U.S. patent application number 10/456852 was filed with the patent office on 2004-04-22 for methods to identify growth differentiation factor (gdf) receptors.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Lee, Se-Jin, McPherron, Alexandra.
Application Number | 20040077053 10/456852 |
Document ID | / |
Family ID | 31497825 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077053 |
Kind Code |
A1 |
Lee, Se-Jin ; et
al. |
April 22, 2004 |
Methods to identify growth differentiation factor (GDF)
receptors
Abstract
The present invention provides receptors for the growth
differentiation factor (GDF) family of growth factors and methods
of identifying such receptors. Also included are methods of
identifying antibodies which bind to the receptors, peptide
fragments of the receptor which inhibit GDF binding, GDF
receptor-binding agents capable of blocking GDF binding to the
receptor. The receptors of the invention allow the identification
of antagonists or agonists useful for agricultural and human
therapeutic purposes.
Inventors: |
Lee, Se-Jin; (Baltimore,
MD) ; McPherron, Alexandra; (Baltimore, MD) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
|
Family ID: |
31497825 |
Appl. No.: |
10/456852 |
Filed: |
June 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10456852 |
Jun 6, 2003 |
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09485046 |
May 5, 2000 |
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6696260 |
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09485046 |
May 5, 2000 |
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PCT/US98/15598 |
Jul 28, 1998 |
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60054461 |
Aug 1, 1997 |
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Current U.S.
Class: |
435/69.4 ;
435/320.1; 435/325; 530/350; 530/388.25; 530/399; 536/23.5 |
Current CPC
Class: |
C12N 15/8509 20130101;
A01K 2267/02 20130101; G01N 33/74 20130101; A01K 2267/03 20130101;
A01K 2267/01 20130101; A01K 67/0276 20130101; A01K 2227/105
20130101; A01K 2217/075 20130101; G01N 2333/495 20130101; C07K
14/71 20130101; G01N 2500/00 20130101 |
Class at
Publication: |
435/069.4 ;
435/320.1; 435/325; 530/388.25; 530/399; 530/350; 536/023.5 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/71; A61K 031/475; C07H 021/04 |
Claims
What is claimed is:
1. A recombinant cell line that expresses growth differentiation
factor-8 (GDF-8) or growth differentiation factor-11 (GDF-11)
receptor polypeptide.
2. The cell line of claim 1, wherein the cell is selected from the
group of species consisting of avian, bovine, ovine, piscine,
murine, human and porcine.
3. An antibody which specifically binds to growth differentiation
factor-8 (GDF-8) receptor polypeptide or fragments thereof.
4. The antibody of claim 7, wherein the antibody is a monoclonal
antibody.
5. An antibody which specifically binds to growth differentiation
factor-11 (GDF-11) receptor polypeptide or fragments thereof.
6. The antibody of claim 7, wherein the antibody is a monoclonal
antibody.
7. An isolated polynucleotide which encodes growth differentiation
factor-8 (GDF-8) receptor.
8. An isolated polynucleotide which encodes growth differentiation
factor-11 (GDF-11) receptor.
9. An expression vector containing in operable linkage the
polynucleotide as in claim 7 or 8.
10. A host cell containing the vector of claim 9.
11. A substantially purified peptide fragment of GDF-8 receptor,
wherein the peptide inhibits GDF-8 binding to GDF-8 receptor.
12. A substantially purified peptide fragment of GDF-11, wherein
the peptide inhibits GDF-11 binding to GDF-11 receptor.
13. A substantially purified GDF-8-binding agent, wherein the
binding agent inhibits GDF-8 binding to GDF-8 receptor.
14. A substantially purified GDF-11-binding agent, wherein the
binding agent inhibits GDF-11 binding to GDF-11 receptor.
15. The agent as in claims 13 or 14, wherein the agent is selected
from a biologic agent and a chemical compound.
16. The agent as in claims 13 or 14, wherein the agent is a
anti-GDF antibody or epitope binding fragment thereof.
17. The agent of claim 16, wherein the antibody is a monoclonal
antibody or a polyclonal antibody.
18. A method for inhibiting GDF binding to a GDF receptor
comprising contacting GDF-receptor with an anti-GDF-antibody.
19. The method of claim 18, wherein the contacting is by in vivo
administration to a subject.
20. The method of claim 19, wherein the anti-GDF-antibody is
administered by intravenous, intramuscular or subcutaneous
injections.
21. The method of claim 20, wherein the anti-GDF-antibody is
administered within a dose range of 0.1 ug/kg to 100 mg/kg.
22. The method of claim 20, wherein the antibody is formulated in a
pharmaceutically acceptable carrier.
23. A method for identifying a GDF receptor polypeptide comprising:
a) incubating components comprising GDF polypeptide and a cell
expressing a receptor or a soluble receptor under conditions
sufficient to allow the GDF to bind to the receptor; b) measuring
the binding of the GDF polypeptide to the receptor; and c)
isolating the receptor.
24. The method of claim 23, wherein the GDF is GDF-8 or GDF-11.
25. A method for identifying a compound which binds to GDF receptor
polypeptide comprising: a) incubating components comprising the
compound and GDF polypeptide under conditions sufficient to allow
the components to interact; and b) measuring the binding or effect
of binding of the compound to GDF receptor polypeptide.
26. The method of claim 25, wherein the compound is a peptide.
27. The method of claim 25, wherein the compound is a
peptidomimetic.
28. The method of claim 25, wherein the GDF receptor is expressed
in a cell.
29. The method of claim 28, wherein the cell is the cell of claim
1.
30. The method of claim 25, wherein measuring the ability of the
compound to bind to GDF receptor is by detection of a reporter
means.
31. The method of claim 30, wherein the reporter means is selected
from the group consisting of a radioisotope, a fluorescent
compound, a bioluminescent compound, a chemiluminescent compound, a
metal chelator, or an enzyme.
32. A transgenic non-human animal having a phenotype characterized
by expression of GDF-receptor polypeptide, the phenotype being
conferred by a transgene contained in the somatic and germ cells of
the animal, the transgene comprising a nucleic acid sequence which
encodes GDF-receptor polypeptide.
33. The transgenic non-human animal of claim 32, wherein the animal
is selected from the group of species consisting of avian, bovine,
ovine, piscine, murine, and porcine.
34. A method for producing a transgenic non-human animal having a
phenotype characterized by expression of GDF-receptor polypeptide
otherwise not naturally occurring in the animal, the method
comprising: (a) introducing at least one transgene into a zygote of
an animal, the transgene(s) comprising a DNA construct encoding
GDF-receptor, (b) transplanting the zygote into a pseudopregnant
animal, (c) allowing the zygote to develop to term, and (d)
identifying at least one transgenic offspring containing the
transgene.
35. The method of claim 34, wherein the introducing of the
transgene into the embryo is by introducing an embryonic stem cell
containing the transgene into the embryo.
36. The method of claim 34, wherein the introducing of the
transgene into the embryo is by infecting the embryo with a
retrovirus containing the transgene.
37. A transgenic non-human animal having a transgene disrupting or
interfering with expression of GDF-receptor chromosomally
integrated into the germ cells of the animal.
38. The transgenic non-human animal of claim 37, wherein the
transgene comprises GDF-receptor antisense polynucleotide.
39. A method for inhibiting the expression of GDF-receptor in a
cell comprising contacting GDF-receptor with an inhibiting
effective amount of an antisense oligonucleotide that binds to a
segment of an mRNA transcribed from a GDF-receptor gene, whereby
the binding of the antisense to the mRNA segment inhibits
GDF-receptor expression.
40. Substantially pure GDF receptor polypeptide.
41. The GDF receptor of claim 40, wherein GDF is GDF-8 or GDF-11.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to ligand-receptor
interactions and more specifically to growth differentiation factor
receptor proteins and the ligands that bind to such receptors and
methods of use therefor.
DESCRIPTION OF RELATED ART
[0002] 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).
[0003] 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.
[0004] The study of receptor-ligand interactions has revealed a
great deal of information about how cells respond to external
stimuli. This knowledge has led to the development of
therapeutically important compounds, such as erythropoietin, colony
stimulating factors and PDGF.
SUMMARY OF THE INVENTION
[0005] The present invention provides receptors for the growth
differentiation factor (GDF) growth factor family. These receptors
are useful for identifying antagonists and agonists for
agricultural and human therapeutic purposes.
[0006] In a first embodiment, the invention provides a recombinant
cell line that expresses growth differentiation factor-8 (GDF-8) or
growth differentiation factor-11 (GDF-11) receptor polypeptide.
Also included are antibodies that bind to GDF receptors,
polynucleotides encoding the receptors and the GDF receptor
proteins themselves.
[0007] Peptide fragments of GDF receptors, such as the GDF-8 or
GDF-11 receptors, are also included. Such peptides may be useful in
inhibiting binding of GDF-8 or GDF-11 to either its own receptor or
another GDF-receptor (e.g., GDF-8 and -11 may bind the same
receptor).
[0008] In another embodiment, the invention provides a
substantially purified GDF-8-binding agent, wherein the binding
agent inhibits GDF-8 binding to GDF-8 receptor. Such agents that
inhibit GDF-11 binding are also included.
[0009] In yet another embodiment, the invention provides a method
for identifying a GDF receptor polypeptide including incubating
components such as GDF polypeptide and a cell expressing a receptor
or a soluble receptor under conditions sufficient to allow the GDF
to bind to the receptor; measuring the binding of the GDF
polypeptide to the receptor; and isolating the receptor.
[0010] The invention also includes a method for identifying a
compound that binds to GDF receptor polypeptide including
incubating components comprising the compound and GDF polypeptide
under conditions sufficient to allow the components to interact and
measuring the binding or effect of binding of the compound to GDF
receptor polypeptide.
[0011] The invention also provides non-human transgenic animals
that have a phenotype characterized by expression of GDF-receptor
polypeptide, the phenotype being conferred by a transgene contained
in the somatic and germ cells of the animal, the transgene
comprising a nucleic acid sequence which encodes GDF-receptor
polypeptide. Methods of producing such transgenic animals are also
included.
[0012] In another embodiment, the invention includes a method for
inhibiting the expression of GDF-receptor in a cell including
contacting GDF-receptor with an inhibiting effective amount of an
antisense oligonucleotide that binds to a segment of an mRNA
transcribed from a GDF-receptor gene, whereby the binding of the
antisense to the mRNA segment inhibits GDF-receptor expression.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1a and 1b are the nucleotide and amino acid sequence of
murine GDF-8.
[0014] FIG. 1c and Id are the nucleotide and amino acid sequence of
human GDF-8.
[0015] FIGS. 2a-2e are the nucleotide and amino acid sequence of
baboon, bovine, chicken, rat, and turkey GDF-8.
[0016] FIGS. 3a and 3b are Northern blots showing expression of
GDF-8 in muscle and in various species, respectively.
[0017] FIGS. 4a and 4b show the nucleotide and amino acid sequence
of murine GDF-11 and expression of GDF-11, respectively.
[0018] FIG. 5 shows an autoradiogram showing GDF-8.
[0019] FIGS. 6 and 7 show binding studies for GDF-8.
[0020] FIGS. 8-11 show 4 myoblast cell lines that do not bind
GDF-8.
[0021] FIG. 12 shows the construction of GDF-11 null mice by
homologous targeting. a) is a map of the GDF-11 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 targeting construct contains a total of 11 kb of
homology with the GDF-11 gene. A probe derived from the region
upstream of the 3' homology fragment and downstream of the first
EcoRI site shown hybridizes to a 6.5 kb EcoR1 fragment in the
GDF-11 gene and a 4.8 kb fragment in a homologously targeted gene.
Abbreviations: X, Xba1; E, EcoR1. b) Geneomic Southern of DNA
prepared from F1 heterozygous mutant mice (lanes 1 and 2) and
offspring derived from a mating of these mice (lanes 3-12).
[0022] FIG. 13 shows kidney abnormalities in GDF-11 knockout mice.
Kidneys of newborn animals were examined and classified according
to the number of normal sized or small kidneys as shown at the top.
Numbers in the table indicate number of animals falling into each
classification according to genotype.
[0023] FIG. 14 shows homeotic transformations in GDF-11 mutant
mice. a) Newborn pups with missing (first and second from left) and
normal looking tails. b-j) Skeleton preparations for newborn
wild-type (b, e, h), heterozygous (c, f, I) and homozygous (d, g,
j) mutant mice. Whole skeleton preparations (b-d), vertebral
columns (e-g), vertebrosternal ribs (h-j) showing transformations
and defects in homozygous and heterozygous mutant mice. Numbers
indicate thoracic segments.
[0024] FIG. 15 is a table summarizing anterior transformations in
wild-type, heterozygous and homozygous GDF-11 mice.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention provides an isolated polynucleotide sequence
encoding the receptors of the invention. The term "isolated" as
used herein includes polynucleotides substantially free of other
nucleic acids, proteins, lipids, carbohydrates or other materials
with which it is naturally associated. Polynucleotide sequences of
the invention include DNA, cDNA and RNA sequences which encode GDF
receptors. It is understood that all polynucleotides encoding all
or a portion of GDF receptors are also included herein, as long as
they encode a polypeptide with GDF receptors activity (e.g., bind
to GDF). Such polynucleotides include naturally occurring,
synthetic, and intentionally manipulated polynucleotides. For
example, portions of the mRNA sequence may be altered due to
alternate RNA splicing patterns or the use of alternate promoters
for RNA transcription. As another example, GDF receptor
polynucleotide may be subjected to site-directed mutagenesis. The
polynucleotide sequence for GDF receptors 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
receptors polypeptide encoded by the nucleotide sequence is
functionally unchanged. Also included are nucleotide sequences
which encode GDF receptors polypeptide.
[0026] The polynucleotide encoding GDF receptors for GDFs such as
GDF-8 or 11 (shown in the figures). When the sequence is RNA, the
deoxyribonucleotides A, G, C, and T are replaced by ribonucleotides
A, G, C, and U, respectively. Also included in the invention are
fragments (portions) of the above-described nucleic acid sequences
that are at least 15 bases in length, which is sufficient to permit
the fragment to selectively hybridize to DNA that encodes the GDF
receptor. "Selective hybridization" as used herein refers to
hybridization under moderately stringent or highly stringent
physiological conditions (See, for example, the techniques
described in Maniatis et al., 1989 Molecular Cloning A Laboratory
Manual, Cold Spring Harbor Laboratory, N.Y., incorporated herein by
reference), which distinguishes related from unrelated nucleotide
sequences.
[0027] 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.
[0028] 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.degree. 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.
[0029] Specifically disclosed herein is a cDNA sequence for GDF
receptors. SEQ ID NO:3 represents the wild-type sequence and SEQ ID
NO:1 represents a cDNA which encodes GDF receptors having a
conservative substitution of Leucine for Alanine at amino acid
residue 127. The result of this conservative variation should not
affect biological activity of GDF receptors polypetide or peptides
containing the variation (see Example 5).
[0030] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
or computer-based 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) antibody screening of expression libraries to detect
cloned DNA fragments with shared structural features; 3) polymerase
chain reaction (PCR) on genomic DNA or cDNA using primers capable
of annealing to the DNA sequence of interest; 4) computer searches
of sequence databases for similar sequences; and 5) differential
screening of a subtracted DNA library.
[0031] Preferably the GDF receptor polynucleotide of the invention
is derived from avian, bovine, ovine, piscine, murine, human or
porcine. 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.
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., 2:879,
1981). Alternatively, a subtractive library, as illustrated herein
is useful for elimination of non-specific cDNA clones.
[0032] 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).
[0033] A cDNA expression library, such as lambda gt11, can be
screened indirectly for GDF receptors peptides having at least one
epitope, using antibodies specific for GDF receptors. Such
antibodies can be either polyclonally or monoclonally derived and
used to detect expression product indicative of the presence of GDF
receptors cDNA.
[0034] Alterations in GDF receptors nucleic acid include intragenic
mutations (e.g., point mutation, nonsense (stop), missense, splice
site and frameshift) and heterozygous or homozygous deletions.
Detection of such alterations can be done by standard methods known
to those of skill in the art including sequence analysis, Southern
blot analysis, PCR based analyses (e.g., multiplex PCR, sequence
tagged sites (STSs)) and in situ hybridization. Such proteins can
be analyzed by standard SDS-PAGE and/or immunoprecipitation
analysis and/or Western blot analysis, for example.
[0035] DNA sequences encoding GDF receptors 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.
[0036] In the present invention, the GDF receptor 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 receptors 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 I, or polyhedrin
promoters).
[0037] Polynucleotide sequences encoding GDF receptors 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 functional
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.
[0038] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the GDF
receptors coding sequence and appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques,
and in vivo recombination/genetic techniques. (See, for example,
the techniques described in Maniatis et al., 1989 Molecular Cloning
A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.)
[0039] A variety of host-expression vector systems may be utilized
to express the GDF receptors coding sequence. These include but are
not limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing the GDF receptors coding sequence; yeast
transformed with recombinant yeast expression vectors containing
the GDF receptors coding sequence; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing the GDF receptors coding sequence; insect cell systems
infected with recombinant virus expression vectors (e.g.,
baculovirus) containing the GDF receptors coding sequence; or
animal cell systems infected with recombinant virus expression
vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing
the GDF receptors coding sequence, or transformed animal cell
systems engineered for stable expression. Since GDF receptors has
not been confirmed to contain carbohydrates, both bacterial
expression systems as well as those that provide for translational
and post-translational modifications may be used; e.g., mammalian,
insect, yeast or plant expression systems.
[0040] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation elements,
including constitutive and inducible promoters, transcription
enhancer elements, transcription terminators, etc. may be used in
the expression vector (see e.g., Bitter et al., 1987, Methods in
Enzymology 153:516-544). For example, when cloning in bacterial
systems, inducible promoters such as pL of bacteriophage .gamma.,
plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be
used. When cloning in mammalian cell systems, promoters derived
from the genome of mammalian cells (e.g., metallothionein promoter)
or from mammalian viruses (e.g., the retrovirus long terminal
repeat; the adenovirus late promoter; the vaccinia virus 7.5K
promoter) may be used. Promoters produced by recombinant DNA or
synthetic techniques may also be used to provide for transcription
of the inserted GDF receptors coding sequence.
[0041] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al.,
Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et
al., 1987, Expression and Secretion Vectors for Yeast, in Methods
in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y.,
Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL
Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene
Expression in Yeast, Methods in Enzymology, Eds. Berger &
Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular
Biology of the Yeast Saccharomyces, 1982, Eds. Strathem et al.,
Cold Spring Harbor Press, Vols. I and II. A constitutive yeast
promoter such as ADH or LEU2 or an inducible promoter such as GAL
may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning
Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press,
Wash., D.C.). Alternatively, vectors may be used which promote
integration of foreign DNA sequences into the yeast chromosome.
[0042] Eukaryotic systems, and preferably mammalian expression
systems, allow for proper post-translational modifications of
expressed mammalian proteins to occur. Eukaryotic cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, phosphorylation, and advantageously,
plasma membrane insertion of the gene product may be used as host
cells for the expression of GDF receptors.
[0043] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the GDF receptors coding
sequence may be ligated to an adenovirus transcription/-translation
control complex, e.g., the late promoter and tripartite leader
sequence. Alternatively, the vaccinia virus 7.5K promoter may be
used. (e.g., see, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA
79: 7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864;
Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79: 4927-4931).
Of particular interest are vectors based on bovine papilloma virus
which have the ability to replicate as extrachromosomal elements
(Sarver, et al., 1981, Mol. Cell. Biol. 1: 486). Shortly after
entry of this DNA into mouse cells, the plasmid replicates to about
100 to 200 copies per cell. Transcription of the inserted cDNA does
not require integration of the plasmid into the host's chromosome,
thereby yielding a high level of expression. These vectors can be
used for stable expression by including a selectable marker in the
plasmid, such as, for example, the neo gene. Alternatively, the
retroviral genome can be modified for use as a vector capable of
introducing and directing the expression of the GDF receptors gene
in host cells (Cone & Mulligan, 1984, Proc. Natl. Acad. Sci.
USA 81:6349-6353). High level expression may also be achieved using
inducible promoters, including, but not limited to, the
metallothionine IIA promoter and heat shock promoters.
[0044] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with the GDF receptors cDNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. The selectable marker in the recombinant
plasmid confers resistance to the selection and allows cells to
stably integrate the plasmid into their chromosomes and grow to
form foci which in turn can be cloned and expanded into cell lines.
For example, following the introduction of foreign DNA, engineered
cells may be allowed to grow for 1-2 days in an enriched media, and
then are switched to a selective media. A number of selection
systems may be used, including but not limited to the herpes
simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:
223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska
& Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and
adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:
817) genes can be employed in tk-, hgprt.sup.- or aprt.sup.- cells
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., 1980, Natl Acad. Sci. USA 77: 3567;
O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78: 2072; neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al.,
1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance to
hygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Recently,
additional selectable genes have been described, namely trpB, which
allows cells to utilize indole in place of tryptophan; hisD, which
allows cells to utilize histinol in place of histidine (Hartman
& Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85: 8047); and ODC
(ornithine decarboxylase) which confers resistance to the omithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-omithine, DFMO
(McConlogue L., 1987, In: Current Communications in Molecular
Biology, Cold Spring Harbor Laboratory ed.).
[0045] 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 receptors 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).
[0046] Cell Lines
[0047] In one embodiment, the present invention relates to stable
recombinant cell lines, the cells of which express GDF receptor
polypeptides and contain DNA that encodes GDF receptors. Suitable
cell types include but are not limited to cells of the following
types: NIH 3T3 (Murine), C2C12, L6, and P19. C2C12 and L6 myoblasts
will differentiate spontaneously in culture and form myotubes
depending on the particular growth conditions (Yaffe and Saxel,
1977; Yaffe, 1968). P19 is an embryonal carcinoma cell line. Such
cells are described, for example, in the Cell Line Catalog of the
American Type Culture Collection (ATCC). These cells can be stably
transformed by a method known to the skilled artisan. See, for
example, Ausubel et al., Introduction of DNA Into Mammalian Cells,
in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, sections 9.5.1-9.5.6
(John Wiley & Sons, Inc. 1995). "Stable" transformation in the
context of the invention means that the cells are immortal to the
extent of having gone through at least 50 divisions.
[0048] GDF receptors can be expressed using inducible or
constituitive regulatory elements for such expression. Commonly
used constituitive or inducible promoters, for example, are known
in the art. The desired protein encoding sequence and an operably
linked promoter may be introduced into a recipient cell either as a
non-replicating DNA (or RNA) molecule, which may either be a linear
molecule or, more preferably, a closed covalent circular molecule.
Since such molecules are incapable of autonomous replication, the
expression of the desired molecule may occur through the transient
expression of the introduced sequence. Alternatively, permanent
expression may occur through the integration of the introduced
sequence into the host chromosome. Therefore the cells can be
transformed stably or transiently.
[0049] An example of a vector that may be employed is one which is
capable of integrating the desired gene sequences into the host
cell chromosome. Cells which have stably integrated the introduced
DNA into their chromosomes can be selected by also introducing one
or more markers which allow for selection of host cells which
contain the expression vector.
[0050] The marker may complement an auxotrophy in the host (such as
leu2, or ura3, which are common yeast auxotrophic markers), biocide
resistance, e.g., antibiotics, or heavy metals, such as copper, or
the like. The selectable marker gene can either be directly linked
to the DNA gene sequences to be expressed, or introduced into the
same cell by co-transfection.
[0051] In a preferred embodiment, the introduced sequence will be
incorporated into a plasmid or viral vector capable of autonomous
replication in the recipient host. Any of a wide variety of vectors
may be employed for this purpose. Factors of importance in
selecting a particular plasmid or viral vector include: the ease
with which recipient cells that contain the vector may be
recognized and selected from those recipient cells which do not
contain the vector; the number of copies of the vector which are
desired in a particular host; and whether it is desirable to be
able to "shuttle" the vector between host cells of different
species.
[0052] For a mammalian host, several possible vector systems are
available for expression. One class of vectors utilize DNA elements
which provide autonomously replicating extra-chromosomal plasmids,
derived from animal viruses such as bovine papilloma virus, polyoma
virus, adenovirus, or SV40 virus. A second class of vectors include
vaccinia virus expression vectors. A third class of vectors relies
upon the integration of the desired gene sequences into the host
chromosome. Cells which have stably integrated the introduced DNA
into their chromosomes may be selected by also introducing one or
more markers (e.g., an exogenous gene) which allow selection of
host cells which contain the expression vector. The marker may
provide for prototropy to an auxotrophic host, biocide resistance,
e.g., antibiotics, or heavy metals, such as copper or the like. The
selectable marker gene can either be directly linked to the DNA
sequences to be expressed, or introduced into the same cell by
co-transformation. Additional elements may also be needed for
optimal synthesis of mRNA. These elements may include splice
signals, as well as transcription promoters, enhancers, and
termination signals. The cDNA expression vectors incorporating such
elements include those described by Okayama, H., Mol. Cell. Biol.,
3:280 (1983), and others.
[0053] Once the vector or DNA sequence containing the construct has
been prepared for expression, the DNA construct may be introduced
(transformed) into an appropriate host. Various techniques may be
employed, such as protoplast fusion, calcium phosphate
precipitation, electroporation or other conventional
techniques.
[0054] Transgenic Animals
[0055] In another embodiment, the present invention relates to
transgenic animals having cells that express GDF receptors. Such
transgenic animals, for example those containing the GDF-8
receptor, may have decreased fat content and increased muscle mass.
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 may be at "normal"
levels, however, the GDF-8 receptor is produced in reduced amounts,
or is completely disrupted, resulting in animals with decreased
binding of GDF-8 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.
[0056] 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
receptor protein. In a particular embodiment, the transgene
comprises antisense polynucleotide sequences to the GDF-8 receptor
protein. Alternatively, the transgene may comprise a non-functional
sequence which replaces or intervenes in the native GDF-8 receptor
gene or the transgene may encode a GDF-8 receptor antagonist.
[0057] 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
[0058] The term "animal" here denotes all mammalian species except
human. It also includes an individual animal in all stages of
development, including embryonic and fetal stages. Farm animals
(pigs, goats, sheep, cows, horses, rabbits and the like), rodents
(such as mice), and domestic pets (for example, cats and dogs) are
included within the scope of the present invention.
[0059] A "transgenic" animal is any animal containing cells that
bear genetic information received, directly or indirectly, by
deliberate genetic manipulation at the subcellular level, such as
by microinjection or infection with recombinant virus. "Transgenic"
in the present context does not encompass classical crossbreeding
or in vitro fertilization, but rather denotes animals in which one
or more cells receive a recombinant DNA molecule. Although it is
highly preferred that this molecule be integrated within the
animal's chromosomes, the present invention also contemplates the
use of extrachromosomally replicating DNA, sequences, such as might
be engineered into yeast artificial chromosomes.
[0060] The term "transgenic animal" also includes a "germ cell
line" transgenic animal. A germ cell line transgenic animal is a
transgenic animal in which the genetic information has been taken
up and incorporated into a germ line cell, therefore conferring the
ability to transfer the information to offspring. If such offspring
in fact possess some or all of that information, then they, too,
are transgenic animals.
[0061] The cDNA that encodes GDF receptors can be fused in proper
reading frame under the transcriptional and translational control
of a vector to produce a genetic construct that is then amplified,
for example, by preparation in a bacterial vector, according to
conventional methods. See, for example, the standard work: Sambrook
et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor
Press 1989), the contents of which are incorporated by reference.
The amplified construct is thereafter excised from the vector and
purified for use in producing transgenic animals.
[0062] 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."
[0063] The transgene to be used in the practice of the subject
invention may be a DNA sequence comprising a modified GDF receptors
coding sequence. In a preferred embodiment, the GDF receptor gene
is disrupted by homologous targeting in embryonic stem cells. For
example, the entire mature C-terminal region of the GDF receptors
gene may be deleted as described in the examples below. Optionally,
the GDF receptors disruption or deletion may be accompanied by
insertion of or replacement with other DNA sequences, such as a
non-functional GDF receptors sequence. In other embodiments, the
transgene comprises DNA antisense to the coding sequence for GDF
receptors. In another embodiment, the transgene comprises DNA
encoding an antibody or receptor peptide sequence which is able to
bind to GDF receptors. Where appropriate, DNA sequences that encode
proteins having GDF receptors 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.
[0064] Antibodies which Bind to GDF Receptors
[0065] In another embodiment, the present invention relates to
antibodies that bind GDF receptors that block GDF binding to the
receptor. For example, such antibodies may be useful for
ameliorating disorders associated with muscle tissue.
[0066] A monoclonal antibody which binds to GDF-8 receptor may have
the effect of increasing the development of skeletal muscles. In
preferred embodiments of the claimed methods, the GDF-8 receptor
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 receptor antibody 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 antibody 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 antibody
may be formulated in a formulation suitable for administration to a
patient. Such formulations are known in the art.
[0067] The dosage regimen will be determined by the attending
physician considering various factors which modify the action of
the GDF-8 receptor 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 receptor 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 receptor 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.
[0068] The preparation of polyclonal antibodies is well-known to
those skilled in the art. See, for example, Green et al.,
Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS
(Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al.,
Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992),
which are hereby incorporated by reference.
[0069] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature
256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et
al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor
Pub. 1988), which are hereby incorporated by reference. Briefly,
monoclonal antibodies can be obtained by injecting mice with a
composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B lymphocytes, fusing the B lymphocytes with myeloma
cells to produce hybridomas, cloning the hybridomas, selecting
positive clones that produce antibodies to the antigen, and
isolating the antibodies from the hybridoma cultures. Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections
2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG),
in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana
Press 1992). Methods of in vitro and in vivo multiplication of
monoclonal antibodies is well-known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo may be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, e.g., osyngeneic mice, to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane (tetramethylpentadecane) prior to
injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
[0070] Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of the
present invention may also be derived from subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in Goldenberg et
al., International Patent Publication WO 91/11465 (1991) and Losman
et al., Int. J. Cancer 46:310 (1990), which are hereby incorporated
by reference.
[0071] Alternatively, a therapeutically useful anti-GDF receptors
antibody may be derived from a "humanized" monoclonal antibody.
Humanized monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then substituting human residues in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989), which is hereby incorporated in its
entirety by reference. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988);
Verhoeyen et al., Science 239: 1534 (1988); Carter et al., Proc.
Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech.
12: 437 (1992); and Singer et al., J. Immunol. 150: 2844 (1993),
which are hereby incorporated by reference.
[0072] Antibodies of the invention also may be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., METHODS: A COMPANION TO
METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann.
Rev. Immunol. 12: 433 (1994), which are hereby incorporated by
reference. Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
[0073] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet. 7:13 (1994); Lonberg et
al., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579
(1994), which are hereby incorporated by reference.
[0074] Antibody fragments of the present invention can be prepared
by proteolytic hydrolysis of the antibody or by expression in E.
coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies by
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fe fragment directly. These
methods are described, for example, by Goldenberg, U.S. Pat. No.
4,036,945 and No. 4,331,647, and references contained therein.
These patents are hereby incorporated in their entireties by
reference. See also Nisonhoff et al., Arch. Biochem. Biophys.
89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman et al.,
METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and
Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
[0075] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0076] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv
fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as E
coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are described, for example, by Whitlow et al.,
METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97
(1991); Bird et al., Science 242:423-426 (1988); Ladner et al, U.S.
Pat. No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993);
and Sandhu, supra.
[0077] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al, METHODS: A COMPANION TO
METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).
[0078] Identification of GDF Receptors
[0079] In another embodiment, the invention provides a method for
identifying a GDF receptor polypeptide comprising incubating
components comprising GDF polypeptide and a cell expressing a
receptor or a soluble receptor under conditions sufficient to allow
the GDF to bind to the receptor; measuring the binding of the GDF
polypeptide to the receptor; and isolating the receptor. The GDF
may be any of the known GDFs (e.g., GDF-1-16), and preferably is
GDF-8 or GDF-I 1. Methods of isolating the receptors are described
in more detail in the Examples section below.
[0080] Variants of GDF Receptors
[0081] The term "GDF receptors variant" as used herein means a
molecule that simulates at least part of the structure of GDF
receptors. GDF receptor variants may also be useful in preventing
GDF binding, thereby ameliorating symptoms of disorders described
above.
[0082] In one embodiment, the present invention relates to peptides
and peptide derivatives that have fewer amino acid residues than
GDF receptors. Such peptides and peptide derivatives could
represent research and diagnostic tools in the study of muscle
wasting diseases and the development of more effective
therapeutics.
[0083] The invention relates not only to peptides and peptide
derivatives of naturally-occurring GDF receptors, but also to GDF
receptor mutants and chemically synthesized derivatives of GDF
receptors that bind GDFs. For example, changes in the amino acid
sequence of GDF receptors are contemplated in the present
invention. GDF receptors can be altered by changing the DNA
encoding the protein. Preferably, only conservative amino acid
alterations are undertaken, using amino acids that have the same or
similar properties. Illustrative amino acid substitutions include
the changes of: alanine to serine; arginine to lysine; asparagine
to glutamine or histidine; aspartate to glutamate; cysteine to
serine; glutamine to asparagine; glutamate to aspartate; glycine to
proline; histidine to asparagine or glutamine; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to
arginine, glutamine, or glutamate; methionine to leucine or
isoleucine; phenylalanine to tyrosine, leucine or methionine;
serine to threonine; threonine to serine; tryptophan to tyrosine;
tyrosine to tryptophan or phenylalanine; valine to isoleucine or
leucine.
[0084] Variants useful for the present invention comprise analogs,
homologs, muteins and mimetics of GDF receptors that retain the
ability to bind to their respective GDFs. Peptides of the GDF
receptors refer to portions of the amino acid sequence of GDF
receptors that also retain this ability. The variants can be
generated directly from GDF receptors itself by chemical
modification, by proteolytic enzyme digestion, or by combinations
thereof. Additionally, genetic engineering techniques, as well as
methods of synthesizing polypeptides directly from amino acid
residues, can be employed.
[0085] Peptides of the invention can be synthesized by such
commonly used methods as t-BOC or FMOC protection of alpha-amino
groups. Both methods involve stepwise syntheses whereby a single
amino acid is added at each step starting from the C terminus of
the peptide (See, Coligan, et al., Current Protocols in Immunology,
Wiley Interscience, 1991, Unit 9). Peptides of the invention can
also be synthesized by the well known solid phase peptide synthesis
methods described Merrifield, J. Am. Chem. Soc., 85:2149, 1962),
and Stewart and Young, Solid Phase Peptides Synthesis, (Freeman,
San Francisco, 1969, pp.27-62), using a copoly(styrene-divinylb-
enzene) containing 0.1-1.0 mMol amines/g polymer. On completion of
chemical synthesis, the peptides can be deprotected and cleaved
from the polymer by treatment with liquid HF-10% anisole for about
1/4-1 hours at 0.degree. C. After evaporation of the reagents, the
peptides are extracted from the polymer with 1% acetic acid
solution which is then lyophilized to yield the crude material.
This can normally be purified by such techniques as gel filtration
on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization
of appropriate fractions of the column will yield the homogeneous
peptide or peptide derivatives, which can then be characterized by
such standard techniques as amino acid analysis, thin layer
chromatography, high performance liquid chromatography, ultraviolet
absorption spectroscopy, molar rotation, solubility, and
quantitated by the solid phase Edman degradation.
[0086] Alternatively, peptides can be produced by recombinant
methods as described below.
[0087] The term "substantially purified" as used herein refers to a
molecule, such as a peptide that is substantially free of other
proteins, lipids, carbohydrates, nucleic acids, and other
biological materials with which it is naturally associated. For
example, a substantially pure molecule, such as a polypeptide, can
be at least 60%, by dry weight, the molecule of interest. One
skilled in the art can purify GDF receptors peptides using standard
protein purification methods and the purity of the polypeptides can
be determined using standard methods including, e.g.,
polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column
chromatography (e.g., high performance liquid chromatography
(HPLC)), and amino-terminal amino acid sequence analysis.
[0088] Non-peptide compounds that mimic the binding and function of
GDF receptors ("mimetics") can be produced by the approach outlined
in Saragovi et al., Science 253: 792-95 (1991). Mimetics are
molecules which mimic elements of protein secondary structure. See,
for example, Johnson et al., "Peptide Turn Mimetics," in
BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., (Chapman and
Hall, New York 1993). The underlying rationale behind the use of
peptide mimetics is that the peptide backbone of proteins exists
chiefly to orient amino acid side chains in such a way as to
facilitate molecular interactions. For the purposes of the present
invention, appropriate mimetics can be considered to be the
equivalent of GDF receptors itself.
[0089] Longer peptides can be produced by the "native chemical"
ligation technique which links together peptides (Dawson, et al.,
Science, 266:776, 1994). Variants can be created by recombinant
techniques employing genomic or cDNA cloning methods. Site-specific
and region-directed mutagenesis techniques can be employed. See
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY vol. 1, ch. 8 (Ausubel et
al. eds., J. Wiley & Sons 1989 & Supp. 1990-93); PROTEIN
ENGINEERING (Oxender & Fox eds., A. Liss, Inc. 1987). In
addition, linker-scanning and PCR-mediated techniques can be
employed for mutagenesis. See PCR TECHNOLOGY (Erlich ed., Stockton
Press 1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 &
2, supra. Protein sequencing, structure and modeling approaches for
use with any of the above techniques are disclosed in PROTEIN
ENGINEERING, loc. cit., and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
vols. 1 & 2, supra.
[0090] GDF Receptor-Binding and Blocking Agents
[0091] In yet another embodiment, the present invention relates to
GDF receptor-binding agents that block binding of GDFs to their
receptors. Such agents could represent research and diagnostic
tools in the study of muscle wasting disorder as described above
and the development of more effective therapeutics. In addition,
pharmaceutical compositions comprising GDF receptor-binding agents
may represent effective therapeutics. In the context of the
invention, the phrase "GDF receptor-binding agent" denotes a
naturally occurring ligand of GDF receptors such as, for example:
GDF-1-16; a synthetic ligand of GDF receptors, or appropriate
derivatives of the natural or synthetic ligands. The determination
and isolation of ligands is well described in the art. See, e.g.,
Lerner, Trends NeuroSci. 17:142-146 (1994) which is hereby
incorporated in its entirety by reference.
[0092] In yet another embodiment, the present invention relates to
GDF receptor-binding agents that interfere with binding between GDF
receptor and a GDF. Such binding agents may interfere by
competitive inhibition, by non-competitive inhibition or by
uncompetitive inhibition. Interference with normal binding between
GDF receptors and one or more GDF can result in a useful
pharmacological effect.
[0093] Screen for Binding and Blocking Compositions
[0094] In another embodiment, the invention provides a method for
identifying a composition which binds to GDF receptors. The method
includes incubating components comprising the composition and GDF
receptors under conditions sufficient to allow the components to
interact and measuring the binding of the composition to GDF
receptors. Compositions that bind to GDF receptors include
peptides, peptidomimetics, polypeptides, chemical compounds and
biologic agents as described above.
[0095] Incubating includes conditions which allow contact between
the test composition and GDF receptors. Contacting includes in
solution and in solid phase. The test ligand(s)/composition may
optionally be a combinatorial library for screening a plurality of
compositions. Compositions identified in the method of the
invention can be further evaluated, detected, cloned, sequenced,
and the like, either in solution or after binding to a solid
support, by any method usually applied to the detection of a
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 ligation assays
(OLAs) (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).
[0096] To determine if a composition can functionally complex with
the receptor protein, induction of the exogenous gene is monitored
by monitoring changes in the protein levels of the protein encoded
for by the exogenous gene, for example. When a composition(s) is
found that can induce transcription of the exogenous gene, it is
concluded that this composition(s) can bind to the receptor protein
coded for by the nucleic acid encoding the initial sample test
composition(s).
[0097] Expression of the exogenous gene can be monitored by a
functional assay or assay for a protein product, for example. The
exogenous gene is therefore a gene which will provide an
assayable/measurable expression product in order to allow detection
of expression of the exogenous gene. Such exogenous genes include,
but are not limited to, reporter genes such as chloramphenicol
acetyltransferase gene, an alkaline phosphatase gene,
beta-galactosidase, a luciferase gene, a green fluorescent protein
gene, guanine xanthine phosphoribosyltransferase, alkaline
phosphatase, and antibiotic resistance genes (e.g., neomycin
phosphotransferase).
[0098] Expression of the exogenous gene is indicative of
composition-receptor binding, thus, the binding or blocking
composition can be identified and isolated. The compositions of the
present invention can be extracted and purified from the culture
media or a cell by using known protein purification techniques
commonly employed, such as extraction, precipitation, ion exchange
chromatography, affinity chromatography, gel filtration and the
like. Compositions can be isolated by affinity chromatography using
the modified receptor protein extracellular domain bound to a
column matrix or by heparin chromatography.
[0099] Also included in the screening method of the invention is
combinatorial chemistry methods for identifying chemical compounds
that bind to GDF receptors. Thus, the screening method is also
useful for identifying variants, binding or blocking agents, etc.,
which functionally, if not physically (e.g., sterically) act as
antagonists or agonists, as desired.
EXAMPLES
[0100] Distribution of Receptors for GDF-8 and GDF-11.
[0101] The purified GDF-8 and GDF-11 proteins will be used
primarily to assay for biological activities. In order to identify
potential target cells for GDF-8 and GDF-11 action cells expressing
their receptors will be searched. For this purpose, the purified
protein will be radioiodinated using the chloramine T method, which
has been used successfully to label other members of this
superfamily, like TGF-.beta. (Cheifetz et al., 1987), activins
(Sugino et al., 1988), and BMPs (Paralkar et al., 1991), for
receptor-binding studies. The mature processed forms of GDF-8 and
GDF-11 each contain multiple tyrosine residues. Two different
approaches will then be taken to attempt to identify receptors for
these proteins.
[0102] One approach will be taken to determine the number,
affinity, and distribution of receptors. Either whole cells grown
in culture, frozen sections of embryos or adult tissues, or total
membrane fractions prepared from tissues or cultured cells will be
incubated with the labeled protein, and the amount or distribution
of bound protein will be determined. For experiments involving cell
lines or membranes, the amount of binding will be determined by
measuring either the amount of radioactivity bound to cells on the
dish after several washes or, in the case of membranes, the amount
of radioactivity sedimented with the membranes after centrifugation
or retained with the membranes on a filter. For experiments
involving primary cultures, where the number of cells may be more
limited, binding sites will be visualized directly by overlaying
with photographic emulsion. For experiments involving frozen
sections, sites of ligand binding will be visualized by exposing
these sections to high resolution Beta-max hyperfilm; if finer
localization is required, the sections will be dipped in
photographic emulsion. For all of these experiments, specific
binding will be determined by adding excess unlabeled protein as
competitor (for example, see Lee and Nathans, 1988).
[0103] A second approach will also be taken to begin to
characterize the receptor biochemically. Membrane preparations or
potential target cells grown in culture will be incubated with
labeled ligand, and receptor/ligand complexes will be covalently
cross-linked using disuccinimidyl suberate, which has been commonly
used to identify receptors for a variety of ligands, including
members of the TGF-.beta. superfamily (for example, see Massague
and Like, 1985). Cross-inked complexes will then be electrophoresed
on SDS polyacrylamide gels to look for bands labeled in the absence
but not in the presence of excess unlabeled protein. The molecular
weight of the putative receptor will be estimated by subtracting
the molecular weight of the ligand. An important question that
these experiments will address is whether GDF-8 and GDF-11 signal
through type I and type II receptors like many other members of the
TGF-B superfamily (for review, see Massague, 1996).
[0104] Once a method for detecting receptors for these molecules
has been achieved, more detailed analysis will be carried out to
determine the binding affinities and specificities. A Scatchard
analysis will be used to determine the number of binding sites and
dissociation constants. By carrying out cross-competition analyses
between GDF-8 and GDF-11 (see FIGS. 1 and 2, respectively for
nucleotide and amino acid sequences), it will be possible to
determine whether they are capable of binding to the same receptor
and their relative affinities. These studies will be critical as
they will give an indication as to whether the molecules signal
through the same or different receptors. Competition experiments
using other TGF-B family members will be performed to determine
specificity. Some of these ligands are available commercially, and
some others are available from Genetics Institute, Inc.
[0105] For these experiments, a variety of embryonic and adult
tissues and cell lines will be tested. Based on the specific
expression of GDF-8 in skeletal muscle and the phenotype of GDF-8
knockout mice, initial studies focus on embryonic and adult muscle
tissue for membrane preparation and for receptor studies using
frozen sections. In addition, myoblasts will be isolated and
cultured from embryos at various days of gestation or satellite
cells from adult muscle as described (Vivarelli and Cossu, 1986;
Cossu et al., 1980). The binding studies on these primary cells
after various days in culture will be performed and binding sites
localized by autoradiography so that the binding sites can be
co-localized with various myogenic markers, such as muscle myosin
(Vivarelli et al., 1988), and correlate binding with the
differentiation state of the cells, such as formation of
multinucleated myotubes. In addition to using primary cells, cell
lines will be utilized to look for receptors. In particular, the
initial focus will be on three cells lines, C2C12, L6, and P19.
C2C12 and L6 myoblasts will differentiate spontaneously in culture
and form myotubes depending on the particular growth conditions
(Yaffe and Saxel, 1977; Yaffe, 1968). P19 embryonal carcinoma cells
can be induced to differentiate into various cell types, including
skeletal muscle cells in the presence of DMSO (Rudnicki and
McBurney, 1987). Receptor binding studies will be carried out on
these cell lines under various growth conditions and at various
stages of differentiation.
[0106] Although the initial studies will focus on muscle cells,
other tissues and cell types will be examined for the presence of
GDF-8 and GDF-11 receptors.
[0107] Recombinant human GDF-8 homodimer was used in these binding
studies. The rh-GDF-8 was expressed using CHO cells and purified to
approximately 90% purity. The autoradiograph (FIG. 5) shows that
the GDF-8 has the expected 25-27 KD molecular eight and upon
reduction is reduced to the 12 KD monomer. Using 1-125 labeled
GDF-8 in a receptor-ligand binding assay, two myoblast cell lines,
L6 and G-8, were found to bind GDF-8. The binding was specific
since non labeled GDF-8 effectively competed the binding of the
labeled ligand. These results are illustrated in FIGS. 6 and 7,
respectively. The dissociation constant (K.sub.d) is 370 pM and L6
myoblasts have a high number (5,000 receptors/cell) of cell surface
binding proteins (FIG. 6). GDF-11 (also called BMP-11) is highly
homologous (>90%) to GDF-8. Receptor binding studies were
performed to determine if GDF-11 also binds the GDF-8 receptor.
FIG. 6 shows that GDF-8 and GDF-11 do bind to the same binding
proteins on L6 myoblasts. It is important to establish whether or
not GDF-8 binds to the known TGF-.beta. receptor. As shown in FIG.
6, TGF-.beta. does not compete the binding of GDF-8, indicating
that the GDF-8 receptor is distinct from the TGF-.beta. receptor.
The GDF-8 receptor is not expressed on all myoblast cell lines.
FIGS. 8-11 are examples of four myoblast cell lines (C2C12, G7,
MLB13MYC c14 and BC3H1) which do not bind GDF-8.
[0108] Cloning the Gene or Genes Encoding Receptors for GDF-8 and
GDF-11.
[0109] As a first step towards understanding the mechanism by which
GDF-8 and GDF-11 exert their biological effects, it is important to
clone the genes encoding their receptors. From the experiments
above, it will be more clear as to whether GDF-8 and GDF-11 bind to
the same receptor or to different receptors. There will also be
considerable information regarding the tissue and cell type
distribution of these receptors. Using this information, two
different approaches will be taken to clone the receptor genes.
[0110] The first approach will be to use an expression cloning
strategy. In fact, this was the strategy that was orginally used by
Mathews and Vale (1991) and Lin et al. (1992) to clone the first
activin and TGF-1 receptors. We will begin by preparing poly
A-selected RNA from the tissue or cell type that expresses the
highest relative number of high affinity binding sites. We will
then use this RNA to prepare a cDNA library in the mammalian
expression vector pcDNA-1. This vector contains a CMV promoter and
an SV40 origin of replication. The library will be plated, and
cells from each plate will be pooled into broth and frozen.
Aliquots from each pool will then be grown for preparation of DNA.
Each individual pool will be transiently transfected into COS cells
in chamber slides, and transfected cells will be incubated with
iodinated GDF-8 or GDF-11. After washing away the unbound protein,
the sites of ligand binding will be visualized by autoradiography.
Once a positive pool is identified, the cells from that pool will
be replated at lower density, and the process will be repeated.
Positive pools will then be plated, and individual colonies will be
picked into grids and re-analyzed as described (Wong et al.,
1985).
[0111] We will attempt to carry out this screen initially using
pool sizes of 1500 colonies. In order to be certain that we will be
able to identify a positive clone in a mixture of this complexity,
we will carry out a control experiment using TGF-13 and a cloned
type II receptor. The coding sequence for the TGF-.beta. type II
receptor will be cloned into the pcDNA-1 vector, and bacteria
transformed with this construct will be mixed with bacteria from
our library at various ratios, including 1:1500. We will then
transfect DNA prepared from this mixture into COS cells, incubate
with iodinated TGF-.beta., and visualize by autoradiography. If we
can see positive signals at a ratio of 1:1500, we will begin
screening pools of 1500 clones. Otherwise, we will use smaller pool
sizes corresponding to ratios at which the procedure is sensitive
enough to identify a positive signal in our control experiments.
While we have no previous experience in expression cloning per se,
we have constructed over 50 cDNA libraries in the past, and many of
these have yielded a high frequency of full-length cDNA clones.
[0112] We will also use a second parallel strategy to attempt to
clone the GDF-8 and GDF-11 receptors. We will take advantage of the
fact that most receptors for members of the TGF-.beta. superfamily
that have been identified belong to the membrane-spanning
serine/threonine kinase family (for review, see Massague, 1996).
Because the cytoplasmic domains of these receptors are related in
sequence, we will attempt to use degenerate PCR to clone members of
this receptor family that are expressed in tissues that contain
binding sites for GDF-8 and GDF-11. In fact, this is the approach
that has been used to identify most of the members of this receptor
family. We have extensive experience using this type of strategy
for identifying ligands in this superfamily, and therefore, we are
quite confident that we will be able to carry out this approach
successfully. The general strategy will be to design degenerate
primers corresponding to conserved regions of the known receptors,
to use these primers for PCR on cDNA prepared from the appropriate
RNA samples (most likely from skeletal muscle), to subclone the PCR
products, and finally to sequence individual subclones. As
sequences are identified, they will be used as hybridization probes
to eliminate duplicate clones from further analysis. We will then
test the receptors that we identify for their ability to bind
purified GDF-8 and GDF-11. Because this screen will yield only
small PCR products, we will obtain full-length cDNA clones for each
receptor from cDNA libraries prepared from the appropriate tissue,
insert these cDNA clones into the pcDNA-1 vector, transfect these
constructs into COS cells, and assay the transfected cells for
their ability to bind iodinated GDF-8 or GDF-11. Ideally, we would
like to test every receptor that we identify in this screen for
their ability to bind these ligands. However, the number of
receptors that we identify may be large, and isolating all of the
full-length cDNAs and testing them may require considerable effort.
Almost certainly some of the receptors that we identify will
correspond to known receptors, and for these, either obtaining
full-length cDNA clones from other investigators or amplifying the
coding seqences by PCR based on the published sequences should be
straightforward. For novel sequences, we will determine their
tissue distribution by Northern analysis and then give the highest
priority to those receptors whose expression pattern most closely
resembles the distribution of GDF-8 and/or GDF-11 binding sites as
determined above.
[0113] In particular, it is known that these receptors fall into
two classes, type I and type II, which can be distinguished based
on the sequence and which are both required for full activity.
Certain ligands cannot bind type I receptors in the absence of type
II receptors while others are capable of binding both receptor
types (for review, see Massague, 1996). The cross-linking
experiments outlined above should give some indication as to
whether both type I and type II receptors are also involved in
signalling GDF-8 and GDF-11. If so, it will be important to clone
both of these receptor subtypes in order to fully understand how
GDF-8 and GDF-11 transmit their signals. Because we cannot predict
whether the type I receptor is capable of interacting with GDF-8
and GDF-11 in the absence of the type II receptor, we will focus
first on cloning the type II receptor(s). Only after we have at
least one type II receptor for these ligands in hand will we
attempt to identify the type I receptors for GDF-8 and GDF-11. Our
general strategy will be to co-transfect the type II receptor with
each of the type I receptors that we identify in the PCR screen and
then assay the transfected cells by crosslinking as described in
Specific Aim 4. If the type I receptor is part of the receptor
complex for GDF-8 or GDF-11, we should be able to detect two
cross-linked receptor species in the transfected cells, one
corresponding to the type I receptor and the other corresponding to
the type II receptor.
[0114] The search for GDF-8 and GDF-11 receptors is further
complicated by the fact at least one member of the TGF-.beta.
superfamily, namely, GDNF, is capable of signalling through a
completely different type of receptor complex involving a
GPI-linked component (GDNFR-alpha) and a receptor tyrosine kinase
(c-ret) (Trupp et al., 1996; Durbec et al., 1996; Treanor et al.,
1996; Jing et al., 1996). Although GDNF is the most
distantly-related member of the TGF-.beta. superfamily, it is
certainly possible that other TGF-.beta. family members may also
signal through an analogous receptor system. If GDF-8 and GDF-11 do
signal through a similar receptor complex, our expression screening
approach should be able to identify at least the GPI-linked
component (indeed GDNFR-alpha was identified using an expression
screening approach) of this complex. However, identifying the
analogous receptor tyrosine kinase would probably require a
substantial amount of additional work, such as biochemical
purification of the complex. In the case of GDNF, the similar
phenotypes of GDNF- and c-ret-deficient mice suggested c-ret as a
potential receptor for GDNF.
[0115] GDF-11 Transgenic Knockout Mice
[0116] The phenotype of GDF-11 knockout mice in several respects
resembles the phenotype of mice carrying a deletion of a receptor
for some members of the TGF-.beta. superfamily, the activin type
IIB receptor (ActRIIB). To determine the biological function of
GDF-11, we disrupted the GDF-11 gene by homologous targeting in
embryonic stem cells. A murine 129 SV/J genomic library was
prepared in lambda FIXII according to the instructions provided by
Stratagene (La Jolla, Calif.). The structure of the GDF-11 gene was
deduced from restriction mapping and partial sequencing of phage
clones isolated from the library. Vectors for preparing the
targeting construct were kindly provided by Philip Soriano and Kirk
Thomas. To ensure that the resulting mice would be null for GDF-11
function, the entire mature C-terminal region was deleted and
replaced by a neo cassette (FIGS. 12a,b). 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. Homologous targeting of the GDF-11 gene was seen in 8/155
gancyclovir/G418 doubly resistant ES cell clones. Following
injection of several targeted clones into C57BL/6J blastocysts, we
obtained chimeras from one ES clone that produced heterozygous pups
when crossed to both C57BL/6J and 129/SvJ females. Crosses of
C57BL/6J/129/SvJ hybrid F1 heterozygotes produced 49 wild-type
(34%), 94 heterozygous (66%) and no homozygous mutant adult
offspring. Similarly, there were no adult homozygous null animals
seen in the 129/SvJ background (32 wild-type (36%) and 56
heterozygous mutant (64%) animals).
[0117] To determine the age at which homozygous mutants were dying,
we genotyped litters of embryos isolated at various gestational
ages from heterozygous females that had been mated to heterozygous
males. At all embryonic stages examined, homozygous mutant embryos
were present at approximately the predicted frequency of 25%. Among
hybrid newborn mice, the different genotypes were also represented
at the expected Mendelian ratio of 1:2:1 (34+/+(28%), 61+/-(50%),
and 28-/-(23%)). Homozygous mutant mice were born alive and were
able to breath and nurse. All homozygous mutants died, however,
within the first 24 hours after birth. The precise cause of death
was unknown, but the lethality may have been related to the fact
that the kidneys in homozygous mutants were either severely
hypoplastic or completely absent. A summary of the kidney
abnormalities in these mice is shown in FIG. 13.
[0118] Anatomical Differences In Knockout Mice
[0119] Homozygous mutant animals were easily recognizable by their
severely shortened or absent tails (FIG. 14a). To further
characterize the tail defects in these homozygous mutant animals,
we examined their skeletons to determine the degree of disruption
of the caudal vertebrae. A comparison of wild-type and mutant
skeleton preparations of late stage embryos and newborn mice,
however, revealed differences not only in the caudal region of the
animals but in many other regions as well. In nearly every case
where differences were noted, the abnormalities appeared to
represent homeotic transformations of vertebral segments in which
particular segments appeared to have a morphology typical of more
anterior segments. These transformations, which are summarized in
FIG. 15, were evident throughout the axial skeleton extending from
the cervical region to the caudal region. Except for the defects
seen in the axial skeleton, the rest of the skeleton, such as the
cranium and limb bones, appeared normal.
[0120] Anterior transformations of the vertebrae in mutant newborn
animals were most readily apparent in the thoracic region, where
there was a dramatic increase in the number of thoracic (T)
segments. All wild-type mice examined showed the typical pattern of
13 thoracic vertebrae each with its associated pair of ribs (FIG.
14(b,e)). In contrast, homozygous mutant mice showed a striking
increase in the number of thoracic vertebrae. All homozygous
mutants examined had 4 to 5 extra pairs of ribs for a total of 17
to 18 (FIG. 14(d,g)) although in over 1/3 of these animals, the
18th rib appeared to be rudimentary. Hence, segments that would
normally correspond to lumbar (L) segments L1 to L.sub.4 or L5
appeared to have been transformed into thoracic segments in mutant
animals.
[0121] Moreover, transformations within the thoracic region in
which one thoracic vertebra had a morphology characteristic of
another thoracic vertebra were also evident. For example, in
wild-type mice, the first 7 pairs of ribs attach to the sternum,
and the remaining 6 are unattached or free (FIG. 14(e,h)). In
homozygous mutants, there was an increase in the number of both
attached and free pairs of ribs to 10-11 and 7-8, respectively
(FIG. 14(g,j)). Therefore, thoracic segments T8, T9, T10, and in
some cases even T11, which all have free ribs in wild-type animals,
were transformed in mutant animals to have a characteristic typical
of more anterior thoracic segments, namely, the presence of ribs
attached to the sternum. Consistent with this finding, the
transitional spinous process and transitional articular processes
which are normally found on T10 in wild-type animals were instead
found on T13 in homozygous mutants (data not shown). Additional
transformations within the thoracic region were also noted in
certain mutant animals. For example, in wild-type mice, the ribs
derived from T1 normally touch the top of the sternum. However, in
2/23 hybrid and 2/3 129/SvJ homozygous mutant mice examined, T2
appeared to have been transformed to have a morphology resembling
that of T1; that is, in these animals, the ribs derived from T2
extended to touch the top of the sternum. In these cases, the ribs
derived from T1 appeared to fuse to the second pair of ribs.
Finally, in 82% of homozygous mutants, the long spinous process
normally present on T2 was shifted to the position of T3. In
certain other homozygous mutants, asymmetric fusion of a pair of
vertebrosternal ribs was seen at other thoracic levels.
[0122] The anterior transformations were not restricted to the
thoracic region. The anterior most transformation that we observed
was at the level of the 6th cervical vertebra (C6). In wild-type
mice, C6 is readily identifiable by the presence of two anterior
tuberculi on the ventral side. In several homozygous mutant mice,
although one of these two anterior tuberculi was present on C6, the
other was present at the position of C7 instead. Hence, in these
mice, C7 appeared to have been partially transformed to have a
morphology resembling that of C6. One other homozygous mutant had 2
anterior tuberculi on C7 but retained one on C6 for a complete C7
to C6 transformation but a partial C6 to C5 transformation.
[0123] Transformations of the axial skeleton also extended into the
lumbar region. Whereas wild-type animals normally have only 6
lumbar vertebrae, homozygous mutants had 8-9. At least 6 of the
lumbar vertebrae in the mutants must have derived from segments
that would normally have given rise to sacral and caudal vertebrae
as the data described above suggest that 4 to 5 lumbar segments
were transformed into thoracic segments. Hence, homozygous mutant
mice had a total of 33-34 presacral vertebrae compared to 26
presacral vertebrae normally present in wild-type mice. The most
common presacral vertebral patterns were C7/T18/L8 and C7/T18/L9
for mutant mice compared to C7/T13/L6 for wild-type mice. The
presence of additional presacral vertebrae in mutant animals was
obvious even without detailed examination of the skeletons as the
position of the hindlimbs relative to the forelimbs was displaced
posteriorly by 7-8 segments.
[0124] Although the sacral and caudal vertebrae were also affected
in homozygous mutant mice, the exact nature of each transformation
was not as readily identifiable. In wild-type mice, sacral segments
S1 and S2 typically have broad transverse processes compared to S3
and S4. In the mutants, there did not appear to be an identifiable
S1 or S2 vertebra. Instead, mutant animals had several vertebrae
that appeared to have morphology similar to S3. In addition, the
transverse processes of all 4 sacral vertebrae are normally fused
to each other although in newborns often only fusions of the first
3 vertebrae are seen. In homozygous mutants, however, the
transverse processes of the sacral vertebrae were usually unfused.
In the caudal most region, all mutant animals also had severely
malformed vertebrae with extensive fusions of cartilage. Although
the severity of the fusions made it difficult to count the total
number of vertebrae in the caudal region, we were able to count up
to 15 transverse processes in several animals. We were unable to
determine whether these represented sacral or caudal vertebrae in
the mutants because we could not establish morphologic criteria for
distinguishing S4 from caudal vertebrae even in wild-type newborn
animals. Regardless of their identities, the total number of
vertebrae in this region was significantly reduced from the normal
number of approximately 30. Hence, although the mutants had
significantly more thoracic and lumber vertebrae than wild-type
mice, the total number of segments was reduced in the mutants due
to the truncation of the tails.
[0125] Heterozygous mice also showed abnormalities in the axial
skeleton although the phenotype was much milder than in homozygous
mice. The most obvious abnormality in heterozygous mice was the
presence of an additional thoracic segment with an associated pair
of ribs (FIG. 14(c,f)). This transformation was present in every
heterozygous animal examined, and in every case, the additional
pair of ribs was attached to the sternum (FIG. 14(i)). Hence, T8,
whose associated rib normally does not touch the sternum, appeared
to have been transformed to a morphology characteristic of a more
anterior thoracic vertebra, and L1 appeared to have been
transformed to a morphology characteristic of a posterior thoracic
vertebra. Other abnormalities indicative of anterior
transformations were also seen to varying degrees in heterozygous
mice. These included a shift of the long spinous process
characteristic of T2 by one segment to T3, a shift of the articular
and spinous processes from T10 to T11, a shift of the anterior
tuberculus on C6 to C7, and transformation of T2 to T1 where the
rib associated with T2 touched the top of the sternum.
[0126] In order to understand the basis for the abnormalities in
axial patterning seen in GDF-11 mutant mice, we examined mutant
embryos isolated at various stages of development and compared them
to wild-type embryos. By gross morphological examination,
homozygous mutant embryos isolated up to day 9.5 of gestation were
not readily distinguishable from corresponding wild-type embryos.
In particular, the number of somites present at any given
developmental age was identical between mutant and wild-type
embryos, suggesting that the rate of somite formation was unaltered
in the mutants. By day 10.5-11.5 p.c., mutant embryos could be
easily distinguished from wild-type embryos by the posterior
displacement of the hindlimb by 7-8 somites. The abnormalities in
tail development were also readily apparent at this stage. Taken
together, these data suggest that the abnormalities observed in the
mutant skeletons represented true transformations of segment
identities rather than the insertion of additional segments, for
example, by an enhanced rate of somitogenesis.
[0127] Alterations in expression of homeobox containing genes are
known to cause transformations in Drosophila and in vertebrates. To
see if the expression patterns of Hox genes (the vertebrate
homeobox containing genes) were altered in GDF-11 null mutants we
determined the expression pattern of 3 representative Hox genes,
Hoxc-6, Hoxc-8 and Hoxc-11, in day 12.5 p.c. wild-type,
heterozygous and homozygous mutant embryos by whole mount in situ
hybridization. The expression pattern of Hoxc-6 in wild-type
embryos spanned prevertebrae 8-15 which correspond to thoracic
segments T1-T8. In homozygous mutants, however, the Hoxc-6
expression pattern was shifted posteriorly and expanded to
prevertebrae 9-18 (T2-T11). A similar shift was seen with the
Hoxc-8 probe. In wild-type embryos, Hoxc-8 was expressed in
prevertebrae 13-18 (T6-T11) but, in homozygous mutant embryos,
Hoxc-8 was expressed in prevertebrae 14-22 (T7-T15). Finally,
Hoxc-11 expression was also shifted posteriorly in that the
anterior boundary of expression changed from prevertebrae 28 tin
wild-type embryos to prevertebrae 36 in mutant embryos. (Note that
because the position of the hindlimb is also shifted posteriorly in
mutant embryos, the Hoxc-11 expression patterns in wild-type and
mutant appeared similar relative to the hindlimbs). These data
provide further evidence that the skeletal abnormalities seen in
mutant animals represent homeotic transformations.
[0128] The phenotype of GDF-11 mice suggested that GDF-11 acts
early during embryogenesis as a global regulator of axial
patterning. To begin to examine the mechanism by which GDF-11
exerts its effects, we determined the expression pattern of GDF-11
in early mouse embryos by whole mount in situ hybridization. At
these stages the primary sites of GDF-11 expression correlated
precisely with the known sites at which mesodermal cells are
generated. Expression of GDF-11 was first detected at day 8.25-8.5
p.c. (8-10 somites) in the primitive streak region, which is the
site at which ingressing cells form the mesoderm of the developing
embryo. Expression was maintained in the primitive streak at day
8.75, but by day 9.5 p.c., when the tail bud replaces the primitive
streak as the source of new mesodermal cells, expression of GDF-11
shifted to the tail bud. Hence at these early stages, GDF-11
appears to be synthesized in the region of the developing embryo
where new mesodermal cells arise and presumably acquire their
positional identity.
[0129] The phenotype of GDF-11 knockout mice in several respects
resembles the phenotype of mice carrying a deletion of a receptor
for some members of the TGF-.beta. superfamily, the activin type
IIB receptor (ActRIIB). As in the case of GDF-11 knockout mice, the
ActRIIB knockout mice have extra pairs of ribs and a spectrum of
kidney defects ranging from hypoplastic kidneys to complete absence
of kidneys. The similarity in the phenotypes of these mice raises
the possibility that ActRIIB may be a receptor for GDF-11. However,
Act RIIB may not be the sole receptor for GDF-11 because the
phenotype of GDF-11 knockout mice is more severe than the phenotype
of ActRIIB mice. For example, whereas the GDF-11 knockout animals
have 4-5 extra pairs of ribs and show homeotic transformations
throughout the axial skeleton, the ActRIIB knockout animals have
only 3 extra pairs of ribs and do not show transformations at other
axial levels. In addition, the data indicate that the kidney
defects in the GDF-11 knockout mice are also more severe than those
in ActRIIB knockout mice. The ActRIIB knockout mice show defects in
left/right axis formation, such as lung isomerism and a range of
heart defects that we have not yet observed in GDF-11 knockout
mice. ActRIIB can bind the activins and certain BMPs, although none
of the knockout mice generated for these ligands show defects in
left/right axis formation.
[0130] If GDF-11 does act directly on mesodermal cells to establish
positional identity, the data presented here would be consistent
with either short range or morphogen models for GDF-11 action. That
is, GDF-11 may act on mesodermal precursors to establish patterns
of Hox gene expression as these cells are being generated at the
site of GDF-11 expression, or alternatively, GDF-11 produced at the
posterior end of the embryo may diffuse to form a morphogen
gradient. Whatever the mechanism of action of GDF-11 may be, the
fact that gross anterior/posterior patterning still does occur in
GDF-11 knockout animals suggests that GDF-11 may not be the sole
regulator of anterior/posterior specification. Nevertheless, it is
clear that GDF-11 plays an important role as a global regulator of
axial patterning and that further study of this molecule will lead
to important new insights into how positional identity along the
anterior/posterior axis is established in the vertebrate
embryo.
[0131] Similar phenotypes are expected in GDF-8 knockout animals.
For example, GDF-8 knockout animals are expected to have increased
number of ribs, kidney defects and anatomical differences when
compared to wild-type.
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Sequence CWU 1
1
16 1 2676 DNA Mus musculus CDS (104)..(1231) 1 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 2 376 PRT Mus musculus 2 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 3 2743 DNA Homo sapiens CDS (59)..(1183) 3 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 4 375 PRT Homo sapiens 4 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 5 1128 DNA
Papio hamadryas CDS (1)..(1125) 5 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 6 375 PRT Papio
hamadryas 6 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 7 1128 DNA Bovine CDS
(1)..(1125) 7 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 8 375 PRT Bovine 8 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 9 1128 DNA Gallus gallus CDS (1)..(1125) 9 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 10 375 PRT Gallus gallus 10 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
11 1131 DNA Rattus norvegicus CDS (1)..(1128) 11 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 12 376 PRT Rattus norvegicus 12 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 13 1128 DNA Meleagris gallopavo CDS (1)..(1125) 13 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 14
375 PRT Meleagris gallopavo 14 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
15 1393 DNA Mus musculus CDS (54)..(1274) 15 ccgcgggact ccggcgtccc
cgccccccag tcctccctcc cctcccctcc agc atg 56 Met 1 gtg ctc gcg gcc
ccg ctg ctg ctg ggc ttc ctg ctc ctc gcc ctg gag 104 Val Leu Ala Ala
Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu Glu 5 10 15 ctg cgg ccc
cgg ggg gag gcg gcc gag ggc ccc gcg gcg gcg gcg gcg 152 Leu Arg Pro
Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala Ala 20 25 30 gcg
gcg gcg gcg gcg gca gcg gcg ggg gtc ggg ggg gag cgc tcc agc 200 Ala
Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser Ser 35 40
45 cgg cca gcc ccg tcc gtg gcg ccc gag ccg gac ggc tgc ccc gtg tgc
248 Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val Cys
50 55 60 65 gtt tgg cgg cag cac agc cgc gag ctg cgc cta gag agc atc
aag tcg 296 Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile
Lys Ser 70 75 80 cag atc ttg agc aaa ctg cgg ctc aag gag gcg ccc
aac atc agc cgc 344 Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro
Asn Ile Ser Arg 85 90 95 gag gtg gtg aag cag ctg ctg ccc aag gcg
ccg ccg ctg cag cag atc 392 Glu Val Val Lys Gln Leu Leu Pro Lys Ala
Pro Pro Leu Gln Gln Ile 100 105 110 ctg gac cta cac gac ttc cag ggc
gac gcg ctg cag ccc gag gac ttc 440 Leu Asp Leu His Asp Phe Gln Gly
Asp Ala Leu Gln Pro Glu Asp Phe 115 120 125 ctg gag gag gac gag tac
cac gcc acc acc gag acc gtc att agc atg 488 Leu Glu Glu Asp Glu Tyr
His Ala Thr Thr Glu Thr Val Ile Ser Met 130 135 140 145 gcc cag gag
acg gac cca gca gta cag aca gat ggc agc cct ctc tgc 536 Ala Gln Glu
Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu Cys 150 155 160 tgc
cat ttt cac ttc agc ccc aag gtg atg ttc aca aag gta ctg aag 584 Cys
His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys 165 170
175 gcc cag ctg tgg gtg tac cta cgg cct gta ccc cgc cca gcc aca gtc
632 Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val
180 185 190 tac ctg cag atc ttg cga cta aaa ccc cta act ggg gaa ggg
acc gca 680 Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly
Thr Ala 195 200 205 ggg gga ggg ggc gga ggc cgg cgt cac atc cgt atc
cgc tca ctg aag 728 Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile
Arg Ser Leu Lys 210 215 220 225 att gag ctg cac tca cgc tca ggc cat
tgg cag agc atc gac ttc aag 776 Ile Glu Leu His Ser Arg Ser Gly His
Trp Gln Ser Ile Asp Phe Lys 230 235 240 caa gtg cta cac agc tgg ttc
cgc cag cca cag agc aac tgg ggc atc 824 Gln Val Leu His Ser Trp Phe
Arg Gln Pro Gln Ser Asn Trp Gly Ile 245 250 255 gag atc aac gcc ttt
gat ccc agt ggc aca gac ctg gct gtc acc tcc 872 Glu Ile Asn Ala Phe
Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser 260 265 270 ctg ggg ccg
gga gcc gag ggg ctg cat cca ttc atg gag ctt cga gtc 920 Leu Gly Pro
Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val 275 280 285 cta
gag aac aca aaa cgt tcc cgg cgg aac ctg ggt ctg gac tgc gac 968 Leu
Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp 290 295
300 305 gag cac tca agc gag tcc cgc tgc tgc cga tat ccc ctc aca gtg
gac 1016 Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr
Val Asp 310 315 320 ttt gag gct ttc ggc tgg gac tgg atc atc gca cct
aag cgc tac aag 1064 Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala
Pro Lys Arg Tyr Lys 325 330 335 gcc aac tac tgc tcc ggc cag tgc gag
tac atg ttc atg caa aaa tat 1112 Ala Asn Tyr Cys Ser Gly Gln Cys
Glu Tyr Met Phe Met Gln Lys Tyr 340 345 350 ccg cat acc cat ttg gtg
cag cag gcc aat cca aga ggc tct gct ggg 1160 Pro His Thr His Leu
Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly 355 360 365 ccc tgt tgt
acc ccc acc aag atg tcc cca atc aac atg ctc tac ttc 1208 Pro Cys
Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe 370 375 380
385 aat gac aag cag cag att atc tac ggc aag atc cct ggc atg gtg gtg
1256 Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val
Val 390 395 400 gat cgc tgt ggc tgc tct taagtgggtc actacaagct
gctggagcaa 1304 Asp Arg Cys Gly Cys Ser 405 agacttggtg ggtgggtaac
ttaacctctt cacagaggat aaaaaatgct tgtgagtatg 1364 acagaaggga
ataaacaggc ttaaagggt 1393 16 407 PRT Mus musculus 16 Met Val Leu
Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu 1 5 10 15 Glu
Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala 20 25
30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser
35 40 45 Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys
Pro Val 50 55 60 Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu
Glu Ser Ile Lys 65 70 75 80 Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys
Glu Ala Pro Asn Ile Ser 85 90 95 Arg Glu Val Val Lys Gln Leu Leu
Pro Lys Ala Pro Pro Leu Gln Gln 100 105 110 Ile Leu Asp Leu His Asp
Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp 115 120 125 Phe Leu Glu Glu
Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser 130 135 140 Met Ala
Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu 145 150 155
160 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu
165 170 175 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro
Ala Thr 180 185 190 Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr
Gly Glu Gly Thr 195 200 205 Ala Gly Gly Gly Gly Gly Gly Arg Arg His
Ile Arg Ile Arg Ser Leu 210 215 220 Lys Ile Glu Leu His Ser Arg Ser
Gly His Trp Gln Ser Ile Asp Phe 225 230 235 240 Lys Gln Val Leu His
Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 245 250 255 Ile Glu Ile
Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr 260 265 270 Ser
Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg 275 280
285 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys
290 295 300 Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu
Thr Val 305 310 315 320 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile
Ala Pro Lys Arg Tyr 325 330 335 Lys Ala Asn Tyr Cys Ser Gly Gln Cys
Glu Tyr Met Phe Met Gln Lys 340 345 350 Tyr Pro His Thr His Leu Val
Gln Gln Ala Asn Pro Arg Gly Ser Ala 355 360 365 Gly Pro Cys Cys Thr
Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 370 375 380 Phe Asn Asp
Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val 385 390 395 400
Val Asp Arg Cys Gly Cys Ser 405
* * * * *