U.S. patent application number 11/005197 was filed with the patent office on 2005-09-22 for modulators of morphogen expression and methods of identifying the same.
This patent application is currently assigned to Curis, Inc.. Invention is credited to Haimanti, Dorai, Oppermann, Herman, Sampath, Kuber T., Shepard, Alyssa A..
Application Number | 20050208533 11/005197 |
Document ID | / |
Family ID | 21951710 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208533 |
Kind Code |
A1 |
Haimanti, Dorai ; et
al. |
September 22, 2005 |
Modulators of morphogen expression and methods of identifying the
same
Abstract
Disclosed are methods and compositions for identifying compounds
having an ability to modulate expression of a morphogen,
particularly OP-1, OP-1 homologues and closely related proteins,
using one or more OP-1-specific, non-coding sequences and a
suitable reporter gene. In preferred embodiments, the OP-1-specific
non-coding DNA sequence comprises a Pax-responsive OP-1-modulating
element.
Inventors: |
Haimanti, Dorai; (Lexington,
MA) ; Oppermann, Herman; (Medway, MA) ;
Sampath, Kuber T.; (Holliston, MA) ; Shepard, Alyssa
A.; (Shrewsbury, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Curis, Inc.
Cambridge
MA
|
Family ID: |
21951710 |
Appl. No.: |
11/005197 |
Filed: |
December 6, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11005197 |
Dec 6, 2004 |
|
|
|
09423821 |
May 15, 2001 |
|
|
|
6828095 |
|
|
|
|
09423821 |
May 15, 2001 |
|
|
|
PCT/US98/11025 |
May 28, 1998 |
|
|
|
60047911 |
May 29, 1997 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C12N 2830/15 20130101;
C07K 14/47 20130101; C12N 15/85 20130101; C12N 2830/30 20130101;
C12N 2830/85 20130101; C07K 14/51 20130101; C07K 14/82 20130101;
C12Q 1/6897 20130101; C12N 2830/008 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 038/17; C07K 014/47 |
Claims
1. A vector comprising a DNA sequence encoding a reporter gene in
operative association with at least one OP-1-specific non-coding
DNA sequence capable of modulating expression of said reporter
gene, wherein said non-coding sequence comprises at least a first
Pax-responsive OP-1 modulating element which is responsive to a
first Pax gene expression product.
2. The vector of claim 1 wherein said non-coding sequence is
selected from the group of DNA sequences corresponding to bases
108-121, 139-154, 157-167, 365-378, 497-511, 598-613, 1123-1140,
1144-1161, 1285-1297, 1750-1762, 2001-2023, 2365-2378, 2931-2944 of
SEQ. ID No. 1, including allelic, species and other sequence
variants thereof.
3. The vector of claim 1 wherein said non-coding sequence
corresponds to part or all of SEQ. ID No. 2 including allelic,
species and other sequence variants thereof.
4. The vector of claim 1 wherein said non-coding sequence comprises
between one and twelve first Pax-responsive OP-1 modulating
elements.
5. The vector of claim 1 wherein said non-coding sequence comprises
at least part of a first Pax-responsive OP-1 modulating
element.
6. The vector of claim 1 further comprising at least a second
non-coding sequence, wherein said second non-coding sequence
comprises at least a second Pax-responsive OP-1 modulating element
which is responsive to a second Pax gene expression product, said
second Pax gene expression product differing from said first Pax
gene expression product.
7. The vector of claim 6 wherein said second non-coding sequence is
selected from the group of DNA sequences corresponding to bases
491-503, 737-747, 891-903, 994-1006 of SEQ. ID. No. 1, including
allelic, species and other sequence variants thereof.
8. The vector of claim 6 wherein said second non-coding sequence
corresponds to part or all of SEQ. ID No. 3 including allelic,
species and other sequence variant thereof.
9. The vector of claim 6 wherein said second non-coding sequence
comprises four or more second Pax-responsive OP-1 modulating
elements.
10. The vector of claim 6 wherein said second non-coding sequence
defines at least part of a second Pax-responsive OP-1 modulating
element.
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. A substantially pure nucleic acid comprising a DNA sequence
corresponding to bases 108-121, 139-154, 157-167, 365-378, 497-511,
598-613, 1123-1140, 1144-1161, 1285 1297, 1750-1762, 2001-2023,
2365-2378, 2931-2944 of SEQ. ID No. 1, or corresponding to bases
491-503, 737-747, 891-903, 994-1006 of SEQ. ID. No. 1, including
allelic, species and other sequence variants of any of the
foregoing.
22. (canceled)
23. (canceled)
24. (canceled)
25. A nucleic acid comprising nucleotides 2606-2690 of SEQ ID NO:
1.
26. A method for increasing OP-1 gene expression comprising the
step of deleting a gene expression silencer contained within the
AatII-PvuII upstream fragment (residues 2606 2690 of SEQ ID NO:
1).
27. A recombinant OP-1 gene lacking a gene expression silencer
contained within the AatII PvuII upstream fragment (residues
2606-2690 of SEQ ID NO: 1).
28. A recombinant cell containing the OP-1 gene of claim 27.
Description
FIELD OF THE INVENTION
[0001] The invention relates generally to the field of drug
screening assays. More particularly, the invention relates to
methods and compositions for identifying molecules that modulate
expression of true tissue morphogenic proteins.
BACKGROUND OF THE INVENTION
[0002] A class of proteins recently has been identified, the
members of which are true tissue morphogenic proteins. The members
of this class of proteins are characterized as competent for
inducing the developmental cascade of cellular and molecular events
that culminate in the formation of new organ-specific tissue,
including any vascular and connective tissue formation, as required
by the naturally occurring tissue. Specifically, the morphogens are
competent for inducing all of the following biological functions in
a morphogenically permissive environment: (1) stimulating
proliferation of progenitor cells; (2) stimulating differentiation
of progenitor cells; (3) stimulating the proliferation of
differentiated cells and (4) supporting the growth and maintenance
of differentiated cells. For example, the morphogenic proteins can
induce the full developmental cascade of bone tissue morphogenesis,
including the migration and proliferation of mesenchymal cells,
proliferation and differentiation of chondrocytes, cartilage matrix
formation and calcification, vascular invasion, osteoblast
proliferation, bone formation, bone remodeling, and hematopoietic
bone marrow differentiation. These proteins also have been shown to
induce true tissue morphogenesis of non-chondrogenic tissue,
including dentin, liver, and nerve tissue.
[0003] A particularly useful tissue morphogenic protein is human
OP-1 (Osteogenic Protein-1), described in U.S. Pat. No. 5,011,691;
U.S. Pat. No. 5,266,683 and Ozkaynak et al. (1990) EMBO J. 9:
2085-2093. Species homologues identified to date include, but are
not limited to, mouse OP-1 (see U.S. Pat. No. 5,266,683) and the
Drosophila homologue 60A, described in Wharton et al. (1991) Proc.
Natl. Acad. Sci. USA 88:9214-9218). Other closely related proteins
include OP-2 (Ozkaynak (1992) J. Biol. Chem. 267:25220-25227 and
U.S. Pat. No. 5,266,683); BMP5, BMP6 (Celeste et al. (1991) Proc.
Natl. Acad. Sci. 87:9843-9847) and Vgr-1 (Lyons et al. (1989).
These disclosures are incorporated herein by reference.
[0004] It previously has been contemplated that these tissue
morphogens can be administered to an animal to regenerate lost or
damaged tissue. Certain complications, however, presently are
encountered during the production, formulation and use in vivo of
therapeutic macromolecules, such as morphogen proteins. For
example, such proteins are typically produced by fermentation or
culture of suitable host cells. Any biological product produced
from such host cells for use in humans presently must be shown to
be essentially free of host cell contaminants, such as secreted or
shed proteins, viral particles or degradation products thereof.
Providing such assurance can add significantly to the cost and
technical difficulty of commercial production of biological
macromolecules. Furthermore, appropriate formulations must be
developed for conferring commercially reasonable shelf life on the
produced macromolecule, without significant loss of biological
efficacy. An additional complicating factor arises when
circumstances warrant an extended course of therapeutic treatment
with the produced and formulated macromolecule: the treated mammal
may develop an immunological response to the macromolecule, and any
such response may interfere with effectiveness thereof. In extreme
circumstances, treatment must be discontinued.
[0005] Alternatively, administering a molecule capable of
modulating expression of the endogenous tissue morphogen is an
effective means for providing morphogen to a site in vivo. For
example, DNA sequences have been identified in the OP-1 gene
promoter that resemble wt-1/Egr-1 consensus sequences, TLC binding
sequences, FTZ binding sequences and steroid binding sequences (see
WO 95/33831, the disclosure of which is incorporated herein by
reference). Thus, molecules to which these regulatory sequences are
responsive are likely modulators of the OP-1 gene and can influence
its expression.
[0006] It is an object of this invention to provide compositions
and methods of identifying compounds which can modulate expression
of an endogenous tissue morphogen, particularly OP-1 and other
members of the larger genus of true tissue morphogens. The
compounds thus identified have utility both in vitro and in vivo.
Useful compounds contemplated include at least those that are
capable of stimulating transcription and/or translation of the OP-1
gene, as well as compounds capable of inhibiting transcription
and/or translation of the OP-1 gene, via OP-1 non-coding DNA
sequences resembling consensus sequences for Pax homeobox genes, in
particular, the Pax 6 or Pax 2 genes.
[0007] These and other objects and features of the invention will
be apparent from the description, drawings and claims which
follow.
SUMMARY OF THE INVENTION
[0008] The invention features compositions and methods for
screening candidate compounds for their ability to modulate the
effective local or systemic levels of endogenous morphogen,
particularly OP-1, in an organism. In one aspect, the method is
practiced by: (1) incubating one or more candidate compounds with
cells transfected with a DNA sequence encoding at least a portion
of a morphogen non-coding DNA sequence that is responsive to a Pax
homeobox gene and which is competent to act on and affect
expression of a reporter gene with which it is operatively
associated; (2) measuring the level of reporter gene expression in
the transfected cell, and (3) comparing the level of reporter gene
expressed in the presence of the candidate compound with the level
of reporter gene expressed in the absence of the candidate
compound. The level of an expressed reporter gene product in a
given cell culture, or a change in that level resulting from
exposure to one or more compound(s) indicates that the compound can
also modulate the level of the morphogen normally associated with
the non-coding sequence. Specifically, an increase in the level of
reporter gene expression is indicative of a candidate compound's
ability also to increase morphogen expression in vivo. Similarly, a
decrease in the level of reporter gene expression is indicative of
a candidate compound's ability also to decrease or otherwise
interfere with morphogen expression in vivo. The above method is
particularly useful for identifying compounds that are capable of
influencing Pax mediated OP-1 gene expression.
[0009] The methods of the invention can therefore be used to
identify compounds showing promise as therapeutics for various in
vivo and ex vivo mammalian applications, as well as to identify
compounds having numerous utilities. For example, compounds that
modulate morphogen expression by stimulating Pax 2 or Pax 6
mediated transcription of a morphogen can be used in vivo to
correct or alleviate a disease condition, to regenerate lost or
damaged tissue, to induce cell proliferation and differentiation,
and/or to maintain cell and tissue viability and/or a
differentiated phenotype in vivo or ex vivo. The compounds also can
be used to maintain the viability of, and the differentiated
phenotype of, cells in culture. The various in vivo, ex vivo, and
in vitro utilities and applications of the morphogenic proteins
described herein are well documented in the art. See, for example,
U.S. Ser. No. 92/01968 (WO 94/03200), filed Mar. 11, 1992; U.S.
Ser. No. 92/07358 (WO 93/04692), filed August 28; PCT US 92/0743
(WO 93/05751), filed Aug. 28, 1992; U.S. Ser. No. 93/07321 (WO
94/03200), filed Jul. 29, 1993; U.S. Ser. No. 93/08808 (WO
94/06449), filed Sep. 16, 1993; U.S. Ser. No. 93/08885 (WO
94/06420), filed Sep. 15, 1993, and U.S. Pat. No. 5,266,683.
[0010] In another aspect, the invention further provides vectors
and cells useful for morphogen, particularly OP-1, therapy. In one
embodiment, the invention features a vector having a reporter gene
operatively associated with at least a portion of one or more OP-1
non-coding sequences responsive to Pax homeobox gene products. The
OP-1 non-coding sequences comprise at least a first Pax responsive
OP-1 modulating element which is responsive to a first Pax gene
expression product. In other embodiments, vectors further comprise
a second non-coding sequence comprising at least a second
Pax-responsive OP-1 modulating element which is responsive to a
second Pax gene expression product; in such embodiments, the first
and second Pax gene expression product differ. OP-1 non-coding
sequences which are Pax responsive OP-1 modulating elements can be
selected from nucleotides 1-3317 of SEQ. ID No. 1. Also anticipated
to be similarly useful are certain of the non-coding sequences of
other species homologues of OP-1 and proteins closely related to
OP-1. For example, other non-coding DNA that is responsive to Pax
gene products or homologues thereof can be used to identify
modulators of specific morphogens, or other factors capable of
modulating morphogen gene expression.
[0011] In another embodiment, the vector can include a non-coding
OP-1-specific sequence selected from at least one of the following
sequence segments of SEQ. ID No. 1 presented below, which defines
approximately 3.3 Kb of 5' non-coding human genomic OP-1 sequence.
Preferred vectors comprise sequence segments including nucleotides
1-3317, as well as shorter fragments of this region of DNA such as
approximately nucleotides 108-121, 139-154, 157-167, 365-378,
491-503, 598-613, 737-747, 891-903, 994-1006, 1123-1140, 1144-1161,
1285-1297, 1750-1762, 2001-2023, 2365-2378, 2931-2944 of SEQ. ID
No. 1, including allelic, species and other sequence variants
thereof. As base 2790 is the mRNA start site, other preferred
sequences include approximately 2790-3317, representing transcribed
but not translated 5' non-coding sequence and shorter fragments of
this DNA region. Other preferred regions of the 5' non-coding
region of SEQ. ID No. 1 include regions comprising a cluster of
several Pax responsive elements, such as, for example,
approximately 1-2073, 1-1297, 1-2691, 1-378, 491-1006, 1750-2023,
1750-2378, 1750-2691, 1750-2944. In certain embodiments, non-coding
sequences correspond to part or all of SEQ. ID No. 2 and/or SEQ. ID
No. 3, including allelic, species and other sequence variants
thereof. In yet other embodiments, vectors comprise non-coding
sequences corresponding to at least one, preferably between one and
twelve and/or four or more first and second Pax-responsive OP-1
modulating elements, respectively. First Pax-responsive sites
correspond approximately to bases 108-121, 139-154, 157-167,
365-378, 497-511, 598-613, 1123-1140, 1144-1161, 1285-1297,
1750-1762, 2001-2023, 2365-2378 and 2931-2944 of SEQ. ID No. 1.
Second Pax-responsive sites correspond approximately to bases
491-503, 737-747, 891-903, and 994-1006 of SEQ. ID No. 1.
[0012] In another aspect, the invention provides a cell comprising
a reporter gene whose regulation is mediated by one or more of the
Pax-responsive OP-1 non-coding sequences defined above. In one
embodiment, the cell is transfected with a reporter gene in
operative association with at least one Pax responsive site. In
another embodiment, the present invention provides a cell
comprising a transfected vector encoding a reporter gene
operatively associated with at least two DNA sequences, the first
comprising at least part of a sequence selected from SEQ. ID No. 2
while the second comprises at least part of a sequence selected
from SEQ. ID No. 3, including allelic, species and other sequence
variants of the foregoing. In yet another embodiment, cells of the
present invention are co-transfected with expression vectors
encoding Pax gene expression products such as, for example, Pax 2
and/or Pax 6.
[0013] In another aspect, the invention provides kits useful in the
design and/or identification of OP-1 expression modulating
compounds. As used herein a "kit" comprises a cell comprising a
reporter gene in operative association with an OP-1 non-coding DNA
sequence and the reagents necessary for detecting expression of the
reporter gene. The portion of OP-1 non-coding DNA chosen can be any
of the various sequences which have been described herein
above.
[0014] Following this disclosure, medium flux screen assays, and
kits therefore, for identifying modulators of morphogen expression,
such as OP-1 expression, are available. These compounds can be
naturally occurring molecules, or they can be designed and
biosynthetically created using a rational drug design and an
established structure/function analysis methodology. The compounds
can be amino acid-based or can be composed in part or wholly of
non-proteinaceous synthetic organic molecules.
[0015] The OP-1 expression modulating compounds thus identified can
be produced in reasonable quantities, including commercially
significant quantities, using standard recombinant expression or
chemical synthesis technology well known and characterized in the
art and/or as described herein. For example, automated means for
the chemical synthesis of nucleic and amino acid sequences are
commercially available. Alternatively, promising candidate
compounds can be modified using standard biological or chemical
methodologies to, for example, enhance the binding affinity of the
compound for a DNA element and the preferred candidate compound
derivative then can be produced in quantity.
[0016] Once a candidate compound has been identified and produced,
it can be further tested for its effect on OP-1 expression. For
example, a compound which upregulates (increases) the production of
OP-1 (e.g., an OP-1 agonist) in a kidney cell line is a candidate
for systemic administration. The candidate compound can be assayed
in an animal model to determine the candidate molecule's efficacy
in vivo. For example, the ability of a compound to upregulate
levels of circulating OP-1 in vivo can be used to correct bone
metabolism diseases such as osteoporosis (See, for example,
PCT/US92/07932, above). Conversely, compounds which down regulate
(decrease) the production of OP-1 (e.g., OP-1 antagonists) are also
contemplated to be useful. Useful in vivo animal models for
systemic administration are disclosed in the art and below.
[0017] As is well known in the art, OP-1 is differentially
expressed in different cell types. Accordingly, it is further
anticipated that a candidate compound will have utility as an
inducer of OP-1 expression in one cell type but not in another.
Thus, the invention further contemplates testing a candidate
compound for its utility in modulating expression of OP-1 in a
tissue specific manner in vivo.
[0018] Thus, in view of this disclosure, one of ordinary skill in
recombinant DNA and tissue culture techniques can design and
construct appropriate DNA vectors and transfect cells with
appropriate DNA sequences for use in the method according to the
invention to assay for compounds which modulate the expression of
OP-1. These identified compounds can be used to modulate OP-1
production and its effective concentrations in both in vivo and in
vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A is a diagrammatical illustration of the 5'
non-coding region of the OP-1 gene in operative association with a
luciferase reporter gene. Certain preferred Pax consensus sites are
depicted along the non-coding region.
[0020] FIG. 1B is a graphical representation of the induction of
luciferase activity in G401 human kidney cells transfected with
OP-1 promoter deletion constructs of (1A) in the presence or
absence of co-transfection with Pax 2 and/or Pax 6 expression
constructs.
[0021] FIG. 2 shows the relative levels of luciferase expression
from constructs containing 5' non-coding regions of the OP-1 gene
in operative association with a luciferase reporter gene.
[0022] FIG. 3 shows the levels of luciferase expression in cells
containing pAS3.3 and pAS3.3d.
DETAILED DESCRIPTION
[0023] As will be more fully described below, we have discovered
specific regions in the OP-1 gene sequence useful in identifying
molecules capable of modulating OP-1 expression in vivo. We
discovered DNA sequences responsible for the regulation of OP-1
gene expression by cloning and characterizing various truncated
sequences isolated from the 5' non-coding sequences of the human
OP-1 gene. The presence of sequences defining Pax 2 or Pax 6
consensus binding sequences as defined herein in the OP-1
non-coding region, together with the observed modulation of OP-1
gene expression mediated by Pax 2 and Pax 6 gene products,
implicate these sequences, or variants thereof, as having utility
in a method for the screening of compounds for their ability to
modulate OP-1 expression. Moreover, Pax responsive sequences
located in 5' non-coding sequences of other morphogen genes provide
a means for identifying compounds that modulate expression of other
such morphogens.
List of Useful Terms and Definitions
[0024] As used herein, "morphogen" means the class of proteins
typified by human osteogenic protein 1 (hOP-1). hOP-1 and
functionally equivalent morphogens are, as defined herein, dimeric
proteins that induce or reinduce mammalian cells, particularly
uncommitted progenitor cells, to undergo a fully integrated
developmental cascade of cellular and molecular events that
culminates in the formation of fully differentiated, functional
tissue of a type appropriate to the context or local biological
environment in which morphogenesis is induced, including any
vascularization, connective tissue formation, enervation and the
like characteristic of tissue naturally-occurring in such a
context. For example, if cells are stimulated by OP-1 in the
context of, for example, bone, liver, nerve, tooth dentin,
periodontal tissue, gastrointestinal tract lining tissue, the
resulting cascade of morphogenesis culminates in the formation of
new or regenerative differentiated tissue appropriate to that local
environment. Morphogenesis therefore differs significantly from
simple reparative healing processes in which scar tissue (e.g.,
fibrous connective tissue) is formed and fills a lesion or other
defect in differentiated, functional tissue.
[0025] Morphogens generally induce all of the following biological
functions in a morphogenically permissive environment: stimulating
proliferation of progenitor cells; stimulating the differentiation
of progenitor cells; stimulating the proliferation of
differentiated cells; and supporting the growth and maintenance of
differentiated cells. The term "progenitor cells" includes
uncommitted cells, preferably of mammalian origin, that are
competent to differentiate into one or more specific types of
differentiated cells, depending on their genomic repertoire and the
tissue specificity of the permissive environment in which
morphogenesis is induced. Preferably, morphogenesis culminates in
the formation of differentiated tissue having structural and
functional properties of a tissue that occurs naturally in the body
of a mammal. Morphogens further can delay or mitigate the onset of
senescence- or quiescence-associated loss of phenotype and/or
tissue function. Morphogens still further can stimulate phenotypic
expression of differentiated cells, including expression of
metabolic and/or functional, e.g., secretory, properties thereof.
In addition, morphogens can induce redifferentiation of transformed
cells under appropriate environmental conditions. As noted above,
morphogens that induce proliferation and differentiation at least
of mammalian bone progenitor cells, and/or support the formation,
growth, maintenance and functional properties of mammalian
endochondral bone tissue, are representative.
[0026] A morphogen as isolated from natural sources in mature,
biologically active form is a glycosylated dimer typically having
an apparent molecular weight of about 30-36 kDa as determined by
SDS-PAGE. When reduced, the 30 kDa protein gives rise to two
glycosylated peptide subunits having apparent molecular weights of
about 16 kDa and 18 kDa. The reduced polypeptides themselves have
no detectable morphogenic activity. Glycosylation, however, is not
required for biological activity. The unglycosylated protein has an
apparent molecular weight of about 27 kDa. When reduced, the 27 kDa
protein gives rise to two unglycosylated polypeptides having
molecular weights of about 14 kDa to 16 kDa. The polypeptides which
together form the biologically active dimer comprise at least six,
preferably at least seven, positionally conserved cysteine residues
as set forth in U.S. Ser. No. 08/396,930, the teachings of which
have been incorporated herein by reference. As described above,
particularly preferred sequences include those comprising the
C-terminal 96 or 102 amino acid sequences of DPP (from Drosophila),
Vg1 (from Xenopus), Vgr-1 (from mouse), the OP1 and OP2 proteins,
proteins (see U.S. Pat. No. 5,011,691 and Oppermann et al., as well
as the proteins referred to as BMP2, BMP3, BMP4 (see WO88/00205,
U.S. Pat. No. 5,013,649 and WO91/18098), BMP5 and BMP6 (see
WO90/11366, PCT/US90/01630), BMP8 and BMP9.
[0027] As stated above, the representative morphogen, for purposes
of the present invention, comprises an OP-1 or an OP-1-related
polypeptide. Sequences of useful OP-1 polypeptides are recited in
U.S. Pat. Nos. 5,011,691; 5,018,753 and 5,266,683; in Ozkaynak et
al. (1990) EMBO J 9:2085-2093; and Sampath et al. (1993) Proc.
Natl. Acad. Sci. USA 90: 6004-6008. Additional useful sequences
occur in the C-terminal domains of DPP (from Drosophila), Vg1 (from
Xenopus), 60A (from Drosophila, see Wharton et al. (1991), Proc.
Natl. Acad. Sci. USA 88:9214-9218), Vgr-1 (from mouse), the OP-1
and OP2 proteins, (see U.S. Pat. No. 5,011,691 by Oppermann et
al.), as well as the proteins referred to as BMP2, BMP3, BMP4 (see
WO88/00205, U.S. Pat. No. 5,013,649 and WO91/18098), BMP5 and BMP6
(see WO90/11366, PCT/US90/01630) and BMP8 and 9. Each of the
foregoing polypeptides, when oxidized and dimerized, is useful as a
morphogen herein. Further, this family of morphogenic proteins
includes longer forms of a given protein, as well as phylogenetic,
e.g., species and allelic variants and biosynthetic mutants
thereof, including addition and deletion mutants and variants, such
as those which may alter the conserved C-terminal cysteine
skeleton, provided that the alteration still allows the protein to
form a dimeric species having a conformation capable of inducing
morphogenesis, e.g., endochondral bone formation when implanted in
a mammal in conjunction with a matrix permissive of bone
morphogenesis. In addition, morphogens as defined herein can
include forms having varying glycosylation patterns and varying
N-termini, can be naturally occurring or biosynthetically derived,
and can be produced by expression of recombinant DNA in prokaryotic
or eukaryotic host cells according to established techniques. The
proteins are active either as homodimers or heterodimers.
[0028] As used herein, the terms "morphogen", "bone morphogen",
"bone morphogenic protein", "BMP", "osteogenic protein" and
"osteogenic factor" embrace the class of proteins typified by human
osteogenic protein 1 (hOP-1). It will be appreciated by the artisan
of ordinary skill in the art, however, that OP-1 merely is
representative of the TGF-.beta. subclass of true tissue morphogens
competent to act as osteogenic proteins, and is not intended to
limit the description. Morphogenic protein is generally understood
to mean a protein which can induce the full cascade of morphogenic
events culminating in at least endochondral bone formation. Other
known, and useful proteins include, OP2, OP3, BMP-2, BMP-3, BMP-3b,
BMP4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13,
BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9,
GDF-10, GDF-11, GDF-12, Vg1, Vgr, 60A, DPP, NODAL, UNIVIN, SCREW,
ADMP, NEURAL and morphogenically active amino acid variants
thereof. As defined herein, morphogenic proteins include
biologically active species variants of any of these proteins,
including conservative amino acid sequence variants, proteins
encoded by degenerate nucleotide sequence variants, and
morphogenically active proteins sharing the conserved seven
cysteine skeleton as defined herein and encoded by a DNA sequence
competent to hybridize to a DNA sequence encoding an osteogenic
protein disclosed herein, including, without limitation, OP-1,
BMP-5, BMP-6, BMP-2, BMP-4 or GDF-5, GDF-6 or GDF-7. Morphogenic
proteins include those sharing the conserved seven cysteine domain
and sharing at least 70% amino acid sequence homology (similarity)
within the C-terminal active domain, as defined herein. That is,
particularly preferred morphogenic proteins are those comprising an
amino acid sequence having at least 70% homology with the
C-terminal 102-106 amino acids, defining the conserved seven
cysteine domain, of human OP-1 and related proteins. Certain
preferred embodiments of the instant invention relate to the
morphogenic protein, OP-1. "Amino acid sequence homology" is
understood herein to mean amino acid sequence similarity.
Homologous sequences share identical or similar amino acid
residues, where similar residues are conservative substitutions
for, or allowed point mutations of, corresponding amino acid
residues in an aligned reference sequence. Thus, a candidate
polypeptide sequence that shares 70% amino acid homology with a
reference sequence is one in which any 70% of the aligned residues
are either identical to, or are conservative substitutions of, the
corresponding residues in a reference sequence. Examples of
conservative variations include the substitution of one hydrophobic
residue, such as isoleucine, valine, leucine or methionine, for
another, or the substitution of one polar residue for another, such
as the substitution of arginine for lysine, glutamic acid for
aspartic acid, or glutamine for asparagine, and the like. The term
"conservative variation" also includes the use of a substituted
amino acid in place of an unsubstituted parent amino acid, provided
that antibodies raised to the substituted polypeptide also
immunoreact with the unsubstituted polypeptide. Conservative
substitutions typically include the substitution of one amino acid
for another with similar characteristics, e.g., substitutions
within the following groups: valine, glycine; glycine, alanine;
valine, isoleucine, leticine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine;
phenylalanine, and tyrosine.
[0029] Naturally occurring proteins identified and/or appreciated
herein to be morphogenic proteins form a distinct subgroup within
the loose evolutionary grouping of sequence-related proteins known
as the TGF-.beta. superfamily or supergene family. The naturally
occurring morphogens share substantial amino acid sequence homology
in their C-terminal regions (domains). Morphogenic proteins
comprise a pair of polypeptides with amino acid sequences each
comprising a sequence that shares a defined relationship with an
amino acid sequence of a reference morphogen. Herein, preferred
morphogenic polypeptides share a defined relationship with a
sequence present in active human OP-1. However, any one or more of
the naturally occurring or biosynthetic sequences disclosed herein
similarly could be used as a reference sequence. Preferred
osteogenic polypeptides share a defined relationship with at least
the C-terminal six cysteine domain of human OP-1. Preferably,
morphogenic polypeptides share a defined relationship with at least
the C-terminal seven cysteine domain of human OP-1. That is,
preferred polypeptides in a dimeric protein with morphogenic
activity each comprise a sequence that corresponds to a reference
sequence or is functionally equivalent thereto.
[0030] Functionally equivalent sequences include functionally
equivalent arrangements of cysteine residues disposed within the
reference sequence, including amino acid insertions or deletions
which alter the linear arrangement of these cysteines, but do not
materially impair their relationship in the folded structure of the
dimeric morphogen protein, including their ability to form such
intra- or inter-chain disulfide bonds as may be necessary for
morphogenic activity. Functionally equivalent sequences further
include those wherein one or more amino acid residues differs from
the corresponding residue of a reference sequence, e.g., the
C-terminal seven cysteine domain (also referred to herein as the
conserved seven cysteine skeleton) of human OP-1, provided that
this difference does not destroy morphogenic activity. Accordingly,
conservative substitutions of corresponding amino acids in the
reference sequence are preferred. Amino acid residues that are
conservative substitutions for corresponding residues in a
reference sequence are those that are physically or functionally
similar to the corresponding reference residues, e.g., that have
similar size, shape, electric charge, chemical properties including
the ability to form covalent or hydrogen bonds, or the like.
Particularly preferred conservative substitutions are those
fulfilling the criteria defined for an accepted point mutation in
Dayhoff et al. (1978), 5 Atlas of Protein Sequence and Structure,
Suppl. 3, ch. 22 (pp.354-352), Natl. Biomed. Res. Found.,
Washington, D.C. 20007, the teachings of which are incorporated by
reference herein.
[0031] Publications disclosing these sequences, as well as their
chemical and physical properties, include: OP-1 and OP-2: U.S. Pat.
No. 5,011,691, U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO
J. 9: 2085-2093; OP-3: WO94/10203 (PCT US93/10520); BMP2, BMP3,
BMP4: WO88/00205, Wozney et al. (1988) Science 242: 1528-1534);
BMP5 and BMP6: Celeste et al. (1991) PNAS 87: 9843-9847; Vgr-1:
Lyons et al. (1989) PNAS 86: 4554-4558; DPP: Padgett et al. (1987)
Nature 325: 81-84; Vg-1: Weeks (1987) Cell 51: 861-867; BMP-9:
WO95/33830 (PCT/US95/07084); BMP10: WO94/26893 (PCT/US94/05290);
BMP-11: WO94/26892 (PCT/US94/105288); BMP12: WO95/16035
PCT/US94/14030); BMP-13: WO95/16035 (PCT/US94/14030); GDF-1:
WO92/00382 PCT/US91/04096) and Lee et al. (1991) PNAS 88:
4250-4254; GDF-8: WO94/21681 (PCT/US94/03019); GDF-9: WO94/15966
(PCT/US94/00685); GDF-10: WO95/10539 (PCT/US94/11440); GDF-11:
WO96/01845 (PCT/US95/08543); BMP-15: WO96/36710 (PCT/US96/06540);
MP121: WO96/01316 (PCT/EP95/02552); GDF-5 (CDMP-1, MP52):
WO94/15949 (PCT/US94/00657) and WO96/14335 (PCT/US94/12814) and
WO93/16099 (PCT/EP93/00350); GDF-6 (CDMP-2, BMP13): WO95/01801
(PCT/US94/07762) and WO96/14335 and WO95/10635 (PCT/US94/14030);
GDF-7 (CDMP-3, BMP12): WO95/10802 (PCT/US94/07799) and WO95/10635
(PCT/US94/14030). In another embodiment, useful proteins include
biologically active biosynthetic constructs, including novel
biosynthetic morphogenic proteins and chimeric proteins designed
using sequences from two or more known morphogens. See also the
biosynthetic constructs disclosed in U.S. Pat. No. 5,011,691, the
disclosure of which is incorporated herein by reference (e.g.,
COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).
[0032] As earlier stated, morphogenic proteins contemplated herein
include those in which the amino acid sequences comprise a sequence
sharing at least 70% amino acid sequence homology or "similarity",
and preferably 80% homology or similarity, with a reference
morphogenic protein selected from the foregoing naturally occurring
proteins. Preferably, the reference protein is human OP-1, and the
reference sequence thereof is the C-terminal seven cysteine domain
present in osteogenically active forms of human OP-1. A polypeptide
suspected of being functionally equivalent to a reference morphogen
polypeptide is aligned therewith using the method of Needleman, et
al. (1970) J. Mol. Biol. 48:443-453, implemented conveniently by
computer programs such as the Align program (DNAstar, Inc.). As
noted above, internal gaps and amino acid insertions in the
candidate sequence are ignored for purposes of calculating the
defined relationship, conventionally expressed as a level of amino
acid sequence homology or identity, between the candidate and
reference sequences. "Amino acid sequence homology" is understood
herein to include both amino acid sequence identity and similarity.
Homologous sequences share identical and/or similar amino acid
residues, where similar residues are conservative substitutions
for, or "allowed point mutations" of, corresponding amino acid
residues in an aligned reference sequence. Thus, a candidate
polypeptide sequence that shares 70% amino acid homology with a
reference sequence is one in which any 70% of the aligned residues
are either identical to, or are conservative substitutions of, the
corresponding residues in a reference sequence. In a currently
preferred embodiment, the reference sequence is OP-1. Morphogenic
proteins useful herein accordingly include allelic, phylogenetic
counterpart and other variants of the preferred reference sequence,
whether naturally-occurring or biosynthetically produced (e.g.,
including "muteins" or "mutant proteins"), as well as novel members
of the general morphogenic family of proteins, including those set
forth and identified above. Certain particularly preferred
morphogenic polypeptides share at least 60% amino acid identity
with the preferred reference sequence of human OP-1, still more
preferably at least 65% amino acid identity therewith.
[0033] As noted above, certain currently preferred morphogenic
polypeptide sequences have greater than 60% identity, preferably
greater than 65% identity, with the amino acid sequence defining
the preferred reference sequence of hOP-1. These particularly
preferred sequences include allelic and phylogenetic counterpart
variants of the OP-1 and OP-2 proteins, including the Drosophila
60A protein. Accordingly, preferred morphogenic proteins include
active proteins comprising pairs of polypeptide chains within the
generic amino acid sequence herein referred to as "OPX" (see below
and SEQ ID NO: 4), which defines the seven cysteine skeleton. As
described therein, each Xaa at a given position independently is
selected from the residues occurring at the corresponding position
in the C-terminal sequence of mouse or human OP-1 or OP-2.
1 Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa
Asp Trp 1 5 10 15 Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys
Glu Gly Glu Cys Xaa Phe Pro 20 25 30 35 Leu Xaa Ser Xaa Met Asn Ala
Thr Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa 40 45 50 55 Xaa Xaa
Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala 60
65 70 Xaa Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa
Lys Xaa Arg 75 80 85 90 Asn Met Val Val Xaa Ala Cys Gly Cys His 95
100
[0034] wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or
Arg); Xaa at res: 11(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa
at res. 19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at res.
26=(Ala or Ser); Xaa at res. 35=(Ala or Ser); Xaa at res. 39=(Asn
or Asp); Xaa at res. 41=(Tyr or Cys); Xaa at res. 50=(Val or Leu);
Xaa at res. 52=(Ser or Thr); Xaa at res. 56=(Phe or Leu); Xaa at
res. 57=(Ile or Met); Xaa at res. 58=(Asn or Lys); Xaa at res.
60=(Glu, Asp or Asn); Xaa at res. 61=(Thr, Ala or Val); Xaa at res.
65=(Pro or Ala); Xaa at res. 71=(Gln or Lys); Xaa at res. 73=(Asn
or Ser); Xaa at res. 75=(Ile or Thr); Xaa at res. 80=(Phe or Tyr);
Xaa at res. 82=(Asp or Ser); Xaa at res. 84=(Ser or Asn); Xaa at
res. 89=(Lys or Arg); Xaa at res. 91=(Tyr or His); and Xaa at res.
97=(Arg or Lys).
[0035] In yet other preferred embodiments, morphogenic protein as
contemplated herein can be further defined by a generic amino acid
sequence. For example, SEQ. ID No. 5 and No. 6 disclosed herein
represent composite amino acid sequences of the following proteins:
human OP-1, human OP-2, human OP-3, human BMP-2, human BMP-3, human
BMP4, human BMP-5, human BMP-6, human BMP-8, human BMP-9, human
BMP10, human BMP-11, Drosophila 60A. Xenopus Vg-1, sea urchin
UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouse GDF-6,
human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouse
GDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN, dpp, Drosophila
SCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse
GDF-10, human GDF-11, mouse GDF-11, human BMP-15, and rat BMP3b.
SEQ. ID NO: 5 accommodates the C-terminal six cysteine skeleton
and, SEQ. ID NO: 6 accommodates the seven cysteine skeleton.
2 SEQ. ID NO: 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly
Xaa Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 50 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa 55 60 65 Xaa Xaa
Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 70 75
80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 85 90 95
[0036] wherein each Xaa is independently selected film a group of
one or more specified amino acids defined as follows: "Res. " means
"residue" and Xaa at res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr,
Phe, His, Arg, Thr, Lys, Gln, Val or Glu); Xaa at res. 3=(Val, Ile,
Leu or Asp); Xaa at res. 4=(Ser, Asp, Glu, Asn or Phe); Xaa at res.
5=(Phe or Glu); Xaa at res. 6=(Arg, Gln, Lys, Ser, Glu, Ala or
Asn); Xaa at res. 7=(Asp, Glu, Leu, Ala or Gln); Xaa at res.
8=(Leu, Val, Met, Ile or Phe); Xaa at res. 9=(Gly, His or Lys); Xaa
at res. 10=(Trp or Met); Xaa at res. 11=(Gln, Leu, His, Glu, Asn,
Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser, Lys, Arg, Glu or
His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ile or Val); Xaa
at res. 15=(Ile or Val), Xaa at res. 16=(Ala, Ser, Tyr or Trp); Xaa
at res. 18=(Glu, Lys, Gin, Met, Pro, Leu, Arg, His or Lys); Xaa at
res. 19=(Gly, Glu, Asp, Lys, Ser, Gin, Arg or Phe); Xaa at res.
20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gin, His, Glu,
Asp, Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at
res. 23=(Tyr, Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu,
Phe or Arg); Xaa at res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys,
Arg, Gin or Gly); Xaa at res. 28=(Glu, Asp, Leu, Val, Lys, Gly,
Thr, Ala or Gin); Xaa at res. 30=(Ala, Ser, Ile, Asn, Pro, Glu,
Asp, Phe, Gin or Leu); Xaa at res. 31=(Phe, Tyr, Leu, Asn, Gly or
Arg); Xaa at res. 32=(Pro, Ser, Ala or Val); Xaa at res. 33=(Leu,
Met, Glu, Phe or Val); Xaa at res. 34=(Asn, Asp, Thr, Gly, Ala,
Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu, Asp, Thr, Leu,
Lys, Gin or His); Xaa at res. 36=(Tyr, His, Cys, Ile, Arg, Asp,
Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe, Val, Gly
or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly, Met,
Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa at
res. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys,
Val, Thr or Gin); Xaa at res. 42=(His, Tyr or Lys); Xaa at res.
43=(Ala, Thr, Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr,
Met or Pro); Xaa at res. 45=(Val, Leu, Met, Ile or His); Xaa at
res. 46=(Gin, Arg or Thr); Xaa at res. 47=(Thr, Ser, Ala, Asn or
His); Xaa at res. 48=(Leu, Asn or Ile); Xaa at res. 49=(Val, Met,
Leu, Pro or Ile); Xaa at res. 50=(His, Asn, Arg, Lys, Tyr or Gin);
Xaa at res. 51=(Phe, Leu, Ser, Asn, Met, Ala, Arg, Glu, Gly or
Gln); Xaa at res. 52=(Ile, Met, Leu, Val, Lys, Gin, Ala or Tyr);
Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala, Gln, Gly, Leu or
Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaa at res.
55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His); Xaa
at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg);
Xaa at res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res.
58=(Pro, Gly, Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala,
Ser, Glu, Arg or Gly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser);
Xaa at res. 61=(Cys, Val or Ser); Xaa at res. 63=(Ala, Val or Thr);
Xaa at res. 65=(Thr, Ala, Glu, Val, Gly, Asp or Tyr); Xaa at res.
66=(Gln, Lys, Glu, Arg or Val); Xaa at res. 67=(Leu, Met, Thr or
Tyr); Xaa at res. 68=(Asn, Ser, Gly, Thr, Asp, Glu, Lys or Val);
Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaa at res. 70=(Ile, Thr,
Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr, Asn or Gly); Xaa
at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr, Phe, Arg,
Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile, Lys, Gln
or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.
77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res.
78=(Ser, Asn, Asp, Tyr, Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa
at res. 79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at
res. 80=(Asn, Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at
res. 81=(Val, Ile, Thr or Ala); Xaa at res. 82=(Ile, Asn, Val, Leu,
Tyr, Asp or Ala); Xaa at res. 83=(Leu, Tyr, Lys or Ile); Xaa at
res. 84=(Lys, Arg, Asn, Tyr, Phe, Thr, Glu or Gly); Xaa at res.
85=(Lys, Arg, His, Gln, Asn, Glu or Val); Xaa at res. 86=(Tyr, His,
Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln, Pro or Lys); Xaa at
res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa at res. 89=(Met or
Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys); Xaa at res
91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gin, Asp, Glu, Val, Ala,
Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr); Xaa
at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu or
Ser). Further, after res. 53 in rBMP3b and mGDF-10 there is an Ile;
after res. 54 in GDF-1 there is a T; after res. 54 in BMP3 there is
a V; after res. 78 in BMP-8 and Dorsalin there is a G; after res.
37 in hGDF-1 there is Pro, Gly, Gly, Pro.
[0037] SEQ ID NO: 6 includes all of SEQ ID NO: 5 and in addition
includes the following sequence (SEQ ID NO: 7) at its
N-terminus:
3 Cys Xaa Xaa Xaa Xaa SEQ ID NO:7 1 5
[0038] Accordingly, beginning with residue 6, each "Xaa" in SEQ. ID
No. 6 is a specified amino acid defined as for SEQ. ID No. 5, with
the distinction that each residue number described for SEQ. ID No.
5 is shifted by five in SEQ. ID No. 6. Thus, "Xaa at res. 1=(Tyr,
Phe, His, Arg, Thr, Lys, Gln, Val or Glu)" in SEQ. ID No. 5 refers
to Xaa at res. 6 in SEQ. ID No. 6. In SEQ. ID No. 6, Xaa at res.
2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa at res. 3=(Lys,
Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln,
Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr,
His, Arg, Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).
[0039] Still further, morphogenically active proteins have
polypeptide chains with amino acid sequences comprising a sequence
encoded by a nucleic acid that hybridizes, under low, medium or
high stringency hybridization conditions, to DNA or RNA encoding
reference morphogen sequences, e.g., C-terminal sequences defining
the conserved seven cysteine domains of OP-1, OP-2, BMP2, BMP4,
BMP5, BMP6, 60A, GDF3, GDF6, GDF7 and the like. As used herein,
high stringency hybridization conditions are defined as
hybridization according to known techniques in 40% fornamide,
5.times.SSPE, 5.times. Denhardt's Solution, and 0.1% SDS at
37.degree. C. overnight, and washing in 0.1.times.SSPE, 0.1% SDS at
50.degree. C. Standard stringency conditions are well characterized
in commercially available, standard molecular cloning texts. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleolide Synthesis (M. J. Gait ed., 1984): Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); and
B. Perbal, A Practical Guide To Molecular Cloning (1984).
[0040] As used herein, "gene expression" is understood to refer to
the production of a gene product encoded by a DNA sequence of
interest, including the transcription of the DNA sequence and/or
translation of the mRNA transcript. It is understood that gene
expression in the context of the present invention is not
necessarily a consequence of direct interaction of specific
proteins or factors with specific DNA sequences of the present
invention, but that the reporter gene product is produced as a
consequence of an inductive effect that is mediated or influenced
by the presence and/or expression of Pax genes in the cells of the
invention.
[0041] As used herein, "operative association" relates to a fusion
of the described non-coding DNA sequences with a reporter gene in
such a reading frame as to be co-transcribed, or at such a relative
positioning as to be competent to modulate expression of the
reporter gene.
[0042] As used herein, "vector" is understood to mean any nucleic
acid comprising a nucleotide sequence of interest and competent to
be incorporated into a host cell and capable of functional
transcription. Such vectors include linear or circular nucleic
acids, plasmids, phagemids, cosmids, YACs (yeast artificial
chromosomes) and the like. Vector as used herein can be used to
construct a test cell, or construct DNAs, cells and/or viruses
suitable for gene therapy applications.
[0043] As used herein, "non-coding sequence" or "non-coding DNA"
includes DNA sequences that are not transcribed into RNA sequence,
and/or RNA sequences that are not translated into protein. This
category of "non-coding sequence" has been defined for ease of
reference in the application, and includes sequences occurring 5'
to the ATG site of the OP-1 gene at nucleotide 3318 of SEQ. ID No.
1. Further, as used herein "5' non-coding sequence" or "5'
non-coding DNA" define a gene locus upstream of or 5' to the
translation start site of a reporter gene. In contrast, coding
sequence means a translated and/or transcribed DNA sequence
encoding a reporter gene or morphogenic protein as defined
herein.
[0044] As used herein, an "OP-1-specific" or "morphogen-specific"
non-coding sequence is understood to define a non-coding sequence
that lies contiguous to an OP-1-specific or morphogen specific
coding sequence at a gene locus under naturally-occurring
conditions.
[0045] "Responsive," as defined herein, is not limited to direct
interaction between elements of the invention (e.g. Pax responsive
OP-1 modulating element and Pax gene products) but includes
indirect effects on gene transcription or translation, for example,
that are mediated or influenced by the Pax gene products.
[0046] As used herein, a "Pax responsive OP-1 modulating element"
is a DNA element that is responsive to the presence of a Pax gene
and/or expression of a Pax gene product. That is, expression of a
gene operatively associated therewith is modulated when present in
a cell together with DNA encoding a Pax gene. For example, when a
reporter gene and Pax gene are co-transfected into a host cell,
reporter gene expression is modulated by the presence and/or
expression of an endogenous Pax gene. As discussed herein, it is
contemplated that a Pax responsive modulating element(s) can be
located in the 5' non-coding sequences of any morphogen as defined
herein. Thus, for purposes of the present invention, a Pax
responsive morphogen modulating element comprises at least all of
the generic features (structural, functional, chemical) associated
with a Pax responsive OP-1 modulating element, and further includes
allelic, species and mutant variants thereof. A "Pax gene product"
is a nucleic acid or protein product encoded by a gene which is a
member of the Pax family of genes.
[0047] "Pax genes" encode a family of developmentally regulated
transcription factors that have been implicated in a number of
human and murine congenital disorders, as well as in tumorigenesis.
These genes, also characterized as "paired box genes," are defined
by the presence of an evolutionarily conserved DNA binding domain,
termed the "paired dornain". As demonstrated below in Example 2,
Pax genes and/or their expression and/or transcription products are
morphogen modulators as that group of compounds is defined
herein.
[0048] Paired box genes were first identified in Drosophila as a
family of related genes encoding a 128-amino acid DNA binding
domain, and are expressed in temporally and spatially restricted
patterns during development The phenotypes associated with Pax gene
mutations demonstrate that these gene products are critical during
organogenesis. Missense mutation within the paired domain of Pax
genes have been associated with congenital disorders in both mouse
and man. Also, Pax genes have been demonstrated to have oncogenic
potential, and, for example, a translocation involving the paired
box portion of Pax 3 has been associated with a human tumor.
[0049] The consensus binding sequences for Pax 6 and Pax 2 paired
domains are very similar. The Pax 6 consensus spans 20 base pairs
and shares a central 10-base pair region of homology with the Pax 2
consensus consisting of TCACGC-TGA, where the dash indicates a
nucleotide difference between the two sequences in this central
base pair region (Epstein et al. (1994) J. Biol. Chem.
269:8355-8361, the disclosure of which is incorporated by reference
herein).
[0050] The respective purified paired domain proteins exhibit high
affinity for their respective consensus sequences. The Pax-6 paired
domain has been shown to bind to this consensus sequence as a
monomer. The paired domain, when binding to such a DNA sequence,
adopts an .alpha.-helical conformation and contacts residues within
a large span of the DNA molecule.
[0051] As used herein, a "Pax 2 consensus binding sequence" or "Pax
2 consensus binding element" is a nucleotide sequence which has
been shown to be bound by the DNA binding protein Pax 2. The
consensus sequence of the Pax 2 binding site has been determined by
homology to be: G T C A C/T G C G/A T G A, as depicted in SEQ. ID
No. 3. (Epstein et al. (1994) J. Biol. Chem. 269:8355-8361). A "Pax
6 consensus binding sequence" or "Pax 6 consensus binding element"
is a nucleotide sequence which has been shown to be bound by the
DNA binding protein Pax 6.. The consensus sequence of the Pax 6
binding site has been determined by homology to be T T C A C G C
A/T T G/C A N T G/T A/C N T/C, as depicted in SEQ. ID No. 2 where
N=A, G, C or T. (Epstein et al. (1994) J. Biol. Chem.
269:8355-8361.) Another suitable consensus sequence for purposes of
the present invention is A-TTCACGCATGA-T or TTCACGCATGA, wherein
the dashes indicate that any nucleotide can reside at that
locus.
[0052] "Pax 2 or Pax 6 mediated transcription or expression" means
transcription or expression of a reporter gene operatively
associated with a Pax responsive modulating element, or functional
equivalent thereof, as described elsewhere herein. Such
transcription or expression occurs in the presence of Pax genes
and/or Pax gene expression products, but their presence is not
required. "Pax 2 or Pax 6 mediated transcription or expression" can
involve, for example, the direct physical interaction of Pax 2 or
Pax 6 gene products with the Pax 2 or Pax 6 consensus binding
sequences, as defined herein above. Alternatively, "Pax mediated
transcription or expression" can be modulated by compounds that can
mimic the transcription or expression events otherwise associated
with Pax family proteins. Such compounds are contemplated herein
generally as morphogen modulators, and more specifically as Pax
analogs.
[0053] As discussed elsewhere herein, "allelic, species and other
sequence variants thereof" includes point mutations, insertions and
deletions such as those that are naturally occurring or which can
be genetically engineered into an OP-1 non-coding DNA sequence
using routine methods and which do not substantially affect the
regulation of a reporter gene by the OP-1 non-coding sequence. For
example, one of ordinary skill in the art can use site directed
mutagenesis to modify, as by deletion, for example, one or more of
the OP-1 non-coding sequences described herein without
substantially affecting the regulation of OP-1 or a reporter gene
by the modification. Such modifications are considered to be within
the scope of the disclosure provided herein.
[0054] As discussed elsewhere herein, "analog" or "variant" is
intended to mean a DNA or a protein which mimics or performs
similar functions relative to a naturally-occurring or
representative DNA or protein. For example, a DNA that is
responsive to Pax 2 or Pax 6 in the same manner as those described
herein, yet are structurally different therefrom, are considered
DNA analogs or DNA variants within the scope of this invention.
Similarly, any expression product which can modulate OP-1
expression in the same manner as Pax 2 and/or Pax 6 as disclosed
herein, are considered Pax 2 and/or Pax 2 analogs or variants
within the scope of this invention.
[0055] "Co-transfection" refers to the simultaneous or sequential
transfection of two or more vectors into a given cell.
[0056] Where a cell line is to be established, particularly where
the transfected DNA is to be incorporated into the cell's genome,
lines that can be immortalized are especially desirable. As used
herein, "immortalized" cell lines are viable for multiple passages
without significant reduction in growth rate or protein production.
It is contemplated that the cells of the present invention have
utility for cell mediated gene therapy, where cells producing high
levels of a reporter gene such as OP-1 are desired. It is further
contemplated that the Pax consensus binding sequences or other
useful sequences can be altered or adapted for viral mediated gene
therapy using routine methods.
[0057] The Pax genes and/or gene products as used herein are
capable of stimulating the transcription of a morphogen gene and
are herein also referred to as "morphogen stimulators", "morphogen
modulating compounds" or "OP-1 modulating compounds."
[0058] A "candidate compound" is any test substance that can be
used to treat the cells of the present invention in vitro, in vivo
or ex vivo to determine their usefulness as OP-1 modulating
compounds. Accordingly, and in keeping with the earlier definition,
a candidate compound which can modulate OP-1 expression in the
manner of Pax 2 and/or Pax 6 as disclosed herein, is also a
morphogen stimulator as well as a Pax 2 and/or Pax 6 analog.
Conversely, a candidate compound which acts contrary to Pax 2
and/or Pax 6 as disclosed herein, is a morphogen suppressor or Pax
2/Pax 6 antagonist.
The OP-1 Upstream Region Contains a Silencer of Gene Expression
[0059] The presence of a sequence within the OP-1 upstream region
that reduces OP-1 gene expression (a silencer fragment) was
identified in expression assays described below in Example 9. The
approximately 0.1 kb AatII-PvuII fragment (approximately
nucleotides 2606-2690 of SEQ ID NO: 1) reduces OP-1 expression by
approximately three fold (between approximately two fold and five
fold in the experiments described in Example 9).
[0060] In a preferred embodiment of the invention, expression of
OP-1 is increased by removing the silencer fragment from the OP-1
upstream region. In one embodiment, the silencer fragment is
removed from a plasmid or other recombinant construct containing
the OP-1 gene. In an alternative embodiment, a recombinant cell
line is made wherein one or both genomic copies of the OP-1 gene
are modified to remove the silencer fragment, using methods known
in the art.
[0061] In another embodiment of the invention, the silencer
fragment is used to modulate the expression of another (non-OP-1)
gene. One or more copies of the silencer fragment are inserted in
the promoter region of the gene in order to reduce expression. The
silencer fragment is inserted into the promoter region of a gene
that is contained in a plasmid, a recombinant vector, or in the
genome itself.
[0062] The silencer fragment contained within the AatII-PvuII
fragment can be more precisely identified by standard deletion
analysis of the fragment using the expression assay described in
Examples 2 and 9. Truncated silencer fragments, identified by a
further deletion analysis, are also useful in the invention.
[0063] A naturally occurring sequence variant of the silencer
fragment (or of a truncated silencer fragment), or a nucleic acid
which hybridizes to the silencer fragment (or to a truncated
siencer fragment) under high stringency hybridization conditions,
is also useful in the invention.
Exemplary Cells, Vectors, Reporter Genes and Assays For Use in
Screening Compounds Which Modulate OP-1 Gene Expression
[0064] A. Useful Cells
[0065] Any eukaryotic cell, including an immortalized cell line
suitable for long term culturing conditions, is contemplated to be
useful for the methods and cells of the invention. Useful cells
should be easy to transfect, are capable of stably maintaining
foreign DNA with an unrearranged sequence, and have the necessary
cellular components for efficient transcription and translation of
the protein, including any elements required for post-translational
modification and secretion, if necessary. Where the cell is to be
transfected with a non-dominating selection gene, the cell genotype
preferably is deficient for the endogenous selection gene.
Preferably, the cell line also has simple media composition
requirements, and rapid generation times. Useful cell lines are
mammalian cell lines, including myeloma, HeLa, fibroblast,
embryonic and various tissue cell lines, e.g., kidney, liver, lung
and the like. The cells may be derived from tissue or subcultured
from established cell lines. As used herein, "derived" means the
cells are from the cultured tissue itself or are a cell line whose
parent cells are of the tissue itself. Cell lines particularly
useful in practicing the present invention are, for example, Y79
human retinoblastoma cells, G401 human kidney cells, ROS human
osteoblastic cells, MCF-7 human mammary cancer cells and LLCBK1
porcine proximal tubule cells. A large number of cell lines now are
available through the American Type Culture Collection (Rockville,
Md.) or through the European Collection of Animal Cell Cultures
(Porton Down, Salisbury, SP4 0JG, U.K.)
[0066] Where the expression of a reporter gene that is controlled
by non-coding sequences of the morphogen OP-1 is to be analyzed,
particularly useful cells and cell lines are envisioned to include
eukaryotic, preferably mammalian cells of a tissue and cell type
known to express OP-1 and/or closely related proteins. See, for
example, Ozkaynak, et al. (1991), Biochem. Biophys. Res. Commun.
179: 116-123 for a detailed description of tissues known to express
OP-1. Such cells, include, without limitation, cells of uro-genital
cell origin, including kidney, bladder and ovary cells, lung,
liver, bone, nerve, mammary gland and cardiac cells, cells of
gonadal origin, cells of gastrointestinal origin, glial cells and
other cell lines known to express endogenous genes encoding
morphogenic proteins. Preferred cell lines are of epithelial
origin.
[0067] Cell cultures of kidney, adrenals, urinary bladder, brain,
or other organs, can be prepared as described widely in the
literature. For example, kidneys can be explanted from neonatal or
new born or young or adult rodents (mouse or rat) and used in organ
culture as whole or sliced (1-4 mm) tissues. Primary tissue
cultures and established cell lines, also derived from kidney,
adrenals, urinary, bladder, brain, mammary, or other tissues can be
established in multiwell plates (6 well or 24 well) according to
conventional cell culture techniques, and are cultured in the
absence or presence of serum for a period of time (1-7 days). Cells
can be cultured, for example, in DMEM containing 10% fetal calf
serum or in serum-deprived medium or in defined medium (e.g.,
containing insulin, transferrin, glucose, albumin, or other growth
factors). Test compounds are added to the cultured cells and OP-1
biosynthesis monitored and measured at various time points using
the methods described previously herein. Suitable cell lines
include cell lines that have been shown to contain high levels of
OP-1 mRNA, indicating that the OP-1 promoter is active in the
cells. Cells and their culture fluids are assayed using techniques
well known in the art, either for mRNA levels, using Northern blot
analysis and OP-1 mRNA specific probes, or for protein levels,
using OP-1 specific antibodies. For example, OP-1 protein can be
measured on a tissue section or cell directly using standard
immunofluorescence techniques or in culture fluids and body fluids
using a sandwich immunoassay. Other methods for detecting,
measuring and purifying proteins are well know in the art.
[0068] Alternatively, in one aspect the present invention may be
practiced in yeast cells such as S. cerevisiae to identify or
further characterize protein-protein interactions of the identified
intracellular OP-1 modulating factors of the present invention. For
example, the yeast two hybrid system, as described in Kalpana et
al. (1993) 90 Proc. Natl. Acad Sci. U.S.A. (22): 10593-10597 and
already well known to those skilled in the art, is useful for
delineating domains or critical residues for an interaction between
two proteins, for example, a protein X and a protein Y. Briefly,
hybrid genes are constructed for expression in yeast comprising (a)
a DNA binding domain fused to a protein X and (b) an activator
domain fused to a protein Y. Transcriptional activation in these
experiments is only restored when protein X interacts with protein
Y leading to close contact of both the binding domain and the
activation domain. Transcription activation is then monitored by a
reporter gene resident in the yeast strain.
[0069] B. Exemplary Vectors/Vector Construction Considerations
[0070] Useful vectors for use in the invention include, but are not
limited to plasmids, cosmids, phagemids, yeast artificial
chromosomes or other large vectors. Vectors that can be maintained
within the nucleus or integrated into the genome by homologous
recombination are also useful.
[0071] Selected portions of non-coding OP-1 sequence can be cloned
into a useful vector using standard molecular cloning techniques,
as exemplified below. Restriction endonuclease sites will be
utilized when possible, and can be engineered into the sequence
when needed. Restriction endonuclease sites can be engineered into
the non-coding sequence using the common techniques such as site
directed mutagenesis and PCR with primers including the desired
restriction endonuclease site.
[0072] Also envisioned is a nucleic acid construct comprising a
small fragment of 5' non-coding OP-1 sequence in combination with
additional conserved elements such as one or more Pax 6 binding
sequences and/or Pax 2 binding sequences in operative association
with a reporter gene.
[0073] A range of useful 5' non-coding fragments is provided
herein, and as will be apparent to those of ordinary skill in the
art, smaller fragments of OP-1 sequence also are useful. Such
smaller fragments can be identified by deleting bases from one or
both ends of the provided 5' non-coding fragments, using techniques
that are well known in the art and testing the truncated constructs
for their ability to modulate reporter gene expression. In this
way, the shortest modulating sequences can be identified.
[0074] C. Transfection Considerations
[0075] Any routine method for incorporating nucleic acids into
cells of interest is contemplated in the method of the invention.
For example, calcium phosphate (CaPO.sub.4), followed by glycerol
shock is a standard means used in the art for introducing vectors,
particularly plasmid DNA into mammalian cells. A representative
method is disclosed in Cockett et al., (1990) Biotechnology 8:
662-667, incorporated herein by reference. Other methods that may
be used include electroporation, protoplast fusion (particularly
useful in myeloma transfections), microinjections, lipofections and
DEAE-dextran mediated uptake. Methods for these procedures are
described in F. M. Ausubel, ed., Current Protocols in Molecular
Biology, John Wiley & Sons, New York (1989). An important
aspect of the invention is the DNA sequences with which the cell is
transfected, rather than the mechanical or chemical process by
which the DNA incorporation is accomplished.
[0076] As will be appreciated by those having skill in the art,
optimal DNA concentrations per transfection and other standard
conditions will vary according to the transfection protocol. For
calcium phosphate transfection, for example, preferably 5-10 .mu.g
plasmid DNA per plasmid type is transfected. In addition, the DNA
to be transfected preferably is essentially free of contaminants
that may interfere with DNA incorporation. A standard means used in
the art for purifying DNA is by ethidium bromide banding.
[0077] D. Exemplary Reporter Genes
[0078] Reporter genes are characterized as being easy to transfect
into a suitable host cell, easy to detect using an established
assay protocol, and genes whose expression can be tightly
regulated. Other reporter genes contemplated to have utility
include, without limitation, the luciferase gene, the Green
Fluorescent Protein (GFP) gene, human growth hormone, GAL4 and
.beta.-galactosidase.
[0079] A well-recognized reporter system is the firefly luciferase
reporter system. See, for example Gould, S. J., and Subramani, S.
(1988) Anal. Biochem., 7:404-408 for a description of the reporter
gene and general methodology. The luciferase assay is fast and has
increased sensitivity. Further, the half-life of luciferase protein
is short and therefore allows for accurate kinetic studies of
luciferase production. The system also is particularly useful in
bulk transfections or if the promoter of interest is weak. In this
assay transfected cells are grown under standard conditions, and
when cultured under assay conditions both ATP and the substrate
luciferin is added to the cell lysate. The enzyme luciferase
catalyzes a rapid, ATP dependent oxidation of the substrate which
then emits light. The total light output is measured using a
luminometer according to manufacturer's instructions (e.g.,
Promega) and is proportional to the amount of luciferase present
over a wide range of enzyme concentrations. For example, a vector
such as pGL2-LUC (Promega) is particularly useful.
[0080] A second well-known reporter system is based on immunologic
detection of hGH, it is quick and easy to use. (Selden, R.,
Burke-Howie, K. Rowe, M. E., Goodman, H. M., and Moore, D. D.
(1986), Mol. Cell. Biol., 6:3173-3179 incorporated herein by
reference). hGH is assayed in the media, rather than in cell
extracts. This allows direct monitoring over by a single population
of transfected cells over time.
[0081] Additional useful reporter genes are any well characterized
genes the expression of which is readily assayed, and examples of
such reporter genes can be found in, for example, F. A. Ausubel et
al., Eds., Current Protocols in Molecular Biology, John Wiley &
Sons, New York, (1989). As will be appreciated by those having
ordinary skill in the art, the listed reporter genes are only a few
of the possible reporter genes, and it is only for ease of
description that all available reporter genes are not listed. It
will be apparent to those of ordinary skill in the art that genes
encoding other detectable proteins of interest, such as the gene
encoding human OP-1 shown in SEQ. ID No. 1, or other morphogen
whose enhanced expression is desirable, is also within the scope of
a suitable reporter gene.
[0082] As indicated above and as will be appreciated by those
having ordinary skill in the art, particular details of the
conventional means for transfection, expression, and assay of
recombinant genes are well documented and are understood by those
having ordinary skill in the art. The instant invention enables and
discloses vectors, cells and a method for screening compounds to
determine the capability of compounds to modulate the expression of
OP-1 via the non-coding sequences of the OP-1 genomic DNA.
[0083] Further details on the various technical aspects of each of
the steps used in recombinant production of foreign genes in
mammalian expression systems can be found in a number of texts and
laboratory manuals in the art, such as, for example, F. M. Ausubel
et al., Ed., Current Protocols in Molecular Biology, John Wiley
& Sons, New York, (1989).
[0084] In view of this disclosure and the examples provided below,
a method for identifying molecules which can affect OP-1 expression
in a particular cell type in vivo now is provided.
EXAMPLE 1
Cloning of Human OP-1 Gene Non-coding Sequences
[0085] Human OP-1 upstream non-coding sequence was obtained by
screening the human genomic library, HL1067J. (Clontech). The
library was screened by an initial plating of 750,000 plaques
(approximately 50,000 plaques/plate). Hybridizations were done in
40% formamide, 5.times.SSPE, 5.times. Denhardt's solution, and 0.1%
SDS at 37.degree. C. using a .sup.32P-labeled probe made from a
human 0.47 kb EcoRI OP-1 cDNA fragment containing mainly 5'
non-coding and exon 1 sequences. Nonspecific counts were removed in
0.1.times.SSPE, 0.1% SDS by shaking at 50.degree. C.
[0086] A 7 kb EcoRI fragment from the human genomic clone, lambda
3, was isolated and sequenced and contained 5 kb of OP-1 upstream
non-coding sequence.
[0087] All sequencing was done according to Sanger et al. (1977)
Proc. Natl. Acad. Sci. 74:5463-5467, using exonuclease III-mediated
unidirectional deletion (Ozkaynak et al., (1987) BioTechniques,
5:770-773), subcloning of restriction fragments, and synthetic
primers. Compressions were resolved by performing the reactions at
70.degree. C. with Taq polymerase and using 7-deaza-GTP (U.S.
Biochemical Corp., Cleveland, Ohio).
[0088] As will be discussed in more detail below, the 1-3317 region
of SEQ. ID No. 1 contains a plurality of conserved DNA sequences
which share homology with Pax homeobox consensus sequences of
Epstein et al. (1994) J. Biol. Chem. 269:8355-8361, and have been
identified and designated herein as Pax responsive OP-1 modulating
elements, comprising approximately nucleotides 108-121, 139-154,
157-167, 365-378, 491-503, 497-511, 737-747, 891-903, 994-1006,
1123-1140, 1144-1161, 1285-1297, 598-613, 1750-1762, 2001-2023,
2365-2378, 2931-2944 of SEQ. ID No. 1. Particularly preferred Pax 6
responsive OP-1 modulating elements reside at approximately
nucleotides 108-121, 139-154, 365-378, 497-511, 598-613, 1750-1762,
2365-2378 and 2931-2944. Other preferred Pax 6 elements include
approximately 157-167, 1123-1140, 1144-1161, 1285-1297 and
2001-2023. Particularly preferred Pax 2 responsive OP-1 modulating
elements reside at approximately nucleotides 491-503, 737-747,
891-903 and 994-1006.
[0089] Still other preferred Pax 6 responsive OP-1 modulating
elements reside at approximately 104-120, 105-121, 142-158,
362-378, 497-513, 1750-1766, 2362-2378, 2928-2944, 1128-1144,
1125-1141, 1143-1159, 2000-2016, 2003-20019, 2007-2023, 601-617,
595-611, and 1746-1762. Still other preferred Pax 2 responsive OP-1
modulating elements reside at approximately 493-503, 891-901 and
996-1006.
[0090] The transcription initiation site for the human OP-1 gene is
at base 2790 of SEQ. ID No. 1. The OP-1 protein translation
initiation site is nucleotide 3318 of SEQ. ID No. 1.
[0091] As described above, osteogenic protein-1 plays a critical
role in modulating mesenchymal differentiation and inducing the
process of cartilage and bone formation. It is also required for
kidney development and is shown to prevent kidney damage from
ischemia/reperfusion injury in rats. As presented below in Example
2, functional analysis of the human OP-1 gene promoter has been
carried out. The OP-1 promoter and 5' truncated versions of it were
placed upstream of the luciferase coding region which served as the
reporter gene. In certain embodiments, the results of the
luciferase assays after transient transfection of these constructs
into a human kidney cell line, G401, indicate that this upstream
sequence has promoter activity. This activity resides about 300 to
700 nucleotides upstream of the first ATG of the OP-1 gene. This
promoter activity is constitutive and is not influenced by
transactivating agents, e.g., Pax 2 and Pax 6 (see FIG. 1B) which
are involved in epithelial/mesenchymal interactions during tissue
morphogenesis. In other embodiments drawn to the region between 700
to 3300 nucleotides upstream of the OP-1 gene, however, the data
indicate the presence of potential silencer elements of the
promoter. Moreover, this region of the promoter can be influenced
by a host of factors, including Pax 2, Pax 6 and Retinoic Acid, as
illustrated in Example 2.
[0092] Both transient and permanent G401 cell lines containing the
below-described constructs have been isolated and tested. Other
host cell lines including Human Retinoblastoma Y79, Rat
Osteosarcoma ROS and Porcine Proximal Tubule LLCPK1 have been
tested in transient transfection assays. These data are expected to
illustrate similar Pax-mediated regulatory considerations and
tissue specificity, thereby permitting even further identification
of tissue- and developmental-specific morphogen modulators.
EXAMPLE 2
Analysis of OP-1 Gene Expression
[0093] In one aspect, this invention presents a method in which
OP-1 non-coding sequences are assayed while in operative
association with a reporter gene and modulator compounds such as
Pax 2 or Pax 6 or analogs thereof, are thereby tested for their
influence on the expression of OP-1. For example, non-coding
sequences which are involved in the modulation of OP-1 expression
via Pax gene products or functional equivalents or analogs thereof
will be identified by (1) transfecting a cell with one or more Pax
protein expression vectors and an expression vector comprising OP-1
non-coding sequences in operative association with a reporter gene;
(2) culturing the transfectants with one or more candidate
compounds; (3) measuring the level of reporter gene expression; and
(4) comparing this level of expression to the level of reporter
gene expression in the absence of the compound(s). Alternatively,
non-coding sequences can be assayed for their responsiveness to
compounds which can mimic Pax 2 and/or Pax 6, i.e., Pax 2 and/or
Pax 6 analogs. The protocol of the present invention is based on a
procedure for identifying compounds which alter, either directly or
indirectly, endogenous levels of morphogen expression.
[0094] It is also contemplated that candidate compounds may be
administered in vivo to modulate the level of an endogenous
protein, such as OP-1. This can be accomplished by detection of the
expression product either at the protein or RNA level.
[0095] Cultured cells are transfected with portions of OP-1
non-coding sequences in operative association with a reporter gene,
and such transfected cells are maintained with the vector remaining
as a plasmid in the cell nucleus, or the vector can be integrated
into the host cell genome, preferably at the OP-1 genomic locus.
Cell samples for testing the level of reporter gene expression are
collected periodically and evaluated for reporter gene expression
using the appropriate assay for the given reporter gene as
indicated in the section describing reporter gene assays, or,
alternatively, a portion of the cell culture itself can be
collected periodically and used to prepare polyA(+) RNA for mRNA
analysis. For example, to ascertain the particular time point at
which OP-1 is produced following treatment with a candidate
morphogen, cells treated with the candidate OP-1 modulating
compound, cells treated with compound are collected periodically
and evaluated for OP-1 production, as described above. To monitor
de novo OP-1 synthesis, some cultures are labeled according to
conventional procedures with an
.sup.35S-methionine/.sup.35S-cysteine mixture for 6-24 hours and
then evaluated to quantitate OP-1 synthesis by conventional
immunoassay methods. Alternatively, anti-OP-1 antibodies can be
labeled and incubated with the cells or cell lysates, and the bound
complexes detected and quantitated by conventional means, such as
those described herein above. Tissues can also be examined directly
for the synthesis of OP-1 mRNA using the art-recognized technique
of in situ hybridization.
[0096] Once candidate compounds are identified, they can be
produced in reasonable, useful quantities using standard
methodologies known in the art. Amino acid-based molecules can be
encoded by synthetic nucleic acid molecules, and expressed in a
recombinant expression system as described herein above or in the
art. Alternatively, such molecules can be chemically synthesized,
e.g., by means of an automated peptide synthesizer, for example.
Non-amino acid-based molecules can be produced by standard organic
chemical synthesis procedures.
[0097] Morphogen agonists are anticipated to have utility in any
application where tissue morphogenesis is desired, such as in the
regeneration of damaged tissue resulting from mechanical or
chemical trauma, degenerative diseases, tissue destruction
resulting from chronic inflammation, cirrhosis, inflammatory
diseases, cancer and the like, and in the regeneration of tissues,
organs and limbs. Morphogen antagonists are envisioned to have
utility in applications where tissue morphogenesis is to be limited
as, for example, in the treatment of malignant transformations
including, but not limited to, osteosarcomas, Paget's disease, and
fibrodysplasia ossificans progressiva (See, for example, Roush
(1996) Science 273:1170). The ability to detect OP-1 protein in
solution provides a valuable tool for diagnostic assays, allowing
us to monitor the levels of OP-1 free in the body, e.g., in serum,
urine, spinal or peritoneal fluid, breast exudate, and other body
fluids in order to assess the presence of OP-1 modulating activity.
The present invention therefor provides a means of identifying
naturally occurring or synthetic proteins or other factors or drugs
that have OP-1 modulating activities. Moreover, compounds that
induce the expression of Pax 2 or Pax 6 are also particularly
useful.
[0098] OP-1 is expressed in a variety of different cell types,
including renal, bone, lung, heart, uterine, cardiac and neural
tissue. Candidate compounds can be identified which have a
modulating effect on cells of one tissue type but not another,
and/or wherein the effect is modulated differently in the different
cells. The assay described below can be used to evaluate the effect
of a candidate compound in a particular cell type known to express
OP-1 under physiological conditions.
[0099] The present invention therefor also provides a test cell or
cell line which expresses an exogenously introduced OP-1 protein,
which is responsive to morphogen modulators such as Pax 2 and/or
Pax 6 or analogs thereof as contemplated herein. Said cell or cell
line can be also used as described herein, to replace cell lines
that produce OP-1 endogenously, for the screening assays described
herein above. Further, the invention provides a test cell that is
transfected with DNA encoding Pax gene products (e.g., Pax 2 and/or
Pax 6) for the modulation of endogenous or exogenous OP-1 gene
expression.
[0100] While a readily assayable, well characterized, non OP-1
reporter gene is preferred in the screening method disclosed
herein, as will be appreciated by those having ordinary skill in
the art, OP-1 coding sequence also may be used in the method of the
invention providing an improved method for the production of high
levels of OP-1, exploiting the high level of induction of gene
expression of the present invention. The OP-1 expression preferably
is determined by an immunoassay or by Northern or dot blot or other
means for measuring mRNA transcript. See, for example, WO 95/11983,
published May 4, 1995 for a detailed description on assaying
changes in OP-1 levels in a cell or fluid. Further, the cells of
the present invention can be used in the analysis of the
transcription factors, for example, DNA-protein and/or
protein-protein interactions involved in OP-1 transcription
regulation or that of other morphogens.
[0101] Provided below is an exemplary protocol for carrying out the
method of the invention, using the luciferase gene as the reporter
gene and a mammalian cell line known to express OP-1. The example
is non-limiting, and other cells, reporter genes and morphogen
non-coding sequences such as, but not limited to, an OP-1
non-coding sequence, are envisioned.
[0102] The OP-1 genomic DNA nucleotides 1-3317 of SEQ. ID No. 1 was
used to prepare a series of deletion constructs carrying the
luciferase reporter gene and portions of the OP-1 gene non-coding
region (FIG. 1A). The pGL2-Basic plasmid comprising a nucleotide
sequence encoding the detectable enzyme luciferase (Promega,
Madison Wis.) was employed as the basic vector. The OP-1 promoter
sequence (corresponding to nucleotides 1 to 3305 of SEQ. ID No. 1)
was then inserted into the pGL2-Basic plasmid (construct pAS3.3,
FIG. 1A). Nucleotide +1 is 30 nucleotides downstream of the Hind
III site present in the OP-1 genomic sequence. There are therefore
an additional 30 base pairs of DNA sequence upstream of position +1
in the OP-1 promoter, comprising AAGCTTGATG CCTGCACAGT CAGCCCTCAG,
wherein AAGCTT corresponds to a Hind III site.
[0103] The genomic sequence was digested with Hind III and BamHI.
The BamHI site was introduced into the OP-1 promoter at nucleotides
3306 to 3311 of SEQ. ID No. 1 by site-directed mutagenesis
according to methods well known in the art using the following
reverse complement primer: 5'CAT CGC GCC GGA TCC ACG CGC TAC CCG
GGC 3' wherein GGATCC corresponds to a BamHI site.
[0104] Thus the mutagenesis generated the following change in the
OP-1 sequence:
4 native seq: 5' GCC CGG GTA GCG CGT AGA GCC GGC GCG ATG 3' altered
seq: 5' GCC CGG GTA GCG CGT GGA TCC GGC GCG ATG 3'
[0105] This resulted in the following changes in the OP-1 promoter
in construct pAS3.3: GCCCGGGTAG CGCGTGGATC TAAGTAAGCT TGGCATTCCG
GTACTGTTGG TAAAATG wherein the G represents nucleotide 3306 of SEQ.
ID No. 1 and wherein ATG corresponds to the luciferase ATG
(nucleotides 3318 to 3320 of SEQ. ID No. 1).
[0106] Therefore, the native OP-1 promoter sequences stop at
nucleotide 3305, nucleotide 3306 results from an A to G nucleotide
change, and nucleotides 3307 to 3345 are derived from the
pGL2-Basic vector. Thus, 3335 nucleotides (30 nucleotides upstream
of nucleotide 1 up to 3305 of SEQ. ID No. 1) of cloned 5'
non-coding sequence of the OP-1 gene was placed in
transcriptionally operative association with the luciferase
reporter gene.
[0107] Serial 5' deletions (FIG. 1A) were constructed by digestion
of the pAS3.3 construct with a series of restriction enzymes
followed by religation of the plasmid according to well-known
cloning techniques. For example, the pAS3.3 vector was cleaved with
BgIII (present in the pGL2-Basic vector and in the OP-1 promoter)
and recircularized to obtain pAS2.5; the pAS3.3 vector was cleaved
with Kpnl (present in the pGL2-Basic vector and in the OP-1
promoter) and recircularized to obtain pAS1.2; the pAS3.3 vector
was cleaved with XhoI (present in the pGL2-Basic vector) and SacII,
followed by a T4 DNA polymerase fill-in reaction and recircularized
to obtain pAS0.8; the pAS3.3 vector was cleaved with KpnI (present
in the pGL2-Basic vector) and AatII followed by a T4 DNA polymerase
fill-in reaction and recircularized to obtain pAS0.7; the pAS3.3
vector was cleaved with XhoI (present in the pGL2-Basic vector) and
PvuII followed by a T4 DNA polymerase fill-in reaction and
recircularized to obtain pAS0.6; the pAS3.3 vector was cleaved with
XhoI (present in the pGL2-Basic vector) and AvrII followed by a T4
DNA polymerase fill-in reaction and recircularized to obtain
pAS0.3; and the pAS3.3 vector was cleaved with KpnI (present in the
pGL2-Basic vector) and PstI followed by a T4 DNA polymerase fill-in
reaction and recircularized to obtain pAS0.1.
[0108] The vectors were transfected into G401 human kidney cells,
either alone or together with the cDNA for human Pax 2 and/or for
human Pax 6, previously cloned into a pCMV.beta. expression vector
(Clonetech), with the removal of the .beta.-galactosidase gene by
standard methods (Epstein, et al. (1994) J. Biol. Chem.
269:8355-8361), the disclosure of which is incorporated herein by
reference. Briefly, cells were plated in 60 mm petri dishes
(5.times.10.sup.5 cell/dish) in McCoy's 5A medium containing 10%
fetal bovine serum. Twenty-four hours later, the above-described
vectors were transfected into the cultured cells, using a
Lipofectamine (BRL/Gibco) method in serum-free medium (Optimem,
BRL) for 6 hours. Cells were transfected with (a) an OP-1 promoter
constructs alone; (b) an OP-1 promoter construct+the Pax 2
construct; (c) an OP-1 promoter construct+the Pax 6 construct; or
(d) an OP-1 promoter construct+the Pax 2 construct+the Pax 6
construct. Control transfected cells contained the pGL-Basic
plasmid without OP-1 non-coding sequences as a negative control and
pSV-Luc, which has an SV40 promoter, as a positive control.
Thereafter, transfected cells were cultured in complete media.
Seventy-two hours later, luciferase activity was measured using the
Promega Luciferase Assay System (Promega, Madison Wis.) and a
luminometer (DynaTech). The results are displayed in FIG. 1B and
discussed below.
[0109] The luciferase production by cells containing the pAS3.3
construct, comprising 3305 nucleotides upstream of the translation
start site of the OP-1 gene, was induced 2.4 fold by
co-transfection with the Pax 2 cDNA and 9.6 fold by co-transfection
with the Pax 6 cDNA (FIG. 1B). The triple transfection of both the
Pax 2 and Pax 6 expression vectors with the pAS3.3 construct
resulted in an additive 11.5 fold induction suggesting that Pax 2
and Pax 6 gene products have an additive effect on OP-1 gene
expression. Cells containing the 5' deletion mutants of the pAS3.3
construct were less responsive to the inductive effect of
co-transfection with Pax 2 or Pax 6 constructs. For example, the
pAS2.5 construct was only induced 1.7 fold by co-transfection with
the Pax 2 construct and this Pax 2 responsiveness was abolished by
further deletion of about 1.3 kb of 5' sequence, with no Pax 2
induction detected for the pAS1.2 construct or the remaining
constructs with further 5' deletions.
[0110] Pax 6 responsiveness was also lost by about 42% upon
deletion of 793 bases from the pAS3.3 construct A 4 to 5.5 fold
induction of luciferase production was demonstrated in the cells
comprising the Pax 6 construct co-transfected with pAS2.5 (+794 to
+3305), pAS1.2 (+2068 to +3305), pAS0.8 (+2501 to +3305), and
pAS0.7 (+2606 to +3305) of SEQ. ID No. 1 but this Pax 6
responsiveness was lost upon deletion of nucleotides +2606 to
+2690. This suggests that this 85 nucleotide region contains
preferred sequences necessary for OP-1 modulation by Pax gene
products or analogs thereof.
[0111] Constructs pAS3.3 and pAS2.5, comprising 3.3 kb 5'
non-coding region (1 to 3305 of SEQ. ID No. 1) and a 793 nucleotide
5' deletion thereof, respectively, produced less luciferase
constitutively than the other deletion mutants, suggesting the
presence of negative regulatory elements in the 5' non-coding
region comprising nucleotides 1 to 2067 (FIG. 1B).
[0112] The complete DNA sequence of the 5' non-coding upstream OP-1
gene (1 to 3317 of SEQ. ID No. 1) was analyzed using a string
search program to identify putative Pax 2 or Pax 6 binding motifs,
relying in part on the Pax 2and Pax 6 consensus sequences described
by Epstein et al. (1994) J. Biol. Chem. 269:8355-8361, and in part
on certain other suitable Pax consensus sequences disclosed herein.
One consensus sequence for Pax 6 is T T C A C G C A/T T G/C A N T
G/T A/C N T/C corresponding to SEQ. ID No. 2 wherein the N is
either A, G, C or T. One consensus sequence for Pax 2 is G T C A
C/T G C G/A T G A and corresponds to SEQ. ID No. 3. The string
search program allowed for partial mismatches and revealed a number
of Pax 6 or Pax 2 responsive regions. Currently preferred Pax 2
responsive regions are located at approximately 491-503, 737-747,
891-903, 994-1006 of SEQ. ID No. 1 and share approximately 69-82%
identity with the Pax 2 consensus sequence of SEQ. ID. No. 3.
[0113] Currently preferred Pax 6 responsive elements include
approximately nucleotides 108-121, 139-154, 365-378, 497-511,
1750-1762, 2365-2378, 2931-2944 of SEQ. ID No. 1. These Pax 6
responsive elements share between 53% and 82% identity with the Pax
6 consensus sequence of SEQ. ID No. 3. Other currently preferred
Pax 6 responsive elements include approximately nucleotides
157-167, 1123-1140, 1144-1161, 1285-1297, and 2001-2023 of SEQ. ID
No. 1.
[0114] It is understood that some of the consensus Pax motifs may
function to regulate OP-1 gene expression, where others may not.
Further, characterization of each putative Pax responsive OP-1
element is accomplished as described below in Example 5. As earlier
discussed, it is contemplated that Pax responsive elements, such as
those described herein, are present in the promoter sequences of
other morphogens as defined herein and thereby can be identified
and exploited as taught herein. Pax responsive elements, as
contemplated by the present invention, modulate the in vivo, ex
vivo and in vitro expression of morphogens and thus are useful for
identifying particular compounds that can modulate particular
morphogens.
[0115] It is contemplated that the present invention further
provides a useful method for identifying nucleic acids and their
respective encoded compounds capable of modulating Pax mediated
OP-1 gene regulation. In one embodiment, one or more candidate
nucleic acids which encode a candidate compound or compounds
capable of intracellular modulation of OP-1 gene expression,
mediated directly or indirectly by Pax responsive elements located
in the OP-1 gene 5' non-coding region (nucleotides 1-3317 of SEQ.
ID No. 1), are also transfected into the cells of the invention
comprising Pax responsive OP-1 5' non-coding sequence/reporter gene
vectors. "Candidate nucleic acid" or "candidate DNA" or "candidate
RNA" as defined herein, is therefor a nucleic acid (e.g. a DNA or
RNA) that encodes a compound that modulates OP-1 gene expression
via a Pax responsive element In one embodiment, a candidate DNA can
encode a transcription factor that shares functional similarity
with a Pax gene product or is a Pax analog or agonist.
Alternatively, the candidate nucleic acid can encode a compound
that behaves as an inhibitor of OP-1 gene expression or is a Pax
antagonist. In another embodiment, a candidate RNA or RNAs may be
first isolated from a cell or synthesized in vitro by methods well
known to the skilled artisan and then used to transfect the
above-described cell. It is also, contemplated that tissue specific
candidate nucleic acids can be identified and isolated according to
the method of the present invention. Thus, for example, expression
vectors comprising tissue specific candidate DNAs can be
co-transfected into a cell together with an expression vector
containing the OP-1 5' non-coding sequence of nucleotides 1-3317 of
SEQ ID No 1, or truncated versions thereof, in operative
association with a luciferase reporter gene. Alterations in
luciferase activity relative to control transfections is an
indication that the candidate DNA encodes a compound capable of
modulating OP-1 gene regulation. It is contemplated that individual
nucleic acids or pools of nucleic, acids, for example, a cDNA
library or a pool of synthetic RNAs, may be screened for their
ability to modulate OP-1 gene expression via the responsive
elements of the present invention in accordance with the methods of
the invention.
EXAMPLE 3
Further Characterization of the Pax Responsive OP-1 Modulating
Elements
[0116] To characterize the respective roles of certain of the Pax 2
(for example, approximately, nucleotides 491-503, 737-747, 891-903,
994-1006 of SEQ. ID No. 1) and Pax 6 responsive elements (for
example, approximately, nucleotides 108-121, 139-154, 157-167,
365-378, 498-511, 598-613, 1123-1140, 1144-1161, 1285-1297,
1750-1762, 2001-2023,2365-2378, 2931-2944 of SEQ. ID No. 1) in the
modulation of the OP-1 gene expession, mutations of the various Pax
2 and Pax 6 responsive elements are analyzed. A loss or decrease of
Pax mediated OP-1 gene expression by mutation of a portion of each
Pax 2 responsive element sequence indicates a role for that
sequence in Pax mediated OP-1 gene regulation. Conversely, an
increase in Pax mediated OP-1 gene expression of the mutated Pax
responsive elements may indicate, for example, enhanced binding of
Pax transcription factors to the altered DNA recognition sequence
or, alternatively, a loss or decrease in binding of one or more
negative regulatory elements.
[0117] Further, the OP-1 gene promoter is analyzed by DNase
footprinting to further define the Pax responsive regions and to
determine binding specificity and affinity of the Pax gene products
required for the expression of the OP-1 gene. DNase footprinting is
carried out according to techniques well known to those skilled in
the art.
[0118] In addition, the interaction of the OP-1 promoter with
nuclear proteins is further characterized by gel shift assays using
oligonucleotides probes corresponding to. the Pax 2 consensus
sequence (SEQ. ID No. 3) or the Pax 6 consensus sequence (SEQ. ID
No. 2) or variants thereof, with and without mutations in the
oligonucleotide sequences. Briefly, cells are lysed according to
art known methods and nuclear extracts are prepared and incubated
with a radiolabelled oligonucleotide of choice, by methods well
known to the skilled artisan. Binding of one or more of the protein
components of the extract to the oligonucleotide produces
DNA/protein complexes having retarded electrophoretic mobility
relative to the mobility of the uncomplexed oligonucleotide DNA
probe. Completion of radiolabelled oligonucleotide binding with
cold (unlabelled) oligonucleotide is performed to confirm the
specificity of DNA/protein complexes. These assays can demonstrate
alterations in the amount or activity of a nuclear extract
component in response to treatment with candidate compounds.
[0119] Supershift assays, in which the nuclear extract
oligonucleotide complexes are further retarded by complexing with
specific antibody, are used to determine the identity of the Pax
gene product or analog thereof. In these assays, the previously
described protein-oligonucleotide probe complexes are further
incubated with specific antibody and subjected to electrophoresis,
according to art known methods. The DNA-protein complexes from a
test cell induced, for example, by a candidate compound, are
supershifted during gel analysis when treated with an anti-Pax 2 or
anti-Pax 6 antibody, or other Pax family antibodies, for example.
This supershift can be reversed by incubation with the antigenic
peptide.
[0120] The tissue specificity of the Pax-DNA complexes is further
defined by a comparison of gel shift and supershift mobilities
using cell extracts from various tissues and cells. These studies
may reveal the involvement of one or more tissue specific factors
that may participate in Pax mediated regulation of OP-1 gene
transcription.
EXAMPLE 4
In vivo Animal Model for Testing Efficacy of Compounds to Modulate
OP-1 Expression
[0121] It previously has been demonstrated that OP-1 can be an
effective treatment for osteoporosis on the standard ovariectomized
rat model, as indicated by the dose-response increase in alkaline
phosphatase and osteocalcin levels following injection with OP-1.
The osteoporotic rat model provides an in vivo model for evaluating
the efficacy of a candidate compound for modulating morphogen
synthesis. In order to determine the effect of a candidate
morphogen stimulating agent on OP-1 production and, thereby, on
bone production in viva, alkaline phosphatase and osteocalcin
levels are measured under conditions which promote osteoporosis,
e.g., wherein osteoporosis is induced by ovary removal in rats and
in the presence and absence of a candidate compound. A compound
competent to enhance or induce endogenous OP-1 expression should
result in increased osteocalcin and alkaline phosphatase
levels.
[0122] Forty Long-Evans rats (Charles River Laboratories,
Wilmington) weighing about 200 g each are ovariectomized (OVX)
using standard surgical procedures, and ten rats are sham operated.
The ovariectomization of the rats produces an osteoporotic
condition within the rats as a result of decreased estrogen
production. Food and water are provided ad libitum. Eight days
after ovariectomy, the rats, prepared as described above, are
divided into three groups: (a) sham-operated rats; (b)
ovariectomized rats receiving 1 ml of phosphate-buffered saline
(PBS) i.v. in the tail vein; and (c) ovariectomized rats receiving
various dose ranges of the candidate morphogen stimulating agent
either by intravenous injection through the tail vein or direct
administration to kidney tissue.
[0123] The effect of the candidate compound on in vivo bone
formation can be determined by preparing sections of bone tissue
from the ovariectomized rats. Each rat is injected with 5 mg of
tetracycline, which will stain the new bone (visualized as a yellow
color by fluorescence), on the 15th and 21st day of the study and
on day 22 the rats are sacrificed. The body weights, uterine
weights, serum alkaline phosphatase levels, serum calcium levels
and serum osteocalcin levels then were determined for each rat.
Bone sections are prepared and the distance separating each
tetracycline staining is measured to determine the amount of new
bone growth. The levels of OP-1 in serum following injection of the
candidate agent also can be monitored on a periodic basis using
routine methods.
EXAMPLE 5
Determination of OP-1 Protein Production
[0124] Where OP-1 acts as the reporter gene, detection of the gene
product readily can be assayed using antibodies specific to the
protein and standard immunoassay testings. For example, OP-1 may be
detected using a polyclonal antibody specific for OP-1 in an ELISA,
as follows.
[0125] 1 .mu.g/100 .mu.l of affinity-purified polyclonal rabbit IgG
specific for OP-1 is added to each well of a 96-well plate and
incubated at 37.degree. C. for an hour. The wells are washed four
times with 0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH
8.2, containing 0.1% Tween 20. To minimize non-specific binding,
the wells are blocked by filling completely with 1% bovine serum
albumin (BSA) in BSB and incubating for 1 hour at 37.degree. C. The
wells are then washed four times with BSB containing 0.1% Tween 20.
A 100 .mu.l aliquot of an appropriate dilution of each of the test
samples of cell culture supernatant is added to each well in
triplicate and incubated at 37.degree. C. for 30 min. After
incubation, 100 .mu.l biotinylated rabbit anti-OP-1 serum (stock
solution is about 1 mg/ml and diluted 1:400 in BSB containing 1%
BSA before use) is added to each well and incubated at 37.degree.
C. for 30 min. The wells are then washed four times with BSB
containing 0.1% Tween 20. 100 .mu.l streptavidin-alkaline (Southern
Biotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in
BSB containing 0.1% Tween 20 before use) is added to each well and
incubated at 37.degree. C. for 30 min. The plates are washed four
times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50 .mu.l
substrate (ELISA Amplification System Kit, Life Technologies, Inc.,
Bethesda, Md.) is added to each well incubated at room temperature
for 15 min. Then, 50 .mu.l amplifier (from the same amplification
system kit) is added and incubated for another 15 min. at room
temperature. The reaction is stopped by the addition of 50 .mu.l
0.3 M sulfuric acid. The OD at 490 nm of the solution in each well
is recorded. To quantitate OP-1 in culture media, a OP-1 standard
curve is performed in parallel with the test samples.
EXAMPLE 6
Production of OP-1 Polyclonal and Monoclonal Antibodies
[0126] Polyclonal antibody for OP-1 protein may be prepared as
follows. Each rabbit is given a primary immunization of 100
.mu.g/500 .mu.l E. coli produced OP-1 monomer (amino acids 328-431
in SEQ ID NO: 5) in 0.1% SDS mixed with 500 .mu.l Complete Freund's
Adjuvant. The antigen is injected subcutaneously at multiple sites
on the back and flanks of the animal. The rabbit is boosted after a
month in the same manner using incomplete Freund's Adjuvant. Test
bleeds are taken from the ear vein seven days later. Two additional
boosts and test bleeds are performed at monthly intervals until
antibody against OP-1 is detected in the serum using an ELISA
assay. Then, the rabbit is boosted monthly with 100 .mu.g of
antigen and bled (15 ml per bleed) at days seven and ten after
boosting.
[0127] Monoclonal antibody specific for OP-1 protein may be
prepared as follows. A mouse is given two injections of E. coli
produced OP-1 monomer. The first injection contains 100 .mu.g of
OP-1 in complete Freund's adjuvant and is given subcutaneously. The
second injection contains 50 .mu.g of OP-1 in incomplete adjuvant
and is given intraperitoneally. The mouse then receives a total of
230 .mu.g of OP-1 in four intraperitoneal injections at various
times over an eight month period. One week prior to fusion, both
mice are boosted intraperitoneally with 100 .mu.g of OP-1 and 30
.mu.g of the N-terminal peptide (Ser.sub.293-Asn.sub.309-Cys)
conjugated through the added cysteine to bovine serum albumin with
SMCC crosslinking agent. This boost was repeated five days (IP),
four days (IP), three days (IP) and one day (IV) prior to fusion.
The mouse spleen cells are then fused to myeloma cells at a ratio
of 1:1 using PEG 1500 (Boeringer Mannheim), and the cell fusion is
plated and screened for OP-1-specific antibodies using OP-1 as
antigen. The cell fusion and monoclonal screening are then
performed according to standard procedures widely available in the
art.
EXAMPLE 7
Process for Detecting OP-1 in Serum
[0128] Presented below is a sample protocol for identifying OP-1 in
serum. Following this general methodology, OP-1 may be detected in
body fluids, including serum, and can be used in a protocol for
evaluating the efficacy of an OP-1 modulating compound in vivo.
[0129] A monoclonal antibody raised against mammalian,
recombinantly produced OP-1 using standard immunology techniques
well described in the art and described generally in example 6,
above, was immobilized by passing the antibody over an
agarose-activated gel (e.g., Affi-Gel.TM., from Bio-Rad
Laboratories, Richmond, Calif., prepared following manufacturer's
instructions) and used to purify OP-1 from serum. Human serum then
was passed over the column and eluted with 3M K-thiocyanate.
K-thiocyanate fractions then were dialyzed in 6M urea, 20 mM
PO.sub.4, pH 7.0, applied to a C8 HPLC column, and eluted with a 20
minute, 25-50% acetonitrile/0.1% TFA gradient. Mature,
recombinantly produced OP-1 homodimers elute between 20-22 minutes,
and are used as a positive control. Fractions then were collected
and tested for the presence of OP-1 by standard immunoblot using an
OP-1 specific antibody. Using this method OP-1 readily was detected
in human serum. See also, PCT/US92/07432 for a detailed description
of the assay.
EXAMPLE 8
Considerations for Formulations and Methods for Administering
Therapeutic Agents
[0130] Where the OP-1-modulating agent identified herein comprises
part of a tissue or organ preservation solution, any commercially
available preservation solution may be used. For example, useful
solutions known in the art include Collins solution, Wisconsin
solution, Belzer solution, Eurocollins solution and lactated
Ringer's solution. Generally, an organ preservation solution
usually possesses one or more of the following properties: (a) an
osmotic pressure substantially equal to that of the inside of a
mammalian cell, (solutions typically are hyperosmolar and have K+
and/or Mg++ ions present in an amount sufficient to produce an
osmotic pressure slightly higher than the inside of a mammalian
cell); (b) the solution typically is capable of maintaining
substantially normal ATP levels in the cells; and (c) the solution
usually allows optimum maintenance of glucose metabolism in the
cells. Organ preservation solutions also may contain
anticoagulants, energy sources such as glucose, fructose and other
sugars, metabolites, heavy metal chelators, glycerol and other
materials of high viscosity to enhance survival at low
temperatures, free oxygen radical inhibiting agents and a pH
indicator. A detailed description of preservation solutions and
useful components may be found, for example, in U.S. Pat. No.
5,002,965.
[0131] Where the OP-1-modulating agent is to be provided to an
individual, e.g., the donor prior to harvest, or the recipient
prior to or concomitant with transplantation, the therapeutic agent
may be provided by any suitable means, preferably directly (e.g.,
locally, as by injection to the tissue or organ locus) or
systemically (e.g., parenterally or orally).
[0132] Useful solutions for parenteral administration may be
prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical Sciences
(Gemaro, A., ed.), Mack Pub., 1990. Formulations may include, for
example, polyalkylene glycols such as polyethylene glycol, oils of
vegetable original, hydrogenated naphthalenes, and the like.
Formulations for direct administration, in particular, may include
glycerol and other compositions of high viscosity to help maintain
the agent at the desired locus. Biocompatible, preferably
bioresorbable, polymers, including, for example, hyaluronic acid,
collagen, tricalcium phosphate, polybutyrate, lactide and glycolide
polymers and lactide/glycolide copolymers, may be useful excipients
to control the release of the agent in vivo.
[0133] As will be appreciated by those skilled in the art, the
concentration of the compounds described in a therapeutic
composition will vary depending upon a number of factors, including
the dosage of the drug to be administered, the chemical
characteristics (e.g., hydrophobicity) of the compounds employed,
and the route of administration. Where the morphogen-stimulating
agent is part of a preservation solution, the dosage likely will
depend for example, on the size of the tissue or organ to be
transplanted, the overall health status of the organ or tissue
itself, the length of time between harvest and transplantation
(e.g., the duration in storage), the frequency with which the
preservation solution is changed, and the type of storage
anticipated, e.g., low temperature. In general terms, preferred
ranges include a concentration range between about 0.1 ng to 100
.mu.g/kg per tissue or organ weight per day.
[0134] Where the therapeutic agent is to be administered to a donor
or recipient, the preferred dosage of drug to be administered also
is likely to depend on such variables as the type and extent of
progression of the disease, the overall health status of the
particular patient, the relative biological efficacy of the
compound selected, the formulation of the compound excipients, and
its route of administration. In general terms, a suitable compound
of this invention may be provided in an aqueous physiological
buffer solution containing about 0.001% to 10% w/v compound for
parenteral administration. Typical dose ranges are from about 10
ng/kg to about 1 g/kg of body weight per day; and preferred dose
range is from about 0.1 .mu.g/kg to 100 mg/kg of body weight per
day.
[0135] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
EXAMPLE 9
An AatII-PvuII Fragment of the OP-1 Upstream Region Contains an
Expression Silencer
[0136] Luciferase levels were assayed in G401 cells transfected
with the OP-1 upstream deletion constructs described in Example 2.
The G401 cells were transfected with the deletion constructs using
Lipofectamine. Forty-eight to seventy-two hours later, luciferase
activity was measured using the Promega Luciferase Assay System
(Promega, Madison Wis.) and a luminometer (DynaTech). The relative
luciferase activities are shown in FIG. 2. Maximal activity was
observed for the construct containing 0.6 kb of OP-1 upstream DNA.
The addition of approximately 0.1 kb of upstream DNA (the
AatII-PvuII fragment, approximately nucleotides 2606-2690 of SEQ ID
NO: 1) reduces luciferase activity approximately five fold as shown
in FIG. 2 by the AatII construct containing approximately 0.7 kb of
OP-1 upstream DNA.
[0137] The expression silencing property of the AatII-PvuII
fragment (nucleotides 2606-2690 of SEQ ID NO: 1) was further tested
by comparing luciferase expression in pAS3.3 containing cells with
luciferase expression in pAS33d containing cells. The pAS3.3
construct contains 3.3 kb of OP-1 upstream DNA fused to the
luciferase gene. The pAS3.3d construct lacks the AatII-PvuII
fragment, and was made by digesting the pAS3.3 construct with AatII
and PvuII, blunting the AatII overhang, and religating the
vector.
[0138] G401 cells were plated in triplicate in 6 well plates. After
a one day incubation, the cultured cells were transfected with the
deletion constructs, using Lipofectamine and approximately 1 .mu.g
of either pAS3.3 or pAS3.3d. Forty-eight to seventy-two hours
later, luciferase activity was measured as described previously.
The results are shown in FIG. 3. Cells containing pAS3.3 expressed
approximately three-fold (between two and four-fold) more
luciferase than cells containing pAS3.3d. The AatII-PvuII fragment
(nucleotides 2606-2690 of SEQ ID NO: 1) therefore contains a
silencer of gene expression.
[0139] The luciferase production by cells containing the pAS3.3
construct, comprising 3305 nucleotides upstream of the translation
start site of the OP-1 gene, was induced 2.4 fold by
co-transfection with the Pax 2 cDNA and 9.6 fold by co-transfection
with the Pax 6 cDNA (FIG. 1B). The triple transfection of both the
Pax 2 and Pax 6 expression vectors with the pAS3.3 construct
resulted in an additive 11.5 fold induction suggesting that Pax 2
and Pax 6 gene products have an additive effect on OP-1 gene
expression. Cells containing the 5' deletion mutants of the pAS3.3
construct were less responsive to the inductive effect of
co-transfection with Pax 2 or Pax 6 constructs. For example, the
pAS2.5 construct was only induced 1.7 fold by co-transfection with
the Pax 2 construct and this Pax 2 responsiveness was abolished by
further deletion of about 1.3 kb of 5' sequence, with no Pax 2
induction detected for the pAS 1.2 construct or the remaining
constructs with further 5' deletions.
Sequence CWU 1
1
15 1 17415 DNA Homo sapiens 1 tcaaccggtc tctttaggtt ttggctgtgc
ttattactat tcattcaaca ggtactaatt 60 gagcacctgc tgtgtgccag
gctcagaata ggctcaggtg agatgcacaa agaagggtaa 120 actagaatcc
ttgcttagac actgacggat cagttgtttc atatgtaaat tgtagcacca 180
agacctgctg cccctgcccc cagcctcacc tgcttgtgaa gatccctcca aaagatttga
240 gagtagataa aaagcagaga ctactactga agaacagggc tgctttggct
ccttattatt 300 tcagactttg gaagaaaatg acctcctttt tctctactgg
cactgagtgc atagctgacc 360 tagcaagcca ggcctggagg gcgtgtgcag
ggctggggac cgagcctggt ttctgttccc 420 tgctctgcag ctcaagcact
tgctgttcct ccacctggga tgcctttccc tggaaaagcc 480 tgtctctttc
ttgtctttca ggactcaggt cagtggcatc tcctccaaaa actccccttc 540
ccaccctcca tcacctcacc ctgtttatct gcgcccccgc ccccactgcc tgtcacttat
600 tgcaggctga agtgacccag gctctccagt tgtacactct cagatggacc
ctggacgact 660 gtggcactcc tgcaatttcc ccagtctccc tggggtagga
ttcctgcttg ccaggatgcc 720 cacctttcct tctccctcct gcatgtcctc
ctctgcctgg cttctgaatt gtttccagag 780 agagtgatag acaagatctg
cctctccttc agtccctgaa tcttatttaa ggctcttgct 840 ttgcttccct
ggcctggagg cggctccttg atggagtctg ccatgtgggt tcgctcatgg 900
ccatgtcttc ctgcccagca tggtgcttgg ccctgggact ggccacataa tatctgggcc
960 aggtgcaaaa ttagtacggg gcagggggta ctttgttcat aggtgattca
gaaccacata 1020 tggtgacctc agagtaggaa accaagtgtg gggcccttaa
gagctggggg gccctgtacg 1080 actgtccagg ttgcaggccc cacagctcgc
ctcctgatat cctgtgctcc atgcttgtct 1140 gttgaaggaa ggagtgaatg
gatgaagagc aggtggtggg gggtggtttg agggccttgc 1200 tggtgggtgg
gtagaggccc ctccctggca tggggctcaa gacctgttcc atcccacagc 1260
ctggggctgt gtgtaaatgg ccaggacctg caggctggca tttttctgct ccttgcctgg
1320 ctctggctcc cctttctcca cccatgtggc ccctcaggct gccatctagt
ccaaaagtcc 1380 caagggagac ccagaggcca cttggcaaac tacttctgct
ccagaaaact gtagaagacc 1440 ataattctct tccccagctc tcctgctcca
ggaaggacag ccccaaagtg aggcttagca 1500 gagcccctcc cagacaagcg
cccccgcttc cccaacctca gcccttccca gttcatccca 1560 aaggccctct
ggggacccac tctctcaccc agccccagga gggaaggaga caggatgaac 1620
ttttaccccg ctgccctcac tgccactctg ggtgcagtaa ttcccttgag atcccacacc
1680 ggcagaggga ccggtgggtt ctgagtggtc tggggactcc ctgtgacagc
gtgcatggct 1740 cggtattgat tgagggatga atggatgagg agagacagga
gaggaggccg atggggaggt 1800 ctcaggcaca gacccttgga ggggaagagg
atgtgaagac cagcggctgg ctccccaggc 1860 actgccacga ggagggctga
tgggaagccc tagtggtggg gctggggtgt ctggtctcag 1920 gctgaggggt
ggctggaaag atacagggcc ccgaagagga ggaggtggga agaacccccc 1980
cagctcacac gcagttcact tattcactca acaaatcgtg actgcgcacg tacagtggct
2040 accaggcgct gggttcaagg cactgcgggt accagaggtg cggagaagat
cgctgatccg 2100 ggccccagtg ctctgggtgt ctagcggggg taagaaggca
ataaagaagg cacggagtaa 2160 ctcaaacagc aattccagac agcaagagaa
actacaggaa agaaaacaaa cgtgcgaggg 2220 gcgaggcgag gaaacaacct
cagcttggca ggtcttggag gtctctggga ggagaaagca 2280 gcgtctgatg
ggggcgggag gtggtgagtg gggagaggtc caggcggagg gaatggcgag 2340
cgagagacag gctggcaacg gcttcaggga ggcgcggagg ggtcagcgtg gctggcttaa
2400 aaggatacat gggactaggg gcaagaccgg ctcaaggtca ccgcttccag
gaccttctat 2460 ttccgcgcca cctccgcgct cccccaactt ttcccaccgc
ggtccgcagc ccacccgtcc 2520 tgctcgggcc gccttcctgg tccggaccgc
gagtgccgag agggcagggc cggctccgat 2580 tcctccagcc gcatccccgc
gacgtcccgc caggctctag gcaccccgtg ggcactcagt 2640 aaacatttgt
cgagcgctct agagggaatg aatgaaccca ctgggcacag ctggggggag 2700
ggcggggccg agggcaggtg ggaggccgcc ggcgcgggag gggcccctcg aagcccgtcc
2760 tcctcctcct cctcctccgc ccaggcccca gcgcgtacca ctctggcgct
cccgaggcgg 2820 cctcttgtgc gatccagggc gcacaaggct gggagagcgc
cccggggccc ctgctatccg 2880 cgccggaggt tggaagaggg tgggttgccg
ccgcccgagg gcgagagcgc cagaggagcg 2940 ggaagaagga gcgctcgccc
gcccgcctgc ctcctcgctg cctccccggc gttggctctc 3000 tggactccta
ggcttgctgg ctgctcctcc cacccgcgcc cgcctcctca ctcgcctttt 3060
cgttcgccgg ggctgctttc caagccctgc ggtgcgcccg ggcgagtgcg gggcgagggg
3120 cccggggcca gcaccgagca gggggcgggg gtccgggcag agcgcggccg
gccggggagg 3180 ggccatgtct ggcgcgggcg cagcggggcc cgtctgcagc
aagtgaccga gcggcgcgac 3240 ggccgcctgc cccctctgcc acctggggcg
gtgcgggccc ggagcccgga gcccgggtag 3300 cgcgtagagc cggcgcgatg
cacgtgcgct cactgcgagc tgcggcgccg cacagcttcg 3360 tggcgctctg
ggcacccctg ttcctgctgc gctccgccct ggccgacttc agcctggaca 3420
acgaggtgca ctcgagcttc atccaccggc gcctccgcag ccaggagcgg cgggagatgc
3480 agcgcgagat cctctccatt ttgggcttgc cccaccgccc gcgcccgcac
ctccagggca 3540 agcacaactc ggcacccatg ttcatgctgg acctgtacaa
cgccatggcg gtggaggagg 3600 gcggcgggcc cggcggccag ggcttctcct
acccctacaa ggccgtcttc agtacccagg 3660 gcccccctct ggccagcctg
caagatagcc atttcctcac cgacgccgac atggtcatga 3720 gcttcgtcaa
cctcggtgag taagggcagg cgagggtacg ccgtctcctt tcgggggcac 3780
tttgagactg ggagggaggg agccgcttct tctatgcagc ccgcccagct ttccgctcct
3840 ggctgaaatc gcagtgcctg cccgagggtc tcccacccac agccctatga
ctcccaagct 3900 gtgtgcgccc ccaggtcggg ccgctgggtc ggtgagcctg
taggggttac tgggaaggag 3960 ggatcctccg aagtcccctc catgttacgc
cgccggccgc atctctgggg ctggaggcaa 4020 gggcgttcaa agcgcggggc
tcggtcatgt gagctgtccc gggccggcgc cggtccgtga 4080 cctggatgta
aagggccctt cccggcgagg ctgccttgcc gcccttcctg ggcccctctc 4140
agccctgcct ggctctggca tcgcggccgt cgcaccccct taccctccct gtcaagccct
4200 acctgtcccc tcgtggtgcg cccgccttag gctaccggcc gctccgagcc
ttggggcccc 4260 tctccgggcg ccgatgcccc attctctctt ggctggagct
ggggaagaaa cggtgccatt 4320 gctaattttc tttgttttct ttctttgttt
atttttttct tttttctttt tttttctttt 4380 cttttctttt cttttttttt
ttttttgaga cggagtttca ctcttgctcg cccagactgg 4440 agtgcaatgg
cgcgatctct gctcaccgca acctctgcct cccgggttca agcgattctc 4500
gtgcctcagc ctcccgagta gctgggatta caggcatgcg caccatgcct ggctaatttt
4560 gtattttagt agagacaggg tttctccatg ttaggcaggc tggtctcgaa
ctcccgatct 4620 caggtgatcc tcccgcctca gcctcccaaa gtggtgctgg
gattacaggc gtgaagctgt 4680 gccctgccgc tagtcttcta ttttaagtat
ttagtggtag gtcccgggcc ggcagaatct 4740 attttcagca tttaccacgt
gtggcgcgca aaccacaggt tttggcgatt gggttgcgcg 4800 ggatctcaga
gctgacgacc gcgggggcct gggggtcccg gtttccgact ggagccgcga 4860
cgaccccggc gacggcagcc tggggctgca gccgagggcc ggggagctcc ccctccatat
4920 gtgcgcgcac attctccaga cttgctcaaa ctaacccccc ggagcagcgc
acgggctggg 4980 actgatgatc aaatatttgg tttccgagat aacacacccc
gatagcgctg tttcctgagc 5040 cgctttcatt ctacttgtgt aacttgctgc
gaaaacccga accaagtcaa gacagcaaac 5100 tcacgcccac gggcctgtgt
caacatggaa ataatgatac tgaagcccca cgctgggcac 5160 ctggggcgtg
gactgggggc gcgggggaag cgcagatccg ccttcatgct tcccctcctc 5220
ctgataaggt ccctggagtt cccgggagcc attgtctgta cttaataata actaaatcca
5280 actagtgaac caagcttcag cgaggcaagg ggagggaggt ttagatgcca
aaattacctt 5340 caaaaaagtt taaattatac taagcagcca gttaagaagg
aagcagcaat atatgacctg 5400 atttagaacc atctccaaga tgtatgaggt
ggaaagaagc aaggtgcaga tgagtgggct 5460 gcatgtgtgc ttgtatatca
tcgtgtcctc ctggaggaag acaccaggaa ctggagagag 5520 attttactgg
aggggtatat ggcgggggca tagctggggc ttacggagtg ggaggtgggg 5580
tctgattttt cgtcgtctgc acttctgtat ttgtgatttt tttaaaacaa tgtgtattta
5640 ttaactatac caaaaaataa aggaaaattc caaatacata catataaata
atgaaccgca 5700 gagctctgtc gccctcctga agcctggggt tagccagggc
cctttctctg gtgggggatt 5760 tatagcatct tcccttctgt tgggtacccc
ggactcccac tgaatgtgca ggtcccagtg 5820 gctgccttca gagcctggct
ggaatcatta aaaaggtatt tgtaatctct ggcttctgca 5880 gaaggccctg
caaaccaaga gcaaaaaagc ccccagtgct tatgggccgg cagtgtgggc 5940
taggcccggg gctccctgtc cccaagagaa agaccaggtt gctcggaggg tgcctctggg
6000 aactttggtg cgggctattt gctcccccca tggcggcagg agcaagctgg
gacttgtttg 6060 ggaaggccac agctgggtgg ttttcctcct ctggctgtac
atacaccttt caatccattt 6120 ctttcatctt gaaaggacaa agaccggctt
gtctgagcct cttaatcagt caggctggct 6180 ttgggctttg gggaccctga
ctttctcagg tctagctttc tgggacatca ctccaaatta 6240 gatggcagag
tggcttttaa cagagcgcac tgaccttgtt ttctttctct ctctgtccct 6300
aaactcgagg tcattagtta ggtgaagacc tgggctgcag tttggcgaga cacttcctgt
6360 agatgcttct aatgttggcc tttaatttct gctaagcagc agcacacaaa
taaatggcct 6420 gtcccttcta tcctgttgta gcttggaatt tctccatagg
agggacttgg gggtggcagt 6480 agggttggag agggttgggg ggaggtgtag
gagacttgtc tggccactga gtttgctgag 6540 aaagtactgc tatagtgttt
ttccttggat tgcaaatcat gttgatctga actgctgatt 6600 tgaagtggat
tgagaggatg gaacaataga aggaggatat ggctcaggac agtcaagtac 6660
tggaagaggg aaaggtacaa agaggtgttg gcactgaatg accctgaaca gggctgccct
6720 ggaaatatca gaggtgagtg acaaagagaa ctctagtcga aggtctggaa
gtcaattatt 6780 gtctccagct tttgtcccac cctaagggat ggagcatgaa
cttcatgcat gtaacatccc 6840 tccaggagcg ctgaggttct gggaattccc
agtgctggct accatgccat tcttttctca 6900 ttcactcaag agcgtattgg
gatatgcgtg catgaaagca atgtaattat gggcacaacc 6960 tcaaaacctg
ctctaatttt tttttttttt ggagatggag tctcgctcca tcacccaggc 7020
tggagtgcaa tggcgcgatc tcagctcact gcaagctcag acctccaggg ttcacaccat
7080 tctcctgcct cagcctcccg agtagctggg aatacaggcg cccgcaccat
gcgcggctaa 7140 tttttttgta tttttagtag agacggggtt tcactgtgtt
agccaggatg gtctcgatct 7200 cctgacctcg tgatccaccc gcctcggcct
cccaaagttc tgggattaca ggcgtgacag 7260 ccgtgcccgg aatctgctct
aattttttaa agatatcatt tgcaaacttt gggcacttga 7320 gtcactcagt
aagatattat ttacaacccc accatagatt caaacctctg tcctagaatg 7380
ttgtcgagtt aggcatctgg cttgcagcaa cagctggctt tcctgtctat gctgtctcct
7440 tccagggagg atgtttcacc cttcatattg aggaaatggg cacagagaac
ccatttctct 7500 tactcatcat gtaacttcag tgggatggtc agatctatct
ttaacctggc cactcttcca 7560 caagctcaca ctgactccag caagatctta
aactagaagg caggagttca aatcctagct 7620 ggtgcagtgg ccaaatctcg
gctcacagca ccttctgcct cctgggctca agcgatcctc 7680 tgacctcagt
ctcccaagta gctgggacca taggcatgca ccactatgcc tggctaattt 7740
ttgtattttt gtaatttttt gtagagacag agtttcacca tgttgcccag cccagtcttg
7800 aactcctgga ctcaagcaat cttcccacct ttgcctacca gagtgccggg
attacaggtg 7860 tgagccatca tgctagttgc gcacagttgg gcgaaactga
cagatgagaa agcagaacct 7920 cgtgagtcca ctcagtaaga gactccctac
tttctttctg agtctttgtt tctcatcaat 7980 tgaatggcaa taaacaactt
ggtggcccaa gagttgatga caacagtcct ataagattat 8040 acatgtaaaa
gaaacagagt attctacaaa tatcagttat tgatagttca ataggcaacc 8100
tgacattacc ttttcttgga acttgatgaa caactcagaa actcattaat atcaaaccca
8160 atggtgagca cttggtcttt atttatggct gtaagagaag aaattgaatt
aactctatgt 8220 aaatgccaac taagaacatc gaagtctgaa atcaacagtt
ttcctcgctc atacgacaca 8280 cccaaactca agcagtggtt ccaagcccct
ttggaaaata ccatgggcta acgactttaa 8340 aagcttagaa gtgaattcta
cttacttatt acttaaaagt ggttctcaaa cttcaaggtg 8400 aatcaaaatc
atctgtagag cttgttaaaa cacaggttgc tggtccaccc caagagtgtc 8460
ttgagtcagt aggtctcaag tagggctcaa gaatatgcat ttctaatgag ctccaggtga
8520 gtctaagtgt tagtcgtcgg tcttgggacc acaactttgg gaacaattga
tttagaagaa 8580 ctcaaagatc agaaaggggt ggaatatttt taaaattgtg
gtaaaatacg cataaacaga 8640 aaaggtacaa ttttaaccac ttagagagag
gtgggatcta agaacagaaa ttgttatgcc 8700 atcaaaggtg agttcagata
agcattatta aatggtatct atggataaac ttcaggggcc 8760 ctgtggagca
acccaatgct gggatggggt ccaggtgtgc tatggtttgg atgtggtttg 8820
tccctacaaa aactcatgtt gaaatttaat tgccagtgta acattattga gaggttatgg
8880 acttttaaga ggcatttggg tcatgaggga tccaccttca gggattagtg
cagtctccag 8940 ggagtgagtg agttcccatt ctagtgggac tggattagtt
accatacagt ggttgttata 9000 aagtgaggct gcttctggtg ttttatctgt
ttgcaggcac ttccttcccc ttccacttct 9060 ctgccaggtt aggatgcagc
atgaggccct caccagaagc tgaccagatg tggctgcctg 9120 atcttgaact
tcccagtccc cagaaccatg agctaaataa accttttttc tctataaatt 9180
acgcagtcta gagtattcta ttatagcaac acaagacaga ctaagacaca gtggtagaaa
9240 gaacactact gacttctccc atactctggc ctatggacaa gagtgacaga
cagacaagag 9300 tgaatatcag ggccctcagg cacattcctc tctgcccctt
cctcccttct tgcagagtct 9360 ccagtgactg ccagctaatg ctatcataga
ccccaccttt cccctgactt gattggacca 9420 gaagcagcct cctgatccat
ggccaacaat cagattcact ttcaagaatt tgaactaaga 9480 gacactagga
agatggccct tgagctgtga gtcctacact tgaaagttct tagcatcttg 9540
gtcaggtacc caccagggcc atgtgcaaac tgagataatg gggacatgga acaagggtaa
9600 gtggagaggg ctggctggag agagacgggc agaggaaagc cctgccaaga
ggagcagaga 9660 tgagagacct tggagggaga ggtaataaaa ggaggcaaag
atgattttcc atgcttacaa 9720 ctcacagctg aggcctaact atctttatgt
ccataagagg catccttgtg tcgaacctct 9780 cctctttctt gggtcaatgg
gggatggttg caagggacca tcagtaggaa ggcatagtac 9840 actaacccag
tctggggtgg gcttttagac tagtcttcct cccatgctcc tcctcccatt 9900
ggaaccccgg actttcaaga ctgctaccta gcacaccagt gcaccagatg tcactcaaaa
9960 cctcttcagc aatggcccac tcaccttcaa aaaggctgaa gagcagactg
gctgggttct 10020 tcatggtgga ggggcagtct gggaggtttt aaggttgaag
atgaaaactt tcacttttgg 10080 ctcaatggtc tgaaaaagag aaggaccagc
aagtgaactg aagcctcctg gaaagcatct 10140 tgataacagg ggcagagttt
caagatgaga agctgtggca cttactctgg ctttggaaat 10200 gacctctaag
tatctcagtt aattaaagga gtcaaactct agactcgaag gagaagatct 10260
acaattttca ataacatagt ctaccctccc ctccttcccc caccttcacc tcttctttca
10320 tcacaggctt acagggcacc tcttagagcc aggcacggtg ttgggatcag
gaacaaggcc 10380 actgctcaca tccagagcct gtgctactta agaagcttcc
aggacctctt ggatggctgt 10440 ggttagtgcc ctacttttcc cagcaggttg
gatgcagaat catgctcttg tcgttcagga 10500 tgaccatggg gaccatgggt
ctgagcctgt gaccctccag tctacagtgt gttggtgagg 10560 aaggagcagt
tgtcactggg gtcactggca atgggcatgc ctccatctag cttaggcaag 10620
atgcttagac tcagagccag agagtgaaac ccagacacta atgagctgtc ggtgttggtg
10680 tgtgttctct tcctcttcca gtggaacatg acaaggaatt cttccaccca
cgctaccacc 10740 atcgagagtt ccggtttgat ctttccaaga tcccagaagg
ggaagctgtc acggcagccg 10800 aattccggat ctacaaggac tacatccggg
aacgcttcga caatgagacg ttccggatca 10860 gcgtttatca ggtgctccag
gagcacttgg gcaggtgggt gctatacggg tatctgggag 10920 aggtgctgag
tttcctctgg gggcagagga agaaggtggt gagggtttcc ctcccctccc 10980
accccatgag ctctgcttcc catctgttgg ggtagtggag ctgtgacctg ctaacgcgaa
11040 gcccgtgtct ctcctcctct ctcgcaggga atcggatctc ttcctgctcg
acagccgtac 11100 ctctgggcct cggaggaggg ctggctggtg tttgacatca
cagccaccag caaccactgg 11160 gtggtcaatc cgcggcacaa cctgggcctg
cagctctcgg tggagacgct ggatggtgag 11220 tcccccgcca ctgccagtcc
taatgcagcc tgtgctcctg gacttcagga gggtctcagc 11280 agtgctcatg
cttgcttcac tacaaacagg cttccccgcc cctcccaacc agtactccat 11340
gttcagcctt ttgatcctgc agccctgtcc cgctcgtggc cctcctgtaa ctgctcttct
11400 gtgcacttgg ctgcttcctg tccagggcag acgatcaacc ccaagttggc
gggcctgatt 11460 gggcggcacg ggccccagaa caagcagccc ttcatggtgg
ctttcttcaa ggccacggag 11520 gtccacttcc gcagcatccg gtccacgggg
agcaaacagc gcagccagaa ccgctccaag 11580 acgcccaaga accaggaagc
cctcggatgg ccaacgtggc agggtatctt aggtgggagg 11640 gatcacagac
ccaccacagg aacccagcag gccccggcga ccgcaggaga ctgactaaaa 11700
tcattcagtg ctcaccaaga tgctctgagc tctcttcgat tttagcaaac caggagtccg
11760 aagatctaag gagagctggg ggtttgactc cgagagctcg agcagtcccc
aagacctggt 11820 cttgactcac gagttagact ccactcagag gctgactgtc
tccagggtct acacctctaa 11880 gggcgacact gggctcaagc agactgccgt
tttctatatg ggatgagcct tcacagggca 11940 gccagttggg atgggttgag
gtttggctgt agacatcaga aacccaagtc aaatgcgctt 12000 caaccagtag
aaaattcacc agcccgcaga gctaaggttg ggtggacatt agggttggtt 12060
gatccaggag ctcaacagtg tcctctgagc cccagctcct tctgccccac cccaccatct
12120 tcagtgctgc ttcctctcaa ggccacagct gtagttggcc aggggggctt
cattattttt 12180 tgctcctggg cagtaggagg aagagaatga atgtctctcc
atgggtcttt cttaggaatg 12240 tgggaacttt ttccagaagt ctctatgtct
tttagtttgt gttgggtcac ttgcccttcc 12300 tgaaccactt cctgactcct
ggacaggatg tgcactgatg agcttagctt tggggatcta 12360 atagtgactt
tacaaagcct ctttgagaag gtgacattgg aaccaaggct tgagcagaca 12420
caacaaagat tgcagggagg ggcattgcag gtggaggaaa cggcacatgc aagagccctg
12480 cgtgggagtg agcttggtgt ttggtcaatc agttgtcaga gcacaccggg
ccctgtcagc 12540 aggcacagcc tgggcctgct ctgagtatga cagagagccc
ctgggaagtt gtaggtggag 12600 gaaagacagg tcatgactag gaaaaaagca
atccctctgt tgtggggtgg aaggaaggtt 12660 gcagtgtgtg tgagagagag
acaagacaga cagacagaca cttctcaatg tttacaagtg 12720 ctcaggccct
gacccgaatg cttccaaatt tacgtagttc tggaaaaccc cctgtatcat 12780
tttcactact caaagaaacc tcgggagtgt tttcttctga aaggtcatca ggttttgact
12840 ctctgctgtc tcatttcttc ttgctggtgg tggtgatggt tgcttgtccc
aggccctgtc 12900 ccgcatcctc ttgcccctgc agagggatga gtgtgttggg
gcctcacgag ttgaggttgt 12960 tcataagcag atctctttga gcagggcgcc
tgcagtggcc ttgtgtgagg ctggaggggt 13020 ttcgattccc ttatggaatc
caggcagatg tagcatttaa acaacacacg tgtataaaag 13080 aaaccagtgt
ccgcagaagg ttccagaaag tattatggga taagactaca tgagagagga 13140
atggggcatt ggcacctccc ttagtagggc ctttgctggg ggtagaaatg agttttaagg
13200 caggttagac cctcgaactg gcttttgaat cgggaaattt accccccagc
cgttctgtgc 13260 ttcattgctg ttcacatcac tgcctaagat ggaggaactt
tgatgtgtgt gtgtttcttt 13320 ctcctcactg ggctctgctt cttcacttcc
ttgtcaatgc agagaacagc agcaggcacc 13380 agaggcaggc cttgtaagaa
gcacgagctg tatgtcagct tccgagacct gggctggcag 13440 gtaaggggct
ggctgggtct gtcttgggtg tgggccctct ggcgtgggct cccacaggca 13500
gcgggtgctg tgctcagtct tgtttctcat ctctgccagt taagactcca gtatcaagtg
13560 gcctcgctag ggaaggggac ttgggctaag gatacaggga ggcctcatga
aatccgagag 13620 cagaaatgtg gttgagactt gaactcgaac caggaaccca
aacactttgg actctgaacc 13680 ccattctctg catgcacctc attcccatcc
cttggctggc tgcttctcaa gatgatgccg 13740 ggccgtgtgt ttgaatgtag
atacctgggg agccatctcc ccctctgccc tctgacttca 13800 tttaccccat
tcccattccc acgggaggga cggatctccc cagcttggtt caggcgcttg 13860
ttcctgaacc agtcaactgt ttcaggggtg gggtcatgtt actggcacat ggctgccccc
13920 tctggagcca tttgcatgga gtgaggcaaa aggcagggga tgaatctagg
agaggagtga 13980 gggtcatgtg atccacctgc cgtgagctct ggatcgtgat
tctcattcag cagtcacgag 14040 catctcgagc gttctgggcc ctgttctagg
tactggattg gagatgcagc gatgaacact 14100 gcaatgtgtc tgccctgtgg
ggctcaaata tccctggaga gggtattgtc atgaggtcat 14160 cagggcaact
ggtggtattc taccctcagg gagcttgtag ttcagtggga gagtccagaa 14220
tcttccctgg ggattatgcc cagacacact cagggcgtac gtgcacacag ccagctctga
14280 gccctcctgt gagcctgccc tcaggactga tgaccacatc tacctgcagc
tgggacagaa 14340 cccaaactcc aggggcctct gctggaagat tccatgtgct
taagcatcac tgaggagtat 14400 attgattatt gggcaacatt tctgtgccac
ccagacccta gaggcaagga tggcacatgg 14460 atcccttact gaccagtgca
cccggagcca gcatgggtga tgccattatg agttattagc 14520 ctctctggca
ggtgggcaaa ccgaggcatg gaggtttgtt taaggtgaac tgccagtgtg 14580
tgaccaccta gtgggggtag agctgatgat tgcctcacac cggaggctcc ttcctgtgcc
14640 gcgttctgtc cagaagacac agccatggat gtccatttta ggatcagcca
agcccgtggg 14700 gctttccttc atttttattt tatgtttttt tagaaatggg
gtcttgctct gtcacccagg 14760 ctggggtgca gtggtgtgat catacgtcac
cgcagctttg agccgtcttc ccactcagtc 14820 tactaagctt ggactatagg
ccaagactat agagtggtcc ttctttccat tcttttggga 14880 ccatgagagg
ccacccatgt ttcctgcccc tgctgggccc tgctgctcag aaggcatggt 14940
ctgaggcttt caccttggtc gtgagccttc gtggtggttt ctttcagcat ggggttggga
15000 tgctgtgctc aggcttctgc atggtttccc acactctctt
ctcctcctca ggactggatc 15060 atcgcgcctg aaggctacgc gcgctactac
tgtgaggggg agtgtgcctt ccctctgaac 15120 tcctacatga acgccaccaa
ccacgccatc gtgcagacgc tggtgggtgt cacgccatct 15180 tggggtgtgg
tcacctgggc cgggcaggct gcggggccac cagatcctgc tgcctccaag 15240
ctggggcctg agtagatgtc agcccattgc catgtcatga cttttggggg ccccttgcgc
15300 cgttaaaaaa aaatcaaaaa ttgtacttta tgactggttt ggtataaaga
ggagtataat 15360 cttcgaccct ggagttcatt tatttctcct aatttttaaa
gtaactaaaa gttgtatggg 15420 ctcctttgag gatgcttgta gtattgtggg
tgctggttac ggtgcctaag agcactgggc 15480 ccctgcttca ttttccagta
gaggaaacag gtaaacagat gagaaatttc agtgaggggc 15540 acagtgatca
gaagcgggcc agcaggataa tgggatggag agatgagtgg ggacccatgg 15600
gccatttcaa gttaaatttc agtcgggtca ccaggaagat tccatgtgat aatgagatta
15660 acgtgcccag tcacggcgac actcagtagg tgttattcct gctctgccaa
cagcaaccat 15720 agttgataag agctgttagg gattttgtcc ttttgcttag
aatccaaggt tcaaggacct 15780 tggttatgta gctccctgtc atgaacatca
tctgagcctt tcctgcctac tgatcatcca 15840 ccctgccttg aatgcttcta
gtgacagaga gctcactacc aggactactc cctcctttca 15900 tttagtaatc
tgcctccttc ttttcttgtc cctgtcctgt gtgttaagtc ctggagaaaa 15960
atctcatcta tccctttcat ttgattctgc tctttgaggg caggggtttt tgtttctttg
16020 tttgtttttt taagtgttgg ttttccaaag cccttgctcc cctcctcaat
tgaaacttca 16080 aagccctcat tgggattgaa ggtccttagg ctggaaacag
aagagtcctc cccaacctgt 16140 tccctggcct ggatgtgctg tgctgtgcca
gtatcccctg gaaggtgcca ggcatgtctc 16200 cccggctgcc aggggacaca
tctctatcct tctccaaccc ctgccttcat ggcccatgga 16260 acaggagtgc
catcgccctg tgtgcaccta cttccatcag tatttcacca gagatctgca 16320
ggatcaaagt gaattctcca gggattgtga aatgatgcga ttgtggtcat gtttaaaagg
16380 gggcaactgt cttctagaga gtcctgatga aatgcttcca gaggaaatga
gctgatggct 16440 ggaatttgct ttaaaatcat tcaaggtgga gcaggtgggg
aagggtatgg atgtgtaaga 16500 gtttgaaatt gtccatcata aaatgtgtaa
aaagcatgct ggcctatgtc agcagtcaca 16560 gcctggaggt ggtaacagag
tgccagtcac tgatgctcaa gcctggcacc tacagttgct 16620 ggaaacccag
aagtttcacg ttgaaaacaa caggacagtg gaatctctgg ccctgtcttg 16680
aacacgtggc agatctgcta acactgatct tggttggctg ccgtcagctt aggttgagtg
16740 gcggtcttcc cttagtttgc ttagtccccg ctattcccta ttgtcttacc
tcggtctatt 16800 ttgcttatca gtggacctca cgaggcactc ataggcattt
gagtctatgt gtccctgtcc 16860 cacatcctct gtaaggtgca gagaagtcca
tgagcaagat ggagcacttc tagtgggtcc 16920 aagtcaggga cactattcag
caatctacag tgcacagggc agttccccaa cagagaatta 16980 cctggtcctg
aatgtcggat ctggcccctt ccttccccac tgtataatgt gaaaacctct 17040
atgctttgtt ccccttgtct gcaaaacagg gataatccca gaactgagtt gtccatgtaa
17100 agtgcttaga acagggagtg cttggcttgg ggagtgtcac ctgcagtcat
tcattatgcc 17160 cagacaggat gtttctttat agaaacgtgg aggccagtta
gaacgactca ccgcttctca 17220 ccactgccca tgttttggtg tgtgtttcag
gtccacttca tcaacccgga aacggtgccc 17280 aagccctgct gtgcgcccac
gcagctcaat gccatctccg tcctctactt cgatgacagc 17340 tccaacgtca
tcctgaagaa atacagaaac atggtggtcc gggcctgtgg ctgccactag 17400
ctcctccgag aattc 17415 2 17 DNA Artificial Sequence Description of
Artificial Sequence Consensus Sequence 2 ttcacgcwts antkmny 17 3 11
DNA Artificial Sequence Description of Artificial Sequence
Consensus Sequence 3 gtcaygcrtg a 11 4 102 PRT Artificial Sequence
Description of Artificial Sequence Generic sequence OPX 4 Cys Xaa
Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa 1 5 10 15
Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 20
25 30 Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His
Ala 35 40 45 Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa
Val Pro Lys 50 55 60 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa
Ser Val Leu Tyr Xaa 65 70 75 80 Asp Xaa Ser Xaa Asn Val Ile Leu Xaa
Lys Xaa Arg Asn Met Val Val 85 90 95 Xaa Ala Cys Gly Cys His 100 5
97 PRT Artificial Sequence Description of Artificial Sequence
Generic morphogenic sequence 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 50 55 60 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Cys 85 90 95 Xaa 6 102 PRT Artificial Sequence Description of
Artificial Sequence Generic morphogenic sequence 6 Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly 20 25 30
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35
40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 50 55 60 Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100 7 5 PRT
Artificial Sequence Description of Artificial Sequence Generic
morphogenic sequence 7 Cys Xaa Xaa Xaa Xaa 1 5 8 16 DNA Artificial
Sequence Pax 6 consensus sequence 8 annttcacgc atgant 16 9 11 DNA
Artificial Sequence Pax 6 consensus sequence 9 ttcacgcatg a 11 10
30 DNA Homo sapiens 10 aagcttgatg cctgcacagt cagccctcag 30 11 30
DNA Artificial Sequence Reverse compliment primer 11 catcgcgccg
gatccacgcg ctacccgggc 30 12 30 DNA Homo sapiens 12 gcccgggtag
cgcgtagagc cggcgcgatg 30 13 30 DNA Artificial Sequence Mutagenesis
altered OP-1 sequence 13 gcccgggtag cgcgtggatc cggcgcgatg 30 14 57
DNA Artificial Sequence Altered OP-1 promoter in construct pAS3.3
14 gcccgggtag cgcgtggatc taagtaagct tggcattccg gtactgttgg taaaatg
57 15 10 DNA Artificial Sequence nucleic acid 15 tcacgcntga 10
* * * * *