U.S. patent application number 10/013136 was filed with the patent office on 2003-01-30 for follistatin-related protein zfsta2.
Invention is credited to Conklin, Darrell C., Ellsworth, Jeff L..
Application Number | 20030023067 10/013136 |
Document ID | / |
Family ID | 26801536 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030023067 |
Kind Code |
A1 |
Conklin, Darrell C. ; et
al. |
January 30, 2003 |
Follistatin-related protein zfsta2
Abstract
The present invention relates to polynucleotide and polypeptide
molecules for zfsta2, a novel member of the follistatin family. The
polypeptides, and polynucleotides encoding them are useful for
binding to members of the TGF-.beta. family and mediating central
nervous system, reproductive, hematopoietic and bone-related
activities. The present invention also includes antibodies to the
zfsta2 polypeptides.
Inventors: |
Conklin, Darrell C.;
(Seattle, WA) ; Ellsworth, Jeff L.; (Seattle,
WA) |
Correspondence
Address: |
Brian J. Walsh
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
26801536 |
Appl. No.: |
10/013136 |
Filed: |
December 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10013136 |
Dec 6, 2001 |
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09412554 |
Oct 5, 1999 |
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6355788 |
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60104431 |
Oct 15, 1998 |
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Current U.S.
Class: |
536/23.5 ;
435/320.1; 435/325; 435/69.1; 530/350 |
Current CPC
Class: |
C07K 2319/02 20130101;
C07K 14/4703 20130101; A61K 38/00 20130101 |
Class at
Publication: |
536/23.5 ;
530/350; 435/69.1; 435/325; 435/320.1 |
International
Class: |
C12P 021/02; C12N
005/06; C07H 021/04; C07K 014/72 |
Claims
What is claimed is:
1. An isolated polypeptide comprising a follistatin homology
domain, wherein said follistatin homology domain comprises amino
acid residues 65 to 133 of the amino acid sequence of SEQ ID
NO:2.
2. An isolated polypeptide of claim 1, wherein said polypeptide
further comprises an alpha helical linker region that resides in a
carboxyl-terminal position relative to said follistatin homology
domain, wherein said alpha helical linker region comprises amino
acid residues 134 to 174 of the amino acid sequence of SEQ ID
NO:2.
3. An isolated polypeptide of claim 2, wherein said polypeptide
further comprises a calmodulin homology domain that resides in a
carboxyl-terminal position relative to said alpha helical linker
region, wherein said calmodulin homology domain comprises amino
acid residues 175 to 250 of the amino acid sequence of SEQ ID
NO:2.
4. An isolated polypeptide of claim 2, wherein said polypeptide
further comprises two I-set Ig domains that reside in a
carboxyl-terminal position relative to said calmodulin homology
domain, wherein said I-set Ig domains comprise amino acid residues
251 to 431 of the amino acid sequence of SEQ ID NO:2.
5. An isolated polypeptide of claim 2, wherein said polypeptide
further comprises a carboxy-terminal domain that resides in a
carboxyl-terminal position relative to said I-set Ig domains,
wherein said carboxy-terminal domain comprises amino acid residues
433 to 847 of said amino acid sequence of SEQ ID NO:2.
6. An isolated polypeptide of claim 1, wherein said polypeptide
further comprises a hydrophilic linker region that resides in an
amino-terminal position relative to said follistatin homology
domain, wherein said hydrophobic linker region comprises amino acid
residues 21 to 64 of the amino acid sequence of SEQ ID NO:2.
7. An isolated polypeptide of claim 6, wherein said polypeptide
further comprises a secretory signal sequence that resides in an
amino-terminal position relative to said hydrophobic linker region,
wherein said secretory signal sequence comprises amino acid
residues 1 to 20 of the amino acid sequence of SEQ ID NO:2.
8. An isolated polypeptide having an amino acid sequence that is at
least 70% identical to the amino acid sequence of SEQ ID NO:2,
wherein said isolated polypeptide specifically binds with an
antibody to which a polypeptide having the amino acid sequence of
SEQ ID NO:2 specifically binds.
9. An isolated polypeptide of claim 8, wherein said isolated
polypeptide has an amino acid sequence that is at least 80%
identical to the amino acid sequence of SEQ ID NO:2.
10. An isolated polypeptide of claim 8, wherein said isolated
polypeptide has an amino acid sequence that is at least 90%
identical to the amino acid sequence of SEQ ID NO:2.
11. An isolated polypeptide of claim 8, wherein any difference
between said amino acid sequence and said corresponding amino acid
sequence of SEQ ID NO:2 is due to one or more conservative amino
acid substitutions.
12. An isolated polypeptide of claim 8, wherein the amino acid
percent identity is determined using a FASTA program with ktup=1,
gap opening penalty=10, gap extension penalty=1, and substitution
matrix=blosum62, with other parameters set as default.
13. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:2.
14. An isolated polypeptide selected from the group consisting of:
a) a polypeptide consisting of the sequence of amino acid residues
from residue 21 to residue 64 of SEQ ID NO:2; b) a polypeptide
consisting of the sequence of amino acid residues from residue 65
to residue 133 of SEQ ID NO :2; c) a polypeptide consisting of the
sequence of amino acid residues from residue 134 to residue 174 of
SEQ ID NO:2; d) a polypeptide consisting of the sequence of amino
acid residues from residue 175 to residue 250 of SEQ ID NO:2; e) a
polypeptide consisting of the sequence of amino acid residues from
residue 251 to residue 334 of SEQ ID NO:2; f) a polypeptide
consisting of the sequence of amino acid residues from residue 335
to residue 432 of SEQ ID NO:2; g) a polypeptide consisting of the
sequence of amino acid residues from residue 433 to residue 847 of
SEQ ID NO:2; h) a polypeptide consisting of the sequence of amino
acid residues from residue 251 to residue 432 of SEQ ID NO:2; i) a
polypeptide consisting of the sequence of amino acid residues from
residue 65 to residue 174 of SEQ ID NO:2; j) a polypeptide
consisting of the sequence of amino acid residues from residue 65
to residue 250 of SEQ ID NO: 2; k) a polypeptide consisting of the
sequence of amino acid residues from residue 65 to residue 334 of
SEQ ID NO:2; l) a polypeptide consisting of the sequence of amino
acid residues from residue 65 to residue 847 of SEQ ID NO:2; m) a
polypeptide consisting of the sequence of amino acid residues from
residue 134 to residue 250 of SEQ ID NO: 2; n) a polypeptide
consisting of the sequence of amino acid residues from residue 134
to residue 334 of SEQ ID NO:2; o) a polypeptide consisting of the
sequence of amino acid residues from residue 134 to residue 432 of
SEQ ID NO:2; p) a polypeptide consisting of the sequence of amino
acid residues from residue 134 to residue 847 of SEQ ID NO:2; q) a
polypeptide consisting of the sequence of amino acid residues from
residue 175 to residue 334 of SEQ ID NO: 2; r) a polypeptide
consisting of the sequence of amino acid residues from residue 175
to residue 432 of SEQ ID NO:2; and s) a polypeptide consisting of
the sequence of amino acid residues from residue 175 to residue 847
of SEQ ID NO: 2.
15. An isolated polypeptide according to claim 1, further
comprising an affinity tag or binding domain.
16. A fusion protein comprising a secretory signal sequence having
the amino acid sequence of amino acid residues 1-20 of SEQ ID NO:2,
wherein said secretary signal sequence is operably linked to an
additional polypeptide.
17. A fusion protein consisting essentially of a first portion and
a second portion joined by a peptide bond, said first portion
comprising a polypeptide according to claim 1; and said second
portion comprising another polypeptide.
18. An isolated polynucleotide molecule that encodes a polypeptide
according to claim 1.
19. An isolated polynucleotide molecule according to claim 18,
encoding a polypeptide further comprising an alpha helical linker
region that resides in a carboxyl-terminal position relative to
said follistatin homology domain, wherein said alpha helical linker
region comprises amino acid residues 134 to 174 of the amino acid
sequence of SEQ ID NO:2.
20. An isolated polynucleotide of claim 19, wherein said
polynucleotide encodes a polypeptide further comprising a
calmodulin homology domain that resides in a carboxyl-terminal
position relative to said alpha helical linker region, wherein said
calmodulin homology domain comprises amino acid residues 175 to 250
of the amino acid sequence of SEQ ID NO:2.
21. An isolated polynucleotide of claim 20, wherein said
polynucleotide encodes a polypeptide further comprising two I-set
Ig domains that reside in a carboxyl-terminal position relative to
said calmodulin homology domain, wherein said I-set Ig domains
comprise amino acid residues 251 to 431 of the amino acid sequence
of SEQ ID NO:2.
22. An isolated polynucleotide of claim 21, wherein said
polynucleotide encodes a polypeptide further comprising a
carboxy-terminal domain that resides in a carboxyl-terminal
position relative to said I-set Ig domains, wherein said
carboxy-terminal domain comprises amino acid residues 433 to 847 of
said amino acid sequence of SEQ ID NO:2.
23. An isolated polynucleotide according to claim 22, wherein said
polypeptide further comprises an affinity tag or binding
domain.
24. An isolated polynucleotide molecule, wherein said
polynucleotide molecule is a degenerate nucleotide sequence
encoding a polypeptide according to claim 1.
25. An isolated polynucleotide encoding a polypeptide having an
amino acid sequence that is at least 70% identical to the amino
acid sequence of SEQ ID NO:2, wherein said isolated polypeptide
specifically binds with an antibody to which a polypeptide having
the amino acid sequence of SEQ ID NO:2 specifically binds.
26. An isolated polynucteotide of claim 25, wherein said isolated
polypeptide has an amino acid sequence that is at least 80%
identical to the amino acid sequence of SEQ ID NO:2.
27. An isolated polynucleotide of claim 26, wherein said isolated
polypeptide has an amino acid sequence that is at least 90%
identical to the amino acid sequence of SEQ ID NO:2.
28. An isolated polynucleotide of claim 27, wherein any difference
between said amino acid sequence and said corresponding amino acid
sequence of SEQ ID NO:2 is due to one or more conservative amino
acid substitutions.
29. An isolated polynucleotide of claim 27, wherein the amino acid
percent identity is determined using a FASTA program with ktup=1,
gap opening penalty=10, gap extension penalty=1, and substitution
matrix=blosum62, with other parameters set as default.
30. An isolated polynucleotide molecule comprising the nucleotide
sequence of nucleotides 58 to 3006 of SEQ ID NO:1.
31. An isolated polynucleotide molecule of SEQ ID NO:1.
32. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide consisting of nucleotides 58-117 of SEQ ID
NO:1; b) a polynucleotide consisting of nucleotides 118-249 of SEQ
ID NO:1; c) a polynucleotide consisting of nucleotides 250-456 of
SEQ ID NO:1; d) a polynucleotide consisting of nucleotides 457-579
of SEQ ID NO:1; e) a polynucleotiqe consisting of nucleotides
580-810 of SEQ ID NO:1; f) a polynucleotide consisting of
nucleotides 811-1059 of SEQ ID NO:1; g) a polynucleotide consisting
of nucleotides 1060-1353 of SEQ ID NO:1; h) a polynucleotide
consisting of nucleotides 1354-3006 of SEQ ID NO:1; i) a
polynucleotide consisting of nucleotides 250-579 of SEQ ID NO:1; j)
a polynucleotide consisting of nucleotides 250-810 of SEQ ID NO:1;
k) a polynucleotide consisting of nucleotides 250-1059 of SEQ ID
NO:1; l) a polynucleotide consisting of nucleotides 250-1353 of SEQ
ID NO:1; m) a polynucleotide consisting of nucleotides 250-3006 of
SEQ ID NO:1; n) a polynucleotide consisting of nucleotides 457-810
of SEQ ID NO:1; o) a polynucleotide consisting of nucleotides
457-1059 of SEQ ID NO:1; p) a polynucleotide consisting of
nucleotides 457-1353 of SEQ ID NO:1; q) a polynucleotide consisting
of nucleotides 457-3006 of SEQ ID NO:1; r) a polynucleotide
consisting of nucleotides 580-1059 of SEQ ID NO:1; s) a
polynucleotide consisting of nucleotides 580-1353 of SEQ ID NO:1;
t) a polynucleotide consisting of nucleotides 580-3006 of SEQ ID
NO:1; and u) a polynucleotide consisting of nucleotides 811-1353 of
SEQ ID NO:1.
33. A polynucleotide encoding a fusion protein comprising a
secretory signal sequence having the amino acid sequence of amino
acid residues 1-20 of SEQ ID NO:2, wherein said secretory signal
sequence is operably linked to an additional polypeptide.
34. A polynucleotide molecule encoding a fusion protein consisting
essentially of a first portion and a second portion joined by a
peptide bond, said first portion comprising a polypeptide according
to claim 1; and said second portion comprising another
polypeptide.
35. An expression vector comprising the following operably linked
elements: a transcription promoter; a polynucleotide molecule that
encodes a polypeptide according to claim 1; and a transcription
terminator.
36. An expression vector according to claim 35 further comprising a
secretory signal sequence operably linked to said DNA segment.
37. An expression vector according to claim 35, wherein said
polynucleotide encodes a polypeptide covalently linked amino
terminally or carboxy terminally to an affinity tag.
38. A cultured cell into which has been introduced an expression
vector comprising the following operably linked elements: a
transcription promoter; a polynucleotide molecule that encodes a
polypeptide according to claim 1; and a transcription terminator,
wherein said cultured cell expresses said polypeptide encoded by
said polynucleotide segment.
39. A method of producing a polypeptide comprising: culturing a
cell into which has been introduced an expression vector comprising
the following operably linked elements: a transcription promoter; a
polynucleotide molecule that encodes a polypeptide according to
claim 1; and a transcription terminator; whereby said cell
expresses said polypeptide encoded by said polynucleotide segment;
and recovering said expressed polypeptide.
40. An antibody or antibody fragment that specifically binds to a
polypeptide according to claim 1.
41. An antibody according to claim 40, wherein said antibody is
selected from the group consisting of: a) polyclonal antibody; b)
murine monoclonal antibody; c) humanized antibody derived from b);
and d) human monoclonal antibody.
42. An antibody fragment according to claim 41, wherein said
antibody fragment is selected from the group consisting of F(ab'),
F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.
43. An anti-idiotype antibody that specifically binds to said
antibody of claim 40
44. A polypeptide according to claim 1, in combination with a
pharmaceutically acceptable vehicle.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to Provisional Application No.
60/104,431, filed on Oct. 15, 1998. Under 35 U.S.C.
.sctn.119(e)(1), this application claims benefit of said
Provisional Application.
BACKGROUND OF THE INVENTION
[0002] Follistatin is a monomeric, glycosylated protein originally
identified in porcine follicular fluid as a potent inhibitor of
pituitary follicle-stimulating hormone (FSH) synthesis and
secretion, follistatin was later shown to exert some of its
biological effects by specifically binding the FSH-inducer activin.
Other follistatin family members include follistatin related
protein or FRP (Zwijsen et al., Eur. J. Biochem. 225:937-46, 1994),
SPARC, also known as osteonectin or BM-40 or the human ortholog of
mouse TSC-36 (Lane and Sage, ibid.), agrin (Patthy and Nikolics,
TINS 16:76-81, 1993), hevin (Girard and Springer, Immunity
2:113-23, 1995), the Flik protein of chickens (Amthor et al., Dev.
Biology 178:343-62, 1996) and the rat brain protein SC1 (Mendis et
al., Brain Res. 730:95-106, 1996). Follistatins, however, are
thought to be more than "activin binders" since follistatin
deficient mice prepared by gene targeting have a more complex and
different phenotype than activin gene knock-out animals (Mazuk et
al., Nature 374:360-3, 1995 and Mazuk et al., Nature 374:356-9,
1995).
[0003] Activins and inhibins are potent activators and inhibitors,
respectively, of pituitary FSH secretion and are members of the
TGF-.beta. family of peptide growth factors (Mather et al., Proc.
Soc. Exp. Biol. Med. 215:209-22, 1997). The activin and inhibin
family of hormones, while originally described as gonadally
produced regulators of pituitary FSH secretion, are now known to
have a broad range of effects within and outside of the
reproductive system (Mather et al., ibid.). Inhibins consist of a
common alpha subunit which is covalently linked to one of two
different beta subunits (inhibin A:.alpha./B.sub.A; inhibin
B:.alpha./B.sub.B); activins are covalently linked dimers of the
two B-subunits and therefore exist in three different forms
(activin A:B.sub.A/B.sub.A; activin B:B.sub.B/B.sub.B; activin
AB:B.sub.A/B.sub.B) Activin and inhibin bind to follistatin with
high affinity, and although the structure of the activin binding
site has not been completely defined, preliminary data (Inouye et
al., Biochem. Biophys. Res. Commun. 179:352-8, 1991) suggest that
residues in the first amino terminal cysteine-rich follistatin
domain are involved in hormone binding. Activin binding to
follistatin is thus thought to limit its biological effects by
sequestration of the peptide hormone. Thus, the broad range of
biological actions of the activins and inhibins, and possibly other
members of the TGF-.beta. family as well, may be regulated by
binding to proteins of the follistatin family. Different binding
proteins may be involved for each TGF-.beta. family member as
follistatin binds activin with high affinity (nM), inhibin with
lower affinity, and does not appear to bind TGF-.beta. at all
(Mather et al., ibid.). Follistatin family members may regulate the
activity of other growth factors as well, for example, SPARC or
BM-40 have been shown to bind platelet derived growth factor
(PDGF-AB, PDGF-BB) (Lane and Sage, FASEB J. 8:163-73, 1994).
[0004] This application provides a new member of the follistatin
family, zfsta2, which is likely to play a major role in regulating
the biological activities of the TGF-.beta. growth factors. Like
other members of the follistatin family, zfsta2 may play a broad
role in development and differentiation, pathogenesis of
atherosclerosis, regulation of the gonadal-pituitary-hypothalamic
axis, tooth and bone formation, regulation of gonadal hormone
production, spermatogenesis, hypothalmic oxytocin secretion,
proliferation and differentiation of erythroid progenitors,
hematopoiesis, host defense and neuron survival.
[0005] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
SUMMARY OF THE INVENTION
[0006] Within one aspect the invention provides an isolated
polypeptide comprising a follistatin homology domain, wherein the
follistatin homology domain comprises amino acid residues 65 to 133
of the amino acid sequence of SEQ ID NO:2. Within one embodiment
the polypeptide further comprises an alpha helical linker region
that resides in a carboxyl-terminal position relative to the
follistatin homology domain, wherein the alpha helical linker
region comprises amino acid residues 134 to 174 of the amino acid
sequence of SEQ ID NO:2. Within a related embodiment the
polypeptide farther comprises a calmodulin homology domain that
resides in a carboxyl-terminal position relative to the alpha
helical linker region, wherein the calmodulin homology domain
comprises amino acid residues 175 to 250 of the amino acid sequence
of SEQ ID NO:2. Within another embodiment the polypeptide further
comprises two I-set Ig domains that reside in a carboxyl-terminal
position relative to the calmodulin homology domain, wherein the
I-set Ig domains comprise amino acid residues 251 to 431 of the
amino acid sequence of SEQ ID NO:2. Within another embodiment the
polypeptide further comprises a carboxy-terminal domain that
resides in a carboxyl-terminal position relative to the I-set Ig
domains, wherein the carboxy-terminal domain comprises amino acid
residues 433 to 847 of the amino acid sequence of SEQ ID NO:2.
Within a yet another embodiment the polypeptide further comprises a
hydrophilic linker region that resides in an amino-terminal
position relative to the follistatin homology domain, wherein the
hydrophobic linker region comprises amino acid residues 21 to 64 of
the amino acid sequence of SEQ ID NO:2. Within another embodiment
the polypeptide further comprises a secretory signal sequence that
resides in an amino-terminal position relative to the hydrophobic
linker region, wherein the secretory signal sequence comprises
amino acid residues 1 to 20 of the amino acid sequence of SEQ ID
NO:2.
[0007] Within another aspect the invention provides an isolated
polypeptide having an amino acid sequence that is at least 70%
identical to the amino acid sequence of SEQ ID NO:2, wherein the
isolated polypeptide specifically binds with an antibody to which a
polypeptide having the amino acid sequence of SEQ ID NO:2
specifically binds. Within one embodiment the isolated polypeptide
has an amino acid sequence that is at least 80% identical to the
amino acid sequence of SEQ ID NO:2. Within another embodiment the
isolated polypeptide has an amino acid sequence that is at least
90% identical to the amino acid sequence of SEQ ID NO:2. Within a
further embodiment any difference between the amino acid sequence
and the corresponding amino acid sequence of SEQ ID NO:2 is due to
one or more conservative amino acid substitutions. Within another
embodiment the amino acid percent identity is determined using a
FASTA program with ktup=1, gap opening penalty=10, gap extension
penalty=1, and substitution matrix=blosum62, with other parameters
set as default.
[0008] The invention also provides an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:2.
[0009] The invention further provides an isolated polypeptide
selected from the group consisting of: a) a polypeptide consisting
of the sequence of amino acid residues from residue 21 to residue
64 of SEQ ID NO:2; b) a polypeptide consisting of the sequence of
amino acid residues from residue 65 to residue 133 of SEQ ID NO:2;
c) a polypeptide consisting of the sequence of amino acid residues
from residue 134 to residue 174 of SEQ ID NO:2; d) a polypeptide
consisting of the sequence of amino acid residues from residue 175
to residue 250 of SEQ ID NO:2; e) a polypeptide consisting of the
sequence of amino acid residues from residue 251 to residue 334 of
SEQ ID NO:2; f) a polypeptide consisting of the sequence of amino
acid residues from residue 335 to residue 432 of SEQ ID NO:2; g) a
polypeptide consisting of the sequence of amino acid residues from
residue 433 to residue 847 of SEQ ID NO:2; h) a polypeptide
consisting of the sequence of amino acid residues from residue 251
to residue 432 of SEQ ID NO:2; i) a polypeptide consisting of the
sequence of amino acid residues from residue 65 to residue 174 of
SEQ ID NO:2; j) a polypeptide consisting of the sequence of amino
acid residues from residue 65 to residue 250 of SEQ ID NO:2; k) a
polypeptide consisting of the sequence of amino acid residues from
residue 65 to residue 334 of SEQ ID NO:2; l) a polypeptide
consisting of the sequence of amino acid residues from residue 65
to residue 847 of SEQ ID NO:2; m) a polypeptide consisting of the
sequence of amino acid residues from residue 134 to residue 250 of
SEQ ID NO:2; n) a polypeptide consisting of the sequence of amino
acid residues from residue 134 to residue 334 of SEQ ID NO:2; o) a
polypeptide consisting of the sequence of amino acid residues from
residue 134 to residue 432 of SEQ ID NO:2; p) a polypeptide
consisting of the sequence of amino acid residues from residue 134
to residue 847 of SEQ ID NO:2; q) a polypeptide consisting of the
sequence of amino acid residues from residue 175 to residue 334 of
SEQ ID NO:2; r) a polypeptide consisting of the sequence of amino
acid residues from residue 175 to residue 432 of SEQ ID NO:2; and
s) a polypeptide consisting of the sequence of amino acid residues
from residue 175 to residue 847 of SEQ ID NO:2.
[0010] The invention also provides an isolated polypeptide as
described above, further comprising an affinity tag or binding
domain.
[0011] Within another aspect the invention provides a fusion
protein comprising a secretory signal sequence having the amino
acid sequence of amino acid residues 1-20 of SEQ ID NO:2, wherein
the secretory signal sequence is operably linked to an additional
polypeptide.
[0012] The invention also provides a fusion protein consisting
essentially of a first portion and a second portion joined by a
peptide bond, the first portion comprising a polypeptide as
described above; and the second portion comprising another
polypeptide.
[0013] Within another aspect the invention also provides an
isolated polynucleotide molecule that encodes a polypeptide as
described above. Within one embodiment the polypeptide further
comprises an affinity tag or binding domain.
[0014] The invention also provides an isolated polynucleotide
molecule, wherein the polynucleotide molecule is a degenerate
nucleotide sequence encoding a polypeptide as described above.
[0015] The invention further provides an isolated polynucleotide
encoding a polypeptide having an amino acid sequence that is at
least 70% identical to the amino acid sequence of SEQ ID NO:2,
wherein the isolated polypeptide specifically binds with an
antibody to which a polypeptide having the amino acid sequence of
SEQ ID NO:2 specifically binds. Within one-embodiment the isolated
polypeptide has an amino acid sequence that is at least 80%
identical to the amino acid sequence of SEQ ID NO:2. Within another
embodiment the isolated polypeptide has an amino acid sequence that
is at least 90% identical to the amino acid sequence of SEQ ID
NO:2. Within yet another embodiment difference between the amino
acid sequence and the corresponding amino acid sequence of SEQ ID
NO:2 is due to one or more conservative amino acid substitutions.
Within still another embodiment the amino acid percent identity is
determined using a FASTA program with ktup=1, gap opening
penalty=10, gap extension penalty=1, and substitution
matrix=blosum62, with other parameters set as default.
[0016] The invention provides an isolated polynucleotide molecule
comprising the nucleotide sequence of nucleotides 58 to 3006 of SEQ
ID NO:1.
[0017] The invention also provides an isolated polynucleotide
molecule of SEQ ID NO:1.
[0018] The invention further provides an isolated polynucleotide
selected from the group consisting of: a) a polynucleotide
consisting of nucleotides 58-117 of SEQ ID NO:1; b) a
polynucleotide consisting of nucleotides 118-249 of SEQ ID NO:1; c)
a polynucleotide consisting of nucleotides 250-456 of SEQ ID NO:1;
d) a polynucleotide consisting of nucleotides 457-579 of SEQ ID
NO:1; e) a polynucleotide consisting of nucleotides 580-810 of SEQ
ID NO:1; f) a polynucleotide consisting of nucleotides 811-1059 of
SEQ ID NO:1; g) a polynucleotide consisting of nucleotides
1060-1353 of SEQ ID NO:1; h)a polynucleotide consisting of
nucleotides 1354-3006 of SEQ ID NO:1; i) a polynucleotide
consisting of nucleotides 250-579 of SEQ ID NO:1; j) a
polynucleotide consisting of nucleotides 250-810 of SEQ ID NO:1; k)
a polynucleotide consisting of nucleotides 250-1059 of SEQ ID NO:1;
l) a polynucleotide consisting of nucleotides 250-1353 of SEQ ID
NO:1; m) a polynucleotide consisting of nucleotides 250-3006 of SEQ
ID NO:1; n) a polynucleotide consisting of nucleotides 457-810 of
SEQ ID NO:1; o) a polynucleotide consisting of nucleotides 457-1059
of SEQ ID NO:1; p) a polynucleotide consisting of nucleotides
457-1353 of SEQ ID NO:1; q) a polynucleotide consisting of
nucleotides 457-3006 of SEQ ID NO:1; r) a polynucleotide consisting
of nucleotides 580-1059 of SEQ ID NO:1; s) a polynucleotide
consisting of nucleotides 580-1353 of SEQ ID NO:1; t) a
polynucleotide consisting of nucleotides 580-3006 of SEQ ID NO:1;
and u) a polynucleotide consisting of nucleotides 811-1353 of SEQ
ID NO:1.
[0019] The invention also provides a polynucleotide encoding a
fusion protein comprising a secretory signal sequence having the
amino acid sequence of amino acid residues 1-20 of SEQ ID NO:2,
wherein the secretory signal sequence is operably linked to an
additional polypeptide.
[0020] Still further, the invention provides a polynucleotide
molecule encoding a fusion protein consisting essentially of a
first portion and a second portion joined by a peptide bond, the
first portion comprising a polypeptide as described above; and the
second portion comprising another polypeptide.
[0021] Within another aspect the invention provides an expression
vector comprising the following operably linked elements: a
transcription promoter; a polynucleotide molecule that encodes a
polypeptide as described above; and a transcription terminator.
Within one embodiment the expression vector further comprises a
secretory signal sequence operably linked to the DNA segment.
Within another embodiment the polynucleotide encodes a polypeptide
covalently linked amino terminally or carboxy terminally to an
affinity tag.
[0022] Within another aspect the invention provides a cultured cell
into which has been introduced an expression vector comprising the
following operably linked elements: a transcription promoter; a
polynucleotide molecule that encodes a polypeptide as described
above; and a transcription terminator, wherein the cultured cell
expresses the polypeptide encoded by the polynucleotide
segment.
[0023] Within still another aspect the invention provides a method
of producing a polypeptide comprising: culturing a cell into which
has been introduced an expression vector comprising the following
operably linked elements, a transcription promoter; a
polynucleotide molecule that encodes a polypeptide as described
above; and a transcription terminator; whereby the cell expresses
the polypeptide encoded by the polynucleotide segment; and
recovering the expressed polypeptide.
[0024] Within another aspect the invention provides an antibody or
antibody fragment that specifically binds to a polypeptide as
described above. Within one embodiment the antibody is selected
from the group consisting of: a) polyclonal antibody; b) murine
monoclonal antibody; c) humanized antibody derived from b); and
d)human monoclonal antibody. Within another aspect the antibody
fragment is selected from the group consisting of F(ab'), F(ab),
Fab', Fab, Fv, scFv, and minimal recognition unit. Within another
embodiment is an anti-idiotype antibody that specifically binds to
the antibody described above.
[0025] Within another aspect is provided a polypeptide as described
above in combination with a pharmaceutically acceptable
vehicle.
[0026] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0028] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Meth. Enzymol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0029] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0030] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0031] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0032] The term "complements of a polynucleotide molecule" is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5'
CCCGTGCAT 3'.
[0033] The term "contig" denotes a polynucleotide that has a
contiguous stretch of identical or complementary sequence to
another polynucleotide. Contiguous sequences are said to "overlap"
a given stretch of polynucleotide sequence either in their entirety
or along a partial stretch of the polynucleotide. For example,
representative contigs to the polynucleotide sequence
5'-ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3' and
3'-gtcgacTACCGA-5'.
[0034] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0035] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0036] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0037] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95 pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0038] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0039] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0040] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0041] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0042] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0043] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0044] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating
hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0045] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0046] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0047] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0048] All references cited herein are incorporated by reference in
their entirety.
[0049] A new member of the follistatin family of proteins, zfsta2,
has been identified from a human hypothalamic library. The zfsta2
protein has a predicted molecular weight of about 86,000 Da and
exhibits the characteristic amino terminal cysteine-rich
follistatin domain (Hohenester et al., EMBO J. 16:3778-86, 1997)
found in other follistatin family members such as follistatin
related protein or FRP (Zwijsen et al., ibid.), SPARC, also known
as osteonectin or BM-40 or the human ortholog of mouse TSC-36 (Lane
and Sage, ibid.), agrin (Patthy and Nikolics, ibid.), hevin (Girard
and Springer, ibid.), the Flik protein of chickens (Amthor et al.,
ibid.) and the rat brain protein SC1 (Mendis et al., ibid.).
[0050] The present invention is based in part upon the discovery of
a novel DNA sequence that encodes a polypeptide having homology to
the family of follistatins. The zfsta2 polynucleotide sequence is
disclosed in SEQ ID NO:1 and encodes a multi-domain secreted
protein of 847 amino acids (SEQ ID NO:2). Sequence analysis of a
deduced amino acid sequence of zfsta2, as represented by SEQ ID
NO:2, indicates the presence of a 20 amino acid residue signal
sequence (amino acid residues 1-20 of SEQ ID NO:2, nucleotides
58-117 of SEQ ID NO:1), followed by a predominantly hydrophilic
short linker domain that has no known homology (amino acid residues
21-64 of SEQ ID NO:2, nucleotides 118-249 of SEQ ID NO:1), a
follistatin homology domain (amino acid residues 65-133 of SEQ ID
NO:2, nucleotides 250-456 of SEQ ID NO:1), an alpha-helical linker
region (amino acid residues 134-174 of SEQ ID NO:2, nucleotides
457-579 of SEQ ID NO:1), a calmodulin domain (amino acid residue
175-250 of SEQ ID NO:2, nucleotides 580-810 of SEQ ID NO:1), an
I-set IG domain #1 (amino acid residues 251-334 of SEQ ID NO:2,
nucleotides 811-1059 of SEQ ID NO:1), an I-set IG domain #2 (amino
acid residues 335-432 of SEQ ID NO:2, nucleotides 1060-1353 of SEQ
ID NO:1) and a C-terminal domain with no known homology (amino acid
residues 433-847 of SEQ ID NO:2, nucleotides 1354-3006 of SEQ ID
NO:1). Those skilled in the art will recognize that predicted
domain boundaries are approximations based on primary sequence
content, and may vary slightly; however, such estimates are
generally accurate to within .+-.5 amino acid residues.
[0051] The follistatin homology domain is predicted to fold into a
structure similar to that determined for the follistatin homology
domain in SPARC (Swiss-Prot SPRC_HUMAN, PDB 1BMO, also known as
BM-40 or osteonectin, Hohenester et al., 1997). This is a beta
hairpin structure, followed by a small hydrophobic core of
alpha/beta structure. Unlike SPARC, which is glycosylated at Asn99,
there is no predicted glycosylation site in zfsta2. Based on the
disulfide bonding pattern in SPARC, the disulfide pairings in
zfsta2 are as follows: Cys65-Cys76, Cys70-Cys87, Cys89-Cys119,
Cys93-Cys112, and Cys101-Cys133, of SEQ ID NO:2. The zfsta2
follistatin homology domain has 47% identity to the follistatin
domain in human follistatin related protein (Swiss-Prot FRP_HUMAN);
the mouse orthologue of this protein is known as TSC-36 (Swiss-Prot
FRP_MOUSE).
[0052] The follistatin homology domain has substantial sequence
similarity to the Kazal family (Bode and Huber., Eur. J. Biochem.
204, 433-51, 1992) of serine proteinase inhibitors. Serine
proteinase inhibitors regulate the proteolytic activity of target
proteinases by occupying the active site and thereby preventing
occupation by normal substrates. Although serine proteinase
inhibitors fall into several unrelated structural classes, they all
possess an exposed loop (variously termed an "inhibitor loop", a
"reactive core", a "reactive site", a "binding loop") which is
stabilized by intermolecular interactions between residues flanking
the binding loop and the protein core (Bode and Huber, ibid.).
[0053] Interaction between inhibitor and enzyme produces a stable
complex which disassociates very slowly, producing either a virgin
or a modified inhibitor which is cleaved at the scissile bond of
the binding loop. Based on analogy with the crystal structures for
the proteinase inhibitors PEC-60 (PDB 1PCE), and ovomucoid (PDB
1OVO), the putative proteinase binding site in the follistatin
homology domain of zfsta2 comprises the amino acid residues Cys93
(P3), Lys94 (P2), Arg95 (P1), His96 (P1'), and Tyr97 (P2') of SEQ
ID NO:2. The scissile bond of the binding loop will therefore
reside between the P1 and P1' residues Arg95 and His96 of SEQ ID
NO:2.
[0054] The calmodulin homology domain is predicted to fold into a
structure similar to that determined for the EC (EF-hand calcium
binding; calmodulin-like) domain in SPARC (Hohenester et al., EMBO
J. 16:3778-86, 1997). Calmodulin (Swiss-Prot CALM_HUMAN, PDB 1CLI)
is an alpha-helical protein which binds calcium ions through the
loops of helix-loop-helix substructures known as EF hands.
Calmodulin has two structurally similar regions, each containing
two EF hands, linked by a connecting helical segment. As is used
herein "calmodulin homology domain" is meant to describe one of
these two regions. The calmodulin homology domain of zfsta2 is
predicted to contain two EF hand motifs, and hence two suspected
calcium ion binding sites. Based on motif analysis, the loops of
these two EF hands are predicted to reside between Asp amino acid
residue 188 and Leu, amino acid residue, 200 of SEQ ID NO:2, and
between Asp, amino acid residue 226 and Phe, amino acid residue 238
of SEQ ID NO:2. The last residue of the EF hand loop is always
hydrophobic: in zfsta2 these residues are Leu, amino acid residue
200 and Phe, amino acid residue 238 of SEQ ID NO:2. In terms of
sequence homology, the calmodulin homology domain of zfsta2 has 24%
identity at the amino acid level, to the double EF hand segment of
human protein phosphatase PPEF-2 (GenBank accession AF023456). The
zfsta2 calmodulin homology domain has no detectable sequence
homology to the calmodulin domain of SPARC.
[0055] The second EF hand of the calmodulin domain of SPARC is
stabilized by a disulfide bond spanning the EF hand loop. When the
two Cys residues in this EF hand were mutated to Leu residues, a
100-fold decrease in calcium ion affinity was noted (Hohenester et
al., Nat. Struct. Biol., 3:67-73, 1996). The present application
also provides a mutated form of zfsta2 where the second EF hand is
stabilized by replacing Asp, amino acid residue 225 and Ala, amino
acid residue 241 of SEQ ID NO:2, with cysteine residues. This
mutated form may have higher calcium binding affinity.
[0056] Between the follistatin and calmodulin homology domains is a
short segment, called the alpha-helical linker which may form a
short linker peptide between the two segments. This linker is
predicted to have an alpha helical structure from Glu, amino acid
residue 144 through Glu, amino acid residue 166 of SEQ ID NO:2. At
the C-terminus of this linker are three basic residues which could
be the location of a proteolysis site. Processing at this site of
the secreted protein would release domains B and C, containing the
follistatin homology domain, from the rest of the protein.
[0057] Amino acid residue 140 (Cys, SEQ ID NO:2) of the
alpha-helical linker peptide may form a disulfide bond with amino
acid residue 216 (Cys, SEQ ID NO:2), which precedes the second EF
hand in the calmodulin homology domain of zfsta2.
[0058] The I-set IG domains #1 and #2 of zfsta2 are predicted to
fold into a structure similar to that determined for the telokin
peptide (Swiss-Prot KMLS_HUMAN, PDB 1TLK). The telokin peptide
falls into the class of immunoglobulins (Bork et al., J. Mol. Biol.
242:309-20, 1994) which are all beta proteins folding into a
beta-sandwich like structure. These have two beta sheets comprising
3+4 beta strands. Furthermore, the telokin peptide has been
sub-classified as an "I" set immunoglobulin (IG) domain. Other
proteins with I set immunoglobulin domains include titin, vascular
and neural cell adhesion molecules, and twitchin. In zfsta2 domains
I-set IG #1 and #2 there may be two intra-domain disulfide bonds,
one between cysteine residues 270 and 321 of SEQ ID NO:2 in I-set
IG domain #1 and cysteine residues 362 and 413 of SEQ ID NO:2 in
I-set IG domain #2.
[0059] The C-terminal domain of zfsta2 shows no recognizable
sequence or structural similarity to any known protein. This
segment may serve to anchor the protein to the extracellular
matrix, or to the cell surface membrane.
[0060] Northern blot analysis of various human tissues resulted in
a transcript of approximately 5 kb seen in brain, placenta and
spinal cord. RNA Dot Blot analysis indicated expression in the
cerebellum, occipital lobe and pituitary gland.
[0061] The results of chromosomal localization showed that zfsta2
maps 2.84 cR.sub.--3000 from the framework marker WI-5113 on the
chromosome 4 WICGR radiation hybrid map. Proximal and distal
framework markers were WI-5113 and CHLC.GATA4C05.17, respectively.
The use of surrounding markers positions zfsta2 in the 4q28.3
region on the integrated LDB chromosome 4 map.
[0062] The present invention further provides polynucleotide
molecules, including DNA and RNA molecules, encoding zfsta2
proteins. The polynucleotides of the present invention include the
sense strand; the anti-sense strand; and the DNA as
double-stranded, having both the sense and anti-sense strand
annealed together by their respective hydrogen bonds. A
representative DNA sequence encoding a zfsta2 protein is set forth
in SEQ ID NO:1. DNA sequences encoding other zfsta2 proteins can be
readily generated by those of ordinary skill in the art based on
the genetic code. Counterpart RNA sequences can be generated by
substitution of U for T.
[0063] Those skilled in the art will readily recognize that, in
view of the degeneracy of the genetic code, considerable sequence
variation is possible among these polynucleotide molecules. SEQ ID
NO:3 is a degenerate DNA sequence that encompasses all DNAs that
encode the zfsta2 polypeptide of SEQ ID NO:2. Those skilled in the
art will recognize that the degenerate sequence of SEQ ID NO:3 also
provides all RNA sequences encoding SEQ ID NO:2 by substituting U
for T. Thus, zfsta2 polypeptide-encoding polynucleotides comprising
nucleotide 1 to nucleotide 2949 of SEQ ID NO:3 and their RNA
equivalents are contemplated by the present invention. Table 1 sets
forth the one-letter codes used within SEQ ID NO:3 to denote
degenerate nucleotide positions. "Resolutions" are the nucleotides
denoted by a code letter. "Complement" indicates the code for the
complementary nucleotide(s). For example, the code Y denotes either
C or T, and its complement R denotes A or G, A being complementary
to T, and G being complementary to C.
1TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G
G G G C C T T A A R A.vertline.G Y C.vertline.T Y C.vertline.T R
A.vertline.G M A.vertline.C K G.vertline.T K G.vertline.T M
A.vertline.C S C.vertline.G S C.vertline.G W A.vertline.T W
A.vertline.T H A.vertline.C.vertline.T D A.vertline.G.vertline.T B
C.vertline.G.vertline.T V A.vertline.C.vertline.G V
A.vertline.C.vertline.G B C.vertline.G.vertline.T D
A.vertline.G.vertline.T H A.vertline.C.vertline.T N
A.vertline.C.vertline.G.vertline.T N
A.vertline.C.vertline.G.vertline.T
[0064] The degenerate codons used in SEQ ID NO:3, encompassing all
possible codons for a given amino acid, are set forth in Table
2.
2TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG lIe I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR
Asn.vertline.Asp B RAY Glu.vertline.Gln Z SAR Any X NNN
[0065] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequence of SEQ ID NO:2.
Variant sequences can be readily tested for functionality as
described herein.
[0066] One of ordinary skill in the art will also appreciate that
different species can exhibit "preferential codon usage." In
general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980;
Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene
13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol.
158:573-97, 1982. As used herein, the term "preferential codon
usage" or "preferential codons" is a term of art referring to
protein translation codons that are most frequently used in cells
of a certain species, thus favoring one or a few representatives of
the possible codons encoding each amino acid (See Table 2). For
example, the amino acid threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID NO:3 serves as a template for optimizing
expression of polynucleotides in various cell types and species
commonly used in the art and disclosed herein. Sequences containing
preferential codons can be tested and optimized for expression in
various species, and tested for functionality as disclosed
herein.
[0067] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO:1, other polynucleotide probes, primers, fragments and sequences
recited herein or sequences complementary thereto. Polynucleotide
hybridization is well known in the art and widely used for many
applications, see for example, Sambrook et al., Molecular Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
1989; Ausubel et al., eds., Current Protocols in Molecular Biology,
John Wiley and Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide
to Molecular Cloning Techniques, Methods in Enzymology, volume 152,
1987 and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-59, 1990.
Polynucleotide hybridization exploits the ability of single
stranded complementary sequences to form a double helix hybrid.
Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.
[0068] Hybridization will occur between sequences which contain
some degree of complementarity. Hybrids can tolerate mismatched
base pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Hybridization buffers generally contain blocking
agents such as Denhardt's solution (Sigma Chemical Co., St. Louis,
Mo.), denatured salmon sperm DNA, milk powders (BLOTTO), heparin or
SDS, and a Na.sup.+ source, such as SSC (1.times.SSC: 0.15 M NaCl,
15 mM sodium citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). By decreasing the ionic
concentration of the buffer, the stability of the hybrid is
increased. Typically, hybridization buffers contain from between 10
mM-1 M Na.sup.+. Premixed hybridization solutions are also
available from commercial sources such as Clontech Laboratories
(Palo Alto, Calif.) and Promega Corporation (Madison, Wis.) for use
according to manufacturer's instruction. Addition of destabilizing
or denaturing agents such as formamide, tetralkylammonium salts,
guanidinium cations or thiocyanate cations to the hybridization
solution will alter the T.sub.m of a hybrid. Typically, formamide
is used at a concentration of up to 50% to allow incubations to be
carried out at more convenient and lower temperatures. Formamide
also acts to reduce non-specific background when using RNA
probes.
[0069] Stringent hybridization conditions encompass temperatures of
about 5-25.degree. C. below the thermal melting point (T.sub.m) of
the hybrid and a hybridization buffer having up to 1 M Na.sup.+.
Higher degrees of stringency at lower temperatures can be achieved
with the addition of formamide which reduces the T.sub.m of the
hybrid about 1.degree. C. for each 1% formamide in the buffer
solution. Generally, such stringent conditions include temperatures
of 20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0070] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polypeptide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
that influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, see for example (Sambrook et al.,
ibid.; Ausubel et al., ibid.; Berger and Kimmel, ibid. and Wetmur,
ibid.) and are specific for DNA, RNA and DNA-RNA hybrids and
polynucleotide probe sequences of varying length. Sequence analysis
software such as Oligo 4.0 and Primer Premier, as well as sites on
the Internet, are available tools for analyzing a given sequence
and calculating T.sub.m based on user defined criteria. Such
programs can also analyze a given sequence under defined conditions
and suggest suitable probe sequences. Typically, hybridization of
longer polynucleotide sequences, >50 bp, is done at temperatures
of about 20-25.degree. C. below the calculated T.sub.m. For smaller
probes, <50 bp, hybridization is typically carried out at the
T.sub.m or 5-10.degree. C. below. This allows for the maximum rate
of hybridization for DNA-DNA and DNA-RNA hybrids.
[0071] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for preparing DNA
and RNA are well known in the art. In general, RNA is isolated from
a tissue or cell that produces large amounts of zfsta2 RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include brain and
spinal cord. Total RNA can be prepared using guanidinium
isothiocyanate extraction followed by isolation by centrifugation
in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
Poly (A).sup.+ RNA is prepared from total RNA using the method of
Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
Complementary DNA (cDNA) is prepared from poly(A).sup.+ RNA using
known methods. In the alternative, genomic DNA can be isolated.
Polynucleotides encoding zfsta2 polypeptides are then identified
and isolated by, for example, hybridization or PCR.
[0072] A full-length clone encoding a zfsta2 polypeptide can be
obtained by conventional cloning procedures. Complementary DNA
(cDNA) clones are preferred, although for some applications (e.g.,
expression in transgenic animals) it may be preferable to use a
genomic clone, or to modify a cDNA clone to include at least one
genomic intron. Methods for preparing cDNA and genomic clones are
well known and within the level of ordinary skill in the art, and
include the use of the sequence disclosed herein, or parts thereof,
for probing or priming a library. Expression libraries can be
probed with antibodies to zfsta2, receptor fragments, or other
specific binding partners.
[0073] The polynucleotides of the present invention can also be
synthesized using automated equipment. The current method of choice
is the phosphoramidite method. If chemically synthesized double
stranded DNA is required for an application such as the synthesis
of a gene or a gene fragment, then each complementary strand is
made separately. The production of short genes (60 to 80 bp) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 bp), however, special
strategies must be invoked, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length. Gene synthesis methods are well known in the
art. See, for example, Glick and Pasternak, Molecular
Biotechnology, Principles & Applications of Recombinant DNA,
ASM Press, Washington, D.C., 1994; Itakura et al., Annu. Rev.
Biochem. 53: 323-56, 1984; and Climie et al., Proc. Natl. Acad.
Sci. USA 87:633-7, 1990.
[0074] The present invention further provides counterpart
polypeptides and polynucleotides from other species (orthologs).
These species include, but are not limited to mammalian, avian,
amphibian, reptile, fish, insect and other vertebrate and
invertebrate species. Of particular interest are zfsta2
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human zfsta2 can be cloned using
information and compositions provided by the present invention in
combination, with conventional cloning techniques. For example, a
cDNA can be cloned using mRNA obtained from a tissue or cell type
that expresses zfsta2 as disclosed herein. Suitable sources of mRNA
can be identified by probing Northern blots with probes designed
from the sequences disclosed herein. A library is then prepared
from mRNA of a positive tissue or cell line. A zfsta2-encoding cDNA
can then be isolated by a variety of methods, such as by probing
with a complete or partial human cDNA or with one or more sets of
degenerate probes based on the disclosed sequences. A cDNA can also
be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.
Pat. No. 4,683,202), using primers designed from the representative
human zfsta2 sequence disclosed herein. Within an additional
method, the cDNA library can be used to transform or transfect host
cells, and expression of the cDNA of interest can be detected with
an antibody to zfsta2 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
[0075] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human zfsta2
and that allelic variation and alternative splicing are expected to
occur. Allelic variants of this sequence can be cloned by probing
cDNA or genomic libraries from different individuals according to
standard procedures. Allelic variants of the DNA sequence shown in
SEQ ID NO:1, including those containing silent mutations and those
in which mutations result in amino acid sequence changes, are
within the scope of the present invention, as are proteins which
are allelic variants of SEQ ID NO:2. cDNAs generated from
alternatively spliced mRNAs, which retain the properties of the
zfsta2 polypeptide are included within the scope of the present
invention, as are polypeptides encoded by such cDNAs and mRNAs.
Splice variants are known in the follistatin family, follistatin
exists in at least three forms (32,000, 35,000 and 39,000 Da) due
to alternative splicing. Allelic variants and splice variants of
these sequences can be cloned by probing cDNA or genomic libraries
from different individuals or tissues according to standard
procedures known in the art.
[0076] The present invention also provides isolated zfsta2
polypeptides that are substantially homologous to the polypeptides
of SEQ ID NO:2 and their orthologs. The term "substantially
homologous" is used herein to denote polypeptides having 50%,
preferably 60%, more preferably at least 80%, sequence identity to
the sequences shown in SEQ ID NO:2 or their orthologs. Such
polypeptides will more preferably be at least 90% identical, and
most preferably 95% or more identical to SEQ ID NO:2 or its
orthologs. Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:
603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915-9, 1992. Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "blosum 62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: 1 Total number of identical matches [ length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences ] .times.
100
3 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0077] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0078] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant zfsta2. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat. Acad. Sci.
USA 85:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990.
[0079] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2) and a test sequence that have either the highest density of
identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the highest density
of identities are then re-scored by comparing the similarity of all
paired amino acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those residues
that contribute to the highest score. If there are several regions
with scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974),
which allows for amino acid insertions and deletions. Illustrative
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63, 1990.
[0080] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from four to six.
[0081] The BLOSUM62 table is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein
sequence segments, representing highly conserved regions of more
than 500 groups of related proteins (Henikoff and Henikoff, Proc.
Natl. Acad. Sci. USA 89:10915, 1992). Accordingly, the BLOSUM62
substitution frequencies can be used to define conservative amino
acid substitutions that may be introduced into the amino acid
sequences of the present invention. Although it is possible to
design amino acid substitutions based solely upon chemical
properties (as discussed above), the language "conservative amino
acid substitution" preferably refers to a substitution represented
by a BLOSUM62 value of greater than -1. For example, an amino acid
substitution is conservative if the substitution is characterized
by a BLOSUM62 value of 0, 1, 2, or 3. According to this system,
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
[0082] Conservative amino acid changes in a zfsta2 gene can be
introduced by substituting nucleotides for the nucleotides recited
in SEQ ID NO:1. Such "conservative amino acid" variants can be
obtained, for example, by oligonucleotide-directed mutagenesis,
linker-scanning mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22;
and McPherson (ed.), Directed Mutacenesis: A Practical Approach
(IRL Press 1991)). To select for variants having the properties of
the wild-type protein can be done using standard methods, such as
the assays described herein. Alternatively, a variant zfsta2
polypeptide can be identified by the ability to specifically bind
anti-zfsta2 antibodies.
[0083] Variant zfsta2 polypeptides or substantially homologous
zfsta2 polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions (see Table 4) and other substitutions that do not
significantly affect the folding or activity of the polypeptide;
small deletions, typically of one to about 30 amino acids; and
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. Polypeptides comprising
affinity tags can further comprise a proteolytic cleavage site
between the zfsta2 polypeptide and the affinity tag. Preferred such
sites include thrombin cleavage sites and factor Xa cleavage
sites.
4TABLE 4 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serine threonine methionine
[0084] The present invention further provides a variety of other
polypeptide fusions. For example, a zfsta2 polypeptide can be
prepared as a fusion to a dimerizing protein as disclosed in U.S.
Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in
this regard include immunoglobulin constant region domains.
Immunoglobulin-zfsta2 polypeptide fusions can be expressed in
genetically engineered cells to produce a variety of multimeric
zfsta2 analogs. Auxiliary domains can be fused to zfsta2
polypeptides to target them to specific cells, tissues, or
macromolecules. For example, a zfsta2 polypeptide or protein could
be targeted to a predetermined cell type by fusing a zfsta2
polypeptide to a ligand that specifically binds to a receptor on
the surface of the target cell. In this way, polypeptides and
proteins can be targeted for therapeutic or diagnostic purposes. A
zfsta2 polypeptide can be fused to two or more moieties, such as an
affinity tag for purification and a targeting domain. Polypeptide
fusions can also comprise one or more cleavage sites, particularly
between domains. See, Tuan et al., Conn. Tiss. Res. 34:1-9,
1996.
[0085] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991;
Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,
translation is carried out in Xenopus oocytes by microinjection of
mutated mRNA and chemically aminoacylated suppressor tRNAs
(Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third
method, E. coli cells are cultured in the absence of a natural
amino acid that is to be replaced (e.g., phenylalanine) and in the
presence of the desired non-naturally occurring amino acid(s)
(e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine,
or 4-fluorophenylalanine). The non-naturally occurring amino acid
is incorporated into the protein in place of its natural
counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally
occurring amino acid residues can be converted to non-naturally
occurring species by in vitro chemical modification. Chemical
modification can be combined with site-directed mutagenesis to
further expand the range of substitutions (Wynn and Richards,
Protein Sci. 2:395-403, 1993).
[0086] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for zfsta2 amino acid residues.
[0087] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass
et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). In the
latter technique, single alanine mutations are introduced at every
residue in the molecule, and the resultant mutant molecules are
tested for biological activity as disclosed below to identify amino
acid residues that are critical to the activity of the molecule.
See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites
of ligand-receptor interaction can also be determined by physical
analysis of structure, as determined by such techniques as nuclear
magnetic resonance, crystallography, electron diffraction or
photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids. See, for example, de Vos et al., Science
255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992;
Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of
essential amino acids can also be inferred from analysis of
homologies with related follistatins.
[0088] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-olson and Sauer (Science 241:53-7, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et
al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204)
and region-directed mutagenesis (Derbyshire et al., Gene 46:145,
1986; Ner et al., DNA 7:127, 1988).
[0089] Variants of the disclosed zfsta2 DNA and polypeptide
sequences can be generated through DNA shuffling as disclosed by
Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci.
USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly,
variant DNAs are generated by in vitro homologous recombination by
random fragmentation of a parent DNA followed by reassembly using
PCR, resulting in randomly introduced point mutations. This
technique can be modified by using a family of parent DNAs, such as
allelic variants or DNAs from different species, to introduce
additional variability into the process. Selection or screening for
the desired activity, followed by additional iterations of
mutagenesis and assay provides for rapid "evolution" of sequences
by selecting for desirable mutations while simultaneously selecting
against detrimental changes.
[0090] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using modern equipment. These
methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide of interest, and
can be applied to polypeptides of unknown structure.
[0091] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptide
fragments or variants of SEQ ID NO:2 that retain the properties of
the wild-type zfsta2 protein. Such polypeptide fragments may
include the N-terminal region, the follistatin and/or calmodulin
homology domains, I-set IG domains #1 and/or #2, the alpha-helical
linker and the C-terminal region. Amino acid truncations or
additions can also occur.
[0092] For any zfsta2 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above.
[0093] The zfsta2 polypeptides of the present invention, including
full-length polypeptides, biologically active fragments, and fusion
polypeptides, can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells, and
cultured higher eukaryotic cells. Eukaryotic cells, particularly
cultured cells of multicellular organisms, are preferred.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, and Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987.
[0094] In general, a DNA sequence encoding a zfsta2 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0095] To direct a zfsta2 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be that of
zfsta2, or may be derived from another secreted protein (e.g.,
t-PA) or synthesized de novo. The secretory signal sequence is
operably linked to the zfsta2 DNA sequence, i.e., the two sequences
are joined in the correct reading frame and positioned to direct
the newly synthesized polypeptide into the secretory pathway of the
host cell. Secretory signal sequences are commonly positioned 5' to
the DNA sequence encoding the polypeptide of interest, although
certain secretory signal sequences may be positioned elsewhere in
the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat.
No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
[0096] Alternatively, the secretory signal sequence contained in
the polypeptides of the present invention is used to direct other
polypeptides into the secretory pathway. The present invention
provides for such fusion polypeptides. A signal fusion polypeptide
can be made wherein a secretory signal sequence derived from amino
acid residues 1-20 of SEQ ID NO:2 is be operably linked to another
polypeptide using methods known in the art and disclosed herein.
The secretory signal sequence contained in the fusion polypeptides
of the present invention is preferably fused amino-terminally to an
additional peptide to direct the additional peptide into the
secretory pathway. Such constructs have numerous applications known
in the art. For example, these novel secretory signal sequence
fusion constructs can direct the secretion of an active component
of a normally non-secreted protein. Such fusions-may be used in
vivo or in vitro to direct peptides through the secretory
pathway.
[0097] Cultured mammalian cells are suitable hosts within the
present invention: Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al.,
ibid.), and liposome-mediated transfection (Hawley-Nelson et al.,
Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral
vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and
Finer, Nature Med. 2:714-6, 1996). The production of recombinant
polypeptides in cultured mammalian cells is disclosed, for example,
by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.
Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and
Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells
include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651),
BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC
No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and
Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
Additional suitable cell lines are known in the art and available
from public depositories such as the American Type Culture
Collection, Manassas, Va. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable
promoters include those from metallothionein genes (U.S. Pat. Nos.
4,579,821 and 4,601,978) and the adenovirus major late
promoter.
[0098] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
Alternative markers that introduce an altered phenotype, such as
green fluorescent protein, or cell surface proteins such as CD4,
CD8, Class I MHC, placental alkaline phosphatase may be used to
sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0099] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). DNA
encoding the zfsta2 polypeptide is inserted into the baculoviral
genome in place of the AcNPV polyhedrin gene coding sequence by one
of two methods. The first is the traditional method of homologous
DNA recombination between wild-type AcNPV and a transfer vector
containing the zfsta2 flanked by AcNPV sequences. Suitable insect
cells, e.g. SF9 cells, are infected with wild-type AcNPV and
transfected with a transfer vector comprising a zfsta2
polynucleotide operably linked to an AcNPV polyhedrin gene
promoter, terminator, and flanking sequences. See, King and Possee,
The Baculovirus Expression System: A Laboratory Guide, London,
Chapman & Hall; O'Reilly et al., Baculovirus Expression
Vectors: A Laboratory Manual, New York, Oxford University Press.,
1994; and, Richardson, Ed., Baculovirus Expression Protocols.
Methods in Molecular Biology, Totowa, N.J., Humana Press, 1995.
Natural recombination within an insect cell will result in a
recombinant baculovirus which contains zfsta2 driven by the
polyhedrin promoter. Recombinant viral stocks are made by methods
commonly used in the art.
[0100] The second method of making recombinant baculovirus utilizes
a transposon-based system described by Luckow (Luckow et al., J.
Virol. 67:4566-79, 1993). This system is sold in the Bac-to-Bac kit
(Life Technologies, Rockville, Md.). This system utilizes a
transfer vector, pFastBac1.TM. (Life Technologies) containing a Tn7
transposon to move the DNA encoding the zfsta2 polypeptide into a
baculovirus genome maintained in E. coli as a large plasmid called
a "bacmid." The pFastBac1.TM. transfer vector utilizes the AcNPV
polyhedrin promoter to drive the expression of the gene of
interest, in this case zfsta2. However, pFastBac1.TM. can be
modified to a considerable degree. The polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins. See, Hill-Perkins
and Possee, J. Gen. Virol. 71:971-6, 1990; Bonning et al., J. Gen.
Virol. 75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, J.
Biol. Chem. 270:1543-9, 1995. In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native zfsta2 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67
(PharMingen, San Diego, Calif.) can be used in constructs to
replace the native secretory signal sequence. In addition, transfer
vectors can include an in-frame fusion with DNA encoding an epitope
tag at the C- or N-terminus of the expressed zfsta2 polypeptide,
for example, a Glu-Glu epitope tag (Grussenmeyer et al., ibid.).
Using a technique known in the art, a transfer vector containing
zfsta2 is transformed into E. coli, and screened for bacmids which
contain an interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant baculovirus
genome is isolated, using common techniques, and used to transfect
Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that
expresses zfsta2 is subsequently produced. Recombinant viral stocks
are made by methods commonly used the art.
[0101] The recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodoptera
frugiperda. See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. Another suitable cell line is the
High FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
are used to grow and maintain the cells. Suitable media are Sf900
II.TM. (Life Technologies) or ESF 921.TM. (Expression Systems) for
the Sf9 cells; and Ex-cellO400.TM. (JRH Biosciences, Lenexa, Kans.)
or Express FiveO.TM. (Life Technologies) for the T. ni cells. The
cells are grown up from an inoculation density pf approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3. The
recombinant virus-infected cells typically produce the recombinant
zfsta2 polypeptide at 12-72 hours post-infection and secrete it
with varying efficiency into the medium. The culture is usually
harvested 48 hours post-infection. Centrifugation is used to
separate the cells from the medium (supernatant). The supernatant
containing the zfsta2 polypeptide is filtered through micropore
filters, usually 0.45 .mu.m pore size. Procedures used are
generally described in available laboratory manuals (King and
Possee, ibid.; O'Reilly et al., ibid.; Richardson, C. D., ibid.).
Subsequent purification of the zfsta2 polypeptide from the
supernatant can be achieved using methods described herein.
[0102] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0103] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23, 1998, and in WIPO Publication Nos. WO
97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules
for use in transforming P. methanolica will commonly be prepared as
double-stranded, circular plasmids, which are preferably linearized
prior to transformation. For polypeptide production in P.
miethanolica, it is preferred that the promoter and terminator in
the plasmid be that of a P. mnethanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A preferred selectable
marker for use in Pichia mnethanolica is a P. methanolica ADE2
gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase
(AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the
absence of adenine. For large-scale, industrial processes where it
is desirable to minimize the use of methanol, it is preferred to
use host cells in which both methanol utilization genes (AUG1 and
AUG2) are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PBP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (.tau.) of from 1
to 40 milliseconds, most preferably about 20 milliseconds.
[0104] Prokaryotic host cells, including strains of the bacteria
Escherichia coli Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a zfsta2 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic space by
a bacterial secretion sequence. In the former case, the cells are
lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0105] The adenovirus system can also be used for protein
production in vitro. By culturing adenovirus-infected non-293 cells
under conditions where the cells are not rapidly dividing, the
cells can produce proteins for extended periods of time. For
instance, BHK cells are grown to confluence in cell factories, then
exposed to the adenoviral vector encoding the secreted protein of
interest. The cells are then grown under serum-free conditions,
which allows infected cells to survive for several weeks without
significant cell division. Alternatively, adenovirus vector
infected 293 cells can be grown as adherent cells or in suspension
culture at relatively high cell density to produce significant
amounts of protein (see Garnier et al., Cytotechnol. 15:145-55,
1994). With either protocol, an expressed, secreted heterologous
protein can be repeatedly isolated from the cell culture
supernatant. Within the infected 293 cell production protocol,
non-secreted proteins may also be effectively obtained.
[0106] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell. P. methanolica cells
are cultured in a medium comprising adequate sources of carbon,
nitrogen and trace nutrients at a temperature of about 25.degree.
C. to 35.degree. C. Liquid cultures are provided with sufficient
aeration by conventional means, such as shaking of small flasks or
sparging of fermentors. A preferred culture medium for P.
methanolica is YEPD (2% D-glucose, 2% Bacto.TM. Peptone (Difco
Laboratories, Detroit, Mich.), 1% Bacto.TM. yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0107] It is preferred to purify the polypeptides of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified polypeptide
is substantially free of other polypeptides, particularly other
polypeptides of animal origin.
[0108] Expressed recombinant zfsta2 polypeptides (or chimeric
zfsta2 polypeptides) can be purified using fractionation and/or
conventional purification methods and media. Ammonium sulfate
precipitation and acid or chaotrope extraction may be used for
fractionation of samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase high
performance liquid chromatography. Suitable chromatographic media
include derivatized dextrans, agarose, cellulose, polyacrylamide,
specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives
are preferred. Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic,.resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Methods for binding receptor
polypeptides to support media are well known in the art. Selection
of a particular method is a matter of routine design and is
determined in part by the properties of the chosen support. See,
for example, Affinity Chromatography: Principles & Methods,
Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
[0109] The polypeptides of the present invention can be isolated by
exploitation of physical properties of the zfsta2 sequence or
properties of coupled tags or epitopes. For example, immobilized
metal ion adsorption (IMAC) chromatography can be used to purify
histidine-rich proteins, including those comprising polyhistidine
tags. Briefly, a gel is first charged with divalent metal ions to
form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985).
Histidine-rich proteins will be adsorbed to this matrix with
differing affinities, depending upon the metal ion used, and will
be eluted by competitive elution, lowering the pH, or use of strong
chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (Methods in
Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher,
(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, FLAG
tag, Glu-Glu tag, an immunoglobulin domain) may be constructed to
facilitate purification. An exemplary purification method of
protein constructs having an N-terminal or C-terminal affinity tag
involves using an antibody to the affinity tag epitope to Ipurify
the protein using chromatography methods known in the art.
SDS-PAGE, Western analysis, amino acid analysis and N-terminal
sequencing can be done to confirm the identity of the purified
protein.
[0110] Protein refolding (and optionally reoxidation) procedures
may be advantageously used. It is preferred to purify the protein
to >80% purity, more preferably to >90% purity, even more
preferably >95%, and particularly preferred is a
pharmaceutically pure state, that is greater than 99.9% pure with
respect to contaminating macromolecules, particularly other
proteins and nucleic acids, and free of infectious and pyrogenic
agents. Preferably, a purified protein is substantially free of
other proteins, particularly other proteins of animal origin.
[0111] Proteins/polypeptides which bind zfsta2 (such as a
zfsta2-binding receptor) can also be used for purification of
zfsta2. The zfsta2-binding protein/polypeptide is immobilized on a
solid support, such as beads of agarose, cross-linked agarose,
glass, cellulosic resins, silica-based resins, polystyrene,
cross-linked polyacrylamide, or like materials that are stable
under the conditions of use. Methods for linking polypeptides to
solid supports are known in the art, and include amine chemistry,
cyanogen bromide activation, N-hydroxysuccinimide activation,
epoxide activation, sulfhydryl activation, and hydrazide
activation. The resulting medium will generally be configured in
the form of a column, and fluids containing zfsta2 polypeptide are
passed through the column one or more times to allow zfsta2
polypeptide to bind to the ligand-binding or receptor polypeptide.
The bound zfsta2 polypeptide is then eluted using changes in salt
concentration, chaotropic agents (guanidine HCl), or pH to disrupt
ligand-receptor binding.
[0112] Moreover, using methods described in the art, polypeptide
fusions, or hybrid zfsta2 proteins, are constructed using regions
or domains of the inventive zfsta2 in combination with other
polypeptides, in particular, those of other follistatin family
proteins (e.g. FRP, SPARC, agrin or hevin), or heterologous
proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard,
Cur. Opin. Biology, 5:511-5, 1994, and references therein). These
methods allow the determination of the biological importance of
larger domains or regions in a polypeptide of interest. Such
hybrids may alter reaction kinetics, binding, constrict or expand
the substrate specificity, or alter tissue and cellular
localization of a polypeptide, and can be applied to polypeptides
of unknown structure.
[0113] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. For example, part or
all of a domain(s) conferring a biological function may be swapped
between zfsta2 of the present invention with the functionally
equivalent domain(s) from another family member, such as FRP. Such
domains include, but are not limited to, the secretory signal
sequence, follistatin homology domain, calmodulin homology domain,
I-set IG domains #1 and #2, the N or C-terminal domains and the
alpha helical linker, for example. Such fusion proteins would be
expected to have a biological functional profile that is the same
or similar to polypeptides of the present invention or other known
follistatin family proteins described herein, depending on the
fusion constructed. Moreover, such fusion proteins may exhibit
other properties as disclosed herein.
[0114] zfsta2 polypeptides or fragments thereof may also be
prepared through chemical synthesis. zfsta2 polypeptides may be
monomers or multimers; glycosylated or non-glycosylated; pegylated
or non-pegylated; and may or may not include an initial methionine
amino acid residue. Polypeptides, especially polypeptides of the
present invention, can also be synthesized as described by
Merrifield, J. Am. Chem. Soc. 85:2149, 1963, Stewart et al., "Solid
Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co.,
Rockford, Ill., 1984) and Bayer & Rapp Chem. Pest. Prot. 3:3,
1986 and Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach, IRL Press, Oxford, 1989, for example.
[0115] As described above, the disclosed polypeptides can be used
to construct zfsta2 variants and functional fragments of zfsta2.
Such variants and extracellular domain fragments are considered to
be zfsta2 agonists. Another type of zfsta2 agonist is provided by
anti-idiotype antibodies, and fragments thereof, which mimic the
extracellular domain of zfsta2. Moreover, recombinant antibodies
comprising anti-idiotype variable domains that mimic the zfsta2
extracellular domain can be used as agonists (see, for example,
Monfardini et al., Proc. Assoc. Am. Physicians 108:420, 1996).
zfsta2 agonists can also be constructed using combinatorial
libraries. Methods for constructing and screening phage display and
other combinatorial libraries are provided, for example, by Kay et
al., Phage Display of Peptides and Proteins (Academic Press 1996),
Verdine, U.S. Pat. No. 5,783,384, Kay, et. al., U.S. Pat. No.
5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.
[0116] The invention also provides antagonists, which either bind
to zfsta2 polypeptides or, alternatively, to a receptor to which
zfsta2 polypeptides bind, thereby inhibiting or eliminating the
function of zfsta2. Such zfsta2 antagonists would include
antibodies; oligonucleotides which bind either to the zfsta2
polypeptide or to its receptor; natural or synthetic analogs of
zfsta2 polypeptides which retain the ability to bind the receptor
but do not result in either ligand or receptor signaling. Such
analogs could be peptides or peptide-like compounds. Natural or
synthetic small molecules which bind to receptors of zfsta2
polypeptides and prevent signaling are also contemplated as
antagonists. As such, zfsta2 antagonists would be useful as
therapeutics for treating certain disorders where blocking signal
from either a zfsta2 ligand or receptor would be beneficial.
[0117] zfsta2 can also be used to identify inhibitors (antagonists)
of its activity. Test compounds are added to the assays disclosed
herein to identify compounds that inhibit the activity of zfsta2.
In addition to those assays disclosed herein, samples can be tested
for inhibition of zfsta2 activity within a variety of assays
designed to measure receptor binding or the stimulation/inhibition
of zfsta2-dependent cellular responses. For example,
zfsta2-responsive cell lines can be transfected with a reporter
gene construct that is responsive to a zfsta2-stimulated cellular
pathway. Reporter gene constructs of this type are known in the
art, and will generally comprise a zfsta2-DNA response element
operably linked to a gene encoding an assayable protein, such as
luciferase. DNA response elements can include, but are not limited
to, cyclic AMP response elements (CRE), hormone response elements
(HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl.
Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE)
(Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements
are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6;
1988 and Habener, Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone
response elements are reviewed in Beato, Cell 56:335-44; 1989.
Candidate compounds, solutions, mixtures or extracts are tested for
the ability to inhibit the activity of zfsta2 on the target cells
as evidenced by a decrease in zfsta2 stimulation of reporter gene
expression. Assays of this type will detect compounds that directly
block zfsta2 binding to cell-surface receptors, as well as
compounds that block processes in the cellular pathway subsequent
to receptor-ligand binding. In the alternative, compounds or other
samples can be tested for direct blocking of zfsta2 binding to
receptor using zfsta2 tagged with a detectable label (e.g.,
.sup.125I, biotin, horseradish peroxidase, FITC, and the like).
Within assays of this type, the ability of a test sample to inhibit
the binding of labeled zfsta2 to the receptor is indicative of
inhibitory activity, which can be confirmed through secondary
assays. Receptors used within binding assays may be cellular
receptors or isolated, immobilized receptors.
[0118] The invention also provides isolated and purified zfsta2
polynucleotide probes and/or primers. The probes and/or primers can
be RNA or DNA. DNA can be either cDNA or genomic DNA.
Polynucleotide probes and primers are single or double-stranded DNA
or RNA, generally synthetic oligonucleotides, but may be generated
from cloned cDNA or genomic sequences or its complements.
Analytical probes will generally be at least 20 nucleotides in
length, although somewhat shorter probes (14-17 nucleotides) can be
used. PCR primers are at least 5 nucleotides in length, preferably
15 or more nt, more preferably 20-30 nt. Short plolynucleotide
probes can be used when a small region of the gene is targeted for
analysis. For gross analysis of genes, a polynucleotide probe may
comprise an entire Axon or more.
[0119] Such probes can also be used in hybridizations to detect the
presence or quantify the amount of zfsta2 gene or mRNA transcript
in a sample. zfsta2 polynucleotide probes could be used to
hybridize to DNA or RNA targets for diagnostic purposes, using such
techniques such as fluorescent in situ hybridization (FISH) or
immunohistochemistry. Polynucleotide probes could be used to
identify genes encoding zfsta2-like proteins. Such probes can also
be used to screen libraries for related zfsta2 sequences. Such
screening would be carried out under conditions of lower stringency
which would allow identification of sequences which are
substantially homologous, but not requiring complete homology to
the probe sequence. Such methods and conditions are well known in
the art, see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989.
Such stringency conditions are described herein. Libraries may be
made of genomic DNA or cDNA. Polynucleotide probes are also useful
for Southern, Northern, or slot blots, colony and plaque
hybridization and in situ hybridization. Mixtures of different
zfsta2 polynucleotide probes can be prepared which would increase
sensitivity or the detection of low copy number targets, in
screening systems.
[0120] Nucleic acid molecules can be used to detect the expression
of a zfsta2 gene in a biological sample. In a basic assay, a
single-stranded probe molecule is incubated with RNA, isolated from
a biological sample, under conditions of temperature and ionic
strength that promote base pairing between the probe and target
zfsta2 RNA species. After separating unbound probe from hybridized
molecules, the amount of hybrids is detected.
[0121] A method of detecting the presence of zfsta2 RNA in a
biological sample is provided, comprising the steps of:
[0122] a) contacting a zfsta2 nucleic acid probe under stringent
hybridizing conditions with either
[0123] i) test RNA molecules isolated from the biological sample,
or
[0124] ii) nucleic acid molecules synthesized from the isolated RNA
molecules,
[0125] wherein the probe has a nucleotide sequence comprising a
portion of the nucleotide sequence of SEQ ID NOs:1 or 3, or their
complements, and
[0126] b) detecting the formation of hybrids of the nucleic acid
probe and either the test RNA molecules or the synthesized nucleic
acid molecules,
[0127] wherein the presence of the hybrids indicates the presence
of zfsta2 RNA is the biological sample.
[0128] Well-established hybridization methods of RNA detection
include northern analysis and dot/slot blot hybridization (see, for
example, Ausubel ibid. at pages 4-1 to 4-27, and Wu et al. (eds.),
"Analysis of Gene Expression at the RNA Level," .sup.1 in Methods
in Gene Biotechnology, pages 225-39, CRC Press, Inc., 1997).
Nucleic acid probes can be detectably labeled with radioisotopes
such as .sup.32P or .sup.35S. Alternatively, zfsta2 RNA can be
detected with a nonradioactive hybridization method (see, for
example, Isaac (ed.), Protocols for Nucleic Acid Analysis by
Nonradioactive Probes, Humana Press, Inc., 1993). Typically,
nonradioactive detection is achieved by enzymatic conversion of
chromogenic or chemiluminescent substrates. Illustrative
non-radioactive moieties include biotin, fluorescein, and
digoxigenin.
[0129] zfsta2 oligonucleotide probes are also useful for in vivo
diagnosis. As an illustration, .sup.18F-labeled oligonucleotides
can be administered to a subject and visualized by positron
emission tomography (Tavitian et al., Nat. Med. 4:467, 1998).
[0130] Numerous diagnostic procedures take advantage of the
polymerase chain reaction (PCR) to increase isensitivity of
detection methods. Standard techniques for performing PCR are
well-known (see, generally, Mathew (ed.), Protocols in Human
Molecular Genetics, Humana Press, Inc., 1991; White (ed.), PCR
Protocols: Current Methods and Applications, Humana Press, Inc.,
1993; Cotter (ed.), Molecular Diagnosis of Cancer, Humana Press,
Inc., 1996; Hanausek and Walaszek (eds.), Tumor Marker Protocols,
Humana Press, Inc., 1998; Lo (ed.), Clinical Applications of PCR,
Humana Press, Inc., 1998 and Meltzer (ed.), PCR in Bioanalysis,
Humana Press, Inc., 1998). PCR primers can be designed to amplify a
sequence encoding a particular zfsta2 region, such as the
follistatin homology domain, encoded by about nucleotide 250 to
nucleotide 456 of SEQ ID NO:1, and the calmodulin domain, encoded
by about nucleotide 580 to nucleotide 810 of SEQ ID NO:1.
[0131] One variation of PCR for diagnostic assays is reverse
transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is
isolated from a biological sample, reverse transcribed to cDNA, and
the cDNA is incubated with zfsta2 primers (see, for example, Wu et
al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in
Methods in Gene Biotechnology, pages 15-28, CRC Press, Inc. 1997).
PCR is then performed and the products are analyzed using standard
techniques.
[0132] As an illustration, RNA is isolated from a biological sample
using, for example, the guanidinium-thiocyanate cell lysis
procedure described above. Alternatively, a solid-phase technique
can be used to isolate mRNA from a cell lysate. A reverse
transcription reaction can be primed with the isolated RNA using
random oligonucleotides, short homopolymers of dT, or zfsta2
anti-sense oligomers. Oligo-dT primers offer the advantage that
various mRNA nucleotide sequences are amplified that can provide
control target sequences. zfsta2 sequences are amplified by the
polymerase chain reaction using two flanking oligonucleotide
primers that are typically 20 bases in length.
[0133] PCR amplification products can be detected using a variety
of approaches. For example, PCR products can be fractionated by gel
electrophoresis, and visualized by ethidium bromide staining.
Alternatively, fractionated PCR products can be transferred to a
membrane, hybridized with a detectably-labeled zfsta2 probe, and
examined by autoradiography. Additional alternative approaches
include the use of digoxigenin-labeled deoxyribonucleic acid
triphosphates to provide chemiluminescence detection, and the
C-TRAK calorimetric assay.
[0134] Another approach is real time quantitative PCR (Perkin-Elmer
Cetus, Norwalk, Conn.). A fluorogenic probe, consisting of an
oligonucleotide with both a reporter and a quencher dye attached,
anneals specifically between the forward and reverse primers. Using
the 5' endonuclease activity of Taq DNA polymerase, the reporter
dye is separated from the quencher dye and a sequence-specific
signal is generated and increases as amplification increases. The
fluorescence intensity can be continuously monitored and quantified
during the PCR reaction.
[0135] Another approach for detection of zfsta2 expression is
cycling probe technology (CPT), in which a single-stranded DNA
target binds with an excess of DNA-RNA-DNA chimeric probe to form a
complex, the RNA portion is cleaved with RNase H, and the presence
of cleaved chimeric probe is detected (see, for example, Beggs et
al., J. Clin. Microbiol. 34:2985, 1996 and Bekkaoui et al.,
Biotechniques 20:240, 1996). Alternative methods for detection of
zfsta2 sequences can utilize approaches such as nucleic acid
sequence-based amplification (NASBA), cooperative amplification of
templates by cross-hybridization (CATCH), and the ligase chain
reaction (LCR) (see, for example, Marshall et al., U.S. Pat. No.
5,686,272 (1997), Dyer et al. J. Virol. Methods 60:161, 1996;
Ehricht et al., Eur. J. Biochem. 243:358, 1997; and Chadwick et
al., J. Virol. Methods 70:59, 1998). Other standard methods are
known to those of skill in the art.
[0136] Zfsta2 probes and primers can also be used to detect and to
localize zfsta2 gene expression in tissue samples. Methods for such
in situ hybridization are well-known to those of skill in the art
(see, for example, Choo (ed.), In Situ Hybridization Protocols,
Humana Press, Inc., 1994; Wu et al. (eds.), "Analysis of Cellular
DNA or Abundance of mRNA by Radioactive In Situ Hybridization
(RISH)," in Methods in Gene Biotechnology, pages 259-278, CRC
Press, Inc., 1997; and Wu et al. (eds.), "Localization of DNA or
Abundance of mRNA by Fluorescence In Situ Hybridization (RISH)," in
Methods in Gene Biotechnology, pages 279-289, CRC Press, Inc.,
1997).
[0137] Various additional diagnostic approaches are well-known to
those of skill in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics, Humana Press, Inc., 1991;
Coleman and Tsongalis, Molecular Diagnostics, Humana Press, Inc.,
1996; and Elles, Molecular Diagnosis of Genetic Diseases, Humana
Press, Inc., 1996).
[0138] An assay system that uses a ligand-binding receptor (or an
antibody, one member of a complement/anti-complement pair) or a
binding fragment thereof, and a commercially available biosensor
instrument (BIAcore, Pharmacia Biosensor, Piscataway, N.J.) may be
advantageously employed. Such receptor, antibody, member of a
complement/anti-complement pair or fragment is immobilized onto the
surface of a receptor chip. Use of this instrument is disclosed by
Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and
Wells, J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member
or fragment is covalently attached, using amine or sulfhydryl
chemistry, to dextran fibers that are attached to gold film within
the flow cell. A test sample is passed through the cell. If a
ligand, epitope, or opposite member of the
complement/anti-complement pair is present in the sample, it will
bind to the immobilized receptor, antibody or member, respectively,
causing a change in the refractive index of the medium, which is
detected as a change in surface plasmon resonance of the gold film.
This system allows the determination of on- and off-rates, from
which binding affinity can be calculated, and assessment of
stoichiometry of binding.
[0139] Ligand-binding receptor polypeptides can also be used within
other assay systems known in the art. Such systems include
Scatchard analysis for determination of binding affinity (see
Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric
assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et
al., Science 245:821-25, 1991).
[0140] Probes and primers generated from the sequences disclosed
herein can be used to map the zfsta2 gene to human chromosome 4.
Radiation hybrid mapping is a somatic cell genetic technique
developed for constructing high-resolution, contiguous maps of
mammalian chromosomes (Cox et al., Science 250:245-50, 1990).
Partial or full knowledge of a gene's sequence allows one to design
PCR primers suitable for use with chromosomal radiation hybrid
mapping panels. Radiation hybrid mapping panels are commercially
available which cover the entire human genome, such as the Stanford
G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc.,
Huntsville, Ala.). These panels enable rapid, PCR-based chromosomal
localizations and ordering of genes, sequence-tagged sites (STSs),
and other nonpolymorphic and polymorphic markers within a region of
interest. This includes establishing directly proportional physical
distances between newly discovered genes of interest and previously
mapped markers. The precise knowledge of a gene's position can be
useful for a number of purposes, including: 1) determining if a
sequence is part of an existing contig and obtaining additional
surrounding genetic sequences in various forms, such as YACs, BACs
or cDNA clones; 2) providing a possible candidate gene for an
inheritable disease which shows linkage to the same chromosomal
region; and 3) cross-referencing model organisms, such as mouse,
which may aid in determining what function a particular gene might
have.
[0141] Sequence tagged sites (STSs) can also be used independently
for chromosomal localization. An STS is a DNA sequence that is
unique in the human genome and can be used as a reference point for
a particular chromosome or region of a chromosome. An STS is
defined by a pair of oligonucleotide primers that are used in a
polymerase chain reaction to specifically detect this site in the
presence of all other genomic sequences. Since STSs are based
solely on DNA sequence they can be completely described within an
electronic database, for example, Database of Sequence Tagged Sites
(dbSTS), GenBank, (National Center for Biological Information,
National Institutes of Health, Bethesda, Md. http://www.ncbi.nlm.
nih.gov), and can be searched with a gene sequence of interest for
the mapping data contained within these short genomic landmark STS
sequences.
[0142] The present invention also contemplates use of such
chromosomal localization for. diagnostic applications. Briefly, the
zfsta2 gene, a probe comprising zfsta2 DNA or RNA or a subsequence
thereof, can be used to determine if the zfsta2 gene is present on
human chromosome 4 or if a mutation has occurred. Detectable
chromosomal aberrations at the zfsta2 gene locus include, but are
not limited to, aneuploidy, gene copy number changes, insertions,
deletions, restriction site changes and rearrangements. Such
aberrations can be detected using polynucleotides of the present
invention by employing molecular genetic techniques, such as
restriction fragment length polymorphism (RFLP) analysis, short
tandem repeat (STR) analysis employing PCR techniques, and other
genetic linkage analysis techniques known in the art (Sambrook et
al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65,
1995).
[0143] In general, these diagnostic methods comprise the steps of
(a) obtaining a genetic sample from a patient; (b) incubating the
genetic sample with a polynucleotide probe or primer as disclosed
above, under conditions wherein the polynucleotide will hybridize
to complementary polynucleotide sequence, to produce a first
reaction product; and (iii) comparing the first reaction product to
a control reaction product. A difference between the first reaction
product and the control reaction product is indicative of a genetic
abnormality in the patient. Genetic samples for use within the
present invention include genomic DNA, cDNA, and RNA. The
polynucleotide probe or primer can be RNA or DNA, and will comprise
a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or an RNA
equivalent thereof. Suitable assay methods in this regard include
molecular genetic techniques known to those in the art, such as
restriction fragment length polymorphism (RFLP) analysis, short
tandem repeat (STR) analysis employing PCR techniques, ligation
chain reaction (Barany, PCR Methods and Applications 1:5- 16,
1991), ribonuclease protection assays, and other genetic linkage
analysis techniques known in the art (Sambrook et al., ibid.;
Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
Ribonuclease protection assays (see, e.g., Ausubel et al., ibid.,
ch. 4) comprise the hybridization of an RNA probe to a patient RNA
sample, after which the reaction product (RNA-RNA hybrid) is
exposed to RNase. Hybridized regions of the RNA are protected from
digestion. Within PCR assays, a patient's genetic sample is
incubated with a pair of polynucleotide primers, and the region
between the primers is amplified and recovered. Changes in size or
amount of recovered product are indicative of mutations in the
patient. Another PCR-based technique that can be employed is single
strand conformational polymorphism (SSCP) analysis (Hayashi, PCR
Methods and Applications 1:34-8, 1991).
[0144] The invention also provides anti-zfsta2 antibodies.
Antibodies to zfsta2 can be obtained, for example, using as an
antigen the product of a zfsta2 expression vector, or zfsta2
isolated from a natural source. Particularly useful anti-zfsta2
antibodies "bind specifically" with zfsta2. Antibodies are
considered to be specifically binding if the antibodies bind to a
zfsta2 polypeptide, peptide or epitope with a binding affinity
(K.sub.a) of 10.sup.6 M.sup.-1 or greater, preferably 10.sup.7
M.sup.-1 or greater, more preferably 10.sup.8 M.sup.-1 or greater,
and most preferably 10.sup.9 M.sup.-1 or greater. The binding
affinity of an antibody can be readily determined by one of
ordinary skill in the art, for example, by Scatchard analysis
(Scatchard, Ann. NY Acad. Sci. 51:660, 1949). Suitable antibodies
include antibodies that bind with zfsta2 in particular domains,
such as the zfsta2 follistatin homology domain (amino acid residues
65 to about 133 of SEQ ID NO:2), the calmodulin homology domain
(located at about amino acid residues 175 to 250 of SEQ ID NO:2),
or I-set IG domains #1 or #2 (located at about amino acid residues
251 to 334 of SEQ ID NO:2 or amino acid residues 335 to 432 of SEQ
ID NO:2).
[0145] Anti-zfsta2 antibodies can be produced using antigenic
zfsta2 epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, preferably between 15 to about
30 amino acids contained within SEQ ID NO:2. However, peptides or
polypeptides comprising a larger portion of an amino acid sequence
of the invention, containing from 30 to 50 amino acids, or any
length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are useful for inducing
antibodies that bind with zfsta2. It is desirable that the amino
acid sequence of the epitope-bearing peptide is selected to provide
substantial solubility in aqueous solvents (i.e., the sequence
includes relatively hydrophilic residues, while hydrophobic
residues are preferably avoided). Moreover, amino acid sequences
containing proline residues may be also be desirable for antibody
production.
[0146] Polyclonal antibodies to recombinant zfsta2 protein or to
zfsta2 isolated from natural sources can be prepared using methods
well-known to those of skill in the art. See, for example, Green et
al., "Production of Polyclonal Antisera," in Immunochemical
Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and
Williams et al., "Expression of foreign proteins in E. coli using
plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a zfsta2 polypeptide can be increased through the
use of an adjuvant, such as alum (aluminum hydroxide) or Freund's
complete or incomplete adjuvant. Polypeptides useful for
immunization also include fusion polypeptides, such as fusions of
zfsta2 or a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like," such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0147] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
hamsters, guinea pigs, goats or sheep, an anti-zfsta2 antibody of
the present invention may also be derived from a subhuman primate
antibody. General techniques for raising diagnostically and
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465, and in Losman et al., Int. J. Cancer 46:310, 1990.
Antibodies can also be raised in transgenic animals such as
transgenic sheep, cows, goats or pigs, and may be expressed in
yeast and fungi in modified forms as will as in mammalian and
insect cells.
[0148] Alternatively, monoclonal anti-zfsta2 antibodies can be
generated. Rodent monoclonal antibodies to specific antigens may be
obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991), Picksley et al., "Production of
monoclonal antibodies against proteins expressed in E. coli," in
DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.), page 93 (Oxford University Press 1995)).
[0149] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a zfsta2 gene product, 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 which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0150] In addition, an anti-zfsta2 antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal 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 locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain 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, for example, by Green et al., Nat. Genet. 7:13,
1994, Lonberg et al., Nature 368:856, 1994, and Taylor et al., Int.
Immun. 6:579, 1994.
[0151] 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, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0152] For particular uses, it may be desirable to prepare
fragments of anti-zfsta2 antibodies. Such antibody fragments can be
obtained, for example, by proteolytic hydrolysis of the antibody.
Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods. As an illustration,
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 to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230, 1960, Porter, Biochem. J. 73:119, 1959, Edelman et al., in
Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and
by Coligan, ibid.
[0153] 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.
[0154] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by 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
gluteraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437, 1992).
[0155] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are 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 scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97, 1991, also see, Bird et al., Science
242:423, 1988, Ladner et al., U.S. Pat. No. 4,946,778, Pack et al.,
Bio/Technology 11:1271, 1993, and Sandhu, supra.
[0156] As an illustration, a scFV can be obtained by exposing
lymphocytes to zfsta2 polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled zfsta2 protein or peptide).
Genes encoding polypeptides having potential zfsta2 polypeptide
binding domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides which interact with a
known target which can be a protein or polypeptide, such as a
ligand or receptor, a biological or synthetic macromolecule, or
organic or inorganic substances. Techniques for creating and
screening such random peptide display libraries are known in the
art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.
Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner
et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of
Peptides and Proteins (Academic Press, Inc. 1996)) and random
peptide display libraries and kits for screening such libraries are
available commercially, for instance from Clontech (Palo Alto,
Calif.) Invitrogen Inc. (San Diego, Calif.), New England Biolabs,
Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc.
(Piscataway, N.J.). Random peptide display libraries can be
screened using the zfsta2 sequences disclosed herein to identify
proteins which bind to zfsta2.
[0157] 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 2:106, 1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0158] Alternatively, an anti-zfsta2 antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted 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. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522, 1986, Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285, 1992, Sandhu, Crit. Rev. Biotech. 12:437, 1992, Singer et
al., J. Immun. 150:2844, 1993, Sudhir (ed.), Antibody Engineering
Protocols (Humana Press, Inc. 1995), Kelley, "Engineering
Therapeutic Antibodies," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 399-434 (John Wiley &
Sons, Inc. 1996), and by Queen et al., U.S. Pat. No. 5,693,762
(1997).
[0159] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-zfsta2 antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan, ibid. at pages 2.4.1-2.4.7.
Alternatively, monoclonal anti-idiotype antibodies can be prepared
using anti-zfsta2 antibodies or antibody fragments as immunogens
with the techniques, described above. As another alternative,
humanized anti-idiotype antibodies or subhuman primate
anti-idiotype antibodies can be prepared using the above-described
techniques. Methods for producing anti-idiotype antibodies are
described, for example, by, Irie, U.S. Pat. No. 5,208,146, Greene,
et. al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J.
Gen. Virol. 77:1875, 1996.
[0160] Antibodies or polypeptides herein can also be directly or
indirectly conjugated to drugs, toxins, radionuclides and the like,
and these conjugates used for in vivo diagnostic or therapeutic
applications. For instance, polypeptides or antibodies of the
present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule
(receptor or antigen, respectively, for instance). More
specifically, zfsta2 polypeptides or anti-zfsta2 antibodies, or
bioactive fragments or portions thereof, can be coupled to
detectable or cytotoxic molecules and delivered to a mammal having
cells, tissues or organs that express the anti-complementary
molecule.
[0161] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide or antibody, and include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like. Suitable
cytotoxic molecules may be directly or indirectly attached to the
polypeptide or antibody, and include bacterial or plant toxins (for
instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and
the like), as well as therapeutic radionuclides, such as
iodine-131, rhenium-188 or yttrium-90 (either directly attached to
the polypeptide or antibody, or indirectly attached through means
of a chelating moiety, for instance). Polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/anticomplementary pair, where the other member is
bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0162] Expression of zfsta2mRNA is largely confined to spinal cord,
brain, and placenta with low level expression seen in a wide
variety of other tissues. This is consistent with the reported
distribution of follistatin gene transcripts and transcripts of a
number of other follistatin family members. This distribution
suggests that zfsta2 may play a role in neuron regeneration and
repair within the CNS. Injury to the adult mammalian brain or
spinal cord generates a cascade of cellular events leading to
inflammation, proliferation of astrocytes, angiogenesis, and
formation of a glial-mesodermal scar (Logan et al., Brain Res.
587:216-25, 1992; Wang et al., Brain Res. Bull. 36:607-9, 1995 and
Lindholm et al., J. Cell. Biol. 117:395-400, 1992). Production of
scar tissue within the CNS provides a physical barrier for
regeneration of neurons and is thought to limit the ability of the
adult CNS to recover after injury. Scar tissue formation in the CNS
is thought to be dependent on the localized TGF-.beta. stimulated
production of extracellular matrix components, similar to what is
seen for scar tissue formation in the periphery. Indeed, TGF-.beta.
mRNA and protein have been localized to astrocytes at the site of
damage in the CNS (Logan et al., ibid., Wang et al., ibid. and
Lindholm et al., ibid.) suggesting that a follistatin family
member, such as zfsta2, facilitates neuron regeneration and
establishment of new synaptic contacts by sequestering TGF-.beta..
SC1, a member of the follistatin family, is expressed in brain
astrocytes following injury (Mendis et al., Brain Res. 730:95-106,
1996) and follistatin related protein (FRP) is secreted by glioma
cells in culture (Zwijsen et al., Eur. J. Biochem. 225:937-46,
1994).
[0163] Proteins that can sequester TGF.beta. and stimulate neuron
regeneration would be useful in treatment of peripheral
neuropathies by increasing spinal cord and sensory neurite
outgrowth. Such polypeptides, agonists and antagonists can be
included in therapeutic treatment to regenerate neurite outgrowths
following strokes, brain damage caused by head injuries, and
paralysis caused by spinal injuries. Application may also be made
in treating neurodegenerative diseases such as Alzheimer's disease,
Parkinson's disease and multiple sclerosis by stimulating neuronal
outgrowths. Additional applications would include repair of
transected axons which are common in lesions of multiple
sclerosis.
[0164] Zfsta2 polypeptides, agonists or antagonists thereof may be
therapeutically useful for treating brain and spinal cord injuries.
To verify the presence of this capability in zfsta2 polypeptides,
agonists or antagonists of the present invention, such zfsta2
polypeptides, agonists or antagonists are evaluated with respect to
their ability to stimulate neuron regeneration and establish new
synaptic contacts according to procedures known in the art, see for
example Mendis et al., Brain Res. 730:95-106, 1996; Lindholm et
al., J. Cell Biol. 117:395-400, 1992 and Logan et al., Brain Res.
587:216-25, 1992. If desired, zfsta2 polypeptide performance in
this regard can be compared to other follistatins such as SC1 and
FRP and the like. In addition, zfsta2 polypeptides or agonists or
antagonists thereof may be evaluated in combination with one or
more follistatins to identify synergistic effects. If desired,
zfsta2 performance in this regard can be compared to other
anti-inflammatory compounds, such as dexamethasone and
hydrocortisone and the like.
[0165] In addition to its potential role in treatment of injuries
to the CNS, zfsta2 may also have a role in host defense. Human
marrow stromal cells have been shown to be reactive with
anti-activin A antibodies and the production of the B.sub.A-subunit
mRNA is increased in these cells by a number of pro-inflammatory
cytokines/regulators such as interleukin 1.alpha.,
lipopolysaccaride, tumor necrosis factor-.alpha., or
12-O-tetradecanoylphorbol 13-acetate (Shao et al., Cytokine
10:227-35, 1998). In contrast to the stimulatory effects of these
agents, the anti-inflammatory compounds dexamethasone and
hydrocortisone inhibited the constitutive and cytokine-stimulated
expression of activin B.sub.A-mRNA (Shao et al., ibid.).
[0166] Application of the polypeptides of the present invention may
be made to inhibit inflammatory response, stimulate a reduction in
the number and activity of inflammatory cells, and diminish edema
and inflammation. Such anti-inflammatory polypeptides would find
application in the treatment of acute inflammation conditions,
bursitis, chronic inflammatory demyelinating polyneuropathy,
various forms of contact dermatitis, contact vulvovaginitis,
myositis, sepsis and ulcerative colitis. Use as therapeutic agents
could also be made for treating acute renal failure, pancreatitis
and neonatal bronchopulmonary dysplasia. Application can also be
made for ocular injuries, such as corneal injury from burns or
penetration of a foreign body or ocular inflammatory diseases such
as uveitis.
[0167] Application may also be made to alleviate chronic itching
and inflammation associated with dermatological conditions and skin
diseases such as eczema, neurodermatitis, allergy, psoriasis,
xerosis, insect bites, and burns, such as thermal, chemical and
radiation burns, particularly sunburns.
[0168] Symptoms associated with gout, asthma, carpal tunnel
syndrome, systemic lupus erythematosus, multiple sclerosis and
myasthenia gravis may also be alleviated using the compounds of the
present invention. zfsta2 polypeptide, agonist or
antagonist-mediated removal of bioactive activin from sites of
inflammation would be a useful therapy for treatment of a wide
variety of inflammatory disorders. To verify the presence of this
capability in zfsta2 polypeptides, or polypeptide fragments
thereof, such polypeptides and polypeptide fragments are evaluated
with respect to their ability to inhibit acute inflammation. Such
methods are known in the art, in particular, zfsta2 polypeptides
can be tested for anti-inflammatory activity in the
carrageenan-induced rat footpad edema model (Winter et al., J.
Pharmac. Exp. Ther. 141:369-76, 1963 and Miele et al., Nature
335:726-30, 1988). Other models include the endotoxin-induced
uveitis (EIU) model (Chan et al., Arch. Ophthalmol. 109:278-81,
1991), Oxazolone-induced inflammation model (Lloret and Moreno,
Biochem. Pharmocol. 44:1437, 1992), croton oil-induced inflammation
model, PMA-induced inflammation model (Miele et al., ibid.), and
dextran-induced edema assay for anti-inflammatory agents (Ialenti
et al., Agents Actions 29:48-9, 1990 and Rosa and Willoughby, J.
Pharm. Pharmac. 23:297-8, 1971). Efficacy for treating diseases
such as rheumatoid arthritis can be evaluated using indicators
which would include a reduction in inflammation and relief of pain
or stiffness, and in animal models indications would be derived
from macroscopic inspection of joints and change in swelling of
hind paws. If desired, zfsta2 polypeptide performance in this
regard can be compared to other anti-inflammatory agents, in
particular, dexamethasone and hydrocortisone. In addition, zfsta2
polypeptides may be evaluated in combination with one or more
anti-inflammatory agents to identify synergistic effects.
[0169] The recent conformation of the sequence identity of
erythroid differentiation factor (EDF) and B.sub.A subunit of
activins and inhibins (Murata et al., Proc. Natl. Acad. Sci. USA
85:2434, 1988) suggests a role for zfsta2 in regulating
hematopoiesis and differentiation of erythroid progenitors. EDF
exhibits potent differentiation-inducing activity towards cultured
erythroleukemia cells and enhances the growth of normal erythroid
precursor cells in vitro and in vivo (Yu et al., Nature, 330:765,
1987, Shiozaki, et al., Biochem. Biophys. Res. Commun. 165:1155,
1989) and activin A/EDF is expressed in activated macrophages
(Eramaa et al., J. Exp. Med. 176:1449-52, 1992). Continuous
intraperitoneal administration of follistatin to normal mice
resulted in a decrease of erythroid progenitors in bone marrow and
spleen (Shiozaki et al., Proc. Natl. Acad. Sci. USA 89:1553-6,
1992) demonstrating that follistatin modulates murine
erythropoeisis. In humans, moreover, the follistatin related gene
is a target of chromosonal rearrangement in a B-cell chronic
lymphocytic leukemia (Hayette et al., Oncogene 16:2949-54,
1998).
[0170] EDF-binding proteins such as zfsta2 polypeptides, agonists
or antagonists would provide a useful therapeutic for modulating
hematopoiesis and differentiation of erythroid progenitors. To
verify the presence of this capability, zfsta2 polypeptides and
agonists of the present invention are evaluated with respect to
their ability to alter erythropoiesis by decreasing erythroid
progenitors in bone marrow and spleen, according to procedures
known in the art. zfsta2 antagonists can be evaluated with respect
to enhancing hematopoiesis and differentiation of erythroid
progenitors by inactivating follistatin and follistatin-like
molecules. If desired, zfsta2 performance in this regard can be
compared to other follistatins or hematopoietic factors such as
erythropoietin or thrombopoietin and the like. In addition, zfsta2
polypeptides or agonists or antagonists thereof may be evaluated in
combination with one or more follistatins to identify synergistic
effects.
[0171] The pleiotropic actions of activins and inhibins on the
gonadal/hypothalamic/pituitary axis would indicate that
follistatins, such as zfsta2, would be useful in treatment of
fertility disorders such characterized by abnormalities in hormone
production. Activin A, for example, has been shown to stimulate
hypothalamic oxytocin secretion (Sawchenko et al., Nature
334:615-7; 1988). Oxytocin specifically stimulates uterine
contraction near term. Proteins which bind activin A would serve as
useful therapeutics for delaying birth in pre-term pregnancies.
[0172] Folliculogenesis is a physiological event characterized by
morphological and functional changes of the follicle. Of these
events, antrum formation is considered the milestone of this
pathway, a process that is governed by the pituitary hormone FSH.
Since FSH is required for normal function of the ovaries, and
activin is required for activation of FSH synthesis and secretion,
it is not surprising that follistatin is an important regulator of
ovarian function. Follistatin mRNA is present in primordial
follicles and its levels are dramatically-increased in granulosa
cells of the growing secondary or tertiary follicles and then
decreases in the pre-ovulatory follicles (Shimasaki et al., Mol.
Endocrinol. 3:651-9, 1989). Recent in vitro assay systems have also
shown that activin is directly folliculogenic in immature mice but
not in adults, the inhibition of folliculogenesis in adults was,
furthermore, reversed by follistatin (Yokota et al., Endocrinology
138:4572-6, 1997). The balance between activin and follistatin
appears to be critical for normal ovarian function as
overexpression of mouse follistatin in female transgenic mice had a
number of reproductive defects (Guo et al., Mol. Endocrinol. 12:
96-106, 1998). Follistatins, such as zfsta2, play a role in
regulating folliculogenesis by affecting proliferation or
differentiation of follicular cells, affecting cell-cell
interactions, modulating hormones involved in the process, and the
like. The role of sex steroids, such as FSH, on target tissues and
organs, e.g., uterus, breast, adipose, bones and liver, has made
modulators of their activity desirable for therapeutic
applications. Such applications include treatments for precocious
puberty, endometriosis, uterine leiomyomata, hirsutism,
infertility, pre-menstrual syndrome (PMS), amenorrhea, and as
contraceptive agents.
[0173] The level and ratio of gonadotropin and steroid hormones in
the blood can be used to assess the existence of hormonal
imbalances associated with diseases, as well as determine whether
normal hormonal balance has been restored after administration of a
therapeutic agent. Determination of estradiol, progesterone, LH,
and FSH levels, for example, from serum is known by one of skill in
the art. Such assays can be used to monitor the effects on hormone
levels after administration of zfsta2 in vivo, or in a transgenic
mouse model where the zfsta2 gene is expressed or the murine
ortholog is deleted.
[0174] The zfsta2 polypeptides, agonists and antagonists of the
present invention may be used directly or incorporated into
therapies for treating reproductive disorders. As a
hormone-modulating molecule, zfsta2 polypeptides, agonists and
antagonists can have therapeutic application for treating, for
example, breakthrough menopausal bleeding, as part of a therapeutic
regime for pregnancy support, or for treating symptoms associated
with polycystic ovarian syndrome (PCOS), PMS and menopause. In
addition, other in vivo rodent models are known in the art to assay
effects of zfsta2 polypeptides, agonists and antagonists on, for
example, polycystic ovarian syndrome (PCOS).
[0175] Activin, inhibin and follistatin are also found in the
testes. mRNA encoding follistatin is located in many germ cells
including type B spermatogonia, primary spermatocytes and
spermatids at steps 1 to 11 (Meinhardt et al., J. Reprod. Fertil.
112:233-41, 1998). It is also found in Sertoli cells and
endothelial cells but not in Leydig cells. Immunohistochemistry
with anti-follistatin antibodies showed that the protein was
localized to spermatids at all stages and it was also localized to
endothelial and Leydig cells. This widespread localization,
together with follistatin's capacity to neutralize the activity of
activin, suggests that follistatin modulates spermatogenesis and a
range of other testicular functions. The balance between activin
and follistatin plays an important role in normal reproduction in
males was shown in mouse follistatin transgenic mice: males
exhibited variable degrees of Leydig cell hyperplasia,
spermatogenesis was arrested, and seminiferous tubules degenerated
which lead to infertility. This suggests that reproductive
disorders due to an excess of activin or other TGF-beta family
members would be amenable to treatment with members of the
follistatin family. Additionally, follistatin antagonists would be
useful in treatment regimes to enhance male fertility.
[0176] In vivo assays for evaluating the effect of zfsta2
polypeptides, agonists and antagonists on testes are well known in
the art. For example, compounds can be injected intraperitoneally
for a specific time duration. After the treatment period, animals
are sacrificed and testes removed and weighed. Testicles are
homogenized and sperm head counts are made (Meistrich et al., Exp.
Cell Res. 99:72-78, 1976).
[0177] Other activities, for example, chemotaxic activity that may
be associated with proteins of the present invention can be
analyzed. For example, late stage factors in spermatogenesis may be
involved in egg-sperm interactions and sperm motility. Activities,
such as enhancing viability of cryopreserved sperm, stimulating the
acrosome reaction, enhancing sperm motility and enhancing egg-sperm
interactions may be associated with the proteins of the present
invention. Assays evaluating such activities are known
(Rosenberger, J. Androl. 11:89- 96, 1990; Fuchs, Zentralbl Gynakol
11:117-120, 1993; Neurwinger et al., Andrologia 22:335-9, 1990;
Harris et al., Human Reprod. 3:856-60, 1988; and Jockenhovel,
Andrologia 22:171-178, 1990; Lessing et al., Fertil. Steril.
44:406-9 (1985); Zaneveld, In Male Infertility Chapter 11, Comhaire
Ed., Chapman & Hall, London 1996). These activities are
expected to result in enhanced fertility and successful
reproduction.
[0178] zfsta2 polypeptides agonists or antagonists would provide a
useful therapeutic for modulating reproductive hormones. To verify
the presence of this capability, zfsta2 polypeptides, agonists and
antagonists of the present invention are evaluated with respect to
their ability td regulate hormones associated with reproduction,
according to procedures known in the art. For example, Guoqetal,
Mol, Endocrinol. 12:96-106, 1998 describes RIA measurement of serum
LH, FSH, testosterone, estradiol, activin and follistatin. Zfsta2
polypeptides and agonists would be useful for treating male and
female reproductive disorders. Zfsta2 antagonists would also be
useful as contraceptives. If desired, zfsta2 performance in this
regard can be compared to other follistatins and the like. In
addition, zfsta2 polypeptides or agonists or antagonists thereof
may be evaluated in combination with one or more follistatins to
identify synergistic effects.
[0179] Zfsta2 polypeptides, agonists and antagonists of the present
invention may also be used in applications for enhancing
fertilization during assisted reproduction in humans and in
animals. Such assisted reproduction methods are known in the art
and include artificial insemination, in vitro fertilization, embryo
transfer, and gamete intrafallopian transfer. Such methods are
useful for assisting those who may have physiological or metabolic
disorders that prevent or impede natural conception. Such methods
are also used in animal breeding programs, e.g., for livestock,
racehorses, domestic and wild animals, and could be used as methods
for the creation of transgenic animals. Zfsta2 polypeptides,
agonists or antagonists could be used in the induction of
ovulation, either independently or in conjunction with a regimen of
gonadotropins or agents such as clomiphene citrate or bromocriptine
(Speroff et al., Induction of ovulation, Clinical Gynecologic
Endocrinology and Infertility, 5.sup.th ed., Baltimore, Williams
& Wilkins, 1994). Zfsta2 polypeptides, agonists and antagonists
can also be used in stimulation of spermatogenesis, independently
or in conjunction with other gonadotropins or sex steroids such as
testosterone. As such, proteins of the present invention can be
administered to the recipient prior to fertilization or combined
with the sperm, an egg or an egg-sperm mixture prior to in vitro or
in vivo fertilization. Such proteins can also be mixed with oocytes
or sperm prior to cryopreservation to enhance viability of the
preserved tissues for use in assisted reproduction.
[0180] The formation of bone and teeth and is a multi-step process
that is known to be initiated and promoted by members of the
TGF-.beta. superfamily, including TGF-.beta.s and bone morphogenic
proteins (BMPs). Accumulating evidence suggests that activin and
follistatin play regulatory roles in both tooth and bone formation.
The temporal-spatial expression of activin and follistatin in
pre-odontoblasts suggests that activin is required for
proliferation of these cells, while odontoblast terminal
differentiation is mediated, at least partly, by follistatin
inactivation of these proliferative effects (Heikinheimo et al., J.
Dent. Res. 76:1625-36; 1997, Heikinheimo et al., Eur. J. Oral Sci.
106:167-73; 1998). Follistatin is also expressed in bone (Inoue et
al., Calcif. Tiss. Int. 55:395-7, 1994) and activin and follistatin
have been detected by immunohistochemistry in healing fractures in
the rat (Nagamine et al., J. Orthopaed. Res. 16:314-21, 1998).
Follistatin has also been detected in developing bone and the
expression of follistatin and activin A genes during demineralized
bone matrix-induced endochondral bone development suggests a
cooperative interaction between follistatin and activin during bone
formation (Funaba, et al., Endocrinology 137:4250-9, 1996).
[0181] Such therapeutic agents may be used for repair of bone
defects and deficiencies, such as those occurring in closed, open
and non-union fractures; prophylactic use in closed and open
fracture reduction; promotion of bone healing in plastic surgery;
stimulation of bone ingrowth into non-cemented prosthetic joints
and dental implants; elevation of peak bone mass in pre-menopausal
women; treatment of growth deficiencies; treatment of periodontal
disease and defects, and other tooth repair processes; increase in
bone formation during distraction osteogenesis; and treatment of
other skeletal disorders, such as age-related osteoporosis,
post-menopausal osteoporosis, glucocorticoid-induced osteoporosis,
diabetes-associated osteoporosis or disuse osteoporosis and
arthritis. The compounds of the present invention can also be
useful in repair of congenital, trauma-induced or surgical
resection of bone (for instance, for cancer treatment), and in
cosmetic surgery. Further uses include limiting or treating
cartilage defects or disorders and stimulation of wound healing and
tissue repair.
[0182] Well established animal models are available to test in vivo
efficacy of modulators of bone formation. For example, the
hypocalcemic rat or mouse model can be used to determine the effect
of test compounds on serum calcium, and the ovariectomized rat or
mouse can be used as a model system for osteoporosis. Bone changes
seen in these models and in humans during the early stages of
estrogen deficiency are qualitatively similar.
[0183] Molecules that are capable of modulating the effects of
members of the TGF-.beta. family, such as zfsta2 polypeptides,
agonists or antagonists, would provide molecules useful for tooth
and bone formation. To verify the presence of this capability,
zfsta2 polypeptides, agonists and antagonists of the present
invention are evaluated with respect to their ability to stimulate
tooth or bone formation according to procedures known in the art.
If desired, zfsta2 performance in this regard can be compared to
other follistatins and the like. In addition, zfsta2 polypeptides
or agonists or antagonists thereof may be evaluated in combination
with one or more follistatins to identify synergistic effects.
[0184] Follistatin and activin also appear likely to play a role in
the pathogenesis of atherosclerosis. In vascular wall cells,
activin-A has been shown to inhibit endothelial cell growth and
promote smooth muscle cell growth (Kojima et al., Exp. Cell Res.
206:152-6; 1993, McCarthy and Bicknell, J. Biol. Chem.
268:23066-71; 1993) and has been shown to produce a modest
inhibition of scavenger receptor, SRB1, expression and foam cell
formation in THP-1 macrophages (Kozaki et al., Arterioscler.
Thromb. Vasc. Biol. 17:2389-94; 1997). These effects are
antagonized by follistatin. Activin-A, follistatin and bone
morphogenic protein-2, are produced by human atherosclerotic
lesions and expression of the first two has been localized to the
neointima of the diseased arteries (Inoue et al., Biochem. Biophys.
Res. Commun. 205:441-8; 1994). These data suggest that the relative
balance between activin, and its binding protein, follistatin, may
be important in initiation and progression of atherosclerotic
lesions.
[0185] Zfsta2 polypeptides, agonists or antagonists would be useful
for neutralizing the activities of TGF-.beta. family members. Such,
molecules would provide a novel therapy for treatment of restenosis
after angioplasty. Additionally, TGF-.beta. neutralizers would be
useful for the treatment of atherosclerosis. Use of such molecules
would also be applicable for treatment of stroke.
[0186] Follistatin and activin appear to play important roles in
development. Two classes of TGF-.beta. family members are believed
to determine the dorsal/ventral pattern of the mesoderm in early
development in Xenopus laevi. The first are related to activin and
induce the formation of the dorsal mesoderm, which gives rise to
muscle and the notocord (Asashima et al., Roux's Arch. Dev. Biol.
198:330-5, 1990) and the second are related to the bone morphogenic
proteins (BMPs) which inhibit dorsal mesoderm formation and induce
cells to take on ventral fates, such as blood cells (Maeno et al.,
Dev. Biol. 161: 522-9, 1994). Follistatin can block the activities
of activin (Fukui et al., Dev. Biol. 159:131-9, 1993) and BMPs
(Iemura et al., Proc. Natl. Acad. Sci. USA 95:9337-42, 1998) in
these systems. Taken together, these findings suggest that zfsta2,
as a member of the follistatin family of TGF-beta binding proteins,
may useful as a therapy for a wide range of developmental
disorders.
[0187] The effects of zfsta2 can be measured in vitro using
cultured cells or in vivo by administering molecules of the claimed
invention to the appropriate animal model. For instance, zfsta2
transfected or co-transfected expression host cells may be embedded
in an alginate environment and injected (implanted) into recipient
animals. Alginate-poly-L-lysine microencapsulation, permselective
membrane encapsulation and diffusion chambers have been described
as a means to entrap transfected mammalian cells or primary
mammalian cells. These types of non-immunogenic "encapsulations" or
microenvironments permit the transfer of nutrients into the
microenvironment, and also permit the diffusion of proteins and
other macromolecules secreted or released by the captured cells
across the environmental barrier to the recipient animal. Most
importantly, the capsules or microenvironments mask and shield the
foreign, embedded cells from the recipient animal's immune
response. Such microenvironments can extend the life of the
injected cells from a few hours or days (naked cells) to several
weeks (embedded cells).
[0188] Alginate threads provide a simple and quick means for
generating embedded cells. The materials needed to generate the
alginate threads are readily available and relatively inexpensive.
Once made, the alginate threads are relatively strong and durable,
both in vitro and, based on data obtained using the threads, in
vivo. The alginate threads are easily manipulable and the
methodology is scalable for preparation of numerous threads. In an
exemplary procedure, 3% alginate is prepared in sterile H.sub.2O,
and sterile filtered. Just prior to preparation of alginate
threads, the alginate solution is again filtered. An approximately
50% cell suspension (containing about 5.times.10.sup.5 to about
5.times.10.sup.7 cells/ml) is mixed with the 3% alginate solution.
One ml of the alginate/cell suspension is extruded into a 100 mM
sterile filtered CaCl.sub.2 solution over a time period of
.sup..about.15 min, forming a "thread". The extruded thread is then
transferred into a solution of 50 mM CaCl.sub.2, and then into a
solution of 25 mM CaCl.sub.2. The thread is then rinsed with
deionized water before coating the thread by incubating in a 0.01%
solution of poly-L-lysine. Finally, the thread is rinsed with
Lactated Ringer's Solution and drawn from solution into a syringe
barrel (without needle attached). A large bore needle is then
attached to the syringe, and the thread is intraperitoneally
injected into a recipient in a minimal volume of the Lactated
Ringer's Solution.
[0189] An alternative in vivo approach for assaying proteins of the
present invention involves viral delivery systems. Exemplary
viruses for this purpose include adenovirus, herpesvirus, vaccinia
virus and adeno- associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acid (for a
review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and
Douglas and Curiel, Science & Medicine 4:44-53, 1997). The
adenovirus system offers several advantages: adenovirus can (i)
accommodate relatively large DNA inserts; (ii) be grown to
high-titer; (iii) infect a broad range of mammalian cell types; and
(iv) be used with a large number of available vectors containing
different promoters. Also, because adenoviruses are stable in the
bloodstream, they can be administered by intravenous injection.
[0190] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. In an
exemplary system, the essential E1 gene has been deleted from the
viral vector, and the virus will not replicate unless the E1 gene
is provided by the host cell (the human 293 cell line is
exemplary). When intravenously administered to intact animals,
adenovirus primarily targets the liver. If the adenoviral delivery
system has an E1 gene deletion, the virus cannot replicate in the
host cells. However, the host's tissue (e.g., liver) will express
and process (and, if a secretory signal sequence is present,
secrete) the heterologous protein. Secreted proteins will enter the
circulation in the highly vascularized liver, and effects on the
infected animal can be determined.
[0191] Polynucleotides encoding zfsta2 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit zfsta2 activity. If a mammal has a mutated or absent zfsta2
gene, the zfsta2 gene can be introduced into the cells of the
mammal. In one embodiment, a gene encoding a zfst2 polypeptide is
introduced in vivo in a viral vector. Such vectors include an
attenuated or defective DNA virus, such as, but not limited to,
herpes simplex virus (HSV), papillomavirus, Epstein Barr virus
(EBV), adenovirus, adeno-associated virus (AAV), and the like.
Defective viruses, which entirely or almost entirely lack viral
genes, are preferred. A defective virus is not infective after
introduction into a cell. Use of defective viral vectors allows for
administration to cells in a specific, localized area, without
concern that the vector can infect other cells. Examples of
particular vectors include, but are not limited to, a defective
herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al., J. Clin.
Invest. 90:626-30, 1992; and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-8, 1989).
[0192] In another embodiment, a zfsta2 gene can be introduced in a
retroviral vector, e.g., as described in Anderson et al., U.S. Pat.
No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S.
Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Pat.
No. 5,124,263; International Pat. Publication No. WO 95/07358,
published Mar. 16, 1995 by Dougherty et al.; and Kuo et al., Blood
82:845, 1993. Alternatively, the vector can be introduced by
lipofection in vivo using liposomes. Synthetic cationic lipids can
be used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA
85:8027-31, 1988). The use of lipofection to introduce exogenous
genes into specific organs in vivo has certain practical
advantages. Molecular targeting of liposomes to specific cells
represents one area of benefit. More particularly, directing
transfection to particular cells represents one area of benefit.
For instance, directing transfection to particular cell types would
be particularly advantageous in a tissue with cellular
heterogeneity, such as the pancreas, liver, kidney, and brain.
Lipids may be chemically coupled to other molecules for the purpose
of targeting. Targeted peptides (e.g., hormones or
neurotransmitters), proteins such as antibodies, or non-peptide
molecules can be coupled to liposomes chemically.
[0193] It is possible to remove the target cells from the body; to
introduce the vector as a naked DNA plasmid; and then to re-implant
the transformed cells into the body. Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun or use of a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992;
Wu et al., J. Biol. Chem. 263:14621-4, 1988.
[0194] Antisense methodology can be used to inhibit zfsta2 gene
transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
zfsta2-encoding polynucleotide (e.g., a polynucleotide as set froth
in SEQ ID NO:1) are designed to bind to zfsta2-encoding mRNA and to
inhibit translation of such mRNA. Such antisense polynucleotides
are used to inhibit expression of zfsta2 polypeptide-encoding genes
in cell culture or in a subject.
[0195] Transgenic mice, engineered to express the zfsta2 gene, and
mice that exhibit a complete absence of zfsta2 gene function,
referred to as "knockout mice" (Snouwaert et al., Science 257:1083,
1992), may also be generated (Lowell et al., Nature 366:740-42,
1993). These mice may be employed to study the zfsta2 gene and the
protein encoded thereby in an in vivo system.
[0196] The zfsta2 polypeptides are also contemplated for
pharmaceutical use. Pharmaceutically effective amounts of zfsta2
polypeptides, agonists or zfsta2 antagonists of the present
invention can be formulated with pharmaceutically acceptable
carriers for parenteral, oral, nasal, rectal, topical, transdermal
administration or the like, according to conventional methods.
Formulations may further include one or more diluents, fillers,
emulsifiers, preservatives, buffers, excipients, and the like, and
may be provided in such forms as liquids, powders, emulsions,
suppositories, liposomes, transdermal patches and tablets, for
example. Slow or extended-release delivery systems, including any
of a number of biopolymers (biological-based systems), systems
employing liposomes, and polymeric delivery systems, can also be
utilized with the compositions described herein to provide a
continuous or long-term source of the zfsta2 polypeptide or
antagonist. Such slow release systems are applicable to
formulations, for example, for oral, topical and parenteral use.
The term "pharmaceutically acceptable carrier" refers to a carrier
medium which does not interfere with the effectiveness of the
biological activity of the active ingredients and which is not
toxic to the host or patient. One skilled in the art may formulate
the compounds of the present invention in an appropriate manner,
and in accordance with accepted practices, such as those disclosed
in Remington's Pharmaceutical Sciences, Gennaro (ed.), Mack
Publishing Co., Easton, Pa. 1990.
[0197] As used herein a "pharmaceutically effective amount" of a
zfsta2 polypeptide, agonist or antagonist is an amount sufficient
to induce a desired biological result. The result can be
alleviation of the signs, symptoms, or causes of a disease, or any
other desired alteration of a biological system. For example, an
effective amount of a zfsta2 polypeptide is that which provides
either subjective relief of symptoms or an objectively identifiable
improvement as noted by the clinician or other qualified observer.
Effective amounts of the zfsta2 polypeptides can vary widely
depending on the disease or symptom to be treated. The amount of
the polypeptide to be administered and its concentration in the
formulations, depends upon the vehicle selected, route of
administration, the potency of the particular polypeptide, the
clinical condition of the patient, the side effects and the
stability of the compound in the formulation. Thus, the clinician
will employ the appropriate preparation containing the appropriate
concentration in the formulation, as well as the amount of
formulation administered, depending upon clinical experience with
the patient in question or with similar patients. Such amounts will
depend, in part, on the particular condition to be treated, age,
weight, and general health of the patient, and other factors
evident to those skilled in the art. Typically a dose will be in
the range of 0.1-100 mg/kg of subject. Doses for specific compounds
may be determined from in vitro or ex vivo studies in combination
with studies on experimental animals. Concentrations of compounds
found to be effective in vitro or ex vivo provide guidance for
animal studies, wherein doses are calculated to provide similar
concentrations at the site of action.
[0198] The dosages of the present compounds used to practice the
invention include dosages effective to result in the desired
effects. Estimation of appropriate dosages effective for the
individual patient is well within the skill of the ordinary
prescribing physician or other appropriate health care
practitioner. As a guide, the clinician can use conventionally
available advice from a source such as the Physician's Desk
Reference, 48.sup.th Edition, Medical Economics Data Production
Co., Montvale, N.J. 07645-1742 (1994).
[0199] Preferably the compositions are presented for administration
in unit dosage forms. The term "unit dosage form" refers to
physically discrete units suitable as unitary dosed for human
subjects and animals, each unit containing a predetermined quantity
of active material calculated to produce a desired pharmaceutical
effect in association with the required pharmaceutical diluent,
carrier or vehicle. Examples of unit dosage forms include vials,
ampules, tablets, caplets, pills, powders, granules, eyedrops, oral
or ocular solutions or suspensions, ocular ointments, and
oil-in-water emulsions. Means of preparation, formulation and
administration are known to those of skill, see generally
Remington's ibid.
[0200] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Extension of EST Sequence
[0201] The novel zfsta2 polypeptide-encoding polynucleotides of the
present invention were initially identified by querying an EST
database for follistatin homologs. An EST discovered and predicted
to be related to the follistatin family, but lacked complete 5' and
3' regions. To identify the corresponding full length cDNA, a clone
considered likely to contain the missing 3' coding region was used
for sequencing. Using an Invitrogen S.N.A.P..TM. Miniprep kit
(Invitrogen, Corp., San Diego, Calif.) according to manufacturer's
instructions a 5 ml overnight culture in LB+50 .mu.g/ml ampicillin
was prepared. The template was sequenced on an ABIPRISM.TM. model
377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Conn.) using the
ABI PRISM.TM. Dye Terminator Cycle Sequencing Ready Reaction Kit
(Perkin-Elmer Corp.) according to manufacturer's instructions.
Sequencing reactions were carried out in a Hybaid OmniGene
Temperature Cycling System (National Labnet Co., Woodbridge, N.Y.).
SEQUENCHER.TM. 3.0 sequence analysis software (Gene Codes
Corporation, Ann Arbor, Mich.) was used for data analysis. The
resulting 1965 bp sequence provided the 3' end of the zfsta2 cDNA
(SEQ ID NO:7).
[0202] Using fetal brain, brain, spinal cord and retina
Marathon.TM. cDNA libraries (Clontech, Palo Alto, Calif.) as
separate templates and oligonucleotide primer ZC18,415 (SEQ ID
NO:5) to the initial EST and oligonucleotide primer ZC14701 (SEQ ID
NO:6) to an internal sequence of zfsta2 from above, 5' RACE was
carried out at 94.degree. C., for 1.5 minutes, followed by 35
cycles at 94.degree. C. for 5 seconds and 66.degree. C. for 1.5
minutes, followed by a 10 minute extension at 72.degree. C. A band
of approximately 1255 bp (SEQ ID NO:8) was resolved by gel
electrophoresis from each of the templates. 5' RACE fragments from
each of the above reactions were ligated into a TA vector
(Invitrogen Inc, San Diego, Calif.) according to manufacturer's
instructions. The sequence of the 5' end of zfsta2 was confirmed
from a fetal brain PCR fragment by sequence analysis as described
above. The 3' EST-derived sequence and the 5' RACE-derived sequence
were joined together through an overlapping sequence and the
complete cDNA sequence of zfsta2 is disclosed in SEQ ID NO:1.
Example 2
Tissue Distribution
[0203] Human Multiple Tissue Northern Blots (MTN I, MTN II and MTN
III; Clontech) were probed to determine the tissue distribution of
human zfsta2 expression. An approximately 140 bp PCR derived probe
(SEQ ID NO:4) was amplified using fetal brain, brain, spinal cord
and retina Marathon.TM. CDNA libraries (Clontech) as templates and
oligonucleotide ZC12881 (SEQ ID NO:9) and ZC12884 (SEQ ID NO:10) as
primers. The amplification was carried out as follows: 1 cycle at
94.degree. C. for 1.5 minutes, 35 cycles of 94.degree. C. for 15
seconds and 60.degree. C. 30 seconds, followed by 1 cycle at
72.degree. C. for 10 minutes. The PCR products were visualized by
agarose gel electrophoresis and the 140 bp PCR product from fetal
brain was purified using a Gel Extraction Kit (Qiagen, Chatsworth,
Calif.) according to manufacturer's instructions. The probe was
radioactively labeled using the MULTIPRIME DNA labeling kit
(Amersham, Arlington Heights, Ill.) according to the manufacturer's
instructions. The probe was purified using a NUCTRAP push column
(Stratagene). EXPRESSHYB (Clontech) solution was used for
prehybridization and as a hybridizing solution for the Northern
blots. Hybridization and washes were done under appropriately
stringent conditions. A strong transcript of approximately 5 kb was
seen in spinal cord and placenta, and a weaker transcript was
detected in brain.
[0204] A RNA Master Dot Blot (Clontech) that contained RNAs from
various tissues that were normalized to 8 housekeeping genes was
also probed and hybridized as described above. Expression was seen
in the cerebellum, occipital lobe and pituitary gland.
Example 3
Chromosomal Localization
[0205] zfsta2 was mapped to chromosome 4 using the commercially
available "GeneBridge 4Radiation Hybrid Panel" (Research Genetics,
Inc.). The GeneBridge 4 Radiation Hybrid Panel contains PCRable
DNAs from each of 93 radiation hybrid clones, plus two control DNAs
(the HFL donor and the A23 recipient). A publicly available WWW
server (http://www-genome.wi.mit.edu- /cgi-bin/contig/ rhmapper.pl)
allows mapping relative to the Whitehead Institute/MITCenter for
Genome Research's radiation hybrid map of the human genome (the
"WICGR" radiation hybrid map) which was constructed with the
GeneBridge 4 Radiation Hybrid Panel.
[0206] For the mapping of zfsta2 with the GeneBridge 4 RH Panel, 20
.mu.l reactions were set up in a 96-well microtiter plate
(Stratagene, La Jolla, Calif. and used in a "RoboCycler Gradient
96" thermal cycler (Stratagene). Each of the 95 PCR reactions
consisted of 2 .mu.l 10.times.PCR reaction buffer (Clontech), 1.6
.mu.l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif., 1
.mu.l sense primer, ZC 15,570 (SEQ ID NO:11), 1 .mu.l antisense
primer, ZC 15,575 (SEQ ID NO:12), 2 .mu.l RediLoad (Research
Genetics, Inc.), 0.4 .mu.l 50.times.Advantage KlenTaq Polymerase
Mix (Clontech), 25 ng of DNA from an individual hybrid clone or
control and ddH.sub.2O for a total volume of 20 .mu.l. The
reactions were overlaid with an equal amount of mineral oil and
sealed. The PCR cycler conditions were as follows: an initial 1
cycle 5 minute denaturation at 95.degree. C., 35 cycles of a 1
minute denaturation at 95.degree. C., 1 minute annealing at
62.degree. C. and 1.5 minute extension at 72.degree. C., followed
by a final 1 cycle extension of 7 minutes at 72.degree. C. The
reactions were separated by electrophoresis on a 2% agarose gel
(Life Technologies, Gaithersburg, Md.).
[0207] The results showed that zfsta2 maps 2.84 cR.sub.--3000 from
the framework marker WI-5113 on the chromosome 4 WICGR radiation
hybrid map. Proximal and distal framework markers were WI-5113 and
CHLC.GATA4C05.17, respectively. The use of surrounding markers
positions zfsta2 in the 4q28.3 region on the integrated LDB
chromosome 4 map (The Genetic Location Database, University of
Southhampton, WWW
server:http://cedar.genetics.soton.ac.uk/public_html/).
[0208] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
15 1 3192 DNA Homo sapiens CDS (58)...(3006) 1 gaattcggct
tcctggggga ttgtgtgact gttaaaataa ggtgaaaagc aataagg atg 60 Met 1
ttt aag tgc tgg tca gtt gtc ttg gtt ctc gga ttc att ttt ctg gag 108
Phe Lys Cys Trp Ser Val Val Leu Val Leu Gly Phe Ile Phe Leu Glu 5
10 15 tcg gaa gga agg cca acc aaa gaa gga gga tat ggc ctt aaa tcc
tat 156 Ser Glu Gly Arg Pro Thr Lys Glu Gly Gly Tyr Gly Leu Lys Ser
Tyr 20 25 30 cag cct cta atg aga ttg cga cat aag cag gaa aaa aat
caa gaa agt 204 Gln Pro Leu Met Arg Leu Arg His Lys Gln Glu Lys Asn
Gln Glu Ser 35 40 45 tca aga gtc aaa gga ttt atg att cag gat ggc
cct ttt gga tct tgt 252 Ser Arg Val Lys Gly Phe Met Ile Gln Asp Gly
Pro Phe Gly Ser Cys 50 55 60 65 gaa aat aag tac tgt ggt ttg gga aga
cac tgt gtt acc agc aga gag 300 Glu Asn Lys Tyr Cys Gly Leu Gly Arg
His Cys Val Thr Ser Arg Glu 70 75 80 aca ggg caa gca gaa tgt gcc
tgt atg gac ctt tgc aaa cgt cac tac 348 Thr Gly Gln Ala Glu Cys Ala
Cys Met Asp Leu Cys Lys Arg His Tyr 85 90 95 aaa cct gtg tgt gga
tct gac gga gaa ttc tat gaa aac cac tgt gaa 396 Lys Pro Val Cys Gly
Ser Asp Gly Glu Phe Tyr Glu Asn His Cys Glu 100 105 110 gtg cac aga
gct gct tgc ctg aaa aaa caa aag att acc att gtt cac 444 Val His Arg
Ala Ala Cys Leu Lys Lys Gln Lys Ile Thr Ile Val His 115 120 125 aat
gaa gac tgc ttc ttt aaa gga gat aag tgc aag act act gaa tac 492 Asn
Glu Asp Cys Phe Phe Lys Gly Asp Lys Cys Lys Thr Thr Glu Tyr 130 135
140 145 agc aag atg aaa aat atg cta tta gat tta caa aat caa aaa tat
att 540 Ser Lys Met Lys Asn Met Leu Leu Asp Leu Gln Asn Gln Lys Tyr
Ile 150 155 160 atg caa gaa aat gaa aat cct aat ggc gac gac ata tct
cgg aag aag 588 Met Gln Glu Asn Glu Asn Pro Asn Gly Asp Asp Ile Ser
Arg Lys Lys 165 170 175 cta ttg gtg gat caa atg ttt aaa tat ttt gat
gca gac agt aat gga 636 Leu Leu Val Asp Gln Met Phe Lys Tyr Phe Asp
Ala Asp Ser Asn Gly 180 185 190 ctt gta gat att aat gaa cta act cag
gtg ata aaa cag gaa gaa ctt 684 Leu Val Asp Ile Asn Glu Leu Thr Gln
Val Ile Lys Gln Glu Glu Leu 195 200 205 ggc aag gat ctc ttt gat tgt
act ttg tat gtt cta ttg aaa tat gat 732 Gly Lys Asp Leu Phe Asp Cys
Thr Leu Tyr Val Leu Leu Lys Tyr Asp 210 215 220 225 gat ttt aat gct
gac aag cac ctg gct ctt gaa gaa ttt tat aga gca 780 Asp Phe Asn Ala
Asp Lys His Leu Ala Leu Glu Glu Phe Tyr Arg Ala 230 235 240 ttc caa
gtg atc cag ttg agt ctg cca gaa gat cag aaa cta agc atc 828 Phe Gln
Val Ile Gln Leu Ser Leu Pro Glu Asp Gln Lys Leu Ser Ile 245 250 255
act gca gca act gtg gga caa agt gct gtt ctg agc tgt gcc att caa 876
Thr Ala Ala Thr Val Gly Gln Ser Ala Val Leu Ser Cys Ala Ile Gln 260
265 270 gga acc ctg aga cct ccc att atc tgg aaa agg aac aat att att
cta 924 Gly Thr Leu Arg Pro Pro Ile Ile Trp Lys Arg Asn Asn Ile Ile
Leu 275 280 285 aat aat tta gat ttg gaa gac atc aat gac ttt gga gat
gat ggg tcc 972 Asn Asn Leu Asp Leu Glu Asp Ile Asn Asp Phe Gly Asp
Asp Gly Ser 290 295 300 305 ttg tat att act aag gtt acc aca act cac
gtt ggc aat tac acc tgc 1020 Leu Tyr Ile Thr Lys Val Thr Thr Thr
His Val Gly Asn Tyr Thr Cys 310 315 320 tat gca gat ggc tat gaa caa
gtc tat cag act cac atc ttc caa gtg 1068 Tyr Ala Asp Gly Tyr Glu
Gln Val Tyr Gln Thr His Ile Phe Gln Val 325 330 335 aat gtt cct cca
gtc atc cgg gtg tat cca gag agt cag gct aga gag 1116 Asn Val Pro
Pro Val Ile Arg Val Tyr Pro Glu Ser Gln Ala Arg Glu 340 345 350 cct
ggg gta act gcc agt ctt agg tgc cat gca gag ggc ata cca aag 1164
Pro Gly Val Thr Ala Ser Leu Arg Cys His Ala Glu Gly Ile Pro Lys 355
360 365 cct cag ctt ggc tgg ttg aag aat gga att gat att aca cca aag
ctt 1212 Pro Gln Leu Gly Trp Leu Lys Asn Gly Ile Asp Ile Thr Pro
Lys Leu 370 375 380 385 tcc aaa caa ctc acg ctt caa gca aat ggc gca
act gtg gga caa agt 1260 Ser Lys Gln Leu Thr Leu Gln Ala Asn Gly
Ala Thr Val Gly Gln Ser 390 395 400 gct gtt ctg agc tgt gcc att caa
gga acc ctg aga cct ccc att atc 1308 Ala Val Leu Ser Cys Ala Ile
Gln Gly Thr Leu Arg Pro Pro Ile Ile 405 410 415 tgg aaa agg aac aat
att att cta aat aat tta gat ttg gaa gac atc 1356 Trp Lys Arg Asn
Asn Ile Ile Leu Asn Asn Leu Asp Leu Glu Asp Ile 420 425 430 aat gac
ttt gga gat gat ggg tcc ttg tat att act aag gtt acc aca 1404 Asn
Asp Phe Gly Asp Asp Gly Ser Leu Tyr Ile Thr Lys Val Thr Thr 435 440
445 act cac gtt ggc aat tac acc tgc tat gca gat ggc tat gaa caa gtc
1452 Thr His Val Gly Asn Tyr Thr Cys Tyr Ala Asp Gly Tyr Glu Gln
Val 450 455 460 465 tat cag act cac atc ttc caa gtg aat gtt cct cca
gtc atc cgg gtg 1500 Tyr Gln Thr His Ile Phe Gln Val Asn Val Pro
Pro Val Ile Arg Val 470 475 480 tat cca gag agt cag gct aga gag cct
ggg gta act gcc agt ctt agg 1548 Tyr Pro Glu Ser Gln Ala Arg Glu
Pro Gly Val Thr Ala Ser Leu Arg 485 490 495 tgc cat gca gag ggc ata
cca aag cct cag ctt ggc tgg ttg aag aat 1596 Cys His Ala Glu Gly
Ile Pro Lys Pro Gln Leu Gly Trp Leu Lys Asn 500 505 510 gga att gat
att aca cca aag ctt tcc aaa caa ctc acg ctt caa gca 1644 Gly Ile
Asp Ile Thr Pro Lys Leu Ser Lys Gln Leu Thr Leu Gln Ala 515 520 525
aat ggc agt gag gtt cac ata agc aat gtg cgc tat gaa gat act gga
1692 Asn Gly Ser Glu Val His Ile Ser Asn Val Arg Tyr Glu Asp Thr
Gly 530 535 540 545 gca tac act tgt atc gca aag aat gaa gca gga gtg
gat gaa gac atc 1740 Ala Tyr Thr Cys Ile Ala Lys Asn Glu Ala Gly
Val Asp Glu Asp Ile 550 555 560 tct tct ctt ttt gtg gaa gac tct gct
aga aag acc cta gct aac ata 1788 Ser Ser Leu Phe Val Glu Asp Ser
Ala Arg Lys Thr Leu Ala Asn Ile 565 570 575 tta tgg aga gaa gaa ggt
ctg gga att ggg aac atg ttc tat gtt ttt 1836 Leu Trp Arg Glu Glu
Gly Leu Gly Ile Gly Asn Met Phe Tyr Val Phe 580 585 590 tat gaa gat
gga atc aaa gtg ata caa ccc ata gaa tgt gaa ttt cag 1884 Tyr Glu
Asp Gly Ile Lys Val Ile Gln Pro Ile Glu Cys Glu Phe Gln 595 600 605
agg cac att aag cct agt gaa aag ctc ctt gga ttt cag gat gaa gtc
1932 Arg His Ile Lys Pro Ser Glu Lys Leu Leu Gly Phe Gln Asp Glu
Val 610 615 620 625 tgt ccc aaa gct gag gga gat gaa gtt cag agg tgt
gtg tgg gca tca 1980 Cys Pro Lys Ala Glu Gly Asp Glu Val Gln Arg
Cys Val Trp Ala Ser 630 635 640 gct gtt aat gtc aaa gac aag ttc att
tat gtt gca cag cca act ttg 2028 Ala Val Asn Val Lys Asp Lys Phe
Ile Tyr Val Ala Gln Pro Thr Leu 645 650 655 gac aga gtc ctt att gtt
gat gtg cag tcc caa aaa gtt gtt cag gca 2076 Asp Arg Val Leu Ile
Val Asp Val Gln Ser Gln Lys Val Val Gln Ala 660 665 670 gtg agc aca
gac cct gtc cca gtt aaa tta cac tat gac aaa tca cat 2124 Val Ser
Thr Asp Pro Val Pro Val Lys Leu His Tyr Asp Lys Ser His 675 680 685
gat cag gtc tgg gtg cta agc tgg ggt acc ttg gag aag aca tca cca
2172 Asp Gln Val Trp Val Leu Ser Trp Gly Thr Leu Glu Lys Thr Ser
Pro 690 695 700 705 aca cta cag gta att acc ctg gcc agt ggg aat gtg
cct cac cac acg 2220 Thr Leu Gln Val Ile Thr Leu Ala Ser Gly Asn
Val Pro His His Thr 710 715 720 atc cac acc caa cca gtg gga aag caa
ttt gac aga gtg gat gat ttt 2268 Ile His Thr Gln Pro Val Gly Lys
Gln Phe Asp Arg Val Asp Asp Phe 725 730 735 ttc att ccc acc aca aca
ctc att atc acc cat atg agg ttt gga ttt 2316 Phe Ile Pro Thr Thr
Thr Leu Ile Ile Thr His Met Arg Phe Gly Phe 740 745 750 att ctt cat
aaa gat gaa gct gca cta caa aaa att gat ctt gaa acc 2364 Ile Leu
His Lys Asp Glu Ala Ala Leu Gln Lys Ile Asp Leu Glu Thr 755 760 765
atg tca tac atc aag aca att aac ttg aag gac tat aag tgc gtt cct
2412 Met Ser Tyr Ile Lys Thr Ile Asn Leu Lys Asp Tyr Lys Cys Val
Pro 770 775 780 785 cag tca ttg gca tat aca cac ttg gga ggc tac tac
ttc att ggc tgc 2460 Gln Ser Leu Ala Tyr Thr His Leu Gly Gly Tyr
Tyr Phe Ile Gly Cys 790 795 800 aaa cct gac agc acc gga gca gtt tcc
cca cag gtc atg gtg gac ggt 2508 Lys Pro Asp Ser Thr Gly Ala Val
Ser Pro Gln Val Met Val Asp Gly 805 810 815 gta act gac tca gtc att
ggg ttc aat agt gat gtg acg ggc act cca 2556 Val Thr Asp Ser Val
Ile Gly Phe Asn Ser Asp Val Thr Gly Thr Pro 820 825 830 tat gtc tct
cca gat ggc cac tac ctt gtc agc att aat gat gtg aaa 2604 Tyr Val
Ser Pro Asp Gly His Tyr Leu Val Ser Ile Asn Asp Val Lys 835 840 845
ggt ctt gta agg gtt cag tac att acc atc aga gga gaa ata cag gag
2652 Gly Leu Val Arg Val Gln Tyr Ile Thr Ile Arg Gly Glu Ile Gln
Glu 850 855 860 865 gct ttt gat att tac aca aat ctg cac ata tct gat
ctg gca ttt caa 2700 Ala Phe Asp Ile Tyr Thr Asn Leu His Ile Ser
Asp Leu Ala Phe Gln 870 875 880 cca tcc ttt act gaa gcc cac caa tat
aac atc tac ggt agt tca agc 2748 Pro Ser Phe Thr Glu Ala His Gln
Tyr Asn Ile Tyr Gly Ser Ser Ser 885 890 895 aca caa act gat gtg ctc
ttt gtg gag ctc tct tct ggg aag gtc aag 2796 Thr Gln Thr Asp Val
Leu Phe Val Glu Leu Ser Ser Gly Lys Val Lys 900 905 910 atg ata aag
agt ctc aag gaa cca ctc aag gca gaa gaa tgg cct tgg 2844 Met Ile
Lys Ser Leu Lys Glu Pro Leu Lys Ala Glu Glu Trp Pro Trp 915 920 925
aac cgg aaa aac agg caa atc cag gac agt ggc ttg ttt ggt caa tac
2892 Asn Arg Lys Asn Arg Gln Ile Gln Asp Ser Gly Leu Phe Gly Gln
Tyr 930 935 940 945 ctg atg aca cct tcc aag gac tct ctc ttc atc cta
gat gga cga ctc 2940 Leu Met Thr Pro Ser Lys Asp Ser Leu Phe Ile
Leu Asp Gly Arg Leu 950 955 960 aat aaa tta aac tgt gag atc act gaa
gtt gaa aaa gga aat aca gtc 2988 Asn Lys Leu Asn Cys Glu Ile Thr
Glu Val Glu Lys Gly Asn Thr Val 965 970 975 att tgg gtt gga gat gcc
taaaaaccct acgatacaat tattgaatga 3036 Ile Trp Val Gly Asp Ala 980
agcgttttac aatacattgc acttaatcca ttgtttaaat ttacaactta actttccaag
3096 tttatatcct agtcaaacaa aatttacttg gttggtccaa ataaaataaa
ttgtttttga 3156 ctaagaaaaa aaaaaaaaaa aaattcctgc ggccgc 3192 2 983
PRT Homo sapiens 2 Met Phe Lys Cys Trp Ser Val Val Leu Val Leu Gly
Phe Ile Phe Leu 1 5 10 15 Glu Ser Glu Gly Arg Pro Thr Lys Glu Gly
Gly Tyr Gly Leu Lys Ser 20 25 30 Tyr Gln Pro Leu Met Arg Leu Arg
His Lys Gln Glu Lys Asn Gln Glu 35 40 45 Ser Ser Arg Val Lys Gly
Phe Met Ile Gln Asp Gly Pro Phe Gly Ser 50 55 60 Cys Glu Asn Lys
Tyr Cys Gly Leu Gly Arg His Cys Val Thr Ser Arg 65 70 75 80 Glu Thr
Gly Gln Ala Glu Cys Ala Cys Met Asp Leu Cys Lys Arg His 85 90 95
Tyr Lys Pro Val Cys Gly Ser Asp Gly Glu Phe Tyr Glu Asn His Cys 100
105 110 Glu Val His Arg Ala Ala Cys Leu Lys Lys Gln Lys Ile Thr Ile
Val 115 120 125 His Asn Glu Asp Cys Phe Phe Lys Gly Asp Lys Cys Lys
Thr Thr Glu 130 135 140 Tyr Ser Lys Met Lys Asn Met Leu Leu Asp Leu
Gln Asn Gln Lys Tyr 145 150 155 160 Ile Met Gln Glu Asn Glu Asn Pro
Asn Gly Asp Asp Ile Ser Arg Lys 165 170 175 Lys Leu Leu Val Asp Gln
Met Phe Lys Tyr Phe Asp Ala Asp Ser Asn 180 185 190 Gly Leu Val Asp
Ile Asn Glu Leu Thr Gln Val Ile Lys Gln Glu Glu 195 200 205 Leu Gly
Lys Asp Leu Phe Asp Cys Thr Leu Tyr Val Leu Leu Lys Tyr 210 215 220
Asp Asp Phe Asn Ala Asp Lys His Leu Ala Leu Glu Glu Phe Tyr Arg 225
230 235 240 Ala Phe Gln Val Ile Gln Leu Ser Leu Pro Glu Asp Gln Lys
Leu Ser 245 250 255 Ile Thr Ala Ala Thr Val Gly Gln Ser Ala Val Leu
Ser Cys Ala Ile 260 265 270 Gln Gly Thr Leu Arg Pro Pro Ile Ile Trp
Lys Arg Asn Asn Ile Ile 275 280 285 Leu Asn Asn Leu Asp Leu Glu Asp
Ile Asn Asp Phe Gly Asp Asp Gly 290 295 300 Ser Leu Tyr Ile Thr Lys
Val Thr Thr Thr His Val Gly Asn Tyr Thr 305 310 315 320 Cys Tyr Ala
Asp Gly Tyr Glu Gln Val Tyr Gln Thr His Ile Phe Gln 325 330 335 Val
Asn Val Pro Pro Val Ile Arg Val Tyr Pro Glu Ser Gln Ala Arg 340 345
350 Glu Pro Gly Val Thr Ala Ser Leu Arg Cys His Ala Glu Gly Ile Pro
355 360 365 Lys Pro Gln Leu Gly Trp Leu Lys Asn Gly Ile Asp Ile Thr
Pro Lys 370 375 380 Leu Ser Lys Gln Leu Thr Leu Gln Ala Asn Gly Ala
Thr Val Gly Gln 385 390 395 400 Ser Ala Val Leu Ser Cys Ala Ile Gln
Gly Thr Leu Arg Pro Pro Ile 405 410 415 Ile Trp Lys Arg Asn Asn Ile
Ile Leu Asn Asn Leu Asp Leu Glu Asp 420 425 430 Ile Asn Asp Phe Gly
Asp Asp Gly Ser Leu Tyr Ile Thr Lys Val Thr 435 440 445 Thr Thr His
Val Gly Asn Tyr Thr Cys Tyr Ala Asp Gly Tyr Glu Gln 450 455 460 Val
Tyr Gln Thr His Ile Phe Gln Val Asn Val Pro Pro Val Ile Arg 465 470
475 480 Val Tyr Pro Glu Ser Gln Ala Arg Glu Pro Gly Val Thr Ala Ser
Leu 485 490 495 Arg Cys His Ala Glu Gly Ile Pro Lys Pro Gln Leu Gly
Trp Leu Lys 500 505 510 Asn Gly Ile Asp Ile Thr Pro Lys Leu Ser Lys
Gln Leu Thr Leu Gln 515 520 525 Ala Asn Gly Ser Glu Val His Ile Ser
Asn Val Arg Tyr Glu Asp Thr 530 535 540 Gly Ala Tyr Thr Cys Ile Ala
Lys Asn Glu Ala Gly Val Asp Glu Asp 545 550 555 560 Ile Ser Ser Leu
Phe Val Glu Asp Ser Ala Arg Lys Thr Leu Ala Asn 565 570 575 Ile Leu
Trp Arg Glu Glu Gly Leu Gly Ile Gly Asn Met Phe Tyr Val 580 585 590
Phe Tyr Glu Asp Gly Ile Lys Val Ile Gln Pro Ile Glu Cys Glu Phe 595
600 605 Gln Arg His Ile Lys Pro Ser Glu Lys Leu Leu Gly Phe Gln Asp
Glu 610 615 620 Val Cys Pro Lys Ala Glu Gly Asp Glu Val Gln Arg Cys
Val Trp Ala 625 630 635 640 Ser Ala Val Asn Val Lys Asp Lys Phe Ile
Tyr Val Ala Gln Pro Thr 645 650 655 Leu Asp Arg Val Leu Ile Val Asp
Val Gln Ser Gln Lys Val Val Gln 660 665 670 Ala Val Ser Thr Asp Pro
Val Pro Val Lys Leu His Tyr Asp Lys Ser 675 680 685 His Asp Gln Val
Trp Val Leu Ser Trp Gly Thr Leu Glu Lys Thr Ser 690 695 700 Pro Thr
Leu Gln Val Ile Thr Leu Ala Ser Gly Asn Val Pro His His 705 710 715
720 Thr Ile His Thr Gln Pro Val Gly Lys Gln Phe Asp Arg Val Asp Asp
725 730 735 Phe Phe Ile Pro Thr Thr Thr Leu Ile Ile Thr His Met Arg
Phe Gly 740 745 750 Phe Ile Leu His Lys Asp Glu Ala Ala Leu Gln Lys
Ile Asp Leu Glu 755 760 765 Thr Met Ser Tyr Ile Lys Thr Ile Asn Leu
Lys Asp Tyr Lys Cys Val 770 775 780 Pro Gln Ser Leu Ala Tyr Thr His
Leu Gly Gly Tyr Tyr Phe Ile Gly 785 790 795
800 Cys Lys Pro Asp Ser Thr Gly Ala Val Ser Pro Gln Val Met Val Asp
805 810 815 Gly Val Thr Asp Ser Val Ile Gly Phe Asn Ser Asp Val Thr
Gly Thr 820 825 830 Pro Tyr Val Ser Pro Asp Gly His Tyr Leu Val Ser
Ile Asn Asp Val 835 840 845 Lys Gly Leu Val Arg Val Gln Tyr Ile Thr
Ile Arg Gly Glu Ile Gln 850 855 860 Glu Ala Phe Asp Ile Tyr Thr Asn
Leu His Ile Ser Asp Leu Ala Phe 865 870 875 880 Gln Pro Ser Phe Thr
Glu Ala His Gln Tyr Asn Ile Tyr Gly Ser Ser 885 890 895 Ser Thr Gln
Thr Asp Val Leu Phe Val Glu Leu Ser Ser Gly Lys Val 900 905 910 Lys
Met Ile Lys Ser Leu Lys Glu Pro Leu Lys Ala Glu Glu Trp Pro 915 920
925 Trp Asn Arg Lys Asn Arg Gln Ile Gln Asp Ser Gly Leu Phe Gly Gln
930 935 940 Tyr Leu Met Thr Pro Ser Lys Asp Ser Leu Phe Ile Leu Asp
Gly Arg 945 950 955 960 Leu Asn Lys Leu Asn Cys Glu Ile Thr Glu Val
Glu Lys Gly Asn Thr 965 970 975 Val Ile Trp Val Gly Asp Ala 980 3
2949 DNA Artificial Sequence Degenerate oligonucleotide sequence
encoding the zfsta2 polypeptide of SEQ ID NO2. 3 atgttyaart
gytggwsngt ngtnytngtn ytnggnttya thttyytnga rwsngarggn 60
mgnccnacna argarggngg ntayggnytn aarwsntayc arccnytnat gmgnytnmgn
120 cayaarcarg araaraayca rgarwsnwsn mgngtnaarg gnttyatgat
hcargayggn 180 ccnttyggnw sntgygaraa yaartaytgy ggnytnggnm
gncaytgygt nacnwsnmgn 240 garacnggnc argcngartg ygcntgyatg
gayytntgya armgncayta yaarccngtn 300 tgyggnwsng ayggngartt
ytaygaraay caytgygarg tncaymgngc ngcntgyytn 360 aaraarcara
arathacnat hgtncayaay gargaytgyt tyttyaargg ngayaartgy 420
aaracnacng artaywsnaa ratgaaraay atgytnytng ayytncaraa ycaraartay
480 athatgcarg araaygaraa yccnaayggn gaygayathw snmgnaaraa
rytnytngtn 540 gaycaratgt tyaartaytt ygaygcngay wsnaayggny
tngtngayat haaygarytn 600 acncargtna thaarcarga rgarytnggn
aargayytnt tygaytgyac nytntaygtn 660 ytnytnaart aygaygaytt
yaaygcngay aarcayytng cnytngarga rttytaymgn 720 gcnttycarg
tnathcaryt nwsnytnccn gargaycara arytnwsnat hacngcngcn 780
acngtnggnc arwsngcngt nytnwsntgy gcnathcarg gnacnytnmg nccnccnath
840 athtggaarm gnaayaayat hathytnaay aayytngayy tngargayat
haaygaytty 900 ggngaygayg gnwsnytnta yathacnaar gtnacnacna
cncaygtngg naaytayacn 960 tgytaygcng ayggntayga rcargtntay
caracncaya thttycargt naaygtnccn 1020 ccngtnathm gngtntaycc
ngarwsncar gcnmgngarc cnggngtnac ngcnwsnytn 1080 mgntgycayg
cngarggnat hccnaarccn carytnggnt ggytnaaraa yggnathgay 1140
athacnccna arytnwsnaa rcarytnacn ytncargcna ayggngcnac ngtnggncar
1200 wsngcngtny tnwsntgygc nathcarggn acnytnmgnc cnccnathat
htggaarmgn 1260 aayaayatha thytnaayaa yytngayytn gargayatha
aygayttygg ngaygayggn 1320 wsnytntaya thacnaargt nacnacnacn
caygtnggna aytayacntg ytaygcngay 1380 ggntaygarc argtntayca
racncayath ttycargtna aygtnccncc ngtnathmgn 1440 gtntayccng
arwsncargc nmgngarccn ggngtnacng cnwsnytnmg ntgycaygcn 1500
garggnathc cnaarccnca rytnggntgg ytnaaraayg gnathgayat hacnccnaar
1560 ytnwsnaarc arytnacnyt ncargcnaay ggnwsngarg tncayathws
naaygtnmgn 1620 taygargaya cnggngcnta yacntgyath gcnaaraayg
argcnggngt ngaygargay 1680 athwsnwsny tnttygtnga rgaywsngcn
mgnaaracny tngcnaayat hytntggmgn 1740 gargarggny tnggnathgg
naayatgtty taygtnttyt aygargaygg nathaargtn 1800 athcarccna
thgartgyga rttycarmgn cayathaarc cnwsngaraa rytnytnggn 1860
ttycargayg argtntgycc naargcngar ggngaygarg tncarmgntg ygtntgggcn
1920 wsngcngtna aygtnaarga yaarttyath taygtngcnc arccnacnyt
ngaymgngtn 1980 ytnathgtng aygtncarws ncaraargtn gtncargcng
tnwsnacnga yccngtnccn 2040 gtnaarytnc aytaygayaa rwsncaygay
cargtntggg tnytnwsntg gggnacnytn 2100 garaaracnw snccnacnyt
ncargtnath acnytngcnw snggnaaygt nccncaycay 2160 acnathcaya
cncarccngt nggnaarcar ttygaymgng tngaygaytt yttyathccn 2220
acnacnacny tnathathac ncayatgmgn ttyggnttya thytncayaa rgaygargcn
2280 gcnytncara arathgayyt ngaracnatg wsntayatha aracnathaa
yytnaargay 2340 tayaartgyg tnccncarws nytngcntay acncayytng
gnggntayta yttyathggn 2400 tgyaarccng aywsnacngg ngcngtnwsn
ccncargtna tggtngaygg ngtnacngay 2460 wsngtnathg gnttyaayws
ngaygtnacn ggnacnccnt aygtnwsncc ngayggncay 2520 tayytngtnw
snathaayga ygtnaarggn ytngtnmgng tncartayat hacnathmgn 2580
ggngarathc argargcntt ygayathtay acnaayytnc ayathwsnga yytngcntty
2640 carccnwsnt tyacngargc ncaycartay aayathtayg gnwsnwsnws
nacncaracn 2700 gaygtnytnt tygtngaryt nwsnwsnggn aargtnaara
tgathaarws nytnaargar 2760 ccnytnaarg cngargartg gccntggaay
mgnaaraaym gncarathca rgaywsnggn 2820 ytnttyggnc artayytnat
gacnccnwsn aargaywsny tnttyathyt ngayggnmgn 2880 ytnaayaary
tnaaytgyga rathacngar gtngaraarg gnaayacngt nathtgggtn 2940
ggngaygcn 2949 4 147 DNA Artificial Sequence 147 bp probe 4
ggatttatga ttcaggatgg cccttttgga tcttgtgaaa ataagtactg tggtttngga
60 agacactgtg ttacccagca gagagacagg gcaagcagaa tgtgcctgta
tggacctttg 120 caaacgtcac tacaaacctg tgtgtgg 147 5 25 DNA
Artificial Sequence Oligonucleotide ZC18415 5 cctgggggat tgtgtgactg
ttaaa 25 6 24 DNA Artificial Sequence Oligonucleotide ZC14701 6
ctgccatttg cttgaagcgt gagt 24 7 1255 DNA Artificial Sequence 3' end
of zfsta2 nucleotide sequence 7 gaattcggct tcctggggga ttgtgtgact
gttaaaataa ggtgaaaagc aataaggatg 60 tttaagtgct ggtcagttgt
cttggttctc ggattcattt ttctggagtc ggaaggaagg 120 ccaaccaaag
aaggaggata tggccttaaa tcctatcagc ctctaatgag attgcgacat 180
aagcaggaaa aaaatcaaga aagttcaaga gtcaaaggat ttatgattca ggatggccct
240 tttggatctt gtgaaaataa gtactgtggt ttgggaagac actgtgttac
cagcagagag 300 acagggcaag cagaatgtgc ctgtatggac ctttgcaaac
gtcactacaa acctgtgtgt 360 ggatctgacg gagaattcta tgaaaaccac
tgtgaagtgc acagagctgc ttgcctgaaa 420 aaacaaaaga ttaccattgt
tcacaatgaa gactgcttct ttaaaggaga taagtgcaag 480 actactgaat
acagcaagat gaaaaatatg ctattagatt tacaaaatca aaaatatatt 540
atgcaagaaa atgaaaatcc taatggcgac gacatatctc ggaagaagct attggtggat
600 caaatgttta aatattttga tgcagacagt aatggacttg tagatattaa
tgaactaact 660 caggtgataa aacaggaaga acttggcaag gatctctttg
attgtacttt gtatgttcta 720 ttgaaatatg atgattttaa tgctgacaag
cacctggctc ttgaagaatt ttatagagca 780 ttccaagtga tccagttgag
tctgccagaa gatcagaaac taagcatcac tgcagcaact 840 gtgggacaaa
gtgctgttct gagctgtgcc attcaaggaa ccctgagacc tcccattatc 900
tggaaaagga acaatattat tctaaataat ttagatttgg aagacatcaa tgactttgga
960 gatgatgggt ccttgtatat tactaaggtt accacaactc acgttggcaa
ttacacctgc 1020 tatgcagatg gctatgaaca agtctatcag actcacatct
tccaagtgaa tgttcctcca 1080 gtcatccggg tgtatccaga gagtcaggct
agagagcctg gggtaactgc cagtcttagg 1140 tgccatgcag agggcatacc
aaagcctcag cttggctggt tgaagaatgg aattgatatt 1200 acaccaaagc
tttccaaaca actcacgctt caagcaaatg gcagaagccg aattc 1255 8 1896 DNA
Artificial Sequence 5' end of zfsta2 nucleotide sequence 8
aagcttggca cgagggcaac tgtgggacaa agtgctgttc tgagctgtgc cattcaagga
60 accctgagac ctcccattat ctggaaaagg aacaatatta ttctaaataa
tttagatttg 120 gaagacatca atgactttgg agatgatggg tccttgtata
ttactaaggt taccacaact 180 cacgttggca attacacctg ctatgcagat
ggctatgaac aagtctatca gactcacatc 240 ttccaagtga atgttcctcc
agtcatccgg gtgtatccag agagtcaggc tagagagcct 300 ggggtaactg
ccagtcttag gtgccatgca gagggcatac caaagcctca gcttggctgg 360
ttgaagaatg gaattgatat tacaccaaag ctttccaaac aactcacgct tcaagcaaat
420 ggcagtgagg ttcacataag caatgtgcgc tatgaagata ctggagcata
cacttgtatc 480 gcaaagaatg aagcaggagt ggatgaagac atctcttctc
tttttgtgga agactctgct 540 agaaagaccc tagctaacat attatggaga
gaagaaggtc tgggaattgg gaacatgttc 600 tatgtttttt atgaagatgg
aatcaaagtg atacaaccca tagaatgtga atttcagagg 660 cacattaagc
ctagtgaaaa gctccttgga tttcaggatg aagtctgtcc caaagctgag 720
ggagatgaag ttcagaggtg tgtgtgggca tcagctgtta atgtcaaaga caagttcatt
780 tatgttgcac agccaacttt ggacagagtc cttattgttg atgtgcagga
tcaggtctgg 840 gtgctaagct ggggtacctt ggagaagaca tcaccaacac
tacaggtaat taccctggcc 900 agtgggaatg tgcctcacca cacgatccac
acccaaccag tgggaaagca atttgacaga 960 gtggatgatt ttttcattcc
caccacaaca ctcattatca cccatatgag gtttggattt 1020 attcttcata
aagatgaagc tgcactacaa aaaattgatc ttgaaaccat gtcatacatc 1080
aagacaatta acttgaagga ctataagtgc gttcctcagt cattggcata tacacacttg
1140 ggaggctact acttcattgg ctgcaaacct gacagcaccg gagcagtttc
cccacaggtc 1200 atggtggacg gtgtaactga ctcagtcatt gggttcaata
gtgatgtgac gggcactcca 1260 tatgtctctc cagatggcca ctaccttgtc
agcattaatg atgtgaaagg tcttgtaagg 1320 gttcagtaca ttaccatcag
aggagaaata caggaggctt ttgatattta cacaaatctg 1380 cacatatctg
atctggcatt tcaaccatcc tttactgaag cccaccaata taacatctac 1440
ggtagttcaa gcacacaaac tgatgtgctc tttgtggagc tctcttctgg gaaggtcaag
1500 atgataaaga gtctcaagga accactcaag gcagaagaat ggccttggaa
ccggaaaaac 1560 aggcaaatcc aggacagtgg cttgtttggt caatacctga
tgacaccttc caaggactct 1620 ctcttcatcc tagatggacg actcaataaa
ttaaactgtg agatcactga agttgaaaaa 1680 ggaaatacag tcatttgggt
tggagatgcc taaaaaccct acgatacaat tattgaatga 1740 agcgttttac
aatacattgc acttaatcca ttgtttaaat ttacaactta actttccaag 1800
tttatatcct agtcaaacaa aatttacttg gttggtccaa ataaaataaa ttgtttttga
1860 ctaagaaaaa aaaaaaaaaa aaattcctgc ggccgc 1896 9 23 DNA
Artificial Sequence Oligonucleotide ZC12881 9 ggatttatga ttcaggatgg
ccc 23 10 22 DNA Artificial Sequence Oligonucleotide ZC12884 10
ccacacacag gtttgtagtg ac 22 11 18 DNA Artificial Sequence
Oligonucleotide ZC15510 11 tggacggtgt aactgact 18 12 18 DNA
Artificial Sequence Oligonucleotide ZC15575 12 aagcctcctg tatttctc
18 13 12 DNA Artificial Sequence Contig example 13 atggcttagc tt 12
14 12 DNA Artificial Sequence Contig example 14 tagcttgagt ct 12 15
12 DNA Artificial Sequence Contig example 15 gtcgactacc ga 12
* * * * *
References