U.S. patent application number 10/384451 was filed with the patent office on 2003-09-11 for polynucleotide encoding a polypeptide having heparanase activity and expression of same in genetically modified cells.
Invention is credited to Feinstein, Elena, Pecker, Iris, Vlodavsky, Israel.
Application Number | 20030170860 10/384451 |
Document ID | / |
Family ID | 22982587 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170860 |
Kind Code |
A1 |
Pecker, Iris ; et
al. |
September 11, 2003 |
Polynucleotide encoding a polypeptide having heparanase activity
and expression of same in genetically modified cells
Abstract
A polynucleotide (hpa) encoding a polypeptide having heparanase
activity, vectors including same, genetically modified cells
expressing heparanase, a recombinant protein having heparanase
activity and antisense oligonucleotides and constructs for
modulating heparanase expression.
Inventors: |
Pecker, Iris; (Rishon
LeZion, IL) ; Vlodavsky, Israel; (Mevaseret Zion,
IL) ; Feinstein, Elena; (Rehovot, IL) |
Correspondence
Address: |
G.E. EHRLICH (1995) LTD.
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Family ID: |
22982587 |
Appl. No.: |
10/384451 |
Filed: |
March 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10384451 |
Mar 10, 2003 |
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09988113 |
Nov 19, 2001 |
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09988113 |
Nov 19, 2001 |
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09776874 |
Feb 6, 2001 |
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09776874 |
Feb 6, 2001 |
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09258892 |
Mar 1, 1999 |
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09258892 |
Mar 1, 1999 |
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PCT/US98/17954 |
Aug 31, 1998 |
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Current U.S.
Class: |
435/200 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 536/23.2; 702/20 |
Current CPC
Class: |
C12N 9/2402 20130101;
C12Y 302/01166 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/200 ; 435/6;
435/69.1; 435/320.1; 435/325; 536/23.2; 702/20 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; C07H 021/04; C12N 009/24; C12P
021/02; C12N 005/06 |
Claims
What is claimed is:
1. A polynucleotide fragment comprising a polynucleotide sequence
encoding a polypeptide having heparanase catalytic activity,
wherein said polypeptide shares at least 70% homology with SEQ ID
NO:44, as determined using default parameters of a DNA sequence
analysis software package developed by the Genetic Computer Group
(GCG) at the University of Wisconsin.
2. The polynucleotide fragment of claim 1, wherein said
polynucleotide sequence includes nucleotides 63-1691 of SEQ ID
NO:9.
3. The polynucleotide fragment of claim 1, wherein said
polynucleotide sequence includes nucleotides 63-721 of SEQ ID
NO:9.
4. The polynucleotide fragment of claim 1, wherein said
polynucleotide is as set forth in SEQ ID NO:9.
5. The polynucleotide fragment of claim 1, wherein said
polynucleotide sequence includes a segment of SEQ ID NO:9, said
segment encodes said polypeptide having said heparanase catalytic
activity.
6. The polynucleotide fragment of claim 1, wherein said polypeptide
includes an amino acid sequence as set forth in SEQ ID NO:44.
7. The polynucleotide fragment of claim 1, wherein said polypeptide
includes a segment of SEQ ID NO:44 said segment harbors said
heparanase catalytic activity.
8. The polynucleotide fragment of claim 1, wherein said
polynucleotide sequence is selected from the group consisting of
double stranded DNA, single stranded DNA and RNA.
9. A polynucleotide sequence as set forth in SEQ ID NOs:42 or
43.
10. A polynucleotide sequence at least 70% homologous to SEQ ID
NOs:42 or 43, as determined using default parameters of a DNA
sequence analysis software package developed by the Genetic
Computer Group (GCG) at the University of Wisconsin, wherein said
polynucleotide sequence encodes a polypeptide having heparanase
catalytic activity.
11. A vector comprising a polynucleotide sequence encoding a
polypeptide having heparanase catalytic activity, wherein said
polypeptide shares at least 70% homology with SEQ ID NO:44, as
determined using default parameters of a DNA sequence analysis
software package developed by the Genetic Computer Group (GCG) at
the University of Wisconsin.
12. The vector of claim 11, wherein said polynucleotide sequence
includes nucleotides 63-1691 of SEQ ID NO:9.
13. The vector of claim 11, wherein said polynucleotide sequence
includes nucleotides 63-721 of SEQ ID NO:9.
14. The vector of claim 11, wherein said polynucleotide sequence is
as set forth in SEQ ID NO:9.
15. The vector of claim 11, wherein said polynucleotide sequence
includes a segment of SEQ ID NO:9, said segment encodes said
polypeptide having said heparanase catalytic activity.
16. The vector of claim 11, wherein said polypeptide includes an
amino acid sequence as set forth in SEQ ID NO:44.
17. The vector of claim 11, wherein said polypeptide includes a
segment of SEQ ID NO:44, said segment harbors said heparanase
catalytic activity.
18. The vector of claim 11, wherein said polynucleotide sequence is
selected from the group consisting of double stranded DNA, single
stranded DNA and RNA.
19. The vector of claim 11, wherein said vector is a baculovirus
vector.
20. A host cell comprising an exogenous polynucleotide fragment
including a polynucleotide sequence encoding a polypeptide having
heparanase catalytic activity, wherein said polypeptide shares at
least 70% homology with SEQ ID NO:44, as determined using default
parameters of a DNA sequence analysis software package developed by
the Genetic Computer Group (GCG) at the University of
Wisconsin.
21. The host cell of claim 20, wherein said polynucleotide sequence
includes nucleotides 63-1691 of SEQ ID NO:9.
22. The host cell of claim 20, wherein said polynucleotide sequence
includes nucleotides 63-721 of SEQ ID NO:9.
23. The host cell of claim 20, wherein said polynucleotide sequence
is as set forth in SEQ ID NO:9.
24. The host cell of claim 20, wherein said polynucleotide sequence
includes a segment of SEQ ID NO:9, said segment encodes said
polypeptide having said heparanase catalytic activity.
25. The host cell of claim 20, wherein said polypeptide includes an
amino acid sequence as set forth in SEQ ID NO:44.
26. The host cell of claim 20, wherein said polypeptide includes a
segment of SEQ ID NO:44 said segment harbors said heparanase
catalytic activity.
27. The host cell of claim 20, wherein said polynucleotide sequence
is selected from the group consisting of double stranded DNA,
single stranded DNA and RNA.
28. A host cell expressing a recombinant heparanase, wherein said
recombinant heparanase shares at least 70% homology with SEQ ID
NO:44, as determined using default parameter of a DNA sequence
analysis software package developed by the Genetic Computer (Group
(GCG) at the University of Wisconsin.
29. A heparanase overexpression system comprising a cell
overexpressing heparanase catalytic activity, wherein said
heparanase catalytic activity is effected by a heparanase sharing
at least 70% homology with SEQ ID NO:44, as determined using
default parameters of a DNA sequence analysis software package
developed by the Genetic Computer Group (GCG) at the University of
Wisconsin.
30. The host cell of claim 20, wherein said cell is an insect cell.
Description
[0001] This is a divisional of U.S. patent application Ser. No.
09/988,113, filed Nov. 19, 2001, which is a continuation of U.S.
patent application Ser. No. 09/776,874, filed Feb. 6, 2001, which
is a continuation of U.S. patent application Ser. No. 09/258,892,
filed Mar. 1, 1999, which is a continuation-in-part of
PCT/US98/17954, filed Aug. 31, 1998, which claims priority from
U.S. patent application Ser. No. 09/109,386, filed Jul. 2, 1998,
now abandoned, which is a continuation-in-part of U.S. patent
application Ser. No. 08/922,170, filed Sep. 2, 1997, now, U.S. Pat.
No. 5,968,822.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to a polynucleotide, referred
to hereinbelow as hpa, encoding a polypeptide having heparanase
activity, vectors (nucleic acid constructs) including same and
genetically modified cells expressing heparanase. The invention
further relates to a recombinant protein having heparanase activity
and to antisense oligonucleotides, constructs and ribozymes for
down regulating heparanase activity. In addition, the invention
relates to heparanase promoter sequences and their uses.
[0003] Heparan sulfate proteoglycans: Heparan sulfate proteoglycans
(HSPG) are ubiquitous macromolecules associated with the cell
surface and extra cellular matrix (ECM) of a wide range of cells of
vertebrate and invertebrate tissues (1-4). The basic HSPG structure
includes a protein core to which several linear heparan sulfate
chains are covalently attached. These polysaccharide chains are
typically composed of repeating hexuronic and D-glucosamine
disaccharide units that are substituted to a varying extent with N-
and O-linked sulfate moieties and N-linked acetyl groups (1-4).
Studies on the involvement of ECM molecules in cell attachment,
growth and differentiation revealed a central role of HSPG in
embryonic morphogenesis, angiogenesis, neurite outgrowth and tissue
repair (1-5). HSPG are prominent components of blood vessels (3).
In large blood vessels they are concentrated mostly in the intima
and inner media, whereas in capillaries they are found mainly in
the subendothelial basement membrane where they support
proliferating and migrating endothelial cells and stabilize the
structure of the capillary wall. The ability of HSPG to interact
with ECM macromolecules such as collagen, laminin and fibronectin,
and with different attachment sites on plasma membranes suggests a
key role for this proteoglycan in the self-assembly and
insolubility of ECM components, as well as in cell adhesion and
locomotion. Cleavage of the heparan sulfate (HS) chains may
therefore result in degradation of the subendothelial ECM and hence
may play a decisive role in extravasation of blood-borne cells. HS
catabolism is observed in inflammation, wound repair, diabetes, and
cancer metastasis, suggesting that enzymes which degrade HS play
important roles in pathologic processes. Heparanase activity has
been described in activated immune system cells and highly
metastatic cancer cells (6-8), but research has been handicapped by
the lack of biologic tools to explore potential causative roles of
heparanase in disease conditions.
[0004] Involvement of Heparanase in Tumor Cell Invasion and
Metastasis: Circulating tumor cells arrested in the capillary beds
of different organs must invade the endothelial cell lining and
degrade its underlying basement membrane (BM) in order to invade
into the extravascular tissue(s) where they establish metastasis
(9, 10). Metastatic tumor cells often attach at or near the
intercellular junctions between adjacent endothelial cells. Such
attachment of the metastatic cells is followed by rupture of the
junctions, retraction of the endothelial cell borders and migration
through the breach in the endothelium toward the exposed underlying
BM (9). Once located between endothelial cells and the BM, the
invading cells must degrade the subendothelial glycoproteins and
proteoglycans of the BM in order to migrate out of the vascular
compartment. Several cellular enzymes (e.g., collagenase IV,
plasminogen activator, cathepsin B, elastase, etc.) are thought to
be involved in degradation of BM (10). Among these enzymes is an
endo-.beta.-D-glucuroni- dase (heparanase) that cleaves HS at
specific intrachain sites (6, 8, 11). Expression of a HS degrading
heparanase was found to correlate with the metastatic potential of
mouse lymphoma (11), fibrosarcoma and melanoma (8) cells. Moreover,
elevated levels of heparanase were detected in sera from metastatic
tumor bearing animals and melanoma patients (8) and in tumor
biopsies of cancer patients (12).
[0005] The control of cell proliferation and tumor progression by
the local microenvironment, focusing on the interaction of cells
with the extracellular matrix (ECM) produced by cultured corneal
and vascular endothelial cells, was investigated previously by the
present inventors. This cultured ECM closely resembles the
subendothelium in vivo in its morphological appearance and
molecular composition. It contains collagens (mostly type III and
IV, with smaller amounts of types I and V), proteoglycans (mostly
heparan sulfate- and dermatan sulfate-proteoglycans, with smaller
amounts of chondroitin sulfate proteoglycans), laminin,
fibronectin, entactin and elastin (13, 14). The ability of cells to
degrade HS in the cultured ECM was studied by allowing cells to
interact with a metabolically sulfate labeled ECM, followed by gel
filtration (Sepharose 6B) analysis of degradation products released
into the culture medium (11). While intact HSPG are eluted next to
the void volume of the column (Kav<0.2,
Mr.about.0.5.times.10.sup.6), labeled degradation fragments of HS
side chains are eluted more toward the V.sub.t of the column
(0.5<kav<0.8, Mr=5-7.times.10.sup.3) (11).
[0006] The heparanase inhibitory effect of various
non-anticoagulant species of heparin that might be of potential use
in preventing extravasation of blood-borne cells was also
investigated by the present inventors. Inhibition of heparanase was
best achieved by heparin species containing 16 sugar units or more
and having sulfate groups at both the N and O positions. While
O-desulfation abolished the heparanase inhibiting effect of
heparin, O-sulfated, N-acetylated heparin retained a high
inhibitory activity, provided that the N-substituted molecules had
a molecular size of about 4,000 daltons or more (7). Treatment of
experimental animals with heparanase inhibitors (e.g.,
non-anticoagulant species of heparin) markedly reduced (>90%)
the incidence of lung metastases induced by B 16 melanoma, Lewis
lung carcinoma and mammary adenocarcinoma cells (7, 8, 16). Heparin
fractions with high and low affinity to anti-thrombin III exhibited
a comparable high anti-metastatic activity, indicating that the
heparanase inhibiting activity of heparin, rather than its
anticoagulant activity, plays a role in the anti-metastatic
properties of the polysaccharide (7).
[0007] Heparanase activity in the urine of cancer patients: In an
attempt to further elucidate the involvement of heparanase in tumor
progression and its relevance to human cancer, urine samples for
heparanase activity were screened (16a). Heparanase activity was
detected in the urine of some, but not all, cancer patients. High
levels of heparanase activity were determined in the urine of
patients with an aggressive metastatic disease and there was no
detectable activity in the urine of healthy donors. Heparanase
activity was also found in the urine of 20% of normal and
microalbuminuric insulin dependent diabetes mellitus (IDDM)
patients, most likely due to diabetic nephropathy, the most
important single disorder leading to renal failure in adults.
[0008] Possible involvement of heparanase in tumor angiogenesis:
Fibroblast growth factors are a family of structurally related
polypeptides characterized by high affinity to heparin (17). They
are highly mitogenic for vascular endothelial cells and are among
the most potent inducers of neovascularization (17, 18). Basic
fibroblast growth factor (bFGF) has been extracted from the
subendothelial ECM produced in vitro (19) and from basement
membranes of the cornea (20), suggesting that ECM may serve as a
reservoir for bFGF. Immunohistochemical staining revealed the
localization of bFGF in basement membranes of diverse tissues and
blood vessels (21). Despite the ubiquitous presence of bFGF in
normal tissues, endothelial cell proliferation in these tissues is
usually very low, suggesting that bFGF is somehow sequestered from
its site of action. Studies on the interaction of bFGF with ECM
revealed that bFGF binds to HSPG in the ECM and can be released in
an active form by HS degrading enzymes (15, 20, 22). It was
demonstrated that heparanase activity expressed by platelets, mast
cells, neutrophils, and lymphoma cells is involved in release of
active bFGF from ECM and basement membranes (23), suggesting that
heparanase activity may not only function in cell migration and
invasion, but may also elicit an indirect neovascular response.
These results suggest that the ECM HSPG provides a natural storage
depot for bFGF and possibly other heparin-binding growth promoting
factors (24, 25). Displacement of bFGF from its storage within
basement membranes and ECM may therefore provide a novel mechanism
for induction of neovascularization in normal and pathological
situations.
[0009] Recent studies indicate that heparin and HS are involved in
binding of bFGF to high affinity cell surface receptors and in bFGF
cell signaling (26, 27). Moreover, the size of HS required for
optimal effect was similar to that of HS fragments released by
heparanase (28). Similar results were obtained with vascular
endothelial cells growth factor (VEGF) (29), suggesting the
operation of a dual receptor mechanism involving HS in cell
interaction with heparin-binding growth factors. It is therefore
proposed that restriction of endothelial cell growth factors in ECM
prevents their systemic action on the vascular endothelium, thus
maintaining a very low rate of endothelial cells turnover and
vessel growth. On the other hand, release of bFGF from storage in
ECM as a complex with HS fragment, may elicit localized endothelial
cell proliferation and neovascularization in processes such as
wound healing, inflammation and tumor development (24, 25).
[0010] Expression of heparanase by cells of the immune system:
Heparanase activity correlates with the ability of activated cells
of the immune system to leave the circulation and elicit both
inflammatory and autoimmune responses. Interaction of platelets,
granulocytes, T and B lymphocytes, macrophages and mast cells with
the subendothelial ECM is associated with degradation of HS by a
specific heparanase activity (6). The enzyme is released from
intracellular compartments (e.g., lysosomes, specific granules,
etc.) in response to various activation signals (e.g., thrombin,
calcium ionophore, immune complexes, antigens, mitogens, etc.),
suggesting its regulated involvement in inflammation and cellular
immunity.
[0011] Some of the Observations Regarding the Heparanase Enzyme
were Reviewed in Reference No. 6 and are Listed Hereinbelow:
[0012] First, a proteolytic activity (plasminogen activator) and
heparanase participate synergistically in sequential degradation of
the ECM HSPG by inflammatory leukocytes and malignant cells.
[0013] Second, a large proportion of the platelet heparanase exists
in a latent form, probably as a complex with chondroitin sulfate.
The latent enzyme is activated by tumor cell-derived factor(s) and
may then facilitate cell invasion through the vascular endothelium
in the process of tumor metastasis.
[0014] Third, release of the platelet heparanase from
.alpha.-granules is induced by a strong stimulant (i.e., thrombin),
but not in response to platelet activation on ECM.
[0015] Fourth, the neutrophil heparanase is preferentially and
readily released in response to a threshold activation and upon
incubation of the cells on ECM.
[0016] Fifth, contact of neutrophils with ECM inhibited release of
noxious enzymes (proteases, lysozyme) and oxygen radicals, but not
of enzymes (heparanase, gelatinase) which may enable diapedesis.
This protective role of the subendothelial ECM was observed when
the cells were stimulated with soluble factors but not with
phagocytosable stimulants.
[0017] Sixth, intracellular heparanase is secreted within minutes
after exposure of T cell lines to specific antigens.
[0018] Seventh, mitogens (Con A, LPS) induce synthesis and
secretion of heparanase by normal T and B lymphocytes maintained in
vitro. T lymphocyte heparanase is also induced by immunization with
antigen in vivo.
[0019] Eighth, heparanase activity is expressed by pre-B lymphomas
and B-lymphomas, but not by plasmacytomas and resting normal B
lymphocytes.
[0020] Ninth, heparanase activity is expressed by activated
macrophages during incubation with ECM, but there was little or no
release of the enzyme into the incubation medium. Similar results
were obtained with human myeloid leukemia cells induced to
differentiate to mature macrophages.
[0021] Tenth, T-cell mediated delayed type hypersensitivity and
experimental autoimmunity are suppressed by low doses of heparanase
inhibiting non-anticoagulant species of heparin (30).
[0022] Eleventh, heparanase activity expressed by platelets,
neutrophils and metastatic tumor cells releases active bFGF from
ECM and basement membranes. Release of bFGF from storage in ECM may
elicit a localized neovascular response in processes such as wound
healing, inflammation and tumor development.
[0023] Twelfth, among the breakdown products of the ECM generated
by heparanase is a tri-sulfated disaccharide that can inhibit
T-cell mediated inflammation in vivo (31). This inhibition was
associated with an inhibitory effect of the disaccharide on the
production of biologically active TNF.alpha. by activated T cells
in vitro (31).
[0024] Other potential therapeutic applications: Apart from its
involvement in tumor cell metastasis, inflammation and
autoimmunity, mammalian heparanase may be applied to modulate:
bioavailability of heparin-binding growth factors (15); cellular
responses to heparin-binding growth factors (e.g., bFGF, VEGF) and
cytokines (IL-8) (31a, 29); cell interaction with plasma
lipoproteins (32); cellular susceptibility to certain viral and
some bacterial and protozoa infections (33, 33a, 33b); and
disintegration of amyloid plaques (34). Heparanase may thus prove
useful for conditions such as wound healing, angiogenesis,
restenosis, atherosclerosis, inflammation, neurodegenerative
diseases and viral infections. Mammalian heparanase can be used to
neutralize plasma heparin, as a potential replacement of protamine.
Anti-heparanase antibodies may be applied for immunodetection and
diagnosis of micrometastases, autoimmune lesions and renal failure
in biopsy specimens, plasma samples, and body fluids. Common use in
basic research is expected.
[0025] The identification of the hpa gene encoding for heparanase
enzyme will enable the production of a recombinant enzyme in
heterologous expression systems. Availability of the recombinant
protein will pave the way for solving the protein structure
function relationship and will provide a tool for developing new
inhibitors.
[0026] Viral Infection: The presence of heparan sulfate on cell
surfaces have been shown to be the principal requirement for the
binding of Herpes Simplex (33) and Dengue (33a) viruses to cells
and for subsequent infection of the cells. Removal of the cell
surface heparan sulfate by heparanase may therefore abolish virus
infection. In fact, treatment of cells with bacterial heparitinase
(degrading heparan sulfate) or heparinase (degrading heparan)
reduced the binding of two related animal herpes viruses to cells
and rendered the cells at least partially resistant to virus
infection (33). There are some indications that the cell surface
heparan sulfate is also involved in HIV infection (33b).
[0027] Neurodegenerative diseases: Heparan sulfate proteoglycans
were identified in the prion protein amyloid plaques of
Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape
(34). Heparanase may disintegrate these amyloid plaques which are
also thought to play a role in the pathogenesis of Alzheimer's
disease.
[0028] Restenosis and Atherosclerosis: Proliferation of arterial
smooth muscle cells (SMCs) in response to endothelial injury and
accumulation of cholesterol rich lipoproteins are basic events in
the pathogenesis of atherosclerosis and restenosis (35). Apart from
its involvement in SMC proliferation (i.e., low affinity receptors
for heparin-binding growth factors), HS is also involved in
lipoprotein binding, retention and uptake (36). It was demonstrated
that HSPG and lipoprotein lipase participate in a novel catabolic
pathway that may allow substantial cellular and interstitial
accumulation of cholesterol rich lipoproteins (32). The latter
pathway is expected to be highly atherogenic by promoting
accumulation of apoB and apoE rich lipoproteins (i.e. LDL, VLDL,
chylomicrons), independent of feed back inhibition by the cellular
sterol content. Removal of SMC HS by heparanase is therefore
expected to inhibit both SMC proliferation and lipid accumulation
and thus may halt the progression of restenosis and
atherosclerosis.
[0029] Gene Therapy:
[0030] The ultimate goal in the management of inherited as well as
acquired diseases is a rational therapy with the aim to eliminate
the underlying biochemical defects associated with the disease
rather then symptomatic treatment. Gene therapy is a promising
candidate to meet these objectives. Initially it was developed for
treatment of genetic disorders, however, the consensus view today
is that it offers the prospect of providing therapy for a variety
of acquired diseases, including cancer, viral infections, vascular
diseases and neurodegenerative disorders.
[0031] The gene-based therapeutic can act either intracellularly,
affecting only the cells to which it is delivered, or
extracellularly, using the recipient cells as local endogenous
factories for the therapeutic product(s). The application of gene
therapy may follow any of the following strategies: (i)
prophylactic gene therapy, such as using gene transfer to protect
cells against viral infection; (ii) cytotoxic gene therapy, such as
cancer therapy, where genes encode cytotoxic products to render the
target cells vulnerable to attack by the normal immune response;
(iii) biochemical correction, primarily for the treatment of single
gene defects, where a normal copy of the gene is added to the
affected or other cells.
[0032] To allow efficient transfer of the therapeutic genes, a
variety of gene delivery techniques have been developed based on
viral and non-viral vector systems. The most widely used and most
efficient systems for delivering genetic material into target cells
are viral vectors. So far, 329 clinical studies (phase I, I/II and
II) with over 2,500 patients have been initiated Worldwide since
1989 (50).
[0033] The approach of gene addition pose serious barriers. The
expression of many genes is tightly regulated and context
dependent, so achieving the correct balance and function of
expression is challenging. The gene itself is often quite large,
containing many exons and introns. The delivery vector is usually a
virus, which can infect with a high efficiency but may, on the
other hand, induce immunological response and consequently
decreases effectiveness, especially upon secondary administration.
Most of the current expression vector-based gene therapy protocols
fail to achieve clinically significant transgene expression
required for treating genetic diseases. Apparently, it is difficult
to deliver enough virus to the right cell type to elicit an
effective and therapeutic effect (51)
[0034] Homologous recombination, which was initially considered to
be of limited use for gene therapy because of its low frequency in
mammalian cells, has recently emerged as a potential strategy for
developing gene therapy. Different approaches have been used to
study homologous recombination in mammalian cells; some involve DNA
repair mechanisms. These studies aimed at either gene disruption or
gene correction and include RNA/DNA chimeric oligonucleotides,
small or large homologous DNA fragments, or adeno-associated viral
vectors. Most of these studies show a reasonable frequency of
homologous recombination, which warrants further in vivo testing
(52). Homologous recombination-based gene therapy has the potential
to develop into a powerful therapeutic modality for genetic
diseases. It can offer permanent expression and normal regulation
of corrected genes in appropriate cells or organs and probably can
be used for treating dominantly inherited diseases such as
polycystic kidney disease.
[0035] Genomic Sequences Function in Regulation of Gene
Expression:
[0036] The efficient expression of therapeutic genes in target
cells or tissues is an important component of efficient and safe
gene therapy. The expression of genes is driven by the promoter
region upstream of the coding sequence, although regulation of
expression may be supplemented by farther upstream or downstream
DNA sequences or DNA in the introns of the gene. Since this
important information is embedded in the DNA, the description of
gene structure is crucial to the analysis of gene regulation.
Characterization of cell specific or tissue specific promoters, as
well as other tissue specific regulatory elements enables the use
of such sequences to direct efficient cell specific, or
developmental stage specific gene expression. This information
provides the basis for targeting individual genes and for control
of their expression by exogenous agents, such as drugs.
Identification of transcription factors and other regulatory
proteins required for proper gene expression will point at new
potential targets for modulating gene expression, when so desired
or required.
[0037] Efficient expression of many mammalian genes depends on the
presence of at least one intron. The expression of mouse
thymidylate synthase (TS) gene, for example, is greatly influenced
by intron sequences. The addition of almost any of the introns from
the mouse TS gene to an intronless TS minigene leads to a large
increase in expression (42). The involvement of intron 1 in the
regulation of expression was demonstrated for many other genes. In
human factor IX (hFIX), intron 1 is able to increase the expression
level about 3 fold mare as compared to that of the HFIX cDNA (43).
The expression enhancing activity of intron 1 is due to efficient
functional splicing sequences, present in the precursor mRNA. By
being efficiently assembled into spliceosome complexes, transcripts
with splicing sequences may be better protected in the nucleus from
random degradations, than those without such sequences (44).
[0038] A forward-inserted intron 1-carrying HFIX expression
cassette suggested to be useful for directed gene transfer, while
for retroviral-mediated gene transfer system, reversely-inserted
intron 1-carrying HFIX expression cassette was considered (43).
[0039] A highly conserved cis-acting sequence element was
identified in the first intron of the mouse and rat c-Ha-ras, and
in the first exon of Ha- and Ki-ras genes of human, mouse and rat.
This cis-acting regulatory sequence confers strong transcription
enhancer activity that is differentially modulated by steroid
hormones in metastatic and nonmetastatic subpopulations.
Perturbations in the regulatory activities of such cis-acting
sequences may play an important role in governing oncogenic potency
of Ha-ras through transcriptional control mechanisms (45).
[0040] Intron sequences affect tissue specific, as well as
inducible gene expression. A 182 bp intron 1 DNA segment of the
mouse Col2a1 gene contains the necessary information to confer
high-level, temporally correct, chondrocyte expression on a
reporter gene in intact mouse embryos, while Col2a1 promoter
sequences are dispensable for chondrocyte expression (46). In
Col1A1 gene the intron plays little or no role in constitutive
expression of collagen in the skin, and in cultured cells derived
from the skin, however, in the lungs of young mice, intron deletion
results in decrease of expression to less than 50% (47).
[0041] A classical enhancer activity was shown in the 2 kb intron
fragment in bovine beta-casein gene. The enhancer activity was
largely dependent on the lactogenic hormones, especially prolactin.
It was suggested that several elements in the intron-1 of the
bovine beta-casein gene cooperatively interact not only with each
other but also with its promoter for hormonal induction (48).
[0042] Identification and characterization of regulatory elements
in genomic non-coding sequences, such as introns, provides a tool
for designing and constructing novel vectors for tissue specific,
hormone regulated or any other defined expression pattern, for gene
therapy. Such an expression cassette was developed, utilizing
regulatory elements from the human cytokeratin 18 (K18) gene,
including 5' genomic sequences and one of its introns. This
cassette efficiently expresses reporter genes, as well as the human
cystic fibrosis transmembrane conductance regulator (CFTR) gene, in
cultured lung epithelial cells (49).
[0043] Alternative Splicing:
[0044] Alternative splicing of pre mRNA is a powerful and versatile
regulatory mechanism that can effect quantitative control of gene
expression and functional diversification of proteins. It
contributes to major developmental decisions and also to a
fine-tuning of gene function. Genetic and biochemical approaches
have identified cis-acting regulatory elements and trans-acting
factors that control alternative splicing of specific mRNAs. This
mechanism results in the generation of variant isoforms of various
proteins from a single gene. These include cell surface molecules
such as CD44, receptors, cytokines such as VEGF and enzymes.
Products of alternatively spliced transcripts differ in their
expression pattern, substrate specificity and other biological
parameters.
[0045] The FGF receptor RNA undergoes alternative splicing which
results in the production of several isoforms, which exhibit
different ligand binding specificities. The alternative splicing is
regulated in a cell specific manner (53).
[0046] Alternative spliced mRNAs are often correlated with
malignancy. An increase in specific splice variant of tyrosinase
was identified in murine melanomas (54). Multiple splicing variants
of estrogen receptor are present in individual human breast tumors.
CD44 has various isoform, some are characteristic of malignant
tissues.
[0047] Identification of tumor specific alternative splice variants
provide new tool for cancer diagnostics. CD44 variants have been
used for detection of malignancy in urine samples from patients
with urothelial cancer by competitive RT-PCR (55). CD44 exon 6 was
suggested as prognostic indicator of metastasis in breast cancer
(56).
[0048] Different enzymes or polypeptides generated by alternative
splicing may have different function or catalytic specificity. The
identification and characterization of the enzyme forms, which are
involved in pathological processes, is crucial for the design of
appropriate and efficient drugs.
[0049] Modulation of Gene Expression--Antisense Technology:
[0050] An antisense oligonucleotide (e.g., antisense
oligodeoxyribonucleotide) may bind its target nucleic acid either
by Watson-Crick base pairing or Hoogsteen and anti-Hoogsteen base
pairing (64). According to the Watson-Crick base pairing,
heterocyclic bases of the antisense oligonucleotide form hydrogen
bonds with the heterocyclic bases of target single-stranded nucleic
acids (RNA or single-stranded DNA), whereas according to the
Hoogsteen base pairing, the heterocyclic bases of the target
nucleic acid are double-stranded DNA, wherein a third strand is
accommodated in the major groove of the B-form DNA duplex by
Hoogsteen and anti-Hoogsteen base pairing to form a triple helix
structure.
[0051] According to both the Watson-Crick and the Hoogsteen base
pairing models, antisense oligonucleotides have the potential to
regulate gene expression and to disrupt the essential functions of
the nucleic acids in cells. Therefore, antisense oligonucleotides
have possible uses in modulating a wide range of diseases in which
gene expression is altered.
[0052] Since the development of effective methods for chemically
synthesizing oligonucleotides, these molecules have been
extensively used in biochemistry and biological research and have
the potential use in medicine, since carefully devised
oligonucleotides can be used to control gene expression by
regulating levels of transcription, transcripts and/or
translation.
[0053] Oligodeoxyribonucleotides as long as 100 base pairs (bp) are
routinely synthesized by solid phase methods using commercially
available, fully automated synthesis machines. The chemical
synthesis of oligoribonucleotides, however, is far less routine.
Oligoribonucleotides are also much less stable than
oligodeoxyribonucleotides, a fact which has contributed to the more
prevalent use of oligodeoxyribonucleotides in medical and
biological research, directed at, for example, the regulation of
transcription or translation levels.
[0054] Gene expression involves few distinct and well regulated
steps. The first major step of gene expression involves
transcription of a messenger RNA (mRNA) which is an RNA sequence
complementary to the antisense (i.e., -) DNA strand, or, in other
words, identical in sequence to the DNA sense (i.e., +) strand,
composing the gene. In eukaryotes, transcription occurs in the cell
nucleus.
[0055] The second major step of gene expression involves
translation of a protein (e.g., enzymes, structural proteins,
secreted proteins, gene expression factors, etc.) in which the mRNA
interacts with ribosomal RNA complexes (ribosomes) and amino acid
activated transfer RNAs (tRNAs) to direct the synthesis of the
protein coded for by the mRNA sequence.
[0056] Initiation of transcription requires specific recognition of
a promoter DNA sequence located upstream to the coding sequence of
a gene by an RNA-synthesizing enzyme--RNA polymerase. This
recognition is preceded by sequence-specific binding of one or more
transcription factors to the promoter sequence. Additional proteins
which bind at or close to the promoter sequence may trans
upregulate transcription via cis elements known as enhancer
sequences. Other proteins which bind to or close to the promoter,
but whose binding prohibits the action of RNA polymerase, are known
as repressors.
[0057] There are also evidence that in some cases gene expression
is downregulated by endogenous antisense RNA repressors that bind a
complementary mRNA transcript and thereby prevent its translation
into a functional protein.
[0058] Thus, gene expression is typically upregulated by
transcription factors and enhancers and downregulated by
repressors.
[0059] However, in many disease situation gene expression is
impaired. In many cases, such as different types of cancer, for
various reasons the expression of a specific endogenous or
exogenous (e.g., of a pathogen such as a virus) gene is
upregulated. Furthermore, in infectious diseases caused by
pathogens such as parasites, bacteria or viruses, the disease
progression depends on expression of the pathogen genes, this
phenomenon may also be considered as far as the patient is
concerned as upregulation of exogenous genes.
[0060] Most conventional drugs function by interaction with and
modulation of one or more targeted endogenous or exogenous
proteins, e.g., enzymes. Such drugs, however, typically are not
specific for targeted proteins but interact with other proteins as
well. Thus, a relatively large dose of drug must be used to
effectively modulate a targeted protein.
[0061] Typical daily doses of drugs are from 10.sup.-5-10.sup.-1
millimoles per kilogram of body weight or 10.sup.-3-10 millimoles
for a 100 kilogram person. If this modulation instead could be
effected by interaction with and inactivation of mRNA, a dramatic
reduction in the necessary amount of drug could likely be achieved,
along with a corresponding reduction in side effects. Further
reductions could be effected if such interaction could be rendered
site-specific. Given that a functioning gene continually produces
mRNA, it would thus be even more advantageous if gene transcription
could be arrested in its entirety.
[0062] Given these facts, it would be advantageous if gene
expression could be arrested or downmodulated at the transcription
level.
[0063] The ability of chemically synthesizing oligonucleotides and
analogs thereof having a selected predetermined sequence offers
means for downmodulating gene expression. Three types of gene
expression modulation strategies may be considered.
[0064] At the transcription level, antisense or sense
oligonucleotides or analogs that bind to the genomic DNA by strand
displacement or the formation of a triple helix, may prevent
transcription (64).
[0065] At the transcript level, antisense oligonucleotides or
analogs that bind target mRNA molecules lead to the enzymatic
cleavage of the hybrid by intracellular RNase H (65). In this case,
by hybridizing to the targeted mRNA, the oligonucleotides or
oligonucleotide analogs provide a duplex hybrid recognized and
destroyed by the RNase H enzyme. Alternatively, such hybrid
formation may lead to interference with correct splicing (66). As a
result, in both cases, the number of the target mRNA intact
transcripts ready for translation is reduced or eliminated.
[0066] At the translation level, antisense oligonucleotides or
analogs that bind target mRNA molecules prevent, by steric
hindrance, binding of essential translation factors (ribosomes), to
the target mRNA, a phenomenon known in the art as hybridization
arrest, disabling the translation of such mRNAs (67).
[0067] Thus, antisense sequences, which as described hereinabove
may arrest the expression of any endogenous and/or exogenous gene
depending on their specific sequence, attracted much attention by
scientists and pharmacologists who were devoted at developing the
antisense approach into a new pharmacological tool (68).
[0068] For example, several antisense oligonucleotides have been
shown to arrest hematopoietic cell proliferation (69), growth (70),
entry into the S phase of the cell cycle (71), reduced survival
(72) and prevent receptor mediated responses (73). For use of
antisense oligonucleotides as antiviral agents the reader is
referred to reference 74.
[0069] For efficient in vivo inhibition of gene expression using
antisense oligonucleotides or analogs, the oligonucleotides or
analogs must fulfill the following requirements (i) sufficient
specificity in binding to the target sequence; (ii) solubility in
water; (iii) stability against intra- and extracellular nucleases;
(iv) capability of penetration through the cell membrane; and (v)
when used to treat an organism, low toxicity.
[0070] Unmodified oligonucleotides are impractical for use as
antisense sequences since they have short in vivo half-lives,
during which they are degraded rapidly by nucleases. Furthermore,
they are difficult to prepare in more than milligram quantities. In
addition, such oligonucleotides are poor cell membrane penetraters
(75).
[0071] Thus it is apparent that in order to meet all the above
listed requirements, oligonucleotide analogs need to be devised in
a suitable manner. Therefore, an extensive search for modified
oligonucleotides has been initiated.
[0072] For example, problems arising in connection with
double-stranded DNA (dsDNA) recognition through triple helix
formation have been diminished by a clever "switch back" chemical
linking, whereby a sequence of polypurine on one strand is
recognized, and by "switching back", a homopurine sequence on the
other strand can be recognized. Also, good helix formation has been
obtained by using artificial bases, thereby improving binding
conditions with regard to ionic strength and pH.
[0073] In addition, in order to improve half-life as well as
membrane penetration, a large number of variations in
polynucleotide backbones have been done, nevertheless with little
success.
[0074] Oligonucleotides can be modified either in the base, the
sugar or the phosphate moiety. These modifications include, for
example, the use of methylphosphonates, monothiophosphates,
dithiophosphates, phosphoramidates, phosphate esters, bridged
phosphorothioates, bridged phosphoramidates, bridged
methylenephosphonates, dephospho internucleotide analogs with
siloxane bridges, carbonate bridges, carboxymethyl ester bridges,
carbonate bridges, carboxymethyl ester bridges, acetamide bridges,
carbamate bridges, thioether bridges, sulfoxy bridges, sulfono
bridges, various "plastic" DNAs, .alpha.-anomeric bridges and
borane derivatives. For further details the reader is referred to
reference 76.
[0075] International patent application WO 89/12060 discloses
various building blocks for synthesizing oligonucleotide analogs,
as well as oligonucleotide analogs formed by joining such building
blocks in a defined sequence. The building blocks may be either
"rigid" (i.e., containing a ring structure) or "flexible" (i.e.,
lacking a ring structure). In both cases, the building blocks
contain a hydroxy group and a mercapto group, through which the
building blocks are said to join to form oligonucleotide analogs.
The linking moiety in the oligonucleotide analogs is selected from
the group consisting of sulfide (--S--), sulfoxide (--SO--), and
sulfone (--SO.sub.2--). However, the application provides no data
supporting the specific binding of an oligonucleotide analog to a
target oligonucleotide.
[0076] International patent application WO 92/20702 describe an
acyclic oligonucleotide which includes a peptide backbone on which
any selected chemical nucleobases or analogs are stringed and serve
as coding characters as they do in natural DNA or RNA. These new
compounds, known as peptide nucleic acids (PNAs), are not only more
stable in cells than their natural counterparts, but also bind
natural DNA and RNA 50 to 100 times more tightly than the natural
nucleic acids cling to each other (77). PNA oligomers can be
synthesized from the four protected monomers containing thymine,
cytosine, adenine and guanine by Merrifield solid-phase peptide
synthesis. In order to increase solubility in water and to prevent
aggregation, a lysine amide group is placed at the C-terminal.
[0077] Thus, antisense technology requires pairing of messenger RNA
with an oligonucleotide to form a double helix that inhibits
translation. The concept of antisense-mediated gene therapy was
already introduced in 1978 for cancer therapy. This approach was
based on certain genes that are crucial in cell division and growth
of cancer cells. Synthetic fragments of genetic substance DNA can
achieve this goal. Such molecules bind to the targeted gene
molecules in RNA of tumor cells, thereby inhibiting the translation
of the genes and resulting in dysfunctional growth of these cells.
Other mechanisms has also been proposed. These strategies have been
used, with some success in treatment of cancers, as well as other
illnesses, including viral and other infectious diseases. Antisense
oligonucleotides are typically synthesized in lengths of 13-30
nucleotides. The life span of oligonucleotide molecules in blood is
rather short. Thus, they have to be chemically modified to prevent
destruction by ubiquitous nucleases present in the body.
Phosphorothioates are very widely used modification in antisense
oligonucleotide ongoing clinical trials (57). A new generation of
antisense molecules consist of hybrid antisense oligonucleotide
with a central portion of synthetic DNA while four bases on each
end have been modified with 2'O-methyl ribose to resemble RNA. In
preclinical studies in laboratory animals, such compounds have
demonstrated greater stability to metabolism in body tissues and an
improved safety profile when compared with the first-generation
unmodified phosphorothioate (Hybridon Inc. news). Dosens of other
nucleotide analogs have also been tested in antisense
technology.
[0078] RNA oligonucleotides may also be used for antisense
inhibition as they form a stable RNA-RNA duplex with the target,
suggesting efficient inhibition. However, due to their low
stability RNA oligonucleotides are typically expressed inside the
cells using vectors designed for this purpose. This approach is
favored when attempting to target a mRNA that encodes an abundant
and long-lived protein (57). Recent scientific publications have
validated the efficacy of antisense compounds in animal models of
hepatitis, cancers, coronary artery restenosis and other diseases.
The first antisense drug was recently approved by the FDA. This
drug Fomivirsen, developed by Isis, is indicated for local
treatment of cytomegalovirus in patients with AIDS who are
intolerant of or have a contraindication to other treatments for
CMV retinitis or who were insufficiently responsive to previous
treatments for CMV retinitis (Pharmacotherapy News Network).
[0079] Several antisense compounds are now in clinical trials in
the United States. These include locally administered antivirals,
systemic cancer therapeutics. Antisense therapeutics has the
potential to treat many life-threatening diseases with a number of
advantages over traditional drugs. Traditional drugs intervene
after a disease-causing protein is formed.
[0080] Antisense therapeutics, however, block mRNA
transcription/translati- on and intervene before a protein is
formed, and since antisense therapeutics target only one specific
mRNA, they should be more effective with fewer side effects than
current protein-inhibiting therapy.
[0081] A second option for disrupting gene expression at the level
of transcription uses synthetic oligonucleotides capable of
hybridizing with double stranded DNA. A triple helix is formed.
Such oligonucleotides may prevent binding of transcription factors
to the gene's promoter and therefore inhibit transcription.
Alternatively, they may prevent duplex unwinding and, therefore,
transcription of genes within the triple helical structure.
[0082] Another approach is the use of specific nucleic acid
sequences to act as decoys for transcription factors. Since
transcription factors bind specific DNA sequences it is possible to
synthesize oligonucleotides that will effectively compete with the
native DNA sequences for available transcription factors in vivo.
This approach requires the identification of gene specific
transcription factor (57).
[0083] Indirect inhibition of gene expression was demonstrated for
matrix metalloproteinase genes (MMP-1, -3, and -9), which are
associated with invasive potential of human cancer cells. E1AF is a
transcription activator of MMP genes. Expression of E1AF antisense
RNA in HSC3AS cells showed decrease in mRNA and protein levels of
MMP-1, -3, and -9. Moreover, HSC3AS showed lower invasive potential
in vitro and in vivo. These results imply that transfection of
antisense inhibits tumor invasion by down-regulating MMP genes
(58).
[0084] Ribozymes:
[0085] Ribozymes are being increasingly used for the
sequence-specific inhibition of gene expression by the cleavage of
mRNAs encoding proteins of interest. The possibility of designing
ribozymes to cleave any specific target RNA has rendered them
valuable tools in both basic research and therapeutic applications.
In the therapeutics area, ribozymes have been exploited to target
viral RNAs in infectious diseases, dominant oncogenes in cancers
and specific somatic mutations in genetic disorders. Most notably,
several ribozyme gene therapy protocols for HIV patients are
already in Phase 1 trials (62). More recently, ribozymes have been
used for transgenic animal research, gene target validation and
pathway elucidation. Several ribozymes are in various stages of
clinical trials. ANGIOZYME was the first chemically synthesized
ribozyme to be studied in human clinical trials. ANGIOZYME
specifically inhibits formation of the VEGF-r (Vascular Endothelial
Growth Factor receptor), a key component in the angiogenesis
pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms
have demonstrated the importance of anti-angiogenesis therapeutics
in animal models. HEPTAZYME, a ribozyme designed to selectively
destroy Hepatitis C Virus (HCV) RNA, was found effective in
decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme
Pharmaceuticals, Incorporated--WEB home page).
[0086] Gene Disruption in Animal Models:
[0087] The emergence of gene inactivation by homologous
recombination methodology in embryonic stem cells has
revolutionized the field of mouse genetics. The availability of a
rapidly growing number of mouse null mutants has represented an
invaluable source of knowledge on mammalian development, cellular
biology and physiology, and has provided many models for human
inherited diseases. Animal models are required for an effective
drug delivery development program and evaluation of gene therapy
approach. The improvement of the original knockout strategy, as
well as exploitation of exogenous enzymatic systems that are active
in the recombination process, has been considerably extended the
range of genetic manipulations that can be produced. Additional
methods have been developed to provide versatile research tools:
Double replacement method, sequential gene targeting, conditional
cell type specific gene targeting, single copy integration method,
inducible gene targeting, gene disruption by viral delivery,
replacing one gene with another, the so called knock-in method and
the induction of specific balanced chromosomal translocation. It is
now possible to introduce a point mutation as a unique change in
the entire genome, therefore allowing very fine dissection of gene
function in vivo. Furthermore, the advent of methods allowing
conditional gene targeting opens the way for analysis of
consequence of a particular mutation in a defined organ and at a
specific time during the life of the experimental animal (59).
[0088] DNA Vaccination:
[0089] Observations in the early 1990s that plasmid DNA could
directly transfect animal cells in vivo sparked exploration of the
use of DNA plasmids to induce immune response by direct injection
into animal of DNA encoding antigenic protein. When a DNA vaccine
plasmid enters the eukaryotic cell, the protein it encodes is
transcribed and translated within the cell. In the case of
pathogens, these proteins are presented to the immune system in
their native form, mimicking the presentation of antigens during a
natural infection. DNA vaccination is particularly useful for the
induction of T cell activation. It was applied for viral and
bacterial infectious diseases, as well as for allergy and for
cancer. The central hypothesis behind active specific immunotherapy
for cancer is that tumor cells express unique antigens that should
stimulate the immune system. The first DNA vaccine against tumor
was carcino-embrionic antigen (CEA). DNA vaccinated animals
expressed immunoprotection and immunotherapy of human
CEA-expressing syngeneic mouse colon and breast carcinoma (61). In
a mouse model of neuroblastoma, DNA immunization with HuD resulted
in tumor growth inhibition with no neurological disease (60).
Immunity to the brown locus protein, gp.sup.75 tyrosinase-related
protein-1, associated with melanoma, was investigated in a
syngeneic mouse model.
[0090] Priming with human gp75 DNA broke tolerance to mouse gp75.
Immunity against mouse gp75 provided significant tumor protection
(60).
[0091] Glycosyl Hydrolases:
[0092] Glycosyl hydrolases are a widespread group of enzymes that
hydrolyze the o-glycosidic bond between two or more carbohydrates
or between a carbohydrate and a noncarbohydrate moiety. The
enzymatic hydrolysis of glycosidic bond occurs by using major one
or two mechanisms leading to overall retention or inversion of the
anomeric configuration. In both mechanisms catalysis involves two
residues: a proton donor and a nucleophile. Glycosyl hydrolyses
have been classified into 58 families based on amino acid
similarities. The glycosyl hydrolyses from families 1, 2, 5, 10,
17, 30, 35, 39 and 42 act on a large variety of substrates,
however, they all hydrolyze the glycosidic bond in a general acid
catalysis mechanism, with retention of the anomeric configuration.
The mechanism involves two glutamic acid residues, which are the
proton donors and the nucleophile, with an aspargine always
preceding the proton donor. Analyses of a set of known 3D
structures from this group revealed that their catalytic domains,
despite the low level of sequence identity, adopt a similar
(.alpha./.beta.) 8 fold with the proton donor and the nucleophile
located at the C-terminal ends of strands .beta.4 and .beta.7,
respectively. Mutations in the functional conserved amino acids of
lysosomal glycosyl hydrolases were identified in lysosomal storage
diseases.
[0093] Lysosomal glycosyl hydrolases including
.beta.3-glucuronidase, .beta.-manosidase,
.beta.-glucocerebrosidase, .beta.-galactosidase and .alpha.-L
iduronidase, are all exo-glycosyl hydrolases, belong to the GH-A
clan and share a similar catalytic site. However, many
endo-glucanases from various organisms, such as bacterial and
fungal xylenases and cellulases share this catalytic domain.
[0094] Genomic Sequence of Hpa Gene and its Implications:
[0095] It is well established that heparanase activity is
correlated with cancer metastasis. This correlation was
demonstrated at the level of enzymatic activity as well as the
levels of protein and hpa cDNA expression in highly metastatic
cancer cells as compared with non-metastatic cells. As such,
inhibition of heparanase activity is desirable, and has been
attempted by several means. The genomic region, encoding the hpa
gene and the surrounding, provides a new powerful tool for
regulation of heparanase activity at the level of gene expression.
Regulatory sequences may reside in noncoding regions both upstream
and downstream the transcribed region as well as in intron
sequences. A DNA sequence upstream of the transcription start site
contains the promoter region and potential regulatory elements.
Regulatory factors, which interact with the promoter region may be
identified and be used as potential drugs for inhibition of cancer,
metastasis and inflammation. The promoter region can be used to
screen for inhibitors of heparanase gene expression. Furthermore,
the hpa promoter can be used to direct cell specific, particularly
cancer cell specific, expression of foreign genes, such as
cytotoxic or apoptotic genes, in order to specifically destroy
cancer cells.
[0096] Cancer and yet unknown related genetic disorders may involve
rearrangements and mutations in the heparanase gene, either in
coding or non-coding regions. Such mutations may affect expression
level or enzymatic activity. The genomic sequence of hpa enables
the amplification of specific genomic DNA fragments, identification
and diagnosis of mutations.
[0097] There is thus a widely recognized need for, and it would be
highly advantageous to have genomic, cDNA and composite
polynucleotides encoding a polypeptide having heparanase activity,
vectors including same, genetically modified cells expressing
heparanase and a recombinant protein having heparanase activity, as
well as antisense oligonucleotides, constructs and ribozymes which
can be used for down regulation heparanase activity.
SUMMARY OF THE INVENTION
[0098] Cloning of the human hpa gene which encodes heparanase, and
expression of recombinant heparanase by transfected host cells is
reported herein, as well as downregulation of heparanase activity
by antisense technology.
[0099] A purified preparation of heparanase isolated from human
hepatoma cells was subjected to tryptic digestion and
microsequencing. The YGPDVGQPR (SEQ ID NO:8) sequence revealed was
used to screen EST databases for homology to the corresponding back
translated DNA sequence. Two closely related EST sequences were
identified and were thereafter found to be identical. Both clones
contained an insert of 1020 bp which included an open reading frame
of 973 bp followed by a 27 bp of 3' untranslated region and a Poly
A tail. Translation start site was not identified.
[0100] Cloning of the missing 5' end of hpa was performed by PCR
amplification of DNA from placenta Marathon RACE cDNA composite
using primers selected according to the EST clones sequence and the
linkers of the composite. A 900 bp PCR fragment, partially
overlapping with the identified 3' encoding EST clones was
obtained. The joined cDNA fragment (hpa), 1721 bp long (SEQ ID
NO:9), contained an open reading frame which encodes a polypeptide
of 543 amino acids (SEQ ID NO:10) with a calculated molecular
weight of 61,192 daltons.
[0101] Cloning an extended 5' sequence was enabled from the human
SK-hep1 cell line by PCR amplification using the Marathon RACE. The
5' extended sequence of the SK-hep1 hpa cDNA was assembled with the
sequence of the hpa cDNA isolated from human placenta (SEQ ID
NO:9). The assembled sequence contained an open reading frame, SEQ
ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs:14 and 15,
a polypeptide of 592 amino acids with a calculated molecular weight
of 66,407 daltons.
[0102] The ability of the hpa gene product to catalyze degradation
of heparan sulfate in an in vitro assay was examined by expressing
the entire open reading frame of hpa in insect cells, using the
Baculovirus expression system. Extracts and conditioned media of
cells infected with virus containing the hpa gene, demonstrated a
high level of heparan sulfate degradation activity both towards
soluble ECM-derived HSPG and intact ECM. This degradation activity
was inhibited by heparin, which is another substrate of heparanase.
Cells infected with a similar construct containing no hpa gene had
no such activity, nor did non-infected cells. The ability of
heparanase expressed from the extended 5' clone towards heparin was
demonstrated in a mammalian expression system.
[0103] The expression pattern of hpa RNA in various tissues and
cell lines was investigated using RT-PCR. It was found to be
expressed only in tissues and cells previously known to have
heparanase activity.
[0104] A panel of monochromosomal human/CHO and human/mouse somatic
cell hybrids was used to localize the human heparanase gene to
human chromosome 4. The newly isolated heparanase sequence can be
used to identify a chromosome region harboring a human heparanase
gene in a chromosome spread.
[0105] A human genomic library was screened and the human locus
harboring the heparanase gene isolated, sequenced and
characterized. Alternatively spliced heparanase mRNAs were
identified and characterized. The human heparanase promoter has
been isolated, identified and positively tested for activity. The
mouse heparanase promoter has been isolated and identified as well.
Antisense heparanase constructs were prepared and their influence
on cells in vitro tested. A predicted heparanase active site was
identified. And finally, the presence of sequences hybridizing with
human heparanase sequences was demonstrated for a variety of
mammalians and for an avian.
[0106] According to one aspect of the present invention there is
provided an isolated nucleic acid comprising a genomic,
complementary or composite polynucleotide sequence encoding a
polypeptide having heparanase catalytic activity.
[0107] According to further features in preferred embodiments of
the invention described below, the polynucleotide or a portion
thereof is hybridizable with SEQ ID NOs: 9, 13, 42, 43 or a portion
thereof at 68.degree. C. in 6.times.SSC, 1% SDS, 5.times. Denharts,
10% dextran sulfate, 100 .mu.g/ml salmon sperm DNA, and .sup.32p
labeled probe and wash at 68.degree. C. with 3.times.SSC and 0.1%
SDS.
[0108] According to still further features in the described
preferred embodiments the polynucleotide or a portion thereof is at
least 60% identical with SEQ ID NOs: 9, 13, 42, 43 or portions
thereof as determined using the Bestfit procedure of the DNA
sequence analysis software package developed by the Genetic
Computer Group (GCG) at the university of Wisconsin (gap creation
penalty--12, gap extension penalty--4).
[0109] According to still further features in the described
preferred embodiments the polypeptide is as set forth in SEQ ID
NOs:10, 14, 44 or portions thereof.
[0110] According to still further features in the described
preferred embodiments the polypeptide is at least 60% homologous to
SEQ ID NOs: 10, 14, 44 or portions thereof as determined with the
Smith-Waterman algorithm, using the Bioaccelerator platform
developed by Compugene (gapop: 10.0, gapext: 0.5, matrix:
blosum62).
[0111] According to additional aspects of the present invention
there are provided a nucleic acid construct (vector) comprising the
isolated nucleic acid described herein and a host cell comprising
the construct.
[0112] According to a further aspect of the present invention there
is provided an antisense oligonucleotide comprising a
polynucleotide or a polynucleotide analog of at least 10 bases
being hybridizable in vivo, under physiological conditions, with a
portion of a polynucleotide strand encoding a polypeptide having
heparanase catalytic activity.
[0113] According to an additional aspect of the present invention
there is provided a method of in vivo downregulating heparanase
activity comprising the step of in vivo administering the antisense
oligonucleotide herein described.
[0114] According to yet an additional aspect of the present
invention there is provided a pharmaceutical composition comprising
the antisense oligonucleotide herein described and a
pharmaceutically acceptable carrier.
[0115] According to still an additional aspect of the present
invention there is provided a ribozyme comprising the antisense
oligonucleotide described herein and a ribozyme sequence.
[0116] According to a further aspect of the present invention there
is provided an antisense nucleic acid construct comprising a
promoter sequence and a polynucleotide sequence directing the
synthesis of an antisense RNA sequence of at least 10 bases being
hybridizable in vivo, under physiological conditions, with a
portion of a polynucleotide strand encoding a polypeptide having
heparanase catalytic activity.
[0117] According to further features in preferred embodiments of
the invention described below, the polynucleotide strand encoding
the polypeptide having heparanase catalytic activity is as set
forth in SEQ ID NOs: 9, 13, 42 or 43.
[0118] According to still further features in the described
preferred embodiments the polypeptide having heparanase catalytic
activity is as set forth in SEQ ID NOs: 10, 14 or 44.
[0119] According to still a further aspect of the present invention
there is provided a method of in vivo downregulating heparanase
activity comprising the step of in vivo administering the antisense
nucleic acid construct herein described.
[0120] According to yet a further aspect of the present invention
there is provided a pharmaceutical composition comprising the
antisense nucleic acid construct herein described and a
pharmaceutically acceptable carrier.
[0121] According to a further aspect of the present invention there
is provided a nucleic acid construct comprising a polynucleotide
sequence functioning as a promoter, the polynucleotide sequence is
derived from SEQ ID NO:42 and includes at least nucleotides
2535-2635 thereof or from SEQ ID NO:43 and includes at least
nucleotides 320-420.
[0122] According to a further aspect of the present invention there
is provided a method of expressing a polynucleotide sequence
comprising the step of ligating the polynucleotide sequence into
the nucleic acid construct described above, downstream of the
polynucleotide sequence derived from SEQ ID NOs:42 or 43.
[0123] According to a further aspect of the present invention there
is provided a recombinant protein comprising a polypeptide having
heparanase catalytic activity.
[0124] According to further features in preferred embodiments of
the invention described below, the polypeptide includes at least a
portion of SEQ ID NOs:10, 14 or 44.
[0125] According to still further features in the described
preferred embodiments the protein is encoded by a polynucleotide
hybridizable with SEQ ID NOs: 9, 13, 42, 43 or a portion thereof at
68.degree. C. in 6.times.SSC, 1% SDS, 5.times. Denharts, 10%
dextran sulfate, 100 .mu.g/ml salmon sperm DNA, and .sup.32p
labeled probe and wash at 68.degree. C. with 3.times.SSC and 0.1%
SDS.
[0126] According to still further features in the described
preferred embodiments the protein is encoded by a polynucleotide at
least 60% identical with SEQ ID NOs: 9, 13, 42, 43 or portions
thereof as determined using the Bestfit procedure of the DNA
sequence analysis software package developed by the Genetic
Computer Group (GCG) at the university of Wisconsin (gap creation
penalty--12, gap extension penalty--4).
[0127] According to a further aspect of the present invention there
is provided a pharmaceutical composition comprising, as an active
ingredient, the recombinant protein herein described.
[0128] According to a further aspect of the present invention there
is provided a method of identifying a chromosome region harboring a
heparanase gene in a chromosome spread comprising the steps of (a)
hybridizing the chromosome spread with a tagged polynucleotide
probe encoding heparanase; (b) washing the chromosome spread,
thereby removing excess of non-hybridized probe; and (c) searching
for signals associated with the hybridized tagged polynucleotide
probe, wherein detected signals being indicative of a chromosome
region harboring a heparanase gene.
[0129] According to a further aspect of the present invention there
is provided a method of in vivo eliciting anti-heparanase
antibodies comprising the steps of administering a nucleic acid
construct including a polynucleotide segment corresponding to at
least a portion of SEQ ID NOs:9, 13 or 43 and a promoter for
directing the expression of said polynucleotide segment in vivo.
Accordingly, there is provided also a DNA vaccine for in vivo
eliciting anti-heparanase antibodies comprising a nucleic acid
construct including a polynucleotide segment corresponding to at
least a portion of SEQ ID NOs:9, 13 or 43 and a promoter for
directing the expression of said polynucleotide segment in
vivo.
[0130] The present invention can be used to develop new drugs to
inhibit tumor cell metastasis, inflammation and autoimmunity. The
identification of the hpa gene encoding for heparanase enzyme
enables the production of a recombinant enzyme in heterologous
expression systems. Additional features, advantages, uses and
applications of the present invention in biological science and in
diagnostic and therapeutic medicine are described hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0131] The invention herein described, by way of example only, with
reference to the accompanying drawings, wherein:
[0132] FIG. 1 presents nucleotide sequence and deduced amino acid
sequence of hpa cDNA. A single nucleotide difference at position
799 (A to T) between the EST (Expressed Sequence Tag) and the PCR
amplified cDNA (reverse transcribed RNA) and the resulting amino
acid substitution (Tyr to Phe) are indicated above and below the
substituted unit, respectively. Cysteine residues and the poly
adenylation consensus sequence are underlined. The asterisk denotes
the stop codon TGA.
[0133] FIG. 2 demonstrates degradation of soluble sulfate labeled
HSPG substrate by lysates of High Five cells infected with pFhpa2
virus. Lysates of High Five cells that were infected with pFhpa2
virus (.circle-solid.) or control pF2 virus (.quadrature.) were
incubated (18 h, 37.degree. C.) with sulfate labeled ECM-derived
soluble HSPG (peak I). The incubation medium was then subjected to
gel filtration on Sepharose 6B. Low molecular weight HS degradation
fragments (peak II) were produced only during incubation with the
pFhpa2 infected cells, but there was no degradation of the HSPG
substrate () by lysates of pF2 infected cells.
[0134] FIGS. 3a-b demonstrate degradation of soluble sulfate
labeled HSPG substrate by the culture medium of pFhpa2 and pFhpa4
infected cells. Culture media of High Five cells infected with
pFhpa2 (3a) or pFhpa4 (3b) viruses (.circle-solid.), or with
control viruses (.quadrature.) were incubated (18 h, 37.degree. C.)
with sulfate labeled ECM-derived soluble HSPG (peak I, ). The
incubation media were then subjected to gel filtration on Sepharose
6B. Low molecular weight HS degradation fragments (peak II) were
produced only during incubation with the hpa gene containing
viruses. There was no degradation of the HSPG substrate by the
culture medium of cells infected with control viruses.
[0135] FIG. 4 presents size fractionation of heparanase activity
expressed by pFhpa2 infected cells. Culture medium of pFhpa2
infected High Five cells was applied onto a 50 kDa cut-off
membrane. Heparanase activity (conversion of the peak I substrate,
() into peak II HS degradation fragments) was found in the high
(>50 kDa) (.circle-solid.), but not low (<50 kDa)
(.smallcircle.) molecular weight compartment.
[0136] FIGS. 5a-b demonstrate the effect of heparin on heparanase
activity expressed by pFhpa2 and pFhpa4 infected High Five cells.
Culture media of pFhpa2 (5a) and pFhpa4 (5b) infected High Five
cells were incubated (18 h, 37.degree. C.) with sulfate labeled
ECM-derived soluble HSPG (peak I, ) in the absence (.circle-solid.)
or presence (A) of 10 .mu.g/ml heparin. Production of low molecular
weight HS degradation fragments was completely abolished in the
presence of heparin, a potent inhibitor of heparanase activity (6,
7).
[0137] FIGS. 6a-b demonstrate degradation of sulfate labeled intact
ECM by virus infected High Five and Sf21 cells. High Five (6a) and
Sf21 (6b) cells were plated on sulfate labeled ECM and infected (48
h, 28.degree. C.) with pFhpa4 (.circle-solid.) or control pF1
(.quadrature.) viruses. Control non-infected Sf21 cells (R) were
plated on the labeled ECM as well. The pH of the cultured medium
was adjusted to 6.0-6.2 followed by 24 h incubation at 37.degree.
C. Sulfate labeled material released into the incubation medium was
analyzed by gel filtration on Sepharose 6B. HS degradation
fragments were produced only by cells infected with the hpa
containing virus.
[0138] FIG. 7a-b demonstrate degradation of sulfate labeled intact
ECM by virus infected cells. High Five (7a) and Sf21 (7b) cells
were plated on sulfate labeled ECM and infected (48 h, 28.degree.
C.) with pFhpa4 (.circle-solid.) or control pF1 (.quadrature.)
viruses. Control non-infected Sf21 cells (R) were plate on labeled
ECM as well. The pH of the cultured medium was adjusted to 6.0-6.2,
followed by 48 h incubation at 28.degree. C. Sulfate labeled
degradation fragments released into the incubation medium was
analyzed by gel filtration on Sepharose 6B. HS degradation
fragments were produced only by cells infected with the hpa
containing virus.
[0139] FIGS. 8a-b demonstrate degradation of sulfate labeled intact
ECM by the culture medium of pFhpa4 infected cells. Culture media
of High Five (8a) and Sf21 (8b) cells that were infected with
pFhpa4 (.circle-solid.) or control pF1 (.epsilon.) viruses were
incubated (48 h, 37.degree. C., pH 6.0) with intact sulfate labeled
ECM. The ECM was also incubated with the culture medium of control
non-infected Sf21 cells (R). Sulfate labeled material released into
the reaction mixture was subjected to gel filtration analysis.
Heparanase activity was detected only in the culture medium of
pFhpa4 infected cells.
[0140] FIGS. 9a-b demonstrate the effect of heparin on heparanase
activity in the culture medium of pFhpa4 infected cells. Sulfate
labeled ECM was incubated (24 h, 37.degree. C., pH 6.0) with
culture medium of pFhpa4 infected High Five (9a) and Sf21 (9b)
cells in the absence (.circle-solid.) or presence (V) of 10
.mu.g/ml heparin. Sulfate labeled material released into the
incubation medium was subjected to gel filtration on Sepharose 6B.
Heparanase activity (production of peak II HS degradation
fragments) was completely inhibited in the presence of heparin.
[0141] FIGS. 10a-b demonstrate purification of recombinant
heparanase on heparin-Sepharose. Culture medium of Sf21 cells
infected with pFhpa4 virus was subjected to heparin-Sepharose
chromatography. Elution of fractions was performed with 0.35-2 M
NaCl gradient (). Heparanase activity in the eluted fractions is
demonstrated in FIG. 10a (.circle-solid.). Fractions 15-28 were
subjected to 15% SDS-polyacrylamide gel electrophoresis followed by
silver nitrate staining. A correlation is demonstrated between a
major protein band (MW.about.63,000) in fractions 19-24 and
heparanase activity.
[0142] FIGS. 11a-b demonstrate purification of recombinant
heparanase on a Superdex 75 gel filtration column. Active fractions
eluted from heparin-Sepharose (FIG. 10a) were pooled, concentrated
and applied onto Superdex 75 FPLC column. Fractions were collected
and aliquots of each fraction were tested for heparanase activity
(c, FIG. 11a) and analyzed by SDS-polyacrylamide gel
electrophoresis followed by silver nitrate staining (FIG. 11b). A
correlation is seen between the appearance of a major protein band
(MW.about.63,000) in fractions 4-7 and heparanase activity.
[0143] FIGS. 12a-e demonstrate expression of the hpa gene by RT-PCR
with total RNA from human embryonal tissues (12a), human
extra-embryonal tissues (12b) and cell lines from different origins
(12c-e). RT-PCR products using hpa specific primers (I), primers
for GAPDH housekeeping gene (II), and control reactions without
reverse transcriptase demonstrating absence of genomic DNA or other
contamination in RNA samples (III). M-DNA molecular weight marker
VI (Boehringer Mannheim). For 12a: lane 1--neutrophil cells
(adult), lane 2--muscle, lane 3--thymus, lane 4--heart, lane
5--adrenal. For 12b: lane 1--kidney, lane 2--placenta (8 weeks),
lane 3--placenta (11 weeks), lanes 4-7--mole (complete hydatidiform
mole), lane 8--cytotrophoblast cells (freshly isolated), lane
9--cytotrophoblast cells (1.5 h in vitro), lane 10--cytotrophoblast
cells (6 h in vitro), lane 11--cytotrophoblast cells (18 h in
vitro), lane 12--cytotrophoblast cells (48 h in vitro). For 12c:
lane 1--JAR bladder cell line, lane 2--NCITT testicular tumor cell
line, lane 3--SW-480 human hepatoma cell line, lane 4--HTR
(cytotrophoblasts transformed by SV40), lane 5--HPTLP-I
hepatocellular carcinoma cell line, lane 6--EJ-28 bladder carcinoma
cell line. For 12d: lane 1--SK-hep-1 human hepatoma cell line, lane
2--DAMI human megakaryocytic cell line, lane 3--DAMI cell line+PMA,
lane 4--CHRF cell line+PMA, lane 5--CHRF cell line. For 12e: lane
1--ABAE bovine aortic endothelial cells, lane 2--1063 human ovarian
cell line, lane 3--human breast carcinoma MDA435 cell line, lane
4--human breast carcinoma MDA231 cell line.
[0144] FIG. 13 presents a comparison between nucleotide sequences
of the human hpa and a mouse EST cDNA fragment (SEQ ID NO:12) which
is 80% homologous to the 3' end (starting at nucleotide 1066 of SEQ
ID NO:9) of the human hpa. The aligned termination codons are
underlined.
[0145] FIG. 14 demonstrates the chromosomal localization of the hpa
gene. PCR products of DNA derived from somatic cell hybrids and of
genomic DNA of hamster, mouse and human of were separated on 0.7%
agarose gel following amplification with hpa specific primers. Lane
1--Lambda DNA digested with BstEII, lane 2--no DNA control, lanes
3-29, PCR amplification products. Lanes 3-5--human, mouse and
hamster genomic DNA, respectively. Lanes 6-29, human
monochromosomal somatic cell hybrids representing chromosomes 1-22
and X and Y, respectively. Lane 30--Lambda DNA digested with
BstEII. An amplification product of approximately 2.8 Kb is
observed only in lanes 5 and 9, representing human genomic DNA and
DNA derived from cell hybrid carrying human chromosome 4,
respectively. These results demonstrate that the hpa gene is
localized in human chromosome 4.
[0146] FIG. 15 demonstrates the genomic exon-intron structure of
the human hpa locus (top) and the relative positions of the lambda
clones used as sequencing templates to sequence the locus (below).
The vertical rectangles represent exons (E) and the horizontal
lines therebetween represent introns (I), upstream (U) and
downstream (D) regions. Continuous lines represent DNA fragments,
which were used for sequence analysis. The discontinuous line in
lambda 6 represent a region, which overlaps with lambda 8 and hence
was not analyzed. The plasmid contains a PCR product, which bridges
the gap between L3 and L6.
[0147] FIG. 16 presents the nucleotide sequence of the genomic
region of the hpa gene. Exon sequences appear in upper case and
intron sequences in lower case. The deduced amino acid sequence of
the exons is printed below the nucleotide sequence. Two predicted
transcription start sites are shown in bold.
[0148] FIG. 17 presents an alignment of the amino acid sequences of
human heparanase, mouse and partial sequences of rat homologues.
The human and the mouse sequences were determined by sequence
analysis of the isolated cDNAs. The rat sequence is derived from
two different EST clones, which represent two different regions (5'
and 3') of the rat hpa cDNA. The human sequence and the amino acids
in the mouse and rat homologues, which are identical to the human
sequence, appear in bold.
[0149] FIG. 18 presents a heparanase Zoo blot. Ten micrograms of
genomic DNA from various sources were digested with EcoRI and
separated on 0.7% agarose--TBE gel. Following electrophoresis, the
was gel treated with HCl and than with NaOH and the DNA fragments
were downward transferred to a nylon membrane (Hybond N+, Amersham)
with 0.4 N NaOH. The membrane was hybridized with a 1.6 Kb DNA
probe that contained the entire hpa cDNA. Lane order: H--Human;
M--Mouse; Rt--Rat; P--Pig; Cw--Cow; Hr--Horse; S--Sheep;
Rb--Rabbit; D--Dog; Ch--Chicken; F--Fish. Size markers (Lambda
BsteII) are shown on the left
[0150] FIG. 19 demonstrates the secondary structure prediction for
heparanase performed using the PHD server--Profile network
Prediction Heidelberg. H--helix, E--extended (beta strand), The
glutamic acid predicted as the proton donor is marked by asterisk
and the possible nucleophiles are underlined.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0151] The present invention is of a polynucleotide or nucleic
acid, referred to hereinbelow interchangeably as hpa, hpa cDNA or
hpa gene or identified by its SEQ ID NOs, encoding a polypeptide
having heparanase activity, vectors or nucleic acid constructs
including same and which are used for over-expression or antisense
inhibition of heparanase, genetically modified cells expressing
same, recombinant protein having heparanase activity, antisense
oligonucleotides and ribozymes for heparanase modulation, and
heparanase promoter sequences which can be used to direct the
expression of desired genes.
[0152] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0153] Cloning of the human and mouse hpa genes, cDNAs and genomic
sequence (for human), encoding heparanase and expressing
recombinant heparanase by transfected cells is reported herein.
These are the first mammalian heparanase genes to be cloned.
[0154] A purified preparation of heparanase isolated from human
hepatoma cells was subjected to tryptic digestion and
microsequencing.
[0155] The YGPDVGQPR (SEQ ID NO:8) sequence revealed was used to
screen EST databases for homology to the corresponding back
translated DNA sequences. Two closely related EST sequences were
identified and were thereafter found to be identical.
[0156] Both clones contained an insert of 1020 bp which includes an
open reading frame of 973 bp followed by a 3' untranslated region
of 27 bp and a Poly A tail, whereas a translation start site was
not identified.
[0157] Cloning of the missing 5' end was performed by PCR
amplification of DNA from placenta Marathon RACE cDNA composite
using primers selected according to the EST clones sequence and the
linkers of the composite.
[0158] A 900 bp PCR fragment, partially overlapping with the
identified 3' encoding EST clones was obtained. The joined cDNA
fragment (hpa), 1721 bp long (SEQ ID NO:9), contained an open
reading frame which encodes, as shown in FIG. 1 and SEQ ID NO:11, a
polypeptide of 543 amino acids (SEQ ID NO:10) with a calculated
molecular weight of 61,192 daltons.
[0159] A single nucleotide difference at position 799 (A to T)
between the EST clones and the PCR amplified cDNA was observed.
This difference results in a single amino acid substitution (Tyr to
Phe) (FIG. 1). Furthermore, the published EST sequences contained
an unidentified nucleotide, which following DNA sequencing of both
the EST clones was resolved into two nucleotides (G and C at
positions 1630 and 1631 in SEQ ID NO:9, respectively).
[0160] The ability of the hpa gene product to catalyze degradation
of heparan sulfate in an in vitro assay was examined by expressing
the entire open reading frame in insect cells, using the
Baculovirus expression system.
[0161] Extracts and conditioned media of cells infected with virus
containing the hpa gene, demonstrated a high level of heparan
sulfate degradation activity both towards soluble ECM-derived HSPG
and intact ECM, which was inhibited by heparin, while cells
infected with a similar construct containing no hpa gene had no
such activity, nor did non-infected cells.
[0162] The expression pattern of hpa RNA in various tissues and
cell lines was investigated using RT-PCR. It was found to be
expressed only in tissues and cells previously known to have
heparanase activity.
[0163] Cloning an extended 5' sequence was enabled from the human
SK-hep1 cell line by PCR amplification using the Marathon RACE. The
5' extended sequence of the SK-hep1 hpa cDNA was assembled with the
sequence of the hpa cDNA isolated from human placenta (SEQ ID
NO:9). The assembled sequence contained an open reading frame, SEQ
ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs:14 and 15,
a polypeptide of 592 amino acids, with a calculated molecular
weight of 66,407 daltons. This open reading frame was shown to
direct the expression of catalytically active heparanase in a
mammalian cell expression system. The expressed heparanase was
detectable by anti heparanase antibodies in Western blot
analysis.
[0164] A panel of monochromosomal human/CHO and human/mouse somatic
cell hybrids was used to localize the human heparanase gene to
human chromosome 4. The newly isolated heparanase sequence can
therefore be used to identify a chromosome region harboring a human
heparanase gene in a chromosome spread.
[0165] The hpa cDNA was then used as a probe to screen a a human
genomic library. Several phages were positive. These phages were
analyzed and were found to cover most of the hpa locus, except for
a small portion which was recovered by bridging PCR. The hpa locus
covers about 50,000 bp. The hpa gene includes 12 exons separated by
11 introns.
[0166] RT-PCR performed on a variety of cells revealed
alternatively spliced hpa transcripts.
[0167] The amino acid sequence of human heparanase was used to
search for homologous sequences in the DNA and protein databases.
Several human EST's were identified, as well as mouse sequences
highly homologous to human heparanase. The following mouse EST's
were identified AA177901, AA674378, AA67997, AA047943, AA690179,
A1122034, all sharing an identical sequence and correspond to amino
acids 336-543 of the human heparanase sequence. The entire mouse
heparanase cDNA was cloned, based on the nucleotide sequence of the
mouse EST's using Marathon cDNA libraries. The mouse and the human
hpa genes share an average homology of 78% between the nucleotide
sequences and 81% similarity between the deduced amino acid
sequences. hpa homologous sequences from rat were also uncovered
(EST's A1060284 and A1237828).
[0168] Homology search of heparanase amino acid sequence against
the DNA and the protein databases and prediction of its protein
secondary structure enabled to identify candidate amino acids that
participate in the heparanase active site.
[0169] Expression of hpa antisense in mammalian cell lines resulted
in about five fold decrease in the number of recoverable cells as
compared to controls.
[0170] Human Hpa cDNA was shown to hybridize with genomic DNAs of a
variety of mammalian species and with an avian.
[0171] The human and mouse hpa promoters were identified and the
human promoter was tested positive in directing the expression of a
reporter gene.
[0172] Thus, according to the present invention there is provided
an isolated nucleic acid comprising a genomic, complementary or
composite polynucleotide sequence encoding a polypeptide having
heparanase catalytic activity.
[0173] The phrase "composite polynucleotide sequence" refers to a
sequence which includes exonal sequences required to encode the
polypeptide having heparanase activity, as well as any number of
intronal sequences. The intronal sequences can be of any source and
typically will include conserved splicing signal sequences. Such
intronal sequences may further include cis acting expression
regulatory elements.
[0174] The term "heparanase catalytic activity" or its equivalent
term "heparanase activity" both refer to a mammalian
endoglycosidase hydrolyzing activity which is specific for heparan
or heparan sulfate proteoglycan substrates, as opposed to the
activity of bacterial enzymes (heparinase I, II and III) which
degrade heparin or heparan sulfate by means of .beta.-elimination
(37).
[0175] According to a preferred embodiment of the present invention
the polynucleotide or a portion thereof is hybridizable with SEQ ID
NOs: 9, 13, 42, 43 or a portion thereof at 68.degree. C. in
6.times.SSC, 1% SDS, 5.times. Denharts, 10% dextran sulfate, 100
.mu.g/ml salmon sperm DNA, and .sup.32p labeled probe and wash at
68.degree. C. with 3, 2, 1, 0.5 or 0.1.times. SSC and 0.1% SDS.
[0176] According to another preferred embodiment of the present
invention the polynucleotide or a portion thereof is at least 60%,
preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, most
preferably, 95-100% identical with SEQ ID NOs: 9, 13, 42, 43 or
portions thereof as determined using the Bestfit procedure of the
DNA sequence analysis software package developed by the Genetic
Computer Group (GCG) at the university of Wisconsin (gap creation
penalty--12, gap extension penalty--4--which are the default
parameters).
[0177] According to another preferred embodiment of the present
invention the polypeptide encoded by the polynucleotide sequence is
as set forth in SEQ ID NOs:10, 14, 44 or portions thereof having
heparanase catalytic activity. Such portions are expected to
include amino acids Asp-Glu 224-225 (SEQ ID NO:10), which can serve
as proton donors and glutamic acid 343 or 396 which can serve as a
nucleophile.
[0178] According to another preferred embodiment of the present
invention the polypeptide encoded by the polynucleotide sequence is
at least 60%, preferably at least 65%, more preferably at least
70%, more preferably at least 75%, more preferably at least 80%,
more preferably at least 85%, more preferably at least 90%, most
preferably, 95-100% homologous (both similar and identical acids)
to SEQ ID NOs:10, 14, 44 or portions thereof as determined with the
Smith-Waterman algorithm, using the Bioaccelerator platform
developed by Compugene (gapop: 10.0, gapext: 0.5, matrix: blosum62,
see also the description to FIG. 17).
[0179] Further according to the present invention there is provided
a nucleic acid construct comprising the isolated nucleic acid
described herein. The construct may and preferably further include
an origin of replication and trans regulatory elements, such as
promoter and enhancer sequences.
[0180] The construct or vector can be of any type. It may be a
phage which infects bacteria or a virus which infects eukaryotic
cells. It may also be a plasmid, phagemid, cosmid, bacmid or an
artificial chromosome.
[0181] Further according to the present invention there is provided
a host cell comprising the nucleic acid construct described herein.
The host cell can be of any type. It may be a prokaryotic cell, an
eukaryotic cell, a cell line, or a cell as a portion of an
organism. The polynucleotide encoding heparanase can be permanently
or transiently present in the cell. In other words, genetically
modified cells obtained following stable or transient transfection,
transformation or transduction are all within the scope of the
present invention. The polynucleotide can be present in the cell in
low copy (say 1-5 copies) or high copy number (say 5-50 copies or
more). It may be integrated in one or more chromosomes at any
location or be present as an extrachromosomal material.
[0182] The present invention is further directed at providing a
heparanase over-expression system which includes a cell
overexpressing heparanase catalytic activity. The cell may be a
genetically modified host cell transiently or stably transfected or
transformed with any suitable vector which includes a
polynucleotide sequence encoding a polypeptide having heparanase
activity and a suitable promoter and enhancer sequences to direct
over-expression of heparanase. However, the overexpressing cell may
also be a product of an insertion (e.g., via homologous
recombination) of a promoter and/or enhancer sequence downstream to
the endogenous heparanase gene of the expressing cell, which will
direct over-expression from the endogenous gene.
[0183] The term "over-expression" as used herein in the
specification and claims below refers to a level of expression
which is higher than a basal level of expression typically
characterizing a given cell under otherwise identical
conditions.
[0184] According to another aspect the present invention provides
an antisense oligonucleotide comprising a polynucleotide or a
polynucleotide analog of at least 10, preferably 11-15, more
preferably 16-17, more preferably 18, more preferably 19-25, more
preferably 26-35, most preferably 35-100 bases being hybridizable
in vivo, under physiological conditions, with a portion of a
polynucleotide strand encoding a polypeptide having heparanase
catalytic activity. The antisense oligonucleotide can be used for
downregulating heparanase activity by in vivo administration
thereof to a patient. As such, the antisense oligonucleotide
according to the present invention can be used to treat types of
cancers which are characterized by impaired (over) expression of
heparanase, and are dependent on the expression of heparanase for
proliferating or forming metastases.
[0185] The antisense oligonucleotide can be DNA or RNA or even
include nucleotide analogs, examples of which are provided in the
Background section hereinabove. The antisense oligonucleotide
according to the present invention can be synthetic and is
preferably prepared by solid phase synthesis. In addition, it can
be of any desired length which still provides specific base pairing
(e.g., 8 or 10, preferably more, nucleotides long) and it can
include mismatches that do not hamper base pairing under
physiological conditions.
[0186] Further according to the present invention there is provided
a pharmaceutical composition comprising the antisense
oligonucleotide herein described and a pharmaceutically acceptable
carrier. The carrier can be, for example, a liposome loadable with
the antisense oligonucleotide.
[0187] According to a preferred embodiment of the present invention
the antisense oligonucleotide further includes a ribozyme sequence.
The ribozyme sequence serves to cleave a heparanase RNA molecule to
which the antisense oligonucleotide binds, to thereby downregulate
heparanase expression.
[0188] Further according to the present invention there is provided
an antisense nucleic acid construct comprising a promoter sequence
and a polynucleotide sequence directing the synthesis of an
antisense RNA sequence of at least 10 bases being hybridizable in
vivo, under physiological conditions, with a portion of a
polynucleotide strand encoding a polypeptide having heparanase
catalytic activity. Like the antisense oligonucleotide, the
antisense construct can be used for downregulating heparanase
activity by in vivo administration thereof to a patient. As such,
the antisense construct, like the antisense oligonucleotide,
according to the present invention can be used to treat types of
cancers which are characterized by impaired (over) expression of
heparanase, and are dependent on the expression of heparanase for
proliferating or forming metastases.
[0189] Thus, further according to the present invention there is
provided a pharmaceutical composition comprising the antisense
construct herein described and a pharmaceutically acceptable
carrier. The carrier can be, for example, a liposome loadable with
the antisense construct.
[0190] Formulations for topical administration may include, but are
not limited to, lotions, ointments, gels, creams, suppositories,
drops, liquids, sprays and powders. Conventional pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like
may be necessary or desirable. Coated condoms, stents, active pads,
and other medical devices may also be useful. Compositions for oral
administration include powders or granules, suspensions or
solutions in water or non-aqueous media, sachets, capsules or
tablets. Thickeners, diluents, flavorings, dispersing aids,
emulsifiers or binders may be desirable. Formulations for
parenteral administration may include, but are not limited to,
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives.
[0191] Dosing is dependent on severity and responsiveness of the
condition to be treated, but will normally be one or more doses per
day, week or month with course of treatment lasting from several
days to several months or until a cure is effected or a diminution
of disease state is achieved. Persons ordinarily skilled in the art
can easily determine optimum dosages, dosing methodologies and
repetition rates.
[0192] Further according to the present invention there is provided
a nucleic acid construct comprising a polynucleotide sequence
functioning as a promoter, the polynucleotide sequence is derived
from SEQ ID NO:42 and includes at least nucleotides 2135-2635,
preferably 2235-2635, more preferably 2335-2635, more preferably
2435-2635, most preferably 2535-2635 thereof, or SEQ ID NO:43 and
includes at least nucleotides 1-420, preferably 120-420, more
preferably 220-420, most preferably 320-420, thereof. These
nucleotides are shown in the example section that follows to direct
the synthesis of a reporter gene in transformed cells. Thus,
further according to the present invention there is provided a
method of expressing a polynucleotide sequence comprising the step
of ligating the polynucleotide sequence downstream to either of the
promoter sequences described herein. Heparanase promoters can be
isolated from a variety of mammalian an other species by cloning
genomic regions present 5' to the coding sequence thereof. This can
be readily achievable by one ordinarily skilled in the art using
the heparanase polynucleotides described herein, which are shown in
the Examples section that follows to participate in efficient cross
species hybridization.
[0193] Further according to the present invention there is provided
a recombinant protein comprising a polypeptide having heparanase
catalytic activity. The protein according to the present invention
include modifications known as post translational modifications,
including, but not limited to, proteolysis (e.g., removal of a
signal peptide and of a pro- or preprotein sequence), methionine
modification, glycosylation, alkylation (e.g., methylation),
acetylation, etc. According to preferred embodiments the
polypeptide includes at least a portion of SEQ ID NOs:10, 14 or 44,
the portion has heparanase catalytic activity. According to
preferred embodiments of the present invention the protein is
encoded by any of the above described isolated nucleic acids.
Further according to the present invention there is provided a
pharmaceutical composition comprising, as an active ingredient, the
recombinant protein described herein.
[0194] The recombinant protein may be purified by any conventional
protein purification procedure close to homogeneity and/or be mixed
with additives. The recombinant protein may be manufactured using
any of the genetically modified cells described above, which
include any of the expression nucleic acid constructs described
herein. The recombinant protein may be in any form. It may be in a
crystallized form, a dehydrated powder form or in solution. The
recombinant protein may be useful in obtaining pure heparanase,
which in turn may be useful in eliciting anti-heparanase
antibodies, either poly or monoclonal antibodies, and as a
screening active ingredient in an anti-heparanase inhibitors or
drugs screening assay or system.
[0195] Further according to the present invention there is provided
a method of identifying a chromosome region harboring a human
heparanase gene in a chromosome spread. the method is executed
implementing the following method steps, in which in a first step
the chromosome spread (either interphase or metaphase spread) is
hybridized with a tagged polynucleotide probe encoding heparanase.
The tag is preferably a fluorescent tag. In a second step according
to the method the chromosome spread is washed, thereby excess of
non-hybridized probe is removed. Finally, signals associated with
the hybridized tagged polynucleotide probe are searched for,
wherein detected signals being indicative of a chromosome region
harboring the human heparanase gene. One ordinarily skilled in the
art would know how to use the sequences disclosed herein in
suitable labeling reactions and how to use the tagged probes to
detect, using in situ hybridization, a chromosome region harboring
a human heparanase gene.
[0196] Further according to the present invention there is provided
a method of in vivo eliciting anti-heparanase antibodies comprising
the steps of administering a nucleic acid construct including a
polynucleotide segment corresponding to at least a portion of SEQ
ID NOs:9, 13 or 43 and a promoter for directing the expression of
said polynucleotide segment in vivo. Accordingly, there is provided
also a DNA vaccine for in vivo eliciting anti-heparanase antibodies
comprising a nucleic acid construct including a polynucleotide
segment corresponding to at least a portion of SEQ ID NOs:9, 13 or
43 and a promoter for directing the expression of said
polynucleotide segment in vivo. The vaccine optionally further
includes a pharmaceutically acceptable carrier, such as a virus,
liposome or an antigen presenting cell. Alternatively, the vaccine
is employed as a naked DNA vaccine The present invention can be
used to develop treatments for various diseases, to develop
diagnostic assays for these diseases and to provide new tools for
basic research especially in the fields of medicine and
biology.
[0197] Specifically, the present invention can be used to develop
new drugs to inhibit tumor cell metastasis, inflammation and
autoimmunity. The identification of the hpa gene encoding for the
heparanase enzyme enables the production of a recombinant enzyme in
heterologous expression systems.
[0198] Furthermore, the present invention can be used to modulate
bioavailability of heparin-binding growth factors, cellular
responses to heparin-binding growth factors (e.g., bFGF, VEGF) and
cytokines (e.g., IL-8), cell interaction with plasma lipoproteins,
cellular susceptibility to viral, protozoa and some bacterial
infections, and disintegration of neurodegenerative plaques.
Recombinant heparanase offers a potential treatment for wound
healing, angiogenesis, restenosis, atherosclerosis, inflammation,
neurodegenerative diseases (such as, for example,
Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease, Scrape
and Alzheimer's disease) and certain viral and some bacterial and
protozoa infections. Recombinant heparanase can be used to
neutralize plasma heparin, as a potential replacement of
protamine.
[0199] As used herein, the term "modulate" includes substantially
inhibiting, slowing or reversing the progression of a disease,
substantially ameliorating clinical symptoms of a disease or
condition, or substantially preventing the appearance of clinical
symptoms of a disease or condition. A "modulator" therefore
includes an agent which may modulate a disease or condition.
Modulation of viral, protozoa and bacterial infections includes any
effect which substantially interrupts, prevents or reduces any
viral, bacterial or protozoa activity and/or stage of the virus,
bacterium or protozoon life cycle, or which reduces or prevents
infection by the virus, bacterium or protozoon in a subject, such
as a human or lower animal.
[0200] As used herein, the term "wound" includes any injury to any
portion of the body of a subject including, but not limited to,
acute conditions such as thermal burns, chemical burns, radiation
bums, bums caused by excess exposure to ultraviolet radiation such
as sunburn, damage to bodily tissues such as the perineum as a
result of labor and childbirth, including injuries sustained during
medical procedures such as episiotomies, trauma-induced injuries
including cuts, those injuries sustained in automobile and other
mechanical accidents, and those caused by bullets, knives and other
weapons, and post-surgical injuries, as well as chronic conditions
such as pressure sores, bedsores, conditions related to diabetes
and poor circulation, and all types of acne, etc.
[0201] Anti-heparanase antibodies, raised against the recombinant
enzyme, would be useful for immunodetection and diagnosis of
micrometastases, autoimmune lesions and renal failure in biopsy
specimens, plasma samples, and body fluids. Such antibodies may
also serve as neutralizing agents for heparanase activity.
[0202] The genomic heparanase sequences described herein can be
used to construct knock-in and knock-out constructs. Such
constructs include a fragment of 10-20 Kb of a heparanase locus and
a negative and a positive selection markers and can be used to
provide heparanase knock-in and knock-out animal models by methods
known to the skilled artisan. Such animal models can be used for
studying the function of heparanase in developmental processes, and
in normal as well as pathological processes. They can also serve as
an experimental model for testing drugs and gene therapy protocols.
The complementary heparanase sequence (cDNA) can be used to derive
transgenic animals, overexpressing heparanase for same.
Alternatively, if cloned in the antisense orientation, the
complementary heparanase sequence (cDNA) can be used to derive
transgenic animals under-expressing heparanase for same.
[0203] The heparanase promoter sequences described herein and other
cis regulatory elements linked to the heparanase locus can be used
to regulated the expression of genes. For example, these promoters
can be used to direct the expression of a cytotoxic protein, such
as TNF, in tumor cells. It will be appreciated that heparanase
itself is abnormally expressed under the control of its own
promoter and other cis acting elements in a variety of tumors, and
its expression is correlated with metastasis. It is also abnormally
highly expressed in inflammatory cells. The introns of the
heparanase gene can be used for the same purpose, as it is known
that introns, especially upstream introns include cis acting
element which affect expression. A heparanase promoter fused to a
reporter protein can be used to study/monitor its activity.
[0204] The polynucleotide sequences described herein can also be
used to provide DNA vaccines which will elicit in vivo anti
heparanase antibodies. Such vaccines can therefore be used to
combat inflammatory and cancer.
[0205] Antisense oligonucleotides derived according to the
heparanase sequences described herein, especially such
oligonucleotides supplemented with ribozyme activity, can be used
to modulate heparanase expression. Such oligonucleotides can be
from the coding region, from the introns or promoter specific.
Antisense heparanase nucleic acid constructs can similarly
function, as well known in the art.
[0206] The heparanase sequences described herein can be used to
study the catalytic mechanism of heparanase. Carefully selected
site directed mutagenesis can be employed to provide modified
heparanase proteins having modified characteristics in terms of,
for example, substrate specificity, sensitivity to inhibitors,
etc.
[0207] While studying heparanase expression in a variety of cell
types alternatively spliced transcripts were identified. Such
transcripts if found characteristic of certain pathological
conditions can be used as markers for such conditions. Such
transcripts are expected to direct the synthesis of heparanases
with altered functions.
[0208] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0209] Generally, the nomenclature used herein and the laboratory
procedures in recombinant DNA technology described below are those
well known and commonly employed in the art. Standard techniques
are used for cloning, DNA and RNA isolation, amplification and
purification. Generally enzymatic reactions involving DNA ligase,
DNA polymerase, restriction endonucleases and the like are
performed according to the manufacturers' specifications. These
techniques and various other techniques are generally performed
according to Sambrook et al., Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989), which is incorporated herein by reference. Other general
references are provided throughout this document. The procedures
therein are believed to be well known in the art and are provided
for the convenience of the reader. All the information contained
therein is incorporated herein by reference.
[0210] The following protocols and experimental details are
referenced in the Examples that follow:
[0211] Purification and characterization of heparanase from a human
hepatoma cell line and human placenta: A human hepatoma cell line
(Sk-hep-1) was chosen as a source for purification of a human
tumor-derived heparanase. Purification was essentially as described
in U.S. Pat. No. 5,362,641 to Fuks, which is incorporated by
reference as if fully set forth herein. Briefly, 500 liter,
5.times.10.sup.11 cells were grown in suspension and the heparanase
enzyme was purified about 240,000 fold by applying the following
steps: (i) cation exchange (CM-Sephadex) chromatography performed
at pH 6.0, 0.3-1.4 M NaCl gradient; (ii) cation exchange
(CM-Sephadex) chromatography performed at pH 7.4 in the presence of
0.1% CHAPS, 0.3-1.1 M NaCl gradient; (iii) heparin-Sepharose
chromatography performed at pH 7.4 in the presence of 0.1% CHAPS,
0.35-1.1 M NaCl gradient; (iv) ConA-Sepharose chromatography
performed at pH 6.0 in buffer containing 0.1% CHAPS and 1 M NaCl,
elution with 0.25 M .alpha.-methyl mannoside; and (v) HPLC cation
exchange (Mono-S) chromatography performed at pH 7.4 in the
presence of 0.1% CHAPS, 0.25-1 M NaCl gradient.
[0212] Active fractions were pooled, precipitated with TCA and the
precipitate subjected to SDS polyacrylamide gel electrophoresis
and/or tryptic digestion and reverse phase HPLC. Tryptic peptides
of the purified protein were separated by reverse phase HPLC (C8
column) and homogeneous peaks were subjected to amino acid sequence
analysis.
[0213] The purified enzyme was applied to reverse phase HPLC and
subjected to N-terminal amino acid sequencing using the amino acid
sequencer (Applied Biosystems).
[0214] Cells: Cultures of bovine corneal endothelial cells (BCECs)
were established from steer eyes as previously described (19, 38).
Stock cultures were maintained in DMEM (1 g glucose/liter)
supplemented with 10% newborn calf serum and 5% FCS. bFGF (1 ng/ml)
was added every other day during the phase of active cell growth
(13, 14).
[0215] Preparation of dishes coated with ECM: BCECs (second to
fifth passage) were plated into 4-well plates at an initial density
of 2.times.10.sup.5 cells/ml, and cultured in sulfate-free Fisher
medium plus 5% dextran T-40 for 12 days. Na.sub.2.sup.35SO.sub.4
(25 .mu.Ci/ml) was added on day 1 and 5 after seeding and the
cultures were incubated with the label without medium change. The
subendothelial ECM was exposed by dissolving (5 min., room
temperature) the cell layer with PBS containing 0.5% Triton X-100
and 20 mM NH.sub.4OH, followed by four washes with PBS. The ECM
remained intact, free of cellular debris and firmly attached to the
entire area of the tissue culture dish (19, 22).
[0216] To prepare soluble sulfate labeled proteoglycans (peak I
material), the ECM was digested with trypsin (25 .mu.g/ml, 6 h,
37.degree. C.), the digest was concentrated by reverse dialysis and
the concentrated material was applied onto a Sepharose 6B gel
filtration column. The resulting high molecular weight material
(Kav<0.2, peak I) was collected. More than 80% of the labeled
material was shown to be composed of heparan sulfate proteoglycans
(11, 39).
[0217] Heparanase activity: Cells (1.times.10.sup.6/35-mm dish),
cell lysates or conditioned media were incubated on top of
.sup.35S-labeled ECM (18 h, 37.degree. C.) in the presence of 20 mM
phosphate buffer (pH 6.2). Cell lysates and conditioned media were
also incubated with sulfate labeled peak I material (10-20 .mu.l).
The incubation medium was collected, centrifuged (18,000.times.g,
4.degree. C., 3 min.), and sulfate labeled material analyzed by gel
filtration on a Sepharose CL-6B column (0.9.times.30 cm). Fractions
(0.2 ml) were eluted with PBS at a flow rate of 5 ml/h and counted
for radioactivity using Bio-fluor scintillation fluid. The excluded
volume (V.sub.o) was marked by blue dextran and the total included
volume (V.sub.t) by phenol red. The latter was shown to comigrate
with free sulfate (7, 11, 23). Degradation fragments of HS side
chains were eluted from Sepharose 6B at 0.5<Kav<0.8 (peak II)
(7, 11, 23). A nearly intact HSPG released from ECM by
trypsin--and, to a lower extent, during incubation with PBS
alone--was eluted next to V.sub.o (Kav<0.2, peak I). Recoveries
of labeled material applied on the columns ranged from 85 to 95% in
different experiments (11). Each experiment was performed at least
three times and the variation of elution positions (Kav values) did
not exceed +/-15%.
[0218] Cloning of hpa cDNA: cDNA clones 257548 and 260138 were
obtained from the I.M.A.G.E Consortium (2130 Memorial Parkway SW,
Hunstville, Ala. 35801). The cDNAs were originally cloned in EcoRI
and NotI cloning sites in the plasmid vector pT3T7D-Pac. Although
these clones are reported to be somewhat different, DNA sequencing
demonstrated that these clones are identical to one another.
Marathon RACE (rapid amplification of cDNA ends) human placenta
(poly-A) cDNA composite was a gift of Prof. Yossi Shiloh of Tel
Aviv University. This composite is vector free, as it includes
reverse transcribed cDNA fragments to which double, partially
single stranded adapters are attached on both sides. The
construction of the specific composite employed is described in
reference 39a.
[0219] Amplification of hp3 PCR fragment was performed according to
the protocol provided by Clontech laboratories. The template used
for amplification was a sample taken from the above composite. The
primers used for amplification were:
[0220] First step: 5'-primer: API: 5'-CCATCCTAATACGACTCACT
ATAGGGC-3', SEQ ID NO: 1; 3'-primer: HPL229: 5'-GTAGTGATGCCA
TGTAACTGAATC-3', SEQ ID NO:2.
[0221] Second step: nested 5'-primer: AP2: 5'-ACTCACTATAGGGCTCG
AGCGGC-3', SEQ ID NO:3; nested 3'-primer: HPL171:
5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:4. The HPL229 and HPL171
were selected according to the sequence of the EST clones. They
include nucleotides 933-956 and 876-897 of SEQ ID NO:9,
respectively.
[0222] PCR program was 94.degree. C.-4 min., followed by 30 cycles
of 94.degree. C.-40 sec., 62.degree. C.-1 min., 72.degree. C.-2.5
min. Amplification was performed with Expand High Fidelity
(Boehringer Mannheim). The resulting ca. 900 bp hp3 PCR product was
digested with BfrI and PvuII. Clone 257548 (phpa1) was digested
with EcoRI, followed by end filling and was then further digested
with BfrI. Thereafter the PvuII-BfrI fragment of the hp3 PCR
product was cloned into the blunt end-BfrI end of clone phpa1 which
resulted in having the entire cDNA cloned in pT3T7-pac vector,
designated phpa2.
[0223] RT-PCR: RNA was prepared using TRI-Reagent (Molecular
research center Inc.) according to the manufacturer instructions.
1.25 .mu.g were taken for reverse transcription reaction using
MuMLV Reverse transcriptase (Gibco BRL) and Oligo (dT).sub.15
primer, SEQ ID NO:5, (Promega). Amplification of the resultant
first strand cDNA was performed with Taq polymerase (Promega). The
following primers were used:
[0224] HPU-355: 5'-TTCGATCCCAAGAAGGAATCAAC-3', SEQ ID NO:6,
nucleotides 372-394 in SEQ ID NOs:9 or 11.
[0225] HPL-229: 5'-GTAGTGATGCCATGTAACTGAATC-3', SEQ ID NO:7,
nucleotides 933-956 in SEQ ID NOs:9 or 11.
[0226] PCR program: 94.degree. C.-4 min., followed by 30 cycles of
94.degree. C.-40 sec., 62.degree. C.-1 min., 72.degree. C.-1
min.
[0227] Alternatively, total RNA was prepared from cell cultures
using Tri-reagent (Molecular Research Center, Inc.) according to
the manufacturer recommendation. Poly A+ RNA was isolated from
total RNA using mRNA separator (Clontech). Reverse transcription
was performed with total RNA using Superscript II (GibcoBRL). PCR
was performed with Expand high fidelity (Boehringer Mannheim).
Primers used for amplification were as follows:
1 Hpu-685, 5'-GAGCAGCCAGGTGAGCCCAAGAT-3', SEQ ID NO:24 Hpu-355,
5'-TTCGATCCCAAGAAGGAATCAAC-3', SEQ ID NO:25 Hpu 565,
5'-AGCTCTGTAGATGTGCTATACAC-3', SEQ ID NO:26 Hpl 967,
5'-TCAGATGCAAGCACCAACTTTG- GC-3', SEQ ID NO:27 Hpl 171,
5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:28 Hpl 229,
5'-GTAGTGATGCCATGTAACTGAATC-3', SEQ ID NG:29
[0228] PCR reaction was performed as follows: 94.degree. C. 3
minutes, followed by 32 cycles of 94.degree. C. 40 seconds,
64.degree. C. 1 minute, 72.degree. C. 3 minutes, and one cycle
72.degree. C., 7 minutes.
[0229] Expression of recombinant heparanase in insect cells: Cells,
High Five and Sf21 insect cell lines were maintained as monolayer
cultures in SF900II-SFM medium (GibcoBRL).
[0230] Recombinant Baculovirus: Recombinant virus containing the
hpa gene was constructed using the Bac to Bac system (GibcoBRL).
The transfer vector pFastBac was digested with SalI and NotI and
ligated with a 1.7 kb fragment of phpa2 digested with XhoI and
NotI. The resulting plasmid was designated pFasthpa2. An identical
plasmid designated pFasthpa4 was prepared as a duplicate and both
independently served for further experimentations. Recombinant
bacmid was generated according to the instructions of the
manufacturer with pFasthpa2, pFasthpa4 and with pFastBac. The
latter served as a negative control. Recombinant bacmid DNAs were
transfected into Sf21 insect cells. Five days after transfection
recombinant viruses were harvested and used to infect High Five
insect cells, 3.times.10.sup.6 cells in T-25 flasks. Cells were
harvested 2-3 days after infection. 4.times.10.sup.6 cells were
centrifuged and resuspended in a reaction buffer containing 20 mM
phosphate citrate buffer, 50 mM NaCl. Cells underwent three cycles
of freeze and thaw and lysates were stored at -80.degree. C.
Conditioned medium was stored at 4.degree. C.
[0231] Partial purification of recombinant heparanase: Partial
purification of recombinant heparanase was performed by
heparin-Sepharose column chromatography followed by Superdex 75
column gel filtration. Culture medium (150 ml) of Sf21 cells
infected with pFhpa4 virus was subjected to heparin-Sepharose
chromatography. Elution of 1 ml fractions was performed with 0.35-2
M NaCl gradient in presence of 0.1% CHAPS and 1 mM DTT in 10 mM
sodium acetate buffer, pH 5.0. A 25 .mu.l sample of each fraction
was tested for heparanase activity. Heparanase activity was eluted
at the range of 0.65-1.1 M NaCl (fractions 18-26, FIG. 10a). 5
.mu.l of each fraction was subjected to 15% SDS-polyacrylamide gel
electrophoresis followed by silver nitrate staining. Active
fractions eluted from heparin-Sepharose (FIG. 10a) were pooled and
concentrated (.times.6) on YM3 cut-off membrane. 0.5 ml of the
concentrated material was applied onto 30 ml Superdex 75 FPLC
column equilibrated with 10 mM sodium acetate buffer, pH 5.0,
containing 0.8 M NaCl, 1 mM DTT and 0.1% CHAPS. Fractions (0.56 ml)
were collected at a flow rate of 0.75 ml/min. Aliquots of each
fraction were tested for heparanase activity and were subjected to
SDS-polyacrylamide gel electrophoresis followed by silver nitrate
staining (FIG. 11b).
[0232] PCR amplification of genoinic DNA: 94.degree. C. 3 minutes,
followed by 32 cycles of 94.degree. C. 45 seconds, 64.degree. C. 1
minute, 68.degree. C. 5 minutes, and one cycle at 72.degree. C., 7
minutes. Primers used for amplification of genomic DNA
included:
[0233] GHpu-L3 5'-AGGCACCCTAGAGATGTTCCAG-3', SEQ ID NO:30
[0234] GHpl-L6 5'-GAAGATTTCTGTTTCCATGACGTG-3', SEQ ID NO:31.
[0235] Screening of genomic libraries: A human genomic library in
Lambda phage EMBLE3 SP6/T7 (Clontech, Paulo Alto, Calif.) was
screened. 5.times.10.sup.5 plaques were plated at 5.times.10.sup.4
pfu/plate on NZCYM agar/top agarose plates. Phages were absorbed on
nylon membranes in duplicates (Qiagen). Hybridization was performed
at 65.degree. C. in 5.times.SSC, 5.times. Denhart's, 10% dextran
sulfate, 100 .mu.g/ml Salmon sperm, .sup.32p labeled probe
(10.sup.6 cpm/ml). A 1.6 kb fragment, containing the entire hpa
cDNA was labeled by random priming (Boehringer Mannheim). Following
hybridization membranes were washed once with 2.times.SSC, 0.1% SDS
at 65.degree. C. for 20 minutes, and twice with 0.2.times.SSC, 0.1%
SDS at 65.degree. C. for 15 minutes. Hybridizing plaques were
picked, and plated at 100 pfu/plate. Hybridization was performed as
above and single isolated positive plaques were picked.
[0236] Phage DNA was extracted using a Lambda DNA extraction kit
(Qiagen). DNA was digested with XhoI and EcoRI, separated on 0.7%
agarose gel and transferred to nylon membrane Hybond N+ (Amersham).
Hybridization and washes were performed as above.
[0237] cDNA Sequence analysis: Sequence determinations were
performed with vector specific and gene specific primers, using an
automated DNA sequencer (Applied Biosystems, model 373A). Each
nucleotide was read from at least two independent primers.
[0238] Genomic sequence analysis: Large-scale sequencing was
performed by Commonwealth Biotechnology Incorporation.
[0239] Isolation of mouse hpa: Mouse hpa cDNA was amplified from
either Marathon ready cDNA library of mouse embryo or from mRNA
isolated from mouse melanoma cell line BL6, using the Marathon RACE
kit from Clontech. Both procedures were performed according to the
manufacturer's recommendation.
[0240] Primers used for PCR amplification of mouse hpa:
[0241] Mhp1773 5'-CCACACTGAATGTAATACTGAAGTG-3', SEQ ID NO:32
[0242] MHp1736 5'-CGAAGCTCTGGAACTCGGCAAG-3', SEQ ID NO:33
[0243] MHp183 5'-GCCAGCTGCAAAGGTGTTGGAC-3', SEQ ID NO:34
[0244] Mhp1152 5'-AACACCTGCCTCATCACGACTTC-3', SEQ ID NO:35
[0245] Mhp114 5'-GCCAGGCTGGCGTCGATGGTGA-3', SEQ ID NO:36
[0246] MHp1103 5'-GTCGATGGTGATGGACAGGAAC-3', SEQ ID NO:37
[0247] Ap1 5'-GTAATACGACTCACTATAGGGC-3', SEQ ID NO:38-(Genome
walker)
[0248] Ap2 5'-ACTATAGGGCACGCGTGGT-3', SEQ ID NO:39-(Genome
walker)
[0249] Ap1 5'-CCATCCTAATACGACTCACTATAGGGC-3', SEQ ID
NO:40-(Marathon RACE)
[0250] Ap2 5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID NO:41-(Marathon
RACE)
[0251] Southern analysis of genomic DNA: Genomic DNA was extracted
from animal or from human blood using Blood and cell culture DNA
maxi kit (Qiagene). DNA was digested with EcoRI, separated by gel
electrophoresis and transferred to a nylon membrane Hybond N+
(Amersham). Hybridization was performed at 68.degree. C. in
6.times.SSC, 1% SDS, 5.times. Denharts, 10% dextran sulfate, 100
.mu.g/ml salmon sperm DNA, and .sup.32p labeled probe. A 1.6 kb
fragment, containing the entire hpa cDNA was used as a probe.
Following hybridization, the membrane was washed with 3.times.SSC,
0.1% SDS, at 68.degree. C. and exposed to X-ray film for 3 days.
Membranes were then washed with 1.times.SSC, 0.1% SDS, at
68.degree. C. and were reexposed for 5 days.
[0252] Construction of hpa promoter-GFP expression vector: Lambda
DNA of phage L3, was digested with SacI and BglII, resulting in a
1712 bp fragment which contained the hpa promoter (877-2688 of SEQ
ID NO:42). The pEGFP-1 plasmid (Clontech) was digested with BglII
and SacI and ligated with the 1712 bp fragment of the hpa promoter
sequence. The resulting plasmid was designated phpEGL. A second hpa
promoter-GFP plasmid was constructed containing a shorter fragment
of the hpa promoter region: phpEGL was digested with HindIII, and
the resulting 1095 bp fragment (nucleotides 1593-2688 of SEQ ID
NO:42) was ligated with HindIII digested pEGFP-1. The resulting
plasmid was designated phpEGS.
[0253] Computer analysis of sequences: Homology searches were
performed using several computer servers, and various databases.
Blast 2.0 service, at the NCBI server was used to screen the
protein database swplus and DNA databases such as GenBank, EMBL,
and the EST databases. Blast 2.0 search was performed using the
basic search option of the NCBI server. Sequence analysis and
alignments were done using the DNA sequence analysis software
package developed by the Genetic Computer Group (GCG) at the
university of Wisconsin. Alignments of two sequences were performed
using Bestfit (gap creation penalty--12, gap extension penalty--4).
Protein homology search was performed with the Smith-Waterman
algorithm, using the Bioaccelerator platform developed by
Compugene. The protein database swplus was searched using the
following parameters: gapop: 10.0, gapext: 0.5, matrix: blosum62.
Blocks homology was performed using the Blocks WWW server developed
at Fred Hutchinson Cancer Research Center in Seattle, Wash., USA.
Secondary structure prediction was performed using the PHD
server--Profile network Prediction Heidelberg. Fold recognition
(threading) was performed using the UCLA-DOE structure prediction
server. The method used for prediction was gonnet+predss. Alignment
of three sequences was performed using the pileup application (gap
creation penalty--5, gap extension penalty--1). Promoter analysis
was performed using TSSW and TSSG programs (BCM Search Launcher
Human Genome Center, Baylor College of Medicine, Houston Tex.).
Example 1
Cloning of Human Hpa cDNA
[0254] Purified fraction of heparanase isolated from human hepatoma
cells (SK-hep-1) was subjected to tryptic digestion and
microsequencing. EST (Expressed Sequence Tag) databases were
screened for homology to the back translated DNA sequences
corresponding to the obtained peptides. Two EST sequences
(accession Nos. N41349 and N45367) contained a DNA sequence
encoding the peptide YGPDVGQPR (SEQ ID NO:8). These two sequences
were derived from clones 257548 and 260138 (I.M.A.G.E Consortium)
prepared from 8 to 9 weeks placenta cDNA library (Soares). Both
clones which were found to be identical contained an insert of 1020
bp which included an open reading frame (ORF) of 973 bp followed by
a 3' untranslated region of 27 bp and a Poly A tail. No translation
start site (AUG) was identified at the 5' end of these clones.
[0255] Cloning of the missing 5' end was performed by PCR
amplification of DNA from a placenta Marathon RACE cDNA composite.
A 900 bp fragment (designated hp3), partially overlapping with the
identified 3' encoding EST clones was obtained.
[0256] The joined cDNA fragment, 1721 bp long (SEQ ID NO:9),
contained an open reading frame which encodes, as shown in FIG. 1
and SEQ ID NO: 11, a polypeptide of 543 amino acids (SEQ ID NO: 10)
with a calculated molecular weight of 61,192 daltons. The 3' end of
the partial cDNA inserts contained in clones 257548 and 260138
started at nucleotide G.sup.721 of SEQ ID NO:9 and FIG. 1.
[0257] As further shown in FIG. 1, there was a single sequence
discrepancy between the EST clones and the PCR amplified sequence,
which led to an amino acid substitution from Tyr.sup.246 in the EST
to Phe.sup.246 in the amplified cDNA. The nucleotide sequence of
the PCR amplified cDNA fragment was verified from two independent
amplification products. The new gene was designated hpa.
[0258] As stated above, the 3' end of the partial cDNA inserts
contained in EST clones 257548 and 260138 started at nucleotide 721
of hpa (SEQ ID NO:9). The ability of the hpa cDNA to form stable
secondary structures, such as stem and loop structures involving
nucleotide stretches in the vicinity of position 721 was
investigated using computer modeling. It was found that stable stem
and loop structures are likely to be formed involving nucleotides
698-724 (SEQ ID NO:9). In addition, a high GC content, up to 70%,
characterizes the 5' end region of the hpa gene, as compared to
about only 40% in the 3' region. These findings may explain the
immature termination and therefore lack of 5' ends in the EST
clones.
[0259] To examine the ability of the hpa gene product to catalyze
degradation of heparan sulfate in an in vitro assay the entire open
reading frame was expressed in insect cells, using the Baculovirus
expression system. Extracts of cells, infected with virus
containing the hpa gene, demonstrated a high level of heparan
sulfate degradation activity, while cells infected with a similar
construct containing no hpa gene had no such activity, nor did
non-infected cells. These results are further demonstrated in the
following Examples.
Example 2
Degradation of Soluble ECM-Derived HSPG
[0260] Monolayer cultures of High Five cells were infected (72 h,
28.degree. C.) with recombinant Bacoluvirus containing the pFasthpa
plasmid or with control virus containing an insert free plasmid.
The cells were harvested and lysed in heparanase reaction buffer by
three cycles of freezing and thawing. The cell lysates were then
incubated (18 h, 37.degree. C.) with sulfate labeled, ECM-derived
HSPG (peak I), followed by gel filtration analysis (Sepharose 6B)
of the reaction mixture.
[0261] As shown in FIG. 2, the substrate alone included almost
entirely high molecular weight (Mr) material eluted next to V.sub.o
(peak I, fractions 5-20, Kav<0.35). A similar elution pattern
was obtained when the HSPG substrate was incubated with lysates of
cells that were infected with control virus. In contrast,
incubation of the HSPG substrate with lysates of cells infected
with the hpa containing virus resulted in a complete conversion of
the high Mr substrate into low Mr labeled degradation fragments
(peak II, fractions 22-35, 0.5<Kav<0.75).
[0262] Fragments eluted in peak II were shown to be degradation
products of heparan sulfate, as they were (i) 5- to 6-fold smaller
than intact heparan sulfate side chains (Kav approx. 0.33) released
from ECM by treatment with either alkaline borohydride or papain;
and (ii) resistant to further digestion with papain or
chondroitinase ABC, and susceptible to deamination by nitrous acid
(6, 11). Similar results (not shown) were obtained with Sf21 cells.
Again, heparanase activity was detected in cells infected with the
hpa containing virus (pFhpa), but not with control virus (pF). This
result was obtained with two independently generated recombinant
viruses. Lysates of control not infected High Five cells failed to
degrade the HSPG substrate.
[0263] In subsequent experiments, the labeled HSPG substrate was
incubated with medium conditioned by infected High Five or Sf21
cells.
[0264] As shown in FIGS. 3a-b, heparanase activity, reflected by
the conversion of the high Mr peak I substrate into the low Mr peak
II which represents HS degradation fragments, was found in the
culture medium of cells infected with the pFhpa2 or pFhpa4 viruses,
but not with the control pF1 or pF2 viruses. No heparanase activity
was detected in the culture medium of control non-infected High
Five or Sf21 cells.
[0265] The medium of cells infected with the pFhpa4 virus was
passed through a 50 kDa cut off membrane to obtain a crude
estimation of the molecular weight of the recombinant heparanase
enzyme. As demonstrated in FIG. 4, all the enzymatic activity was
retained in the upper compartment and there was no activity in the
flow through (<50 kDa) material. This result is consistent with
the expected molecular weight of the hpa gene product.
[0266] In order to further characterize the hpa product the
inhibitory effect of heparin, a potent inhibitor of heparanase
mediated HS degradation (40) was examined.
[0267] As demonstrated in FIGS. 5a-b, conversion of the peak I
substrate into peak II HS degradation fragments was completely
abolished in the presence of heparin.
[0268] Altogether, these results indicate that the heparanase
enzyme is expressed in an active form by insect cells infected with
Baculovirus containing the newly identified human hpa gene.
Example 3
Degradation of HSPG in Intact ECM
[0269] Next, the ability of intact infected insect cells to degrade
HS in intact, naturally produced ECM was investigated. For this
purpose, High Five or Sf21 cells were seeded on metabolically
sulfate labeled ECM followed by infection (48 h, 28.degree. C.)
with either the pFhpa4 or control pF2 viruses. The pH of the medium
was then adjusted to pH 6.2-6.4 and the cells further incubated
with the labeled ECM for another 48 h at 28.degree. C. or 24 h at
37.degree. C. Sulfate labeled material released into the incubation
medium was analyzed by gel filtration on Sepharose 6B.
[0270] As shown in FIGS. 6a-b and 7a-b, incubation of the ECM with
cells infected with the control pF2 virus resulted in a constant
release of labeled material that consisted almost entirely
(>90%) of high Mr fragments (peak I) eluted with or next to
V.sub.o. It was previously shown that a proteolytic activity
residing in the ECM itself and/or expressed by cells is responsible
for release of the high Mr material (6). This nearly intact HSPG
provides a soluble substrate for subsequent degradation by
heparanase, as also indicated by the relatively large amount of
peak I material accumulating when the heparanase enzyme is
inhibited by heparin (6, 7, 12, FIG. 9). On the other hand,
incubation of the labeled ECM with cells infected with the pFhpa4
virus resulted in release of 60-70% of the ECM-associated
radioactivity in the form of low Mr sulfate-labeled fragments (peak
II, 0.5<Kav<0.75), regardless of whether the infected cells
were incubated with the ECM at 28.degree. C. or 37.degree. C.
Control intact non-infected Sf21 or High Five cells failed to
degrade the ECM HS side chains.
[0271] In subsequent experiments, as demonstrated in FIGS. 8a-b,
High Five and Sf21 cells were infected (96 h, 28.degree. C.) with
pFhpa4 or control pF1 viruses and the culture medium incubated with
sulfate-labeled ECM. Low Mr HS degradation fragments were released
from the ECM only upon incubation with medium conditioned by pFhpa4
infected cells. As shown in FIG. 9, production of these fragments
was abolished in the presence of heparin. No heparanase activity
was detected in the culture medium of control, non-infected cells.
These results indicate that the heparanase enzyme expressed by
cells infected with the pFhpa4 virus is capable of degrading HS
when complexed to other macromolecular constituents (i.e.
fibronectin, laminin, collagen) of a naturally produced intact ECM,
in a manner similar to that reported for highly metastatic tumor
cells or activated cells of the immune system (6, 7).
Example 4
Purification of Recombinant Human Heparanase
[0272] The recombinant heparanase was partially purified from
medium of pFhpa4 infected Sf21 cells by Heparin-Sepharose
chromatography (FIG. 10a) followed by gel filtration of the pooled
active fractions over an FPLC Superdex 75 column (FIG. 11a). A
.about.63 kDa protein was observed, whose quantity, as was detected
by silver stained SDS-polyacrylamide gel electrophoresis,
correlated with heparanase activity in the relevant column
fractions (FIGS. 10b and 11b, respectively). This protein was not
detected in the culture medium of cells infected with the control
pF1 virus and was subjected to a similar fractionation on
heparin-Sepharose (not shown).
Example 5
Expression of the Human Hpa cDNA in Various Cell Types, Organs and
Tissues
[0273] Referring now to FIGS. 12a-e, RT-PCR was applied to evaluate
the expression of the hpa gene by various cell types and tissues.
For this purpose, total RNA was reverse transcribed and amplified.
The expected 585 bp long cDNA was clearly demonstrated in human
kidney, placenta (8 and 11 weeks) and mole tissues, as well as in
freshly isolated and short termed (1.5-48 h) cultured human
placental cytotrophoblastic cells (FIG. 12a), all known to express
a high heparanase activity (41). The hpa transcript was also
expressed by normal human neutrophils (FIG. 12b). In contrast,
there was no detectable expression of the hpa mRNA in embryonic
human muscle tissue, thymus, heart and adrenal (FIG. 12b). The hpa
gene was expressed by several, but not all, human bladder carcinoma
cell lines (FIG. 12c), SK hepatoma (SK-hep-1), ovarian carcinoma
(OV 1063), breast carcinoma (435, 231), melanoma and megakaryocytic
(DAMI, CHRF) human cell lines (FIGS. 12d-e).
[0274] The above described expression pattern of the hpa transcript
was determined to be in a very good correlation with heparanase
activity levels determined in various tissues and cell types (not
shown).
Example 6
Isolation of an Extended 5' End of Hpa cDNA from Human SK-hep1 Cell
Line
[0275] The 5' end of hpa cDNA was isolated from human SK-hep1 cell
line by PCR amplification using the Marathon RACE (rapid
amplification of cDNA ends) kit (Clontech). Total RNA was prepared
from SK-hep1 cells using the TRI-Reagent (Molecular research center
Inc.) according to the manufacturer instructions. Poly A+ RNA was
isolated using the mRNA separator kit (Clonetech).
[0276] The Marahton RACE SK-hep1 cDNA composite was constructed
according to the manufacturer recommendations. First round of
amplification was performed using an adaptor specific primer AP1:
5'-CCATCCTAATACG ACTCACTATAGGGC-3', SEQ ID NO:1, and a hpa specific
antisense primer hpl-629: 5'-CCCCAGGAGCAGCAGCATCAG-3', SEQ ID
NO:17, corresponding to nucleotides 119-99 of SEQ ID NO:9. The
resulting PCR product was subjected to a second round of
amplification using an adaptor specific nested primer AP2:
5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID NO:3, and a hpa specific
antisense nested primer hpl-666 5'-AGGCTTCGAGCGCAGCAGCAT-3', SEQ ID
NO:18, corresponding to nucleotides 83-63 of SEQ ID NO:9. The PCR
program was as follows: a hot start of 94.degree. C. for 1 minute,
followed by 30 cycles of 90.degree. C.-30 seconds, 68.degree. C.-4
minutes. The resulting 300 bp DNA fragment was extracted from an
agarose gel and cloned into the vector pGEM-T Easy (Promega). The
resulting recombinant plasmid was designated pHPSK1. The nucleotide
sequence of the pHPSK1 insert was determined and it was found to
contain 62 nucleotides of the 5' end of the placenta hpa cDNA (SEQ
ID NO:9) and additional 178 nucleotides upstream, the first 178
nucleotides of SEQ ID NOs:13 and 15.
[0277] A single nucleotide discrepancy was identified between the
SK-hep1 cDNA and the placenta cDNA. The "T" derivative at position
9 of the placenta cDNA (SEQ ID NO:9), is replaced by a "C"
derivative at the corresponding position 187 of the SK-hep1 cDNA
(SEQ ID NO:13).
[0278] The discrepancy is likely to be due to a mutation at the 5'
end of the placenta cDNA clone as confirmed by sequence analysis of
sevsral additional cDNA clones isolated from placenta, which like
the SK-hep1 cDNA contained C at position 9 of SEQ ID NO:9.
[0279] The 5' extended sequence of the SK-hep1 hpa cDNA was
assembled with the sequence of the hpa cDNA isolated from human
placenta (SEQ ID NO:9). The assembled sequence contained an open
reading frame which encodes, as shown in SEQ ID NOs:14 and 15, a
polypeptide of 592 amino acids with a calculated molecular weight
of 66,407 daltons. The open reading frame is flanked by 93 bp 5'
untranslated region (UTR).
Example 7
Isolation of the Upstream Genomic Region of the Hpa Gene
[0280] The upstream region of the hpa gene was isolated using the
Genome Walker kit (Clontech) according to the manufacturer
recommendations. The kit includes five human genomic DNA samples
each digested with a different restriction endonuclease creating
blunt ends: EcoRV, ScaI, DraI, PvuII and SspI.
[0281] The blunt ended DNA fragments are ligated to partially
single stranded adaptors. The Genomic DNA samples were subjected to
PCR amplification using the adaptor specific primer and a gene
specific primer. Amplification was performed with Expand High
Fidelity (Boehringer Mannheim).
[0282] A first round of amplification was performed using the ap1
primer: 5'-G TAATACGACTCACTATAGGGC-3', SEQ ID NO:19, and the hpa
specific antisense primer hpl-666: 5'-AGGCTTCGAGCGCAGCAGCAT-3', SEQ
ID NO:18, corresponding to nucleotides 83-63 of SEQ ID NO:9. The
PCR program was as follows: a hot start of 94.degree. C.-3 minutes,
followed by 36 cycles of 94.degree. C.-40 seconds, 67.degree. C.-4
minutes.
[0283] The PCR products of the first amplification were diluted
1:50. One .mu.l of the diluted sample was used as a template for a
second amplification using a nested adaptor specific primer ap2:
5'-ACTATAGGGCACGCGTGGT-3', SEQ ID NO:20, and a hpa specific
antisense primer hpl-690, 5'-CTTGGGCTCACC TGGCTGCTC-3', SEQ ID
NO:21, corresponding to nucleotides 62-42 of SEQ ID NO:9. The
resulting amplification products were analyzed using agarose gel
electrophoresis. Five different PCR products were obtained from the
five amplification reactions. A DNA fragment of approximately 750
bp which was obtained from the SspI digested DNA sample was gel
extracted. The purified fragment was ligated into the plasmid
vector pGEM-T Easy (Promega). The resulting recombinant plasmid was
designated pGHP6905 and the nucleotide sequence of the hpa insert
was determined.
[0284] A partial sequence of 594 nucleotides is shown in SEQ ID
NO:16. The last nucleotide in SEQ ID NO: 13 corresponds to
nucleotide 93 in SEQ ID:13. The DNA sequence in SEQ ID NO:16
contains the 5' region of the hpa cDNA and 501 nucleotides of the
genomic upstream region which are predicted to contain the promoter
region of the hpa gene.
Example 8
Expression of the 592 Amino Acids HPA Polypeptide in a Human 293
Cell Line
[0285] The 592 amino acids open reading frame (SEQ ID NOs:13 and
15) was constructed by ligation of the 110 bp corresponding to the
5' end of the SK-hep1 hpa cDNA with the placenta cDNA. More
specifically the Marathon RACE--PCR amplification product of the
placenta hpa DNA was digested with SacI and an approximately 1 kb
fragment was ligated into a SacI-digested pGHP6905 plasmid. The
resulting plasmid was digested with EarI and AatII. The Earl sticky
ends were blunted and an approximately 280 bp EarI/blunt-AatII
fragment was isolated. This fragment was ligated with pFasthpa
digested with EcoRI which was blunt ended using Klenow fragment and
further digested with AatII. The resulting plasmid contained a 1827
bp insert which includes an open reading frame of 1776 bp, 31 bp of
3' UTR and 21 bp of 5' UTR. This plasmid was designated
pFastLhpa.
[0286] A mammalian expression vector was constructed to drive the
expression of the 592 amino acids heparanase polypeptide in human
cells. The hpa cDNA was excised prom pFastLhpa with BssHII and
NotI. The resulting 1850 bp BssHII-NotI fragment was ligated to a
mammalian expression vector pSI (Promega) digested with MluI and
NotI. The resulting recombinant plasmid, pSIhpaMet2 was transfected
into a human 293 embryonic kidney cell line.
[0287] Transient expression of the 592 amino-acids heparanase was
examined by western blot analysis and the enzymatic activity was
tested using the gel shift assay. Both these procedures are
described in length in U.S. pat. application Ser. No. 09/071,739,
filed May 1, 1998, which is incorporated by reference as if fully
set forth herein. Cells were harvested 3 days following
transfection. Harvested cells were re-suspended in lysis buffer
containing 150 mM NaCl, 50 mM Tris pH 7.5, 1% Triton X-100, 1 mM
PMSF and protease inhibitor cocktail (Boehringer Mannheim). 40
.mu.g protein extract samples were used for separation on a
SDS-PAGE. Proteins were transferred onto a PVDF Hybond-P membrane
(Amersham). The membrane was incubated with an affinity purified
polyclonal anti heparanase antibody, as described in U.S. pat.
application Ser. No. 09/071,739. A major band of approximately 50
kDa was observed in the transfected cells as well as a minor band
of approximately 65 kDa. A similar pattern was observed in extracts
of cells transfected with the pShpa as demonstrated in U.S. pat.
application Ser. No. 09/071,739. These two bands probably represent
two forms of the recombinant heparanase protein produced by the
transfected cells. The 65 kDa protein probably represents a
heparanase precursor, while the 50 kDa protein is suggested herein
to be the processed or mature form.
[0288] The catalytic activity of the recombinant protein expressed
in the pShpaMet2 transfected cells was tested by gel shift assay.
Cell extracts of transfected and of mock transfected cells were
incubated overnight with heparin (6 .mu.g in each reaction) at
37.degree. C., in the presence of 20 mM phosphate citrate buffer pH
5.4, 1 mM CaCl.sub.2, 1 mM DTT and 50 mM NaCl. Reaction mixtures
were then separated on a 10% polyacrylamide gel. The catalytic
activity of the recombinant heparanase was clearly demonstrated by
a faster migration of the heparin molecules incubated with the
transfected cell extract as compared to the control. Faster
migration indicates the disappearance of high molecular weight
heparin molecules and the generation of low molecular weight
degradation products.
Example 9
Chromosomal Localization of the Hpa Gene
[0289] Chromosomal mapping of the hpa gene was performed utilizing
a panel of monochromosomal human/CHO and human/mouse somatic cell
hybrids, obtained from the UK HGMP Resource Center (Cambridge,
England).
[0290] 40 ng of each of the somatic cell hybrid DNA samples were
subjected to PCR amplification using the hpa primers: hpu565
5'-AGCTCTGTAGATGTGC TATACAC-3', SEQ ID NO:22, corresponding to
nucleotides 564-586 of SEQ ID NO:9 and an antisense primer hpl171
5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:23, corresponding to
nucleotides 897-876 of SEQ ID NO:9.
[0291] The PCR program was as follows: a hot start of 94.degree.
C.-3 minutes, followed by 7 cycles of 94.degree. C.-45 seconds,
66.degree. C.-1 minute, 68.degree. C.-5 minutes, followed by 30
cycles of 94.degree. C.-45 seconds, 62.degree. C.-1 minute,
68.degree. C.-5 minutes, and a 10 minutes final extension at
72.degree. C.
[0292] The reactions were performed with Expand long PCR
(Boehringer Mannheim). The resulting amplification products were
analyzed using agarose gel electrophoresis. As demonstrated in FIG.
14, a single band of approximately 2.8 Kb was obtained from
chromosome 4, as well as from the control human genomic DNA. A 2.8
kb amplification product is expected based on amplification of the
genomic hpa clone (data not shown). No amplification products were
obtained neither in the control DNA samples of hamster and mouse
nor in somatic hybrids of other human chromosome.
Example 10
Human Genomic Clone Encoding Heparanase
[0293] Five plaques were isolated following screening of a human
genomic library and were designated L3-1, L5-1, L8-1, L10-1 and
L6-1. The phage DNAs were analyzed by Southern hybridization and by
PCR with hpa specific and vector specific primers Southern analysis
was performed with three fragments of hpa cDNA: a PvuII-BamHI
fragment (nucleotides 32-450, SEQ ID NO:9), a BamHI-NdeI fragment
(nucleotides 451-1102, SEQ ID NO:9) and an NdeI-XhoI fragment
(nucleotides 1103-1721, SEQ ID NO:9).
[0294] Following Southern analysis, phages L3, L6, L8 were selected
for further analysis. A scheme of the genomic region and the
relative position of the three phage clones is depicted in FIG. 15.
A 2 kb DNA fragment containing the gap between phages L6 and L3 was
PCR amplified from human genomic DNA with two gene specific primers
GHpuL3 and GHplL6. The PCR product was cloned into the plasmid
vector pGEM-T-easy (Promega).
[0295] Large scale DNA sequencing of the three Lambda clones and
the amplified fragment was performed with Lambda purified DNA by
primer walking. A nucleotide sequence of 44,898 bp was analyzed
(FIG. 16, SEQ ID NO:42). Comparison of the genomic sequence with
that of hpa cDNA revealed 12 exons separated by 11 introns (FIGS.
15 an 16). The genomic organization of the hpa gene is depicted in
FIG. 15 (top). The sequence include the coding region from the
first ATG to the stop codon which spans 39,113 nucleotides, 2742
nucleotides upstream of the first ATG and 3043 nucleotides
downstream of the stop codon. Splice site consensus sequences were
identified at exon/intron junctions.
Example 11
Alternative Splicing
[0296] Several minor RT-PCR products were obtained from various
cell types, following amplification with hpa specific primers. Each
one found to contain a deletion of one or two exons. Some of these
PCR products contain ORFs, which encode potential shorter
proteins.
[0297] Table 1 below summarizes the alternative spliced products
isolated from various cell lines.
[0298] Fragments of similar sizes were obtained following
amplification with two cell lines, placenta and platelets.
2 Cell type Nucleotides deleted Exons deleted ORF Platelets
1047-1267 8, 9 + Platelets 1154-1267 9 - Platelets 289-435, 562-735
2, 4 - Sk-hep1, platelets, Zr75 562-735 4 + Sk-hep1 (hepatoma)
561-904 4, 5 - Zr75 (breast carcinoma) 96-203 1 (partial) +
Example 12
Mouse and Rat Hpa
[0299] EST databases were screened for sequences homologous to the
hpa gene. Three mouse EST's were identified (accession No.
Aa177901, from mouse spleen, Aa067997 from mouse skin, Aa47943 from
mouse embryo), assembled into a 824 bp cDNA fragment which contains
a partial open reading frame (lacking a 5' end) of 629 bp and a 3'
untranslated region of 195 bp (SEQ ID NO:12). As shown in FIG. 13,
the coding region is 80% similar to the 3' end of the hpa cDNA
sequence. These EST's are probably cDNA fragments of the mouse hpa
homolog that encodes for the mouse heparanase.
[0300] Searching for consensus protein domains revealed an amino
terminal homology between the heparanasd and several precursor
proteins such as Procollagen Alpha 1 precursor, Tyrosine-protein
kinase-RYK, Fibulin-1, Insulin-like growth factor binding protein
and several others. The amino terminus is highly hydrophobic and
contains a potential trans-membrane domain. The homology to known
signal peptide sequences suggests that it could function as a
signal peptide for protein localization.
[0301] The amino acid sequence of human heparanase was used to
search for homologous sequences in the DNA and protein databases.
Several human EST's were identified, as well as mouse sequences
highly homologous to human heparanase. The following mouse EST's
were identified AA177901, AA674378, AA67997, AA047943, AA690179,
A1122034, all sharing an identical sequence and correspond to amino
acids 336-543 of the human heparanase sequence. The entire mouse
heparanase cDNA was cloned, based on the nucleotide sequence of the
mouse EST's. PCR primers were designed and a Marathon RACE was
performed using a Marathon cDNA library from 15 days mouse embryo
(Clontech) and from BL6 mouse melanoma cell line. The mouse hpa
homologous cDNA was isolated following several amplification steps.
A 1.1 kb fragment was amplified from mouse embryo Marathon cDNA
library. The first cycle of amplification was performed with
primers mhpl773 and Ap1 and the second cycle with primers mhpl736
and AP2. A 1.1 kb fragment was then amplified from BL6 Marathon
cDNA library. The first cycle of amplification was performed with
the primers mhpl152 and Ap1, and the second with mhpl83 and AP2.
The combined sequence was homologous to nucleotides 157-1702 of the
human hpa cDNA, which encode amino acids 33-543. The 5' end of the
mouse hpa gene was isolated from a mouse genomic DNA library using
the Genome Walker kit (Clontech). An 0.9 kb fragment was amplified
from a DraI digested Genome walker DNA library. The first cycle of
amplification was performed with primers mhpl114 and Ap1 and the
second with primers mhpl103 and AP2. The assembled sequence (SEQ ID
NOs:43, 45) is 2396 nucleotides long. It contains an open reading
frame of 1605 nucleotides, which encode a polypeptide of 535 amino
acids (SEQ ID NOs:44, 45), 196 nucleotides of 3' untranslated
region (UTR), and anupstream sequence which includes the promoter
region and the 5'-UTR of the mouse hpa cDNA. According to two
promoter predicting programs TSSW and TSSG, the transcription start
site is localized to nucleotide 431 of SEQ ID NOs:43, 45, 163
nucleotides upstream of the first ATG codon. The 431 upstream
genomic sequence contains the promoter region. A TATA box is
predicted at position 394 of SEQ ID NOs:43, 45. The mouse and the
human hpa genes share an average homology of 78% between the
nucleotide sequences and 81% similarity between the deduced amino
acid sequences.
[0302] Search for hpa homologous sequences, using the Blast 2.0
server revealed two EST's from rat: AI060284 (385 nucleotides, SEQ
ID NO:46) which is homologous to the amino terminus (68% similarity
to amino acids 12-136) of human heparanase and AI237828 (541
nucleotides, SEQ ID NO:47) which is homologous to the carboxyl
terminus (81% similarity to amino acids 500-543) of human
heparanase, and contains a 3'-UTR. A comparison between the human
heparanase and the mouse and rat homologous sequences is
demonstrated in FIG. 17.
Example 13
Prediction of Heparanase Active Site
[0303] Homology search of heparanase amino acid sequence against
the DNA and the protein databases revealed no significant
homologies. The protein secondary structure as predicted by the PHD
program consists of alternating alpha helices and beta sheets. The
fold recognition server of UCLA predicted alpha/beta barrel
structure, with under-threshold confidence.
[0304] Five of 15 proteins, which were predicted to have most
similar folds, were glycosyl hydrolases from various organisms:
lxyza--xylanase from Clostridium Thermocellum,
lpbga--6-phospho-beta-.delta.-galactosidas- e from Lactococcus
Lactis, lamy--alpha-amylase from Barley, lecea--endocellulase from
Acidothermus Cellulolyticus and lqbc--hexosaminidase alpha chain,
glycosyl hydrolase.
[0305] Protein homology search using the bioaccelerator pulled out
several proteins, including glycosyl hydrolyses such as
beta-fructofuranosidase from Vicia faba (broad bean) and from
potato, lactase phlorizin hydrolase from human, xylanases from
Clostridium thermocellum and from Streptomyces halstedii and
cellulase from Clostridium thermocellum. Blocks 9.3 database pulled
out the active site of glycosyl hydrolases family five, which
includes cellulases from various bacteria and fungi. Similar active
site motif is shared by several lysosomal acid hydrolases (63) and
other glycosyl hydrolases. The common mechanism shared by these
enzymes involves two glutamic acid residues, a proton donor and a
nucleophile.
[0306] Despite the lack of an overall homology between the
heparanase and other glycosyl hydolases, the amino acid couple
Asp-Glu (NE), which is characteristic of the proton donor of
glycosyl hydrolyses of the GH-A clan, was found at positions
224-225 of the human heparanase protein sequence. As in other clan
members, this NE couple is located at the end of a .beta.
sheet.
[0307] Considering the relative location of the proton donor and
the predicted secondary structure, the glutamic acid that functions
as nucleophile is most likely located at position 343, or at
positon 396. Identification of the active site and the amino acids
directly involved in hydrolysis opens the way for expression of the
defined catalytic domain. In addition, it will provide the tools
for rational design of enzyme activity either by modification of
the microenviroment or catalytic site itself.
Example 14
Expression of Hpa Antisense in Mammalian Cell Lines
[0308] A mammalian expression vector Hpa2Kepcdna3 was constructed
in order to express hpa antisense in mammalian cells. hpa cDNA (1.7
kb EcoRI fragment) was cloned into the plasmid pcDNA3 in 3'>5'
(antisense) orientation. The construct was used to transfect
MBT2-T50 and T24P cell lines. 2.times.10.sup.5 cells in 35 mm
plates were transfected using the Fugene protocol (Boehringer
Mannheim). 48 hours after transfection cells were trypsinized and
seeded in six well plates. 24 hours later G418 was added to
initiate selection. The number of colonies per 35 mm plate
following 3 weeks:
3 Antisense No insert T24P 15 60 MBT-T50 1 6
[0309] The lower number of colonies obtained after transfection
with hpa antisense, as compared with the control plasmid suggests
that the introduction of hpa antisense interfere with cell growth.
This experiment demonstrates the use of complementary antisense hpa
DNA sequence to control heparanase expression in cells. This
approach may be used to inhibit expression of heparanase in vivo,
in, for example, cancer cells and in other pathological processes
in which heparanase is involved.
Example 15
Zoo Blot
[0310] Hpa cDNA was used as a probe to detect homologous sequences
in human DNA and in DNA of various animals. The autoradiogram of
the Southern analysis is presented in FIG. 18. Several bands were
detected in human DNA, which correlated with the accepted pattern
according to the genomic hpa sequence. Several intense bands were
detected in all mammals, while faint bands were detected in
chicken. This correlates with the phylogenetic relation between
human and the tested animals. The intense bands indicate that hpa
is conserved among mammals as well as in more genetically distant
organisms. The multiple bands patterns suggest that in all animals,
like in human, the hpa locus occupy large genomic region.
Alternatively, the various bands could represent homologous
sequences and suggest the existence of a gene family, which can be
isolated based on their homology to the human hpa reported herein.
This conservation was actually found, between the isolated human
hpa cDNA and the mouse homologue.
Example 16
Characterization of the Hpa Promoter
[0311] The DNA sequence upstream of the hpa first ATG was subjected
to computational analysis in order to localize the predicted
transcription start site and to identify potential transcription
factors binding sites. Recognition of human PolII promoter region
and start of transcription were predicted using the TSSW and TSSG
programs. Both programs identified a promoter region upstream of
the coding region. TSSW pointed at nucleotide 2644 and TSSG at 2635
of SEQ ID NO:42. These two predicted transcription start sites are
located 4 and 13 nucleotides upstream of the longest hpa cDNA
isolated by RACE.
[0312] A hpa promoter-GFP reporter vector was constructed in order
to investigate the regulation of hpa transcription. Two constructs
were made, containing 1.8 kb and 1.1 kb of the hpa promoter region.
The reporter vector was transfected into T50-mouse bladder
carcinoma cells. Cells transfected with both constructs exhibited
green fluorescence, which indicated the promoter activity of the
genomic sequence upstream of the hpa-coding region. This reporter
vector, enables the monitoring of hpa promoter activity, at various
conditions and in different cell types and to characterize the
factors involved regulation of hpa expression.
[0313] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
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Sequence CWU 1
1
47 1 27 DNA Artificial sequence Synthetic oligonucleotide 1
ccatcctaat acgactcact atagggc 27 2 24 DNA Artificial sequence
Synthetic oligonucleotide 2 gtagtgatgc catgtaactg aatc 24 3 23 DNA
Artificial sequence Synthetic oligonucleotide 3 actcactata
gggctcgagc ggc 23 4 22 DNA Artificial sequence Synthetic
oligonucleotide 4 gcatcttagc cgtctttctt cg 22 5 15 DNA Artificial
sequence Synthetic oligonucleotide 5 tttttttttt ttttt 15 6 23 DNA
Artificial sequence Synthetic oligonucleotide 6 ttcgatccca
agaaggaatc aac 23 7 24 DNA Artificial sequence Synthetic
oligonucleotide 7 gtagtgatgc catgtaactg aatc 24 8 9 PRT Artificial
sequence Peptide derived from tryptic digestion of human heparenase
8 Tyr Gly Pro Asp Val Gly Gln Pro Arg 1 5 9 1721 DNA Homo sapiens 9
ctagagcttt cgactctccg ctgcgcggca gctggcgggg ggagcagcca ggtgagccca
60 agatgctgct gcgctcgaag cctgcgctgc cgccgccgct gatgctgctg
ctcctggggc 120 cgctgggtcc cctctcccct ggcgccctgc cccgacctgc
gcaagcacag gacgtcgtgg 180 acctggactt cttcacccag gagccgctgc
acctggtgag cccctcgttc ctgtccgtca 240 ccattgacgc caacctggcc
acggacccgc ggttcctcat cctcctgggt tctccaaagc 300 ttcgtacctt
ggccagaggc ttgtctcctg cgtacctgag gtttggtggc accaagacag 360
acttcctaat tttcgatccc aagaaggaat caacctttga agagagaagt tactggcaat
420 ctcaagtcaa ccaggatatt tgcaaatatg gatccatccc tcctgatgtg
gaggagaagt 480 tacggttgga atggccctac caggagcaat tgctactccg
agaacactac cagaaaaagt 540 tcaagaacag cacctactca agaagctctg
tagatgtgct atacactttt gcaaactgct 600 caggactgga cttgatcttt
ggcctaaatg cgttattaag aacagcagat ttgcagtgga 660 acagttctaa
tgctcagttg ctcctggact actgctcttc caaggggtat aacatttctt 720
gggaactagg caatgaacct aacagtttcc ttaagaaggc tgatattttc atcaatgggt
780 cgcagttagg agaagattat attcaattgc ataaacttct aagaaagtcc
accttcaaaa 840 atgcaaaact ctatggtcct gatgttggtc agcctcgaag
aaagacggct aagatgctga 900 agagcttcct gaaggctggt ggagaagtga
ttgattcagt tacatggcat cactactatt 960 tgaatggacg gactgctacc
agggaagatt ttctaaaccc tgatgtattg gacattttta 1020 tttcatctgt
gcaaaaagtt ttccaggtgg ttgagagcac caggcctggc aagaaggtct 1080
ggttaggaga aacaagctct gcatatggag gcggagcgcc cttgctatcc gacacctttg
1140 cagctggctt tatgtggctg gataaattgg gcctgtcagc ccgaatggga
atagaagtgg 1200 tgatgaggca agtattcttt ggagcaggaa actaccattt
agtggatgaa aacttcgatc 1260 ctttacctga ttattggcta tctcttctgt
tcaagaaatt ggtgggcacc aaggtgttaa 1320 tggcaagcgt gcaaggttca
aagagaagga agcttcgagt ataccttcat tgcacaaaca 1380 ctgacaatcc
aaggtataaa gaaggagatt taactctgta tgccataaac ctccataacg 1440
tcaccaagta cttgcggtta ccctatcctt tttctaacaa gcaagtggat aaataccttc
1500 taagaccttt gggacctcat ggattacttt ccaaatctgt ccaactcaat
ggtctaactc 1560 taaagatggt ggatgatcaa accttgccac ctttaatgga
aaaacctctc cggccaggaa 1620 gttcactggg cttgccagct ttctcatata
gtttttttgt gataagaaat gccaaagttg 1680 ctgcttgcat ctgaaaataa
aatatactag tcctgacact g 1721 10 543 PRT Homo sapiens 10 Met Leu Leu
Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu 1 5 10 15 Leu
Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro 20 25
30 Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro
35 40 45 Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp
Ala Asn 50 55 60 Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly
Ser Pro Lys Leu 65 70 75 80 Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala
Tyr Leu Arg Phe Gly Gly 85 90 95 Thr Lys Thr Asp Phe Leu Ile Phe
Asp Pro Lys Lys Glu Ser Thr Phe 100 105 110 Glu Glu Arg Ser Tyr Trp
Gln Ser Gln Val Asn Gln Asp Ile Cys Lys 115 120 125 Tyr Gly Ser Ile
Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp 130 135 140 Pro Tyr
Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe 145 150 155
160 Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe
165 170 175 Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala
Leu Leu 180 185 190 Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala
Gln Leu Leu Leu 195 200 205 Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile
Ser Trp Glu Leu Gly Asn 210 215 220 Glu Pro Asn Ser Phe Leu Lys Lys
Ala Asp Ile Phe Ile Asn Gly Ser 225 230 235 240 Gln Leu Gly Glu Asp
Tyr Ile Gln Leu His Lys Leu Leu Arg Lys Ser 245 250 255 Thr Phe Lys
Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg 260 265 270 Arg
Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu 275 280
285 Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr
290 295 300 Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile
Phe Ile 305 310 315 320 Ser Ser Val Gln Lys Val Phe Gln Val Val Glu
Ser Thr Arg Pro Gly 325 330 335 Lys Lys Val Trp Leu Gly Glu Thr Ser
Ser Ala Tyr Gly Gly Gly Ala 340 345 350 Pro Leu Leu Ser Asp Thr Phe
Ala Ala Gly Phe Met Trp Leu Asp Lys 355 360 365 Leu Gly Leu Ser Ala
Arg Met Gly Ile Glu Val Val Met Arg Gln Val 370 375 380 Phe Phe Gly
Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro 385 390 395 400
Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr 405
410 415 Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu
Arg 420 425 430 Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr
Lys Glu Gly 435 440 445 Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn
Val Thr Lys Tyr Leu 450 455 460 Arg Leu Pro Tyr Pro Phe Ser Asn Lys
Gln Val Asp Lys Tyr Leu Leu 465 470 475 480 Arg Pro Leu Gly Pro His
Gly Leu Leu Ser Lys Ser Val Gln Leu Asn 485 490 495 Gly Leu Thr Leu
Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met 500 505 510 Glu Lys
Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser 515 520 525
Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 530 535
540 11 1721 DNA Homo sapiens CDS (63)..(1691) 11 ctagagcttt
cgactctccg ctgcgcggca gctggcgggg ggagcagcca ggtgagccca 60 ag atg
ctg ctg cgc tcg aag cct gcg ctg ccg ccg ccg ctg atg ctg 107 Met Leu
Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu 1 5 10 15 ctg
ctc ctg ggg ccg ctg ggt ccc ctc tcc cct ggc gcc ctg ccc cga 155 Leu
Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg 20 25
30 cct gcg caa gca cag gac gtc gtg gac ctg gac ttc ttc acc cag gag
203 Pro Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu
35 40 45 ccg ctg cac ctg gtg agc ccc tcg ttc ctg tcc gtc acc att
gac gcc 251 Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile
Asp Ala 50 55 60 aac ctg gcc acg gac ccg cgg ttc ctc atc ctc ctg
ggt tct cca aag 299 Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu
Gly Ser Pro Lys 65 70 75 ctt cgt acc ttg gcc aga ggc ttg tct cct
gcg tac ctg agg ttt ggt 347 Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro
Ala Tyr Leu Arg Phe Gly 80 85 90 95 ggc acc aag aca gac ttc cta att
ttc gat ccc aag aag gaa tca acc 395 Gly Thr Lys Thr Asp Phe Leu Ile
Phe Asp Pro Lys Lys Glu Ser Thr 100 105 110 ttt gaa gag aga agt tac
tgg caa tct caa gtc aac cag gat att tgc 443 Phe Glu Glu Arg Ser Tyr
Trp Gln Ser Gln Val Asn Gln Asp Ile Cys 115 120 125 aaa tat gga tcc
atc cct cct gat gtg gag gag aag tta cgg ttg gaa 491 Lys Tyr Gly Ser
Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu 130 135 140 tgg ccc
tac cag gag caa ttg cta ctc cga gaa cac tac cag aaa aag 539 Trp Pro
Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys 145 150 155
ttc aag aac agc acc tac tca aga agc tct gta gat gtg cta tac act 587
Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr 160
165 170 175 ttt gca aac tgc tca gga ctg gac ttg atc ttt ggc cta aat
gcg tta 635 Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn
Ala Leu 180 185 190 tta aga aca gca gat ttg cag tgg aac agt tct aat
gct cag ttg ctc 683 Leu Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn
Ala Gln Leu Leu 195 200 205 ctg gac tac tgc tct tcc aag ggg tat aac
att tct tgg gaa cta ggc 731 Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn
Ile Ser Trp Glu Leu Gly 210 215 220 aat gaa cct aac agt ttc ctt aag
aag gct gat att ttc atc aat ggg 779 Asn Glu Pro Asn Ser Phe Leu Lys
Lys Ala Asp Ile Phe Ile Asn Gly 225 230 235 tcg cag tta gga gaa gat
tat att caa ttg cat aaa ctt cta aga aag 827 Ser Gln Leu Gly Glu Asp
Tyr Ile Gln Leu His Lys Leu Leu Arg Lys 240 245 250 255 tcc acc ttc
aaa aat gca aaa ctc tat ggt cct gat gtt ggt cag cct 875 Ser Thr Phe
Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro 260 265 270 cga
aga aag acg gct aag atg ctg aag agc ttc ctg aag gct ggt gga 923 Arg
Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly 275 280
285 gaa gtg att gat tca gtt aca tgg cat cac tac tat ttg aat gga cgg
971 Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg
290 295 300 act gct acc agg gaa gat ttt cta aac cct gat gta ttg gac
att ttt 1019 Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu
Asp Ile Phe 305 310 315 att tca tct gtg caa aaa gtt ttc cag gtg gtt
gag agc acc agg cct 1067 Ile Ser Ser Val Gln Lys Val Phe Gln Val
Val Glu Ser Thr Arg Pro 320 325 330 335 ggc aag aag gtc tgg tta gga
gaa aca agc tct gca tat gga ggc gga 1115 Gly Lys Lys Val Trp Leu
Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly 340 345 350 gcg ccc ttg cta
tcc gac acc ttt gca gct ggc ttt atg tgg ctg gat 1163 Ala Pro Leu
Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp 355 360 365 aaa
ttg ggc ctg tca gcc cga atg gga ata gaa gtg gtg atg agg caa 1211
Lys Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln 370
375 380 gta ttc ttt gga gca gga aac tac cat tta gtg gat gaa aac ttc
gat 1259 Val Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn
Phe Asp 385 390 395 cct tta cct gat tat tgg cta tct ctt ctg ttc aag
aaa ttg gtg ggc 1307 Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe
Lys Lys Leu Val Gly 400 405 410 415 acc aag gtg tta atg gca agc gtg
caa ggt tca aag aga agg aag ctt 1355 Thr Lys Val Leu Met Ala Ser
Val Gln Gly Ser Lys Arg Arg Lys Leu 420 425 430 cga gta tac ctt cat
tgc aca aac act gac aat cca agg tat aaa gaa 1403 Arg Val Tyr Leu
His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu 435 440 445 gga gat
tta act ctg tat gcc ata aac ctc cat aac gtc acc aag tac 1451 Gly
Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr 450 455
460 ttg cgg tta ccc tat cct ttt tct aac aag caa gtg gat aaa tac ctt
1499 Leu Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr
Leu 465 470 475 cta aga cct ttg gga cct cat gga tta ctt tcc aaa tct
gtc caa ctc 1547 Leu Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys
Ser Val Gln Leu 480 485 490 495 aat ggt cta act cta aag atg gtg gat
gat caa acc ttg cca cct tta 1595 Asn Gly Leu Thr Leu Lys Met Val
Asp Asp Gln Thr Leu Pro Pro Leu 500 505 510 atg gaa aaa cct ctc cgg
cca gga agt tca ctg ggc ttg cca gct ttc 1643 Met Glu Lys Pro Leu
Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe 515 520 525 tca tat agt
ttt ttt gtg ata aga aat gcc aaa gtt gct gct tgc atc 1691 Ser Tyr
Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 530 535 540
tgaaaataaa atatactagt cctgacactg 1721 12 824 DNA Mus musculus 12
ctggcaagaa ggtctggttg ggagagacga gctcagctta cggtggcggt gcacccttgc
60 tgtccaacac ctttgcagct ggctttatgt ggctggataa attgggcctg
tcagcccaga 120 tgggcataga agtcgtgatg aggcaggtgt tcttcggagc
aggcaactac cacttagtgg 180 atgaaaactt tgagccttta cctgattact
ggctctctct tctgttcaag aaactggtag 240 gtcccagggt gttactgtca
agagtgaaag gcccagacag gagcaaactc cgagtgtatc 300 tccactgcac
taacgtctat cacccacgat atcaggaagg agatctaact ctgtatgtcc 360
tgaacctcca taatgtcacc aagcacttga aggtaccgcc tccgttgttc aggaaaccag
420 tggatacgta ccttctgaag ccttcggggc cggatggatt actttccaaa
tctgtccaac 480 tgaacggtca aattctgaag atggtggatg agcagaccct
gccagctttg acagaaaaac 540 ctctccccgc aggaagtgca ctaagcctgc
ctgccttttc ctatggtttt tttgtcataa 600 gaaatgccaa aatcgctgct
tgtatatgaa aataaaaggc atacggtacc cctgagacaa 660 aagccgaggg
gggtgttatt cataaaacaa aaccctagtt taggaggcca cctccttgcc 720
gagttccaga gcttcgggag ggtggggtac acttcagtat tacattcagt gtggtgttct
780 ctctaagaag aatactgcag gtggtgacag ttaatagcac tgtg 824 13 1899
DNA Homo sapiens 13 gggaaagcga gcaaggaagt aggagagagc cgggcaggcg
gggcggggtt ggattgggag 60 cagtgggagg gatgcagaag aggagtggga
gggatggagg gcgcagtggg aggggtgagg 120 aggcgtaacg gggcggagga
aaggagaaaa gggcgctggg gctcggcggg aggaagtgct 180 agagctctcg
actctccgct gcgcggcagc tggcgggggg agcagccagg tgagcccaag 240
atgctgctgc gctcgaagcc tgcgctgccg ccgccgctga tgctgctgct cctggggccg
300 ctgggtcccc tctcccctgg cgccctgccc cgacctgcgc aagcacagga
cgtcgtggac 360 ctggacttct tcacccagga gccgctgcac ctggtgagcc
cctcgttcct gtccgtcacc 420 attgacgcca acctggccac ggacccgcgg
ttcctcatcc tcctgggttc tccaaagctt 480 cgtaccttgg ccagaggctt
gtctcctgcg tacctgaggt ttggtggcac caagacagac 540 ttcctaattt
tcgatcccaa gaaggaatca acctttgaag agagaagtta ctggcaatct 600
caagtcaacc aggatatttg caaatatgga tccatccctc ctgatgtgga ggagaagtta
660 cggttggaat ggccctacca ggagcaattg ctactccgag aacactacca
gaaaaagttc 720 aagaacagca cctactcaag aagctctgta gatgtgctat
acacttttgc aaactgctca 780 ggactggact tgatctttgg cctaaatgcg
ttattaagaa cagcagattt gcagtggaac 840 agttctaatg ctcagttgct
cctggactac tgctcttcca aggggtataa catttcttgg 900 gaactaggca
atgaacctaa cagtttcctt aagaaggctg atattttcat caatgggtcg 960
cagttaggag aagattatat tcaattgcat aaacttctaa gaaagtccac cttcaaaaat
1020 gcaaaactct atggtcctga tgttggtcag cctcgaagaa agacggctaa
gatgctgaag 1080 agcttcctga aggctggtgg agaagtgatt gattcagtta
catggcatca ctactatttg 1140 aatggacgga ctgctaccag ggaagatttt
ctaaaccctg atgtattgga catttttatt 1200 tcatctgtgc aaaaagtttt
ccaggtggtt gagagcacca ggcctggcaa gaaggtctgg 1260 ttaggagaaa
caagctctgc atatggaggc ggagcgccct tgctatccga cacctttgca 1320
gctggcttta tgtggctgga taaattgggc ctgtcagccc gaatgggaat agaagtggtg
1380 atgaggcaag tattctttgg agcaggaaac taccatttag tggatgaaaa
cttcgatcct 1440 ttacctgatt attggctatc tcttctgttc aagaaattgg
tgggcaccaa ggtgttaatg 1500 gcaagcgtgc aaggttcaaa gagaaggaag
cttcgagtat accttcattg cacaaacact 1560 gacaatccaa ggtataaaga
aggagattta actctgtatg ccataaacct ccataacgtc 1620 accaagtact
tgcggttacc ctatcctttt tctaacaagc aagtggataa ataccttcta 1680
agacctttgg gacctcatgg attactttcc aaatctgtcc aactcaatgg tctaactcta
1740 aagatggtgg atgatcaaac cttgccacct ttaatggaaa aacctctccg
gccaggaagt 1800 tcactgggct tgccagcttt ctcatatagt ttttttgtga
taagaaatgc caaagttgct 1860 gcttgcatct gaaaataaaa tatactagtc
ctgacactg 1899 14 592 PRT Homo sapiens 14 Met Glu Gly Ala Val Gly
Gly Val Arg Arg Arg Asn Gly Ala Glu Glu 1 5 10 15 Arg Arg Lys Gly
Arg Trp Gly Ser Ala Gly Gly Ser Ala Arg Ala Leu 20 25 30 Asp Ser
Pro Leu Arg Gly Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro 35 40 45
Lys Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu 50
55 60 Leu Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro
Arg 65 70 75 80 Pro Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe
Thr Gln Glu 85 90 95 Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser
Val Thr Ile Asp Ala
100 105 110 Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser
Pro Lys 115 120 125 Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr
Leu Arg Phe Gly 130 135 140 Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp
Pro Lys Lys Glu Ser Thr 145 150 155 160 Phe Glu Glu Arg Ser Tyr Trp
Gln Ser Gln Val Asn Gln Asp Ile Cys 165 170 175 Lys Tyr Gly Ser Ile
Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu 180 185 190 Trp Pro Tyr
Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys 195 200 205 Phe
Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr 210 215
220 Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu
225 230 235 240 Leu Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala
Gln Leu Leu 245 250 255 Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile
Ser Trp Glu Leu Gly 260 265 270 Asn Glu Pro Asn Ser Phe Leu Lys Lys
Ala Asp Ile Phe Ile Asn Gly 275 280 285 Ser Gln Leu Gly Glu Asp Tyr
Ile Gln Leu His Lys Leu Leu Arg Lys 290 295 300 Ser Thr Phe Lys Asn
Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro 305 310 315 320 Arg Arg
Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly 325 330 335
Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg 340
345 350 Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile
Phe 355 360 365 Ile Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser
Thr Arg Pro 370 375 380 Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser
Ala Tyr Gly Gly Gly 385 390 395 400 Ala Pro Leu Leu Ser Asp Thr Phe
Ala Ala Gly Phe Met Trp Leu Asp 405 410 415 Lys Leu Gly Leu Ser Ala
Arg Met Gly Ile Glu Val Val Met Arg Gln 420 425 430 Val Phe Phe Gly
Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp 435 440 445 Pro Leu
Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly 450 455 460
Thr Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu 465
470 475 480 Arg Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr
Lys Glu 485 490 495 Gly Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn
Val Thr Lys Tyr 500 505 510 Leu Arg Leu Pro Tyr Pro Phe Ser Asn Lys
Gln Val Asp Lys Tyr Leu 515 520 525 Leu Arg Pro Leu Gly Pro His Gly
Leu Leu Ser Lys Ser Val Gln Leu 530 535 540 Asn Gly Leu Thr Leu Lys
Met Val Asp Asp Gln Thr Leu Pro Pro Leu 545 550 555 560 Met Glu Lys
Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe 565 570 575 Ser
Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 580 585
590 15 1899 DNA Homo sapiens CDS (94)..(1869) 15 gggaaagcga
gcaaggaagt aggagagagc cgggcaggcg gggcggggtt ggattgggag 60
cagtgggagg gatgcagaag aggagtggga ggg atg gag ggc gca gtg gga ggg
114 Met Glu Gly Ala Val Gly Gly 1 5 gtg agg agg cgt aac ggg gcg gag
gaa agg aga aaa ggg cgc tgg ggc 162 Val Arg Arg Arg Asn Gly Ala Glu
Glu Arg Arg Lys Gly Arg Trp Gly 10 15 20 tcg gcg gga gga agt gct
aga gct ctc gac tct ccg ctg cgc ggc agc 210 Ser Ala Gly Gly Ser Ala
Arg Ala Leu Asp Ser Pro Leu Arg Gly Ser 25 30 35 tgg cgg ggg gag
cag cca ggt gag ccc aag atg ctg ctg cgc tcg aag 258 Trp Arg Gly Glu
Gln Pro Gly Glu Pro Lys Met Leu Leu Arg Ser Lys 40 45 50 55 cct gcg
ctg ccg ccg ccg ctg atg ctg ctg ctc ctg ggg ccg ctg ggt 306 Pro Ala
Leu Pro Pro Pro Leu Met Leu Leu Leu Leu Gly Pro Leu Gly 60 65 70
ccc ctc tcc cct ggc gcc ctg ccc cga cct gcg caa gca cag gac gtc 354
Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro Ala Gln Ala Gln Asp Val 75
80 85 gtg gac ctg gac ttc ttc acc cag gag ccg ctg cac ctg gtg agc
ccc 402 Val Asp Leu Asp Phe Phe Thr Gln Glu Pro Leu His Leu Val Ser
Pro 90 95 100 tcg ttc ctg tcc gtc acc att gac gcc aac ctg gcc acg
gac ccg cgg 450 Ser Phe Leu Ser Val Thr Ile Asp Ala Asn Leu Ala Thr
Asp Pro Arg 105 110 115 ttc ctc atc ctc ctg ggt tct cca aag ctt cgt
acc ttg gcc aga ggc 498 Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu Arg
Thr Leu Ala Arg Gly 120 125 130 135 ttg tct cct gcg tac ctg agg ttt
ggt ggc acc aag aca gac ttc cta 546 Leu Ser Pro Ala Tyr Leu Arg Phe
Gly Gly Thr Lys Thr Asp Phe Leu 140 145 150 att ttc gat ccc aag aag
gaa tca acc ttt gaa gag aga agt tac tgg 594 Ile Phe Asp Pro Lys Lys
Glu Ser Thr Phe Glu Glu Arg Ser Tyr Trp 155 160 165 caa tct caa gtc
aac cag gat att tgc aaa tat gga tcc atc cct cct 642 Gln Ser Gln Val
Asn Gln Asp Ile Cys Lys Tyr Gly Ser Ile Pro Pro 170 175 180 gat gtg
gag gag aag tta cgg ttg gaa tgg ccc tac cag gag caa ttg 690 Asp Val
Glu Glu Lys Leu Arg Leu Glu Trp Pro Tyr Gln Glu Gln Leu 185 190 195
cta ctc cga gaa cac tac cag aaa aag ttc aag aac agc acc tac tca 738
Leu Leu Arg Glu His Tyr Gln Lys Lys Phe Lys Asn Ser Thr Tyr Ser 200
205 210 215 aga agc tct gta gat gtg cta tac act ttt gca aac tgc tca
gga ctg 786 Arg Ser Ser Val Asp Val Leu Tyr Thr Phe Ala Asn Cys Ser
Gly Leu 220 225 230 gac ttg atc ttt ggc cta aat gcg tta tta aga aca
gca gat ttg cag 834 Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr
Ala Asp Leu Gln 235 240 245 tgg aac agt tct aat gct cag ttg ctc ctg
gac tac tgc tct tcc aag 882 Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu
Asp Tyr Cys Ser Ser Lys 250 255 260 ggg tat aac att tct tgg gaa cta
ggc aat gaa cct aac agt ttc ctt 930 Gly Tyr Asn Ile Ser Trp Glu Leu
Gly Asn Glu Pro Asn Ser Phe Leu 265 270 275 aag aag gct gat att ttc
atc aat ggg tcg cag tta gga gaa gat tat 978 Lys Lys Ala Asp Ile Phe
Ile Asn Gly Ser Gln Leu Gly Glu Asp Tyr 280 285 290 295 att caa ttg
cat aaa ctt cta aga aag tcc acc ttc aaa aat gca aaa 1026 Ile Gln
Leu His Lys Leu Leu Arg Lys Ser Thr Phe Lys Asn Ala Lys 300 305 310
ctc tat ggt cct gat gtt ggt cag cct cga aga aag acg gct aag atg
1074 Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg Arg Lys Thr Ala Lys
Met 315 320 325 ctg aag agc ttc ctg aag gct ggt gga gaa gtg att gat
tca gtt aca 1122 Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu Val Ile
Asp Ser Val Thr 330 335 340 tgg cat cac tac tat ttg aat gga cgg act
gct acc agg gaa gat ttt 1170 Trp His His Tyr Tyr Leu Asn Gly Arg
Thr Ala Thr Arg Glu Asp Phe 345 350 355 cta aac cct gat gta ttg gac
att ttt att tca tct gtg caa aaa gtt 1218 Leu Asn Pro Asp Val Leu
Asp Ile Phe Ile Ser Ser Val Gln Lys Val 360 365 370 375 ttc cag gtg
gtt gag agc acc agg cct ggc aag aag gtc tgg tta gga 1266 Phe Gln
Val Val Glu Ser Thr Arg Pro Gly Lys Lys Val Trp Leu Gly 380 385 390
gaa aca agc tct gca tat gga ggc gga gcg ccc ttg cta tcc gac acc
1314 Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu Leu Ser Asp
Thr 395 400 405 ttt gca gct ggc ttt atg tgg ctg gat aaa ttg ggc ctg
tca gcc cga 1362 Phe Ala Ala Gly Phe Met Trp Leu Asp Lys Leu Gly
Leu Ser Ala Arg 410 415 420 atg gga ata gaa gtg gtg atg agg caa gta
ttc ttt gga gca gga aac 1410 Met Gly Ile Glu Val Val Met Arg Gln
Val Phe Phe Gly Ala Gly Asn 425 430 435 tac cat tta gtg gat gaa aac
ttc gat cct tta cct gat tat tgg cta 1458 Tyr His Leu Val Asp Glu
Asn Phe Asp Pro Leu Pro Asp Tyr Trp Leu 440 445 450 455 tct ctt ctg
ttc aag aaa ttg gtg ggc acc aag gtg tta atg gca agc 1506 Ser Leu
Leu Phe Lys Lys Leu Val Gly Thr Lys Val Leu Met Ala Ser 460 465 470
gtg caa ggt tca aag aga agg aag ctt cga gta tac ctt cat tgc aca
1554 Val Gln Gly Ser Lys Arg Arg Lys Leu Arg Val Tyr Leu His Cys
Thr 475 480 485 aac act gac aat cca agg tat aaa gaa gga gat tta act
ctg tat gcc 1602 Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly Asp Leu
Thr Leu Tyr Ala 490 495 500 ata aac ctc cat aac gtc acc aag tac ttg
cgg tta ccc tat cct ttt 1650 Ile Asn Leu His Asn Val Thr Lys Tyr
Leu Arg Leu Pro Tyr Pro Phe 505 510 515 tct aac aag caa gtg gat aaa
tac ctt cta aga cct ttg gga cct cat 1698 Ser Asn Lys Gln Val Asp
Lys Tyr Leu Leu Arg Pro Leu Gly Pro His 520 525 530 535 gga tta ctt
tcc aaa tct gtc caa ctc aat ggt cta act cta aag atg 1746 Gly Leu
Leu Ser Lys Ser Val Gln Leu Asn Gly Leu Thr Leu Lys Met 540 545 550
gtg gat gat caa acc ttg cca cct tta atg gaa aaa cct ctc cgg cca
1794 Val Asp Asp Gln Thr Leu Pro Pro Leu Met Glu Lys Pro Leu Arg
Pro 555 560 565 gga agt tca ctg ggc ttg cca gct ttc tca tat agt ttt
ttt gtg ata 1842 Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser Tyr Ser
Phe Phe Val Ile 570 575 580 aga aat gcc aaa gtt gct gct tgc atc
tgaaaataaa atatactagt 1889 Arg Asn Ala Lys Val Ala Ala Cys Ile 585
590 cctgacactg 1899 16 594 DNA Homo sapiens 16 attactatag
ggcacgcgtg gtcgacggcc cgggctggta ttgtcttaat gagaagttga 60
taaagaattt tgggtggttg atctctttcc agctgcagtt tagcgtatgc tgaggccaga
120 ttttttcagg caaaagtaaa atacctgaga aactgcctgg ccagaggaca
atcagatttt 180 ggctggctca agtgacaagc aagtgtttat aagctagatg
ggagaggaag ggatgaatac 240 tccattggag gctttactcg agggtcagag
ggatacccgg cgccatcaga atgggatctg 300 ggagtcggaa acgctgggtt
cccacgagag cgcgcagaac acgtgcgtca ggaagcctgg 360 tccgggatgc
ccagcgctgc tccccgggcg ctcctccccg ggcgctcctc cccaggcctc 420
ccgggcgctt ggatcccggc catctccgca cccttcaagt gggtgtgggt gatttcgtaa
480 gtgaacgtga ccgccaccgg ggggaaagcg agcaaggaag taggagagag
ccgggcaggc 540 ggggcggggt tggattggga gcagtgggag ggatgcagaa
gaggagtggg aggg 594 17 21 DNA Artificial sequence Synthetic
oligonucleotide 17 ccccaggagc agcagcatca g 21 18 21 PRT Artificial
sequence Synthetic oligonucleotide 18 Ala Gly Gly Cys Thr Thr Cys
Gly Ala Gly Cys Gly Cys Ala Gly Cys 1 5 10 15 Ala Gly Cys Ala Thr
20 19 22 DNA Artificial sequence Synthetic oligonucleotide 19
gtaatacgac tcactatagg gc 22 20 19 DNA Artificial sequence Synthetic
oligonucleotide 20 actatagggc acgcgtggt 19 21 21 DNA Artificial
sequence Synthetic oligonucleotide 21 cttgggctca cctggctgct c 21 22
23 DNA Artificial sequence Synthetic oligonucleotide 22 agctctgtag
atgtgctata cac 23 23 22 DNA Artificial sequence Synthetic
oligonucleotide 23 gcatcttagc cgtctttctt cg 22 24 23 DNA Artificial
sequence Synthetic oligonucleotide 24 gagcagccag gtgagcccaa gat 23
25 23 DNA Artificial sequence Synthetic oligonucleotide 25
ttcgatccca agaaggaatc aac 23 26 23 DNA Artificial sequence
Synthetic oligonucleotide 26 agctctgtag atgtgctata cac 23 27 24 DNA
Artificial sequence Synthetic oligonucleotide 27 tcagatgcaa
gcagcaactt tggc 24 28 22 DNA Artificial sequence Synthetic
oligonucleotide 28 gcatcttagc cgtctttctt cg 22 29 24 DNA Artificial
sequence Synthetic oligonucleotide 29 gtagtgatgc catgtaactg aatc 24
30 22 DNA Artificial sequence Synthetic oligonucleotide 30
aggcacccta gagatgttcc ag 22 31 24 DNA Artificial sequence Synthetic
oligonucleotide 31 gaagatttct gtttccatga cgtg 24 32 25 DNA
Artificial sequence Synthetic oligonucleotide 32 ccacactgaa
tgtaatactg aagtg 25 33 22 DNA Artificial sequence Synthetic
oligonucleotide 33 cgaagctctg gaactcggca ag 22 34 22 DNA Artificial
sequence Synthetic oligonucleotide 34 gccagctgca aaggtgttgg ac 22
35 23 DNA Artificial sequence Synthetic oligonucleotide 35
aacacctgcc tcatcacgac ttc 23 36 22 DNA Artificial sequence
Synthetic oligonucleotide 36 gccaggctgg cgtcgatggt ga 22 37 22 DNA
Artificial sequence Synthetic oligonucleotide 37 gtcgatggtg
atggacagga ac 22 38 22 DNA Artificial sequence Synthetic
oligonucleotide 38 gtaatacgac tcactatagg gc 22 39 19 DNA Artificial
sequence Synthetic oligonucleotide 39 actatagggc acgcgtggt 19 40 27
DNA Artificial sequence Synthetic oligonucleotide 40 ccatcctaat
acgactcact atagggc 27 41 23 DNA Artificial sequence Synthetic
oligonucleotide 41 actcactata gggctcgagc ggc 23 42 44848 DNA Homo
sapiens 42 ggatcttggc tcactgcaat ctctgcctcc catgcaattc ttatgcatca
gcctcctgag 60 tagcttggat tataggtctg cgccaccact cctggctaca
ccatgttgcc caggctggtc 120 ttgaactctt gggctctagt gatccacccg
ccttggcctc ccaaagtgct gggattacag 180 gtgtgagcca tcacacccgg
ccccccgttt ccatattagt aactcacatg tagaccacaa 240 ggatgcacta
tttagaaaac ttgcaatggt ccacttttca aatcacccaa acatgttaaa 300
gaaattggta tgactgggca tggcacagtg gctcatgcct gcaatcctag cattttgtga
360 ggctgagacg ggcagatcac gaggtcagga gattgagacc atcctgacag
acatggtgaa 420 atcccatctc tactaaaaat acaaaacaat tagccggggg
tgatggcagg cccctgtagt 480 cccagctact cgggaggctg aggcaggaga
atggcgtgaa tccaggaggc agagcttgca 540 gtgagccgag atggtgccac
tgcactccag cctgggcgac agagcgagac tccgtctcaa 600 aaaaaaaaaa
aaagaaagaa attggtatga ctgttgactc acaacaggag tcaggggcat 660
ggggtggggt gtaagattaa tgtcatgaca aatgtggaaa agaaacttct gtttttccaa
720 ctccacgtct gctaccatat tattacactc ttctggtagt gtggtgttta
tgtgtgaatt 780 ttttttcata tgtatacagt aattgtagga tatgaacctg
attctagttg caaaactcac 840 tatgagctta gcttttaagt tgcttaagaa
taggtagatc tatgcaaata atgataatta 900 ttattattat tttaagagag
ggtctcactt tgtcacccag gctggagtgc agtggtgtga 960 ttaagggtca
ctgcaacctc cacctcccag gctcaaataa acctcccacc tcagcctccc 1020
cagtagctgg aaccacaggc acgggccacc acgcctggct aattttttgt attttttgta
1080 gagatggggt ttcatcatgt tgcccaggct gttcttgaat tcctcggctc
aagcaatcct 1140 cccaccttgg cctcccaaaa tgctggcatc acaggcatga
tggcatcact ggcatcacat 1200 accatgcctg gcctgattta tgcaaattag
atatgcattt caaaataatc tatttttatt 1260 tgttgcctta ttggtggtac
aatctcaagt ggaaaaatct aagggttttg gtgttatttg 1320 cttactcaac
caatatttat tagactctta ctaagcacca acatgatcac atgcctgagc 1380
tatggctagc atagcgtgtg agacaaactt aatctctgtt ttggtggagc atataatcta
1440 gtagatgaag ccaatgttga gcaacatcac aatactaaca aattgaggat
gctacgagag 1500 tgtctaacaa attgaggatg ctacgagagt gtctaacaaa
ttgaggatgc tatgagagtg 1560 tgtcatggag agctgcctgg agattgagag
aaagcttcct tgagggaagt tacatttcag 1620 ctgaaacaca ctgccatctg
ctcgaggttt tgtaactgca ttcacatccc gattctgaca 1680 cttcacatcc
cgattctgac acttcaccca gttactgtct cagagcttgg gtccgcatgt 1740
gtaaaacaag gacagtatgc acttggcagg gttgtgagaa gggaagagaa cacaagtaaa
1800 gcacctgtat caggcataca gtaggcacta agcgtgcgat gcttgctatg
attatacatc 1860 agtgtaagca tcaaggaaaa gctgaagaaa agtctgacca
acagcgaaag ataaatgcgc 1920 agaggagaaa tttggcaaag gctccaaatt
caggggcagt ccgtactcta cactttgtat 1980 gggggcttca ggtcctgagt
tccagacatt ggagcaacta accctttaag attgctaaat 2040 attgtcttaa
tgagaagttg ataaagaatt ttgggtggtt gatctctttc cagctgcagt 2100
ttagcgtatg ctgaggccag attttttcaa gcaaaagtaa aatacctgag aaactgcctg
2160 gccagaggac aatcagattt tggctggctc aagtgacaag caagtgttta
taagctagat 2220 gggagaggaa gggatgaata
ctccattgga ggttttactc gagggtcaga gggatacccg 2280 gcgccatcag
aatgggatct gggagtcgga aacgctgggt tcccacgaga gcgcgcagaa 2340
cacgtgcgtc aggaagcctg gtccgggatg cccagcgctg ctccccgggc gctcctcccc
2400 gggcgctcct ccccaggcct cccgggcgct tggatcccgg ccatctccgc
acccttcaag 2460 tgggtgtggg tgatttcgta agtgaacgtg accgccaccg
aggggaaagc gagcaaggaa 2520 gtaggagaga gccgggcagg cggggcgggg
ttggattggg agcagtggga gggatgcaga 2580 agaggagtgg gagggatgga
gggcgcagtg ggaggggtga ggaggcgtaa cggggcggag 2640 gaaaggagaa
aagggcgctg gggctcggcg ggaggaagtg ctagagctct cgactctccg 2700
ctgcgcggca gctggcgggg ggagcagcca ggtgagccca agatgctgct gcgctcgaag
2760 cctgcgctgc cgccgccgct gatgctgctg ctcctggggc cgctgggtcc
cctctcccct 2820 ggcgccctgc cccgacctgc gcaagcacag gacgtcgtgg
acctggactt cttcacccag 2880 gagccgctgc acctggtgag cccctcgttc
ctgtccgtca ccattgacgc caacctggcc 2940 acggacccgc ggttcctcat
cctcctgggg taagcgccag cctcctggtc ctgtcccctt 3000 tcctgtcctc
ctgacaccta tgtctgcccc gccagcggct ctccttcttt tgcgcggaaa 3060
caacttcaca ccggaacctc cccgcctgtc tctccccacc ccacttcccg cctctcattc
3120 tccctctccc tcccttactc tcagacccca aaccgctttt tggggggtat
catttaaaaa 3180 atagatttag gggttacaag tgcagttctg ttccatgggt
atattgcatt gtggtggcat 3240 ctgggctctt agtgtaactg tcacccgaat
gttgtacatt gtatctaata ggtaatttct 3300 catccctcat ccctctccca
ccctcccacc ttttggagtc tccagtgtct actattccac 3360 taagtccatg
tgtacacatt gtttagcgcc cactctaaat gagccttttt gtttcattca 3420
ttctgtaagt gttgaatagg caccacctaa ggtcaggtat aagtggaaat ttgaaaaaga
3480 aactgcccac ttgccccagt acttccctag ccaagaggag ggaaaccagg
caggtgcacc 3540 tgaaggcctg tgagtgcttg atttgctgtg cagtgtagga
caagtaagat tgtgcatagc 3600 cttctgtatt taagactgtg ttaggaagat
ttctctttct tttcttttct ttttcttttt 3660 tcttttcttt ttttttttta
ggcagatgaa aagggcgtca cagaacagga ataaaaatct 3720 aaatattcaa
taaatgagac ctaggagact actgcagtga cttacaaagt cctaataaaa 3780
agatgtctct ccaaaatggg gctgcaaaat gtggtgctgc cttatcagct ctaagttttt
3840 tccttacctg agaaagaagg aacctgatgc aggttcaggg ctcctgcccc
atgaatgcag 3900 gctgactcca agatggggag ctacagggac aatcccaggt
cttctaggcc tcttatttag 3960 gccctgggag cctccagaga tggccacatc
ttgaccagcc cagatagagg gaaagatcac 4020 cattatctca cctctgtgtc
aaatacctag atgctgtcct ccctgagccc acactatagt 4080 tgccagcgct
aatttaatgg gtagtgtact ggttaagaga tggacagacc atcctggctt 4140
gactctcagc tctggcaaag atgagtgact tggtttttcc atatctcttg gccacaccaa
4200 ccttgatttc ttcagctgta gaatggaatt tctcaagctt gcctcaagga
ttattgcccg 4260 aggatttgat gatatggtaa gagcttctca gtgtttgacc
catagtaagt gtttgacgtt 4320 tcaaacgaat tgtttctttc taggacatgg
tgagcatttg gtagccattc accggttttc 4380 tgtttctttg gatcatagtt
aacctctcct tttccttctg gcactacaat tttctggtgg 4440 ggaagaatcc
ttactttctg cccttcccct taaggatagg aagctgatac taggcagcaa 4500
ctagttgggg gataggaaga ttgttccaga gaaatgctga accatagggc tccagatcac
4560 aggaccccag tcttagcttg ctggggtgtg gggtgggggg gggcggttac
tgaacatggg 4620 tatgaagtag atgtccattt actgaaatgt gaggacctga
ggcctcttct attgctgtag 4680 ccagcatatt ccccaacctc tccccaagaa
aggacagatg ggggttcccc cctggagtaa 4740 caggtccaaa agaaaaaaca
tacagtggga cttccaggat ctgggcctga tcacccagca 4800 gtcaagctcc
ccgcaattga ctaacacccc cctaacacgt agaaattcca atctgcaatt 4860
tagtgaggat gataccttta ttcttcttaa atacatctct tcatttccca gagcaccctt
4920 ttttcccctc ctctgcacct ttttgttaaa gactggagta taatgaaata
ccaagagagc 4980 ataacatgtg atacataaaa ctttttttct ggtttacaaa
acagttcatt cttgtccata 5040 cgtgcttctc tccaaggctg gctgctgtct
gttccagccc gcttcgcttg gagaggccat 5100 ctgccatacc tgctccccag
acgcatcgac aagcacaccc agagtgttat ctgctaagac 5160 ctaaaagagg
gaggaacccc ctctcctcat ctaagaccta gcttctaaat tagagtgtga 5220
gggtccatct ccccaggagg ggcacagggc ccaaacagcc cagccatctc agaagacaac
5280 actaagcttt gtaggggtcc acagtagagg agagtaagac gcctgttgtt
taatttatta 5340 cagttcctca aaagtgaaga tgtgtgggcg ggatggcaag
agctgagcag acgaaagctg 5400 aaggaataag gaaagagagg aggacacaaa
cagctgacac ttcctcagtt cttgtcattt 5460 gcctggccct gttctaagca
ccttctaggt attaatccat ttagtcttgg ctacaacact 5520 gtgagtaact
agttttgtca cccccatttt aaaaatgaag aaagtgaggc tcagggaggt 5580
taagtaactt ggccacagtt tgaaactaga ctctgatcac atgagataat agtgcccata
5640 aaaagggaaa gcagattata ttttttaaag gaaagagagt aggatatggt
agaaaaagat 5700 tgtttggaaa ggaattgaga gattgatata atgaaaagaa
gcattcacat gagagtaaca 5760 gtatcagggc ccaaaccttc atctaaggta
cttcaaagag gcctaagcaa acttagtcac 5820 tggcgtggtt ctagtctcca
tgatggcaaa tacattgtgt acagcccaac tccacacaaa 5880 acttaaatac
caatgataga gcaatctaaa atttgaaaga aaaaatcttt caatttgtcg 5940
tcttcccaga gggacttaat caagaaacca atcaaaatac ttcctaagcc taactgtgtg
6000 cagaactcca aagagagccc agccctaaat caacactgtc caatggaaat
ataatataat 6060 gtgggcctca tatgcaaggt catatgtaat tttaaatttt
ctagtagcca tattaaaaag 6120 gtaaaaagaa acaagtgaaa ttaattttaa
taattttatt tagttcaata gatccaaaat 6180 gttttctcag catgtaatca
atataaaaat attaatgagg tatttattat tccttttctc 6240 aaaccaagtc
tattctataa tctggcgtgt attatttaca gcacttctca gactatattt 6300
ctttctttct tttttttttc cgagacaatt ttgctcttgt cacccaagct agagtacaat
6360 ggcgttacct cggctcactg caacctccgc ctcccgggtt caagttattc
tcctgcctca 6420 gtctcccaag tagctgggac tagaggcatg caccaccacg
cctggctaat tgtgtatttt 6480 tagtagagac agggtttcac catgttggcc
aggctaatct caaactcctg agctcaggtg 6540 atatgcccac ctcggcctcc
caaagtgttg ggattacagg cgtgagccac tgcacccggc 6600 ctcagattaa
ctatatttca agcgttcagt agccacatgt agctagtgct atggtagtgg 6660
acagtacaga tctgcatttc aattaagaca cgtatacaag catagttcac taatgcacgg
6720 taaaaaaaag tatagtgctg agtcggtggt agaaatccta aatactgcag
agcaaaagtg 6780 gtacgaacag caatctcagt gataatgcaa ccatgcttgc
ttttcattgc aatttgctta 6840 ttttccttca gcaaagttca tccatttttg
ccaattcaat aaatatttac tgataaaaac 6900 tttcaatatt agattcttgc
atcttcatag acagagttgc ttttcacatt tagaaaatta 6960 cttatcaatg
ttaaacacac gttttgataa ccagtgttgg aaagaggtgc agactcccca 7020
tgtgcctatt gatggcagaa atattcacag ccaaagggaa acaaagggct ggggacaatc
7080 acacacctca tgtctcctaa ctcctgggaa gtgctgtccc tctgattgag
ctcttattat 7140 tgccttcccc actaaccctg tccactgtgc cctggagccc
tttgcagggt tacctgctct 7200 gtcctcctca cagaatatct cctctacctc
cttgtccaag ctacaacttg gctattctct 7260 gatgacactg tcttccctgt
agcccttttg agtaatggct gcatattctc ccatagtcca 7320 gttcttttcc
tgttctccag tctggcttct ggatgacagc ccactagttt gaactccata 7380
ctgctatagt tcaagtccct tttgacttgt taccttgggc aaattacctc cttttgttca
7440 ggttccttgt ttgtaaaatg acgataataa tgccatttgc ttcagtgggt
tattttgaaa 7500 ttgagtgaaa gaaggcgggt agcttcccta cacgctcagt
gtagactagc ctgatgtgca 7560 ttacgggtga tgccatgact cagtgtgttt
tcctcatctc cacatctggc tctcatccag 7620 tgctcctgct tacggcactc
tgtccccctc ttacttactc ccccttatta actgaagact 7680 ggcactgatc
tcacagtttc ctctccactt cctagtctca ccatcatcct agatgacttc 7740
aagtcaccta gataaactgt ctcagtttct tcactcacat ttttttataa cagataatgt
7800 tacactcaag ttgtaacaga accagcttat ccagctcatg aaatgtatgc
atttcatctc 7860 aactctgtat tcagtgacat cctgtgggta tctggaaatc
agccatggtg agaatattta 7920 ccatggaaat tggcaaatac taaaaagcag
agcacctttt tttctgagag ccagaccata 7980 gctcttctac tccatagcac
ccatcataac aatttttaaa tacctccact gaacagcttc 8040 ttcctctctc
tacttcttcc atatctgatt tgagcttctt aatttatcat gtgaaccact 8100
cttgtaataa taaccccaaa tccctgttcc attgttcttc ctgctaaaat actaaacctg
8160 gtttagtcca accatatttt ctctctttgg aatctacagg gtggcccaaa
aacctggaaa 8220 tggaaaaata ttacttatta attttaatgt atattaataa
gccattttaa tgcttcattt 8280 ccagtctcag tggccaccct gtatagctgg
gctattgagc tcttgcggga ggagggagtg 8340 gacagtctcc cagccacaca
gactgatgtt gcaccaaaca ttttttagct tccagacttc 8400 cctggccctt
agtgttaccc ttaactctcc atttctctgc ctttcacatt ctctactttt 8460
taaaaatctc tgactccacc ttcaccttat cattcttagc acatgaccat acttctgctt
8520 cccaaagaaa atgagcaatt acttcctttt ccttttcctc ctgtcatcaa
atctgcagac 8580 atgtcatgcc taagtccagc tttcctcctt tctctgatct
cagtctgctt cttccatttc 8640 tgccctgaat cccgtcccct ccccaacccc
caaggacttc gctctatcag tcacctcttc 8700 cctctcctgt atcttcaact
cctcccattt tactggcttc ttcctcaagc ctttccccaa 8760 gcctttccca
tctcaattac ctcctcgcac atgcctctgc agaaaccacc ccgtttcttc 8820
cctcccctcg gcagcctgtt cttcctgttc tgccctcatg atggcaccat cattgtgtca
8880 ctaaaatcaa tctctccgac atcatcaatg gccttccttt gttgggaaac
ctaataaaca 8940 ctttatctta tttggtcttt gttatgggtt gaatgaggtt
accccgaaat ccatattaga 9000 agtcctaacc cccagtacct cagaatgtga
ctttatttgg gaatagggtc attgcagacg 9060 ttattagtta ggatgaggtc
atactggaat gtgatgggct gcttatctaa tatgactgat 9120 gtccttataa
caaggagaaa tttggagaca gacacgcaca tagggagaat accatgtgat 9180
gacaggagtt atggagttgg agtcaaaaag ctatgggaac ttaggagaaa gacctggaac
9240 aaatcctttc ctgcgcctag agagggagta tggccctgcc actaccttga
attcaacgtt 9300 tcggcttttc aaaactgtaa gacaatacat ttctgttgtt
caaaccaatt agtttgcagt 9360 actctgcgac tgcagcccta acaaactaat
acagtctctt ggaggcattt ggcaaggttg 9420 acaatggaag cactttctta
cccctttagg tctgtcgcct ttcttgttgg ggggtgtttt 9480 ctaacaattc
ctctccatct ctctctctct agtttgtctt aaacattggt gttcttcaga 9540
cttctgacct aggccttctt ttcacttcac atattcccct gggtggtctc acccacttcc
9600 agaaattact taaattactg ctcatgcagt actgtgctgg aaactgttta
acaactggct 9660 ctctgggaag aggggagact ggttgatggt ttttgctgat
ttctgtggtg taaatactcc 9720 ctccatggcc aattccaaac tgccaacagt
ttaacaactg gctcacaaat tttctccaaa 9780 tttaacattt ggctttcaca
ggccaacaac gtggtacagc caactccagc acacctctgc 9840 ttttgtgtca
gagagaagta acttattttt gtacaaaagg taaaataaaa acacctgcag 9900
gccccctttt tttccttaac aaactgctct agaaatagaa tagctgaagc ttcttttatg
9960 cattcatctg ttatttccat gtcactgtgg tggtgggatt atttttcctt
tatttttctt 10020 gtatatggtt gaaatactgt acctttgatc agttttagtt
ttatggcatg ttttgcaccc 10080 atattaaatc tagtttttgt cagagggcgt
caatattatt ttctcaaaac aagaaaatat 10140 ttcattgcaa aggagacaaa
caaaaaggtc cttaatacca aaactttgaa atgtgatttc 10200 ttgtacttgg
cagtgtccaa gtggtaaacc caaacagtat tgggttttca ttttgttcag 10260
gaaagtcttt gtctggcagc gacttaccct tacatcaggc gggccttgct cattcattca
10320 cttaagtatt tattaaacac cagcggtgtg ccaagtactt atctaggtat
cgggtagatt 10380 ctgataagtc agtcaggtcc ctgctctcag ggagcttgca
gcagagatgg gggctgcaat 10440 agagagtaag ccaaggaaat gaaaaaggaa
gttgatttca gagagtgatg aatgctatga 10500 agaaaatgaa ggcagcgcag
tgtgatggag agtgacccaa ggtggtacag tttgtacctc 10560 taaggaccag
actgtgaccc aggtcactca cagatgcccg tcatgtgatg ccacagcaac 10620
ttttccaggt gctcgtttcc tcccacttcc cagtctcttg cccagccgcg actgcttaca
10680 aatacagcta gaggaatcta aatgaggttc ctctatcatc aaacccaatc
aaaatgccaa 10740 ggaacagaat cagtgcctgg ctgaaggcag tggaacaggg
ccagcctgga gtggttctct 10800 ctgaggaagt tcctcatctt ggttttaggg
ccataccttg tgacctgtga gctaggggtt 10860 gccagtccct gacatttcta
ctgaggactc gcctgtctat attcccggcc tgtatgtgtc 10920 tcctgagttc
cagacacaca gggcgaagcg cctgatggat ggaagtatgt tttttggtgt 10980
tccattggta tctcaaattc tacaaaactt agtgcccctt ctcctccctg ttcctcccca
11040 tcttcagtct atcacctgtt cctcatccag caaatgatat taccatcttc
caaggagctt 11100 cccaggagta atccttgact cctcctcaac atccaattaa
taatcaaatc taggccaggt 11160 acaatagctc acgcctataa tcccagcact
ttgggaggct gaggcaggtg gatcatttga 11220 ggccaggagt tcaagaccag
cctggccaac aaggtgaaac ctgtctcatt taaaaaaagt 11280 tattttaaaa
actcaaatct attatttcta cctctaagtg tgtcttgaat ttatccatct 11340
ctctccatct ctgagctgtt accttacctc agtccatcac gttttgtcta cgttaacatg
11400 accagagtct tgttcttagt ctggtgaggt cactccagct gcttcagatc
cttccatggc 11460 tcaccgttgc cctcatataa agttggcact cctggacatg
tggcttacgg ggccctccgt 11520 gatgtggccc tatttgcttc tccattctgt
tctctcccag cctctctgcc cccatctcta 11580 ggcaccaacc acacccttct
gctcgtcaat ggtgccagct tctcttctat ctctggtctt 11640 tggacagact
tttcccttca cctggaatgc tttcttcaat cctaccccac tctctttaat 11700
ctagataagg tttattcttt ttgaatgtct agcagtgaaa ccatttcccc tgaaaaacct
11760 tctctaacca accccctacc ctcagcccaa ggtctagatt aggagtccct
ctgaatgttt 11820 ccatagcatt tttaaagaat tgcctattta cttgttcgta
tctatcacta aactacaaat 11880 tgtatgagaa cagccactat ctctgcctgg
ttcaccattc atctccagca actagcataa 11940 tgcctggcag agtcagcctg
caacaaatat ttgttgaata aattaacaga tggctttatc 12000 tccttaagta
aatcttgctt ttttcaccta ttaaaacaga cgcacaggcc aggtgtggtg 12060
gcccatgcct gtaatcccag cactttggca ggctgaggtg ggcggatcac ctgaggtcag
12120 gagttcaaga ccagcctggc caacatggtg aaaccccatc tctaataaaa
atacaaaaat 12180 tagctgggca tggtggtggg tgcgtatagt cccagctact
agggaggctg aggcaagaga 12240 atcgcttgaa cccaggaggc agaggtggca
gtgagccgag atcatgccac tgtactccag 12300 cctggatgac agagaccctg
tctcaaaaca cacacacaca cacacacaca cacacacaca 12360 cacacacaca
cacacacacc aagttgtata atttaaaata taacgtgctt gttatggaac 12420
acttgtaaaa tacaggaaag taatgaaaaa gtctaccatc tagctcacca cataatgacc
12480 attgctatca tcctggcata attctctcct gtatataaat atatattctt
ttattgttaa 12540 aattacacta tgagtactat ttatttattt tactgtggca
aaatgcgcaa aacataaaat 12600 cttgccattt taaggtatgc agtttggtgc
attcaccaca ctcacattgt tgtgcaaata 12660 tcaccactat ctatctcaga
acttcttcgt cttcccaaac tgaaactctg tacccattaa 12720 acaatagtgc
atcctctgtt ttcccctccc tacaatttat ttttatttgg gtttgtacca 12780
aactgaaaat agctgcttct tccttactta gttcagatta gcatttccat ttatttagcc
12840 gtggttttga ggatgccatg acagatgcca tccttcctag agctctttgg
ggctgtcagg 12900 tatttcagtc agggtgaatt cgggttgata acattttaaa
atctcacttt attctgaggt 12960 tcctagtgtc agagcccacc gtatttttag
ggactcccaa gttacaaaca aaaatatggt 13020 gaggaggaat cactgaagtt
ttaacacaag agacttacat tttgttcaat ttctatcttt 13080 tagtttattt
cctaagcata aagaaatact ttgaaaattt tacatagcat tatacatatt 13140
taattaagca tgagcacatc ttaaaacttt aaattttaga tcagatcttt aattcctagg
13200 atattaagag gtactggcaa tttggccagg tgtggtggtt cacgcctata
atcccaacac 13260 tttgggaggg tgaagtgggc gaattgctag agcccaggag
gtggaggctg caatggcctg 13320 agatcacgcc atcgtactcc agcctggatg
atgagaatga aatcctgtct caaaaaaaaa 13380 aaaaaaaaaa aaaagaagaa
gaagaagtat tggcaatcag tgctccagga ataatttcct 13440 gacttgaaat
aaacctacat gtagacaaac taattaggcc attccaagag ttgctagcat 13500
tggtttaata tgttttcaga gcattccagg aagcagtgtg gccagcattg catgtttgat
13560 acttcagaaa tgtatgacag gtgtttctct tacccaggtc ttctgttttc
ttagttttgc 13620 tcatgtaaat atttatgaac atcctcatct ttttgaggga
agggattata gatcattcta 13680 attccatttt ctagcatttg gtaccattct
aagcacatga taggcaccca tttggagcat 13740 ttttggcttg acagaatatg
catttagaat tgttcaaatt agaggtgtca gtgatgggaa 13800 ttagaatact
atataattct aagtcatttg acttaaatac aaaagaatga ttttccttgg 13860
tggggaatgg tgaagggagg caggagttaa gaagaggaga agagatccta agtcatttat
13920 aaacttctct ggaaagacag gtgtgtgaag actttttaaa aagtcattca
ccaaattgtg 13980 tgtgtgtgtg tgtgtgtgtt ttaaatagac tttatttttt
agagcagttt taggttcaca 14040 gcaaaattga atgcaaggac agagatttcc
cataaacccc ctgcccacac acatgcatag 14100 cctccctcat tatcaacatc
cccaccagag aggtgtttgt tctagttgat gaacctacac 14160 tgacacatca
ttatcaccca aagtccatag ttcacggcag ggttcactgt cggtgtacat 14220
tctatgggtt tgagcaaatg tataatgaca tgtatccacc attatagtaa catacagagt
14280 attttcagtg ccctgcaaat cccctgttct ccacctattc atccctccct
ctctgcattt 14340 ccacccccag cccctggtaa ccgctgatct ttttactgtc
ccatagtttc ggacgatcta 14400 tttttcagac agacacagag ctgtctttcc
cttagtttct attctatcat ttctttctcc 14460 ccatccatca taaaaggcta
tgagtttttt ttaagtgttg aacaccatcc tacttgtcaa 14520 gttaaaacat
aagctcctgg ctgggtacag tggctcatgc ctgtaatctc agcattttgg 14580
gaggctgtgg cagaagcatc acttgaagcc agaagtttga gaccagcctg ggcaacatag
14640 caagacccca tccctccaca cacaaacaca cacacacaca cacacacaca
cacacacaca 14700 cacacacaca cacaaaaaca agctcttgcc agaattagag
ctacaaattg ccctcaggtt 14760 cctagaagat cagtccttca attagattca
gattgagatg cttcctcttt taaacaatga 14820 ttccctttct atcatgccca
ataagaaaac aaataaaaat taaacaatac tgcctgtaat 14880 ctcagctacc
caggaggcag aagcagaact gcttcaaccc ggcaagcaga agttgcagtg 14940
aagtgagatc gcgccactgc actccagcct gggaaacaga gcaagattct gtctcaaaaa
15000 caaaacaatg tgatttcctc ctctaagtcc tgcacaggga aatgttaaga
aataggtcca 15060 ccaggaaaga aggaagtaag aatgtttgac tagattgtct
tggaaaaaat agttatactt 15120 tcttgcttgt cttcctaaca gttctccaaa
gcttcgtacc ttggccagag gcttgtctcc 15180 tgcgtacctg aggtttggtg
gcaccaagac agacttccta attttcgatc ccaagaagga 15240 atcaaccttt
gaagagagaa gttactggca atctcaagtc aaccagggtg aaaattttta 15300
aagattcact ctatatttta attaacgtca gtccgtcatg agaatgcttt gagaaaactg
15360 ttatttctca cacctaacaa ttaatgagat taacttcctc tcccctcatc
tgacctgtgg 15420 aggaatctga acaagaggag gaggcagtgg gcaggtttcc
ttatcatgat gtttgtcatg 15480 ttcagtgtga ggcctcacaa aaaaaaaaaa
aaaaaaaaaa ggcgtcctgg atataactga 15540 gagctcattg tacagtaaat
attaataaaa cagtgattgt agctgaagga tagaactgct 15600 tggagggagc
aagtgggtag aatcgcgtca aactaaagag catttctagc caaagacaca 15660
atgatagatt gaaggatatt tattctaaat atagaatatg ggtgaacgag atctgtggac
15720 ttctgggctc caacgttaga ttctgatttt agcaagcttg tcaggggatt
ctgatattga 15780 aaggctgtgg ccttcacctg agaaacctgc cctagggggc
catgaaaatt tgtcctgtct 15840 ttcagaagtg ctatcagaca tcaaatggaa
gttaaatcgt atcttaacaa ttactaggat 15900 gggcgcagtg actcacacct
gtaatcccaa cactttggga ggctgaggca ggaggatcac 15960 ttgagcccag
gagttcggga ccagcctggg caacatagag agacgttgtc tctatttttt 16020
aataatttaa agagaaaaaa atactgaaaa tattgtatac accactgaat tataataatg
16080 tgtatataat gtatatattc attatgagga atatttgatt atttcatata
ttatatcttt 16140 tccttctgtt tattttatcc agttatgaag tatttagaac
aattcatcag taattggggc 16200 taaattgaca gaatagtaat cagagaaaat
agaaaaagac agatgggtta tctttgaata 16260 ccaggttgga gttgtttatg
ggtttgtttt ttgttttggg ggcgtttttt tagacagagt 16320 cccactctgt
tgcccaggct ggagtgcagt ggcacaagca tggcccactg catccttgac 16380
ctcttgggct caagcaatct tcccacctta gcctcctgag tagctgggac cacaggtgca
16440 tgtcaccaca cccagctaat ttttttattt tttgtagaga cagtctttct
atgttatcca 16500 ggctgatctc aaactcctgc actcaagtga tccccctgcc
ttggcgtccc aaagtattgg 16560 gattataggc atagccacca cacccaacct
agtttctatt tagacttggc cctttcccac 16620 cagtcatttg tgtccaaaag
atctcataaa tgtagacagg aaactgtcct ttgctcatca 16680 gttttcttca
tcctgtgtct agggggatgg tcggtggggg aaactggggt tatgcaagtt 16740
cctctgaaac atcctctgtg agcccaggga tggatgaggc accagccgcc agcgagtcag
16800 tgtgcagctt tccagaaagg aagtcatcag ccagtcagcc ggccctggca
gccagcaccc 16860 ggcaaccctg ctgtcttgtg ataaagaaat ggtctgcctg
acaggatggt gtggattttt 16920 cttttttctt tttttttttt ttgagacagg
gtctggctct gtcgcccagg ctggagtgca 16980 atggcgggat cttggctcac
tgcagcctct gcctcccagg ctcaaggcat cctcccacct 17040 cggtctcccg
agtagctggg accacaggca cacaccacca cgcccaacta agttttcgta 17100
tttttagtag aggcagggtt ttactatgtt gtccaggcta gtctcaaact cctgagctca
17160 agctatccat ctgccttggc ctcccaaaga gctggaatta caagcgtgag
ccactgtgcc 17220 tgaccagggt ggattttttc aagtgcacat gttgtggtcc
cagaagctct gatggtacca 17280 aattccaagc gaaaaaaagt
caatggttcc cacccatcct acctcccatg atggcaagag 17340 gaaatcacca
cactgcagat acagtccatg taaaacaaat tgctatggat tttgaaagtg 17400
aaccttaaga gaactgcact atgttttctt cattagagtt ctctggtaat ttccagcttt
17460 tttttttttt ttttttagac agtgtctcgc tttgtcgccc agtgtcaccc
aggctggagt 17520 gcagtgacgt gatctcggct cactgcaacc tccgcctcgt
gggttgaagt gattctcctg 17580 cctcagcctc ctgagtagct gtattttagt
agagacgagg tttcaccatt tggccaggct 17640 ggtctcgaac tcctgacctc
aagtgattcg cccatctcag cctcccaaag tgctgggatt 17700 acaggtgtga
gccactgcac ccggccagta atttcaagct tctgaggagc cctttgaatt 17760
gttaaataac ttgtagctat gtccaacata tccatgttca gtgtatgttc gatatttctt
17820 aggaaacctg cccttggttg ttttctttgt ggtaattcat gagccggcaa
atttgacatg 17880 tgttacagaa tatacctttt ctctgctctc ctacctcata
accagaactt aattatcctg 17940 ctttagtcac ataaatagct aactaaataa
atatatgaga tttcagtctg ctcactgtga 18000 aaatagacct tctaaatgat
ctcttccact tgcagatatt tgcaaatatg gatccatccc 18060 tcctgatgtg
gaggagaagt tacggttgga atggccctac caggagcaat tgctactccg 18120
agaacactac cagaaaaagt tcaagaacag cacctactca agtaagaaat gaaaggcacc
18180 ctagagatgt tccagcccca aagatatttg aataggttgg actcgggcac
caatctagca 18240 agtcctacgg aagttgtata aagctgaaaa tactgaagca
tttcccaaat gggaaatcct 18300 aaactcaaaa cttgcttttt ggtttttttg
tttgtttgtt ttttcttcat ctgacattgc 18360 ttagtagtca cagaatgaaa
gataaatcaa tcattcatga tctaacaatg accttcagtg 18420 ctctaaaaaa
ctacggagtc aaggaaaaca tgaatatatt cctcatgtaa aattaaaata 18480
cagacatata aagggcaaaa catgaacatc attcatacct tgaggtccgt ccccctccca
18540 gaaataaccc ccagtatgcc ttggtttaga gcattaagca ggagggccct
gagtcactcc 18600 agacagtctt gaccaccaag cagcattctc tttttgtttc
ctctgtggct tttgcaaaca 18660 cagggctagc tcagctaccc attagtatgt
tttcagtcac taaaacagtc ttccagtctt 18720 caaattagga tgacattgtc
acatggggct ttaaagcaag tgaaacaagg aacccccttt 18780 tttttttttt
ttgagatgga atctcactct tgtcgcccag cctggagtgc aatggcgcaa 18840
tcttggctca ctgcaacctc cacctcccag gttcaagaga ttctcctgcc ttagcctcct
18900 attcattatg aggaatattt gattattcag ttcctgtagg gtaaagatat
tacccccgat 18960 catattattg attattgagt agctgagatt acaggtgcct
gccaccacga ccggctaatt 19020 ttttgtattt tttagtagag acagggtttc
accatgttgg ccaggctcca ggctcgtctc 19080 gaactcctga cctcaggtga
tccacccacc tcagcctccc aaagttctgg gattacaggc 19140 gtgagccacc
actcctggcc acaatccttt tttaactatg aaatatattt ttatctgaag 19200
tttgatgttt atacccaact gagggatgat gttcccatat ctcagttaaa gaaataacct
19260 gctcagatac ttcaagctct tcttttgact tttgaaaata aatgatcttg
aagttactat 19320 actttgtttg ggttagttaa cattatttaa agtatattat
tttaattaat tatctttgta 19380 agattttact gtatactacc tggagttcaa
tgtatcagat ggatttcaaa tttatgtaca 19440 ttttttatgt atatggtaca
gaaaaaaatg tgatccataa gaaatcagaa aatagcgcat 19500 atgctaatag
ctaatgttgt cctctaaaaa acttattttt gcatttttaa gagggggata 19560
tactctgaca ctttaataag tgtaattaat tattgactgg aatttggcat gaggcagggc
19620 catttcagat cccattaaag gaatgacaca taccagagaa ccacagaagt
aaggccacat 19680 ttgtaataaa tcattatagc tctgctagga gaagacccag
ttgtattagg taattaatgg 19740 atttgctctt aaaacacatg tcccggaaga
tataggtgag tcttgggggg ccgcattaaa 19800 cattatacca atgtatctta
catttctaag aaagttttac tactttacag gatctttctg 19860 ttaccaaaat
ggaaggtttc caactccagg acttggcttt catagttcct acaccagggg 19920
aaatgccttc ctttgctaac tatgcaacca ggttagttag tgtaagtcca gccaccctgt
19980 tggcaatgct aaaaggtaca acaaacacag aattttattt gcatttgtaa
acatttgatt 20040 tctggctcga aattttcagt tttcatgggc acgtcatgga
aacagaaatc ttctgtgttt 20100 agtttgggca cctactcatt gtagtgacaa
atatttcaga agccaatagg ggattccaca 20160 aattgttctg aacctgtggc
tgagactggt aatggctgag tgacatgggg acataccaca 20220 aaagaagagg
tagcaaaagg ctgctgagat aaggacatgt tcattgctta gctagtggcc 20280
tgcaccctta aaacacatgt cccaggctgg gtgctgtggc tcacgcctgt aatcccagca
20340 ctttgggagg ctgaggcggg tggattacct gaggtcagga gttcgagacc
aacctggcca 20400 acatagtgaa acctcatttc tactaaaaat acaaaaatta
gccaggcatg gtggcgggcg 20460 cctgtagtcc cagctactca ggaggcaggc
aggagaatta cttgaatctg ggaggcagag 20520 gttgtggtga gccgagattg
cgccaccgca cgctagcctg ggcgacaaag tgagactctg 20580 tctcaaaaaa
acaaaaacaa aaaacaaaca aacaaaaaac aacaacaaca aaaaaacggg 20640
tatcccagaa gatacaggta agttttctaa cacaggtcct cttgtatggt gcgttccact
20700 taagtagaag atgacaaaaa catttgtcat gagaatatag actcacattt
taaacctgtt 20760 tgagcaggaa aaggaagcaa tgttacagat gtaattctgg
gtgtgactgc agaaaggatg 20820 actcccttat taaagtagtc atcctgagtg
agctaactct ttgtacttcc tcttctcctc 20880 ctgttcccct catcacccca
ttcttccgtt gcctacaccc aggcccacat tggatgctga 20940 catagactta
catggtacag tccaagggaa agatctgcca tttttttcaa tgtgtcatct 21000
tggttatctt cattccaagg atctctccac tctttataca gtaagagatg agagtctgga
21060 aaggattggg aataagataa tgaattgtaa gttttaaatt gttcttcgta
ttttggggaa 21120 ggagtaggct aggtggtcct tctgtttttt ttttgttttt
ttttttaaag tagatgtggc 21180 cagacgtggt ggctcacgcc tgtaatccca
gcactttgag aggctgaggc aggtggatca 21240 cttgatgtca ggagttcaag
accagcctgg ccaacacagt gaaaccccgt ctttactaaa 21300 aatacaaaaa
ctagccgggc ttggtggcgt ccacctgtag tcccagctac tgcagaggtg 21360
gaggcaggag aatcacttga acccgggagg tggaggttgc agtgagccaa gatcatgcca
21420 ttgtactcca gcctgggcga cagaacaata ctctgtctca aaaaaaaaga
gaaaagaaaa 21480 gaaaaaaaga atggatttga actcagtcgt caatagcctc
tattccagga gatgttacag 21540 ttgattatgt tatagggggt gtataataga
atttcgagct atgtaaattc caagtgcatt 21600 tggaagaatg aagaaatgga
ggaagggtaa agtatgagtg caagcattcc aggttttttg 21660 aaaatgctat
aatctttgtt cagggctagt acaaagtgct atttagctgt aagggttttt 21720
tgtgatttac agacagtttt cacatgtgtc atttcaacct tggttttatg gcgaaggcat
21780 gtgatggtgc ttgtcccagg actttagatc catatctgag gttcctgtcg
ggcaaagata 21840 ttacccctga tcatattata gtctataagt gggagagttg
tgcctggagc tcaagtctta 21900 tgatttctga tccagggcac ttcctacaac
atgattttgc aatataaaag cctataatgt 21960 gtgactaaag caggtcactc
accccttgta acagactcta gtaatggtac tgccaccaaa 22020 cggctgcgtg
atattgggca aagacttacc ttatttgaat ctcagtttcc tcctagaaaa 22080
atgagggtgg aggttaagca taggctgatg atcctaaagc ctccatactg ccctaaactg
22140 tggctctaag atccagtaga atgctgggtc acaggactct agggagcttt
tcaaacccaa 22200 atgtctgtca ttccttgatg gtaggcagca gtttatggaa
gtgggcgaca cagcaaatat 22260 caaaatacct aaagcagctt gcaagagttg
tttctgccta gtggtcttta tagttaatat 22320 taaatagtta attttttttt
tttttgagac agagtcttgc tctgttaccc aggctgcagt 22380 gcagtggcac
aatctcggct cactgcaacc tccacctccc gggtttgagc aattctgtct 22440
cagcctccca agtagctggg actacaggtg catgccactg cacccagcta atttttgtat
22500 ttttagtaga gacggggttt caccatattg ggcaggctgg tctcgaactc
ttgacctcag 22560 gtgatccacc tgcctcagcc tcccaaagtg ctgggattac
aggcatgagc cactgcaccc 22620 agcttaaata gctaatattt aatattattc
tatagttatt caagtaattc aggccaaaga 22680 cttagaaaca aaacaaaaag
ccacttttaa ggagaaaggg tgtaagtttg ccagatagat 22740 agagatcttt
cttttttaac tacaagagtt caggaatgaa ttactcttta acaaacgact 22800
atagatatac atgaaaattg gaaggactta ttatgcatat gataatcaat ttaaagacaa
22860 cacttaaaat tatattgttg ccactctcaa aaagtggtaa tagaacagct
aatggtttaa 22920 aaagcagagt acagaagttc ccaaacttat ggcaccttaa
tatcgcagaa aactttttaa 22980 agcatgccta ggccacaaaa aatacctgta
ttttgattat taaattgtaa ggtctacaca 23040 acctaatagt aataggtcca
atagtaatgc tgtccaatag atgttgatgt ttttttcctt 23100 gcaaacttaa
aagatcctac agtgcctctg taaatagcac tgcctggtta gagttgaatt 23160
tcagataaat aatttttttc atgttaatta tttttctttt ctttactttt ttttttgttt
23220 ttttgttttt ttgttttttt ttttgagaca gggtctcatt ctgttgccca
ggctgctgtg 23280 caatggcatg atcatggctc actgcagcct tgacctccct
gggctcaggt gatcctccca 23340 cctcagcctc ccaagtagct agctgggact
acaggtgctt accatcatgc ccggctaatt 23400 tttgtgtttt ttgtagagat
gtggttttgc catgttgccc aggctggtct tgaactcctg 23460 ggctcaagtg
atccgcccgc ctcggcctcc caaagtgcta ggatgacagg catgagccac 23520
tgcacctggc ccctgggcga agtatttctt aatggttaca taggacatac actaaacatt
23580 atttattgtc tatatgaagt tcaagtttaa ctaggtgccc tgcactttta
gttgctaaat 23640 cctgtagctg tacccatgca ttcactggtg ctccccagct
tgccttgcac agagtttgga 23700 aaccatagtc ctataactct aggccaattt
tttaatgtaa aatttgattc attttaaatt 23760 aataaataat aacaggaatt
tttttaaaaa ttgttttaaa tataattaaa attatcaaaa 23820 tattttttaa
ctgaacttgt gactagagat atttagatta tgaagagtgg ggtttatgct 23880
aactaatgac agtctggcta tgcatgtgga gcactgagct ataaattgtg gcttccccaa
23940 ttctcctgat gtcacttgaa caaaacctaa gtgtcagacc agagcttctg
gtatcttcca 24000 tgggatttca ttcaacagct ggagcaaatg aagtcagatt
gatttttttt aatttgtcca 24060 attttgttgt ctcaaaaaca taattataat
catttattag aactagaatt tcttcagttt 24120 aacaacagaa atagttattc
attatgaaaa gcgaatctgg aggccttcat tgtggtgcca 24180 atctaaccat
taaattgtga cgtttttctt ttaggaagct ctgtagatgt gctatacact 24240
tttgcaaact gctcaggact ggacttgatc tttggcctaa atgcgttatt aagaacagca
24300 gatttgcagt ggaacagttc taatgctcag ttgctcctgg actactgctc
ttccaagggg 24360 tataacattt cttgggaact aggcaatggt gagtacccca
gggaacaatt cattaataag 24420 gagattcccc actagcatta tttcttttct
tttctttttc ttttcttttt tttttttttt 24480 gagacagagt ctcgcactgc
tgcccaggct ggagtgcagt ggcgccacct cggctcactt 24540 gaagctctgc
ctcccaaaac gccattctcc tgcctcagcc tcccgagtag ctgggactac 24600
aggcacccgc caccgcgccc ggctaatttt tttttttttt tttttttttt tttttttgca
24660 tttttagtag agacggggtt tcaccgtgtt agccaggatg gtcttgatct
cctgacctcg 24720 tgatctgccc tcctcggcct cccaaagtgc tgggattaca
ggcgtgagcc accaggcccg 24780 gctagcatta tttcttatga cacttttttt
ttttttttga gacggagtct cgctctgtcg 24840 cccaggctgg agtgcagtgg
cgccatctcg gctcactgca agctccacct cccaggttca 24900 cgccattctc
ctgcctcagc ctcccgagta gctgggacta cacgcacccg ccaccacgcc 24960
cggctaattt ttttgtattt ttagtagaga cggggtttca ccgtgttagc caggatggtc
25020 tctatatcct gaccccatga tctgcccgcc tcggcctccc aaagtggtgg
gattacaggc 25080 gtgagccact gcgcccggcc aacactcttt ttattattag
caaatatact tctgcctggg 25140 cacattcttg caagtgctca acaatgcaac
ttttggaagt gcatgtggca gaaactcctg 25200 ctgtatttat tccagaacct
attattgcta atcccagttt atgttacatt tgaagtgaga 25260 accagttgga
gccagcaacg ttcccagctc caaagttccc ttgagatttt cagaatcact 25320
taaccctatt atgcttggca acctggactc agcaaaactg ggaagtcagc agtttgtttt
25380 attcatccct tcctttctca gtttctcaaa tgtgtcagtt aatctcagta
accccattgc 25440 aaccttcatt acctgcccaa gcggtctaga acttgccagt
atagaatcct acgtgggtca 25500 agctcctgac tgtctccttc ttcactcttt
ttttgcaaag aacttgtaaa ttttaactat 25560 aagtattcat gattcgccac
atttattcaa aacatagagt gctttttcca catatcagcc 25620 aatggaaata
aggattaaat gggaaatgaa atgtagtaat aggataagca caagtcttct 25680
tcctgctcaa actttttttt tttttttttt cagacaagat cttgctctgt tacccaggct
25740 ggagtgcagt ggcgtgttca tagctcaatg taacctccaa ctcctgggct
catgcaatct 25800 ctcacacctc agccccctga ttagctagga ctacactatg
cctagccaat tttttttctt 25860 ttgtctggtt gtgttgccca ggctgtctcg
atctcctggc ctcaagtaat cctcctgcct 25920 cggccttcta aagtgctggg
attataggca tgagccactg tgcccggtct caaacctttt 25980 tttccaaagt
aaatgaagtt attagatatg gaatatagtc tagttcccag atatccatat 26040
ccattggttt attaccctca ttattaactt caaattgttt aatagaccct catatctcag
26100 ttatacagtt aaaatttttg ttttgttttt ctggagtatc ttatttataa
ctatgagttt 26160 tactttactt atttatttta ttttttgaga cagacgcttg
ctctgtcact caggctggag 26220 tgcggttgcg tgatcatggc tcactatggc
ctcgaccttc tgggctcaag tgatcctctc 26280 cctcagcctc ccaagctgag
actacaggca tgcaccacca catctagcta attttttttt 26340 ttccccatgg
aacaaggctt tactatgtta cccagagtgg tctcaaactc ctggcctcag 26400
gggatcctcc tgtctcagcc taccaaaatg ctgggattac aggcatgagc catagcgcca
26460 gacctggttt tacttttctt gactttgaat tacaagtttt tgtaatttgg
aaaatgtttt 26520 gttgctttta aatactgctg tatgtttgct tttaaataca
acatttctcg atatatattt 26580 tgagaattgc tgtctttcag aacctaacag
tttccttaag aaggctgata ttttcatcaa 26640 tgggtcgcag ttaggagaag
attttattca attgcataaa cttctaagaa agtccacctt 26700 caaaaatgca
aaactctatg gtcctgatgt tggtcagcct cgaagaaaga cggctaagat 26760
gctgaagagg taggaactag aggatgcaga atcactttac ttttcttctt tttccttttg
26820 agacagagtc tcactctgtc agccagactg gagtgcagtg gtacaatcat
ggctcactgc 26880 aacttcgacc tcccaggctc aagcaatcct cccatctcag
tcccacaaat agctgggact 26940 acaggtgcac atcaccacac ctggctactt
taaaaaaatt tttttgtaga gatggggtct 27000 ccctgtgttg cccaggctgg
tctcttgaat tcctgtgctc aagccatcct tccacctcag 27060 cctcccagag
tgccaggatt acaggcatga gccaccacac ccagccacca cttttcttaa 27120
aaaaaaaaaa agattctctc tggtagacaa tcctcaatag tccacatgtt attaaacaat
27180 ctgctgcctg aatacatgat ttaccaaaaa aaggaaattt tgacgggttc
agaatatcaa 27240 gggatctgag gcaaatgtca cctatgataa aatttgctat
caaaattagg aagtttgtgt 27300 ttacctgatc ctaaagcagt aaccagccca
tttctaggga ataaaactct catgcgtata 27360 ttgtgcatat atatgtatta
tatgactgag tgataataaa attttttttc tagcttcctg 27420 aaggctggtg
gagaagtgat tgattcagtt acatggcatc agtaagtatg tctcctattc 27480
ttaatactag gaaagtaagg ctagctttat ttattaccta gtattcaaaa agttagttca
27540 tttaactgcc aattgactgc agttcaaata agaaacaaat agtgtctcaa
gtagcactgt 27600 actccaattt taatattaat aaaaaaaatt ttaagttatt
ttaaataatg tagtggtttc 27660 tataaagatc actttataca gaagaacagt
gccaattaac ccatggaaca tataagtagc 27720 taaaaccaat tgcttgccaa
agaaccagta acccaggagt acatgtcctt gccactgtgt 27780 tttttcaaga
cagagtaact gatttctagt tacttgcata gaatggactc ctcctcataa 27840
ctcccttcca tcttggtctt tccctagtag aacttctacc tttttttagt aacaggtgag
27900 tgggagaggt aagaaggaga ataaggtcag caattaacct aaaagcagaa
agtaaaattt 27960 gttatttttt ttctgaatat tttctgtgta atttagctac
tatttgaatg gacggactgc 28020 taccagggaa gattttctaa accctgatgt
attggacatt tttatttcat ctgtgcaaaa 28080 agttttccag gtaatagtct
ttttaaactt tttaatgtaa aaccagaatc cttattttat 28140 agtctagcta
gttctaaatt ctataggtat gtatatttac atgtttttct aattttagag 28200
aacaagcact atgacttatc cactgttagt tttcccctta gcattgggtc ttaccccatg
28260 tacgtgatta gaaatttgaa atatttccaa tagcctttag tagaattaac
tcacatagat 28320 gataagaatg ggttggttca cttcatgttc cttccacagc
ctactatttc aataaaagaa 28380 agtttcccaa gacctaaatg actatgaaca
tattttataa ctatatagga ggggtgggtc 28440 taggaataca aagttttgaa
tgctgttaat cttcaacacc acagttgaaa ccacaggtca 28500 gcttttttgc
aattaccatg gatacttttc tgttctatag gtggttgaga gcaccaggcc 28560
tggcaagaag gtctggttag gagaaacaag ctctgcatat ggaggcggag cgcccttgct
28620 atccgacacc tttgcagctg gctttatgtg agtgaagcag cgctggcctt
aggggtcaga 28680 gtgcagctct tctccatcct tctattctgc tgaaatagct
ccccagccaa aaagcagatc 28740 aaagaccgtt tcagtggctg agccccaaaa
ttcatgccag attttgcaag aaaatgattt 28800 actaaagctt gagggacatc
tttaacaagt gttccaaatt aatcactata aggatgaatt 28860 gtttcagaaa
ttttggcctt taattatggc ccataaatat gtcaagtagt ccttactcta 28920
aagaagtaca ctgtaaaaga atgcatatag ccggatatgg tagttccctg taatcccaat
28980 actttgggag gccaaggtgg gaggattgct tgagcccagg agtttgaggc
tgcagtgagt 29040 tatgatggtg ccactgcact ctagactggg caacagagtg
agactgtctt tttttttccc 29100 ctctgtcacc cagactggag ggcagtggca
cgatctcacc tcactgcaac ctctgcctcc 29160 cggattgaag cgattctcct
gcctcagcgt cctgagtagc tgggactaca ggagtatcac 29220 cgcactgggc
taatttttgt atttttagta gagacggggt tttgacatgt tgcccaggct 29280
ggtctgaaac ccatgagctc aagtgatctg cctacctcag ccttccaaaa tgctgggatt
29340 acggacatga gctaccacgc ccggccacac cctgtctctt aaaaaaaaaa
aaaatgcaag 29400 ttagagcata ttacagcttt gtctctcagg aggatactta
gtgtatgtag ctataattca 29460 tagattccca agaagtttag agcctaaagt
atgaggtccc accagagggg ctatcattaa 29520 atttaaagat ttgttaaatc
atctcattgt ccaacaccac aaacttgatt gctttaaaat 29580 actggtttag
ttacatttag taactctatt agtgctttta atctatactg ctatatcctc 29640
acattgagat tttttttctt ttctcttcca tcttcattct tttttctctc atcctcattc
29700 ttataagcct agaatacatc acaaatcctt tatgcccatg gaagcaagag
gaataaagaa 29760 tggagatgtt tgttttgcca ttaactaaag atctggggtg
tcggggagaa gggggataga 29820 gaaggagaag tgggaagagg tgtccataat
agcttaggtg caattctgct tattttacat 29880 tttacccccg ctgactgcca
ctttttcttc agccctcaca cattgtttgt gcagggacct 29940 cataggacca
ggaattgtct atagaggtgg gaatttgtct caccctgaaa gggatacctc 30000
tagcatggta atagtcttct aggatttgtt atcatatgga aagatgtaaa gggagggatt
30060 ctgctgctgc tgctgctgct gcatgcagtt gccatttcat ttaaatgact
tatttataat 30120 tgatgacact tttctggctt cctgttaatt cctccctcaa
agatcaataa accagaacca 30180 ggcatggtgg catgcacttg tggtcctgta
accacccaac aggttcacct tgcctgctgt 30240 ctagatagag ccaattatca
agacagggga attgcaaagg agaaagagta atttatgcag 30300 agccagctgt
gcaggagacc agagttttat tattactcaa atcagtctcc ccgaacattc 30360
gaggatcaga gcttttaagg ataatttggc cggtaggggc ttaggaagtg gagagtgctg
30420 gttggtcagg ttggagatgg aatcacaggg agtggaagtg aggttttctt
gctgtcttct 30480 gttcctggat gggatggcag aactggttgg gccagattac
cggtctgggt ggtctcaaat 30540 gatccaccca gttcagggtc tgcaagatat
ctcaagcact gatcttaggt tttacaacag 30600 tgatgttatc cccaggaaca
atttggggag gttcagactc ttggagccag aggctgcatt 30660 atccctaaac
cgtaatctct aatgttgtag ctaatttgtt agtcctgcaa aggtagactt 30720
gtccccaggc aagaaggggg tcttttcaga aaagggctat tatcattttt gtttcagagt
30780 caaaccatga actgaatttc ttcccaaagt tagttcagcc tacacccagg
aatgaagaag 30840 gacagcttaa aggttagaag caagatggag tcaatgaggt
ctgatctctt tcactgtcat 30900 aatttcctca gttataattt ttgcaaaggc
ggtttcagtc ccagctactt gggaggctga 30960 gacaggagga ttaatggagc
ccaggagttt gaggttgcag agagctatga tcacgccact 31020 gcactccagc
ctgggtgaca gagtgagacc ctgtctctaa ataaataaat aagtaaataa 31080
ataaatacat aaataaaatc aagatggtgt gcaattagaa ttgagcgatt ttgtttccaa
31140 acctcaagaa agcttggtct tgctctgtcc caggtggctg gataaattgg
gcctgtcagc 31200 ccgaatggga atagaagtgg tgatgaggca agtattcttt
ggagcaggaa actaccattt 31260 agtggatgaa aacttcgatc ctttacctgt
aagtgaccat tattttccta attctagtgg 31320 agtagattaa agtcaactca
ggacctctgg tgttaacctc ctatgaacag tcagtcctct 31380 cagtaactag
ccaaatcatg agatgatgaa ttagaaggag ccttagatag catccaatct 31440
aacatttttt tgtgtgtttg aagagaagaa atcaagagct aggaataact ttttaaaggt
31500 aagccatttg cagtatagtg tggattttgt ttaaaagggg ataatttgaa
attttatgac 31560 tcattataca agacaaaata agttggattt tcaaatgttt
tacaaagtaa atcaaagtta 31620 taattgccta cagtacgcaa agcttcaaaa
cattttttat gttatgaaat tgtaatttat 31680 ttaaccttaa aatgagccag
taccatgtgt ttgcttaaaa atctcatgct aagaatttac 31740 tatgttgtta
ataatcttca agatatttat gaataaagtc ttatttctaa tccttcctcc 31800
aactgtatct ggtgctaaat caggaaatgt ttcttcccaa aaagcctcgt ggaagatctg
31860 tatgtctaaa tatatgtcag ggataataca gatgtagccc tgcgaagcat
gaccttgatt 31920 tttatagtct aaaatgtcat ttgcagatat ctattttcta
agaataattc ctaaaagaat 31980 tatttgaatg ttgtaggaaa gctaagaaat
tttgcaaaga gcgtacgtga aaatataagc 32040 taggcttttg tggtttgtgg
atagacttcc caacaaaatt gctttttatc tatagtgatc 32100 caagcttgtg
gaacatatta gtcatctttt tttagaaaat tcttagaaaa gtgatcttgc 32160
aaaaatggaa tttatctttc cccaagtata ttctgtcatg tatagagtta aactaagcat
32220 agtaatttca ccagacaaac attcaaaatc tactcctgac ctttttatct
catccaaatt 32280 ttcccagggc ccagacataa acctttgcct tacgaactct
ttgtatatgc actaaatatg 32340 cttctccttc aaggttctca
gtcagctaga aaaatgtgca agagtaaatg gtacccttct 32400 cacttgtaga
tccaagagaa ttagacttaa actcactcta catgtctgtg actttatttt 32460
atttgcatga cagtcctgtg aggtggcaag gcaggtatct tggatccatt ttttagataa
32520 ggaagttcaa attgagaaga ggttgcatga tttacaggaa gccatactgt
agtcctatgt 32580 tactcttaaa aatcccattc aaatcctgct tctgaggcct
gcatactttc taccctacca 32640 gtcattgacc catgcttatg tctcctttga
aaacattgat tccactcttg tctccagtga 32700 aaaagtggaa tttaagcaga
gaaacaaaag ccatttgtct tgttaagtct actttccctc 32760 tactttcaag
aaggaaagtt ggggtatgtg ttgaatggtg atttatttat ttatttatta 32820
ttttaaaaat tgatacaagg tcttactgta ttgtgcaggc tggtctcaaa ctcctgggct
32880 caagtgatca tcccacctca gcctcccagt gttgggatta cagcatgaac
cattgtgccc 32940 accaccgatc cgcagttttt taagaaaaac ttttactata
gaaaatttta atcatataca 33000 aaatacagag gaaagtatat gaacccactt
taggagacta gaatatgcca ccccaaaata 33060 tgccactttg gcataaggat
tatttcgagc taaaggcaac tgggaagaaa cacatagaag 33120 aaaagttctc
tgtccttctc catttgccta aaagcaggac atgaatctta aaagtccccc 33180
tccttccctt tctaccagga aaaacaagag ttaatcactg aagataactt cagaccctta
33240 tcagtgtaga gatggcacta gaagaatcta tattacatac tcatttattt
tccttcccac 33300 aacttgccac cccagagact aaaaatcctt ttcctttgtc
atgtctcttg tccaaaaatt 33360 tgctctataa gctggagttc taagccacct
ctttgagaat tacttgttcc ctggtatttt 33420 ctgttaacat acatgtatta
atatacatgt taacaagctt ctgtttgttt ttctcctgtt 33480 ttctgtcttg
ttacagaggt ccatcccaac taagaactaa agagtaggag gaaaatataa 33540
tttcctcctg catactttga tcttgtttaa tccgtaaccc ttcccacttt tcacctccta
33600 cctattagat tactttgaag caaatttcag atatattact ttatctataa
atatttcagt 33660 atgtgctagg tgtggtggct cacacctgta atcccaacac
tttgggaagc tgaggcagga 33720 ggatcacttg agcccaggag ttcaagacca
gctacggcaa caaaaaatca aaaacttatc 33780 tgggcatggt ggcacatgcc
tgtggtccca gctacatgag aggctgaggc aggaggatcg 33840 ctttagccca
ggaggttgag gctgcagtaa gctgcattca caccactgca ctccagcctg 33900
ggtgacagag taagaccatg tctcaaaaaa atacatattt tagtatgtat cctttttgta
33960 aaaacacaat acttttatca tactttaaat aataacaata attccttagt
atcaccaaat 34020 attttgtcag tgtctcacat tttccttatt gtctaaaata
ttgttgatag ttattcaaat 34080 cagaatccaa acaaggtcca tatattacat
ttggttgaca agtctcttaa gtttgttcat 34140 ctttaagttc ttcctccctc
tctttcatct cttgtaattt attaatgtga aaaaacaggt 34200 aatttgttct
atagtatttc ctacattata gagtttgcta catttattcc ctatgatatc 34260
atttagcatg ttcctctgtc ccctgtgttt cctgtaaact ggtagttata cctagaagct
34320 tgagtttatt caggttttta attgtatttt ttttgcaaga attctttatt
atctgcttct 34380 ggaagcacag aatgtctggt tgtgtctggt tttgatcttg
acagctactg atgaccattg 34440 cctaatccat tactttattg gggtgggggg
aataaggttt taaaataaat tttttttaaa 34500 gattttttta actgttattt
tgagacagtg tctcatttcg tttcccaggc tggagtgcag 34560 tggcacaatc
acggctcact gcagccttga cctcctggga tcaggtgatc ttctcacctc 34620
agcctcctgg gtacctggaa ctacaggtgc acaccaccac acctggctaa ttttttgtat
34680 tttgtgtaca gaaggggttt catcatgttt cccagactgg tcttgaactc
ctgggttcaa 34740 gtgatctacc cacttcagct tcccaaaatc ctgggattac
actttggcca ccgtgcctgg 34800 cctaaatgaa attatttgtc tctaaacaga
cagaagtttt actttaaaaa tttgtctttg 34860 tgtgtacatg tgtttgtgta
tgtgtgtgtg tctaaaagtt tggctttgag ctttgctttg 34920 aattcttgga
tgaacaataa ccaagaatac ttaaactctg atcattcttg acagatatcc 34980
cctacaggct atggcctttt gaattgtgtc ctccagtgat aaaaagcagc aagcacgata
35040 ctgctctcag attcatggtg gtcacatgtg aggtgaaaaa aaaaaaaaag
atgaatccta 35100 tttaaatgcc cccaggataa cagtgatact ctttgtagga
taactatttg cttgccactg 35160 gtttcattaa ataaggacat aagtaaagat
ctatttttgt ctctttctcc ccaaccacca 35220 caactaggat tattggctat
ctcttctgtt caagaaattg gtgggcacca aggtgttaat 35280 ggcaagcgtg
caaggttcaa agagaaggaa gcttcgagta taccttcatt gcacaaacac 35340
tgacaagtaa gtatgaaaca caccctttac caatcatcaa gttttagtgg gtaagcctgt
35400 aactttactc aaacaccctg ttgcatgtgt ctatacattg cataagtata
ggcagttgca 35460 atttagtaaa gttttataca acgattttat tttattttat
ttttagaaga aaaatgctac 35520 ttttgttgtt gttgtttttt gagacggggc
ctcgctcgtc acccaggctg gagtgcagtg 35580 gtgcaatctc agctcactgc
aacctccgcc tcccgggttc aagtgattct tgaagaggag 35640 aacaataata
acaacaatat tattttcaaa agttgtgacc gcagtttctg gagttgagaa 35700
gacatcgaga tttttgtagc ctcatactct tgctttaggt agcaaaaaat gttcctaaat
35760 ctcaggaata ttctctagat aggtttcaat ctatcattcc tgataagatg
atgctgaaat 35820 actaattcta gccaaaaaag accagctacc atttccgatt
gttggggact gggaactctg 35880 gatagtgagg accccagtag gaagtagcga
ggggaatggt ttgaatggat aaattcataa 35940 aaaatgtcag tagatttaat
tttcttatac atttcagtct ttttataagg ctaggaaaag 36000 cccctgtttt
tatggtttat aatttgaatt cacatgaacc cacaaaattt gccttttacc 36060
ttcctatgtc tgaaaatgga tagtctggct ggcctcttaa caacccagct ggcagagctg
36120 tgaggatctc agtgtgctct agcccagaca ttggtagcat gaacggcaac
atttttaatt 36180 gtgttttcaa aataggagca cactagcggt ctaaaacgat
cataaaagaa ggatactaag 36240 agggcccact gtcattatgg atcctaatac
ttaggatgca ttatggattg tcattatgga 36300 tactaatact taggatcaca
tttgtaattg agtttttaat tgcttaaatt agatacatat 36360 ttctattaag
ttaacctctt tgcttttagt ccaaggtata aagaaggaga tttaactctg 36420
tatgccataa acctccataa tgtcaccaag tacttgcggt taccctatcc tttttctaac
36480 aagcaagtgg ataaatacct tctaagacct ttgggacctc atggattact
ttccaagtaa 36540 gtaattttcc ttgttcattc caaactttca ataaatttat
tggtgtttat cagaatagag 36600 agtttggaca gggagcaaaa gacaaagtca
actatatcaa gttctaataa ttcttaatat 36660 tcaggaaatt tatgtatgaa
tacttactaa tatgagtata actcatccta agagtctaaa 36720 gcaaaaggat
gtgaacacaa actagcagtt atcttagaga ataagtttgc atttcaaaat 36780
aacttgacat atcaagatcc actcaacgca tttaaattat ttactctaaa aagacataat
36840 tcttggtaac acattcacta aagcaaaata tacctttata taattgctat
caaaggtatg 36900 tgggttggta taaaatatca taccatgtga gatcagtgtg
attcctttac agcattaatt 36960 tttattggtt agagtaagaa aaagaatagc
tagagtatat ttcttaagta gattctcata 37020 cactttggtt tcaaaaacca
attattgact acatcttata aaagcctgta ttcaatggag 37080 tgccaaaaaa
tgactatgag tcttaaagag ttaggcatat aaatatttta aggtttctgt 37140
tcaatgtatg ttggaaggag ttcctttctc atgactattc tcatattgga gcataaaaag
37200 agtttacagg cttggcgcag tggctcatgc ctgtaatccc aatactttgg
gaagctgaag 37260 caggcagatc acttcagccc aggagtttga gaccagcctg
ggcaatatgg caaaactctc 37320 tctacaaaat ataccaaaat tagccaggcg
tggtggtgca tgcctgtagt cccagctact 37380 tgggaagctg aggtgggagg
attgcttgag cccagggggg tcatggctgc agtgagctgt 37440 gatggtgcct
ctgtcaccca gcctgggtga cagagtgaga ccctgtctca aaaaaataaa 37500
taaataaaaa ttaagagttt acaaaattct caccatctcc tcccatcttt gcaaatgcca
37560 cataagtgat gtgttccagg actattagcc tcggaacctg aggcagtaca
gtaagcacgc 37620 tttctccaaa gtcctgtccc ccacagacaa acattattta
cactgggtac tgctctttta 37680 ttttttcccc tctatgcttt attttactat
aactataatc atataacatg taataggaaa 37740 aaggcagggt cgggggagag
atccagaagt cttcccaaga gcctttccaa catagcctct 37800 gtagacattt
tttctttctt cttttttttt tttttttttt ttctgagaca gagtctcact 37860
ctgttgtcca ggctagagtg cagtggcgtg atctaggctc actgcaacct ccgcctcctg
37920 ggttcaagca attctcccac ctcagcctcc ctagtagctg ggattagagg
catgcatcac 37980 cacgcctggc taatttttgt atttttagta gagatgaggt
ttcaccatgt gggccaggct 38040 ggtcttgaac tcctgacctc aagtgatcca
cctgccttag cctcccaaag tgctaggatt 38100 acacgagtga gccaccgtgc
cctgccccta ttacattctg atcacacatt tcatgtttta 38160 taattggaaa
actggtgaaa ttatagacaa tgttttgttc ccctaaattc tctttgatga 38220
gtatatatta cttacactct tctgtcttta aaattttgca aaatagtatc ctagataagt
38280 ttatgagtgc acagtctgta cgcttactca tattaatgac ctcggagagt
taaacaacag 38340 tcacctttaa aaattattac tatcattatc attatttttg
aggcgggggt ctcattctgt 38400 ctcccaggct ggagagtagt ggtgcggtca
cagctcactg cagccaccgc tacctgggct 38460 caagtgatcc ttcctcctca
gccttctgag tagctgagac cacaggctta tgctaccaca 38520 cctggctaat
tttttaactt tttgtagaga cgatgtctca ttatgttgcc caggctggtc 38580
tcaaactcct aagctcaagt gatcttcctc agcctcccaa agtgctggga ttacaggcat
38640 gaaaaactgc acccagccct aaaaattatt agggtcctgc atagtaagac
tttaataaat 38700 atttaaatga acatctggtt tttttaaaaa aaaaatagag
acaaggtctc actatattgc 38760 ccaagctggt ctcgaactcc tggactcacg
caatcctgct gccttagccg cccaaagtgc 38820 tgggattaca ggcatgaccc
acctcatctg ggctgagtga acatattttt aacataaagg 38880 ccgtatttta
tatttatctc atacattttg cccagcatcc ccatttccgc cgaatctgtt 38940
gcttgctaat tccttccagc ttcatttcat ctgaaatttg acaaacatct tctatttctt
39000 tgtcgtcatg ttattgactt cagaatataa aataaaacac tatacccaaa
ttaaacccca 39060 ccctcattgc ccagcctgat gtgaaaataa tcagcataca
ttaagcttac ccttgatata 39120 tgtgtagcat cttttagata aatatacagc
tgattaagca atatagcctg atggtataat 39180 atcttgccca tgtacctcat
cttatctcca gcaggattaa ttcacagtga tcagatttac 39240 ctttaaactt
tgtagcaaaa tatcctctcc aaaagcatat ctaaaacttt tgtgtgtact 39300
cttgcaagtt tcttaatttc atgcagaaca ggctcttacc actgttagct ggagatattt
39360 tcaagaccta tttttgtttg tggtttcctg atgatggtca tggcatttcc
cccttcactc 39420 catctaaaaa ttgaggtgat acaggctttt aaacaaaacc
aactcatata gactgagtac 39480 aactgcaatg caggcatgct aacctctgct
acaatcatgg gcgtgctatt gatatgtctt 39540 aagttacaga acacagggct
gagcgtctca ttaggtcaaa atgtaaacca gtttttctgc 39600 tcactgatgc
ttaatgagga cagggtgtga gagatttctt taaggaaaac aaatatataa 39660
taatgctaca tggaaaaata tctaacatta gagaattaag taaataaact aatatactca
39720 caccatggaa tcttgtgcag acattaaaat tatgtagtgg atggatgttt
aatggtgtga 39780 gaaaaagtta ggatgtgctg gggtgggggg aagaatcaag
ttttaagaaa atacagtata 39840 cccatactta agtaaaaaaa aaaaaaaagg
tatgtacagt catgtgttgc ttaatgatgg 39900 ggatacattc cgagaaatgt
gtcgataggt gatttcatcc ttgtgtgaac atcatagagt 39960 gaacttacac
aaacctagat ggtctagcct actatgtatc taggctatat gactagcctg 40020
ttgctcctag gctacaaacc tgtaaagcat gttactgtag cgaatataca aatacttaac
40080 acaatggcaa gctatcattg tgttaagtag ttgtgtatct aaacatatct
aaaacataga 40140 aaactaatgt gttgtgctac aatgttacaa tgactatgac
attgctaggc aataggaatt 40200 ataattttat ccttttatgg aaccacactt
atatatgcgg tccatggtgg accaaaacat 40260 ccttatgtgg catatgactg
tatacatgta cacaaaaaat agatgaaaga atgaatatac 40320 atcaaaatat
ttaaaatggt tataatgact taggttactt ttatttatct tagtaataat 40380
aatgatgata gataatactt ttatagtgtt tactatataa aagacactgt tataagtgtt
40440 ctacatactt tacatgtatt acctaaatga tataaatata actctgacag
taactaatct 40500 tatacgttct cttttctttt tttttttttt ctttttttag
acagaatctt gctctaccag 40560 gctggagtgc agggtgcaat ctcggctcac
tgcaacctcc gcctcccagg ttcaaacgat 40620 tctcatgtct cagcctcctg
agtagctggg actacaggca cacaccacca tgcccggcta 40680 atttttgtat
ttttgggtag agatggagtt ttgccatgtt ggccaggctg atcttgaact 40740
cctggcctca agtgatctgc ctgcctcagc ctcccaaagt gctgggatta caggtgtgaa
40800 ccactgtgct cggcctaatc ttacaagttt tcaatattta aagagtgcta
actttgttga 40860 caatataaaa catatttgag aaaaagagat ataagcatct
tatttagaat tatgaaaata 40920 tcaatagacc tacagccgac taaagctttt
cttcataagc tcttgcctat attgattcgc 40980 tcctgtgaat atgcattaat
ttgatttaaa taataagtat gtataagaaa taacactttt 41040 ccttaatttt
taagaacgtt caacagtttt taatttgaat tccaatagtg aaatacatag 41100
aaaatataaa attttctgta gtttagccaa attgtttttg tttcaccaca gcattctacc
41160 aaaatttctt aataacagta agaaaatgaa tgcatacctc ctgcagggag
aggggagtta 41220 ggcagtttat gggcatagtt acaagtgaga aatttcattg
gctaccattt acgctaaatt 41280 cataaaaact gcattcaatt ctatatatct
attttcttta cataaaaaag gtttcaatta 41340 ttggccatta aataaaatag
ccaccattcc agaagttgtg tcatgtttat cctttttata 41400 ccaccatcat
attgcctatt atatagattg tgtgtgttcc attttctgta atgggccaga 41460
cagtaagtat ttctggcttt ggagtccata tggtctctat cataactact catctctgcc
41520 attgtagctt aaagattatc taggtcaaat gcctaagtga tatagtgttg
aaatacaagt 41580 tatataatat aggctgccac aaaaaaaaat ttatttggtc
taaaaaagat ttcatgactt 41640 ttgtagcagc atgggtgggg catgcaccac
ttggttaact cggtgtatct ttctcctttg 41700 cagatctgtc caactcaatg
gtctaactct aaagatggtg gatgatcaaa ccttgccacc 41760 tttaatggaa
aaacctctcc ggccaggaag ttcactgggc ttgccagctt tctcatatag 41820
tttttttgtg ataagaaatg ccaaagttgc tgcttgcatc tgaaaataaa atatactagt
41880 cctgacactg aatttttcaa gtatactaag agtaaagcaa ctcaagttat
aggaaaggaa 41940 gcagatacct tgcaaagcaa ctagtgggtg cttgagagac
actgggacac tgtcagtgct 42000 agatttagca cagtattttg atctcgctag
gtagaacact gctaataata atagctaata 42060 ataccttgtt ccaaatactg
cttagcattt tgcatgtttt acttttatct aaagttttgt 42120 tttgttttat
tatttattta tttatttatt ttgagacaga atctctctct gtcacccagg 42180
ctggagtgcc atggtgcgat cttggctcac tgcaacttta agcaattctc ctgcctcagc
42240 ttcctgagta gctgggatta taggcgtgtg ccaccacgcc cagctacttt
ctatattttt 42300 tgtagagatg gagtttcgcc atattggcca agctggtctc
gaactcctgt cctcgaactc 42360 ctgtcctcaa gtgatccacc cgcctcagcc
tctcaaagtg ctgggattac aggtgtgagc 42420 caccacaccc agcagtgttt
tatttttgag acagggtatc attctgttgc ccaggcttga 42480 gtgcagtggt
gcaatcatag atcactgcag ccttttaact cctgggctca agtcatcctc 42540
ctgcttagcc tcccaagtag ctaggaccac agacacatgc catcacactt ggctattttt
42600 aaaaaatttt ttgtagagat ggggtctcgc tatgttaccc aaactggtcc
tgaactcctg 42660 gactcaattg atcctcccac cttggccttc caggtgctgg
gatttctttg ggagtacagc 42720 atggtacagc aggagatcat ttgatgttac
ctctgtgcag tgttgctagt cagcgaaaga 42780 ctataatacc tgtggggaca
gcgattagcc accacaacca gtctttattt aaagttatta 42840 aaaatggctg
ggcgcagtgg ctcacacctg taatcctagc actttgggag gccgaggcag 42900
atggatcacc tgacgtgagg aatttgagac cagcctggcc aacatggtga aaccccatct
42960 ctactaaaaa atacaaaaat tagctgggtg tggtcctgta gtcccagcta
cttgggaggc 43020 tggggcagga gaattacttg aacccaggag gcagaggttg
cagtgagccg agattgtgcc 43080 actgcactcc agcctgggtg acagagagag
attccatctc aaaaaaacaa gttattaaaa 43140 atgtatatga atgctcctaa
tatggtcagg aagcaaggaa gcgaaggata tattatgagt 43200 tttaagaagg
tgcttagctg tatatttatc tttcaaaatg tattagaaga ttttagaatt 43260
ctttccttca tgtgccatct ctacaggcac ccatcagaaa aagcatactg ccgttaccgt
43320 gaaactggtt gtaaaagaga aactatctat ttgcacctta aaagacagct
agattttgct 43380 gattttcttc tttcggtttt ctttgtcagc aataatatgt
gagaggacag attgttagat 43440 atgatagtat aaaaaatggt taatgacaat
tcagaggcga ggagattctg taaacttaaa 43500 attactataa atgaaattga
tttgtcaaga ggataaattt tagaaaacac ccaatacctt 43560 ataactgtct
gttaatgctt gctttttctc tacctttctt ccttgtttca gttgggaagc 43620
ttttggctgc aagtaacaga aactcctaat tcaaatggct taagcaataa ggaaatgtat
43680 attcccacat aactagacgt tcaaacaggc caggctccag cacttcagta
cgtcaccagg 43740 gatctgggtt cttcccagct ctctgctctg ccatctttag
cgctggcttc attctcagac 43800 tctggtagca tgatggctgt agctgtttca
tgggcccctt caaacctcat agcaaccaga 43860 ggaagaaaat gagccatttt
ttgagtctcc ttcatagact tgaataactc tttttcagag 43920 cttctcacag
caaacctctc ctcatgtctc ctcatgtctt attgttcaga aatgggtaat 43980
gtggccattt caccagtcac tgccaacaac aacgaggttc ctataattgt ctctgagtaa
44040 ccctttggaa tggagagggt gttggtcagt ctacaaactg aacactgcag
ttctgcgctt 44100 tttaccagtg aaaaaatgta attattttcc cctcttaagg
attaatattc ttcaaatgta 44160 tgcctgttat ggatatagta tctttaaaat
tttttatttt aatagcttta ggggtacaca 44220 ctttttgctt acaggggtga
attgtgtagt ggtgaagact cggcttttaa tgtacttgtc 44280 acctgagtga
tgtacattgt acccaatagg taatttttca tccattaccc tccttccgcc 44340
ctcttccctt ctgagtctcc aacatccctt ataccactgt gtatgttctt gtgtacctac
44400 agctaagctt ccacttataa gtgagaacat gcagtatttg gttttccatt
cctgagttac 44460 ttcccttagg ataacagccc ccagttccgt ccaagttgct
gcaaaataca ttattcttct 44520 ttatggctga gtaatagtcc atggtacata
tataccacat tttctttatc cacttatcag 44580 ttgatggaca cttaggttaa
ttccattcaa tttcattcaa tttaagtata tttgtaagga 44640 gctaaagctg
aaaattaaat tttagatctt tcaatactct taaattttat atgtaagtgg 44700
tttttatatt ttcacatttg aaataaagta atttttataa ccttgatatt gtatgactat
44760 tcttttagta atgtaaagcc tacagactcc tacatttgga accactagtg
tgttgtttca 44820 ccccttgtta tactatcagg atcctcga 44848 43 2396 DNA
Mus musculus 43 tttctagttg cttttagcca atgtcggatc aggtttttca
agcgacaaag agatactgag 60 atcctgggca gaggacatcc tagctcggtc
agatttgggc aggctcaagt gaccagtgtc 120 ttaaggcaga agggagtcgg
ggtagggtct ggctgaaccc tcaaccgggg cttttaactc 180 agggtctagt
cctggcgcca aatggatggg acctagaaaa ggtgacagag tgcgcaggac 240
accaggaagc tggtcccacc cctgcgcggc tcccgggcgc tccctcccca ggcctccgag
300 gatcttggat tctggccacc tccgcaccct ttggatgggt gtggatgatt
tcaaaagtgg 360 acgtgaccgc ggcggagggg aaagccagca cggaaatgaa
agagagcgag gaggggaggg 420 cggggagggg agggcgctag ggagggactc
ccgggagggg tgggagggat ggagcgctgt 480 gggagggtac tgagtcctgg
cgccagaggc gaagcaggac cggttgcagg gggcttgagc 540 cagcgcgccg
gctgccccag ctctcccggc agcgggcggt ccagccaggt gggatgctga 600
ggctgctgct gctgtggctc tgggggccgc tcggtgccct ggcccagggc gcccccgcgg
660 ggaccgcgcc gaccgacgac gtggtagact tggagtttta caccaagcgg
ccgctccgaa 720 gcgtgagtcc ctcgttcctg tccatcacca tcgacgccag
cctggccacc gacccgcgct 780 tcctcacctt cctgggctct ccaaggctcc
gtgctctggc tagaggctta tctcctgcat 840 acttgagatt tggcggcaca
aagactgact tccttatttt tgatccggac aaggaaccga 900 cttccgaaga
aagaagttac tggaaatctc aagtcaacca tgatatttgc aggtctgagc 960
cggtctctgc tgcggtgttg aggaaactcc aggtggaatg gcccttccag gagctgttgc
1020 tgctccgaga gcagtaccaa aaggagttca agaacagcac ctactcaaga
agctcagtgg 1080 acatgctcta cagttttgcc aagtgctcgg ggttagacct
gatctttggt ctaaatgcgt 1140 tactacgaac cccagactta cggtggaaca
gctccaacgc ccagcttctc cttgactact 1200 gctcttccaa gggttataac
atctcctggg aactgggcaa tgagcccaac agtttctgga 1260 agaaagctca
cattctcatc gatgggttgc agttaggaga agactttgtg gagttgcata 1320
aacttctaca aaggtcagct ttccaaaatg caaaactcta tggtcctgac atcggtcagc
1380 ctcgagggaa gacagttaaa ctgctgagga gtttcctgaa ggctggcgga
gaagtgatcg 1440 actctcttac atggcatcac tattacttga atggacgcat
cgctaccaaa gaagattttc 1500 tgagctctga tgcgctggac acttttattc
tctctgtgca aaaaattctg aaggtcacta 1560 aagagatcac acctggcaag
aaggtctggt tgggagagac gagctcagct tacggtggcg 1620 gtgcaccctt
gctgtccaac acctttgcag ctggctttat gtggctggat aaattgggcc 1680
tgtcagccca gatgggcata gaagtcgtga tgaggcaggt gttcttcgga gcaggcaact
1740 accacttagt ggatgaaaac tttgagcctt tacctgatta ctggctctct
cttctgttca 1800 agaaactggt aggtcccagg gtgttactgt caagagtgaa
aggcccagac aggagcaaac 1860 tccgagtgta tctccactgc actaacgtct
atcacccacg atatcaggaa ggagatctaa 1920 ctctgtatgt cctgaacctc
cataatgtca ccaagcactt gaaggtaccg cctccgttgt 1980 tcaggaaacc
agtggatacg taccttctga agccttcggg gccggatgga ttactttcca 2040
aatctgtcca actgaacggt caaattctga agatggtgga tgagcagacc ctgccagctt
2100 tgacagaaaa acctctcccc gcaggaagtg cactaagcct gcctgccttt
tcctatggtt 2160 tttttgtcat aagaaatgcc aaaatcgctg cttgtatatg
aaaataaaag gcatacggta 2220 cccctgagac aaaagccgag gggggtgtta
ttcataaaac aaaaccctag tttaggaggc 2280 cacctccttg ccgagttcca
gagcttcggg agggtggggt acacttcagt attacattca 2340 gtgtggtgtt
ctctctaaga agaatactgc aggtggtgac agttaatagc actgtg 2396 44 535 PRT
Mus musculus 44 Met Leu Arg Leu Leu Leu Leu Trp Leu Trp Gly Pro Leu
Gly Ala Leu 1 5
10 15 Ala Gln Gly Ala Pro Ala Gly Thr Ala Pro Thr Asp Asp Val Val
Asp 20 25 30 Leu Glu Phe Tyr Thr Lys Arg Pro Leu Arg Ser Val Ser
Pro Ser Phe 35 40 45 Leu Ser Ile Thr Ile Asp Ala Ser Leu Ala Thr
Asp Pro Arg Phe Leu 50 55 60 Thr Phe Leu Gly Ser Pro Arg Leu Arg
Ala Leu Ala Arg Gly Leu Ser 65 70 75 80 Pro Ala Tyr Leu Arg Phe Gly
Gly Thr Lys Thr Asp Phe Leu Ile Phe 85 90 95 Asp Pro Asp Lys Glu
Pro Thr Ser Glu Glu Arg Ser Tyr Trp Lys Ser 100 105 110 Gln Val Asn
His Asp Ile Cys Arg Ser Glu Pro Val Ser Ala Ala Val 115 120 125 Leu
Arg Lys Leu Gln Val Glu Trp Pro Phe Gln Glu Leu Leu Leu Leu 130 135
140 Arg Glu Gln Tyr Gln Lys Glu Phe Lys Asn Ser Thr Tyr Ser Arg Ser
145 150 155 160 Ser Val Asp Met Leu Tyr Ser Phe Ala Lys Cys Ser Gly
Leu Asp Leu 165 170 175 Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Pro
Asp Leu Arg Trp Asn 180 185 190 Ser Ser Asn Ala Gln Leu Leu Leu Asp
Tyr Cys Ser Ser Lys Gly Tyr 195 200 205 Asn Ile Ser Trp Glu Leu Gly
Asn Glu Pro Asn Ser Phe Trp Lys Lys 210 215 220 Ala His Ile Leu Ile
Asp Gly Leu Gln Leu Gly Glu Asp Phe Val Glu 225 230 235 240 Leu His
Lys Leu Leu Gln Arg Ser Ala Phe Gln Asn Ala Lys Leu Tyr 245 250 255
Gly Pro Asp Ile Gly Gln Pro Arg Gly Lys Thr Val Lys Leu Leu Arg 260
265 270 Ser Phe Leu Lys Ala Gly Gly Glu Val Ile Asp Ser Leu Thr Trp
His 275 280 285 His Tyr Tyr Leu Asn Gly Arg Ile Ala Thr Lys Glu Asp
Phe Leu Ser 290 295 300 Ser Asp Ala Leu Asp Thr Phe Ile Leu Ser Val
Gln Lys Ile Leu Lys 305 310 315 320 Val Thr Lys Glu Ile Thr Pro Gly
Lys Lys Val Trp Leu Gly Glu Thr 325 330 335 Ser Ser Ala Tyr Gly Gly
Gly Ala Pro Leu Leu Ser Asn Thr Phe Ala 340 345 350 Ala Gly Phe Met
Trp Leu Asp Lys Leu Gly Leu Ser Ala Gln Met Gly 355 360 365 Ile Glu
Val Val Met Arg Gln Val Phe Phe Gly Ala Gly Asn Tyr His 370 375 380
Leu Val Asp Glu Asn Phe Glu Pro Leu Pro Asp Tyr Trp Leu Ser Leu 385
390 395 400 Leu Phe Lys Lys Leu Val Gly Pro Arg Val Leu Leu Ser Arg
Val Lys 405 410 415 Gly Pro Asp Arg Ser Lys Leu Arg Val Tyr Leu His
Cys Thr Asn Val 420 425 430 Tyr His Pro Arg Tyr Gln Glu Gly Asp Leu
Thr Leu Tyr Val Leu Asn 435 440 445 Leu His Asn Val Thr Lys His Leu
Lys Val Pro Pro Pro Leu Phe Arg 450 455 460 Lys Pro Val Asp Thr Tyr
Leu Leu Lys Pro Ser Gly Pro Asp Gly Leu 465 470 475 480 Leu Ser Lys
Ser Val Gln Leu Asn Gly Gln Ile Leu Lys Met Val Asp 485 490 495 Glu
Gln Thr Leu Pro Ala Leu Thr Glu Lys Pro Leu Pro Ala Gly Ser 500 505
510 Ala Leu Ser Leu Pro Ala Phe Ser Tyr Gly Phe Phe Val Ile Arg Asn
515 520 525 Ala Lys Ile Ala Ala Cys Ile 530 535 45 2396 DNA Mus
musculus CDS (594)..(2198) 45 tttctagttg cttttagcca atgtcggatc
aggtttttca agcgacaaag agatactgag 60 atcctgggca gaggacatcc
tagctcggtc agatttgggc aggctcaagt gaccagtgtc 120 ttaaggcaga
agggagtcgg ggtagggtct ggctgaaccc tcaaccgggg cttttaactc 180
agggtctagt cctggcgcca aatggatggg acctagaaaa ggtgacagag tgcgcaggac
240 accaggaagc tggtcccacc cctgcgcggc tcccgggcgc tccctcccca
ggcctccgag 300 gatcttggat tctggccacc tccgcaccct ttggatgggt
gtggatgatt tcaaaagtgg 360 acgtgaccgc ggcggagggg aaagccagca
cggaaatgaa agagagcgag gaggggaggg 420 cggggagggg agggcgctag
ggagggactc ccgggagggg tgggagggat ggagcgctgt 480 gggagggtac
tgagtcctgg cgccagaggc gaagcaggac cggttgcagg gggcttgagc 540
cagcgcgccg gctgccccag ctctcccggc agcgggcggt ccagccaggt ggg atg 596
Met 1 ctg agg ctg ctg ctg ctg tgg ctc tgg ggg ccg ctc ggt gcc ctg
gcc 644 Leu Arg Leu Leu Leu Leu Trp Leu Trp Gly Pro Leu Gly Ala Leu
Ala 5 10 15 cag ggc gcc ccc gcg ggg acc gcg ccg acc gac gac gtg gta
gac ttg 692 Gln Gly Ala Pro Ala Gly Thr Ala Pro Thr Asp Asp Val Val
Asp Leu 20 25 30 gag ttt tac acc aag cgg ccg ctc cga agc gtg agt
ccc tcg ttc ctg 740 Glu Phe Tyr Thr Lys Arg Pro Leu Arg Ser Val Ser
Pro Ser Phe Leu 35 40 45 tcc atc acc atc gac gcc agc ctg gcc acc
gac ccg cgc ttc ctc acc 788 Ser Ile Thr Ile Asp Ala Ser Leu Ala Thr
Asp Pro Arg Phe Leu Thr 50 55 60 65 ttc ctg ggc tct cca agg ctc cgt
gct ctg gct aga ggc tta tct cct 836 Phe Leu Gly Ser Pro Arg Leu Arg
Ala Leu Ala Arg Gly Leu Ser Pro 70 75 80 gca tac ttg aga ttt ggc
ggc aca aag act gac ttc ctt att ttt gat 884 Ala Tyr Leu Arg Phe Gly
Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp 85 90 95 ccg gac aag gaa
ccg act tcc gaa gaa aga agt tac tgg aaa tct caa 932 Pro Asp Lys Glu
Pro Thr Ser Glu Glu Arg Ser Tyr Trp Lys Ser Gln 100 105 110 gtc aac
cat gat att tgc agg tct gag ccg gtc tct gct gcg gtg ttg 980 Val Asn
His Asp Ile Cys Arg Ser Glu Pro Val Ser Ala Ala Val Leu 115 120 125
agg aaa ctc cag gtg gaa tgg ccc ttc cag gag ctg ttg ctg ctc cga
1028 Arg Lys Leu Gln Val Glu Trp Pro Phe Gln Glu Leu Leu Leu Leu
Arg 130 135 140 145 gag cag tac caa aag gag ttc aag aac agc acc tac
tca aga agc tca 1076 Glu Gln Tyr Gln Lys Glu Phe Lys Asn Ser Thr
Tyr Ser Arg Ser Ser 150 155 160 gtg gac atg ctc tac agt ttt gcc aag
tgc tcg ggg tta gac ctg atc 1124 Val Asp Met Leu Tyr Ser Phe Ala
Lys Cys Ser Gly Leu Asp Leu Ile 165 170 175 ttt ggt cta aat gcg tta
cta cga acc cca gac tta cgg tgg aac agc 1172 Phe Gly Leu Asn Ala
Leu Leu Arg Thr Pro Asp Leu Arg Trp Asn Ser 180 185 190 tcc aac gcc
cag ctt ctc ctt gac tac tgc tct tcc aag ggt tat aac 1220 Ser Asn
Ala Gln Leu Leu Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn 195 200 205
atc tcc tgg gaa ctg ggc aat gag ccc aac agt ttc tgg aag aaa gct
1268 Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe Trp Lys Lys
Ala 210 215 220 225 cac att ctc atc gat ggg ttg cag tta gga gaa gac
ttt gtg gag ttg 1316 His Ile Leu Ile Asp Gly Leu Gln Leu Gly Glu
Asp Phe Val Glu Leu 230 235 240 cat aaa ctt cta caa agg tca gct ttc
caa aat gca aaa ctc tat ggt 1364 His Lys Leu Leu Gln Arg Ser Ala
Phe Gln Asn Ala Lys Leu Tyr Gly 245 250 255 cct gac atc ggt cag cct
cga ggg aag aca gtt aaa ctg ctg agg agt 1412 Pro Asp Ile Gly Gln
Pro Arg Gly Lys Thr Val Lys Leu Leu Arg Ser 260 265 270 ttc ctg aag
gct ggc gga gaa gtg atc gac tct ctt aca tgg cat cac 1460 Phe Leu
Lys Ala Gly Gly Glu Val Ile Asp Ser Leu Thr Trp His His 275 280 285
tat tac ttg aat gga cgc atc gct acc aaa gaa gat ttt ctg agc tct
1508 Tyr Tyr Leu Asn Gly Arg Ile Ala Thr Lys Glu Asp Phe Leu Ser
Ser 290 295 300 305 gat gcg ctg gac act ttt att ctc tct gtg caa aaa
att ctg aag gtc 1556 Asp Ala Leu Asp Thr Phe Ile Leu Ser Val Gln
Lys Ile Leu Lys Val 310 315 320 act aaa gag atc aca cct ggc aag aag
gtc tgg ttg gga gag acg agc 1604 Thr Lys Glu Ile Thr Pro Gly Lys
Lys Val Trp Leu Gly Glu Thr Ser 325 330 335 tca gct tac ggt ggc ggt
gca ccc ttg ctg tcc aac acc ttt gca gct 1652 Ser Ala Tyr Gly Gly
Gly Ala Pro Leu Leu Ser Asn Thr Phe Ala Ala 340 345 350 ggc ttt atg
tgg ctg gat aaa ttg ggc ctg tca gcc cag atg ggc ata 1700 Gly Phe
Met Trp Leu Asp Lys Leu Gly Leu Ser Ala Gln Met Gly Ile 355 360 365
gaa gtc gtg atg agg cag gtg ttc ttc gga gca ggc aac tac cac tta
1748 Glu Val Val Met Arg Gln Val Phe Phe Gly Ala Gly Asn Tyr His
Leu 370 375 380 385 gtg gat gaa aac ttt gag cct tta cct gat tac tgg
ctc tct ctt ctg 1796 Val Asp Glu Asn Phe Glu Pro Leu Pro Asp Tyr
Trp Leu Ser Leu Leu 390 395 400 ttc aag aaa ctg gta ggt ccc agg gtg
tta ctg tca aga gtg aaa ggc 1844 Phe Lys Lys Leu Val Gly Pro Arg
Val Leu Leu Ser Arg Val Lys Gly 405 410 415 cca gac agg agc aaa ctc
cga gtg tat ctc cac tgc act aac gtc tat 1892 Pro Asp Arg Ser Lys
Leu Arg Val Tyr Leu His Cys Thr Asn Val Tyr 420 425 430 cac cca cga
tat cag gaa gga gat cta act ctg tat gtc ctg aac ctc 1940 His Pro
Arg Tyr Gln Glu Gly Asp Leu Thr Leu Tyr Val Leu Asn Leu 435 440 445
cat aat gtc acc aag cac ttg aag gta ccg cct ccg ttg ttc agg aaa
1988 His Asn Val Thr Lys His Leu Lys Val Pro Pro Pro Leu Phe Arg
Lys 450 455 460 465 cca gtg gat acg tac ctt ctg aag cct tcg ggg ccg
gat gga tta ctt 2036 Pro Val Asp Thr Tyr Leu Leu Lys Pro Ser Gly
Pro Asp Gly Leu Leu 470 475 480 tcc aaa tct gtc caa ctg aac ggt caa
att ctg aag atg gtg gat gag 2084 Ser Lys Ser Val Gln Leu Asn Gly
Gln Ile Leu Lys Met Val Asp Glu 485 490 495 cag acc ctg cca gct ttg
aca gaa aaa cct ctc ccc gca gga agt gca 2132 Gln Thr Leu Pro Ala
Leu Thr Glu Lys Pro Leu Pro Ala Gly Ser Ala 500 505 510 cta agc ctg
cct gcc ttt tcc tat ggt ttt ttt gtc ata aga aat gcc 2180 Leu Ser
Leu Pro Ala Phe Ser Tyr Gly Phe Phe Val Ile Arg Asn Ala 515 520 525
aaa atc gct gct tgt ata tgaaaataaa aggcatacgg tacccctgag 2228 Lys
Ile Ala Ala Cys Ile 530 535 acaaaagccg aggggggtgt tattcataaa
acaaaaccct agtttaggag gccacctcct 2288 tgccgagttc cagagcttcg
ggagggtggg gtacacttca gtattacatt cagtgtggtg 2348 ttctctctaa
gaagaatact gcaggtggtg acagttaata gcactgtg 2396 46 385 DNA Rattus
norvegicus 46 cggccgctgc tgctgctgtg gctctggggg cggctccgtg
ccctgaccca aggcactccg 60 gcggggaccg cgccgaccaa agacgtggtg
gacttggagt tttacaccaa gaggctattc 120 caaagcgtga gtccctcgtt
cctgtccatc accatcgacg ccagtctggc caccgaccct 180 cggttcctca
ccttcctgag ctctccacgg cttcgagccc tgtctagagg cttatctcct 240
gcgtacttga gatttggcgg caccaagact gacttcctta tttttgatcc caacaacgaa
300 cccacctctg aagaaagaag ttactggcaa tctcaagaca acaatgatat
ttgcgggtct 360 gaccgggtct ccgctgacgt gttga 385 47 541 DNA Rattus
norvegicus misc_feature (507)..(507) Any nucleotide 47 aaatcaggac
atatccttca cttatttgcc tcttggtcat attggaggca tttgtattca 60
tttttaataa ccctcaaaat agtgcatgca aagtgctaag cgtcatttgc cacatggtgc
120 cattaactgt caccacctgc agtggtctac ttagagaaca ccgcactgga
tgttaacact 180 gaagcgcgtg ccccgccctc ccgaggctct ggatccagcg
ttgaagcttg ccccgccctc 240 ccgaggctct ggatccagca ctggagcatg
ccccgccctc ccgaggctct ggagcttgct 300 aaggagtccg ctccctaccg
ctggggtttt gctttattct tatgaatgac acccctgacc 360 gctttcgtct
caggggtact gtaatgcctt ttattttcat atacaagctg cgattttggc 420
atttcttatg acaaaaaacc cataggaaaa ggcgggcacg cttagtgagc ttcctgcggg
480 gagaggtttt tctgttagag ctggcanggt ctgctcatcg accatcttca
ggcctcgtgc 540 c 541
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