala Pro Lys Arg 290 295 300 Tyr
Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln 305 310
315 320 Lys Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly
Ser 325 330 335 Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile
Asn Met Leu 340 345 350 Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly
Lys Ile Pro Ala Met 355 360 365 Val Val Asp Arg Cys Gly Cys Ser 370
375 26 1128 DNA Meleagris gallopavo CDS (1)...(1125) 26 atg caa aag
cta gca gtc tat gtt tat att tac ctg ttc atg cag att 48 Met Gln Lys
Leu Ala
Val Tyr Val Tyr Ile Tyr Leu Phe Met Gln Ile 1 5 10 15 tta gtt cat
ccg gtg gct ctt gat ggc agt agt cag ccc aca gag aac 96 Leu Val His
Pro Val Ala Leu Asp Gly Ser Ser Gln Pro Thr Glu Asn 20 25 30 gct
gaa aaa gac gga ctg tgc aat gct tgc acg tgg aga cag aat act 144 Ala
Glu Lys Asp Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40
45 aaa tcc tcc aga ata gaa gcc ata aaa att caa atc ctc agc aaa ctg
192 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu
50 55 60 cgc ctg gaa caa gca cct aac att agc agg gac gtt att aaa
caa ctt 240 Arg Leu Glu Gln Ala Pro Asn Ile Ser Arg Asp Val Ile Lys
Gln Leu 65 70 75 80 tta ccc aaa gct cct ccg ctg cag gaa ctg att gat
cag tat gac gtc 288 Leu Pro Lys Ala Pro Pro Leu Gln Glu Leu Ile Asp
Gln Tyr Asp Val 85 90 95 cag aga gac gac agt agc gat ggc tct ttg
gaa gac gat gac tat cat 336 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu
Glu Asp Asp Asp Tyr His 100 105 110 gcc aca acc gaa acg att atc aca
atg cct acg gag tct gat ttt ctt 384 Ala Thr Thr Glu Thr Ile Ile Thr
Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 gta caa atg gag gga aaa
cca aaa tgt tgc ttc ttt aag ttt agc tct 432 Val Gln Met Glu Gly Lys
Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 aaa ata caa tat
aac aaa gta gta aag gca caa tta tgg ata tac ttg 480 Lys Ile Gln Tyr
Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 agg
caa gtc caa aaa cct aca acg gtg ttt gtg cag atc ctg aga ctc 528 Arg
Gln Val Gln Lys Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170
175 att aaa ccc atg aaa gac ggt aca aga tat act gga att cga tct ttg
576 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu
180 185 190 aaa ctt gac atg aac cca ggc act ggt atc tgg cag agt att
gat gtg 624 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile
Asp Val 195 200 205 aag aca gtg ttg caa aat tgg ctc aaa cag cct gaa
tcc aat tta ggc 672 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu
Ser Asn Leu Gly 210 215 220 atc gaa ata aaa gct ttt gat gag aat gga
cga gat ctt gct gta aca 720 Ile Glu Ile Lys Ala Phe Asp Glu Asn Gly
Arg Asp Leu Ala Val Thr 225 230 235 240 ttc cca gga cca ggt gaa gat
gga ctg aac cca ttt tta gag gtc aga 768 Phe Pro Gly Pro Gly Glu Asp
Gly Leu Asn Pro Phe Leu Glu Val Arg 245 250 255 gtt aca gac aca cca
aaa cgg tcc cgc aga gat ttt ggc ctt gac tgc 816 Val Thr Asp Thr Pro
Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 gac gag cac
tca acg gaa tct cga tgt tgt cgc tac ccg ctg aca gtg 864 Asp Glu His
Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 gat
ttt gaa gct ttt gga tgg gac tgg att ata gca cct aaa aga tac 912 Asp
Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295
300 aaa gcc aat tac tgc tct gga gaa tgt gaa ttc gta ttt cta cag aaa
960 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys
305 310 315 320 tac ccg cac act cac ctg gta cac caa gca aat cca aga
ggc tca gca 1008 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro
Arg Gly Ser Ala 325 330 335 ggc cct tgc tgc aca ccc acc aag atg tcc
cct ata aac atg ctg tat 1056 Gly Pro Cys Cys Thr Pro Thr Lys Met
Ser Pro Ile Asn Met Leu Tyr 340 345 350 ttc aat gga aaa gaa caa ata
ata tat gga aag ata cca gcc atg gtt 1104 Phe Asn Gly Lys Glu Gln
Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 gta gat cgt tgc
ggg tgc tca tga 1128 Val Asp Arg Cys Gly Cys Ser 370 375 27 375 PRT
Meleagris gallopavo 27 Met Gln Lys Leu Ala Val Tyr Val Tyr Ile Tyr
Leu Phe Met Gln Ile 1 5 10 15 Leu Val His Pro Val Ala Leu Asp Gly
Ser Ser Gln Pro Thr Glu Asn 20 25 30 Ala Glu Lys Asp Gly Leu Cys
Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40 45 Lys Ser Ser Arg Ile
Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 Arg Leu Glu
Gln Ala Pro Asn Ile Ser Arg Asp Val Ile Lys Gln Leu 65 70 75 80 Leu
Pro Lys Ala Pro Pro Leu Gln Glu Leu Ile Asp Gln Tyr Asp Val 85 90
95 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His
100 105 110 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp
Phe Leu 115 120 125 Val Gln Met Glu Gly Lys Pro Lys Cys Cys Phe Phe
Lys Phe Ser Ser 130 135 140 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala
Gln Leu Trp Ile Tyr Leu 145 150 155 160 Arg Gln Val Gln Lys Pro Thr
Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met Lys
Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 Lys Leu Asp
Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 Lys
Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215
220 Ile Glu Ile Lys Ala Phe Asp Glu Asn Gly Arg Asp Leu Ala Val Thr
225 230 235 240 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu
Glu Val Arg 245 250 255 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp
Phe Gly Leu Asp Cys 260 265 270 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 Ile Ile Ala Pro Lys Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys
Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 Tyr Pro
His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340
345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met
Val 355 360 365 Val Asp Arg Cys Gly Cys Ser 370 375 28 1128 DNA
Porcine CDS (1)...(1125) 28 atg caa aaa ctg caa atc tat gtt tat att
tac ctg ttt atg ctg att 48 Met Gln Lys Leu Gln Ile Tyr Val Tyr Ile
Tyr Leu Phe Met Leu Ile 1 5 10 15 gtt gct ggt ccc gtg gat ctg aat
gag aac agc gag caa aag gaa aat 96 Val Ala Gly Pro Val Asp Leu Asn
Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30 gtg gaa aaa gag ggg ctg
tgt aat gca tgt atg tgg aga caa aac act 144 Val Glu Lys Glu Gly Leu
Cys Asn Ala Cys Met Trp Arg Gln Asn Thr 35 40 45 aaa tct tca aga
cta gaa gcc ata aaa att caa atc ctc agt aaa ctt 192 Lys Ser Ser Arg
Leu Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 cgc ctg
gaa aca gct cct aac att agc aaa gat gct ata aga caa ctt 240 Arg Leu
Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln Leu 65 70 75 80
ttg ccc aaa gct cct cca ctc cgg gaa ctg att gat cag tac gat gtc 288
Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85
90 95 cag aga gat gac agc agt gat ggc tcc ttg gaa gat gat gat tat
cac 336 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr
His 100 105 110 gct acg acg gaa acg atc att acc atg cct aca gag tct
gat ctt cta 384 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser
Asp Leu Leu 115 120 125 atg caa gtg gaa gga aaa ccc aaa tgc tgc ttc
ttt aaa ttt agc tct 432 Met Gln Val Glu Gly Lys Pro Lys Cys Cys Phe
Phe Lys Phe Ser Ser 130 135 140 aaa ata caa tac aat aaa gta gta aag
gcc caa ctg tgg ata tat ctg 480 Lys Ile Gln Tyr Asn Lys Val Val Lys
Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 aga ccc gtc aag act cct
aca aca gtg ttt gtg caa atc ctg aga ctc 528 Arg Pro Val Lys Thr Pro
Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 atc aaa ccc atg
aaa gac ggt aca agg tat act gga atc cga tct ctg 576 Ile Lys Pro Met
Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 aaa ctt
gac atg aac cca ggc act ggt att tgg cag agc att gat gtg 624 Lys Leu
Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205
aag aca gtg ttg caa aat tgg ctc aaa caa cct gaa tcc aac tta ggc 672
Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210
215 220 att gaa atc aaa gct tta gat gag aat ggt cat gat ctt gct gta
acc 720 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val
Thr 225 230 235 240 ttc cca gga cca gga gaa gat ggg ctg aat ccc ttt
tta gaa gtc aag 768 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 agg aga
gat ttt gga ctc gac tgt 816 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 tct cga
tgc tgt cgt tac cct cta act gtg 864 Asp Glu His Ser Thr Glu Ser Arg
Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 gat ttt gaa gct ttt gga
tgg gac tgg att att gca ccc aaa aga tat 912 Asp Phe Glu Ala Phe Gly
Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 aag gcc aat tac
tgc tct gga gag tgt gaa ttt gta ttt tta caa aaa 960 Lys Ala Asn Tyr
Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 tac
cct cac act cat ctt gtg cac caa gca aac ccc aga ggt tca gca 1008
Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325
330 335 ggc ccc tgc tgt act ccc aca aag atg tct cca atc aat atg cta
tat 1056 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met
Leu Tyr 340 345 350 ttt aat ggc aaa gaa caa ata ata tat ggg aaa att
cca gcc atg gta 1104 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys
Ile Pro Ala Met Val 355 360 365 gta gat cgc tgt ggg tgc tca tga
1128 Val Asp Arg Cys Gly Cys Ser 370 375 29 375 PRT Porcine 29 Met
Gln Lys Leu Gln Ile Tyr Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10
15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn
20 25 30 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Met Trp Arg Gln
Asn Thr 35 40 45 Lys Ser Ser Arg Leu Glu Ala Ile Lys Ile Gln Ile
Leu Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys
Asp Ala Ile Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg
Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser
Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu
Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125 Met Gln
Val Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140
Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145
150 155 160 Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu
Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly
Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile
Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu
Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu
Asp Glu Asn Gly His Asp Leu Ala Val Thr 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 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 Ile Ile Ala Pro Lys
Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val
Phe Leu Gln Lys 305 310 315 320 Tyr Pro His Thr His Leu Val His Gln
Ala Asn Pro Arg Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr
Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu
Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 Val Asp Arg
Cys Gly Cys Ser 370 375 30 1128 DNA Ovine CDS (1)...(1125) 30 atg
caa aaa ctg caa atc ttt gtt tat att tac cta ttt atg ctg ctt 48 Met
Gln Lys Leu Gln Ile Phe Val Tyr Ile Tyr Leu Phe Met Leu Leu 1 5 10
15 gtt gct ggc cca gtg gat ctg aat gag aac agc gag cag aag gaa aat
96 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn
20 25 30 gtg gaa aaa aag ggg ctg tgt aat gca tgc ttg tgg aga caa
aac aat 144 Val Glu Lys Lys Gly Leu Cys Asn Ala Cys Leu Trp Arg Gln
Asn Asn 35 40 45 aaa tcc tca aga cta gaa gcc ata aaa atc caa atc
ctc agt aag ctt 192 Lys Ser Ser Arg Leu Glu Ala Ile Lys Ile Gln Ile
Leu Ser Lys Leu 50 55 60 cgc ctg gaa aca gct cct aac atc agc aaa
gat gct ata aga caa ctt 240 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys
Asp Ala Ile Arg Gln Leu 65 70 75 80 ttg ccc aag gct cct cca ctc cgg
gaa ctg att gat cag tac gat gtc 288 Leu Pro Lys Ala Pro Pro Leu Arg
Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 cag aga gat gac agc agc
gac ggc tcc ttg gaa gac gat gac tac cac 336 Gln Arg Asp Asp Ser Ser
Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 gtt acg acg gaa
acg gtc att acc atg ccc acg gag tct gat ctt cta 384 Val Thr Thr Glu
Thr Val Ile Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125 gca gaa
gtg caa gaa aaa ccc aaa tgt tgc ttc ttt aaa ttt agc tct 432 Ala Glu
Val Gln Glu Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140
aag ata caa cac aat aaa gta gta aag gcc caa ctg tgg ata tat ctg 480
Lys Ile Gln His Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145
150 155 160 aga cct gtc aag act cct aca aca gtg ttt gtg caa atc ctg
aga ctc 528 Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu
Arg Leu 165 170 175 atc aaa ccc atg aaa gac ggt aca agg tat act gga
atc cga tct ctg 576 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly
Ile Arg Ser Leu 180 185 190 aaa ctt gac atg aac cca ggc act ggt att
tgg cag agc att gat gtg 624 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile
Trp Gln Ser Ile Asp Val 195 200 205 aag aca gtg ttg caa aac tgg ctc
aaa caa cct gaa tcc aac tta ggc 672 Lys Thr Val Leu Gln Asn Trp Leu
Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 att gaa atc aaa gct tta
gat gag aat ggt cat gat ctt gct gta acc 720 Ile Glu Ile Lys Ala Leu
Asp Glu Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 ttc cca gaa
cca gga gaa gaa gga ctg aat cct ttt tta gaa gtc aag 768 Phe Pro Glu
Pro Gly Glu Glu Gly Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 gta
aca gac aca cca aaa aga tct agg aga gat ttt ggg ctt gat tgt 816 Val
Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265
270 gat gag cac tcc aca gaa tct cga tgc tgt cgt tac cct cta act gtg
864 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
275 280 285 gat ttt gaa gct ttt gga tgg gat tgg att att gca cct aaa
aga tat 912 Asp Phe Glu
Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 aag
gcc aat tac tgc tct gga gaa tgt gaa ttt tta ttt ttg caa aag 960 Lys
Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Leu Phe Leu Gln Lys 305 310
315 320 tat cct cat acc cat ctt gtg cac caa gca aac ccc aaa ggt tca
gcc 1008 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Lys Gly
Ser Ala 325 330 335 ggc cct tgc tgt act cct aca aag atg tct cca att
aat atg cta tat 1056 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro
Ile Asn Met Leu Tyr 340 345 350 ttt aat ggc aaa gaa caa ata ata tat
ggg aag att cca ggc atg gta 1104 Phe Asn Gly Lys Glu Gln Ile Ile
Tyr Gly Lys Ile Pro Gly Met Val 355 360 365 gta gat cgc tgt ggg tgc
tca tga 1128 Val Asp Arg Cys Gly Cys Ser 370 375 31 375 PRT Ovine
31 Met Gln Lys Leu Gln Ile Phe Val Tyr Ile Tyr Leu Phe Met Leu Leu
1 5 10 15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys
Glu Asn 20 25 30 Val Glu Lys Lys Gly Leu Cys Asn Ala Cys Leu Trp
Arg Gln Asn Asn 35 40 45 Lys Ser Ser Arg Leu Glu Ala Ile Lys Ile
Gln Ile Leu Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile
Ser Lys Asp Ala Ile Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro
Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp
Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Val Thr
Thr Glu Thr Val Ile Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125
Ala Glu Val Gln Glu Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130
135 140 Lys Ile Gln His Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr
Leu 145 150 155 160 Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln
Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr
Thr Gly Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr
Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn
Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys
Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 Phe
Pro Glu Pro Gly Glu Glu 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 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 Ile Ile Ala
Pro Lys Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu
Phe Leu Phe Leu Gln Lys 305 310 315 320 Tyr Pro His Thr His Leu Val
His Gln Ala Asn Pro Lys Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr
Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly
Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val 355 360 365 Val
Asp Arg Cys Gly Cys Ser 370 375 32 480 DNA Rattus norvegicus CDS
(1)...(390) 32 gaa gat ggg ctg aat ccc ttt tta gaa gtc aaa gta aca
gac aca ccc 48 Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys Val Thr
Asp Thr Pro 1 5 10 15 aag agg tcc cgg aga gac ttt ggg ctt gac tgc
gat gaa cac tcc acg 96 Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys
Asp Glu His Ser Thr 20 25 30 gaa tcg cgg tgc tgt cgc tac ccc ctc
acg gtc gat ttc gaa gcc ttt 144 Glu Ser Arg Cys Cys Arg Tyr Pro Leu
Thr Val Asp Phe Glu Ala Phe 35 40 45 gga tgg gac tgg att att gca
ccc aaa aga tat aag gct aat tac tgc 192 Gly Trp Asp Trp Ile Ile Ala
Pro Lys Arg Tyr Lys Ala Asn Tyr Cys 50 55 60 tct gga gag tgt gaa
ttt gtg ttc tta caa aaa tat ccg cat act cat 240 Ser Gly Glu Cys Glu
Phe Val Phe Leu Gln Lys Tyr Pro His Thr His 65 70 75 80 ctt gtg cac
caa gca aac ccc aga ggc tcg gca ggc cct tgc tgc acg 288 Leu Val His
Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr 85 90 95 cca
aca aaa atg tct ccc att aat atg cta tat ttt aat ggc aaa gaa 336 Pro
Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu 100 105
110 caa ata ata tat ggg aaa att cca gcc atg gta gta gac cgg tgt ggg
384 Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly
115 120 125 tgc tcg tgagctttgc attagcttta aaatttccca aatcgtggaa
ggtcttcccc 440 Cys Ser 130 tcgatttcga aactgtgaat ttatgtacca
caggctgtag 480 33 130 PRT Rattus norvegicus 33 Glu Asp Gly Leu Asn
Pro Phe Leu Glu Val Lys Val Thr Asp Thr Pro 1 5 10 15 Lys Arg Ser
Arg Arg Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr 20 25 30 Glu
Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe 35 40
45 Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys
50 55 60 Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His
Thr His 65 70 75 80 Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala Gly
Pro Cys Cys Thr 85 90 95 Pro Thr Lys Met Ser Pro Ile Asn Met Leu
Tyr Phe Asn Gly Lys Glu 100 105 110 Gln Ile Ile Tyr Gly Lys Ile Pro
Ala Met Val Val Asp Arg Cys Gly 115 120 125 Cys Ser 130 34 790 DNA
Gallus gallus CDS (1)...(678) 34 tta gta gta aag gca caa tta tgg
ata tac ttg agg caa gtc caa aaa 48 Leu Val Val Lys Ala Gln Leu Trp
Ile Tyr Leu Arg Gln Val Gln Lys 1 5 10 15 cct aca acg gtg ttt gtg
cag atc ctg aga ctc att aag ccc atg aaa 96 Pro Thr Thr Val Phe Val
Gln Ile Leu Arg Leu Ile Lys Pro Met Lys 20 25 30 gac ggt aca aga
tat act gga att cga tct ttg aaa ctt gac atg aac 144 Asp Gly Thr Arg
Tyr Thr Gly Ile Arg Ser Leu Lys Leu Asp Met Asn 35 40 45 cca ggc
act ggt atc tgg cag agt att gat gtg aag aca gtg ctg caa 192 Pro Gly
Thr Gly Ile Trp Gln Ser Ile Asp Val Lys Thr Val Leu Gln 50 55 60
aat tgg ctc aaa cag cct gaa tcc aat tta ggc atc gaa ata aaa gct 240
Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly Ile Glu Ile Lys Ala 65
70 75 80 ttt gat gag act gga cga gat ctt gct gtc aca ttc cca gga
cca gga 288 Phe Asp Glu Thr Gly Arg Asp Leu Ala Val Thr Phe Pro Gly
Pro Gly 85 90 95 gaa gat gga ttg aac cca ttt tta gag gtc aga gtt
aca gac aca ccg 336 Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Arg Val
Thr Asp Thr Pro 100 105 110 aaa cgg tcc cgc aga gat ttt ggc ctt gac
tgt gat gag cac tca acg 384 Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp
Cys Asp Glu His Ser Thr 115 120 125 gaa tcc cga tgt tgt cgc tac ccg
ctg aca gtg gat ttc gaa gct ttt 432 Glu Ser Arg Cys Cys Arg Tyr Pro
Leu Thr Val Asp Phe Glu Ala Phe 130 135 140 gga tgg gac tgg att ata
gca cct aaa aga tac aaa gcc aat tac tgc 480 Gly Trp Asp Trp Ile Ile
Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys 145 150 155 160 tcc gga gaa
tgc gaa ttt gtg ttt cta cag aaa tac ccg cac act cac 528 Ser Gly Glu
Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr His 165 170 175 ctg
gta cac caa gca aat ccc aga ggc tca gca ggc cct tgc tgc aca 576 Leu
Val His Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr 180 185
190 ccc acc aag atg tcc cct ata aac atg ctg tat ttc aat gga aaa gaa
624 Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu
195 200 205 caa ata ata tat gga aag ata cca gcc atg gtt gta gat cgt
tgc ggg 672 Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg
Cys Gly 210 215 220 tgc tca tgaggctgtc gtgagatcca ccattcgata
aattgtggaa gccaccaaaa 728 Cys Ser 225 aaaaaagcta tatcccctca
tccatctttg aaactgtgaa attacgtacg ctaggcattg 788 cc 790 35 226 PRT
Gallus gallus 35 Leu Val Val Lys Ala Gln Leu Trp Ile Tyr Leu Arg
Gln Val Gln Lys 1 5 10 15 Pro Thr Thr Val Phe Val Gln Ile Leu Arg
Leu Ile Lys Pro Met Lys 20 25 30 Asp Gly Thr Arg Tyr Thr Gly Ile
Arg Ser Leu Lys Leu Asp Met Asn 35 40 45 Pro Gly Thr Gly Ile Trp
Gln Ser Ile Asp Val Lys Thr Val Leu Gln 50 55 60 Asn Trp Leu Lys
Gln Pro Glu Ser Asn Leu Gly Ile Glu Ile Lys Ala 65 70 75 80 Phe Asp
Glu Thr Gly Arg Asp Leu Ala Val Thr Phe Pro Gly Pro Gly 85 90 95
Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Arg Val Thr Asp Thr Pro 100
105 110 Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys Asp Glu His Ser
Thr 115 120 125 Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe
Glu Ala Phe 130 135 140 Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr
Lys Ala Asn Tyr Cys 145 150 155 160 Ser Gly Glu Cys Glu Phe Val Phe
Leu Gln Lys Tyr Pro His Thr His 165 170 175 Leu Val His Gln Ala Asn
Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr 180 185 190 Pro Thr Lys Met
Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu 195 200 205 Gln Ile
Ile Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly 210 215 220
Cys Ser 225 36 123 PRT Homo sapiens 36 Arg Pro Arg Arg Asp Ala Glu
Pro Val Leu Gly Gly Gly Pro Gly Gly 1 5 10 15 Ala Cys Arg Ala Arg
Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp 20 25 30 His Arg Trp
Val Ile Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gln 35 40 45 Gly
Gln Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro 50 55
60 Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro
65 70 75 80 Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser
Pro Ile 85 90 95 Ser Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val
Leu Arg Gln Tyr 100 105 110 Glu Asp Met Val Val Asp Glu Cys Gly Cys
Arg 115 120 37 118 PRT Homo sapiens 37 Arg Glu Lys Arg Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser 1 5 10 15 Ser Cys Lys Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp 20 25 30 Asn Asp Trp
Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His 35 40 45 Gly
Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His 50 55
60 Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
65 70 75 80 Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu
Tyr Leu 85 90 95 Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln
Asp Met Val Val 100 105 110 Glu Gly Cys Gly Cys Arg 115 38 118 PRT
Homo sapiens 38 Lys Arg Ser Pro Lys His His Ser Gln Arg Ala Arg Lys
Lys Asn Lys 1 5 10 15 Asn Cys Arg Arg His Ser Leu Tyr Val Asp Phe
Ser Asp Val Gly Trp 20 25 30 Asn Asp Trp Ile Val Ala Pro Pro Gly
Tyr Gln Ala Phe Tyr Cys His 35 40 45 Gly Asp Cys Pro Phe Pro Leu
Ala Asp His Leu Asn Ser Thr Asn His 50 55 60 Ala Ile Val Gln Thr
Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys 65 70 75 80 Ala Cys Cys
Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu 85 90 95 Asp
Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val 100 105
110 Glu Gly Cys Gly Cys Arg 115 39 119 PRT Homo sapiens 39 Ser Arg
Gly Ser Gly Ser Ser Asp Tyr Asn Gly Ser Glu Leu Lys Thr 1 5 10 15
Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp 20
25 30 Gln Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys
Asp 35 40 45 Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala
Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu Val His Leu Met Asn
Pro Glu Tyr Val Pro 65 70 75 80 Lys Pro Cys Cys Ala Pro Thr Lys Leu
Asn Ala Ile Ser Val Leu Tyr 85 90 95 Phe Asp Asp Asn Ser Asn Val
Ile Leu Lys Lys Tyr Arg Asn Met Val 100 105 110 Val Arg Ala Cys Gly
Cys His 115 40 119 PRT Homo sapiens 40 Leu Arg Met Ala Asn Val Ala
Glu Asn Ser Ser Ser Asp Gln Arg Gln 1 5 10 15 Ala Cys Lys Lys His
Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp 20 25 30 Gln Asp Trp
Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu 35 40 45 Gly
Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His 50 55
60 Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro
65 70 75 80 Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser Val
Leu Tyr 85 90 95 Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr
Arg Asn Met Val 100 105 110 Val Arg Ala Cys Gly Cys His 115 41 119
PRT Homo sapiens 41 Ser Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser
Glu Gln Lys Gln 1 5 10 15 Ala Cys Lys Lys His Glu Leu Tyr Val Ser
Phe Arg Asp Leu Gly Trp 20 25 30 Gln Asp Trp Ile Ile Ala Pro Glu
Gly Tyr Ala Ala Phe Tyr Cys Asp 35 40 45 Gly Glu Cys Ser Phe Pro
Leu Asn Ala His Met Asn Ala Thr Asn His 50 55 60 Ala Ile Val Gln
Thr Leu Val His Leu Met Phe Pro Asp His Val Pro 65 70 75 80 Lys Pro
Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr 85 90 95
Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val 100
105 110 Val Arg Ser Cys Gly Cys His 115 42 120 PRT Homo sapiens 42
Glu Gln Thr Leu Lys Lys Ala Arg Arg Lys Gln Trp Ile Glu Pro Arg 1 5
10 15 Asn Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly
Trp 20 25 30 Ser Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala Tyr
Tyr Cys Ser 35 40 45 Gly Ala Cys Gln Phe Pro Met Pro Lys Ser Leu
Lys Pro Ser Asn His 50 55 60 Ala Thr Ile Gln Ser Ile Val Arg Ala
Val Gly Val Val Pro Gly Ile 65 70 75 80 Pro Glu Pro Cys Cys Val Pro
Glu Lys Met Ser Ser Leu Ser Ile Leu 85 90 95 Phe Phe Asp Glu Asn
Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 100 105 110 Thr Val Glu
Ser Cys Ala Cys Arg 115 120 43 116 PRT Homo sapiens 43 Gly Pro Gly
Arg Ala Gln Arg Ser Ala Gly Ala Thr Ala Ala Asp Gly 1 5 10 15 Pro
Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala Glu Arg Ser 20 25
30 Val Leu Ile Pro Glu Thr Tyr Gln Ala Asn Asn Cys Gln Gly Val Cys
35 40 45 Gly Trp Pro Gln Ser Asp Arg Asn Pro Arg Tyr Gly Asn His
Val Val 50 55 60 Leu Leu Leu Lys Met Gln Ala Arg Gly Ala Ala Leu
Ala Arg Pro Pro 65 70 75 80 Cys Cys
Val Pro Thr Ala Tyr Ala Gly Lys Leu Leu Ile Ser Leu Ser 85 90 95
Glu Glu Arg Ile Ser Ala His His Val Pro Asn Met Val Ala Thr Glu 100
105 110 Cys Gly Cys Arg 115 44 122 PRT Homo sapiens 44 Ala Leu Arg
Leu Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala His Ala 1 5 10 15 Asn
Cys His Arg Val Ala Leu Asn Ile Ser Phe Gln Glu Leu Gly Trp 20 25
30 Glu Arg Trp Ile Val Tyr Pro Pro Ser Phe Ile Phe His Tyr Cys His
35 40 45 Gly Gly Cys Gly Leu His Ile Pro Pro Asn Leu Ser Leu Pro
Val Pro 50 55 60 Gly Ala Pro Pro Thr Pro Ala Gln Pro Tyr Ser Leu
Leu Pro Gly Ala 65 70 75 80 Gln Pro Cys Cys Ala Ala Leu Pro Gly Thr
Met Arg Pro Leu His Val 85 90 95 Arg Thr Thr Ser Asp Gly Gly Tyr
Ser Phe Lys Tyr Glu Thr Val Pro 100 105 110 Asn Leu Leu Thr Gln His
Cys Ala Cys Ile 115 120 45 122 PRT Homo sapiens 45 His Arg Arg Arg
Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile 1 5 10 15 Cys Cys
Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn 20 25 30
Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly 35
40 45 Glu Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser
Phe 50 55 60 His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His
Ser Pro Phe 65 70 75 80 Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys
Leu Arg Pro Met Ser 85 90 95 Met Leu Tyr Tyr Asp Asp Gly Gln Asn
Ile Ile Lys Lys Asp Ile Gln 100 105 110 Asn Met Ile Val Glu Glu Cys
Gly Cys Ser 115 120 46 121 PRT Homo sapiens 46 His Arg Ile Arg Lys
Arg Gly Leu Glu Cys Asp Gly Arg Thr Asn Leu 1 5 10 15 Cys Cys Arg
Gln Gln Phe Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn 20 25 30 Asp
Trp Ile Ile Ala Pro Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly 35 40
45 Ser Cys Pro Ala Tyr Leu Ala Gly Val Pro Gly Ser Ala Ser Ser Phe
50 55 60 His Thr Ala Val Val Asn Gln Tyr Arg Met Arg Gly Leu Asn
Pro Gly 65 70 75 80 Thr Val Asn Ser Cys Cys Ile Pro Thr Lys Leu Ser
Thr Met Ser Met 85 90 95 Leu Tyr Phe Asp Asp Glu Tyr Asn Ile Val
Lys Arg Asp Val Pro Asn 100 105 110 Met Ile Val Glu Glu Cys Gly Cys
Ala 115 120 47 115 PRT Homo sapiens 47 His Arg Arg Ala Leu Asp Thr
Asn Tyr Cys Phe Ser Ser Thr Glu Lys 1 5 10 15 Asn Cys Cys Val Arg
Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly 20 25 30 Trp Lys Trp
Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu 35 40 45 Gly
Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val 50 55
60 Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys
65 70 75 80 Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr
Val Gly 85 90 95 Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile
Val Arg Ser Cys 100 105 110 Lys Cys Ser 115 48 115 PRT Homo sapiens
48 Lys Lys Arg Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp
1 5 10 15 Asn Cys Cys Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp
Leu Gly 20 25 30 Trp Lys Trp Ile His Glu Pro Lys Gly Tyr Asn Ala
Asn Phe Cys Ala 35 40 45 Gly Ala Cys Pro Tyr Leu Trp Ser Ser Asp
Thr Gln His Ser Arg Val 50 55 60 Leu Ser Leu Tyr Asn Thr Ile Asn
Pro Glu Ala Ser Ala Ser Pro Cys 65 70 75 80 Cys Val Ser Gln Asp Leu
Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly 85 90 95 Lys Thr Pro Lys
Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys 100 105 110 Lys Cys
Ser 115 49 115 PRT Homo sapiens 49 Lys Lys Arg Ala Leu Asp Thr Asn
Tyr Cys Phe Arg Asn Leu Glu Glu 1 5 10 15 Asn Cys Cys Val Arg Pro
Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly 20 25 30 Trp Lys Trp Val
His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser 35 40 45 Gly Pro
Cys Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val 50 55 60
Leu Gly Leu Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys 65
70 75 80 Cys Val Pro Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr
Val Gly 85 90 95 Arg Thr Pro Lys Val Glu Gln Leu Ser Asn Met Val
Val Lys Ser Cys 100 105 110 Leu Cys Ser 115 50 4 PRT Artificial
Sequence proteolytic cleavage site 50 Arg Xaa Xaa Arg 1 51 4 PRT
Eukaryotes proteolytic processing site 51 Arg Ser Arg Arg 1
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