U.S. patent application number 10/252340 was filed with the patent office on 2003-04-24 for ps20 amino acid sequences.
Invention is credited to Rowley, David R..
Application Number | 20030077745 10/252340 |
Document ID | / |
Family ID | 21731118 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077745 |
Kind Code |
A1 |
Rowley, David R. |
April 24, 2003 |
ps20 amino acid sequences
Abstract
Urogenital sinus derived growth inhibitory factor is a protein
having growth-inhibitory and antiprotease properties. The present
invention relates to amino acid and nucleotide sequences for
urogenital sinus derived growth inhibitory factor.
Inventors: |
Rowley, David R.; (Houston,
TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
21731118 |
Appl. No.: |
10/252340 |
Filed: |
September 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10252340 |
Sep 23, 2002 |
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09335318 |
Jun 17, 1999 |
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09335318 |
Jun 17, 1999 |
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08761248 |
Dec 6, 1996 |
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5958735 |
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60008348 |
Dec 7, 1995 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/6.16; 435/7.5; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/81 20130101;
Y10S 530/834 20130101; A61K 38/00 20130101; C07K 14/4703 20130101;
C07K 14/811 20130101 |
Class at
Publication: |
435/69.1 ; 435/6;
435/7.5; 435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C12P 021/02; C12N
005/06; C12Q 001/68; G01N 033/53; C07H 021/04; C07K 014/705 |
Goverment Interests
[0002] The following invention was supported in part through NIH
Grant Nos. DK45 909, CA 58093, and SPORE CA 58204. The United
States Government may have certain rights in the invention.
Claims
What is claimed is:
1. An isolated DNA molecule encoding a urogenital sinus derived
growth inhibitory factor.
2. The DNA molecule of claim 1 wherein said DNA is cDNA.
3. The DNA molecule of claim 1 wherein said DNA is genomic DNA.
4. The DNA molecule of claim 1 wherein said DNA is human DNA.
5. The DNA molecule of claim 4 wherein said DNA is genomic DNA.
6. The DNA molecule of claim 1 wherein said DNA is mouse DNA.
7. The mouse DNA of claim 6 wherein said DNA is mouse genomic
DNA.
8. The mouse genomic DNA of claim 7 wherein said mouse genomic DNA
comprises the sequence of SEQ ID NOS. 16 or 17.
9. The DNA molecule of claim 1 wherein said DNA is rat genomic
DNA.
10. An isolated DNA molecule encoding a WAP four-disulfide core
domain 1 gene.
11. The DNA molecule of claim 10, wherein said gene is WFDC1.
12. The DNA molecule of claim 10 wherein said gene is Wfdc1.
13. A recombinant urogenital sinus derived growth inhibitory
factor.
14. The factor of claim 13 wherein said factor is a human
protein.
15. The factor of claim 13 wherein said factor is a rat
protein.
16. The factor of claim 13 wherein said factor is a mouse
protein.
17. An antibody immunologically recognizing urogenital sinus
derived growth inhibitory factor.
18. The antibody of claim 17 wherein said antibody is a polyclonal
antibody.
19. The antibody of claim 17 where said antibody is a monoclonal
antibody.
20. A method of detecting the presence of DNA or RNA encoding a
urogenital sinus derived growth inhibitory factor in a sample
comprising: labeling any of the DNA molecules of claims 1-12, or a
fragment thereof; contacting said labeled DNA or fragment with a
sample containing DNA in a manner conducive to DNA-DNA or DNA-RNA
hybridization; and, determining if said hybridization takes place
by detecting the presence of said labeled DNA or fragment.
21. The method of claim 20 wherein said labeling is
radiolabeling.
22. The method of claim 20 wherein said labeling is fluorescent
labeing.
23. A kit for detecting the presence of DNA or RNA encoding a
urogenital sinus derived growth inhibitory factor comprising:
labeled DNA as in any of claims 1-12; and hybridization
reagents.
24. A method of detecting the presence of a urogenital sinus
derived growth inhibitory factor in a sample comprising: labeling
any of the antibodies of claims 17-19, or an immunologically
reactive fragment thereof; contacting said labeled antibody or
fragment with a sample containing protein in a manner conducive to
antibody-antigen cross reactivity; and determining if said cross
reactivity takes place by detecting the presence of said labeled
antibody or fragment.
25. The method of claim 24 wherein the labeling is
radiolabeling.
26. The method of claim 25 wherein the labeling is fluorescent
labeling.
27. A method of detecting the presence or localization of DNA or
RNA encoding a urogenital sinus derived growth inhibitory factor in
a chromosome, comprising: labeling any of the DNA molecules of
claims 1-12, or a fragment thereof; contacting said labelled DNA or
fragment with a chromosome in a manner conducive to DNA-DNA or
DNA-RNA hybridization; and determining the presence or localization
of said chromosonal DNA encoding said factor on said
chromosome.
28. The method of claim 27 wherein the labeling is
radiolabeling.
29. The method of claim 27 wherein the labeling is fluorescent
labeling.
30. The method of claim 27 wherein the labeling is biotin
labeling.
31. A biologically functional vector comprising a DNA molecule
encoding a urogenital sinus derived growth inhibitory factor.
32. The vector of claim 31 wherein said DNA molecule is a human
DNA.
33. The vector of claim 31 wherein said DNA molecule is a rat
DNA.
34. The vector of claim 31 wherein said DNA molecule is a mouse
DNA.
35. A host cell containing any of the vectors of claims 31-34.
36. The cell of claim 39 wherein said cell is prokaryotic.
37. The cell of claim 39 wherein said cell is eukaryotic.
38. A method of making a recombinant urogenital sinus derived
growth inhibitory factor comprising: culturing a cell as in any of
claims 35-37 in a medium; harvesting said cell from said culture or
harvesting said cell culture medium; extracting said factor from
said cell or said medium.
39. A method of treating a stromal cell comprising: contacting said
cell with a urogenital sinus derived growth inhibitory factor.
40. The method of claim 39 wherein said stromal cell is a vascular
cells.
41. The method of claim 39 wherein said stromal cell is a tumor
cells.
42. The method of claim 39 wherein said stromal cell is an organ
smooth muscle cell.
43. The method of claim 39 wherein said stromal cell is a stroma
cell involved in fibrosis, wound repair or reactive stroma.
44. A method of genetic therapy comprising: introducing into a
patient a vector capable of transfecting said patient with a gene
encoded in said vector; said gene encoding a urogenital sinus
derived growth inhibitory factor.
45. The method of claim 44 wherein said transfection is
permanent.
46. The method of claim 44 wherein said transfection is
temporary.
47. The method of claim 44 wherein said therapy is vascular
therapy.
48. The method of claim 44 wherein said therapy is cancer
therapy.
49. An affinity chromatographic medium comprising an antibody
against a recombinant urogenital sinus derived growth inhibitory
factor.
Description
CROSS REFERENCE TO PROVISIONAL APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/008,348 filed Dec. 7, 1995. U.S. Provisional
Application No. 60/008,348 was converted to U.S. patent application
Ser. No. 08/761,248 on Dec. 6, 1996. This application is a
divisional and continuation in part of U.S. patent application Ser.
No. 08/761,248 (now U.S. Pat. No. ______).
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to a urogenital sinus derived
growth inhibitory factor, ps20. More specifically, the present
invention relates to uses of the factor and to the amino acid and
nucleotide sequence of said factor. The present invention also
relates to antibodies which bind to said factor.
[0005] 2. Description of the Prior Art
[0006] Epithelial differentiation patterns are induced by stromal
cells in most eukaryotic tissues including lung, breast, stomach,
skin, eye, and prostate gland. In prostate development, ductal
morphogenesis and epithelial acini differentiation result from
stromal induction. In heterotypical tissue-tissue recombinants,
mesenchyme from fetal urogenital sinus (anlagen of the prostate
gland) induces bladder epithelial cells to undergo ductal
morphogenesis and differentiation to a prostatic epithelial
phenotype, capable of expressing prostate specific proteins.
[0007] Prostate ductal morphogenesis is characterized by a
stromal-induced epithelial cell proliferation. Following this,
epithelial cytodifferentiation (to the secretory phenotype) is
correlated with a cellular quiescence which also requires stromal
interaction. In most cells, differentiated gene expression is
associated with a reduced cell proliferation. In cell culture,
growth stimulatory activities (to epithelial cells) have been
observed in the conditioned medium from prostate stromal cells,
including bFGF and NGF-like factors.
[0008] Progress has been made in the identification of keratinocyte
growth factor (KGF) as a likely candidate stromal-derived factor.
KGF expression is restricted to stroma and is androgen regulated in
the prostate. KGF mediates, in part, the stromal induction of
seminal vesicle epithelium proliferation. TGF-.beta. and TGF-.beta.
receptors are negatively regulated by androgen in the prostate.
TGF-.beta.s are expressed in development, however, their role in
prostate development is not clear. Growth inhibitory activities
secreted from prostate stromal cultures have been reported by the
inventor's laboratory group and others, not attributed to known
inhibitory factors, including the TGF-.beta.s. This growth
inhibitory activity is attributable to the urogenital derived sinus
growth inhibitory factor (UGIF) ps20 protein, which also induces
protein synthesis and alters phenotypic morphology of target
epithelial cells. As with all other growth regulatory proteins,
ps20 is not specific to prostate, but is also expressed in
mesenchymal and smooth muscle cells in other tissue. The
developmental pattern of prostatic ductal morphogenesis followed by
epithelial differentiation likely involves the timed expression of
a variety of positive and negative growth regulatory factors.
[0009] Studies with rat and human prostatic smooth muscle cell
lines show androgen-stimulated proliferation with physiological
concentrations (5-10 mM) of androgen. These observations together
indicate urogenital sinus mesenchyme and adult smooth muscle cells
may express genes fundamental to stromal-epithelial interactions in
the prostate gland.
[0010] Progress has been limited in identification of
stromal-derived regulatory proteins and their mechanisms due to
technical difficulties in the isolation and culture of androgen
responsive stromal cell lines, difficulties in the biochemical
analysis of secreted or extracellular matrix proteins, and the
relative unavailability of tissue-specific stromal cell cDNA
libraries.
[0011] Benign prostatic hyperplasia (BPH) and prostate cancer are
disorders of prostatic epithelial growth and differentiation. BPH
disorders are perhaps most relevant to stromal-epithelial
interactions. BPH initiates from localized stromal cell
proliferation. The initiation of BPH has been termed a
"reawakening" of the inductive potential of the prostate stroma and
a spontaneous reversion of the stroma to an embryonic state.
Accordingly, the abnormal proliferation of stromal cells in the
periurethral region can induce the ingrowth and abnormal formation
of acini from adjacent epithelial cells.
[0012] During prostate carcinogenesis, carcinoma progression
patterns involve stromal-epithelial interactions. Prostatic
carcinoma is typified by progression from an androgen responsive
state to an androgen insensitive state which no longer responds to
anti-androgen therapy. Some evidence exists to suggest that
progression to androgen insensitivity results from altered gene
expression in stromal cells. In the Dunning rat prostate carcinoma,
the type of stroma can induce the adjacent epithelium to exhibit
exon switching of FGF receptors (FGFRc2 IIIb to IIIc) which imparts
androgen insensitive proliferation to these epithelial cells.
Dunning tumor prostate carcinoma cell proliferation was inhibited
by 7-fold when recombined with normal seminal vesicle or urogenital
sinus mesenchyme. The recombined carcinoma cells showed an
alteration in phenotypic morphology. When recombined with normal
mesenchyme, carcinoma cells exhibited a tall, columnar cell shape,
typical of a differentiated secretory epithelium as compared to the
typical squamous/cuboidal undifferentiated phenotype in wild-type
Dunning tumor. In this regard, it is of interest that smooth muscle
is absent from Dunning prostatic tumor. In addition, the pattern of
carcinoma formation can be influenced by the origin of the
associated stromal cells. Recombination of bladder transitional
cell carcinoma with normal urogenital sinus mesenchyme resulted in
the formation of a glandular adenocarcinoma phenotype typical of
prostate. Tissue-tissue recombination studies to produce prostatic
tumors in mice requires transformation of mesenchyme (with myc and
ras) to produce prostatic adenocarcinoma typical of the human
phenotype. Conversely, the inoculation of fibrosarcoma tumorigenic
stromal cells with non-tumorigenic normal epithelial cells into
nude mice resulted in a mixed carcinoma-fibrosarcoma. Together
these studies indicate prostatic carcinoma epithelium is responsive
to the stromal environment and that progression and overall
phenotype of prostate carcinoma is dependent to some degree on
stromal interaction. It follows that key proteins involved in
mechanisms of stromal-epithelial interactions will be of
significance to the study of prostate proliferation diseases.
[0013] Balance of protease and protease inhibitor function is
involved in modeling of tissues, extracellular matrix (ECM)
compositions, and growth factor activation processes. Proteases
play a significant role in embryogenesis, extracellular matrix
modeling/remodeling and in tumorigenesis involving abnormal
proliferation, promotion of tumor invasion, and formation of
metastasis. It is well-established that metalloproteinases are
overexpressed in most neoplastic diseases including breast cancer,
colon cancer, neuroblastomas, and prostate cancer. Cysteine
proteases, including Cathepsins B and D, are elevated in many
cancer metastases, including prostate cancer. Significant to tumor
progression, due to their induced cascade of effects are the
plasminogen activator (PA) proteases. Plasminogen activators are
serine proteases which convert inactive plasminogen to the active
form, plasmin. Plasmin in turn exhibits broad proteolytic
trypsin-like effects on ECM components including glycoproteins,
proteoglycans (including heparin and heparin-sulfates), and
gelatins. Plasmin also activates a variety of EMC bound growth
factors from latent to active forms including the TGF-.beta.s. The
urokinase-type PA and tissue-type PA have been the most extensively
studied PAs. Urokinase PA is primarily involved in tissue
modeling-remodeling activities, whereas tissue PA is most active in
blood clot lysis.
[0014] Since plasminogen is present in all tissues and fluids,
local effects of plasmin are mediated by local expression of PA.
Urokinase PA is secreted as an inactive pro-form which binds with
high affinity to a membrane-anchored specific receptor where it is
cleaved to the active form and remains (on cell surface) for
several hours. On the cell surface, urokinase PA has a focal effect
resulting in local acceleration of plasmin activation by
approximately 40-fold. Plasmin activity is elevated in the focal
environment to the cell surface expressing active urokinase. Focal
plasmin effects degradation of ECM and activates metalloproteinases
(procollagenases, prostromelysin, elastase). Accordingly, secretion
of small amounts of urokinase PA results in a focal plasmin cascade
to effect a spectrum of other enzymes and factors. Urokinase PA is
inhibited by PA inhibitors (PAIs) which are serine protease
inhibitors. Local actions of PAs (and other proteases) have been
implicated in a wide array of developmental processes through
highly regulated mechanisms. PAs, PAIs, and proteases are each
regulated by hormones and growth factors.
[0015] In addition to functions in development, urokinase PA is
elevated in most tumor metastases. Elevated urokinase PA leads to
down stream activation of proteases and growth factors with
increased tumor invasion, increased tumor volume, and increased
cell proliferation rate. In the prostate, the study of proteases
and inhibitors have focused primarily on carcinoma progression. PA
activity is higher in prostate carcinoma than in normal tissue and
the urokinase PA form is primarily associated with progression.
Urokinase PA is elevated in prostate bone metastasis relative to
primary tumor site. Urokinase PA is overexpressed in Dunning,
Nobel, Lobund-Wistar, and Fisher-334 prostatic tumors. Moreover,
urokinase PA is the predominant PA secreted by the PC-3 and DU-145
human prostatic carcinoma cell lines and these cell lines exhibit
the urokinase PA cell surface receptor. Studies by the inventor
have used the PC-3 cell line to identify and purify ps20 secreted
from fetal urogenital sinus mesenchymal cells. Metastasis of PC-3
in nude mice was shown to be blocked by mutated urokinase PA or
urokinase PA receptor blocking antibodies.
[0016] Direct evidence shows growth inhibition of cancer cells by
urokinase PA inhibitors and other protease inhibitors. A synthetic
urokinase PA inhibitor (p-aminobenzamidine) inhibited the
progression of DU-145 human prostate carcinoma in SCID mice and
cell proliferation in culture in a dose-dependent manner (64%
decreased tumor volume). The protease inhibitor actinonin inhibited
mammary tumor progression (both non-metastatic and metastatic
types) in collagen gels. Batimastat, a matrix metalloproteinase
inhibitor, inhibited organ invasion in lung (72% decrease in tumor
volume) of two human colon carcinomas. In human prostate, decreased
expression of acid cysteine proteinase inhibitor (ACPI)(cathepsin
inhibitor) was observed in BPH tissue relative to normal. No
expression of ACPI was found in human prostatic adenocarcinoma
tissue. Accordingly, balances of proteases and protease inhibitors
may affect proliferation in human BPH and carcinoma.
[0017] U.S. Pat. No. 5,196,334, incorporated by reference herein,
describes the isolation and partial characterization of urogenital
sinus derived growth inhibitory factor, UGIF (ps20). However, the
amino acid and nucleotide sequence of ps20 has not heretofore been
described. Additionally, antibodies to ps20 have also not
heretofore been described. Moreover, the gene sequences from a
variety of different sources including human have not heretofor
been proven.
SUMMARY OF THE INVENTION
[0018] The present invention relates to the amino acid and
nucleotide sequences of a urogenital sinus derived growth
inhibitory factor. Accordingly, provided herein is an amino acid
sequence which codes for urogenital sinus derived growth inhibitory
factor, UGIF (ps20). The ps20 of the present invention has protease
inhibitory function. Also provided herein is a nucleotide sequence
which codes for the urogenital sinus derived growth inhibitory
factor protein. Also provided herein are antibodies which bind to
urogenital sinus derived growth inhibiting factor, ps20.
[0019] In partcular, the present invention relates to an isolated
DNA molecule encoding a urogenital sinus derived growth inhibitory
factor. The inventor has provided numerous species of this factor
in the examples. The DNA may be any form of DNA, such as cDNA or
genomic DNA. It also may be derived from any biological source in
which the factor is present, including human DNA, mouse DNA, or rat
DNA as shown in the examples. In particular, the DNA may be any of
those shown in the Sequence Listing for cDAN or genomic DNA derived
from human, mouse or rat biological sources.
[0020] The present invention also relates to an isolated DNA
molecule encoding a WAP four-disulfide core domain 1 gene. As
discussed herein, this designation identifies the family of genes
which encodes urogenital sinus derived growth inhibitory factor
(also known as "ps20."). In particular, the gene may be the human
gene WFDC1, or it may be the mouse gene Wfdc1.
[0021] The present invention also relates to the polypeptide family
known as recombinant urogenital sinus derived growth inhibitory
factor. The term "recombinant" is given its normal meaning to those
of skill in the art of gene cloning, and would include any
genetically isolated gene or DNA fragment which is isolated
independent from the native DNA in which it is found in nature.
Such isolation may involve cloning vectors, or it may involve
fragmentation of the DNA. Regardless of the manner that the
recombinant protein is produced, it may be derived from any
biological source in which it is found, and includes the human,
mouse and rat proteins.
[0022] With the availability of highly purified recombinant
proteins made possible by this invention, the invention discloses
antibodies immunologically recognizing urogenital sinus derived
growth inhibitory factor. Such antibodies may particularly be
polyclonal or monoclonal antibodies.
[0023] Also provided by the present invention are methods of
detecting the presence of DNA encoding a urogenital sinus derived
growth inhibitory factor in a sample. These methods comprise
labeling any of the DNA molecules encoding the factor, or a
fragment of the factor. The labeled DNA or fragment is then
contacted with a sample containing a target. The method is carried
out under conditions known well to those of skill in the art of
DNA-DNA hybridization to allow the target DNA to hybridize to the
labeled DNA. The method then calls for determining whether or not
hybridization has taken place by detecting the presence of the
labeled DNA or labeled fragment is hybridized to the target DNA.
Labeled DNA or labeled RNA derived from the DNA template may be
used to detect messenger RNA (mRNA) through the use of Northern
blot analysis, ribonuclease protection assay (RPA) and in situ
localization of mRNA in cells using appropriately prepared tissue
sections. Moreover, labeled or unlabeled DNA or derived RNA may be
used in preparation of a microarray chip used for the detection of
the gene or cDNA or mRNA derived from this gene. In some cases the
labeling will be done using radioactive markers and in others,
fluorescent markers, or combinations of such markers. In a
particular embodiment, a kit for detecting the presence of DNA
encoding a urogenital sinus derived growth inhibitory factor
utilizing such methods will include labeled DNA and hybridization
reagents.
[0024] Similarly, the present invention disclsoes methods of
detecting the presence of a urogenital sinus derived growth
inhibitory factor in a protein sample comprising labeling any of
the antibodies of the invention (or immunologically reactive
fragments of these antibodies). Then, the labeled antibody or
fragment is contacted with a sample containing protein in a manner
known by those of skill in the art of immunology to be conducive to
antibody-antigen cross reactivity. Finally, the method provides for
determining if cross reactivity takes place by detecting the
presence of the labeled antibody or fragment. The labeling again
may be radiolabeling or fluorescent labeling, among others.
[0025] There is also provided by the present invention a method of
detecting the presence or localization of DNA encoding a urogenital
sinus derived growth inhibitory factor in a chromosome. The method
comprises labeling any of the DNA molecules of the invention (or a
fragment of same), such as by radiolabeling, fluorescent labeling
or biotin labeling. Then, the labeled DNA or fragment is contacted
with a chromosome in a manner known by those of skill in the art to
be conducive to DNA-DNA hybridization. Once hybridiazation with the
chromosome is accomplished, the presence or localization of the
hybridized DNA within the chromosomal DNA encoding the factor is
determined.
[0026] The present invention also provides biologically functional
vectors comprising DNA molecules encoding a urogenital sinus
derived growth inhibitory factor. These vectors may have DNA
derived from any suitable biological source including but not
limited to human DNA, mouse DNA or rat DNA. These vectors may be
incorporated into a host cell, which cell may be either prokaryotic
or eukaryotic.
[0027] Such cells having such vectors provide methods of making a
recombinant urogenital sinus derived growth inhibitory factor..
These methods comprise culturing such a cell in a medium,
harvesting the cell from the culture or harvesting the cell culture
medium (dependning upon the greatest source of the recombinant
factor. Then, the factor is extracted from the cell or the medium
using techniques known well to those of skill in the art of
fermenting recombinant proteins.
[0028] Also provided by the invention are methods of treating
stromal cells to affect their growth and differentiation
characterisitics or phenotype. A stromal cell for purposes of the
invention is a fibroblast, myofibroblast, smooth muscle cell, or
any precursor cell to such a cell type, or any cell type that
differentiates into such a cell type. These methods comprise
contacting smooth muscle cells with a urogenital sinus derived
growth inhibitory factor. The smooth muscle cells may be any such
cells including but not limited to such cells which are vascular
cells, tumor cells, and any organ smooth muscle cells (such as
those in the prostate gland).
[0029] Also provided are methods of genetic therapy comprising
treating a patient with a gene therapy vector capable of
transfecting the patient with a gene encoding the factors of the
invention. The therapy can be one designed to permanently transfect
the patient, or can be designed to only affect a temporary
transfection for a time suitable to achieve the desired therapeutic
effects. The therapy may one designed to affect any suitable
therapy, including but not limited to vascular therapy or cancer
therapy.
[0030] These and other advantages of the present invention will
become apparent from the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1. Rat ps20 cDNA and the deduced amino acid
sequence.
[0032] FIG. 2. Alignment of four disulfide core domain of ps20 with
other family members.
[0033] FIG. 3. Western analysis with ps20 peptide antibody.
[0034] FIG. 4. Immunohistochemical localization.
[0035] FIG. 5. Immunolocalization of ps20 in human benign prostatic
hyperplasia.
[0036] FIG. 6. Immunolocalization of ps20 in human poorly
differentiated carcinoma.
[0037] FIG. 7. Sequence ID No. 1, rat ps20 cDNA nucleotide
sequence.
[0038] FIG. 8. Sequence ID No. 2, rat ps20 amino acid sequence.
[0039] FIG. 9. Sequence ID No. 3, human ps20 cDNA nucleotide
sequence.
[0040] FIG. 10. Sequence ID No. 4, human ps20 amino acid
sequence.
[0041] FIG. 11. Human ps20 cDNA nucleotide sequence and deduced
amino acid sequence.
[0042] FIG. 12. A. Localization of human PS20 on chromosome
16q24.2-.3. B. Ideogram showing localization of ps20 relative to
banding pattern.
[0043] FIG. 13. A. Genomic organization of mouse PS20 gene. B.
Sequence of the mouse PS20 gene.
[0044] FIG. 14. Comparison of human, rat, and mouse ps20 amino acid
sequences.
[0045] FIG. 15. ps20 alters morphology of PS-1 adult rat prostate
stromal cells in the presence of serum.
[0046] FIG. 16. ps20 promotes spheroid formation in stromal cells:
COS and PS-1.
[0047] FIG. 17. ps20 promotes migration of stable transfectant COS
cell lines.
[0048] FIG. 18. TGF-.beta.1 induces expression of smooth muscle
marker proteins in PS-1 adult rat prostate stromal cells.
[0049] FIG. 19. ps20 inhibits expression of polymerized smooth
muscle .alpha.-actin in PS-1 cells.
[0050] FIG. 20. ps20 reduces levels of monomeric SM .alpha.-actin
in PS-1 cells.
[0051] FIG. 21. ps20 inhibits TGF-.beta.1 induced expression of
smooth muscle markers in PS-1 cells.
[0052] FIG. 22. The inhibition of TGF-.beta.1 mediated
differentiation of PS-1 cells by ps20 is dose-dependent and
species-independent.
[0053] FIG. 23. ps20 mRNA expression in adult rat prostate smooth
muscle cells is stimulated by TGF-.beta.1.
DETAILED DESCRIPTION OF THE INVENTION
[0054] The isolation and characterization of ps20 is disclosed in
U.S. Pat. No. 5,196,334, and U.S. Pat. No. 5,496,800, incorporated
by reference herein. The present invention provides nucleotide and
amino acid sequences of ps20. As used herein, the term "nucleotide
sequence" includes polynucleotides and/or oligonucleotides and
refers to a plurality of joined nucleotide units formed from
naturally-occurring bases and cyclofuranosyl groups joined by
native phosphodiester bonds. This term effectively refers to
naturally-occurring species or synthetic species formed from
naturally-occurring subunits. "Nucleotide sequence" also refers to
purine and pyrimidine groups and moieties which function similarly
but which have non naturally-occurring portions. Thus, nucleotide
sequences may have altered sugar moieties or inter-sugar linkages.
Exemplary among these are the phosphorothioate and other sulfur
containing species. They may also contain altered base units or
other modifications, provided that biological activity is retained.
Nucleotide sequences may also include species which include at
least some modified base forms. Thus, purines and pyrimidines other
than those normally found in nature may be so employed. Similarly,
modifications on the cyclofuranose portions of the nucleotide
subunits may also occur as long as biological function is not
eliminated by such modifications.
[0055] As will become apparent, cloning and sequencing of ps20
reveals that the protein has protease inhibitory function.
Therefore, ps20 and its nucleotide sequence may be used in a number
of applications wherein protease inhibition is desirable. These
include, but are not limited to inhibition or regulation of cell
proliferation, inhibition or regulation of cancer cell
proliferation, inhibition or regulation of cancer metastasis,
regulation of biological activities of other growth factors that
are activated by protease action, regulation of extracellular
matrix proteins resulting in alteration in cell proliferation
and/or cell differentiation (altered gene expression) and/or cell
morphologies or any other cell function regulated by extracellular
matrix, activation or inactivation of growth factor activities
associated with protease action. Diseases or conditions responsive
to ps20 include, but are not limited to prostate cancer and
metastasis, breast cancer and metastasis, ovarian cancer and
metastasis, transitional cell carcinoma and metastasis, renal cell
carcinoma and metastasis, bronchogenic carcinoma (lung cancer) and
metastasis, colorectal carcinoma and metastasis, endometrial
(uterine) carcinoma and metastasis, malignant melanoma and
metastasis hepatocellular carcinoma (kidney cancer) and metastasis,
pancreatic cancer and metastasis, testicular seminomas and
nonseminoma germ cell tumors and metastasis, cervical cancer and
metastasis, esophageal squamous cell carcinoma and metastasis,
gastric carcinoma and metastasis, atherosclerosis, restenosis after
angioplasty, vascular smooth muscle proliferation associated with
vascular wall injury, benign prostatic carcinoma, wound healing,
and chronic inflammation. Those skilled in the art will be able to
ascertain suitable doses of ps20 to achieve the desired protease
inhibition function using known pharmacokinetic techniques.
[0056] Additionally, the nucleotide sequence that codes for ps20
may be used as a diagnostic tool for assessing risk of developing
prostatic diseases, such as, but not limited to, prostate cancer
and benign prostatic hyperplasia. The DNA sequence coding for the
ps20 may be isolated and compared to the sequence found in normal
and/or at-risk individuals. In the diagnostic assay, "at risk"
individuals are those who have or may develop prostatic disease.
Additionally ps20 and its nucleotide sequence can be used to
prevent a number of diseases or disorders. Those skilled in the art
will be able to determine appropriate preventative doses. Also, the
ps20 nucleotide sequence may also be used to screen for
cardiovascular diseases such as, but not limited to,
arteriosclerosis and restenosis. The nucleotide sequence of the
present invention may be used to construct recombinant proteins
having the amino acid sequence of rat, mouse or human ps20, for
example. As used herein, the protein UGIF is referred to also as
"ps20." The gene encoding that protein is named (HUGO Nonenclature
Committee, University College, LONDON, UK). WAP four-disulfide core
domain 1." It is given the symbol "WFDC1" in humans and "Wfdc1" in
mice. Additionally, proteins having minor modifications may also be
constructed using the nucleotide sequence of the present invention.
Also provided are vectors comprising the nucleotide sequence of the
present invention.
[0057] Also made possible by the present invention are methods of
treating cardio vascular disease and other diseases and disorders
where there are alterations in smooth muscle biology or phenotype
from that of normal, undiseased tissue.
[0058] Also provided herein are antibodies to ps20. The antibodies
can be used to identify and enumerate ps20-bearing cells, or can be
used to isolate or quantitate the amount of ps20 in body fluids.
For this purpose, antibodies can be used in standard assays known
to those skilled in the art. In general, antibody is contacted with
a sample under conditions which allow the antibody to bind ps20.
Quantitation is conducted by conventional techniques known to those
skilled in the art. These include, but are not limited to
histochemical techniques EMIT, ELISA, latex agglutination
immunoassays, FPIA and other immunoassay techniques. Useful
antibodies to ps20 include polyclonal or monoclonal antibodies.
[0059] The following examples serve to illustrate specific
embodiments of the invention, but should not be considered as a
limitation on the scope of the invention.
EXAMPLE 1
Cloning and Sequencing of ps20
[0060] This example describes the cloning and nucleotide sequence
of rat ps20. To clone ps20 cDNA, a cDNA library was prepared from
the rat prostate smooth muscle PS-1 cell line, which was a high
expresser of ps20 as determined by Western analysis. The PS-1 cell
line is described in U.S. application, Ser. No. 07/928,867 (now
U.S. Pat. No. 5,496,800). The cDNA library was constructed in
lambda ZAP Express vector from oligo d(T) primed cDNA. The ZAP
Express vector was chosen based on versatility, ease of excision
and recircularization to produce pBK-CMV phagemid subclones. The
library exhibited anticipated representation of B-actin cDNA (as
control) and IgG lectin-binding protein cDNA in test screening.
[0061] As an initial approach, degenerate primers were prepared
based on the 5' ends of the amino terminal sequence as determined
from purified ps20 protein. Due to degeneracy of PCR probes, a
nested PCR approach amplifying from 5' vector primer to a ps20
degenerate primer followed by a nested amplification using ps20
forward and reverse degenerate primers allowed for amplification of
authentic ps20 sequence (amino terminal end). The final PCR product
had a predicted size of 81 bp, was cloned directly into pCR II
plasmid by TA cloning. clones isolated, and clone 3438pCRII
sequenced. The corresponding sequence was confirmed as ps20 by a
direct match of the deduced amino acid sequence with the amino
terminal sequence determined from purified ps20. Non-degenerate
forward and reverse PCR primers were developed based on clone
3438pCRII sequence, and used in nested PCR reactions to amplify and
clone the 3' and 5' ends of ps20 cDNA. The 5' clone (clone
T340pCRII) was 184 bp, and overlapped (by 50 bp) with clone
3438pCRII sequence plus an additional 120 bp of 5' sequence. The 3'
clone (clone 42T7pCRII) was 868 bp in length, contained a 3' poly A
tail, and overlapped (by 54 pb) with clone 3438pCRII sequence.
[0062] Screening of PS-1 cDNA library was based on PCR to score
positive plates followed by plaque hybridization with clone
42T7pCRII labeled insert (862 bp) as a probe to score individual
colonies. A total of 1.2 million clones were screened with one
positive clone detected in every 1,020,000 colonies. Clone
1025rps20pBK-CMV-1B was sequenced from both directions (sequence
shown in FIG. 1), matched sequence from an additional separate
clone 1025rps20pBK-CMV-2B, and confirmed as ps20 by deduced amino
acid sequence identical to native ps20. Clone 1025rps20pBK-CMV-IB
was 1029 bp in length and contained a 3' poly (A) tail in agreement
with Northern analysis of U4F cells and rat dorsolateral prostate
which showed a single, identical sized species at approximately 1.1
kb as shown in FIG. 1. Sequence analysis indicated an open reading
frame of 636 nucleotides beginning at nucleotide 52 (ATG), ending
at nucleotide 688 (UGA stop codon), and coding for a deduced 212
amino acid protein. A hydrophobic leader sequence was predicted for
amino acid 1-26 with a perfect signal peptidase cleavage site
between Gly (#26) and Thr (#27) (-1 and +1 respectively) following
the rules of von Heijne. (Von Heijne, G. 1984. J. Mol. Biol.
173:243-251). Thr (#27) (position +1 of mature secreted protein)
through His (#54) were an exact match with Thr (#1) through His
(#28) determined from the amino terminal of purified native ps20.
Hydopathy analysis (Tmpred) suggested no transmembrane domain,
predicting a secreted protein. No potential post-translational
modifications were indicated with the exceptions of 5 potential
casein kinase II sites. The cDNA clone predicts a mature, secreted
protein of 20.7 kDa (identical to purified native 21 kDa ps20) and
an intracellular molecular weight of approximately 23.6 kDa
(including hydrophobic signal peptide) in close agreement with
detection by Western analysis indicating an intracellular Mr of 29
kDa under these conditions. Subsequent refinements by the inventor
suggest a Mr of 21-23 kDa under non-reducing conditions and a Mr of
27-29 kDa under reducing conditions. Larsen, et al. (1998) J. BIOL.
CHEM. 273: 4574-4584.
[0063] Using clone 1025rps20pBK-CMV-2B as a labeled probe, a lambda
gt11 I library prepared from normal human prostate gland (human
prostate 5'-STRETCH cDNA, Clontech) was screened (1.2 million
clones) with standard plaque hybridization techniques. Eight clones
were isolated as potential full length, based on PCR screening of
5' and 3' ends. Of these, 5 were likely full length (1-1.2 kb)
based on comparison to rat ps20 cDNA and the previous determination
of human ps20 protein exhibiting an identical molecular weight to
rat ps20.
[0064] The nucleotide sequence of rat ps20 is shown in FIG. 1 (SEQ
ID NO. 5). Additionally, FIG. 7 depicts the nucleotide sequence of
rat ps20 and which is referred to herein as SEQ ID No. 1. The
sequence of FIG. 7 contains extra nucleotides in the 3' uncoding
region which is not depicted in the sequence shown in FIG. 1. Also
shown in FIG. 1 is the deduced amino acid sequence of rat ps20 (SEQ
ID NO. 6). The rat ps20 amino acid sequence is also shown in FIG. 8
and is referred to herein as EQ ID No. 2. The underlined portion
represents a signal peptide (amino acids 1-26). The ps20 protein is
encoded by a single 1.1 kb transcript expressed in U4F mesenchymal
cell cultures and rat adult prostate tissue. The transcript codes
for a 23.6 kDa protein having a predicted signal peptide leader
sequence (aa 1-26) with a prototypical signal peptidase cleavage
site prior to the first amino acid of the secreted purified
protein.
[0065] Analysis of deduced amino acid sequence revealed that ps20
has a WAP-type four disulfide core domain, classifying ps20 as a
novel member of the WAP-type four disulfide core domain protein
family (ergo, the name of the gene family noted above). Cysteines
58-96 which participate in the WAP--type four disulfide core domain
are also underlined in FIG. 1. The members of the WAP-type four
disulfide core domain family are relatively small proteins
containing a conserved 8 cysteine motif in the protein core
involved in disulfide bonds. The majority of family members with
known biological activity function as protease inhibitors. The
family members having core domains most closely related to ps20
include: Chelonianin, 39.4% identity to ps20 (subtilisin protease
inhibitor isolated from red sea turtle egg white);
Antileukoproteinase 1, 35.4% identity to ps20 (HUSI-1, a secreted
serine protease inhibitor); WAP, 35.3% identity to ps20 (whey
acidic protein, a suspected protease inhibitor found in milk);
WDNM1 protein, 33.3% identity to ps20 (a mammary gland
metastasis-suppressor gene with predicted protease inhibitor
function); HE4, 33.3% identity to ps20 (a predicted protease
inhibitor secreted into epididymis); Kallman syndrome protein,
31.2% identity to ps20 (predicted protease inhibitor localized in
extracellular matrix and required for proper olfactory and
GnRH-synthesizing neuronal development); Elafin, 29.2% identity to
ps20 (a secreted elastase-specific serine protease inhibitor); and
Caltrin-like protein II, 27.1% identity to ps20 (a secreted protein
from seminal vesicle inhibiting calcium transport into
spermatozoa). FIG. 2 depicts the alignment of four disulfide core
domain of ps20 with other family members. (1. SEQ ID NO. 7; 2. SEQ
ID NO. 8; 3. SEQ ID NO. 9;4. SEQ ID NO. 10; 5. SEQ ID NO. 11; 6.
SEQ ID NO. 12; 7. SEQ ID NO. 13; . SEQ ID NO. 14; 9. SEQ ID NO.
15.) Alignment scores were computed by a fasta scoring method.
(EERIE).
[0066] Functional significance of this protein family points to
roles in tissue modeling, cell differentiation and cancer
metastasis control. WAP may play a role in terminal differentiation
and development of mammary acinar epithelial cells. Directed
expression of a WAP transgene by MMTV has resulted in impaired
mammary gland development and a hyperplasia/dysplasia of the
coagulating gland (anterior prostate gland) in male reproductive
tract. This observation is of significance since it was observed
that there was an increased staining intensity of ps20 in human BPH
as compared to normal human prostate tissue.
[0067] Kallman syndrome produces an agenesis of olfactory bulbs
referred to as "olfactogenital dysplasia" and a hypogonadotropic
hypogonadism. The defective gene in Kallman syndrome is termed
ADMLX and encodes a secreted protein containing the WAP-type
four-disulfide core domain as well as fibronectin type III repeats.
This protein may function in cell adhesion and as a protease
inhibitor. ADMLX may participate in migration of GNRH neurons and
the axonal extension of olfactory neurons, thereby inducing a
differentiation pattern.
[0068] The WDNM1 gene is novel member of the four disulfide core
protein family with proposed metastasis-suppressor functions. WDNM1
is down-regulated by 20-fold in rat metastatic mammary
adenocarcinoma in comparison to non-metastatic mammary carcinomas.
WDNM1 has been suggested to function as a protease inhibitor and
hence, modulation of WDNM1 protein could result in unregulated
protease activity, commonly associated with metastatic spread of
carcinomas.
EXAMPLE 2
Preparation of Antibodies to ps20
[0069] This example describes the preparation and characterization
of antibodies specific to ps20. ps20 in rat and human tissues was
localized with immunohistochemistry. A synthetic peptide was made
based on positions 1-14 of ps20 peptide sequence and used as
immunogen in female New Zealand rabbits following modifications of
the procedures of Vitukiatus. Vitukiatis et al. J. Clin. Endocr.
33:988-991.
[0070] A 14 amino acid synthetic peptide corresponding to and
unique to the amino terminus of purified ps20 was synthesized on an
Applied Biosystems 430A Peptide Synthesizer:
N-Thr-Trp-Glu-Ala-Met-Leu-Pro-Val-Ar- g-Leu-Ala-Glu-Lys-Ser-C. For
initial immunization, ps20 peptide was solubilized in sterile,
tissue culture grade H.sub.2O (400 .mu.g/ml) and mixed with
Freund's complete adjuvant (1:1 ratio) and injected in 500 ml (100
.mu.g) aliquots at multiple sites (4-5) in the neck (subcutaneous)
and in the subscapular muscle tissue in the back (intramusclular)
of three female New Zealand rabbits. At three weeks post primary
immunization, sera samples were prepared and analyzed by solid
phase enzyme linked immunoabsorbance assay (ELISA). Each antibody
positive rabbit received a booster of 100 .mu.g peptide in 500
.mu.l of Freund's incomplete adjuvant injected subcutaneously into
multiple sites of the back and neck, and an additional 100 .mu.g
intramusclular in the subscapular region. Serum samples were tested
for ps20-specific antibody every 2 weeks and immunoglobulin subtype
determined by ELISA analysis. Sera was tested for ps20 antibody at
three weeks following the initial booster and antibody positive
rabbits received a secondary booster following identical
procedures. Sera from rabbits producing high titer antisera
(activity at 1:106 dilution) was pooled and IgG was precipitated by
ammonium sulfate (50% saturation), resolubilized in PBS, and
dialyzed overnight against PBS at 4.degree. C. The specificity of
the antibody was confirmed by immunoreactivity with a 20 kDa
protein in concentrated, partially purified preparations of
conditioned medium from U4F cells, from which ps20 was
purified.
[0071] Immunoreactive IgG was purified by peptide column
chromatography. Peptide (10 mg) corresponding to the first 14 amino
acids of purified ps20 was generated as described above, and
coupled to 1 g CNBr-activated Sepharose 4B following standard
procedures as described in Methods in Molecular Biology, Vol. 34,
"Immunocytochemical Methods and Protocols", Lorette C. Javois
(ed.), Chapters 19-23, pgs.155-193, Humana Press, Totowa, N.J. 1994
and poured into a 2 ml Poly-Prep column (Bio Rad). IgG preparations
were diluted in PBS buffer (200 mM sodium borate, 160 mM sodium
chloride, pH 8.0) and chromatographed through the column two times
sequentially. The column was washed extensively in PBS (10-15
column volumes) and bound antibodies eluted with glycine-Cl buffer
(0.05 M glycine, 0.15 M NaCl, pH 2.28). Fractions (2 ml) were
eluted and collected directly in tubes containing 0.5 ml
neutralizing buffer (0.5M phosphate, pH 7.7). Fractions were
assayed for protein content (absorbance at 280 nm) and peak
fractions pooled and assayed for immunoreactivity by solid phase
enzyme linked immunoabsorbance (ELISA) assay. Antibody production
was scored by ELISA. High titer antisera was detected in 3
rabbits.
[0072] The inventor has also made similar anti-peptide antibodies
to regions of the protein near the carboxyl terminus and middle
regions (data not shown). Such anti-peptide antibodies exhibit
characteristics (albeit, unique binding affinities) to that
described in detail for the amino terminus anti-peptide antibody
here. In all cases, these antibodies have the capacity to function
in diagnostic assays and affinity purification methods against the
protein target.
[0073] An IgG fraction of ps20 antisera was analyzed for
specificity by Western analysis and affinity purified antibody
prepared by gel chromatography using peptide 1-14 covalently
attached to sepharose 4B. Western analysis indicated mono-specific
reactivity with the ps20 (20-21 kDa) secreted form (minus signal
peptide) in conditioned medium. See, Rowley, et al. 1995. J. Biol.
Chem. 270:22058-22065. Western analysis from U4F cell extracts
showed mono-specific reactivity with a 29 kDa protein (FIG. 3)
representing the unprocessed cellular form (includes a 26 amino
acid hydrophobic leader sequence) predicted to increase the
apparent backbone (24.6 kDa) size in SDS-PAGE and Western analysis.
(A) U4F fetal rat urogenital sinus mesenchymal cells. Lane 1:
Coomasie stain, 2: ps20 antisera IgG fraction, 3: preimmune sera,
4: affinity purified ps20 antibody, 5: no primary antibody. The
ps20 antisera IgG fraction (lane 2) specifically recognized a
single species of apparent 29 kDa size under these conditions. The
affinity purified antibody (lane 4) recognized the 29 kDa band
exclusively. For refinements in these Mr; see above discussion.
Preimmune sera (lane 3) recognized all non-specific cross-reactive
bands. Secondary antibody alone showed no banding pattern (lane 5).
Western analysis of adult rat prostate smooth muscle and human
prostate smooth muscle extracts showed identical immunoreactivity
to a 29 kDa band from both rat and human cell lines. These studies
indicated rat and human ps20 forms were identical in size and
immunoreactivity as predicted by nearly identical amino acid
sequence.
EXAMPLE 3
Immunohistochemical Localization
[0074] This example shows the immunohistochemical localization of
ps20 in prostate specimens.
[0075] FIGS. 4E & F, 5, and 6 show ps20 localization in human
prostate specimens. FIG. 4 shows ps20 immunohistochemical
localization in rat prostate gland with affinity purified antibody.
Immunohistochemical localization was conducted by the procedure
described in Methods in Molecular Biology, Vol.34,
"Immunocytochemical Methods and Protocols", Lorette C. Javois
(ed.), Chapters 19-23, pgs. 155-193, Humana Press, Totowa, N.J.
1994. Basically, two month old and six month old male Sprague
Dawley rats were sacrificed and whole tissues fixed in formalin
O.N. Fixed tissues were embedded in paraffin and cut into 5 .mu.m
thick sections that were applied to poly-L-lysine coated slides and
baked at 37 C prior to staining.
[0076] Formalin-fixed, paraffin-embedded sections of human prostate
gland from 31 patients were obtained from Methodist Hospital,
Houston (18), and from Texas Children's Hospital, Houston (13).
Sections of 5 .mu.m thickness were cut from the paraffin embedded
blocks and applied to poly-L-lysine coated slides. Slides from
twenty four adult patients ages 53 to 72 generally characterized
as: carcinoma (9), BPH (3), severe BPH (2), stromal BPH (3), and
normal (6) were stained for ps20. Slides from four patients of less
than one year of age and four patients with ages between 10-14
years were stained.
[0077] Tissue sections were deparaffinized by immersion in Hemo D
1.times.10 min. and 1.times.5 min.; rehydrated by 5 min. graded
washes in 100%, 95%, and 70% ethanol; permeablized by immersion in
1.times.PBS/0.1% Triton-X-100 for 5 min.; and treated 5 min. with
3% peroxide (H.sub.2O.sub.2) (diluted from 30% Sigma) to minimize
endogenous peroxidase activity. Primary antibody incubations were
performed at the concentrations and conditions described for
immunocytochemistry, with the exception that affinity purified ps20
antibody was incubated with tissue sections O.N. at 37.degree. C.
Immunoreactivity was visualized by a 45 min. incubation with either
biotinylated goat anti-rabbit or goat anti-mouse secondary
antibodies (Sigma), diluted 1:15; followed by a 30 min. incubation
with ExtrAvidin-conjugated peroxdidase (Sigma), diluted 1:15;
concluding with a 7 min. incubation with diaminobenzidine (DAB) and
mounting with Gel Mount. Staining of slides with hemotoxalin and
eosin (H & E) were performed as described in Methods in
Molecular Biology, Vol. 34, "Immunocytochemical Methods and
Protocols", Lorette C. Javois (ed.), Chapters 19-23, pgs. 155-193,
Humana Press, Totowa, N.J. 1994. Slides were analyzed by light
microscopy (Labophot-2, Nikon) and photographed on Ectachrome 400
slide or Royal Gold 25 print film (Eastman Kodak).
[0078] In FIG. 4, Panel (A) depicts the affinity purified ps20
antibody, Panel (B) the negative control, Panel (C) the smooth
muscle a-actin (SM .alpha.actin), and Panel (D) hematoxylin and
eosin staining patterns. ps20 antisera (A) and the ps20 IgG
fraction showed the same specific immunolocalization in rat
prostate periacinar smooth muscle. Negative controls, including no
primary antibody (B), preimmune sera, and antibody preabsorbed with
ps20 peptide, showed the same lack of specific staining. ps20
immunolocalized to a subset of SM a-actin positive cells.
Immunolocalization is specific to smooth muscle, but is not
specific to prostate. Strong staining was observed in the smooth
muscle of other male reproductive tract tissues, including the vas
deferens and seminal vesicle. Moderate staining was observed in the
tunica media of arteries and the smooth muscle of colon and small
intestine. No apparent staining was observed in the brain, lung,
bladder, or testis. Immunolocalization of ps20 in human prostate
Panel (E) showed a localization corresponding to a subset of SM
a-actin positive cells as shown in Panel (F). Exclusive
localization was observed in the periacinar smooth muscle cells
immediately adjacent to epithelial acini. Significant reactivity
was not observed in any other cell type. A survey of other tissues
including seminal vesicle, vas deferens, stomach, intestine, lung,
salivary gland, heart, brain and testis showed ps20 expression was
preferential to smooth muscle. Highest reactivity was noted in male
reproductive tract tissues (vas deferens, prostate, seminal
vesicle) with moderate staining in smooth muscle of gut and tunica
media of arteries. No reactivity was noted in testis, lung, or
brain. Of interest, ps20 reactivity was observed in the tunica
media of arteries in the prostate gland. Localization was specific
to smooth muscle cells (a-actin positive stromal cells). The human,
unlike rat, does not have a precise periacinar ring of smooth
muscle cells around epithelial acini. Rather, human prostate stroma
is a mix of smooth muscle and fibroblasts. The ps20 positive cells
correlated with a-actin positive cells all sections. Of interest
were the staining patterns observed in prostatic disease.
[0079] FIGS. 5 and 6 show ps20 localization in BPH and prostatic
carcinoma consistently high relative to normal. In FIG. 5, Panel
(A) depicts ps20 antisera IgG fraction, Panel (B) preimmune sera,
Panel (C) SM a-actin, Panel (D) hematoxylin and eosin staining.
Immunolocalization of ps20 in regions of BPH is similar to that in
normal regions of the adult human prostate or is slightly elevated
in comparison to normal. Strong to elevated staining was observed
in patients having both glandular and stromal BPH. The staining
pattern shown here is representative of sample evaluated (5
patients diagnosed with glandular BPH and 3 with stromal BPH.) In
contrast, staining intensity of ps20 was very heterogeneous and
generally lower in carcinoma samples. In FIG. 6, Panel (A) depicts
ps20 antisera IgG fraction, Panel (B) preimmune sera, Panel (C) SM
a-actin, Panel (D) hematoxylin and eosin staining.
Immunolocalization of ps20 in regions of carcinoma exhibited a
heterogeneous staining pattern relative to normal in nine carcinoma
patients evaluated. ps20 staining was reduced in stroma surrounding
some poorly differentiated nodules, as shown in Panel (B). In
particular, ps20 staining intensity was low or absent altogether in
some (not all) stromal regions adjacent to poorly differentiated
carcinoma nodule located in the peripheral, subcapsular region as
shown in FIG. 6.
EXAMPLE 4
Human ps20 Sequence
[0080] This example describes the nucleotide and deduced amino acid
sequence of human ps20. A commercially available (Clonetech, Palo
Alto, Calif.) cDNA library was used. The library was prepared from
normal human prostate gland. Screening was done using standard
plaque hybridization procedures as described in Current Protocols
in Molecular Biology, Vol. 1, Ausubel, F. M.; Brent, R.; Kingston,
R. E.; Moore, D. D; Seidman, J. G.; Struhl, K.; and Smith, J. A.
(eds.), John Wiley & Sons. NY, N.Y. 1995. Plaques were
transferred to Nytran 0.45 .mu.M membranes (Schleicher and Schuell,
Keene, N.H.), DNA cross lined by the Stratalinker UV cross linker
(Statagene), and membranes pre-hybridized 2 h in at 42.degree. C.
in the hybridization solution (50% formamide, 2.times.PIPES buffer,
0.5% (w/v) SDS, 100 .mu.g/ml sonicated salmon sperm DNA). Clones
42T7pCRII insert was purified from an agarose gel slice by Spin-X
columns (Corning Costar Corp., Cambridge, Mass.), 150 ng labeled
with .alpha.[.sup.32P]-dCTP (Amersham, Cleveland, Ohio) by random
priming with Klenow Enzyme (labeling grade, Boehringer, Mannehiem,
Indianapolis, Ind.). Unincorporated nucleotides were removed by
NucTrap Probe Purification Column (Stratagene), and denatured probe
hybridized with filters at 1-3.times.10.sup.6 counts/ml overnight
at 42.degree. C. in hybridization cocktail. Filters were washed
3.times.10 min at room temperature in 2.times.SSC, 0.1% SDS, and
2.times.20 min at 55.degree. C. in 0.2.times.SSC, 0.1% SDS and
exposed to X-OMAT AR film (Eastman Kodak, Rochester, N.Y.).
Positive plaques were screened by PCR, phagemids excised from
lambda arms by the Rapid Excision Kit (Stratagene), and DNA
prepared either by mini alkaline lysis or by Oiagen tip-500
(Oiagen, Chatsworth, Calif.). The probe was rat ps20 cDNA clones
labeled with .sup.32p Colonies of DNA were lifted via filters and
hybridized to the rat ps20 cDNA clones. Positive colonies were
selected and purified by second and third round screening.
[0081] Positive colonies were sequenced and compared to the rat
sequence. Positive clones H.sub.1T.sub.2100 and H.sub.6B.sub.2-3
were sequenced using both dideoxy sequencing (as described in
Current Protocols in Molecular Biology Vol 1, see above) and
automated sequencing using the IBI model 377 automated sequenator.
Clone H.sub.1T.sub.2100 was 1124 bp in size and contained
nucleotide 1-1124 sequence which included the entire coding region
(sequence encoding the mature ps20 protein). Clone H.sub.6B.sub.2-3
was approximately 1000 bp in size and contained the entire 3'
untranslated region (including the poly A tail) and the coding
sequence (overlap with clone H.sub.1T.sub.2100) minus the first few
amino acids. The human nucleotide sequence and derived amino acid
sequence (the human cDNA sequence encoded a 220 amino acid protein)
were compared to the rat ps20 sequences using the MacVector 4.1
sequence analysis program. Based on these analyses, the human and
rat amino acid sequences (Positions 1-212, rat) were well conserved
in sequence with a 82.1% direct match and a 90.6% overall
similarity when considering conservative substitutions of amino
acids. The human ps20 protein contains an extra seven amino acids
in the amino terminal leader peptide sequence and an added amino
acid at position #52.
[0082] The nucleotide sequence of human ps20 is shown as SEQ ID No.
3 in FIG. 9. The derived amino acid sequence of human ps20 is shown
as SEQ ID No. 4 in FIG. 10.
EXAMPLE 5
Chromosomal Localization of ps20 Using Human and Mouse Gene
[0083] The purpose of the present example was to identify the
chromosomal location of the human gene encoding ps20 and to
determine whether it maps to a site implicated in cancer or
disease. The inventor first cloned the human ps20 cDNA from a
prostate cDNA library and used it to isolate the human gene from a
P1 human genomic library (as described above). By fluorescent in
situ hybridization (FISH), the inventor mapped the human gene
(WFDC1) to chromosome 16q24.3, a region of LOH in several human
cancers, including prostate and breast. To facilitate future
studies of ps20 function in vivo in mouse model systems, the
inventor also cloned the mouse ps20 gene (Wfdc1) and sequenced it
to determine its genomic structure. The mouse and human ps20 genes
both consist of seven exons, suggesting the gene encoding ps20 is
most similar to the largest gene in the WAP Signature domain
family, the KAL gene, which is genetically linked to Kallman
Syndrome. The amino acid sequences of mouse, rat, and human ps20
protein are highly conserved. Due to its location on chromosome
16q24.3, combined with its growth inhibitory properties, the
inventor has identified ps20 as a novel candidate tumor suppressor
gene.
[0084] Screening of Human ps20 cDNA. The Human Prostate 5'-STRETCH
Lambda gt11 cDNA Library (Clontech, Palo Alto, Calif.) was screened
by moderate stringency hybridization with a partial rat ps20 cDNA
clone as probe. Ten 150 mm plates with approximately 45,000
pfu/plate were prepared, transferred to Nytran 0.45 mm membranes
(Schleicher and Schuell, Keene, N.H.), and DNA cross linked by UV
irradiation (Stratalinker, Stratagene, La Jolla, Calif.). Membranes
were pre-hybridized 2 h at 42 C in hybridization solution (50%
formamide, 2.times.PIPES buffer, 0.5% (w/v) SDS, 100 .mu.g/ml
sonicated salmon sperm DNA). The insert from clone 42T7-1pCRII
(Larsen et al. 1998. J Biol Chem. 273:4574-4584.), corresponding to
the 864 nt of C-terminal ps20 sequence, was excised and purified,
and 150 ng a-[32P]dCTP (Amersham, Cleveland, Ohio) labeled by
random priming with random hexamer primers and Klenow Polymerase
(Boehringer Mannheim, Indianapolis, Ind.). The labeled probe was
incubated with membranes at >1.times.106 cpm/ml in hybridization
cocktail ON at 42 C undergoing constant rotation. Filters were
washed 2.times.20 min at RT in 2.times.SSC, 0.1% SDS and 2.times.20
min at 55 C in 0.2.times.SSC, 0.1% SDS and exposed to X-OMAT AR
film (Eastman Kodak, Rochester, N.Y.). Eight positive colonies were
obtained and DNA prepared for each (Lambda DNA Maxi Prep Kit,
Qiagen, Chatsworth, Calif.). Inserts were excised from the lambda
arms by EcoRI restriction digestion and subcloned into the vector
pBKCMV (Stratagene, LaJolla, Calif.). Five clones were confirmed to
be partial human ps20 clones by sequencing and comparison with
rps20 cDNA sequence.
[0085] Cloning of human ps20 genomic clone. Insert from human ps20
cDNA clone H1-1pBK-CMV was purified and used to screen a human P1
library (Ad10SacBII) by two sequential rounds of plaque
hybridization (Genome Systems, St. Louis, Mo.). The putative clone
was transferred into host NS1356 and DNA prepared by a modified
alkaline lysis method (100 KB column method), as recommended by the
manufacturer (Genome Systems, St. Louis, Mo.). The human ps20
genomic clone, H1pAd10SacBII (75-100 kB), was confirmed to contain
the human ps20 gene by Southern blotting with a H1-1 cDNA probe and
by sequencing with primers corresponding to human cDNA sequence
(Genosys, The Woodlands, Tex.). Sequencing indicated the human
genomic clone contains seven exons.
[0086] Fluorescence in Situ Hybridization. Chromosome spreads were
prepared using phytohemagglutinin stimulated human peripheral blood
lymphocytes, which were synchronized with methotrexate.
Bromodeoxyuridine incorporation was used to optimize banding. Cells
were harvested by standard cytogenetic techniques. Nick-translation
of probes, in situ hybridization (Johnson et al. 1991. Am J Med
Genet. 39:97-101.)(Lawrence et al. 1998. Cancer Epidemiol
Biomarkers Prev. 7:29-35.), and gene localization analysis (Wydner
et al. 1996 Genomics. 32:474-478.) were performed as described in
detail previously. Metaphase preparations were hybridized with the
human ps20 genomic P1 clone, H1pAd10SacBII, labeled with
digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, Ind.). In
some experiments the spreads were simultaneously hybridized with a
second probe for telomeres labeled with biotin. Signals were
detected by immunofluorecence with rhodamine-anti-digoxigenin and
fluorescein isothiocyanate-conjugated avidin (Boehringer Mannheim)
on DAPI (4,6-diamino-2-phenyl-indole)-banded chromosomes.
Preparations were photographed with a Zeiss Axioplan fluorescence
microscope equipped with a Photometrics CCD camera, filter wheel,
and dual and triple bandpass filters (Chroma, San Juan Capistrano,
Calif.), which avoids any optical shift between filters. Red and
green hybridization signals were visualized on blue DAPI-banded
chromosomes. Cytogenetic analysis was performed on images of
contrast-reversed chromosomes to convert DAPI-bright bands to black
and white G-dark bands, and signals as bars relative to the
chromosome, with the bars approximating the precision of signal
placement, as previously described (Wydner et al. 1996. Genomics.
32:474-478.)
[0087] Cloning of the mouse PS20 gene. Mouse genomic clones
containing ps20 sequence were isolated by plaque hybridization from
the mouse 129 SV Lamda Fix II genomic library with partial rat ps20
cDNA sequence. The mouse 129 SV Lamda Fix II library was
constructed by cloning mouse genomic DNA partially digested with
Sau3A into the Sau3A site in the Lambda Fix II vector. Initially,
the library was screened with [32P]dCTP--labeled 42T7 (partial
rps20 3' end sequence) and one clone (MGLclone 2) was obtained. To
obtain genomic clones containing 5' ps20 sequence, the partial
rps20 5' end clone, T340 was used to identify two other mouse ps20
genomic clones, MGLclone 1 and MGLclone 3. Inserts were excised
from the lambda library by digestion with XhoI and cloned into a
modified pCR2.1 vector (lacking sequences including the 5' EcoRI
site through the BstXI site) at the XhoI site. Clones were screened
for the presence of ps20 sequences by Southern analysis with the
42T7 and T340 probes. Complete sequencing demonstrated MGLclone 1
(SEQ ID No. 17) contains promoter sequence, exon 1, and part of
intron 1. MGLClone 3 was partially sequenced and confirmed to
contain exon 1 sequence and intron 1 (.about.10 kb). Complete
sequencing of MGLClone 2 (SEQ ID No. 16) indicated it contained
part of intron 1 exons 2 through 7. Primers corresponding to the 3'
end of MGLClone 3 and the 5' end of MGLClone 2 were used to amplify
a fragment from 129 mouse genomic DNA by PCR under standard
conditions. Sequencing of the fragment confirmed that MGLClone 3
and 2 are contiguous and separated by a Sau3A site.
[0088] Sequencing and analysis. Multiple pass DNA sequencing was
performed using an Applied Biosystems Model 377 Sequencer Version
2.1.1 using AmpliTaq polymerase (Perkin Elmer, Branchburg, N.J.), 3
pmol primer (pBKT3-1, pBKT7-1, and ps20 specific primers), and 1 mg
double-stranded DNA per reaction. Sequencing of the human genomic
clone was accomplished using two-fold more primer and five-fold
more DNA than for standard plasmid DNA sequencing Sequences were
assembled using MacVector, 4.1 and AssemblyLign, 1.0 (Eastman
Kodak, Rochester, N.Y.).
[0089] PCR primer sequences were designed using MacVector, 4.1
software. Nucleotide sequence searches were performed on all
available databases using the BLASTN and TBLASTN (blast enhanced
alignment utility) algorithms (Altschul et al. 1990 Journal of
Molecular Biology. 215:403-410.) and modifications thereof.
Secretory peptide prediction was confirmed by PSORT II (WWW
Version). Analysis of ORFs was determined by WebGene and multiple
sequence alignments performed with ALIGN (EERIE). Secondary
structure predictions of deduced amino acid sequence were made by
SOPMA (Self Optimized Prediction Method from Alignment) (Geourjon
and Deleage, 1993. CABIOS. 9:87-91.) and SBASE (Pongor et al. 1994.
Nucleic Acids Research. 22:3610-3615) and confirmed by BLASTX and
TBLASTX searches of the available databases. Analysis of splice
sites was performed using Splice View.
[0090] Cloning of Human ps20 cDNA.
[0091] The inventors screened a human prostate cDNA library with
rps20 cDNA sequence as a probe in order isolate the cDNA encoding
the ps20 homolog. Eight putative clones were obtained and
sequenced, of which five were confirmed to be partial human ps20
cDNA clones by comparison with rat ps20 cDNA sequence. The three
non-ps20 clones were very low homology DNA sequences, not closely
similar enough to be related family members (data not shown). The
two longest human ps20 cDNA cloncs, H1-1 and H1-6pBKCMV, were
sequenced and compiled to generate the human ps20 cDNA 1366
nucleotide (nt) composite full length sequence (SEQ ID No. 3).
Clone H1-1 corresponds to nt 1-1124 and H1-6 to nt 205-1366 of full
length human ps20 sequence. Comparison of the human and rat ps20
cDNAs indicated the sequences are 58.1% identical. The most
significant differences in the rat and human ps20 cDNA sequences
are that the human 5' untranslated region (UTR) is 104 nt longer
and the 3' UTR is 211 nt longer than the rat. Comparison of the
human ps20 DNA sequence with existing databases revealed
significant homology of human ps20 cDNA with a 436 nt human EST
(GenBank H52970) cloned from a human fetal male 20 weeks
post-conception liver and spleen cDNA library.
[0092] Human ps20 is highly homologous to rat ps20 on the protein
level. FIG. 11 shows the human ps20 cDNA sequence with the
predicted protein sequence. The 1366 nt human ps20 cDNA sequence
contains a start codon at nucleotide (nt) 155, a stop codon at nt
817 (asterisk), and a polyadenylation signal (underline). The open
reading frame predicts expression of a 220 amino acid protein with
a predicted signal peptide cleavage site immediately following Ala
31 (arrow). The deduced protein sequence is numbered beginning with
the first amino acid of the signal peptide. The Cys residues
comprising the WAP signature domain are noted (grey highlight). The
open reading frame (ORF) presented initiates at the second ATG. The
first ATG in the human ps20 cDNA sequence is at nt 18, but the
context is poor with a C in the -3 and an A in the +4 position and
the predicted ORF is short and not homologous to rat ps20 protein.
The second ATG was presumed to be the actual translation start
site. The second ATG (nt 155) through the stop codon at nt 817,
predicts a peptide of 220 amino acids in length 82.1% identical and
90.6% similar to the rat protein. The upstream ATG in the human
ps20 cDNA appears unique to the human sequence; whether it is
functional or not is not known. Upstream ATGs are sometimes
involved in translational control mechanisms. Few vertebrate mRNAs
have upstream ATGs (5-10%), although those that do are often
involved in growth control. Interestingly, another WAP signature
domain family member, Elafin, also has two ATGs, of which only the
second appears to be functional. Like rat ps20, human ps20 contains
a WAP Signature domain, which may be critical for its function.
[0093] Genomic Cloning of Human ps20 and FISH Mapping to Human
Chromosome 16q24.3
[0094] Given the potential role of ps20 in growth control, it was
of particular interest to identify its chromosomal location and to
assess whether it corresponded to sites implicated in cancer or
disease. The inventors isolated the human PS20 gene from a human P1
library by plaque hybridization with the human cDNA as probe. That
the single clone obtained, of insert size 75-100 kb, contained ps20
sequence was confirmed by Southern analysis and by direct
sequencing with human ps20 specific cDNA primers (data not shown).
Using fluorescence in situ hybridization and the digoxigenin-labled
H1pAd10SacBII P1 clone, normal metaphase chromosomes were probed to
determine the gene's location. Chromosomal location was determined
from analysis of 20 digital images of metaphase spreads that
typically exhibited signal on both sister chromatids of two
homologous chromosomes. The chromosome was identified as chromosome
16, with no evidence of additional sites elsewhere in the genome
(FIG. 12A). The analysis of ps20 location on banded chromosomes
clearly indicated the gene resides in band 16q24 (FIG. 12B). This
site was of significant interest because of its frequent loss in
certain cancers, with band 16q24.3 strongly implicated in prostate
and breast cancers. Close scrutiny of over 20 chromosomes showed
that ps20 signal most frequently localized to the distal portion of
16q24, encompassing minor bands 16q24.2 and 16q24.3. While there
was some technical variation in the placement of the signal, for a
number of chromosomes the signals were so telomeric that they
actually appeared to be off the end of the chromosome, as reflected
in the ideogram in FIG. 2E. Because of the importance of potential
of the precise sub-band localization, 16q24.3, the inventor
co-hybridized telomeric and ps20 probes to assess further the
proximity of the gene to the very end of the chromosome. As shown
in FIG. 12D, on metaphase chromosomes the signals were generally
co-localized and not separated along the length and rarely across
the width of the chromosome. In interphase cells where DNA is more
distended and provides higher resolution, separate but very
closely-spaced signals were resolved (e.g. nucleus in FIG. 12C).
The presence of multiple telomere signals per nucleus precluded
precise determination of average interphase distance. However,
based on earlier work correlating known DNA distances to interphase
and metaphase distance, even a conservative estimate of the
interphase separation between ps20 and the telomere observed here
indicates the ps20 gene is likely less than 2 Mb from the telomere.
These results support localization of the ps20 gene in the most
telomeric sub-band, 16q24.3.
[0095] Genomic cloning and structure of mouse ps20 gene (Wfdc1).
Mouse ps20 gene was isolated and the genomic structure determined
by sequencing. The ps20 mouse genomic clone was isolated by
screening a mouse genomic lambda library with rat ps20 cDNA
sequence by plaque hybridization. Three clones were isolated and
confirmed to contain ps20 sequence by Southern hybridization (data
not shown). By sequencing, MGLClone 1 (SEQ ID No. 17) was found to
contain ps20 exon 1 sequence, part of intron 1 sequence, and an
additional 6.3 kb of upstream sequences. Partial sequencing of
MGLClone 3 indicated it overlaps with MGLClone 1 sequence at its 5'
end and contains exon 1 sequence and intron 1 (estimated size 8-12
kb). Complete sequencing of MGLClone 2 (SEQ ID No. 16) revealed
ps20 exons 2-7 and an additional 2 kb of downstream sequence.
MGLClones 2 and 3 were determined to be contiguous following PCR
amplification of intervening sequence between from mouse total
genomic DNA and sequencing of the PCR product. MGLClone 2 and 3
were separated by a Sau3A site, which was the site used to
construct the library. FIG. 13A summarizes the genomic organization
of the mouse ps20 gene and the contributions of the three clones.
Exons are labeled in Roman numerals with coding sequence in black
and non-coding sequence in open boxes. Exons and introns are drawn
to scale. The three contributing Lamda clones are shown with clones
1 and 2 drawn to scale. Shown in FIG. 13B is the mouse ps20 gene
sequence with corresponding amino acids indicated under the exon
sequences and the location and size of intron sequences indicated.
5.5 kb of Wfdc1 promoter sequence is omitted from the figure. Exons
are labeled (Roman numerals) and amino acid sequence shown in
single amino acid code below exon nucleotide sequence. Introns are
not shown, but their locations and sized (of total intron sequence)
noted. The WAP Signature domain is underlined. The nucleotide
sequence is labeled with the transcription start site designated
+1. The mouse genomic sequence has been submitted to GenBank in two
parts: The complete PS20 gene spans approximately 26 kb. Sequencing
of exon-intron borders of the human genomic clone, H1pAd10SacBII,
confirmed that the human ps20 gene has the same genomic structure
(data not shown).
[0096] Comparison of rat, mouse, and human cDNA and protein
sequences. Shown in FIG. 14 is an alignment of human ps20 and rat
ps20 amino acid sequences together with the putative mouse ps20
amino acid sequence deduced from the mouse genomic sequence. The
mouse ps20 protein sequence, deduced from the genomic clone by
comparison with rat cDNA sequence, is shown aligned with both rat
and human ps20 amino acid sequences. The sequences are highly
homologous. The signal sequence cleavage sites are indicated
(arrow). The full length human ps20 protein is 82.1% identical and
90.6% similar to rat ps20 protein. Cleavage of hps20 following Ala
31, generates a mature protein 88.0% identical and 93.5% similar to
the rat ps20. Amino acids representing conservative amino acid
substitutions are indicated. The WAP signature domain is indicated
(underline).Rat ps20 protein has a signal sequence that was
demonstrated to be functional since mature rat ps20 protein was
expressed and secreted by COS cells transfected with a rat cDNA
expression construct. Both human and mouse ps20 proteins also have
highly hydrophobic putative signal sequences with predicted
cleavage sites indicated. The different species of ps20 are most
dissimilar in the signal sequences.
1TABLE 1 Percent identity among ps20 cDNAs and proteins cDNA full
length protein mature protein rat and mouse 88.0% 93.4% 95.7% rat
and human 58.0% 79.1% 86.1% human and mouse 58.0% 80.5% 87.7%
[0097] Table 1 shows the homologies among the rat, human, and mouse
cDNAs and proteins. The rat and mouse proteins are highly
homologous: the mature proteins (lacking signal peptide) are 95.7%
identical. Human ps20 is not as homologous: human ps20 is 86.1%
identical to the rat and 87.7% identical to the mouse mature
protein. The putative functional motif, the WAP Signature domain,
is contained within the region of highest homology among the three
species FIG. 14 (underlined), suggesting the WAP domain may be
critical for ps20 function. The only other apparent potential
functional motif in ps20 protein is an additional cysteine-rich
region downstream of the WAP motif (aa 144-180 of human ps20)
consisting of four cysteines. This cysteine-rich region shares no
homology with other proteins and is unknown significance, yet is
highly conserved in all three species. In general, the amino acid
sequence of ps20 is highly conserved in human and rodent species,
particularly in the WAP Signature motif.
[0098] Relevance of cloned sequences to diagnostics and treatment.
The cytogenetic localization of a gene can provide important clues
as to a possible involvement in specific diseases, especially
particular types of cancer. There are numerous sites throughout the
genome that show recurrent breaks or LOH (loss of heterozygosity)
in cancer, however such breaks or deletions are not necessarily
observed consistently or at high frequency in patients with that
cancer. However, the band 16q24 is deleted at a particularly high
frequency in certain cancers, providing strong evidence that this
site harbors one or more tumor suppressor genes (TSG). The human
ps20 cDNA was cloned from a prostate library and the protein is
expressed in human prostate. The inventor has mapped the PS20 gene
to within 2 cM of the telomere, and therefore within the
16q24.3-qter region of LOH. Because there is some discrepancy in
the boundaries of the region(s) of most frequent LOH in prostate
cancer on chromosome 16q24, there may be more than one TSG within
the region. PS20, located at 16q24.3-ter, maps to a previously
reported region of LOH in prostate cancer and, owing to its
localization and growth inhibitory properties, can be considered a
candidate TSG.
EXAMPLE 6
Effects of ps20 on Stromal Cell Phenotype
[0099] ps20 Induces Alterations in Cell Shape.
[0100] The protein ps20 affected COS cell growth when stably
expressed by smooth muscle cells. Thus ps20 may have an autocrine
function on cells which express it in vivo. To determine if
prostate stromal cells, which express ps20, are affected by ps20 in
their growth, the PS-1 adult rat prostate stromal cell line was
treated with ps20-His. PS-1 cells were seeded in Bfs (serum
containing) medium at low density with no treatment (FIG. 15a) or
in the presence of ps20-His (FIG. 15c) or vehicle (FIG. 15b). PS-1
cells (passage 13) were grown at low density in Bfs media in the
presence of vehicle or 10 .mu.l/ml hps20-His, grown for 48 h, and
fixed/stained with crystal violet (CV). In the presence of
recombinant human ps20 protein (hps20-His), PS-1 adult rate
prostate stromal cells demonstrated an altered morphology. Cells
become more scattered and contained greater numbers of filopodia
and long cellular extensions. Cells were fixed and stained with
crystal violet to facilitate analysis of cell shape. The ps20-His
treated cultures displayed more filllopodia and other cellular
extensions, analogous to the ps20 treated COS cell cultures. In
general, PS-1 cell cultures are more spindly than COS cells and do
not pack as closely or in a cobblestone pattern, yet the ps20
treated cells did appear to be more spread out on the culture dish
and to have more cellular extensions than the untreated cells. To
quantitate the change in cell shape, the cells were photographed
and images analyzed by NIH Image software. The average cell area of
ps20 treated PS-1 cells is less than vehicle treated or untreated
cells (data not shown). These studies suggest that ps20-His induces
shape changes on prostate stromal cells similar, though not
identical, to shape changes induced on carcinoma cell lines.
[0101] Growth effects of ps20 on the PS-1 cell line. To determine
if ps20 is growth inhibitory to PS-1 cells, PS-1 cells were treated
with ps20-His, vehicle control, TGF-.beta.1, or FGF-2 for 24 hours,
pulsed with [3H]-thymidine, and [3H]-thymidine incorporation
recorded as an indirect measure of cell growth. As shown in FIG.
16, PS-1 cells were not growth inhibited under these conditions
(data not shown). However, when PS-1 cells are seeded at lower
plate densities, slight to moderate inhibition may occur (data not
shown). TGF-.beta.1 was not growth inhibitory. Additional
experiments over a period of 72 hours confirmed that ps20 does not
affect PS-1 cell growth over longer time periods (data not shown).
Cell counting experiments confirmed that [3H]-thymidine
incorporation approximated cell growth characteristics (data not
shown). FGF-2 was mitogenic to PS-1 cell growth and on vascular
cell lines. These data suggest the effects of ps20 on cell shape
and cell growth involve independent, cell specific pathways.
[0102] ps20 Promotes Spheroid Formation.
[0103] 1. COS Cells
[0104] Stable transfectant COS cell lines were observed to "pile"
differently than either mock transfectant lines or COS parent cell
lines when cultures were allowed to become post-confluent. COS
cells are not completely contact inhibited. Once COS cells reach
confluence, they continue to grow and form mounds of piled cells
(FIG. 16a). At high density, COS cells stably transfected with ps20
cDNA piled up differently than COS cells or mock transfected COS
cells. Ps20 stable transfectant cell lines (COS rps20-3) formed
spheroids at high density, while COS and mock transfectant cells
formed "pilles." The ps20 transfectant cells piled on each other to
form three dimensional spheroids (FIG. 16c). The spheroids seemed
to be anchored with long cellular stress cables. The mock
transfectant lines formed piles resembling those made by the parent
cell line (FIG. 16b). These data suggest ps20 alters COS cell-cell
adhesion and/or cell-matrix adhesion.
[0105] 2. PS-1 Cells
[0106] Prostate stromal cells, as well as other smooth muscle cell
lines, are known to form spheroids. To determine if PS-1 cells
preferentially formed spheroids in the presence of ps20, PS-1 cells
were seeded in the presence of ps20-His, vehicle, or were left
untreated. The cultures treated with ps20-His formed spheroids
(FIG. 16f), but mock treated (FIG. 16e), or untreated cells did not
(FIG. 16d), although the cells did pile up. In independent
experiments, spheroids did form in untreated cultures, but to a
lesser extent than in ps20 treated cultures. PS-1 cells express
endogenous ps20 and can form spheroids under ideal conditions.
Therefore, spheroid formation in the absence of ps20 was not
unexpected.
[0107] ps20 promotes migration. Requirements for hillcock formation
have been studied in mesangial cells and have been shown to
include: ability to migrate, alterations in matrix synthesis,
alterations in cell-matrix contacts. The promotion of spheroids by
ps20 suggests ps20 stimulates one or more of the component
activities required for spheroid formation. To specifically
determine if ps20 affects cell migration, COS cells, mock
transfectant, and ps20 transfectant cells were assayed for
migration ability in a migration/wounding assay (Clyman, et al.
1992. EXP CELL RES. 200:272-284; Jones, et al. 1996. PROC NATL ACAD
SCI USA. 93:2482-2487). The assay was performed by seeding cells at
high density on glass coverslips so that they would quickly form a
monolayer and thereby minimize differences in cell growth rates. A
scrape was made (in triplicate) on each cell monolayer. Coverslips
were fixed and stained with crystal violet at timepoints throughout
the course of the assay. Random regions near the center of the
coverslip were photographed and analyzed by NIH Image. A cell
density within the wounded area was determined as an indirect
measure of the number of cells that had migrated into the wound
site. Cell number was previously shown to correlate with cell
number in this assay.
[0108] The ps20 transfectant cell lines were observed to fill in
the wound site faster than mock transfected or the parent cell line
over a 60 hr period, as shown in FIG. 17. ps20 stable transfectant
COS cell lines were compared with COS (untransfected) and mock
transfectanat COS cell lines in an in vitro migration/wounding
assay. As shown, cells were seeded in a monolayer, wounded,
fixed/stained, photographed, and cell density in the remaining area
estimated by image analysis with NIH Image at multiple timepoints.
rps20 stable transfectant cell lines invaded the wounded area
faster than mock transfectant cell lines. Results are typical of
n=3 experiments. Given the time period of the assay, cells would
have time to undergo more than one replication, suggesting
differences in proliferation rates could be a factor in this assay.
Note, however, that the migration rate for ps20 transfectant lines
is consistently higher than controls at all timepoints. Since ps20
transfected COS cells are inhibited in proliferation, the enhanced
migration promoted by ps20 cannot be explained by an differences in
proliferation rate. Additionally, since the average cell area of
ps20 transfectant cell lines is less than for the mock or
non-transfected cells, the actual number of ps20 transfectant cells
is underestimated and, therefore, effects of ps20 on migration are
greater than is reported by this method. Migration is optimal at an
intermediate cell attachment strength, which has been demonstrated
for human smooth muscle cells on fibronectin and collagen,
indicating that ps20 alters attachment strength, either to increase
it or decrease it. Another factor to consider with this migration
assay, is the fact that the substrate is glass in the presence of
serum. Others have reported that serum does act as a substrate and
largely behaves like a vitronectin substrate due to the 10 fold
higher levels (200-300 (g/ml vs 30 g/ml) of vitronectin than
fibronectin in serum. Since cells can migrate on existing
substrates or can synthesize their own substrate, ps20 could alter
attachment strength to the extracellular matrix either by affecting
extracellular matrix receptors (integrins) or by altering synthesis
of extracellular matrix by the COS cells.
[0109] ps20 Inhibits Polymerized SM a-actin Filaments in PS-1
Cells.
[0110] The inventor previously demonstrated that the adult rat
prostate stromal cell line PS-1 expresses smooth muscle markers,
including SM .alpha.-actin, h1-calponon, desmin, and androgen
receptor, a marker of smooth muscle in the prostate in fully
defined media lacking serum (FIG. 18). PS-1 cells grown in fully
defined media lacking serum (MO). After 24 h, PS-1 cells express
very little of the smooth muscle markers, SM .alpha.-actin
(FITC)(left) and calponin (Cy-3)(right). After 96 h In the presence
of TGF-.beta.1 (50 pM), PS-1 cells express smooth muscle markers,
SM .alpha.-actin and calponin. Note that in the absence of
exogenously added TGF-.beta.1, PS-1 cells express increasing levels
of smooth muscle markers with time, though not to the extent as
seen in the presence of TGF-.beta.1.
[0111] At early passage, PS-1 cells express all of the above
markers, immediately after attachment, but, as has been reported
for most smooth muscle cell lines, the cells dedifferentiate with
successive passaging. In the absence of exogenously added
TGF-.beta.1, PS-1 cells at intermediate passage (P. 20-35)
myodifferentiate and express smooth muscle markers, but on a longer
time scale and not to the same extent. However, PS-1 will
spontaneously differentiate over time, or the process can be
accelerated by TGF-.beta.1, as has been reported with other
prostate stromal cell lines.
[0112] Coordinate with the myodifferentiation of PS-1 cells,
measured by visualization of SM .alpha.-actin and calponin
filaments, is a change in cell shape. To investigate whether ps20
induced shape changes are related to those induced by TGF-.beta.1,
PS-1 cells (p.20) were grown for 72 h in fully defined media
lacking serum in the presence of 50 pM TGF-.beta.1 (FIG. 19b),
vehicle control (FIG. 19a), or ps20-His (FIG. 19c). PS-1 cells
(p.20) were grown for 72 h in fully defined media (MO)+recombinant
ps20 or TGF-.beta.1 and labeled with anti(SM)-.alpha.-actin (FITC)
and propidium iodine (PI). 3.84% of untreated PS-1 cells express
polymerized SM .alpha.-actin filaments, while 56.5% of TGF-.beta.1
treated, and 0.00% of hps20-His treated cultures express
polymerized SM .alpha.-actin filaments. In the presence of ps20,
the expression of smooth muscle markers by PS-1 cells is inhibited.
Data shown is representative of n>3 experiments.
[0113] Cells were fixed, double-labeled with a smooth muscle
marker, anti-SM .alpha.-actin antibody (FITC), and propidium iodide
to visualize total cells. Cells treated with TGF-.beta.1 were flat
and polygonal shaped with abundant stress fibers labeling with SM
.alpha.-actin, while untreated or ps20 treated cells were rounder
with virtually no SM .alpha.-actin staining detectable. SM
.alpha.-actin positive cells were counted as a percentage of total
cells, which is shown graphically in FIG. 19d: 3.84% of untreated
PS-1 cells expressed polymerized SM .alpha.-actin filaments, while
56.5% of TGF-.beta.1 treated and 0.00% of hps20-His treated
cultures expressed polymerized SM .alpha.-actin filaments. The
prevention of polymerized SM .alpha.-actin filaments by ps20 is
consistent either with reduction of levels of monomeric SM
.alpha.-actin available in the cell or inhibition of monomer
polymerization into filaments.
[0114] ps20 Inhibits Monomeric SM .alpha.-actin Levels.
[0115] To determine if ps20 affected levels of total SM
.alpha.-actin protein available in the cell, total levels of SM
.alpha.-actin monomeric protein were measured in cell extracts by
immunoblot analysis with anti-SM .alpha.-actin antibody. Cell
extracts were made from PS-1 cells treated with ps20, controls, or
TGF-.beta.1 in fully defined media for 72 hours. In cell extracts,
levels of monomeric SM .alpha.-actin decreased with ps20 treatment
(FIG. 20a). Quantitation of protein levels by densitometry is shown
graphically in FIG. 20b. FIG. 20a shows a Western analysis of
monomeric SM .alpha.-actin in PS-1 cell extracts. PS-1 cells (p.20)
were seeded and allowed to attach overnight in Bfs media, then
allowed to grow for 72 h in fully defined media lacking serum in
the presence of (lane 1) vehicle control, (lane 2) hps20-His (2
ng/ml), (lane 3) hps20-His (20 ng/ml), (lane 4) TGF-.beta.1 (50
pM), or (lane 5) .beta.-Gal-His negative control. Cell extracts
were harvested in RIPA buffer and equal volumes of each extract
resolved by 15% SDS-PAGE, followed by Western analysis with anti-SM
.alpha.-actin antibody. Duplicate samples were loaded on a
duplicate gel and Coomassie stained to control for protein loading
(data not shown). Levels of monomeric SM .alpha.-actin were
decreased in hps20-His, particularly at the highest
concentration.
[0116] FIG. 20b shows image analysis of SM .alpha.-actin Western
blot. The Western blot in FIG. 20a and a duplicate Coommassie
stained gel were scanned and analyzed with NIH Imaging software.
The density of each band was calculated and values plotted relative
to a strongly Coomassie staining band representative of protein
loading. Numbers correspond to lanes in A.
[0117] These data suggest ps20 is not simply inhibiting
polymerization of monomeric SM .alpha.-actin into filaments. From
these data alone, it cannot be determined if ps20 is affecting the
transcription of message, translation of protein, or protein
stability.
[0118] Although during phenotypic modulation of vascular smooth
muscle cells (VSM) SM .alpha.-actin filaments decrease, levels of
monomeric SM .alpha.-actin mRNA are not altered. To determine if
levels of SM .alpha.-actin are affected during the
myodifferentiation of PS-1 cells, total RNA was made from PS-1
cells under conditions identical for those used to make protein
extracts. SM .alpha.-actin levels were analyzed by Northern
analysis with a SM .alpha.-actin specific probe. Levels of SM
.alpha.-actin remained constant at 72 hours (data not shown) under
all conditions. Together, these data suggest ps20 affects SM
.alpha.-actin at the level of protein turn-over, although effects
on polymerization into filaments cannot be ruled out.
[0119] ps20 inhibits TGF-.beta.1 mediated induction of SM
.alpha.-actin filaments. TGF-.beta.1 mediated effects can be
modulated by other growth factors and cytokines. To determine if
ps20 can inhibit TGF-.beta.1 mediated differentiation, PS-1 cells
were simultaneously treated with 5 pM (sub-maximal) TGF-.beta.1 and
hps20-His (20 (1/ml) (FIG. 21c), FGF-2 (10 mg/ml) (FIG. 21d),
.beta.-Gal-His negative control (FIG. 21b), or vehicle (FIG. 21a)
for 72 h in serum-free media. In the presence of 5 pM (sub-maximal)
TGF-.beta.1, PS-1 cells were examined for expression of
SM-.alpha.-actin (G) in the presence of vehicle, .beta.-Gal-His
negative control, hps20-His or bFGF (10 mg/ml) at 72 h in
serum-free media (MO). Representative cell morphologies of each are
shown at left: cells are labeled with immunoflurescent antibodies
to: SM-.alpha.-actin (FITC-G) and b-actin (Cy3-R). SM-.alpha.-actin
positive cells were counted as a percentage of total cells and data
shown in a histogram. The SM-.alpha.-actin positive cells
represented: 25% (vehicle treated cells), 34% (.beta.-Gal negative
control treated cells), 3.2% (hps20-His, 20 .mu.l/ml), and 2.83%
(FGF-2, or bFGF). ps20-His inhibited SM .alpha.-actin filaments in
the presence of 5 pM (sub-maximal) TGF-.beta.1. As shown with the
other smooth muscle cell lines, bFGF similarly inhibited
TGF-.beta.1 induced expression of polymerized SM .alpha.-actin
filaments and demonstrated a morphology similar to ps20 treatment.
The mechanism of bFGF effects on SM .alpha.-actin is not known.
[0120] SM .alpha.-actin filaments were labeled by direct
immunofluorecence. SM .alpha.-actin (FITC-G) and b-actin (Cy3-R).
SM .alpha.-actin positive cells were counted as a percentage of
total cells and data, shown in the histogram in FIG. 7e. SM
.alpha.-actin positive cells were counted as a percentage of total
cells: vehicle 25% (FIG. 21e), (-Gal-His control 34%, hps20-His
3.2%, and FGF-2 2.83%. FGF-2 inhibited TGF-.beta.1 mediated
induction of polymerized SM .alpha.-actin filaments, as previously
reported with human prostate stromal cells. Similarly, ps20-His
inhibited SM .alpha.-actin filaments in the presence of 5 pM
TGF-.beta.1 and produced a morphology similar to bFGF that of
bFGF.
[0121] In the presence of 5 pM (sub-maximal) TGF-.beta.1, PS-1
cells were examined for expression of SM .alpha.-actin in the
presence of vehicle or increasing concentrations of .beta.-Gal-His
negative control, rps20His (rat), or hps20-His (human) at 72 h in
serum-free media (Mo), as shown in the previous figure (FIG. 21).
Both rps20-His and hps20-His induced a dose-dependent decrease in
the percentage of SM .alpha.-actin positive cells detected in the
presence of serum-free media, demonstrating the effects of ps20 are
dose-dependent and species independent (FIG. 22: In the presence of
5 pM (sub-maximal) TGF-.beta.1, PS-1 cells were examined for
expression of SM-.alpha.-actin in the presence of vehicle or
increasing concentrations of .beta.-Gal-His negative control,
rps20His (rat), or hps20-His (human) at 72 h in serum-free media
(MO), as shown in FIG. 21. Both rps20-His and hps20-His induced a
dose-dependent decrease in the percentage of SM-.alpha.-actin
positive cells detected in the presence of serum-free media,
demonstrating the effects of ps20 are dose-dependent and species
independent.)
[0122] ps20 mRNA expression is stimulated by TGF-.beta.1. PS-1
cells, like rat prostate smooth muscle in vivo express ps20, yet
factors affecting ps20 mRNA expression are unknown. To address the
question of regulation of ps20 mRNA, PS-1 cells were grown under
fully-defined media conditions for 24 hours in the presence of 10
nM DHT (dihydrotestosterone), 25 pM TGF.beta.1, 10 nM DHT+25 pM
TGF.beta.1, or 50 pM TGF.beta.1. Total RNA was isolated from cells
grown under these conditions and ps20 mRNA expression analyzed by
Northern analysis (FIG. 23). Middle panel: .alpha.-.sup.32P-labeled
rat ps20 cDNA probe was used in Northern analysis to probe total
RNA isolated from Adult rat prostate smooth muscle (PS-1) cells
grown under fully defined media conditions for 24 hours in the
presence of 10 nM DHT, 25 pM TGF.beta.1, 10 nM DHT+25 pM
TGF.beta.1, or 50 pM TGF.beta.1. Under these conditions, ps20 mRNA
expression was not stimulated by DHT, but was stimulated by
TGF.beta.1 in a concentration dependent manner. An additional
enhancement of TGF.beta.1 stimulation was observed in the presence
of DHT. Upper panel: .alpha.-.sup.32P-labeled rat ddp 17, an
androgen regulated transcript, was used to probe an identical blot
as a positive control for androgen regulation. Lower panel: RNA
transferred to the blot was stained with methylene blue as a
loading control.
[0123] Under these conditions, ps20 mRNA expression was not
detectable (data not shown), without stimulation, nor was
expression stimulated by DHT (FIG. 21, lane 2). But ps20 mRNA
expression was stimulated by TGF.beta.1 in a concentration
dependent manner (FIG. 21, lanes 3 and 5). An additional
enhancement of TGF.beta.1 stimulation was observed in the presence
of DHT (Upper panel). As a positive control for androgen
stimulation, an androgen regulated transcript (ddp17), was used to
probe an identical blot as a positive control for androgen
regulation. The blot was stained with methylene blue as a loading
control (lower panel).
[0124] Many other variations and modifications may be made in the
methods herein described, by those having experience in this art,
without departing from the concept of the present invention.
Accordingly, it should be clearly understood that the compositions
and methods described in the foregoing description are illustrative
only, and not intended as a limitation on the scope of the
invention.
Sequence CWU 1
1
19 1 1071 DNA Rattus sp. misc_feature (1020)..(1071) n is equal to
generic nucleotide 1 cggcacgagg aggtcactcg tgcagaagga aagcctgcca
ccagcctcgg gatgggtagc 60 tgcgacagga aagccctctg ggctctgagc
ttcctactgc tgctactggg ctccagctct 120 gttcagggca cttgggaggc
aatgttgccg gtcaggctgg ctgagaagtc ccaagctgaa 180 gaggttgcag
caacaggctc ccggcagccc cacgcagacc gctgcccacc accgccacgg 240
acgctacccc cgggtgcctg tcaggccaca cgctgccagt ctgactctga gtgcccacga
300 cacagacgct gctgttacaa cggctgtgcc tatgcctgcc tggaggcggt
gccaccgcca 360 ccagttctag actggctggt gcagcccaaa ccacggtggc
ttggtggcaa tggctggctg 420 ctggatggtc cggaggaagt gttacaagca
gaggcctgca gcaccactga ggatggggca 480 gagccactcc tctgtccctc
aggctatgag tgccacatcc tgcagccagg ggatgcggcc 540 cagggcatac
ccaaccatgg acggtgtgtt aagcaacgtc gacaagcaga ggggcgggtc 600
ctgcgacaga agcttcacaa ggagtaccca gaaggtgact ccaagtatgt ggcagagcct
660 gggaagggac aacagaggca ctttccatga agtggagact ggctgccttt
gtggggcctt 720 tcctgtgctt tccacacact accccttgga aacagcaaaa
gaatttgacc ctagacgtca 780 aactccattc cacagaacgg gactccagag
ctcctgggaa acgggacttc agactcccaa 840 ccccagagtg gcccagcctg
gtgcggcggt aacttggcgg aagcccctga ccacctctgg 900 gtccccgctc
agcatccttg tcacaggaac ccgcagcttc taggtgactt tgcagatttt 960
gcctgcagaa ggcatatatt catctctttt tttccccgaa taaatctgcc caccatgtag
1020 cagaaataag ttcctttatc aggctcaagt ccnaaaaaaa aaaaaaaaaa a 1071
2 212 PRT Rattus sp. 2 Met Gly Ser Cys Asp Arg Lys Ala Leu Trp Ala
Leu Ser Phe Leu Leu 1 5 10 15 Leu Leu Leu Gly Ser Ser Ser Val Gln
Gly Thr Trp Glu Ala Met Leu 20 25 30 Pro Val Arg Leu Ala Glu Lys
Ser Gln Ala Glu Glu Val Ala Ala Thr 35 40 45 Gly Ser Arg Gln Pro
His Ala Asp Arg Cys Pro Pro Pro Pro Arg Thr 50 55 60 Leu Pro Pro
Gly Ala Cys Gln Ala Thr Arg Cys Gln Ser Asp Ser Glu 65 70 75 80 Cys
Pro Arg His Arg Arg Cys Cys Tyr Asn Gly Cys Ala Tyr Ala Cys 85 90
95 Leu Glu Ala Val Pro Pro Pro Pro Val Leu Asp Trp Leu Val Gln Pro
100 105 110 Lys Pro Arg Trp Leu Gly Gly Asn Gly Trp Leu Leu Asp Gly
Pro Glu 115 120 125 Glu Val Leu Gln Ala Glu Ala Cys Ser Thr Thr Glu
Asp Gly Ala Glu 130 135 140 Pro Leu Leu Cys Pro Ser Gly Tyr Glu Cys
His Ile Leu Gln Pro Gly 145 150 155 160 Asp Ala Ala Gln Gly Ile Pro
Asn His Gly Arg Cys Val Lys Gln Arg 165 170 175 Arg Gln Ala Glu Gly
Arg Val Leu Arg Gln Lys Leu His Lys Glu Tyr 180 185 190 Pro Glu Gly
Asp Ser Lys Tyr Val Ala Glu Pro Gly Lys Gly Gln Gln 195 200 205 Arg
His Phe Pro 210 3 1366 DNA Homo sapiens 3 gtgctggacg cggacacatg
atccgaggga ccctgctggg tggaactaag aaagtccagc 60 agactgtgca
cgctcctgtc cccactcaca ggcccacgca gcgagggggg cccctcttct 120
gtgtgcgtct ggaaggtcgc tgcccaggga ggaaatgcct ttaaccggcg tggggccggg
180 cagctgcagg aggcagatca tccgggctct gtgcctcttg ctacttctcc
tccacgccgg 240 ctctgccaag aatatctgga aacgggcatt gcctgcgagg
ctggccgaga aatcccgtgc 300 cgaggaggcg ggcgcgcccg gcggcccccg
gcagccccga gcagaccgct gcccgccgcc 360 tccgcggacg ctgccccccg
gcgcctgcca ggccgcgcgc tgtcaggcgg actccgagtg 420 cccgcggcac
cggcgctgct gctacaacgg atgcgcctac gcctgcctag aagctgtgcc 480
gcccccgcca gtcttagact ggctggtgca gccgaaacct cgatggcttg gtggcaatgg
540 ctggctcctg gatggccctg aggaggtgtt acaagcagag gcgtgcagca
ccacggagga 600 tggggccgaa cccctgctct gtccctcggg ctatgagtgc
cacatcctga gcccaggtga 660 cgtggccgaa ggtatcccca accgtgggca
gtgcgtcaag cagcgccggc aagcagatgg 720 gcgaatccta cgacacaaac
tttacaaaga atatccagaa ggtgactcaa agaatgtggc 780 agaacctgga
aggggacaac agaggcactt tcagtaaagc aacggcaagc agctaggttg 840
caagaacatt cctctacttt ctgctaagcc ttggaaacag ttgggaaaag tagtttgacc
900 ctcacagttc acattcagct cagcagagca agaccccaga gatgcttaga
gacaggacac 960 ctggccatca aacccagttt ggcccagcct ggttgggtga
ctttgtggga gccacttaac 1020 agctctgggt ccctgtttta ccatcctggg
agcaaggccc tgcagctcca cgagaccttt 1080 accccgggaa gaagccgccg
cccatgaaag catttctgaa gcccctttct aagacaaggc 1140 tcagcatctt
gatatttttg acagattcct cccaagtctg gctctgggag gtatgtaccc 1200
atctcaaatg ttcccaagat aaattcatcc ttcaggaaat ggaaatgaac ttgcttacta
1260 atgtgtgatt cctagttgta gccaccggat gtgctgaggc ctaaatgtta
gcaggtggga 1320 ggaggccaca gaacaataaa aacaaccaaa taaaaaaaaa aaaaaa
1366 4 220 PRT Homo sapiens 4 Met Pro Leu Thr Gly Val Gly Pro Gly
Ser Cys Arg Arg Gln Ile Ile 1 5 10 15 Arg Ala Leu Cys Leu Leu Leu
Leu Leu Leu His Ala Gly Ser Ala Lys 20 25 30 Asn Ile Trp Lys Arg
Ala Leu Pro Ala Arg Leu Ala Glu Lys Ser Arg 35 40 45 Ala Glu Glu
Ala Gly Ala Pro Gly Gly Pro Arg Gln Pro Arg Ala Asp 50 55 60 Arg
Cys Pro Pro Pro Pro Arg Thr Leu Pro Pro Gly Ala Cys Gln Ala 65 70
75 80 Ala Arg Cys Gln Ala Asp Ser Glu Cys Pro Arg His Arg Arg Cys
Cys 85 90 95 Tyr Asn Gly Cys Ala Tyr Ala Cys Leu Glu Ala Val Pro
Pro Pro Pro 100 105 110 Val Leu Asp Trp Leu Val Gln Pro Lys Pro Arg
Trp Leu Gly Gly Asn 115 120 125 Gly Trp Leu Leu Asp Gly Pro Glu Glu
Val Leu Gln Ala Glu Ala Cys 130 135 140 Ser Thr Thr Glu Asp Gly Ala
Glu Pro Leu Leu Cys Pro Ser Gly Tyr 145 150 155 160 Glu Cys His Ile
Leu Ser Pro Gly Asp Val Ala Glu Gly Ile Pro Asn 165 170 175 Arg Gly
Gln Cys Val Lys Gln Arg Arg Gln Ala Asp Gly Arg Ile Leu 180 185 190
Arg His Lys Leu Tyr Lys Glu Tyr Pro Glu Gly Asp Ser Lys Asn Val 195
200 205 Ala Glu Pro Gly Arg Gly Gln Gln Arg His Phe Gln 210 215 220
5 1042 DNA Rattus sp. 5 cggcacgagg aggtcactcg tgcagaagga aagcctgcca
ccagcctcgg gatgggtagc 60 tgcgacagga aagccctctg ggctctgagc
ttcctactgc tgctactggg ctccagctct 120 gttcagggca cttgggaggc
aatgttgccg gtcaggctgg ctgagaagtc ccaagctgaa 180 gaggttgcag
caacaggctc ccggcagccc cacgcagacc gctgcccacc accgccacgg 240
acgctacccc cgggtgcctg tcaggccaca cgctgccagt ctgactctga gtgcccacga
300 cacagacgct gctgttacaa cggctgtgcc tatgcctgcc tggaggcggt
gccaccgcca 360 ccagttctag actggctggt gcagcccaaa ccacggtggc
ttggtggcaa tggctggctg 420 ctggatggtc cggaggaagt gttacaagca
gaggcctgca gcaccactga ggatggggca 480 gagccactcc tctgtccctc
aggctatgag tgccacatcc tgcagccagg ggatgcggcc 540 cagggcatac
ccaaccatgg acggtgtgtt aagcaacgtc gacaagcaga ggggcgggtc 600
ctgcgacaga agcttcacaa ggagtaccca gaaggtgact ccaagtatgt ggcagagcct
660 gggaagggac aacagaggca ctttccatga agtggagact ggctgccttt
gtggggcctt 720 tcctgtgctt tccacacact accccttgga aacagcaaaa
gaatttgacc ctagacgtca 780 aactccattc cacagaacgg gactccagag
ctcctgggaa acgggacttc agactcccaa 840 ccccagagtg gcccagcctg
gtgcggcggt aacttggcgg aagcccctga ccacctctgg 900 gtccccgctc
agcatccttg tcacaggaac ccgcagcttc taggtgactt ttgcagattt 960
tgcctgcaga aggcatatat tcatctcttt ttttccccga ataaatctgc ccaccatgta
1020 gcagaaatta aaaaaaaaaa aa 1042 6 212 PRT Rattus sp. 6 Met Gly
Ser Cys Asp Arg Lys Ala Leu Trp Ala Leu Ser Phe Leu Leu 1 5 10 15
Leu Leu Leu Gly Ser Ser Ser Val Gln Gly Thr Trp Glu Ala Met Leu 20
25 30 Pro Val Arg Leu Ala Glu Lys Ser Gln Ala Glu Glu Val Ala Ala
Thr 35 40 45 Gly Ser Arg Gln Pro His Ala Asp Arg Cys Pro Pro Pro
Pro Arg Thr 50 55 60 Leu Pro Pro Gly Ala Cys Gln Ala Thr Arg Cys
Gln Ser Asp Ser Glu 65 70 75 80 Cys Pro Arg His Arg Arg Cys Cys Tyr
Asn Gly Cys Ala Tyr Ala Cys 85 90 95 Leu Glu Ala Val Pro Pro Pro
Pro Val Leu Asp Trp Leu Val Gln Pro 100 105 110 Lys Pro Arg Trp Leu
Gly Gly Asn Gly Trp Leu Leu Asp Gly Pro Glu 115 120 125 Glu Val Leu
Gln Ala Glu Ala Cys Ser Thr Thr Glu Asp Gly Ala Glu 130 135 140 Pro
Leu Leu Cys Pro Ser Gly Tyr Glu Cys His Ile Leu Gln Pro Gly 145 150
155 160 Asp Ala Ala Gln Gly Ile Pro Asn His Gly Arg Cys Val Lys Gln
Arg 165 170 175 Arg Gln Ala Glu Gly Arg Val Leu Arg Gln Lys Leu His
Lys Glu Tyr 180 185 190 Pro Glu Gly Asp Ser Lys Tyr Val Ala Glu Pro
Gly Lys Gly Gln Gln 195 200 205 Arg His Phe Pro 210 7 48 PRT Rattus
norvegicus 7 Cys Pro Pro Pro Pro Arg Thr Leu Pro Pro Gly Ala Cys
Gln Ala Thr 1 5 10 15 Arg Cys Gln Ser Asp Ser Glu Cys Pro Arg His
Arg Arg Cys Cys Tyr 20 25 30 Asn Gly Cys Ala Tyr Ala Cys Leu Glu
Ala Val Pro Pro Pro Pro Val 35 40 45 8 44 PRT Homo sapiens 8 Cys
Pro Pro Lys Lys Ser Ala Gln Cys Leu Arg Tyr Lys Lys Pro Glu 1 5 10
15 Cys Gln Ser Asp Trp Gln Cys Pro Gly Lys Lys Arg Cys Cys Pro Asp
20 25 30 Thr Cys Gly Ile Lys Cys Leu Asp Pro Val Thr Pro 35 40 9 42
PRT Cavia porcellus 9 Cys Pro Arg Val Met Ile Tyr Cys Pro Ala Arg
His Pro Pro Asn Lys 1 5 10 15 Cys Thr Ser Asp Tyr Asp Cys Pro Lys
Pro Gln Lys Cys Cys Pro Gly 20 25 30 Tyr Cys Gly Lys Gln Cys Tyr
Gln Pro Glu 35 40 10 42 PRT Homo sapiens 10 Cys Pro Ile Ile Leu Ile
Arg Cys Ala Met Leu Asn Pro Pro Asn Arg 1 5 10 15 Cys Leu Lys Asp
Thr Asp Cys Pro Gly Ile Lys Lys Cys Cys Glu Gly 20 25 30 Ser Cys
Gly Met Ala Cys Phe Val Pro Gln 35 40 11 38 PRT Homo sapiens 11 Cys
Pro Glu Leu Gln Ala Asp Gln Asn Cys Thr Gln Glu Cys Val Ser 1 5 10
15 Asp Ser Glu Cys Ala Asp Asn Leu Lys Cys Cys Ser Ala Gly Cys Ala
20 25 30 Thr Phe Cys Pro Asn Asp 35 12 44 PRT Caretta caretta 12
Cys Pro Lys Thr Ser Gly Pro Gly Ile Cys Leu His Gly Cys Asp Ser 1 5
10 15 Asp Ser Asp Cys Lys Glu Gly Gln Lys Cys Cys Phe Asp Gly Cys
Gly 20 25 30 Tyr Ile Cys Leu Thr Val Ala Pro Ser Gly Ser Pro 35 40
13 43 PRT Homo sapiens 13 Cys Pro Ala Pro Glu Lys Ala Ser Gly Phe
Ala Ala Ala Cys Val Glu 1 5 10 15 Ser Cys Glu Val Asp Asn Glu Cys
Ser Gly Val Lys Lys Cys Cys Ser 20 25 30 Asn Gly Cys Gly His Thr
Cys Gln Val Pro Lys 35 40 14 51 PRT Rattus norvegicus 14 Cys Pro
Trp Asn Pro Ile Gln Met Ile Ala Ala Gly Pro Cys Pro Lys 1 5 10 15
Asp Asn Pro Cys Ser Ile Asp Ser Asp Cys Ser Gly Thr Met Lys Cys 20
25 30 Cys Lys Asn Gly Cys Ile Met Ser Cys Met Asp Pro Glu Pro Lys
Ser 35 40 45 Pro Thr Val 50 15 42 PRT Rattus norvegicus 15 Cys Pro
Lys Asn Pro Pro Arg Ser Ile Gly Thr Cys Val Glu Leu Cys 1 5 10 15
Ser Gly Asp Gln Ser Cys Pro Asn Ile Gln Lys Cys Cys Ser Asn Gly 20
25 30 Cys Gly His Val Cys Lys Ser Pro Val Phe 35 40 16 11141 DNA
Mus sp. misc_feature (10980)..(11040) n is equal to generic
nucleotides 16 tctgtctacc gtgtgggtca aactaatcat aggctaggca
gccagcacct ttaccctcga 60 atccatcttt tttttttttt aattttcttc
ttttttaaaa ttaattaatt aattttatat 120 atgtgagtac actgtcgctg
tcttcagaca caccagaaga gggcatcaaa tcccattcca 180 gatggttgtg
agccaccgtg tagctgctgg gaattgaact cagcacctct ggaagagcag 240
caggttctct taaacgctga gccatctctg cagtccccca ccagctgtct tagttaaggt
300 cactactgct gcgatgaaat accgtgacca aagccacttg gagaggaaag
ggtttatttt 360 gcacacagtt ccatacaagg aacggttcat catccaaaac
agcgagggca ggaacctgga 420 ggcaggagct gctgcagagg ccacggaggg
gtgctgctta caggattgct ttactcttag 480 aatccaggac ctccagtgca
gggatggcac cgcccacatt gggcggggcc ctcttccatc 540 aatcactagt
taagaaaaca ctctacggag ctggcccaca gcacagcccg atctggtgga 600
ggcattccct taaccaaggt ttcctctctt caggttacta tattttatgt caagtcagat
660 atatacgtgc ccagtacacc agctctgtca gtctggtgtt tgccacatca
cttgaaggaa 720 acatgacagt gtccgagggt tcagtccata gttggctggc
tccgtggcca tgggccttgg 780 caaggcagaa catcctggca gtgggaatgt
gaagtcggac aaggttgctt acgtcatgtc 840 agtgaggacg ctggggacag
gaagggaccg agtatctcct tcacaggcat aggtgggccc 900 atgacgtact
ccctccaaca acaccctccc ccctaaagtt tccgccaccc tccctgaaca 960
gcgccacctt ctggccatca agtgttctac ttgtgagatg tatccaagat ggtgtaaaca
1020 cgttctcttg tgtatgatgt atgaattctc agagatcccg atacatggat
gtaccataaa 1080 caaaactatg ttcctataga aatgtacacg tacatgtatc
tgcatgtttg tttacatgta 1140 tatacatgta tatgcagata tatgaagaca
gatgagagct gtctcatagc gggtcatact 1200 ttgcatgcag actgctgtcc
aggcacacac tccaaaactg ccttccttct atagctcgct 1260 cctccctccc
tccctccctc cctccctccc tccctccctc cctcccttcc tctctccctc 1320
cctcttgaag ccatctcttt gaggactcgc agagaaagag caagtcaaac ccagaccttc
1380 cccactcttt gcctagataa ctgttttccc agggaggagg ctcaggctga
aagcctaggt 1440 caacgccctt tcactatgtt ccacaggctg aagaggttgc
agcgacaggc tcccggcagc 1500 cccatgcaga ccgctgccca ccaccgccaa
ggacactgcc cccgggtgcc tgtcaggcca 1560 cacgctgcca ggctgactct
gagtgcccac gacacaggcg ctgctgctac aatggctgtg 1620 cctatgcctg
cctggaggcg gtgccacctc cgccaggtaa gctgagccag ggtcaggggg 1680
ttggaggtcg ggtgggccca acgctgacct agggaagaga aagagctgag ctgtatttgt
1740 gctgagctac ccgctaccta ggctgggtgt tcacctgcag gcttcagata
tcccggcagc 1800 tcctgctagg tgcagctgcc tcacctgtat gcacggagtc
cccagcatgg actgtaccca 1860 tttaggaaca ccattcacct ccatacaatc
ccaccctttg aagggcagtc caacctatag 1920 gatggctcag cccatagaag
agtaagggat aagaggctaa gggtaccaac tccctacggt 1980 caaaggagag
aaaacacgca tctaagatat cacctgacct tccacatgca cacacacaca 2040
cacacacaca cacacacaca cacacacact gaaaataaaa ttcaatatac atgtgtacct
2100 gtatatccag ggtgggacac agctcagttg gtagcctgct tatactctgc
cttagcactt 2160 ggcctatggg gttgggagac aggaggataa gggtgtcccg
gacgctgcag gcctgtggtg 2220 tgtcctatag ggcagtgtgt ggggagccat
cttcacctct tctatgggat gacacacctc 2280 tccccaccca gggctggtca
catgacccca ccccaggaag acctccctcc ctctcaccta 2340 gttctctgtc
agttctagac tggctggtgc agcccaaacc acgatggctg ggtggcaacg 2400
gctggctgct ggacggtcct gaggaagtgt tacaaggtac ctgcccctgg gcatgccagg
2460 tccccagatc agaaccagcc tttctcacta gactgtccat gggccacaag
gatgctggct 2520 gggagcccag tctggttcct atagcttgtg cagagaaaga
gtgaggcttt ccctgacagt 2580 gggcctcctt cctgggtaga gaatgagaag
gaagagggtc tgggacccct aaagtcttaa 2640 agggccttgt ggccacctct
ggagaaaggt tccttatgtg gatagaaagt gaaggatgtt 2700 tcttaaagga
accacatacg ggcctgaggt ttcctgggga cagtgtactt gtgtctgtgc 2760
atctgggcat gtggctttgc acgccctcat gcggaccatg ttggtcaggg tatgctacta
2820 tgcacacacc cgttcttgtg gctgtggatg aacatatgtg tgtctgtgga
taagcacgtg 2880 tgtgtgaaag catgtgtata tataggtgtt gattgtgggt
ggttgtagtg cgtacacatg 2940 tgtctgtagc tgtgtgtgcg tgtttcaggt
atgccagtgt acacgcattc acatgttgtt 3000 ctctgtacga tggtgcattt
agggtgctgc gactgtgtgc gcgtgcaggc gtgctgatgg 3060 gaggtggcct
ctctgggtgc acatgtgtgt ataaacgtgg cttgatgtca gagtgtgcat 3120
gtgtgcgctg tgtgagaggc acagcaccgt ttagcatctg gatggggttg gaaactgccc
3180 tttctggatg gggtgtactg tctgagactt ggcctgtgtc cctccacagc
agagacttgc 3240 agcaccactg aggatggggc agagccactc ctctgtccct
caggctatga gtgccatatc 3300 ctgcagccag gggacgaagc ccagggcata
cccaaccatg gacagtgtgt taagcaacgc 3360 cggcaagcag gtgagcgctg
ctggtccaaa ctgccctgga ccggccccaa ccccattgcc 3420 agagaacacc
ttgtcaccca agactggctc agagtccttg agcctcactt cctgtccctc 3480
tgtgctctgc tccctcctgt aactccagct ctgcctgtgt ctgctacctg ggtggctttg
3540 gggtcgattt ctttgcctct ctgagcctca gtttcttccc ctgtaaaatg
ggaccacttc 3600 ctgttgtcat agggtcaggg gaaaacatgc agagttctgg
gagtggagcc aggcaggcac 3660 aggtgttggg cgacaccaga tcttgtgtat
ccatctctag atcagtcttt aaccccttag 3720 acttgaattt ctaaaatcta
ttattgtgta tggagtatga tgagggaaga gcaagcaggt 3780 gctccgaggc
ccacgtgtgc tctgtggcca actttgggga gtccctcctc tcccttcagc 3840
ttctcatggg ctctggaact ggaactcaga tcatcaggct ggggcgcact gcagacacct
3900 ttacctggtg atccatccaa gagacacaaa cctcacagtt tgaattatct
gagaatcctt 3960 gcttagatgg cagtgggtta tcattaacag gtgtcactgg
aggtcgtggg taaagcagtg 4020 aatggcaagg tcccagcctg tgtgaaatgg
gctgagagca gagtggtcaa gctgctaatt 4080 gaggttctat agggaacagc
caggttaaca cacacacaca aggaccatca tggctctgca 4140 cggagtagcg
tcttcccatt tatcaatgcc agtccctaga taccaggtga tggagagggg 4200
ggtggacctg cagatatata acccccgaag caggcagcca tctagtgctg caggatagag
4260 ggatgccttc catgagaaac cctggcatcc ctagggatgc ttcatacatc
ggaaaccccc 4320 catccctgac tctttctcac aaggtctcat gtagcccagg
ctggcccaga ccctctactt 4380 attgtagcta aggatggcct tagacttctg
attcttctgc cctcacctgc tgggattaca 4440 ggcctataat accataagcc
accatgctga gtttatatac aaggcgatag agcacagggc 4500 tttgtgcatt
gaggcaacac tctgccagct gagctgtacc ccagctccag gatggccttt 4560
ctggtctcct gatcttgctc atttgattct atgatttctc agaccagttc agacaagttc
4620 tagagagtct gaatccaaat ggactcattt cctaaggaag ctaagcatgg
agaatgggct 4680
cttcaacaag accacaccac ggatgcccaa aatcaagaat gaaatgcaat ggagacaagt
4740 ctgcagccca gaacatcaga aaccactcac agcctgctgc ttccttcctg
ggtgccctcg 4800 ctctctcact agactttctt agtctcagtt tttctatctg
taaaatgggc tttggggtac 4860 tgattactgc atgggcagat tctaaggctt
gaatcgatgt ctgtggggtg cttatgtctc 4920 accggacaca cagcagctct
gcagtgagtg tgagccacct ctggccctgg cacatcaggt 4980 caggatcctg
cccaaccatg agctgcacag gcacctgtgt gtgcatgccc gagtgtgctc 5040
tgcgtgcctc ctcgcccgca ggaacacctc cagaccccca gatggaaatg aattcttccc
5100 gtcttgacct cagggctgtc acttagaaat aattgctgat aattatatct
gagtttggaa 5160 acatcttaaa gcccctcaac ttcccttagg acaaagctct
cagtagagta caagaacaca 5220 tgtcatggta actgagagac actgttacaa
gagacatagt ttttttaaac cacatattca 5280 gagtaacctc aggaatatgg
aagagcttca ggaacataac tggggccacc atcgaggccc 5340 agcactatta
aaacaattct cccccccccc atagaaatga gctactgggg tcatttgaga 5400
atttattgct agggctagag agatgggtga gaacccttgt ggagcaagga tagggacctg
5460 agtctacatt tttaaaaaca tgctgggtgt ggccaggtgt gatattccag
ctgtgctgct 5520 gtgtgtctgg tcacccatct agcgccaggt tcaaaggacc
ctacctcaaa ggaacaaagt 5580 agacaatgaa aagcaggata ctaatgtctt
ctctggcctc caagctcata caggcataca 5640 ctcatgcaca cacacatgca
tgaatgcaca cacaagcacg cacacacaca tgcatgcgca 5700 catatcacac
acacacagaa taaactatgg gctaaagacc accctaaggc acagtctttc 5760
tttttactgc tctcctgggc caatggacag agagctttat tgtccagaca gcagttccca
5820 aactctatgg tctcaggtct cttttgatcc tcaaattaag gccaaaacat
ttgcccagag 5880 ctgtaagtgg aaactgacta gcacagcccc tgagctccta
aaagctttct ctttttgctt 5940 gttgcgcaga agggcgggtc ctgcgacaga
ggcttcacaa ggaatatcca ggtgagagga 6000 agaaggcgct ctttccctga
agaagacaca agaaagggga gaaggcatat gtgtgactga 6060 atacatttca
aaagaccagg gctgagctag agagaacaaa atggcaatta ggtttgagaa 6120
ataaagtcga gacctgtcat gatgatgagt gtgagccgga gaggggctca gggcagtcat
6180 gtccttacgc tcttgtcaga gtctggggat gggcacagtt gtctctccta
ccctcatgca 6240 gggaattgga tgatcagtta tccatctttc tgattgttca
tttatctaag aatccataca 6300 tacatacatc cgcccattta tccacctatc
cattcttaca tccactttac ccacctgccc 6360 acccacccat ttgcccatcc
ttccatctat cttctatcca gccatctatc cattcatcca 6420 tcccccatct
acccatcctt ccatccattc atccatccat ccatctaccc acccatccat 6480
ccatccatcc atccatccat ccatccatcc atccacccat ccattttttc atccattcct
6540 tcttcctttc ttccctctct tgatcttcct tcctcatctc ttgatctacc
catttcattc 6600 atccatctac cctacctatc cattttcatc tctccatttt
gtctattgac tcacccaccc 6660 acccactcca cgaatccgcc actcacccac
ccactcactc actgtccacc atccacccct 6720 tccccctccc tccatgtagc
atgtctttgt caggcgttgc tcacaggtga ctcagggttg 6780 tggctccatc
tgaaaatgca atgaatctag ttcctgtcag agtagcccag gcaggaaatt 6840
taaataccca taaagcccat ttcttcgtgg tattgacaca caccaaatac catgaggacc
6900 ctttgaacca ggttgtcaga ggatttccta gggagggatc aggaaaactc
aggctatgga 6960 attagaggca ttggcaccag aaggggatgt gttggtgatc
tgggggagga gtgactgtcc 7020 ccgtggctgc ccccgtggct gcctctgtga
ctgtgatgaa catgtgatga atgagccggg 7080 tggattgagt aggtaggaga
gccaaggcta tggctcaaag gtatggcact tagtgtgctt 7140 gaggctcagt
ccccagcata ctaaagaagg aacagagaga tgcaggcaga agccttgctg 7200
aggccagggg agagctacgt gtgcaaactg agaccgatag gattcatttc aagtgggagc
7260 ccggggccag ctgccatgtg gtgccaaggc atccagacat cctgtgacgg
tcacagcagg 7320 ccagggtagg cattcaagca gagcatctgt tctaatggac
tgggcactct tgttgcagaa 7380 ggtgactcca agaatgtggc agagcctggg
aagggacaac agaggcactt tccataaagt 7440 gaaggctggc tggtgagtgg
aggcaagatg gttgctcaaa cagctgtcac acccaagttg 7500 gccacaggag
ggcgacatca gaacatcaca atccctccaa gtgcccagca actcggaagc 7560
tgaccctaaa cccaagacct ggctgaccac tgcctccttt ctaatctgct gccttcaccc
7620 cttctagctt aggccagtgg tgccctgtaa gtcctgtctc ctcttccatt
ctgcaagaca 7680 atgctatagg gaccagctgt ctgactaatg tggccccagg
atcccccagc tcagcttcat 7740 tgtggtatct actgaatccc agtgctcagc
ggcagttggt ggtccttact gtacaccggg 7800 aaacacatcc cacaggcagg
gttgacatcc tattagaagg gagtctaaac cagtcacccg 7860 gtagatcaac
aaggaggcca cagtcataca taccaggggc tgtgcaagta ttgagaagta 7920
ggatgctccc agagagggtg ggggaagcct caggagggga tactgagtcc agaactgaaa
7980 tctacagagg agaaggtgct tcttggggtt gtaggctgga gggcaggatg
gcaccaggtg 8040 gggggaagag aagggtgcat tccccatggt gggtcacttc
agctctgagg agccacaggg 8100 aggctgctga gggaacaagg gatatcaagg
aggctatttg cataggcagc tgcccagtca 8160 aacacccata cagaaacata
caaagattca ggtttgcaac caccagaggg tcccccgcct 8220 gcaaaccaag
aacctgccca gactttaacg ctctcacaag gcaaaaggca gccatgatag 8280
cggcagctcc cctggggtcg tggaaaagga aggcacagtc cagcttttag ggtattcaag
8340 gctaaatccc cggaagattg tggagcgggc aggcaccaat gccaacctgt
ctgctctctg 8400 caaataccag gagccagcct tggtatgtgt cataagtcca
ggcctacaac tttcattaga 8460 gtggctagct caggctcagc atgaggtcgt
gaatcatagc tgaggaaggg cggcagttca 8520 gactctggag gcaatggggc
ccggctgtaa atcccagctc cctgccgtgt agccttaagc 8580 gagtgactca
gttctccaaa cctgttttcc cctctataga atgtatacaa tggtaatgca 8640
tgggtcagta gctaatcagg gaccatggag acctcacaga ggcttgaggg tttcatcttt
8700 gcagaaatac atctttctga tgggtgtagg gtggacagga atttaacagt
cccacccaca 8760 atcaaatcac attttctttg cccttttcct ttatagcctt
tgcggggcct ttcctgtgtc 8820 ttccacacgc taagccttgg aaacagccaa
agaatgaccc tagatgtcac actccattcc 8880 acagaacagc actcaagagc
tcctgggaaa tgggacttca gactcccaag cccagagtgg 8940 cccagcctgg
tgtggtcact tggtggaagc tcctaaccag ccctgggtcc ctgctcagca 9000
tccttgtcat gtgaacctgc agcttctagg tgactttggc agattttgcc tgcggaaggc
9060 atatattcat ccctaatgtt ccccaaataa atctgcccac cgtgttagca
gaagtaagct 9120 actttatcag gcgtgagcca cagagagtcg tgatccttga
gaggcaagac ggtagttcag 9180 ttcaacaagg ctaagtagct ggtgttggct
caaacatcag gagtctaacc ccaagtagat 9240 tgttttaagt atatttaagc
acagcacagt tcggtgaaaa ctctcctggt gataattagc 9300 ccaatcccat
gtgcacaata aagcaaaggc atgactctat caaaactctt tgtaaagtac 9360
aagtgatatc ttccacaaag gtaggtacta tagactgaca ggctcacaga aagggtctgg
9420 ggaaggattc aatgtagtca cagccacaca gatcaccaac caaggtcacc
caccaggctg 9480 tcaaccaagt ccactcccag ccttctgggc agataatagg
ttatgtgtcc cttccttgga 9540 gggaacaact ctatgtgttt aatattcctg
ggtcccttgg tgcttatctg tgagtacaag 9600 gacctccgtg cctcccacag
tctctctgtg tgtgtgtgtg tgtgtgtatg tgtgtgtgtg 9660 tgtgtgtatg
tgtgtgagtg tgtgtgtatg tttgtgtgtg agtgtgtgtg tatgtgtgtg 9720
tatgtatatg tgtgtgtgta tgtgtgtgtg tatgtgtgtg tatgtatatg tgtgtgtgta
9780 tgtgtgtgtg tgtgtatgtg tgtatgtgtg tgtgtgtgta tgtgtgtgtg
tgtgtgtgtg 9840 tatgtgtgtg tgtgcctgcc catggcacat gtgtggaggt
cagaagacaa cttatccagg 9900 ttggctctcc ttcccccata tctcagggat
gaactcaggc tgacaagctg ggtgtgagct 9960 ccttccctgc tgagccatct
caggtctgag tcagggcttt aactatttaa tatccttaaa 10020 tgtgctgtgc
tagaggctac cccttggagg ttatcaagga gaacagtgtg tgtgtgtgtg 10080
gggggggggg gtgtgccatg cttcctgggg ccagatggat tctttgagta cagcagctga
10140 gtcctgggtg ctctcagcaa ggaggggcac tgggaagctg gggagggtgg
gtgagctgga 10200 agctgcctca ggccagtggc cctgcctggg ccaagtccct
tcacaaggac ttccctgtac 10260 gagcagactt aattaaataa ttgctttgtg
ctctggtcct gctgaccttg gcttagtcct 10320 ccttggtggt tttccatcag
atcttctagc tttaacgccc agcccctggt cttgcccccc 10380 ccccttcccc
cctagagcat ccttacaaga tcattaggtt gatctaagga aagagtaggc 10440
aggtccccct tcaggtgacc atagggaagg atgctctgca catatgacca ccagggagga
10500 tcctcttgtg ctgcttgcta cataggacag acacacaatc ctgggtaaag
caggcaggac 10560 cctaggaccc agaaccttcc ttcctaaagg cacagtggta
tactgtctgt gcccttcagt 10620 gctacgaggt aggtgtagaa gaaaaccagt
tcagggctct gggggaggag ccagagaggg 10680 cttcctggag gtggggaatg
tctgagctaa gtttctgagt attggtctct ggggttgcca 10740 tgccgagtga
tcaccagcca agtagctgca gaaacacagc atgtgattga gcctggagta 10800
cgagcctgac cctcgcccag tggccttggg tagagtctct ggcttctccg tcccttggaa
10860 caatgacaac atcacctccc cttgactacc atggtctgcc acaaagaaaa
ggctgtgcaa 10920 gcatgaatcc actgtgggga gggcaggaga aaagagggaa
ggctggaaaa atctagcagg 10980 ggtggagcgg gggggggggg gggtggaggc
ngnaagaaga aagagaggaa gaggagaagg 11040 ggagggggaa gagagatgag
agagaggggg agggagacag ggagaggtgg aaggaagggg 11100 gagggagaag
gagagtggga gggggagggg gagggganan g 11141 17 6766 DNA Mus sp. 17
gcaggcagaa ctctgtgagt tgctagcctg gtctacatag agagtcacac tgtgtctcat
60 agagtcatag aggtcagtca gggatacaca gagagaactg tgtgtccccg
tgttggtgtg 120 taggagagaa cccaatggaa gacaggtgat gcagaggtca
ggtgtgttct tcagacaagc 180 agctcttggg agaactgagt cagcatccca
caagaactac cattaagagc atctttaacc 240 ctcccatagg agcagagacc
tataggcctg tcaggccccg cctcttaaag gagccaccac 300 ctcccaacat
ccctgaatca agtactaatt ctccagcatg ggacagacca tggggacaga 360
tgatgagtgg gggtgaggca gggagcgtgg agggacatga gagccagcgt tgcaggtgag
420 gagaggccac acttctcttt gtgtgagaca aagtccaata tgtagccctg
actgtcttgg 480 aactcactct gcagaccagg ctggctctcc aactcataga
gatctgctca catctgctct 540 gccaaggctt tggatgatgt gagtgtacaa
ccacactcag gttcacagct ttgctatcac 600 tgggattctg atgccacagg
ctcagtgtgg acccagatgt ggtatagtct cagcctctcc 660 tgcatggggg
tcactgaagt attgccaggg tgggagaggg gccgaacaac actgttagag 720
aggctgccga cctgtggttt agctcaaatt cgcttccaga gggaaatgta cacaaggctg
780 ggtcactcac tcggtttggg ggagtccagg aaggcagggc acggctgagg
cctgagaatg 840 cagtgggggt agggtgtggg ggggttggcc aagggtccag
ccagaccaac ctaaccaagg 900 gacttcagcg cttttggcct tggggtgtcc
ctttcccttg gctgagtgtg tgtgggaggg 960 agggggttct ctctctttgt
aacaatctct gaggcaaggg cccagagctg atcctgggac 1020 tggttcccac
agtcaggtcc cggatgggtc agtctgggcc atcaggaggg aggggcaggt 1080
tgctgcaggc tgaggaggaa gccggaagct ccctgcctga gaggacagtg aaccatcagt
1140 aggaagacgc taaggggagg tcagcagccg gccagccggc cagcctggca
gccagggcct 1200 gactcttgga ggacaaggtt gcccagggca acaactgttt
ccattcccag ccgggggcgg 1260 agccagtggg gcggtacctt gtgcaactga
ggaggttttg ccccgctccc cccaccacca 1320 ccttttgccc aaacacagca
aggagccaca gtggttaggc atggctccat catggggctg 1380 atatctcaga
gctcagtctt tctgttctta gctcagagtt agggacaagc cggaagtcac 1440
acagcaacat gattagacac aaggctcaag ttcccaacag acagctgtta acatgtttga
1500 atgtctcttc atatgtgaag gatagggact agggctatgt gatataaact
agacatggtg 1560 ccatctgtaa tctcagtacc caggcggtaa aggcaggagg
attccagggc tggtaagatg 1620 gctcggtaga gaaaagtgct ccctgccaag
tttgacgacc tgagacatgg gacccatgtg 1680 tgccttctga accatgccac
ggtccccctt tccaaaaata aatttaaaaa acattcaatg 1740 tcatcctcag
ctattgaggg gctgcaagcc cagttggggc tgcatgagac actatctgaa 1800
aaacaattaa tttttaaaat aaaagagaac gtgacgtagc ctgagttaaa tctggtcttg
1860 gtgtcaatgc accagcgggg aaaatgttgt cagcgcatgt ttaatacagg
aaaacctcac 1920 agcagcaagc cagcctcgat ttggccaaac aatggctctg
tatggcaccg cccatccact 1980 tgggggcacc cagccagggc tatgggcctc
tgcctgcatc ctctctgacc tccccatgct 2040 ctgggccatt gaaatggacc
taggtctcag cctgtctccc caagctcagc ccctactaat 2100 ctcctggggc
ttctggtgtc agctccaaca tctcctcaag tgtcttgacc tcctgtggtc 2160
atagcacagc agttctaagc acaggctctg cctctctgct gtgtcctggt tccaatcttg
2220 ctctctctga ccctcagcgt cctctgcaag ttggagacac tgtgagcgcc
cacctaacgt 2280 ggctactaca gtaatgagag gaaatagcgc tctccctttg
tgacacaatg tcatcctata 2340 gctttaactt gtaaccctcc tgcctcagcc
tccaggaggc tgcagctgtg tgccaccagg 2400 tgtactgact ggttttgtgt
gtcaactaga cacaggctgg agttatcaca gagaaaggag 2460 cttccgttga
ggaaatgcct ccatgagatc cagctgtaag gcattttctc aattagtgat 2520
cagtagggga gagccccttg tgggtggtgc catctctggg ctggcagtct tgggttctat
2580 aagaaagcaa gctgaacaag ccaggggaag caagccagtg agtaacatcc
ctccatggcc 2640 tctgcatcag ctcctgcttc ctgacctgct tgagttccag
tcctgacttc ctttggtgat 2700 gaacagcagt gtggaaaagt gtaagctgga
taaacccttt cctccccaac ttgcttcttg 2760 tcatcatgtt tgtgtaggaa
cagaaaccct gactaagata ccatgcctag ctttaagtca 2820 gtgattttct
taaagcagcc aggtgttaac cagtaagcag cgtagggttg atagacggga 2880
ggccattgcc cacgtcctta ggatggtgcc tgccatccat atttatcaga acaccaggtt
2940 aacaatctga ctccttttct tggtagtaag ccctggaggt gcagaggtgg
ggcttgggtc 3000 ctctcctttg gtgttcttag gcctgacttg ggtagatatt
caaaggaatg aatgaatgaa 3060 tgaatgaatg gactctgagt cttctacaca
attctgtttc tctctactgg aagtcattca 3120 cacgacagta taagttaaga
gtcagtacac ttttatctgg tctgatcaat agacttaaac 3180 tgtcctgtgt
ccctggagga tggtgaccct tagtgaccct tagtgtgtcc tctgcttcct 3240
gactccgagt ccccaggcac agcactcttt acattttaaa gtttctattt ctctctctct
3300 ctctctctct ctctctctcc cctgagggag ggaagccctt ggcagagaga
cattctccag 3360 gattcacagt cttcgggaag gcatgccaac ggtcggagac
ttcgcaggag gacatcccac 3420 tgggctgggg gaaagttcag gttgcctggg
tcacataaac tggaaaggcc ctctgaaggt 3480 gggacctcga gttctgagtc
tcggcaggaa actgggaaga gggctggacc tgacctcatg 3540 gtgggaccta
gggccctgct gggagcatcc ggtggctgtc atcctgaggc cacgtcaagt 3600
ccctgatcaa aagaggtgct ggctccccag gcggatttgt tctgtttgtt tcttgggcca
3660 ttttccttcc acacaaagta cccagtgtgg ctcagtaccc tgagcggttg
cccttcttgt 3720 cccgaagact ctgcagggtc cttttgtaac ttaccacggt
gaagagttga ctgtgtgtca 3780 catttcggag gctcctggca tctcaaccca
agcgcctaat gtatgcatgg accttcagtg 3840 ttatctaggg gagagaatgt
tgacttagtc acatcttggg actttagact gcagaactag 3900 tttggggggc
catccctcac taaatccgag gatgaggaag aatcttggca gttaagaaga 3960
ttaagcttac cccatcagca acaaatactc actgggtcca ggccaggttc tgggagcagc
4020 tggaggagat gcagacccat gtctttgtct ccggcagggt tccgaccttc
tctcctcagt 4080 gtggtaaatg ctcatgtgtt aagcagctta tggtgacccg
tgtcagcttc ctcaccacgc 4140 cagggttcaa ctccagatgt tgccggtggg
tagagaggga tcccaaagct ttgctttgct 4200 cctctttaat ttccaaatgc
tgggtggata cacacagaca gcttgtcatt agaaactact 4260 gagtgcattc
tgtctcttag agttccaaac taggggcctg atgtggagga tggagccaca 4320
tgggcggctt cccagtgaga taggcaagta tcaggggttc atcagatgtc acagagcaat
4380 gactgaatgt cctagtagcc gtgtgaccaa acaaggaaat gcggggagag
gatgtcagat 4440 accctcatgg ttccgtttga tccaggagca gttttcaatg
catgcacggt cagatgtcaa 4500 ggacacaccc ctgctttcta ccaattttga
gagttctttg gggtcactgt gggagttgaa 4560 gtgattggct tggttggtga
aacacttccc acgcaggcag agacctgagc ctgattcccc 4620 aggacacaca
caaaaaggca ggtgtgggag tgtgtgtctg taaccccagc tctgaggaga 4680
ccaagacaga agggtccctc atgttcactg gccggccagc ccagccatct tagagaagtc
4740 tcagccagtg agaaagtatc tgaggaaacc aaagagaatg gtcggtgctt
gaggaatgtg 4800 agggaggttg acctctgacc tccaccaacg catgtgtgtg
ttctgtacat gatgtctgct 4860 ttacacgtaa acagtcctca ctcagagctg
ctacatggcg catgtttagt gatcctatga 4920 ctacgtgtta gatgccacaa
gtaccaaaat gtaggacggg gaccccagat gatcctacat 4980 caggcacaac
atggtcaact gtattcacct actgcgcatg agcacagtct tatgaatctg 5040
tgactccagg gatggagcat ggcggggggg ggggtgctac aagaacaaga acaatccacc
5100 aggtagagca gactgtctag ttcagaagag gaggggtggg aggcaggggt
ggggctcagc 5160 gctccaatca gtacagatgc acccaatggt ggccaccaca
tgagagcctc tatcaggagc 5220 catactggga aagcagtccc ggcagaaatc
caaggccgtg ctagggccct gccaggcaga 5280 agagccttcc tggtggggaa
ctaggagacc tggtgcagtg aaggcagagg gaggtcagaa 5340 gggcttcaca
gaccatcagc ccagctcagg cagaagccag gccacccacg gagagaccca 5400
ggaactgggc caagagcctg ctgtccaaga atgtaatgta ggcacatgtg gagacgagca
5460 tgggggctgg gaactgctgt cagagcccca ggacccgggt gctcctggtg
gtgggtggga 5520 agacagacct accttcctgg gtgcccattc tgcccgtgcc
acccagcctc acagggtgtg 5580 cctcggagcc ccctcggcaa ataccactgg
ggctgcaggg aaagccaatc ccaccttctc 5640 cagtggagcc gttcatacca
tcctctgggc ctgagctagc tgctccacct cacactgttg 5700 tctgatgtga
gagcaagcgt gaaacctctt tccatctctg tgtccccctt taagaaaaat 5760
ggggatccac acagcagcca cctctgcatg ctgttgggga caacctgttg gggacaggca
5820 aggtgcttgg ttcccgcagc aaccctcagt gcagaggatg ctttcctgtg
caatgccact 5880 ctcccccaga gtcacatcaa agcagggact cagagacaag
ggaggggtct gcttccccac 5940 accagggcca caggctcctg cggccccctg
tcccctccct atagcacccc caaacccagt 6000 tcctggggca gacagtcaca
ggcagcgctg agagcagcac agcccatcct aactggaggc 6060 cgaggaggcc
gccacagcag ctcaatagtt acaggggagt ggcaggtccc tcccggccac 6120
gtcttggctg gtgccccttc ctccctctgc ccggcatctg agctgcccag cttacagtca
6180 ctctagtcac tggagccaca gggtggacta aaaataacct ggagaaccaa
ccactaccca 6240 gggcacaggt gctggctgag ggcacaagat cccagggacc
ctgtgtggag gaccaaggct 6300 gcagcaggca aagttctgtt gctctctcag
gccccaccca ggctatggtc cctcctccct 6360 cttaacccct cttgcctcct
cttccccggc ctgcggagga ggtcactcgt gcagaaggaa 6420 accctgccac
cagcctcggg atgggtaact gcggcagaaa agtccttcgg gctctgagtt 6480
tcctgttgct actgggctcc agttctgccc agggcacttg ggaggcaatg ctgccggcca
6540 ggctggccga gaagtcccga gtaagtgcca ggaggggtgg aggggcctct
gtgatgggag 6600 gtggcccagg ggacagaagt ccctagatgc tgtctatggt
ttcttatcgt ggtgggaatc 6660 tcctgtattg gagctgtagc gggtggtggg
cagacaaggc tctgccttcc tgagagccca 6720 ttgctgccag cagggaggca
agaaacaggt ccacccccca tgtgga 6766 18 14 PRT Rattus norvegicus 18
Thr Trp Glu Ala Met Leu Pro Val Arg Leu Ala Glu Lys Ser 1 5 10 19
211 PRT Mus sp. 19 Met Gly Asn Cys Gly Arg Lys Val Leu Arg Ala Leu
Ser Phe Leu Leu 1 5 10 15 Leu Leu Gly Ser Ser Ser Ala Gln Gly Thr
Trp Glu Ala Met Leu Pro 20 25 30 Ala Arg Leu Ala Glu Lys Ser Arg
Ala Glu Glu Val Ala Ala Thr Gly 35 40 45 Ser Arg Gln Pro His Ala
Asp Arg Cys Pro Pro Pro Pro Arg Thr Leu 50 55 60 Pro Pro Gly Ala
Cys Gln Ala Thr Arg Cys Gln Ala Asp Ser Glu Cys 65 70 75 80 Pro Arg
His Arg Arg Cys Cys Tyr Asn Gly Cys Ala Tyr Ala Cys Leu 85 90 95
Glu Ala Val Pro Pro Pro Pro Val Leu Asp Trp Leu Val Gln Pro Lys 100
105 110 Pro Arg Trp Leu Gly Gly Asn Gly Trp Leu Leu Asp Gly Pro Glu
Glu 115 120 125 Val Leu Gln Ala Glu Thr Cys Ser Thr Thr Glu Asp Gly
Ala Glu Pro 130 135 140 Leu Leu Cys Pro Ser Gly Tyr Glu Cys His Ile
Leu Gln Pro Gly Asp 145 150 155 160 Glu Ala Gln Gly Ile Pro Asn Lys
Gly Gln Cys Val Lys Gln Arg Arg 165 170 175 Gln Ala Glu Gly Arg Val
Leu Arg Gln Arg Leu His Lys Glu Tyr Pro 180 185 190 Glu Gly Asp Ser
Lys Asn Val Ala Glu Pro Gly Lys Gly Gln Gln Arg 195 200 205 His Phe
Pro 210
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