U.S. patent application number 11/978666 was filed with the patent office on 2009-11-05 for polynucleotide encoding a polypeptide having heparanase activity and expression of same in genetically modified cells.
This patent application is currently assigned to Insight Strategy & Marketing Ltd.. Invention is credited to Elena Feinstein, Iris Pecker, Israel Vlodavsky.
Application Number | 20090275106 11/978666 |
Document ID | / |
Family ID | 22982587 |
Filed Date | 2009-11-05 |
United States Patent
Application |
20090275106 |
Kind Code |
A1 |
Pecker; Iris ; et
al. |
November 5, 2009 |
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; (Mavaseret Zion,
IL) ; Feinstein; Elena; (Rechovot, IL) |
Correspondence
Address: |
MARTIN D. MOYNIHAN d/b/a PRTSI, INC.
P.O. BOX 16446
ARLINGTON
VA
22215
US
|
Assignee: |
Insight Strategy & Marketing
Ltd.
Rehovot
IL
Hadasit Maedical Research Services and Development Ltd.
Jerusalem
IL
|
Family ID: |
22982587 |
Appl. No.: |
11/978666 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09776874 |
Feb 6, 2001 |
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11978666 |
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09258892 |
Mar 1, 1999 |
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09776874 |
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PCT/US98/17954 |
Aug 31, 1998 |
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09258892 |
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09109386 |
Jul 2, 1998 |
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PCT/US98/17954 |
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08922170 |
Sep 2, 1997 |
5968822 |
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09109386 |
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Current U.S.
Class: |
435/232 |
Current CPC
Class: |
A61K 38/00 20130101;
C12Y 302/01166 20130101; C12N 9/2402 20130101 |
Class at
Publication: |
435/232 |
International
Class: |
C12N 9/88 20060101
C12N009/88 |
Claims
1-13. (canceled)
14. A compound comprising a polypeptide at least 80% homologous to
SEQ ID NO: 10 and wherein said compound is pure enough to elicit
anti-heparanase antibodies.
15. The compound of claim 14, wherein said polypeptide is at least
85% homologous to SEQ ID NO: 10.
16. The compound of claim 14, wherein said polypeptide is at least
90% homologous to SEQ ID NO: 10.
17. The compound of claim 14, wherein said polypeptide is at least
95% homologous to SEQ ID NO: 10.
18. The compound of claim 14, which has heparanase catalytic
activity or is cleavable so as to acquire said heparanase catalytic
activity.
19. The compound of claim 14, wherein said amino acid sequence has
a phenylalanine residue corresponding to position 246 of SEQ ID
NO:10.
20. The compound of claim 19, wherein said polypeptide is at least
85% homologous to SEQ ID NO: 10.
21. The compound of claim 19, wherein said polypeptide is at least
90% homologous to SEQ ID NO: 10.
22. The compound of claim 19, wherein said polypeptide is at least
95% homologous to SEQ ID NO: 10.
23. The compound of claim 19, which has heparanase catalytic
activity or is cleavable so as to acquire said heparanase catalytic
activity.
Description
[0001] This 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 proteogly
cans (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-glucuronidase (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 B16 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.
[0008] 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.
[0009] 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.
[0010] 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).
[0011] 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.
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 intron1-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. 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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).
[0078] 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. Antisense therapeutics,
however, block mRNA transcription/translation 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.
[0080] 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.
[0081] 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).
[0082] 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).
[0083] Ribozymes:
[0084] 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).
[0085] Gene Disruption in Animal Models:
[0086] 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).
[0087] DNA Vaccination:
[0088] 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. Priming with human gp75 DNA broke tolerance
to mouse gp75. Immunity against mouse gp75 provided significant
tumor protection (60).
[0089] Glycosyl Hydrolases:
[0090] 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.
[0091] Lysosomal glycosyl hydrolases including
.beta.-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.
[0092] Genomic Sequence of hpa Gene and its Implications:
[0093] 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.
[0094] 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.
[0095] 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
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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).
[0107] 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.
[0108] 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).
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] According to a further aspect of the present invention there
is provided a recombinant protein comprising a polypeptide having
heparanase catalytic activity.
[0122] 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.
[0123] 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.
[0124] 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).
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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
[0129] The invention herein described, by way of example only, with
reference to the accompanying drawings, wherein:
[0130] 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.
[0131] 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 ( ) 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.
[0132] 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 ( ), 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.
[0133] 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) (), but not low (<50 kDa) (.smallcircle.) molecular
weight compartment.
[0134] 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 ( ) or presence
(.DELTA.) 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).
[0135] 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 ( ) 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.
[0136] 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 ( ) 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.
[0137] 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 ( ) or control pF1 (.quadrature.) 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.
[0138] 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 ( ) 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.
[0139] 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 ( ). 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.
[0140] 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. 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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
[0147] 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
[0148] 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.
[0149] 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.
[0150] 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.
[0151] A purified preparation of heparanase isolated from human
hepatoma cells was subjected to tryptic digestion and
microsequencing.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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).
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] RT-PCR performed on a variety of cells revealed
alternatively spliced hpa transcripts.
[0164] 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,
AI122034, 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).
[0165] 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.
[0166] Expression of hpa antisense in mammalian cell lines resulted
in about five fold decrease in the number of recoverable cells as
compared to controls.
[0167] Human Hpa cDNA was shown to hybridize with genomic DNAs of a
variety of mammalian species and with an avian.
[0168] The human and mouse hpa promoters were identified and the
human promoter was tested positive in directing the expression of a
reporter gene.
[0169] 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.
[0170] 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.
[0171] 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).
[0172] 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.
[0173] 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). 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.
[0174] 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).
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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
burns, burns 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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
[0206] 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.
[0207] The following protocols and experimental details are
referenced in the Examples that follow:
[0208] 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.
[0209] 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.
[0210] The purified enzyme was applied to reverse phase HPLC and
subjected to N-terminal amino acid sequencing using the amino acid
sequencer (Applied Biosystems).
[0211] 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).
[0212] 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).
[0213] 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).
[0214] 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%.
[0215] 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.
[0216] 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:
[0217] First step: 5'-primer: AP1: 5'-CCATCCTAATACGACTCACT
ATAGGGC-3', SEQ ID NO:1; 3'-primer: HPL229: 5'-GTAGTGATGCCA
TGTAACTGAATC-3', SEQ ID NO:2.
[0218] 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.
[0219] 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.
[0220] 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:
TABLE-US-00001 HPU-355: 5'-TTCGATCCCAAGAAGGAATCAAC-3',, SEQ ID NO:
6 nucleotides 372-394 in SEQ ID NOs: 9 or 11. HPL-229:
5'-GTAGTGATGCCATGTAACTGAATC-3',, SEQ ID NO: 7 nucleotides 933-956
in SEQ ID NOs: 9 or 11.
[0221] 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.
[0222] 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:
TABLE-US-00002 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'-TCAGATGCAAGCAGCAACTTTGGC-3', SEQ ID NO: 27 Hpl 171,
5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO: 28 Hpl 229,
5'-GTAGTGATGCCATGTAACTGAATC-3', SEQ ID NO: 29
[0223] 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.
[0224] 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).
[0225] 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.
[0226] 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).
[0227] PCR amplification of genomic 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:
TABLE-US-00003 GHpu-L3 5'-AGGCACCCTAGAGATGTTCCAG-3', SEQ ID NO: 30
GHpl-L6 5'-GAAGATTTCTGTTTCCATGACGTG-3',. SEQ ID NO: 31
[0228] 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.
[0229] 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.
[0230] 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.
[0231] Genomic sequence analysis: Large-scale sequencing was
performed by Commonwealth Biotechnology Incorporation.
[0232] 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.
[0233] Primers Used for PCR Amplification of Mouse hpa:
TABLE-US-00004 Mhpl773 SEQ ID NO: 32
5'-CCACACTGAATGTAATACTGAAGTG-3', MHpl736 SEQ ID NO: 33
5'-CGAAGCTCTGGAACTCGGCAAG-3', MHpl83 SEQ ID NO: 34
5'-GCCAGCTGCAAAGGTGTTGGAC-3', Mhpl152 SEQ ID NO: 35
5'-AACACCTGCCTCATCACGACTTC-3', Mhpl114 SEQ ID NO: 36
5'-GCCAGGCTGGCGTCGATGGTGA-3', MHpl103 SEQ ID NO: 37
5'-GTCGATGGTGATGGACAGGAAC-3', Ap1 SEQ ID NO: 38
5'-GTAATACGACTCACTATAGGGC-3',-(Genome walker) Ap2 SEQ ID NO: 39
5'-ACTATAGGGCACGCGTGGT-3',-(Genome walker) Ap1 SEQ ID NO: 40
5'-CCATCCTAATACGACTCACTATAGGGC-3',-(Marathon RACE) Ap2 SEQ ID NO:
41 5'-ACTCACTATAGGGCTCGAGCGGC-3',-(Marathon RACE)
[0234] 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.
[0235] 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 BgIII
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.
[0236] 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
[0237] 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.
[0238] 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.
[0239] 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 G721 of SEQ ID NO:9 and FIG. 1.
[0240] 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.
[0241] 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.
[0242] 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
[0243] 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.
[0244] 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).
[0245] 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.
[0246] In subsequent experiments, the labeled HSPG substrate was
incubated with medium conditioned by infected High Five or Sf21
cells.
[0247] 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.
[0248] 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.
[0249] In order to further characterize the hpa product the
inhibitory effect of heparin, a potent inhibitor of heparanase
mediated HS degradation (40) was examined.
[0250] 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.
[0251] 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
[0252] 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.
[0253] 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.
[0254] 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
[0255] 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
[0256] 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).
[0257] 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
[0258] 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).
[0259] 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 hp1-666 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.
[0260] 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.
[0261] 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).
[0262] 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.
[0263] 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
[0264] 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.
[0265] 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).
[0266] 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.
[0267] 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.
[0268] 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
[0269] 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 EarI 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.
[0270] 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.
[0271] 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. patent 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. patent
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. patent
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.
[0272] 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
[0273] 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).
[0274] 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 hpll71
5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:23, corresponding to
nucleotides 897-876 of SEQ ID NO:9.
[0275] 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.
[0276] 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
[0277] 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).
[0278] 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 GHp1L6. The PCR product was cloned into the plasmid
vector pGEM-T-easy (Promega).
[0279] 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
[0280] 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.
TABLE-US-00005 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) + Fragments of similar sizes were obtained following
amplification with two cells lines, placenta and platelets.
Example 12
Mouse and Rat hpa
[0281] 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.
[0282] Searching for consensus protein domains revealed an amino
terminal homology between the heparanase 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.
[0283] 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,
AI122034, 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 mhp1773 and Ap1 and the second cycle with primers mhp1736
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 mhp183 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 mhpll 14 and Ap1 and the
second with primers mhp1103 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.
[0284] 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
[0285] 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.
[0286] Five of 15 proteins, which were predicted to have most
similar folds, were glycosyl hydrolases from various organisms:
1xyza--xylanase from Clostridium Thermocellum,
1pbga--6-phospho-beta-.delta.-galactosidase from Lactococcus
Lactis, 1amy--alpha-amylase from Barley, 1ecea--endocellulase from
Acidothermus Cellulolyticus and 1qbc--hexosaminidase alpha chain,
glycosyl hydrolase.
[0287] 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.
[0288] Despite the lack of an overall homology between the
heparanase and other glycosyl hydrolases, 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.
[0289] 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
position 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
[0290] 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:
TABLE-US-00006 Antisense No insert T24P 15 60 MBT-T50 1 6
[0291] 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
[0292] 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
[0293] 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.
[0294] 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.
[0295] 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.
LIST OF REFERENCES
[0296] 1. Wight, T. N., Kinsella, M. G., and Qwarnstromn, E. E.
(1992). The role of proteoglycans in cell adhesion, migration and
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Sequence CWU 1
1
47127DNAArtificial sequencesynthetic oligonucleotide 1ccatcctaat
acgactcact atagggc 27224DNAArtificial sequencesynthetic
oligonucleotide 2gtagtgatgc catgtaactg aatc 24323DNAArtificial
sequencesynthetic oligonucleotide 3actcactata gggctcgagc ggc
23422DNAArtificial sequencesynthetic oligonucleotide 4gcatcttagc
cgtctttctt cg 22515DNAArtificial sequencesynthetic oligonucleotide
5tttttttttt ttttt 15623DNAArtificial sequencesynthetic
oligonucleotide 6ttcgatccca agaaggaatc aac 23724DNAArtificial
sequencesynthetic oligonucleotide 7gtagtgatgc catgtaactg aatc
2489PRTHomo sapiens 8Tyr Gly Pro Asp Val Gly Gln Pro Arg1
591721DNAHomo sapiens 9ctagagcttt cgactctccg ctgcgcggca gctggcgggg
ggagcagcca ggtgagccca 60agatgctgct gcgctcgaag cctgcgctgc cgccgccgct
gatgctgctg ctcctggggc 120cgctgggtcc cctctcccct ggcgccctgc
cccgacctgc gcaagcacag gacgtcgtgg 180acctggactt cttcacccag
gagccgctgc acctggtgag cccctcgttc ctgtccgtca 240ccattgacgc
caacctggcc acggacccgc ggttcctcat cctcctgggt tctccaaagc
300ttcgtacctt ggccagaggc ttgtctcctg cgtacctgag gtttggtggc
accaagacag 360acttcctaat tttcgatccc aagaaggaat caacctttga
agagagaagt tactggcaat 420ctcaagtcaa ccaggatatt tgcaaatatg
gatccatccc tcctgatgtg gaggagaagt 480tacggttgga atggccctac
caggagcaat tgctactccg agaacactac cagaaaaagt 540tcaagaacag
cacctactca agaagctctg tagatgtgct atacactttt gcaaactgct
600caggactgga cttgatcttt ggcctaaatg cgttattaag aacagcagat
ttgcagtgga 660acagttctaa tgctcagttg ctcctggact actgctcttc
caaggggtat aacatttctt 720gggaactagg caatgaacct aacagtttcc
ttaagaaggc tgatattttc atcaatgggt 780cgcagttagg agaagattat
attcaattgc ataaacttct aagaaagtcc accttcaaaa 840atgcaaaact
ctatggtcct gatgttggtc agcctcgaag aaagacggct aagatgctga
900agagcttcct gaaggctggt ggagaagtga ttgattcagt tacatggcat
cactactatt 960tgaatggacg gactgctacc agggaagatt ttctaaaccc
tgatgtattg gacattttta 1020tttcatctgt gcaaaaagtt ttccaggtgg
ttgagagcac caggcctggc aagaaggtct 1080ggttaggaga aacaagctct
gcatatggag gcggagcgcc cttgctatcc gacacctttg 1140cagctggctt
tatgtggctg gataaattgg gcctgtcagc ccgaatggga atagaagtgg
1200tgatgaggca agtattcttt ggagcaggaa actaccattt agtggatgaa
aacttcgatc 1260ctttacctga ttattggcta tctcttctgt tcaagaaatt
ggtgggcacc aaggtgttaa 1320tggcaagcgt gcaaggttca aagagaagga
agcttcgagt ataccttcat tgcacaaaca 1380ctgacaatcc aaggtataaa
gaaggagatt taactctgta tgccataaac ctccataacg 1440tcaccaagta
cttgcggtta ccctatcctt tttctaacaa gcaagtggat aaataccttc
1500taagaccttt gggacctcat ggattacttt ccaaatctgt ccaactcaat
ggtctaactc 1560taaagatggt ggatgatcaa accttgccac ctttaatgga
aaaacctctc cggccaggaa 1620gttcactggg cttgccagct ttctcatata
gtttttttgt gataagaaat gccaaagttg 1680ctgcttgcat ctgaaaataa
aatatactag tcctgacact g 172110543PRTHomo sapiens 10Met Leu Leu Arg
Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu1 5 10 15Leu Leu Gly
Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro20 25 30Ala Gln
Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro35 40 45Leu
His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn50 55
60Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu65
70 75 80Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly
Gly85 90 95Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser
Thr Phe100 105 110Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln
Asp Ile Cys Lys115 120 125Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu
Lys Leu Arg Leu Glu Trp130 135 140Pro Tyr Gln Glu Gln Leu Leu Leu
Arg Glu His Tyr Gln Lys Lys Phe145 150 155 160Lys Asn Ser Thr Tyr
Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe165 170 175Ala Asn Cys
Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu180 185 190Arg
Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu195 200
205Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly
Asn210 215 220Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile
Asn Gly Ser225 230 235 240Gln Leu Gly Glu Asp Tyr Ile Gln Leu His
Lys Leu Leu Arg Lys Ser245 250 255Thr Phe Lys Asn Ala Lys Leu Tyr
Gly Pro Asp Val Gly Gln Pro Arg260 265 270Arg Lys Thr Ala Lys Met
Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu275 280 285Val Ile Asp Ser
Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr290 295 300Ala Thr
Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile305 310 315
320Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro
Gly325 330 335Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly
Gly Gly Ala340 345 350Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe
Met Trp Leu Asp Lys355 360 365Leu Gly Leu Ser Ala Arg Met Gly Ile
Glu Val Val Met Arg Gln Val370 375 380Phe Phe Gly Ala Gly Asn Tyr
His Leu Val Asp Glu Asn Phe Asp Pro385 390 395 400Leu Pro Asp Tyr
Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr405 410 415Lys Val
Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg420 425
430Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu
Gly435 440 445Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr
Lys Tyr Leu450 455 460Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val
Asp Lys Tyr Leu Leu465 470 475 480Arg Pro Leu Gly Pro His Gly Leu
Leu Ser Lys Ser Val Gln Leu Asn485 490 495Gly Leu Thr Leu Lys Met
Val Asp Asp Gln Thr Leu Pro Pro Leu Met500 505 510Glu Lys Pro Leu
Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser515 520 525Tyr Ser
Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile530 535
540111721DNAHomo sapiensCDS(63)..(1691) 11ctagagcttt cgactctccg
ctgcgcggca gctggcgggg ggagcagcca ggtgagccca 60ag atg ctg ctg cgc
tcg aag cct gcg ctg ccg ccg ccg ctg atg ctg 107Met Leu Leu Arg Ser
Lys Pro Ala Leu Pro Pro Pro Leu Met Leu1 5 10 15ctg ctc ctg ggg ccg
ctg ggt ccc ctc tcc cct ggc gcc ctg ccc cga 155Leu Leu Leu Gly Pro
Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg20 25 30cct gcg caa gca
cag gac gtc gtg gac ctg gac ttc ttc acc cag gag 203Pro Ala Gln Ala
Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu35 40 45ccg ctg cac
ctg gtg agc ccc tcg ttc ctg tcc gtc acc att gac gcc 251Pro Leu His
Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala50 55 60aac ctg
gcc acg gac ccg cgg ttc ctc atc ctc ctg ggt tct cca aag 299Asn Leu
Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys65 70 75ctt
cgt acc ttg gcc aga ggc ttg tct cct gcg tac ctg agg ttt ggt 347Leu
Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly80 85 90
95ggc acc aag aca gac ttc cta att ttc gat ccc aag aag gaa tca acc
395Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser
Thr100 105 110ttt gaa gag aga agt tac tgg caa tct caa gtc aac cag
gat att tgc 443Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln
Asp Ile Cys115 120 125aaa tat gga tcc atc cct cct gat gtg gag gag
aag tta cgg ttg gaa 491Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu
Lys Leu Arg Leu Glu130 135 140tgg ccc tac cag gag caa ttg cta ctc
cga gaa cac tac cag aaa aag 539Trp Pro Tyr Gln Glu Gln Leu Leu Leu
Arg Glu His Tyr Gln Lys Lys145 150 155ttc aag aac agc acc tac tca
aga agc tct gta gat gtg cta tac act 587Phe Lys Asn Ser Thr Tyr Ser
Arg Ser Ser Val Asp Val Leu Tyr Thr160 165 170 175ttt gca aac tgc
tca gga ctg gac ttg atc ttt ggc cta aat gcg tta 635Phe Ala Asn Cys
Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu180 185 190tta aga
aca gca gat ttg cag tgg aac agt tct aat gct cag ttg ctc 683Leu Arg
Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu195 200
205ctg gac tac tgc tct tcc aag ggg tat aac att tct tgg gaa cta ggc
731Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu
Gly210 215 220aat gaa cct aac agt ttc ctt aag aag gct gat att ttc
atc aat ggg 779Asn Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe
Ile Asn Gly225 230 235tcg cag tta gga gaa gat tat att caa ttg cat
aaa ctt cta aga aag 827Ser Gln Leu Gly Glu Asp Tyr Ile Gln Leu His
Lys Leu Leu Arg Lys240 245 250 255tcc acc ttc aaa aat gca aaa ctc
tat ggt cct gat gtt ggt cag cct 875Ser Thr Phe Lys Asn Ala Lys Leu
Tyr Gly Pro Asp Val Gly Gln Pro260 265 270cga aga aag acg gct aag
atg ctg aag agc ttc ctg aag gct ggt gga 923Arg Arg Lys Thr Ala Lys
Met Leu Lys Ser Phe Leu Lys Ala Gly Gly275 280 285gaa gtg att gat
tca gtt aca tgg cat cac tac tat ttg aat gga cgg 971Glu Val Ile Asp
Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg290 295 300act gct
acc agg gaa gat ttt cta aac cct gat gta ttg gac att ttt 1019Thr Ala
Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe305 310
315att tca tct gtg caa aaa gtt ttc cag gtg gtt gag agc acc agg cct
1067Ile Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg
Pro320 325 330 335ggc aag aag gtc tgg tta gga gaa aca agc tct gca
tat gga ggc gga 1115Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala
Tyr Gly Gly Gly340 345 350gcg ccc ttg cta tcc gac acc ttt gca gct
ggc ttt atg tgg ctg gat 1163Ala Pro Leu Leu Ser Asp Thr Phe Ala Ala
Gly Phe Met Trp Leu Asp355 360 365aaa ttg ggc ctg tca gcc cga atg
gga ata gaa gtg gtg atg agg caa 1211Lys Leu Gly Leu Ser Ala Arg Met
Gly Ile Glu Val Val Met Arg Gln370 375 380gta ttc ttt gga gca gga
aac tac cat tta gtg gat gaa aac ttc gat 1259Val Phe Phe Gly Ala Gly
Asn Tyr His Leu Val Asp Glu Asn Phe Asp385 390 395cct tta cct gat
tat tgg cta tct ctt ctg ttc aag aaa ttg gtg ggc 1307Pro Leu Pro Asp
Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly400 405 410 415acc
aag gtg tta atg gca agc gtg caa ggt tca aag aga agg aag ctt 1355Thr
Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu420 425
430cga gta tac ctt cat tgc aca aac act gac aat cca agg tat aaa gaa
1403Arg Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys
Glu435 440 445gga gat tta act ctg tat gcc ata aac ctc cat aac gtc
acc aag tac 1451Gly Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val
Thr Lys Tyr450 455 460ttg cgg tta ccc tat cct ttt tct aac aag caa
gtg gat aaa tac ctt 1499Leu Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln
Val Asp Lys Tyr Leu465 470 475cta aga cct ttg gga cct cat gga tta
ctt tcc aaa tct gtc caa ctc 1547Leu Arg Pro Leu Gly Pro His Gly Leu
Leu Ser Lys Ser Val Gln Leu480 485 490 495aat ggt cta act cta aag
atg gtg gat gat caa acc ttg cca cct tta 1595Asn Gly Leu Thr Leu Lys
Met Val Asp Asp Gln Thr Leu Pro Pro Leu500 505 510atg gaa aaa cct
ctc cgg cca gga agt tca ctg ggc ttg cca gct ttc 1643Met Glu Lys Pro
Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe515 520 525tca tat
agt ttt ttt gtg ata aga aat gcc aaa gtt gct gct tgc atc 1691Ser Tyr
Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile530 535
540tgaaaataaa atatactagt cctgacactg 172112824DNAMus musculus
12ctggcaagaa ggtctggttg ggagagacga gctcagctta cggtggcggt gcacccttgc
60tgtccaacac ctttgcagct ggctttatgt ggctggataa attgggcctg tcagcccaga
120tgggcataga agtcgtgatg aggcaggtgt tcttcggagc aggcaactac
cacttagtgg 180atgaaaactt tgagccttta cctgattact ggctctctct
tctgttcaag aaactggtag 240gtcccagggt gttactgtca agagtgaaag
gcccagacag gagcaaactc cgagtgtatc 300tccactgcac taacgtctat
cacccacgat atcaggaagg agatctaact ctgtatgtcc 360tgaacctcca
taatgtcacc aagcacttga aggtaccgcc tccgttgttc aggaaaccag
420tggatacgta ccttctgaag ccttcggggc cggatggatt actttccaaa
tctgtccaac 480tgaacggtca aattctgaag atggtggatg agcagaccct
gccagctttg acagaaaaac 540ctctccccgc aggaagtgca ctaagcctgc
ctgccttttc ctatggtttt tttgtcataa 600gaaatgccaa aatcgctgct
tgtatatgaa aataaaaggc atacggtacc cctgagacaa 660aagccgaggg
gggtgttatt cataaaacaa aaccctagtt taggaggcca cctccttgcc
720gagttccaga gcttcgggag ggtggggtac acttcagtat tacattcagt
gtggtgttct 780ctctaagaag aatactgcag gtggtgacag ttaatagcac tgtg
824131899DNAHomo sapiens 13gggaaagcga gcaaggaagt aggagagagc
cgggcaggcg gggcggggtt ggattgggag 60cagtgggagg gatgcagaag aggagtggga
gggatggagg gcgcagtggg aggggtgagg 120aggcgtaacg gggcggagga
aaggagaaaa gggcgctggg gctcggcggg aggaagtgct 180agagctctcg
actctccgct gcgcggcagc tggcgggggg agcagccagg tgagcccaag
240atgctgctgc gctcgaagcc tgcgctgccg ccgccgctga tgctgctgct
cctggggccg 300ctgggtcccc tctcccctgg cgccctgccc cgacctgcgc
aagcacagga cgtcgtggac 360ctggacttct tcacccagga gccgctgcac
ctggtgagcc cctcgttcct gtccgtcacc 420attgacgcca acctggccac
ggacccgcgg ttcctcatcc tcctgggttc tccaaagctt 480cgtaccttgg
ccagaggctt gtctcctgcg tacctgaggt ttggtggcac caagacagac
540ttcctaattt tcgatcccaa gaaggaatca acctttgaag agagaagtta
ctggcaatct 600caagtcaacc aggatatttg caaatatgga tccatccctc
ctgatgtgga ggagaagtta 660cggttggaat ggccctacca ggagcaattg
ctactccgag aacactacca gaaaaagttc 720aagaacagca cctactcaag
aagctctgta gatgtgctat acacttttgc aaactgctca 780ggactggact
tgatctttgg cctaaatgcg ttattaagaa cagcagattt gcagtggaac
840agttctaatg ctcagttgct cctggactac tgctcttcca aggggtataa
catttcttgg 900gaactaggca atgaacctaa cagtttcctt aagaaggctg
atattttcat caatgggtcg 960cagttaggag aagattatat tcaattgcat
aaacttctaa gaaagtccac cttcaaaaat 1020gcaaaactct atggtcctga
tgttggtcag cctcgaagaa agacggctaa gatgctgaag 1080agcttcctga
aggctggtgg agaagtgatt gattcagtta catggcatca ctactatttg
1140aatggacgga ctgctaccag ggaagatttt ctaaaccctg atgtattgga
catttttatt 1200tcatctgtgc aaaaagtttt ccaggtggtt gagagcacca
ggcctggcaa gaaggtctgg 1260ttaggagaaa caagctctgc atatggaggc
ggagcgccct tgctatccga cacctttgca 1320gctggcttta tgtggctgga
taaattgggc ctgtcagccc gaatgggaat agaagtggtg 1380atgaggcaag
tattctttgg agcaggaaac taccatttag tggatgaaaa cttcgatcct
1440ttacctgatt attggctatc tcttctgttc aagaaattgg tgggcaccaa
ggtgttaatg 1500gcaagcgtgc aaggttcaaa gagaaggaag cttcgagtat
accttcattg cacaaacact 1560gacaatccaa ggtataaaga aggagattta
actctgtatg ccataaacct ccataacgtc 1620accaagtact tgcggttacc
ctatcctttt tctaacaagc aagtggataa ataccttcta 1680agacctttgg
gacctcatgg attactttcc aaatctgtcc aactcaatgg tctaactcta
1740aagatggtgg atgatcaaac cttgccacct ttaatggaaa aacctctccg
gccaggaagt 1800tcactgggct tgccagcttt ctcatatagt ttttttgtga
taagaaatgc caaagttgct 1860gcttgcatct gaaaataaaa tatactagtc
ctgacactg 189914592PRTHomo sapiens 14Met Glu Gly Ala Val Gly Gly
Val Arg Arg Arg Asn Gly Ala Glu Glu1 5 10 15Arg Arg Lys Gly Arg Trp
Gly Ser Ala Gly Gly Ser Ala Arg Ala Leu20 25 30Asp Ser Pro Leu Arg
Gly Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro35 40 45Lys Met Leu Leu
Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu50 55 60Leu Leu Leu
Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg65 70 75 80Pro
Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu85 90
95Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp
Ala100 105 110Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly
Ser Pro Lys115 120 125Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala
Tyr Leu Arg Phe Gly130 135 140Gly Thr Lys Thr Asp Phe Leu Ile Phe
Asp Pro Lys Lys Glu Ser Thr145 150 155 160Phe Glu Glu Arg Ser Tyr
Trp Gln Ser Gln Val Asn Gln Asp Ile Cys165 170 175Lys Tyr Gly Ser
Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu180 185 190Trp Pro
Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys195 200
205Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr
Thr210 215 220Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly
Leu Asn Ala Leu225 230 235 240Leu Arg Thr Ala Asp Leu Gln Trp Asn
Ser Ser Asn Ala Gln Leu Leu245 250 255Leu Asp Tyr Cys Ser Ser Lys
Gly Tyr Asn Ile Ser Trp Glu Leu Gly260 265 270Asn Glu Pro Asn Ser
Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly275 280 285Ser Gln Leu
Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys290 295 300Ser
Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro305 310
315 320Arg Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly
Gly325 330 335Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu
Asn Gly Arg340 345 350Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp
Val Leu Asp Ile Phe355 360 365Ile Ser Ser Val Gln Lys Val Phe Gln
Val Val Glu Ser Thr Arg Pro370 375 380Gly Lys Lys Val Trp Leu Gly
Glu Thr Ser Ser Ala Tyr Gly Gly Gly385 390 395 400Ala Pro Leu Leu
Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp405 410 415Lys Leu
Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln420 425
430Val Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe
Asp435 440 445Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys
Leu Val Gly450 455 460Thr Lys Val Leu Met Ala Ser Val Gln Gly Ser
Lys Arg Arg Lys Leu465 470 475 480Arg Val Tyr Leu His Cys Thr Asn
Thr Asp Asn Pro Arg Tyr Lys Glu485 490 495Gly Asp Leu Thr Leu Tyr
Ala Ile Asn Leu His Asn Val Thr Lys Tyr500 505 510Leu Arg Leu Pro
Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu515 520 525Leu Arg
Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu530 535
540Asn Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro
Leu545 550 555 560Met Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly
Leu Pro Ala Phe565 570 575Ser Tyr Ser Phe Phe Val Ile Arg Asn Ala
Lys Val Ala Ala Cys Ile580 585 590151899DNAHomo
sapiensCDS(94)..(1869) 15gggaaagcga gcaaggaagt aggagagagc
cgggcaggcg gggcggggtt ggattgggag 60cagtgggagg gatgcagaag aggagtggga
ggg atg gag ggc gca gtg gga ggg 114Met Glu Gly Ala Val Gly Gly1
5gtg agg agg cgt aac ggg gcg gag gaa agg aga aaa ggg cgc tgg ggc
162Val Arg Arg Arg Asn Gly Ala Glu Glu Arg Arg Lys Gly Arg Trp
Gly10 15 20tcg gcg gga gga agt gct aga gct ctc gac tct ccg ctg cgc
ggc agc 210Ser Ala Gly Gly Ser Ala Arg Ala Leu Asp Ser Pro Leu Arg
Gly Ser25 30 35tgg cgg ggg gag cag cca ggt gag ccc aag atg ctg ctg
cgc tcg aag 258Trp Arg Gly Glu Gln Pro Gly Glu Pro Lys Met Leu Leu
Arg Ser Lys40 45 50 55cct gcg ctg ccg ccg ccg ctg atg ctg ctg ctc
ctg ggg ccg ctg ggt 306Pro Ala Leu Pro Pro Pro Leu Met Leu Leu Leu
Leu Gly Pro Leu Gly60 65 70ccc ctc tcc cct ggc gcc ctg ccc cga cct
gcg caa gca cag gac gtc 354Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro
Ala Gln Ala Gln Asp Val75 80 85gtg gac ctg gac ttc ttc acc cag gag
ccg ctg cac ctg gtg agc ccc 402Val Asp Leu Asp Phe Phe Thr Gln Glu
Pro Leu His Leu Val Ser Pro90 95 100tcg ttc ctg tcc gtc acc att gac
gcc aac ctg gcc acg gac ccg cgg 450Ser Phe Leu Ser Val Thr Ile Asp
Ala Asn Leu Ala Thr Asp Pro Arg105 110 115ttc ctc atc ctc ctg ggt
tct cca aag ctt cgt acc ttg gcc aga ggc 498Phe Leu Ile Leu Leu Gly
Ser Pro Lys Leu Arg Thr Leu Ala Arg Gly120 125 130 135ttg tct cct
gcg tac ctg agg ttt ggt ggc acc aag aca gac ttc cta 546Leu Ser Pro
Ala Tyr Leu Arg Phe Gly Gly Thr Lys Thr Asp Phe Leu140 145 150att
ttc gat ccc aag aag gaa tca acc ttt gaa gag aga agt tac tgg 594Ile
Phe Asp Pro Lys Lys Glu Ser Thr Phe Glu Glu Arg Ser Tyr Trp155 160
165caa tct caa gtc aac cag gat att tgc aaa tat gga tcc atc cct cct
642Gln Ser Gln Val Asn Gln Asp Ile Cys Lys Tyr Gly Ser Ile Pro
Pro170 175 180gat gtg gag gag aag tta cgg ttg gaa tgg ccc tac cag
gag caa ttg 690Asp Val Glu Glu Lys Leu Arg Leu Glu Trp Pro Tyr Gln
Glu Gln Leu185 190 195cta ctc cga gaa cac tac cag aaa aag ttc aag
aac agc acc tac tca 738Leu Leu Arg Glu His Tyr Gln Lys Lys Phe Lys
Asn Ser Thr Tyr Ser200 205 210 215aga agc tct gta gat gtg cta tac
act ttt gca aac tgc tca gga ctg 786Arg Ser Ser Val Asp Val Leu Tyr
Thr Phe Ala Asn Cys Ser Gly Leu220 225 230gac ttg atc ttt ggc cta
aat gcg tta tta aga aca gca gat ttg cag 834Asp Leu Ile Phe Gly Leu
Asn Ala Leu Leu Arg Thr Ala Asp Leu Gln235 240 245tgg aac agt tct
aat gct cag ttg ctc ctg gac tac tgc tct tcc aag 882Trp Asn Ser Ser
Asn Ala Gln Leu Leu Leu Asp Tyr Cys Ser Ser Lys250 255 260ggg tat
aac att tct tgg gaa cta ggc aat gaa cct aac agt ttc ctt 930Gly Tyr
Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe Leu265 270
275aag aag gct gat att ttc atc aat ggg tcg cag tta gga gaa gat tat
978Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser Gln Leu Gly Glu Asp
Tyr280 285 290 295att caa ttg cat aaa ctt cta aga aag tcc acc ttc
aaa aat gca aaa 1026Ile Gln Leu His Lys Leu Leu Arg Lys Ser Thr Phe
Lys Asn Ala Lys300 305 310ctc tat ggt cct gat gtt ggt cag cct cga
aga aag acg gct aag atg 1074Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg
Arg Lys Thr Ala Lys Met315 320 325ctg aag agc ttc ctg aag gct ggt
gga gaa gtg att gat tca gtt aca 1122Leu Lys Ser Phe Leu Lys Ala Gly
Gly Glu Val Ile Asp Ser Val Thr330 335 340tgg cat cac tac tat ttg
aat gga cgg act gct acc agg gaa gat ttt 1170Trp His His Tyr Tyr Leu
Asn Gly Arg Thr Ala Thr Arg Glu Asp Phe345 350 355cta aac cct gat
gta ttg gac att ttt att tca tct gtg caa aaa gtt 1218Leu Asn Pro Asp
Val Leu Asp Ile Phe Ile Ser Ser Val Gln Lys Val360 365 370 375ttc
cag gtg gtt gag agc acc agg cct ggc aag aag gtc tgg tta gga 1266Phe
Gln Val Val Glu Ser Thr Arg Pro Gly Lys Lys Val Trp Leu Gly380 385
390gaa aca agc tct gca tat gga ggc gga gcg ccc ttg cta tcc gac acc
1314Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu Leu Ser Asp
Thr395 400 405ttt gca gct ggc ttt atg tgg ctg gat aaa ttg ggc ctg
tca gcc cga 1362Phe Ala Ala Gly Phe Met Trp Leu Asp Lys Leu Gly Leu
Ser Ala Arg410 415 420atg gga ata gaa gtg gtg atg agg caa gta ttc
ttt gga gca gga aac 1410Met Gly Ile Glu Val Val Met Arg Gln Val Phe
Phe Gly Ala Gly Asn425 430 435tac cat tta gtg gat gaa aac ttc gat
cct tta cct gat tat tgg cta 1458Tyr His Leu Val Asp Glu Asn Phe Asp
Pro Leu Pro Asp Tyr Trp Leu440 445 450 455tct ctt ctg ttc aag aaa
ttg gtg ggc acc aag gtg tta atg gca agc 1506Ser Leu Leu Phe Lys Lys
Leu Val Gly Thr Lys Val Leu Met Ala Ser460 465 470gtg caa ggt tca
aag aga agg aag ctt cga gta tac ctt cat tgc aca 1554Val Gln Gly Ser
Lys Arg Arg Lys Leu Arg Val Tyr Leu His Cys Thr475 480 485aac act
gac aat cca agg tat aaa gaa gga gat tta act ctg tat gcc 1602Asn Thr
Asp Asn Pro Arg Tyr Lys Glu Gly Asp Leu Thr Leu Tyr Ala490 495
500ata aac ctc cat aac gtc acc aag tac ttg cgg tta ccc tat cct ttt
1650Ile Asn Leu His Asn Val Thr Lys Tyr Leu Arg Leu Pro Tyr Pro
Phe505 510 515tct aac aag caa gtg gat aaa tac ctt cta aga cct ttg
gga cct cat 1698Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu Arg Pro Leu
Gly Pro His520 525 530 535gga tta ctt tcc aaa tct gtc caa ctc aat
ggt cta act cta aag atg 1746Gly Leu Leu Ser Lys Ser Val Gln Leu Asn
Gly Leu Thr Leu Lys Met540 545 550gtg gat gat caa acc ttg cca cct
tta atg gaa aaa cct ctc cgg cca 1794Val Asp Asp Gln Thr Leu Pro Pro
Leu Met Glu Lys Pro Leu Arg Pro555 560 565gga agt tca ctg ggc ttg
cca gct ttc tca tat agt ttt ttt gtg ata 1842Gly Ser Ser Leu Gly Leu
Pro Ala Phe Ser Tyr Ser Phe Phe Val Ile570 575 580aga aat gcc aaa
gtt gct gct tgc atc tgaaaataaa atatactagt 1889Arg Asn Ala Lys Val
Ala Ala Cys Ile585 590cctgacactg 189916594DNAHomo sapiens
16attactatag ggcacgcgtg gtcgacggcc cgggctggta ttgtcttaat gagaagttga
60taaagaattt tgggtggttg atctctttcc agctgcagtt tagcgtatgc tgaggccaga
120ttttttcagg caaaagtaaa atacctgaga aactgcctgg ccagaggaca
atcagatttt 180ggctggctca agtgacaagc aagtgtttat aagctagatg
ggagaggaag ggatgaatac 240tccattggag gctttactcg agggtcagag
ggatacccgg cgccatcaga atgggatctg 300ggagtcggaa acgctgggtt
cccacgagag cgcgcagaac acgtgcgtca ggaagcctgg 360tccgggatgc
ccagcgctgc tccccgggcg ctcctccccg ggcgctcctc cccaggcctc
420ccgggcgctt ggatcccggc catctccgca cccttcaagt gggtgtgggt
gatttcgtaa 480gtgaacgtga ccgccaccgg ggggaaagcg agcaaggaag
taggagagag ccgggcaggc 540ggggcggggt tggattggga gcagtgggag
ggatgcagaa gaggagtggg aggg 5941721DNAArtificial sequencesynthetic
oligonucleotide 17ccccaggagc agcagcatca g 211821DNAArtificial
sequencesynthetic oligonucleotide 18aggcttcgag cgcagcagca t
211922DNAArtificial sequencesynthetic oligonucleotide 19gtaatacgac
tcactatagg gc 222019DNAArtificial sequencesynthetic oligonucleotide
20actatagggc acgcgtggt 192121DNAArtificial sequencesynthetic
oligonucleotide 21cttgggctca cctggctgct c 212223DNAArtificial
sequencesynthetic oligonucleotide 22agctctgtag atgtgctata cac
232322DNAArtificial sequencesynthetic oligonucleotide 23gcatcttagc
cgtctttctt cg 222423DNAArtificial sequencesynthetic oligonucleotide
24gagcagccag gtgagcccaa gat 232523DNAArtificial sequencesynthetic
oligonucleotide 25ttcgatccca agaaggaatc aac 232623DNAArtificial
sequencesynthetic oligonucleotide 26agctctgtag atgtgctata cac
232724DNAArtificial sequencesynthetic oligonucleotide 27tcagatgcaa
gcagcaactt tggc 242822DNAArtificial sequencesynthetic
oligonucleotide 28gcatcttagc cgtctttctt cg 222924DNAArtificial
sequencesynthetic oligonucleotide 29gtagtgatgc catgtaactg aatc
243022DNAArtificial sequencesynthetic oligonucleotide 30aggcacccta
gagatgttcc ag 223124DNAArtificial sequencesynthetic oligonucleotide
31gaagatttct gtttccatga cgtg 243225DNAArtificial sequencesynthetic
oligonucleotide 32ccacactgaa tgtaatactg aagtg 253322DNAArtificial
sequencesynthetic oligonucleotide 33cgaagctctg gaactcggca ag
223422DNAArtificial sequencesynthetic oligonucleotide 34gccagctgca
aaggtgttgg ac 223523DNAArtificial sequencesynthetic oligonucleotide
35aacacctgcc tcatcacgac ttc 233622DNAArtificial sequencesynthetic
oligonucleotide 36gccaggctgg cgtcgatggt ga 223722DNAArtificial
sequencesynthetic oligonucleotide 37gtcgatggtg atggacagga ac
223822DNAArtificial sequencesynthetic oligonucleotide 38gtaatacgac
tcactatagg gc 223919DNAArtificial sequencesynthetic oligonucleotide
39actatagggc acgcgtggt 194027DNAArtificial sequencesynthetic
oligonucleotide 40ccatcctaat acgactcact atagggc 274123DNAArtificial
sequencesynthetic oligonucleotide 41actcactata gggctcgagc ggc
234244848DNAHomo sapiens 42ggatcttggc tcactgcaat ctctgcctcc
catgcaattc ttatgcatca gcctcctgag 60tagcttggat tataggtctg cgccaccact
cctggctaca ccatgttgcc caggctggtc 120ttgaactctt gggctctagt
gatccacccg ccttggcctc ccaaagtgct gggattacag 180gtgtgagcca
tcacacccgg ccccccgttt ccatattagt aactcacatg tagaccacaa
240ggatgcacta tttagaaaac ttgcaatggt ccacttttca aatcacccaa
acatgttaaa 300gaaattggta tgactgggca tggcacagtg gctcatgcct
gcaatcctag cattttgtga 360ggctgagacg ggcagatcac gaggtcagga
gattgagacc atcctgacag acatggtgaa 420atcccatctc tactaaaaat
acaaaacaat tagccggggg tgatggcagg cccctgtagt 480cccagctact
cgggaggctg aggcaggaga atggcgtgaa tccaggaggc agagcttgca
540gtgagccgag atggtgccac tgcactccag cctgggcgac agagcgagac
tccgtctcaa 600aaaaaaaaaa aaagaaagaa attggtatga ctgttgactc
acaacaggag tcaggggcat 660ggggtggggt gtaagattaa tgtcatgaca
aatgtggaaa agaaacttct gtttttccaa 720ctccacgtct gctaccatat
tattacactc ttctggtagt gtggtgttta tgtgtgaatt 780ttttttcata
tgtatacagt aattgtagga tatgaacctg attctagttg caaaactcac
840tatgagctta gcttttaagt tgcttaagaa taggtagatc tatgcaaata
atgataatta 900ttattattat tttaagagag ggtctcactt tgtcacccag
gctggagtgc agtggtgtga 960ttaagggtca ctgcaacctc cacctcccag
gctcaaataa acctcccacc tcagcctccc 1020cagtagctgg aaccacaggc
acgggccacc acgcctggct aattttttgt attttttgta 1080gagatggggt
ttcatcatgt tgcccaggct gttcttgaat tcctcggctc aagcaatcct
1140cccaccttgg cctcccaaaa tgctggcatc acaggcatga tggcatcact
ggcatcacat 1200accatgcctg gcctgattta tgcaaattag atatgcattt
caaaataatc tatttttatt 1260tgttgcctta ttggtggtac aatctcaagt
ggaaaaatct aagggttttg gtgttatttg 1320cttactcaac caatatttat
tagactctta ctaagcacca acatgatcac atgcctgagc 1380tatggctagc
atagcgtgtg agacaaactt aatctctgtt ttggtggagc atataatcta
1440gtagatgaag ccaatgttga gcaacatcac aatactaaca aattgaggat
gctacgagag 1500tgtctaacaa attgaggatg ctacgagagt gtctaacaaa
ttgaggatgc tatgagagtg 1560tgtcatggag agctgcctgg agattgagag
aaagcttcct tgagggaagt tacatttcag 1620ctgaaacaca ctgccatctg
ctcgaggttt tgtaactgca ttcacatccc gattctgaca 1680cttcacatcc
cgattctgac acttcaccca gttactgtct cagagcttgg gtccgcatgt
1740gtaaaacaag gacagtatgc acttggcagg gttgtgagaa gggaagagaa
cacaagtaaa 1800gcacctgtat caggcataca gtaggcacta agcgtgcgat
gcttgctatg attatacatc 1860agtgtaagca tcaaggaaaa gctgaagaaa
agtctgacca acagcgaaag ataaatgcgc 1920agaggagaaa tttggcaaag
gctccaaatt caggggcagt ccgtactcta cactttgtat 1980gggggcttca
ggtcctgagt tccagacatt ggagcaacta accctttaag attgctaaat
2040attgtcttaa tgagaagttg ataaagaatt ttgggtggtt gatctctttc
cagctgcagt 2100ttagcgtatg ctgaggccag attttttcaa gcaaaagtaa
aatacctgag aaactgcctg 2160gccagaggac aatcagattt tggctggctc
aagtgacaag caagtgttta taagctagat 2220gggagaggaa gggatgaata
ctccattgga ggttttactc gagggtcaga gggatacccg 2280gcgccatcag
aatgggatct gggagtcgga aacgctgggt tcccacgaga gcgcgcagaa
2340cacgtgcgtc aggaagcctg gtccgggatg cccagcgctg ctccccgggc
gctcctcccc 2400gggcgctcct ccccaggcct cccgggcgct tggatcccgg
ccatctccgc acccttcaag 2460tgggtgtggg tgatttcgta agtgaacgtg
accgccaccg aggggaaagc gagcaaggaa 2520gtaggagaga gccgggcagg
cggggcgggg ttggattggg agcagtggga gggatgcaga 2580agaggagtgg
gagggatgga gggcgcagtg ggaggggtga ggaggcgtaa cggggcggag
2640gaaaggagaa aagggcgctg gggctcggcg ggaggaagtg ctagagctct
cgactctccg 2700ctgcgcggca gctggcgggg ggagcagcca ggtgagccca
agatgctgct gcgctcgaag 2760cctgcgctgc cgccgccgct gatgctgctg
ctcctggggc cgctgggtcc cctctcccct 2820ggcgccctgc cccgacctgc
gcaagcacag gacgtcgtgg acctggactt cttcacccag 2880gagccgctgc
acctggtgag cccctcgttc ctgtccgtca ccattgacgc caacctggcc
2940acggacccgc ggttcctcat cctcctgggg taagcgccag cctcctggtc
ctgtcccctt 3000tcctgtcctc ctgacaccta tgtctgcccc gccagcggct
ctccttcttt tgcgcggaaa 3060caacttcaca ccggaacctc cccgcctgtc
tctccccacc ccacttcccg cctctcattc 3120tccctctccc tcccttactc
tcagacccca aaccgctttt tggggggtat catttaaaaa 3180atagatttag
gggttacaag tgcagttctg ttccatgggt atattgcatt gtggtggcat
3240ctgggctctt agtgtaactg tcacccgaat gttgtacatt gtatctaata
ggtaatttct 3300catccctcat ccctctccca ccctcccacc ttttggagtc
tccagtgtct actattccac 3360taagtccatg tgtacacatt gtttagcgcc
cactctaaat gagccttttt gtttcattca 3420ttctgtaagt gttgaatagg
caccacctaa ggtcaggtat aagtggaaat ttgaaaaaga 3480aactgcccac
ttgccccagt acttccctag ccaagaggag ggaaaccagg caggtgcacc
3540tgaaggcctg tgagtgcttg atttgctgtg cagtgtagga caagtaagat
tgtgcatagc 3600cttctgtatt taagactgtg ttaggaagat ttctctttct
tttcttttct ttttcttttt 3660tcttttcttt ttttttttta ggcagatgaa
aagggcgtca cagaacagga ataaaaatct 3720aaatattcaa taaatgagac
ctaggagact actgcagtga cttacaaagt cctaataaaa 3780agatgtctct
ccaaaatggg gctgcaaaat gtggtgctgc cttatcagct ctaagttttt
3840tccttacctg agaaagaagg aacctgatgc aggttcaggg ctcctgcccc
atgaatgcag 3900gctgactcca agatggggag ctacagggac aatcccaggt
cttctaggcc tcttatttag 3960gccctgggag cctccagaga tggccacatc
ttgaccagcc cagatagagg gaaagatcac 4020cattatctca cctctgtgtc
aaatacctag atgctgtcct ccctgagccc acactatagt 4080tgccagcgct
aatttaatgg gtagtgtact ggttaagaga tggacagacc atcctggctt
4140gactctcagc tctggcaaag atgagtgact tggtttttcc atatctcttg
gccacaccaa 4200ccttgatttc ttcagctgta gaatggaatt tctcaagctt
gcctcaagga ttattgcccg 4260aggatttgat gatatggtaa gagcttctca
gtgtttgacc catagtaagt gtttgacgtt 4320tcaaacgaat tgtttctttc
taggacatgg tgagcatttg gtagccattc accggttttc 4380tgtttctttg
gatcatagtt aacctctcct tttccttctg gcactacaat tttctggtgg
4440ggaagaatcc ttactttctg cccttcccct taaggatagg aagctgatac
taggcagcaa 4500ctagttgggg gataggaaga ttgttccaga gaaatgctga
accatagggc tccagatcac 4560aggaccccag tcttagcttg ctggggtgtg
gggtgggggg gggcggttac tgaacatggg 4620tatgaagtag atgtccattt
actgaaatgt gaggacctga ggcctcttct attgctgtag 4680ccagcatatt
ccccaacctc tccccaagaa aggacagatg ggggttcccc cctggagtaa
4740caggtccaaa agaaaaaaca tacagtggga cttccaggat ctgggcctga
tcacccagca 4800gtcaagctcc ccgcaattga ctaacacccc cctaacacgt
agaaattcca atctgcaatt 4860tagtgaggat gataccttta ttcttcttaa
atacatctct tcatttccca gagcaccctt 4920ttttcccctc ctctgcacct
ttttgttaaa gactggagta taatgaaata ccaagagagc 4980ataacatgtg
atacataaaa ctttttttct ggtttacaaa acagttcatt cttgtccata
5040cgtgcttctc tccaaggctg gctgctgtct gttccagccc gcttcgcttg
gagaggccat 5100ctgccatacc tgctccccag acgcatcgac aagcacaccc
agagtgttat ctgctaagac 5160ctaaaagagg gaggaacccc ctctcctcat
ctaagaccta gcttctaaat tagagtgtga 5220gggtccatct ccccaggagg
ggcacagggc ccaaacagcc cagccatctc agaagacaac 5280actaagcttt
gtaggggtcc acagtagagg agagtaagac gcctgttgtt taatttatta
5340cagttcctca aaagtgaaga tgtgtgggcg ggatggcaag agctgagcag
acgaaagctg 5400aaggaataag gaaagagagg aggacacaaa cagctgacac
ttcctcagtt cttgtcattt 5460gcctggccct gttctaagca ccttctaggt
attaatccat ttagtcttgg ctacaacact 5520gtgagtaact agttttgtca
cccccatttt aaaaatgaag aaagtgaggc tcagggaggt 5580taagtaactt
ggccacagtt tgaaactaga ctctgatcac atgagataat agtgcccata
5640aaaagggaaa gcagattata ttttttaaag gaaagagagt aggatatggt
agaaaaagat 5700tgtttggaaa ggaattgaga gattgatata atgaaaagaa
gcattcacat gagagtaaca 5760gtatcagggc ccaaaccttc atctaaggta
cttcaaagag gcctaagcaa acttagtcac 5820tggcgtggtt ctagtctcca
tgatggcaaa tacattgtgt acagcccaac tccacacaaa 5880acttaaatac
caatgataga gcaatctaaa atttgaaaga aaaaatcttt caatttgtcg
5940tcttcccaga gggacttaat caagaaacca atcaaaatac ttcctaagcc
taactgtgtg 6000cagaactcca aagagagccc agccctaaat caacactgtc
caatggaaat ataatataat 6060gtgggcctca tatgcaaggt catatgtaat
tttaaatttt ctagtagcca tattaaaaag 6120gtaaaaagaa acaagtgaaa
ttaattttaa taattttatt tagttcaata gatccaaaat 6180gttttctcag
catgtaatca atataaaaat attaatgagg tatttattat tccttttctc
6240aaaccaagtc tattctataa tctggcgtgt attatttaca gcacttctca
gactatattt 6300ctttctttct tttttttttc cgagacaatt ttgctcttgt
cacccaagct agagtacaat 6360ggcgttacct cggctcactg caacctccgc
ctcccgggtt caagttattc tcctgcctca 6420gtctcccaag tagctgggac
tagaggcatg caccaccacg cctggctaat tgtgtatttt 6480tagtagagac
agggtttcac catgttggcc aggctaatct caaactcctg agctcaggtg
6540atatgcccac ctcggcctcc caaagtgttg ggattacagg cgtgagccac
tgcacccggc 6600ctcagattaa ctatatttca agcgttcagt agccacatgt
agctagtgct atggtagtgg 6660acagtacaga tctgcatttc aattaagaca
cgtatacaag catagttcac taatgcacgg 6720taaaaaaaag tatagtgctg
agtcggtggt agaaatccta aatactgcag agcaaaagtg 6780gtacgaacag
caatctcagt gataatgcaa ccatgcttgc ttttcattgc aatttgctta
6840ttttccttca gcaaagttca tccatttttg ccaattcaat aaatatttac
tgataaaaac 6900tttcaatatt agattcttgc atcttcatag acagagttgc
ttttcacatt tagaaaatta 6960cttatcaatg ttaaacacac gttttgataa
ccagtgttgg aaagaggtgc agactcccca 7020tgtgcctatt gatggcagaa
atattcacag ccaaagggaa acaaagggct ggggacaatc 7080acacacctca
tgtctcctaa ctcctgggaa gtgctgtccc tctgattgag ctcttattat
7140tgccttcccc actaaccctg tccactgtgc cctggagccc tttgcagggt
tacctgctct 7200gtcctcctca cagaatatct cctctacctc cttgtccaag
ctacaacttg gctattctct 7260gatgacactg tcttccctgt agcccttttg
agtaatggct gcatattctc ccatagtcca 7320gttcttttcc tgttctccag
tctggcttct ggatgacagc ccactagttt gaactccata 7380ctgctatagt
tcaagtccct tttgacttgt taccttgggc aaattacctc cttttgttca
7440ggttccttgt ttgtaaaatg acgataataa tgccatttgc ttcagtgggt
tattttgaaa 7500ttgagtgaaa gaaggcgggt agcttcccta cacgctcagt
gtagactagc ctgatgtgca 7560ttacgggtga tgccatgact cagtgtgttt
tcctcatctc cacatctggc tctcatccag 7620tgctcctgct tacggcactc
tgtccccctc ttacttactc ccccttatta actgaagact 7680ggcactgatc
tcacagtttc ctctccactt cctagtctca ccatcatcct agatgacttc
7740aagtcaccta gataaactgt ctcagtttct tcactcacat ttttttataa
cagataatgt 7800tacactcaag ttgtaacaga accagcttat ccagctcatg
aaatgtatgc atttcatctc 7860aactctgtat tcagtgacat cctgtgggta
tctggaaatc agccatggtg agaatattta 7920ccatggaaat tggcaaatac
taaaaagcag agcacctttt tttctgagag ccagaccata 7980gctcttctac
tccatagcac ccatcataac aatttttaaa tacctccact gaacagcttc
8040ttcctctctc tacttcttcc atatctgatt tgagcttctt aatttatcat
gtgaaccact 8100cttgtaataa taaccccaaa tccctgttcc attgttcttc
ctgctaaaat actaaacctg 8160gtttagtcca accatatttt ctctctttgg
aatctacagg gtggcccaaa aacctggaaa 8220tggaaaaata ttacttatta
attttaatgt atattaataa gccattttaa tgcttcattt 8280ccagtctcag
tggccaccct gtatagctgg gctattgagc tcttgcggga ggagggagtg
8340gacagtctcc cagccacaca gactgatgtt gcaccaaaca ttttttagct
tccagacttc 8400cctggccctt agtgttaccc ttaactctcc atttctctgc
ctttcacatt ctctactttt 8460taaaaatctc tgactccacc ttcaccttat
cattcttagc acatgaccat acttctgctt 8520cccaaagaaa atgagcaatt
acttcctttt ccttttcctc ctgtcatcaa atctgcagac 8580atgtcatgcc
taagtccagc tttcctcctt tctctgatct cagtctgctt cttccatttc
8640tgccctgaat cccgtcccct ccccaacccc caaggacttc gctctatcag
tcacctcttc 8700cctctcctgt atcttcaact cctcccattt tactggcttc
ttcctcaagc ctttccccaa 8760gcctttccca tctcaattac ctcctcgcac
atgcctctgc agaaaccacc ccgtttcttc 8820cctcccctcg gcagcctgtt
cttcctgttc tgccctcatg atggcaccat cattgtgtca 8880ctaaaatcaa
tctctccgac atcatcaatg gccttccttt gttgggaaac ctaataaaca
8940ctttatctta tttggtcttt gttatgggtt gaatgaggtt accccgaaat
ccatattaga 9000agtcctaacc cccagtacct cagaatgtga ctttatttgg
gaatagggtc attgcagacg 9060ttattagtta ggatgaggtc atactggaat
gtgatgggct gcttatctaa tatgactgat 9120gtccttataa caaggagaaa
tttggagaca gacacgcaca tagggagaat accatgtgat 9180gacaggagtt
atggagttgg agtcaaaaag ctatgggaac ttaggagaaa gacctggaac
9240aaatcctttc ctgcgcctag agagggagta tggccctgcc actaccttga
attcaacgtt 9300tcggcttttc aaaactgtaa gacaatacat ttctgttgtt
caaaccaatt agtttgcagt 9360actctgcgac tgcagcccta acaaactaat
acagtctctt ggaggcattt ggcaaggttg 9420acaatggaag cactttctta
cccctttagg tctgtcgcct ttcttgttgg ggggtgtttt 9480ctaacaattc
ctctccatct ctctctctct agtttgtctt aaacattggt gttcttcaga
9540cttctgacct aggccttctt ttcacttcac atattcccct gggtggtctc
acccacttcc 9600agaaattact taaattactg ctcatgcagt actgtgctgg
aaactgttta acaactggct 9660ctctgggaag aggggagact ggttgatggt
ttttgctgat ttctgtggtg taaatactcc 9720ctccatggcc aattccaaac
tgccaacagt ttaacaactg gctcacaaat tttctccaaa 9780tttaacattt
ggctttcaca ggccaacaac gtggtacagc caactccagc acacctctgc
9840ttttgtgtca gagagaagta acttattttt gtacaaaagg taaaataaaa
acacctgcag 9900gccccctttt tttccttaac aaactgctct agaaatagaa
tagctgaagc ttcttttatg 9960cattcatctg ttatttccat gtcactgtgg
tggtgggatt atttttcctt tatttttctt 10020gtatatggtt gaaatactgt
acctttgatc agttttagtt ttatggcatg ttttgcaccc 10080atattaaatc
tagtttttgt cagagggcgt caatattatt ttctcaaaac aagaaaatat
10140ttcattgcaa aggagacaaa caaaaaggtc cttaatacca aaactttgaa
atgtgatttc 10200ttgtacttgg cagtgtccaa gtggtaaacc caaacagtat
tgggttttca ttttgttcag 10260gaaagtcttt gtctggcagc gacttaccct
tacatcaggc gggccttgct cattcattca 10320cttaagtatt tattaaacac
cagcggtgtg ccaagtactt atctaggtat cgggtagatt 10380ctgataagtc
agtcaggtcc ctgctctcag ggagcttgca gcagagatgg gggctgcaat
10440agagagtaag ccaaggaaat gaaaaaggaa gttgatttca gagagtgatg
aatgctatga 10500agaaaatgaa ggcagcgcag tgtgatggag agtgacccaa
ggtggtacag tttgtacctc 10560taaggaccag actgtgaccc aggtcactca
cagatgcccg tcatgtgatg ccacagcaac 10620ttttccaggt gctcgtttcc
tcccacttcc cagtctcttg cccagccgcg actgcttaca 10680aatacagcta
gaggaatcta aatgaggttc ctctatcatc aaacccaatc aaaatgccaa
10740ggaacagaat cagtgcctgg ctgaaggcag tggaacaggg ccagcctgga
gtggttctct 10800ctgaggaagt tcctcatctt ggttttaggg ccataccttg
tgacctgtga gctaggggtt 10860gccagtccct gacatttcta ctgaggactc
gcctgtctat attcccggcc tgtatgtgtc 10920tcctgagttc cagacacaca
gggcgaagcg cctgatggat ggaagtatgt tttttggtgt 10980tccattggta
tctcaaattc tacaaaactt agtgcccctt ctcctccctg ttcctcccca
11040tcttcagtct atcacctgtt cctcatccag caaatgatat taccatcttc
caaggagctt 11100cccaggagta atccttgact cctcctcaac atccaattaa
taatcaaatc taggccaggt 11160acaatagctc acgcctataa tcccagcact
ttgggaggct gaggcaggtg gatcatttga 11220ggccaggagt tcaagaccag
cctggccaac aaggtgaaac ctgtctcatt taaaaaaagt 11280tattttaaaa
actcaaatct attatttcta cctctaagtg tgtcttgaat ttatccatct
11340ctctccatct ctgagctgtt accttacctc agtccatcac gttttgtcta
cgttaacatg 11400accagagtct tgttcttagt ctggtgaggt cactccagct
gcttcagatc cttccatggc 11460tcaccgttgc cctcatataa agttggcact
cctggacatg tggcttacgg ggccctccgt 11520gatgtggccc tatttgcttc
tccattctgt tctctcccag cctctctgcc cccatctcta 11580ggcaccaacc
acacccttct gctcgtcaat ggtgccagct tctcttctat ctctggtctt
11640tggacagact tttcccttca cctggaatgc tttcttcaat cctaccccac
tctctttaat 11700ctagataagg tttattcttt ttgaatgtct agcagtgaaa
ccatttcccc tgaaaaacct 11760tctctaacca accccctacc ctcagcccaa
ggtctagatt aggagtccct ctgaatgttt 11820ccatagcatt tttaaagaat
tgcctattta cttgttcgta tctatcacta aactacaaat 11880tgtatgagaa
cagccactat ctctgcctgg ttcaccattc atctccagca actagcataa
11940tgcctggcag agtcagcctg caacaaatat ttgttgaata aattaacaga
tggctttatc 12000tccttaagta aatcttgctt ttttcaccta ttaaaacaga
cgcacaggcc aggtgtggtg 12060gcccatgcct gtaatcccag cactttggca
ggctgaggtg ggcggatcac ctgaggtcag 12120gagttcaaga ccagcctggc
caacatggtg aaaccccatc tctaataaaa atacaaaaat 12180tagctgggca
tggtggtggg tgcgtatagt cccagctact agggaggctg aggcaagaga
12240atcgcttgaa cccaggaggc agaggtggca gtgagccgag atcatgccac
tgtactccag 12300cctggatgac agagaccctg tctcaaaaca cacacacaca
cacacacaca cacacacaca 12360cacacacaca cacacacacc aagttgtata
atttaaaata taacgtgctt gttatggaac 12420acttgtaaaa tacaggaaag
taatgaaaaa gtctaccatc tagctcacca cataatgacc 12480attgctatca
tcctggcata attctctcct gtatataaat atatattctt ttattgttaa
12540aattacacta tgagtactat ttatttattt tactgtggca aaatgcgcaa
aacataaaat 12600cttgccattt taaggtatgc agtttggtgc attcaccaca
ctcacattgt tgtgcaaata 12660tcaccactat ctatctcaga acttcttcgt
cttcccaaac tgaaactctg tacccattaa 12720acaatagtgc atcctctgtt
ttcccctccc tacaatttat ttttatttgg gtttgtacca 12780aactgaaaat
agctgcttct tccttactta gttcagatta gcatttccat ttatttagcc
12840gtggttttga ggatgccatg acagatgcca tccttcctag agctctttgg
ggctgtcagg 12900tatttcagtc agggtgaatt cgggttgata acattttaaa
atctcacttt attctgaggt 12960tcctagtgtc agagcccacc gtatttttag
ggactcccaa gttacaaaca aaaatatggt 13020gaggaggaat cactgaagtt
ttaacacaag agacttacat tttgttcaat ttctatcttt 13080tagtttattt
cctaagcata aagaaatact ttgaaaattt tacatagcat tatacatatt
13140taattaagca tgagcacatc ttaaaacttt aaattttaga tcagatcttt
aattcctagg 13200atattaagag gtactggcaa tttggccagg tgtggtggtt
cacgcctata atcccaacac 13260tttgggaggg tgaagtgggc gaattgctag
agcccaggag gtggaggctg caatggcctg 13320agatcacgcc atcgtactcc
agcctggatg atgagaatga aatcctgtct caaaaaaaaa 13380aaaaaaaaaa
aaaagaagaa gaagaagtat tggcaatcag tgctccagga ataatttcct
13440gacttgaaat aaacctacat gtagacaaac taattaggcc attccaagag
ttgctagcat 13500tggtttaata tgttttcaga gcattccagg aagcagtgtg
gccagcattg catgtttgat 13560acttcagaaa tgtatgacag gtgtttctct
tacccaggtc ttctgttttc ttagttttgc 13620tcatgtaaat atttatgaac
atcctcatct ttttgaggga agggattata gatcattcta 13680attccatttt
ctagcatttg gtaccattct aagcacatga taggcaccca tttggagcat
13740ttttggcttg acagaatatg catttagaat tgttcaaatt agaggtgtca
gtgatgggaa 13800ttagaatact atataattct aagtcatttg acttaaatac
aaaagaatga ttttccttgg 13860tggggaatgg tgaagggagg caggagttaa
gaagaggaga agagatccta agtcatttat 13920aaacttctct ggaaagacag
gtgtgtgaag actttttaaa aagtcattca ccaaattgtg 13980tgtgtgtgtg
tgtgtgtgtt ttaaatagac tttatttttt agagcagttt taggttcaca
14040gcaaaattga atgcaaggac agagatttcc cataaacccc ctgcccacac
acatgcatag 14100cctccctcat tatcaacatc cccaccagag aggtgtttgt
tctagttgat gaacctacac 14160tgacacatca ttatcaccca aagtccatag
ttcacggcag ggttcactgt cggtgtacat 14220tctatgggtt tgagcaaatg
tataatgaca tgtatccacc attatagtaa catacagagt 14280attttcagtg
ccctgcaaat cccctgttct ccacctattc atccctccct ctctgcattt
14340ccacccccag cccctggtaa ccgctgatct ttttactgtc ccatagtttc
ggacgatcta 14400tttttcagac agacacagag ctgtctttcc cttagtttct
attctatcat ttctttctcc 14460ccatccatca taaaaggcta tgagtttttt
ttaagtgttg aacaccatcc tacttgtcaa 14520gttaaaacat aagctcctgg
ctgggtacag tggctcatgc ctgtaatctc agcattttgg 14580gaggctgtgg
cagaagcatc acttgaagcc agaagtttga gaccagcctg ggcaacatag
14640caagacccca tccctccaca cacaaacaca cacacacaca cacacacaca
cacacacaca 14700cacacacaca cacaaaaaca agctcttgcc agaattagag
ctacaaattg ccctcaggtt 14760cctagaagat cagtccttca attagattca
gattgagatg cttcctcttt taaacaatga 14820ttccctttct atcatgccca
ataagaaaac aaataaaaat taaacaatac tgcctgtaat 14880ctcagctacc
caggaggcag aagcagaact gcttcaaccc ggcaagcaga agttgcagtg
14940aagtgagatc gcgccactgc actccagcct gggaaacaga gcaagattct
gtctcaaaaa 15000caaaacaatg tgatttcctc ctctaagtcc tgcacaggga
aatgttaaga aataggtcca 15060ccaggaaaga aggaagtaag aatgtttgac
tagattgtct tggaaaaaat agttatactt 15120tcttgcttgt cttcctaaca
gttctccaaa gcttcgtacc ttggccagag gcttgtctcc 15180tgcgtacctg
aggtttggtg gcaccaagac agacttccta attttcgatc ccaagaagga
15240atcaaccttt gaagagagaa gttactggca atctcaagtc aaccagggtg
aaaattttta 15300aagattcact ctatatttta attaacgtca gtccgtcatg
agaatgcttt gagaaaactg 15360ttatttctca cacctaacaa ttaatgagat
taacttcctc tcccctcatc tgacctgtgg 15420aggaatctga acaagaggag
gaggcagtgg gcaggtttcc ttatcatgat gtttgtcatg 15480ttcagtgtga
ggcctcacaa aaaaaaaaaa aaaaaaaaaa ggcgtcctgg atataactga
15540gagctcattg tacagtaaat attaataaaa cagtgattgt agctgaagga
tagaactgct 15600tggagggagc aagtgggtag aatcgcgtca aactaaagag
catttctagc caaagacaca 15660atgatagatt gaaggatatt tattctaaat
atagaatatg ggtgaacgag atctgtggac 15720ttctgggctc caacgttaga
ttctgatttt agcaagcttg tcaggggatt ctgatattga 15780aaggctgtgg
ccttcacctg agaaacctgc cctagggggc catgaaaatt tgtcctgtct
15840ttcagaagtg ctatcagaca tcaaatggaa gttaaatcgt atcttaacaa
ttactaggat 15900gggcgcagtg actcacacct gtaatcccaa cactttggga
ggctgaggca ggaggatcac 15960ttgagcccag gagttcggga ccagcctggg
caacatagag agacgttgtc tctatttttt 16020aataatttaa agagaaaaaa
atactgaaaa tattgtatac accactgaat tataataatg 16080tgtatataat
gtatatattc attatgagga atatttgatt atttcatata ttatatcttt
16140tccttctgtt tattttatcc agttatgaag tatttagaac aattcatcag
taattggggc 16200taaattgaca gaatagtaat cagagaaaat agaaaaagac
agatgggtta tctttgaata 16260ccaggttgga gttgtttatg ggtttgtttt
ttgttttggg ggcgtttttt tagacagagt 16320cccactctgt tgcccaggct
ggagtgcagt ggcacaagca tggcccactg catccttgac 16380ctcttgggct
caagcaatct tcccacctta gcctcctgag tagctgggac cacaggtgca
16440tgtcaccaca cccagctaat ttttttattt tttgtagaga cagtctttct
atgttatcca 16500ggctgatctc aaactcctgc actcaagtga tccccctgcc
ttggcgtccc aaagtattgg 16560gattataggc atagccacca cacccaacct
agtttctatt tagacttggc cctttcccac 16620cagtcatttg tgtccaaaag
atctcataaa tgtagacagg aaactgtcct ttgctcatca 16680gttttcttca
tcctgtgtct agggggatgg tcggtggggg aaactggggt tatgcaagtt
16740cctctgaaac atcctctgtg agcccaggga tggatgaggc accagccgcc
agcgagtcag 16800tgtgcagctt tccagaaagg aagtcatcag ccagtcagcc
ggccctggca gccagcaccc 16860ggcaaccctg ctgtcttgtg ataaagaaat
ggtctgcctg acaggatggt gtggattttt 16920cttttttctt tttttttttt
ttgagacagg gtctggctct gtcgcccagg ctggagtgca 16980atggcgggat
cttggctcac tgcagcctct gcctcccagg ctcaaggcat cctcccacct
17040cggtctcccg agtagctggg accacaggca cacaccacca cgcccaacta
agttttcgta 17100tttttagtag aggcagggtt ttactatgtt gtccaggcta
gtctcaaact cctgagctca 17160agctatccat ctgccttggc ctcccaaaga
gctggaatta caagcgtgag ccactgtgcc 17220tgaccagggt ggattttttc
aagtgcacat gttgtggtcc cagaagctct gatggtacca 17280aattccaagc
gaaaaaaagt caatggttcc cacccatcct acctcccatg atggcaagag
17340gaaatcacca cactgcagat acagtccatg taaaacaaat tgctatggat
tttgaaagtg 17400aaccttaaga gaactgcact atgttttctt cattagagtt
ctctggtaat ttccagcttt 17460tttttttttt ttttttagac agtgtctcgc
tttgtcgccc agtgtcaccc aggctggagt 17520gcagtgacgt gatctcggct
cactgcaacc tccgcctcgt gggttgaagt gattctcctg 17580cctcagcctc
ctgagtagct gtattttagt agagacgagg tttcaccatt tggccaggct
17640ggtctcgaac tcctgacctc aagtgattcg cccatctcag cctcccaaag
tgctgggatt 17700acaggtgtga gccactgcac ccggccagta atttcaagct
tctgaggagc cctttgaatt 17760gttaaataac ttgtagctat gtccaacata
tccatgttca gtgtatgttc gatatttctt 17820aggaaacctg cccttggttg
ttttctttgt ggtaattcat gagccggcaa atttgacatg 17880tgttacagaa
tatacctttt ctctgctctc ctacctcata accagaactt aattatcctg
17940ctttagtcac ataaatagct aactaaataa atatatgaga tttcagtctg
ctcactgtga 18000aaatagacct tctaaatgat ctcttccact tgcagatatt
tgcaaatatg gatccatccc 18060tcctgatgtg gaggagaagt tacggttgga
atggccctac caggagcaat tgctactccg 18120agaacactac cagaaaaagt
tcaagaacag cacctactca agtaagaaat gaaaggcacc 18180ctagagatgt
tccagcccca aagatatttg aataggttgg actcgggcac caatctagca
18240agtcctacgg aagttgtata aagctgaaaa tactgaagca tttcccaaat
gggaaatcct 18300aaactcaaaa cttgcttttt ggtttttttg tttgtttgtt
ttttcttcat ctgacattgc 18360ttagtagtca cagaatgaaa gataaatcaa
tcattcatga tctaacaatg accttcagtg 18420ctctaaaaaa ctacggagtc
aaggaaaaca tgaatatatt cctcatgtaa aattaaaata 18480cagacatata
aagggcaaaa catgaacatc attcatacct tgaggtccgt ccccctccca
18540gaaataaccc ccagtatgcc ttggtttaga gcattaagca ggagggccct
gagtcactcc 18600agacagtctt gaccaccaag cagcattctc tttttgtttc
ctctgtggct tttgcaaaca 18660cagggctagc tcagctaccc attagtatgt
tttcagtcac
taaaacagtc ttccagtctt 18720caaattagga tgacattgtc acatggggct
ttaaagcaag tgaaacaagg aacccccttt 18780tttttttttt ttgagatgga
atctcactct tgtcgcccag cctggagtgc aatggcgcaa 18840tcttggctca
ctgcaacctc cacctcccag gttcaagaga ttctcctgcc ttagcctcct
18900attcattatg aggaatattt gattattcag ttcctgtagg gtaaagatat
tacccccgat 18960catattattg attattgagt agctgagatt acaggtgcct
gccaccacga ccggctaatt 19020ttttgtattt tttagtagag acagggtttc
accatgttgg ccaggctcca ggctcgtctc 19080gaactcctga cctcaggtga
tccacccacc tcagcctccc aaagttctgg gattacaggc 19140gtgagccacc
actcctggcc acaatccttt tttaactatg aaatatattt ttatctgaag
19200tttgatgttt atacccaact gagggatgat gttcccatat ctcagttaaa
gaaataacct 19260gctcagatac ttcaagctct tcttttgact tttgaaaata
aatgatcttg aagttactat 19320actttgtttg ggttagttaa cattatttaa
agtatattat tttaattaat tatctttgta 19380agattttact gtatactacc
tggagttcaa tgtatcagat ggatttcaaa tttatgtaca 19440ttttttatgt
atatggtaca gaaaaaaatg tgatccataa gaaatcagaa aatagcgcat
19500atgctaatag ctaatgttgt cctctaaaaa acttattttt gcatttttaa
gagggggata 19560tactctgaca ctttaataag tgtaattaat tattgactgg
aatttggcat gaggcagggc 19620catttcagat cccattaaag gaatgacaca
taccagagaa ccacagaagt aaggccacat 19680ttgtaataaa tcattatagc
tctgctagga gaagacccag ttgtattagg taattaatgg 19740atttgctctt
aaaacacatg tcccggaaga tataggtgag tcttgggggg ccgcattaaa
19800cattatacca atgtatctta catttctaag aaagttttac tactttacag
gatctttctg 19860ttaccaaaat ggaaggtttc caactccagg acttggcttt
catagttcct acaccagggg 19920aaatgccttc ctttgctaac tatgcaacca
ggttagttag tgtaagtcca gccaccctgt 19980tggcaatgct aaaaggtaca
acaaacacag aattttattt gcatttgtaa acatttgatt 20040tctggctcga
aattttcagt tttcatgggc acgtcatgga aacagaaatc ttctgtgttt
20100agtttgggca cctactcatt gtagtgacaa atatttcaga agccaatagg
ggattccaca 20160aattgttctg aacctgtggc tgagactggt aatggctgag
tgacatgggg acataccaca 20220aaagaagagg tagcaaaagg ctgctgagat
aaggacatgt tcattgctta gctagtggcc 20280tgcaccctta aaacacatgt
cccaggctgg gtgctgtggc tcacgcctgt aatcccagca 20340ctttgggagg
ctgaggcggg tggattacct gaggtcagga gttcgagacc aacctggcca
20400acatagtgaa acctcatttc tactaaaaat acaaaaatta gccaggcatg
gtggcgggcg 20460cctgtagtcc cagctactca ggaggcaggc aggagaatta
cttgaatctg ggaggcagag 20520gttgtggtga gccgagattg cgccaccgca
cgctagcctg ggcgacaaag tgagactctg 20580tctcaaaaaa acaaaaacaa
aaaacaaaca aacaaaaaac aacaacaaca aaaaaacggg 20640tatcccagaa
gatacaggta agttttctaa cacaggtcct cttgtatggt gcgttccact
20700taagtagaag atgacaaaaa catttgtcat gagaatatag actcacattt
taaacctgtt 20760tgagcaggaa aaggaagcaa tgttacagat gtaattctgg
gtgtgactgc agaaaggatg 20820actcccttat taaagtagtc atcctgagtg
agctaactct ttgtacttcc tcttctcctc 20880ctgttcccct catcacccca
ttcttccgtt gcctacaccc aggcccacat tggatgctga 20940catagactta
catggtacag tccaagggaa agatctgcca tttttttcaa tgtgtcatct
21000tggttatctt cattccaagg atctctccac tctttataca gtaagagatg
agagtctgga 21060aaggattggg aataagataa tgaattgtaa gttttaaatt
gttcttcgta ttttggggaa 21120ggagtaggct aggtggtcct tctgtttttt
ttttgttttt ttttttaaag tagatgtggc 21180cagacgtggt ggctcacgcc
tgtaatccca gcactttgag aggctgaggc aggtggatca 21240cttgatgtca
ggagttcaag accagcctgg ccaacacagt gaaaccccgt ctttactaaa
21300aatacaaaaa ctagccgggc ttggtggcgt ccacctgtag tcccagctac
tgcagaggtg 21360gaggcaggag aatcacttga acccgggagg tggaggttgc
agtgagccaa gatcatgcca 21420ttgtactcca gcctgggcga cagaacaata
ctctgtctca aaaaaaaaga gaaaagaaaa 21480gaaaaaaaga atggatttga
actcagtcgt caatagcctc tattccagga gatgttacag 21540ttgattatgt
tatagggggt gtataataga atttcgagct atgtaaattc caagtgcatt
21600tggaagaatg aagaaatgga ggaagggtaa agtatgagtg caagcattcc
aggttttttg 21660aaaatgctat aatctttgtt cagggctagt acaaagtgct
atttagctgt aagggttttt 21720tgtgatttac agacagtttt cacatgtgtc
atttcaacct tggttttatg gcgaaggcat 21780gtgatggtgc ttgtcccagg
actttagatc catatctgag gttcctgtcg ggcaaagata 21840ttacccctga
tcatattata gtctataagt gggagagttg tgcctggagc tcaagtctta
21900tgatttctga tccagggcac ttcctacaac atgattttgc aatataaaag
cctataatgt 21960gtgactaaag caggtcactc accccttgta acagactcta
gtaatggtac tgccaccaaa 22020cggctgcgtg atattgggca aagacttacc
ttatttgaat ctcagtttcc tcctagaaaa 22080atgagggtgg aggttaagca
taggctgatg atcctaaagc ctccatactg ccctaaactg 22140tggctctaag
atccagtaga atgctgggtc acaggactct agggagcttt tcaaacccaa
22200atgtctgtca ttccttgatg gtaggcagca gtttatggaa gtgggcgaca
cagcaaatat 22260caaaatacct aaagcagctt gcaagagttg tttctgccta
gtggtcttta tagttaatat 22320taaatagtta attttttttt tttttgagac
agagtcttgc tctgttaccc aggctgcagt 22380gcagtggcac aatctcggct
cactgcaacc tccacctccc gggtttgagc aattctgtct 22440cagcctccca
agtagctggg actacaggtg catgccactg cacccagcta atttttgtat
22500ttttagtaga gacggggttt caccatattg ggcaggctgg tctcgaactc
ttgacctcag 22560gtgatccacc tgcctcagcc tcccaaagtg ctgggattac
aggcatgagc cactgcaccc 22620agcttaaata gctaatattt aatattattc
tatagttatt caagtaattc aggccaaaga 22680cttagaaaca aaacaaaaag
ccacttttaa ggagaaaggg tgtaagtttg ccagatagat 22740agagatcttt
cttttttaac tacaagagtt caggaatgaa ttactcttta acaaacgact
22800atagatatac atgaaaattg gaaggactta ttatgcatat gataatcaat
ttaaagacaa 22860cacttaaaat tatattgttg ccactctcaa aaagtggtaa
tagaacagct aatggtttaa 22920aaagcagagt acagaagttc ccaaacttat
ggcaccttaa tatcgcagaa aactttttaa 22980agcatgccta ggccacaaaa
aatacctgta ttttgattat taaattgtaa ggtctacaca 23040acctaatagt
aataggtcca atagtaatgc tgtccaatag atgttgatgt ttttttcctt
23100gcaaacttaa aagatcctac agtgcctctg taaatagcac tgcctggtta
gagttgaatt 23160tcagataaat aatttttttc atgttaatta tttttctttt
ctttactttt ttttttgttt 23220ttttgttttt ttgttttttt ttttgagaca
gggtctcatt ctgttgccca ggctgctgtg 23280caatggcatg atcatggctc
actgcagcct tgacctccct gggctcaggt gatcctccca 23340cctcagcctc
ccaagtagct agctgggact acaggtgctt accatcatgc ccggctaatt
23400tttgtgtttt ttgtagagat gtggttttgc catgttgccc aggctggtct
tgaactcctg 23460ggctcaagtg atccgcccgc ctcggcctcc caaagtgcta
ggatgacagg catgagccac 23520tgcacctggc ccctgggcga agtatttctt
aatggttaca taggacatac actaaacatt 23580atttattgtc tatatgaagt
tcaagtttaa ctaggtgccc tgcactttta gttgctaaat 23640cctgtagctg
tacccatgca ttcactggtg ctccccagct tgccttgcac agagtttgga
23700aaccatagtc ctataactct aggccaattt tttaatgtaa aatttgattc
attttaaatt 23760aataaataat aacaggaatt tttttaaaaa ttgttttaaa
tataattaaa attatcaaaa 23820tattttttaa ctgaacttgt gactagagat
atttagatta tgaagagtgg ggtttatgct 23880aactaatgac agtctggcta
tgcatgtgga gcactgagct ataaattgtg gcttccccaa 23940ttctcctgat
gtcacttgaa caaaacctaa gtgtcagacc agagcttctg gtatcttcca
24000tgggatttca ttcaacagct ggagcaaatg aagtcagatt gatttttttt
aatttgtcca 24060attttgttgt ctcaaaaaca taattataat catttattag
aactagaatt tcttcagttt 24120aacaacagaa atagttattc attatgaaaa
gcgaatctgg aggccttcat tgtggtgcca 24180atctaaccat taaattgtga
cgtttttctt ttaggaagct ctgtagatgt gctatacact 24240tttgcaaact
gctcaggact ggacttgatc tttggcctaa atgcgttatt aagaacagca
24300gatttgcagt ggaacagttc taatgctcag ttgctcctgg actactgctc
ttccaagggg 24360tataacattt cttgggaact aggcaatggt gagtacccca
gggaacaatt cattaataag 24420gagattcccc actagcatta tttcttttct
tttctttttc ttttcttttt tttttttttt 24480gagacagagt ctcgcactgc
tgcccaggct ggagtgcagt ggcgccacct cggctcactt 24540gaagctctgc
ctcccaaaac gccattctcc tgcctcagcc tcccgagtag ctgggactac
24600aggcacccgc caccgcgccc ggctaatttt tttttttttt tttttttttt
tttttttgca 24660tttttagtag agacggggtt tcaccgtgtt agccaggatg
gtcttgatct cctgacctcg 24720tgatctgccc tcctcggcct cccaaagtgc
tgggattaca ggcgtgagcc accaggcccg 24780gctagcatta tttcttatga
cacttttttt ttttttttga gacggagtct cgctctgtcg 24840cccaggctgg
agtgcagtgg cgccatctcg gctcactgca agctccacct cccaggttca
24900cgccattctc ctgcctcagc ctcccgagta gctgggacta cacgcacccg
ccaccacgcc 24960cggctaattt ttttgtattt ttagtagaga cggggtttca
ccgtgttagc caggatggtc 25020tctatatcct gaccccatga tctgcccgcc
tcggcctccc aaagtggtgg gattacaggc 25080gtgagccact gcgcccggcc
aacactcttt ttattattag caaatatact tctgcctggg 25140cacattcttg
caagtgctca acaatgcaac ttttggaagt gcatgtggca gaaactcctg
25200ctgtatttat tccagaacct attattgcta atcccagttt atgttacatt
tgaagtgaga 25260accagttgga gccagcaacg ttcccagctc caaagttccc
ttgagatttt cagaatcact 25320taaccctatt atgcttggca acctggactc
agcaaaactg ggaagtcagc agtttgtttt 25380attcatccct tcctttctca
gtttctcaaa tgtgtcagtt aatctcagta accccattgc 25440aaccttcatt
acctgcccaa gcggtctaga acttgccagt atagaatcct acgtgggtca
25500agctcctgac tgtctccttc ttcactcttt ttttgcaaag aacttgtaaa
ttttaactat 25560aagtattcat gattcgccac atttattcaa aacatagagt
gctttttcca catatcagcc 25620aatggaaata aggattaaat gggaaatgaa
atgtagtaat aggataagca caagtcttct 25680tcctgctcaa actttttttt
tttttttttt cagacaagat cttgctctgt tacccaggct 25740ggagtgcagt
ggcgtgttca tagctcaatg taacctccaa ctcctgggct catgcaatct
25800ctcacacctc agccccctga ttagctagga ctacactatg cctagccaat
tttttttctt 25860ttgtctggtt gtgttgccca ggctgtctcg atctcctggc
ctcaagtaat cctcctgcct 25920cggccttcta aagtgctggg attataggca
tgagccactg tgcccggtct caaacctttt 25980tttccaaagt aaatgaagtt
attagatatg gaatatagtc tagttcccag atatccatat 26040ccattggttt
attaccctca ttattaactt caaattgttt aatagaccct catatctcag
26100ttatacagtt aaaatttttg ttttgttttt ctggagtatc ttatttataa
ctatgagttt 26160tactttactt atttatttta ttttttgaga cagacgcttg
ctctgtcact caggctggag 26220tgcggttgcg tgatcatggc tcactatggc
ctcgaccttc tgggctcaag tgatcctctc 26280cctcagcctc ccaagctgag
actacaggca tgcaccacca catctagcta attttttttt 26340ttccccatgg
aacaaggctt tactatgtta cccagagtgg tctcaaactc ctggcctcag
26400gggatcctcc tgtctcagcc taccaaaatg ctgggattac aggcatgagc
catagcgcca 26460gacctggttt tacttttctt gactttgaat tacaagtttt
tgtaatttgg aaaatgtttt 26520gttgctttta aatactgctg tatgtttgct
tttaaataca acatttctcg atatatattt 26580tgagaattgc tgtctttcag
aacctaacag tttccttaag aaggctgata ttttcatcaa 26640tgggtcgcag
ttaggagaag attttattca attgcataaa cttctaagaa agtccacctt
26700caaaaatgca aaactctatg gtcctgatgt tggtcagcct cgaagaaaga
cggctaagat 26760gctgaagagg taggaactag aggatgcaga atcactttac
ttttcttctt tttccttttg 26820agacagagtc tcactctgtc agccagactg
gagtgcagtg gtacaatcat ggctcactgc 26880aacttcgacc tcccaggctc
aagcaatcct cccatctcag tcccacaaat agctgggact 26940acaggtgcac
atcaccacac ctggctactt taaaaaaatt tttttgtaga gatggggtct
27000ccctgtgttg cccaggctgg tctcttgaat tcctgtgctc aagccatcct
tccacctcag 27060cctcccagag tgccaggatt acaggcatga gccaccacac
ccagccacca cttttcttaa 27120aaaaaaaaaa agattctctc tggtagacaa
tcctcaatag tccacatgtt attaaacaat 27180ctgctgcctg aatacatgat
ttaccaaaaa aaggaaattt tgacgggttc agaatatcaa 27240gggatctgag
gcaaatgtca cctatgataa aatttgctat caaaattagg aagtttgtgt
27300ttacctgatc ctaaagcagt aaccagccca tttctaggga ataaaactct
catgcgtata 27360ttgtgcatat atatgtatta tatgactgag tgataataaa
attttttttc tagcttcctg 27420aaggctggtg gagaagtgat tgattcagtt
acatggcatc agtaagtatg tctcctattc 27480ttaatactag gaaagtaagg
ctagctttat ttattaccta gtattcaaaa agttagttca 27540tttaactgcc
aattgactgc agttcaaata agaaacaaat agtgtctcaa gtagcactgt
27600actccaattt taatattaat aaaaaaaatt ttaagttatt ttaaataatg
tagtggtttc 27660tataaagatc actttataca gaagaacagt gccaattaac
ccatggaaca tataagtagc 27720taaaaccaat tgcttgccaa agaaccagta
acccaggagt acatgtcctt gccactgtgt 27780tttttcaaga cagagtaact
gatttctagt tacttgcata gaatggactc ctcctcataa 27840ctcccttcca
tcttggtctt tccctagtag aacttctacc tttttttagt aacaggtgag
27900tgggagaggt aagaaggaga ataaggtcag caattaacct aaaagcagaa
agtaaaattt 27960gttatttttt ttctgaatat tttctgtgta atttagctac
tatttgaatg gacggactgc 28020taccagggaa gattttctaa accctgatgt
attggacatt tttatttcat ctgtgcaaaa 28080agttttccag gtaatagtct
ttttaaactt tttaatgtaa aaccagaatc cttattttat 28140agtctagcta
gttctaaatt ctataggtat gtatatttac atgtttttct aattttagag
28200aacaagcact atgacttatc cactgttagt tttcccctta gcattgggtc
ttaccccatg 28260tacgtgatta gaaatttgaa atatttccaa tagcctttag
tagaattaac tcacatagat 28320gataagaatg ggttggttca cttcatgttc
cttccacagc ctactatttc aataaaagaa 28380agtttcccaa gacctaaatg
actatgaaca tattttataa ctatatagga ggggtgggtc 28440taggaataca
aagttttgaa tgctgttaat cttcaacacc acagttgaaa ccacaggtca
28500gcttttttgc aattaccatg gatacttttc tgttctatag gtggttgaga
gcaccaggcc 28560tggcaagaag gtctggttag gagaaacaag ctctgcatat
ggaggcggag cgcccttgct 28620atccgacacc tttgcagctg gctttatgtg
agtgaagcag cgctggcctt aggggtcaga 28680gtgcagctct tctccatcct
tctattctgc tgaaatagct ccccagccaa aaagcagatc 28740aaagaccgtt
tcagtggctg agccccaaaa ttcatgccag attttgcaag aaaatgattt
28800actaaagctt gagggacatc tttaacaagt gttccaaatt aatcactata
aggatgaatt 28860gtttcagaaa ttttggcctt taattatggc ccataaatat
gtcaagtagt ccttactcta 28920aagaagtaca ctgtaaaaga atgcatatag
ccggatatgg tagttccctg taatcccaat 28980actttgggag gccaaggtgg
gaggattgct tgagcccagg agtttgaggc tgcagtgagt 29040tatgatggtg
ccactgcact ctagactggg caacagagtg agactgtctt tttttttccc
29100ctctgtcacc cagactggag ggcagtggca cgatctcacc tcactgcaac
ctctgcctcc 29160cggattgaag cgattctcct gcctcagcgt cctgagtagc
tgggactaca ggagtatcac 29220cgcactgggc taatttttgt atttttagta
gagacggggt tttgacatgt tgcccaggct 29280ggtctgaaac ccatgagctc
aagtgatctg cctacctcag ccttccaaaa tgctgggatt 29340acggacatga
gctaccacgc ccggccacac cctgtctctt aaaaaaaaaa aaaatgcaag
29400ttagagcata ttacagcttt gtctctcagg aggatactta gtgtatgtag
ctataattca 29460tagattccca agaagtttag agcctaaagt atgaggtccc
accagagggg ctatcattaa 29520atttaaagat ttgttaaatc atctcattgt
ccaacaccac aaacttgatt gctttaaaat 29580actggtttag ttacatttag
taactctatt agtgctttta atctatactg ctatatcctc 29640acattgagat
tttttttctt ttctcttcca tcttcattct tttttctctc atcctcattc
29700ttataagcct agaatacatc acaaatcctt tatgcccatg gaagcaagag
gaataaagaa 29760tggagatgtt tgttttgcca ttaactaaag atctggggtg
tcggggagaa gggggataga 29820gaaggagaag tgggaagagg tgtccataat
agcttaggtg caattctgct tattttacat 29880tttacccccg ctgactgcca
ctttttcttc agccctcaca cattgtttgt gcagggacct 29940cataggacca
ggaattgtct atagaggtgg gaatttgtct caccctgaaa gggatacctc
30000tagcatggta atagtcttct aggatttgtt atcatatgga aagatgtaaa
gggagggatt 30060ctgctgctgc tgctgctgct gcatgcagtt gccatttcat
ttaaatgact tatttataat 30120tgatgacact tttctggctt cctgttaatt
cctccctcaa agatcaataa accagaacca 30180ggcatggtgg catgcacttg
tggtcctgta accacccaac aggttcacct tgcctgctgt 30240ctagatagag
ccaattatca agacagggga attgcaaagg agaaagagta atttatgcag
30300agccagctgt gcaggagacc agagttttat tattactcaa atcagtctcc
ccgaacattc 30360gaggatcaga gcttttaagg ataatttggc cggtaggggc
ttaggaagtg gagagtgctg 30420gttggtcagg ttggagatgg aatcacaggg
agtggaagtg aggttttctt gctgtcttct 30480gttcctggat gggatggcag
aactggttgg gccagattac cggtctgggt ggtctcaaat 30540gatccaccca
gttcagggtc tgcaagatat ctcaagcact gatcttaggt tttacaacag
30600tgatgttatc cccaggaaca atttggggag gttcagactc ttggagccag
aggctgcatt 30660atccctaaac cgtaatctct aatgttgtag ctaatttgtt
agtcctgcaa aggtagactt 30720gtccccaggc aagaaggggg tcttttcaga
aaagggctat tatcattttt gtttcagagt 30780caaaccatga actgaatttc
ttcccaaagt tagttcagcc tacacccagg aatgaagaag 30840gacagcttaa
aggttagaag caagatggag tcaatgaggt ctgatctctt tcactgtcat
30900aatttcctca gttataattt ttgcaaaggc ggtttcagtc ccagctactt
gggaggctga 30960gacaggagga ttaatggagc ccaggagttt gaggttgcag
agagctatga tcacgccact 31020gcactccagc ctgggtgaca gagtgagacc
ctgtctctaa ataaataaat aagtaaataa 31080ataaatacat aaataaaatc
aagatggtgt gcaattagaa ttgagcgatt ttgtttccaa 31140acctcaagaa
agcttggtct tgctctgtcc caggtggctg gataaattgg gcctgtcagc
31200ccgaatggga atagaagtgg tgatgaggca agtattcttt ggagcaggaa
actaccattt 31260agtggatgaa aacttcgatc ctttacctgt aagtgaccat
tattttccta attctagtgg 31320agtagattaa agtcaactca ggacctctgg
tgttaacctc ctatgaacag tcagtcctct 31380cagtaactag ccaaatcatg
agatgatgaa ttagaaggag ccttagatag catccaatct 31440aacatttttt
tgtgtgtttg aagagaagaa atcaagagct aggaataact ttttaaaggt
31500aagccatttg cagtatagtg tggattttgt ttaaaagggg ataatttgaa
attttatgac 31560tcattataca agacaaaata agttggattt tcaaatgttt
tacaaagtaa atcaaagtta 31620taattgccta cagtacgcaa agcttcaaaa
cattttttat gttatgaaat tgtaatttat 31680ttaaccttaa aatgagccag
taccatgtgt ttgcttaaaa atctcatgct aagaatttac 31740tatgttgtta
ataatcttca agatatttat gaataaagtc ttatttctaa tccttcctcc
31800aactgtatct ggtgctaaat caggaaatgt ttcttcccaa aaagcctcgt
ggaagatctg 31860tatgtctaaa tatatgtcag ggataataca gatgtagccc
tgcgaagcat gaccttgatt 31920tttatagtct aaaatgtcat ttgcagatat
ctattttcta agaataattc ctaaaagaat 31980tatttgaatg ttgtaggaaa
gctaagaaat tttgcaaaga gcgtacgtga aaatataagc 32040taggcttttg
tggtttgtgg atagacttcc caacaaaatt gctttttatc tatagtgatc
32100caagcttgtg gaacatatta gtcatctttt tttagaaaat tcttagaaaa
gtgatcttgc 32160aaaaatggaa tttatctttc cccaagtata ttctgtcatg
tatagagtta aactaagcat 32220agtaatttca ccagacaaac attcaaaatc
tactcctgac ctttttatct catccaaatt 32280ttcccagggc ccagacataa
acctttgcct tacgaactct ttgtatatgc actaaatatg 32340cttctccttc
aaggttctca gtcagctaga aaaatgtgca agagtaaatg gtacccttct
32400cacttgtaga tccaagagaa ttagacttaa actcactcta catgtctgtg
actttatttt 32460atttgcatga cagtcctgtg aggtggcaag gcaggtatct
tggatccatt ttttagataa 32520ggaagttcaa attgagaaga ggttgcatga
tttacaggaa gccatactgt agtcctatgt 32580tactcttaaa aatcccattc
aaatcctgct tctgaggcct gcatactttc taccctacca 32640gtcattgacc
catgcttatg tctcctttga aaacattgat tccactcttg tctccagtga
32700aaaagtggaa tttaagcaga gaaacaaaag ccatttgtct tgttaagtct
actttccctc 32760tactttcaag aaggaaagtt ggggtatgtg ttgaatggtg
atttatttat ttatttatta 32820ttttaaaaat tgatacaagg tcttactgta
ttgtgcaggc tggtctcaaa ctcctgggct 32880caagtgatca tcccacctca
gcctcccagt gttgggatta cagcatgaac cattgtgccc 32940accaccgatc
cgcagttttt taagaaaaac ttttactata gaaaatttta atcatataca
33000aaatacagag gaaagtatat gaacccactt taggagacta gaatatgcca
ccccaaaata 33060tgccactttg gcataaggat tatttcgagc taaaggcaac
tgggaagaaa cacatagaag 33120aaaagttctc tgtccttctc catttgccta
aaagcaggac atgaatctta aaagtccccc 33180tccttccctt tctaccagga
aaaacaagag ttaatcactg aagataactt cagaccctta 33240tcagtgtaga
gatggcacta gaagaatcta tattacatac tcatttattt tccttcccac
33300aacttgccac cccagagact aaaaatcctt ttcctttgtc atgtctcttg
tccaaaaatt 33360tgctctataa gctggagttc taagccacct ctttgagaat
tacttgttcc ctggtatttt 33420ctgttaacat acatgtatta atatacatgt
taacaagctt ctgtttgttt ttctcctgtt 33480ttctgtcttg ttacagaggt
ccatcccaac taagaactaa agagtaggag gaaaatataa 33540tttcctcctg
catactttga tcttgtttaa tccgtaaccc ttcccacttt tcacctccta
33600cctattagat tactttgaag caaatttcag atatattact ttatctataa
atatttcagt 33660atgtgctagg tgtggtggct cacacctgta atcccaacac
tttgggaagc tgaggcagga 33720ggatcacttg agcccaggag ttcaagacca
gctacggcaa
caaaaaatca aaaacttatc 33780tgggcatggt ggcacatgcc tgtggtccca
gctacatgag aggctgaggc aggaggatcg 33840ctttagccca ggaggttgag
gctgcagtaa gctgcattca caccactgca ctccagcctg 33900ggtgacagag
taagaccatg tctcaaaaaa atacatattt tagtatgtat cctttttgta
33960aaaacacaat acttttatca tactttaaat aataacaata attccttagt
atcaccaaat 34020attttgtcag tgtctcacat tttccttatt gtctaaaata
ttgttgatag ttattcaaat 34080cagaatccaa acaaggtcca tatattacat
ttggttgaca agtctcttaa gtttgttcat 34140ctttaagttc ttcctccctc
tctttcatct cttgtaattt attaatgtga aaaaacaggt 34200aatttgttct
atagtatttc ctacattata gagtttgcta catttattcc ctatgatatc
34260atttagcatg ttcctctgtc ccctgtgttt cctgtaaact ggtagttata
cctagaagct 34320tgagtttatt caggttttta attgtatttt ttttgcaaga
attctttatt atctgcttct 34380ggaagcacag aatgtctggt tgtgtctggt
tttgatcttg acagctactg atgaccattg 34440cctaatccat tactttattg
gggtgggggg aataaggttt taaaataaat tttttttaaa 34500gattttttta
actgttattt tgagacagtg tctcatttcg tttcccaggc tggagtgcag
34560tggcacaatc acggctcact gcagccttga cctcctggga tcaggtgatc
ttctcacctc 34620agcctcctgg gtacctggaa ctacaggtgc acaccaccac
acctggctaa ttttttgtat 34680tttgtgtaca gaaggggttt catcatgttt
cccagactgg tcttgaactc ctgggttcaa 34740gtgatctacc cacttcagct
tcccaaaatc ctgggattac actttggcca ccgtgcctgg 34800cctaaatgaa
attatttgtc tctaaacaga cagaagtttt actttaaaaa tttgtctttg
34860tgtgtacatg tgtttgtgta tgtgtgtgtg tctaaaagtt tggctttgag
ctttgctttg 34920aattcttgga tgaacaataa ccaagaatac ttaaactctg
atcattcttg acagatatcc 34980cctacaggct atggcctttt gaattgtgtc
ctccagtgat aaaaagcagc aagcacgata 35040ctgctctcag attcatggtg
gtcacatgtg aggtgaaaaa aaaaaaaaag atgaatccta 35100tttaaatgcc
cccaggataa cagtgatact ctttgtagga taactatttg cttgccactg
35160gtttcattaa ataaggacat aagtaaagat ctatttttgt ctctttctcc
ccaaccacca 35220caactaggat tattggctat ctcttctgtt caagaaattg
gtgggcacca aggtgttaat 35280ggcaagcgtg caaggttcaa agagaaggaa
gcttcgagta taccttcatt gcacaaacac 35340tgacaagtaa gtatgaaaca
caccctttac caatcatcaa gttttagtgg gtaagcctgt 35400aactttactc
aaacaccctg ttgcatgtgt ctatacattg cataagtata ggcagttgca
35460atttagtaaa gttttataca acgattttat tttattttat ttttagaaga
aaaatgctac 35520ttttgttgtt gttgtttttt gagacggggc ctcgctcgtc
acccaggctg gagtgcagtg 35580gtgcaatctc agctcactgc aacctccgcc
tcccgggttc aagtgattct tgaagaggag 35640aacaataata acaacaatat
tattttcaaa agttgtgacc gcagtttctg gagttgagaa 35700gacatcgaga
tttttgtagc ctcatactct tgctttaggt agcaaaaaat gttcctaaat
35760ctcaggaata ttctctagat aggtttcaat ctatcattcc tgataagatg
atgctgaaat 35820actaattcta gccaaaaaag accagctacc atttccgatt
gttggggact gggaactctg 35880gatagtgagg accccagtag gaagtagcga
ggggaatggt ttgaatggat aaattcataa 35940aaaatgtcag tagatttaat
tttcttatac atttcagtct ttttataagg ctaggaaaag 36000cccctgtttt
tatggtttat aatttgaatt cacatgaacc cacaaaattt gccttttacc
36060ttcctatgtc tgaaaatgga tagtctggct ggcctcttaa caacccagct
ggcagagctg 36120tgaggatctc agtgtgctct agcccagaca ttggtagcat
gaacggcaac atttttaatt 36180gtgttttcaa aataggagca cactagcggt
ctaaaacgat cataaaagaa ggatactaag 36240agggcccact gtcattatgg
atcctaatac ttaggatgca ttatggattg tcattatgga 36300tactaatact
taggatcaca tttgtaattg agtttttaat tgcttaaatt agatacatat
36360ttctattaag ttaacctctt tgcttttagt ccaaggtata aagaaggaga
tttaactctg 36420tatgccataa acctccataa tgtcaccaag tacttgcggt
taccctatcc tttttctaac 36480aagcaagtgg ataaatacct tctaagacct
ttgggacctc atggattact ttccaagtaa 36540gtaattttcc ttgttcattc
caaactttca ataaatttat tggtgtttat cagaatagag 36600agtttggaca
gggagcaaaa gacaaagtca actatatcaa gttctaataa ttcttaatat
36660tcaggaaatt tatgtatgaa tacttactaa tatgagtata actcatccta
agagtctaaa 36720gcaaaaggat gtgaacacaa actagcagtt atcttagaga
ataagtttgc atttcaaaat 36780aacttgacat atcaagatcc actcaacgca
tttaaattat ttactctaaa aagacataat 36840tcttggtaac acattcacta
aagcaaaata tacctttata taattgctat caaaggtatg 36900tgggttggta
taaaatatca taccatgtga gatcagtgtg attcctttac agcattaatt
36960tttattggtt agagtaagaa aaagaatagc tagagtatat ttcttaagta
gattctcata 37020cactttggtt tcaaaaacca attattgact acatcttata
aaagcctgta ttcaatggag 37080tgccaaaaaa tgactatgag tcttaaagag
ttaggcatat aaatatttta aggtttctgt 37140tcaatgtatg ttggaaggag
ttcctttctc atgactattc tcatattgga gcataaaaag 37200agtttacagg
cttggcgcag tggctcatgc ctgtaatccc aatactttgg gaagctgaag
37260caggcagatc acttcagccc aggagtttga gaccagcctg ggcaatatgg
caaaactctc 37320tctacaaaat ataccaaaat tagccaggcg tggtggtgca
tgcctgtagt cccagctact 37380tgggaagctg aggtgggagg attgcttgag
cccagggggg tcatggctgc agtgagctgt 37440gatggtgcct ctgtcaccca
gcctgggtga cagagtgaga ccctgtctca aaaaaataaa 37500taaataaaaa
ttaagagttt acaaaattct caccatctcc tcccatcttt gcaaatgcca
37560cataagtgat gtgttccagg actattagcc tcggaacctg aggcagtaca
gtaagcacgc 37620tttctccaaa gtcctgtccc ccacagacaa acattattta
cactgggtac tgctctttta 37680ttttttcccc tctatgcttt attttactat
aactataatc atataacatg taataggaaa 37740aaggcagggt cgggggagag
atccagaagt cttcccaaga gcctttccaa catagcctct 37800gtagacattt
tttctttctt cttttttttt tttttttttt ttctgagaca gagtctcact
37860ctgttgtcca ggctagagtg cagtggcgtg atctaggctc actgcaacct
ccgcctcctg 37920ggttcaagca attctcccac ctcagcctcc ctagtagctg
ggattagagg catgcatcac 37980cacgcctggc taatttttgt atttttagta
gagatgaggt ttcaccatgt gggccaggct 38040ggtcttgaac tcctgacctc
aagtgatcca cctgccttag cctcccaaag tgctaggatt 38100acacgagtga
gccaccgtgc cctgccccta ttacattctg atcacacatt tcatgtttta
38160taattggaaa actggtgaaa ttatagacaa tgttttgttc ccctaaattc
tctttgatga 38220gtatatatta cttacactct tctgtcttta aaattttgca
aaatagtatc ctagataagt 38280ttatgagtgc acagtctgta cgcttactca
tattaatgac ctcggagagt taaacaacag 38340tcacctttaa aaattattac
tatcattatc attatttttg aggcgggggt ctcattctgt 38400ctcccaggct
ggagagtagt ggtgcggtca cagctcactg cagccaccgc tacctgggct
38460caagtgatcc ttcctcctca gccttctgag tagctgagac cacaggctta
tgctaccaca 38520cctggctaat tttttaactt tttgtagaga cgatgtctca
ttatgttgcc caggctggtc 38580tcaaactcct aagctcaagt gatcttcctc
agcctcccaa agtgctggga ttacaggcat 38640gaaaaactgc acccagccct
aaaaattatt agggtcctgc atagtaagac tttaataaat 38700atttaaatga
acatctggtt tttttaaaaa aaaaatagag acaaggtctc actatattgc
38760ccaagctggt ctcgaactcc tggactcacg caatcctgct gccttagccg
cccaaagtgc 38820tgggattaca ggcatgaccc acctcatctg ggctgagtga
acatattttt aacataaagg 38880ccgtatttta tatttatctc atacattttg
cccagcatcc ccatttccgc cgaatctgtt 38940gcttgctaat tccttccagc
ttcatttcat ctgaaatttg acaaacatct tctatttctt 39000tgtcgtcatg
ttattgactt cagaatataa aataaaacac tatacccaaa ttaaacccca
39060ccctcattgc ccagcctgat gtgaaaataa tcagcataca ttaagcttac
ccttgatata 39120tgtgtagcat cttttagata aatatacagc tgattaagca
atatagcctg atggtataat 39180atcttgccca tgtacctcat cttatctcca
gcaggattaa ttcacagtga tcagatttac 39240ctttaaactt tgtagcaaaa
tatcctctcc aaaagcatat ctaaaacttt tgtgtgtact 39300cttgcaagtt
tcttaatttc atgcagaaca ggctcttacc actgttagct ggagatattt
39360tcaagaccta tttttgtttg tggtttcctg atgatggtca tggcatttcc
cccttcactc 39420catctaaaaa ttgaggtgat acaggctttt aaacaaaacc
aactcatata gactgagtac 39480aactgcaatg caggcatgct aacctctgct
acaatcatgg gcgtgctatt gatatgtctt 39540aagttacaga acacagggct
gagcgtctca ttaggtcaaa atgtaaacca gtttttctgc 39600tcactgatgc
ttaatgagga cagggtgtga gagatttctt taaggaaaac aaatatataa
39660taatgctaca tggaaaaata tctaacatta gagaattaag taaataaact
aatatactca 39720caccatggaa tcttgtgcag acattaaaat tatgtagtgg
atggatgttt aatggtgtga 39780gaaaaagtta ggatgtgctg gggtgggggg
aagaatcaag ttttaagaaa atacagtata 39840cccatactta agtaaaaaaa
aaaaaaaagg tatgtacagt catgtgttgc ttaatgatgg 39900ggatacattc
cgagaaatgt gtcgataggt gatttcatcc ttgtgtgaac atcatagagt
39960gaacttacac aaacctagat ggtctagcct actatgtatc taggctatat
gactagcctg 40020ttgctcctag gctacaaacc tgtaaagcat gttactgtag
cgaatataca aatacttaac 40080acaatggcaa gctatcattg tgttaagtag
ttgtgtatct aaacatatct aaaacataga 40140aaactaatgt gttgtgctac
aatgttacaa tgactatgac attgctaggc aataggaatt 40200ataattttat
ccttttatgg aaccacactt atatatgcgg tccatggtgg accaaaacat
40260ccttatgtgg catatgactg tatacatgta cacaaaaaat agatgaaaga
atgaatatac 40320atcaaaatat ttaaaatggt tataatgact taggttactt
ttatttatct tagtaataat 40380aatgatgata gataatactt ttatagtgtt
tactatataa aagacactgt tataagtgtt 40440ctacatactt tacatgtatt
acctaaatga tataaatata actctgacag taactaatct 40500tatacgttct
cttttctttt tttttttttt ctttttttag acagaatctt gctctaccag
40560gctggagtgc agggtgcaat ctcggctcac tgcaacctcc gcctcccagg
ttcaaacgat 40620tctcatgtct cagcctcctg agtagctggg actacaggca
cacaccacca tgcccggcta 40680atttttgtat ttttgggtag agatggagtt
ttgccatgtt ggccaggctg atcttgaact 40740cctggcctca agtgatctgc
ctgcctcagc ctcccaaagt gctgggatta caggtgtgaa 40800ccactgtgct
cggcctaatc ttacaagttt tcaatattta aagagtgcta actttgttga
40860caatataaaa catatttgag aaaaagagat ataagcatct tatttagaat
tatgaaaata 40920tcaatagacc tacagccgac taaagctttt cttcataagc
tcttgcctat attgattcgc 40980tcctgtgaat atgcattaat ttgatttaaa
taataagtat gtataagaaa taacactttt 41040ccttaatttt taagaacgtt
caacagtttt taatttgaat tccaatagtg aaatacatag 41100aaaatataaa
attttctgta gtttagccaa attgtttttg tttcaccaca gcattctacc
41160aaaatttctt aataacagta agaaaatgaa tgcatacctc ctgcagggag
aggggagtta 41220ggcagtttat gggcatagtt acaagtgaga aatttcattg
gctaccattt acgctaaatt 41280cataaaaact gcattcaatt ctatatatct
attttcttta cataaaaaag gtttcaatta 41340ttggccatta aataaaatag
ccaccattcc agaagttgtg tcatgtttat cctttttata 41400ccaccatcat
attgcctatt atatagattg tgtgtgttcc attttctgta atgggccaga
41460cagtaagtat ttctggcttt ggagtccata tggtctctat cataactact
catctctgcc 41520attgtagctt aaagattatc taggtcaaat gcctaagtga
tatagtgttg aaatacaagt 41580tatataatat aggctgccac aaaaaaaaat
ttatttggtc taaaaaagat ttcatgactt 41640ttgtagcagc atgggtgggg
catgcaccac ttggttaact cggtgtatct ttctcctttg 41700cagatctgtc
caactcaatg gtctaactct aaagatggtg gatgatcaaa ccttgccacc
41760tttaatggaa aaacctctcc ggccaggaag ttcactgggc ttgccagctt
tctcatatag 41820tttttttgtg ataagaaatg ccaaagttgc tgcttgcatc
tgaaaataaa atatactagt 41880cctgacactg aatttttcaa gtatactaag
agtaaagcaa ctcaagttat aggaaaggaa 41940gcagatacct tgcaaagcaa
ctagtgggtg cttgagagac actgggacac tgtcagtgct 42000agatttagca
cagtattttg atctcgctag gtagaacact gctaataata atagctaata
42060ataccttgtt ccaaatactg cttagcattt tgcatgtttt acttttatct
aaagttttgt 42120tttgttttat tatttattta tttatttatt ttgagacaga
atctctctct gtcacccagg 42180ctggagtgcc atggtgcgat cttggctcac
tgcaacttta agcaattctc ctgcctcagc 42240ttcctgagta gctgggatta
taggcgtgtg ccaccacgcc cagctacttt ctatattttt 42300tgtagagatg
gagtttcgcc atattggcca agctggtctc gaactcctgt cctcgaactc
42360ctgtcctcaa gtgatccacc cgcctcagcc tctcaaagtg ctgggattac
aggtgtgagc 42420caccacaccc agcagtgttt tatttttgag acagggtatc
attctgttgc ccaggcttga 42480gtgcagtggt gcaatcatag atcactgcag
ccttttaact cctgggctca agtcatcctc 42540ctgcttagcc tcccaagtag
ctaggaccac agacacatgc catcacactt ggctattttt 42600aaaaaatttt
ttgtagagat ggggtctcgc tatgttaccc aaactggtcc tgaactcctg
42660gactcaattg atcctcccac cttggccttc caggtgctgg gatttctttg
ggagtacagc 42720atggtacagc aggagatcat ttgatgttac ctctgtgcag
tgttgctagt cagcgaaaga 42780ctataatacc tgtggggaca gcgattagcc
accacaacca gtctttattt aaagttatta 42840aaaatggctg ggcgcagtgg
ctcacacctg taatcctagc actttgggag gccgaggcag 42900atggatcacc
tgacgtgagg aatttgagac cagcctggcc aacatggtga aaccccatct
42960ctactaaaaa atacaaaaat tagctgggtg tggtcctgta gtcccagcta
cttgggaggc 43020tggggcagga gaattacttg aacccaggag gcagaggttg
cagtgagccg agattgtgcc 43080actgcactcc agcctgggtg acagagagag
attccatctc aaaaaaacaa gttattaaaa 43140atgtatatga atgctcctaa
tatggtcagg aagcaaggaa gcgaaggata tattatgagt 43200tttaagaagg
tgcttagctg tatatttatc tttcaaaatg tattagaaga ttttagaatt
43260ctttccttca tgtgccatct ctacaggcac ccatcagaaa aagcatactg
ccgttaccgt 43320gaaactggtt gtaaaagaga aactatctat ttgcacctta
aaagacagct agattttgct 43380gattttcttc tttcggtttt ctttgtcagc
aataatatgt gagaggacag attgttagat 43440atgatagtat aaaaaatggt
taatgacaat tcagaggcga ggagattctg taaacttaaa 43500attactataa
atgaaattga tttgtcaaga ggataaattt tagaaaacac ccaatacctt
43560ataactgtct gttaatgctt gctttttctc tacctttctt ccttgtttca
gttgggaagc 43620ttttggctgc aagtaacaga aactcctaat tcaaatggct
taagcaataa ggaaatgtat 43680attcccacat aactagacgt tcaaacaggc
caggctccag cacttcagta cgtcaccagg 43740gatctgggtt cttcccagct
ctctgctctg ccatctttag cgctggcttc attctcagac 43800tctggtagca
tgatggctgt agctgtttca tgggcccctt caaacctcat agcaaccaga
43860ggaagaaaat gagccatttt ttgagtctcc ttcatagact tgaataactc
tttttcagag 43920cttctcacag caaacctctc ctcatgtctc ctcatgtctt
attgttcaga aatgggtaat 43980gtggccattt caccagtcac tgccaacaac
aacgaggttc ctataattgt ctctgagtaa 44040ccctttggaa tggagagggt
gttggtcagt ctacaaactg aacactgcag ttctgcgctt 44100tttaccagtg
aaaaaatgta attattttcc cctcttaagg attaatattc ttcaaatgta
44160tgcctgttat ggatatagta tctttaaaat tttttatttt aatagcttta
ggggtacaca 44220ctttttgctt acaggggtga attgtgtagt ggtgaagact
cggcttttaa tgtacttgtc 44280acctgagtga tgtacattgt acccaatagg
taatttttca tccattaccc tccttccgcc 44340ctcttccctt ctgagtctcc
aacatccctt ataccactgt gtatgttctt gtgtacctac 44400agctaagctt
ccacttataa gtgagaacat gcagtatttg gttttccatt cctgagttac
44460ttcccttagg ataacagccc ccagttccgt ccaagttgct gcaaaataca
ttattcttct 44520ttatggctga gtaatagtcc atggtacata tataccacat
tttctttatc cacttatcag 44580ttgatggaca cttaggttaa ttccattcaa
tttcattcaa tttaagtata tttgtaagga 44640gctaaagctg aaaattaaat
tttagatctt tcaatactct taaattttat atgtaagtgg 44700tttttatatt
ttcacatttg aaataaagta atttttataa ccttgatatt gtatgactat
44760tcttttagta atgtaaagcc tacagactcc tacatttgga accactagtg
tgttgtttca 44820ccccttgtta tactatcagg atcctcga 44848432396DNAHomo
sapiens 43tttctagttg cttttagcca atgtcggatc aggtttttca agcgacaaag
agatactgag 60atcctgggca gaggacatcc tagctcggtc agatttgggc aggctcaagt
gaccagtgtc 120ttaaggcaga agggagtcgg ggtagggtct ggctgaaccc
tcaaccgggg cttttaactc 180agggtctagt cctggcgcca aatggatggg
acctagaaaa ggtgacagag tgcgcaggac 240accaggaagc tggtcccacc
cctgcgcggc tcccgggcgc tccctcccca ggcctccgag 300gatcttggat
tctggccacc tccgcaccct ttggatgggt gtggatgatt tcaaaagtgg
360acgtgaccgc ggcggagggg aaagccagca cggaaatgaa agagagcgag
gaggggaggg 420cggggagggg agggcgctag ggagggactc ccgggagggg
tgggagggat ggagcgctgt 480gggagggtac tgagtcctgg cgccagaggc
gaagcaggac cggttgcagg gggcttgagc 540cagcgcgccg gctgccccag
ctctcccggc agcgggcggt ccagccaggt gggatgctga 600ggctgctgct
gctgtggctc tgggggccgc tcggtgccct ggcccagggc gcccccgcgg
660ggaccgcgcc gaccgacgac gtggtagact tggagtttta caccaagcgg
ccgctccgaa 720gcgtgagtcc ctcgttcctg tccatcacca tcgacgccag
cctggccacc gacccgcgct 780tcctcacctt cctgggctct ccaaggctcc
gtgctctggc tagaggctta tctcctgcat 840acttgagatt tggcggcaca
aagactgact tccttatttt tgatccggac aaggaaccga 900cttccgaaga
aagaagttac tggaaatctc aagtcaacca tgatatttgc aggtctgagc
960cggtctctgc tgcggtgttg aggaaactcc aggtggaatg gcccttccag
gagctgttgc 1020tgctccgaga gcagtaccaa aaggagttca agaacagcac
ctactcaaga agctcagtgg 1080acatgctcta cagttttgcc aagtgctcgg
ggttagacct gatctttggt ctaaatgcgt 1140tactacgaac cccagactta
cggtggaaca gctccaacgc ccagcttctc cttgactact 1200gctcttccaa
gggttataac atctcctggg aactgggcaa tgagcccaac agtttctgga
1260agaaagctca cattctcatc gatgggttgc agttaggaga agactttgtg
gagttgcata 1320aacttctaca aaggtcagct ttccaaaatg caaaactcta
tggtcctgac atcggtcagc 1380ctcgagggaa gacagttaaa ctgctgagga
gtttcctgaa ggctggcgga gaagtgatcg 1440actctcttac atggcatcac
tattacttga atggacgcat cgctaccaaa gaagattttc 1500tgagctctga
tgcgctggac acttttattc tctctgtgca aaaaattctg aaggtcacta
1560aagagatcac acctggcaag aaggtctggt tgggagagac gagctcagct
tacggtggcg 1620gtgcaccctt gctgtccaac acctttgcag ctggctttat
gtggctggat aaattgggcc 1680tgtcagccca gatgggcata gaagtcgtga
tgaggcaggt gttcttcgga gcaggcaact 1740accacttagt ggatgaaaac
tttgagcctt tacctgatta ctggctctct cttctgttca 1800agaaactggt
aggtcccagg gtgttactgt caagagtgaa aggcccagac aggagcaaac
1860tccgagtgta tctccactgc actaacgtct atcacccacg atatcaggaa
ggagatctaa 1920ctctgtatgt cctgaacctc cataatgtca ccaagcactt
gaaggtaccg cctccgttgt 1980tcaggaaacc agtggatacg taccttctga
agccttcggg gccggatgga ttactttcca 2040aatctgtcca actgaacggt
caaattctga agatggtgga tgagcagacc ctgccagctt 2100tgacagaaaa
acctctcccc gcaggaagtg cactaagcct gcctgccttt tcctatggtt
2160tttttgtcat aagaaatgcc aaaatcgctg cttgtatatg aaaataaaag
gcatacggta 2220cccctgagac aaaagccgag gggggtgtta ttcataaaac
aaaaccctag tttaggaggc 2280cacctccttg ccgagttcca gagcttcggg
agggtggggt acacttcagt attacattca 2340gtgtggtgtt ctctctaaga
agaatactgc aggtggtgac agttaatagc actgtg 239644535PRTHomo sapiens
44Met Leu Arg Leu Leu Leu Leu Trp Leu Trp Gly Pro Leu Gly Ala Leu1
5 10 15Ala Gln Gly Ala Pro Ala Gly Thr Ala Pro Thr Asp Asp Val Val
Asp20 25 30Leu Glu Phe Tyr Thr Lys Arg Pro Leu Arg Ser Val Ser Pro
Ser Phe35 40 45Leu Ser Ile Thr Ile Asp Ala Ser Leu Ala Thr Asp Pro
Arg Phe Leu50 55 60Thr Phe Leu Gly Ser Pro Arg Leu Arg Ala Leu Ala
Arg Gly Leu Ser65 70 75 80Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys
Thr Asp Phe Leu Ile Phe85 90 95Asp Pro Asp Lys Glu Pro Thr Ser Glu
Glu Arg Ser Tyr Trp Lys Ser100 105 110Gln Val Asn His Asp Ile Cys
Arg Ser Glu Pro Val Ser Ala Ala Val115 120 125Leu Arg Lys Leu Gln
Val Glu Trp Pro Phe Gln Glu Leu Leu Leu Leu130 135 140Arg Glu Gln
Tyr Gln Lys Glu Phe Lys Asn Ser Thr Tyr Ser Arg Ser145 150 155
160Ser Val Asp Met Leu Tyr Ser Phe Ala Lys Cys Ser Gly Leu Asp
Leu165 170 175Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Pro Asp Leu
Arg Trp Asn180 185 190Ser Ser Asn Ala Gln Leu Leu Leu Asp Tyr Cys
Ser Ser Lys Gly Tyr195 200 205Asn Ile Ser Trp Glu Leu Gly Asn Glu
Pro Asn Ser Phe Trp Lys Lys210 215 220Ala His Ile Leu Ile Asp Gly
Leu Gln Leu Gly Glu Asp Phe Val Glu225 230 235 240Leu His Lys Leu
Leu Gln Arg Ser Ala Phe Gln Asn Ala Lys Leu Tyr245 250 255Gly Pro
Asp Ile Gly Gln Pro Arg Gly Lys Thr Val Lys Leu
Leu Arg260 265 270Ser Phe Leu Lys Ala Gly Gly Glu Val Ile Asp Ser
Leu Thr Trp His275 280 285His Tyr Tyr Leu Asn Gly Arg Ile Ala Thr
Lys Glu Asp Phe Leu Ser290 295 300Ser Asp Ala Leu Asp Thr Phe Ile
Leu Ser Val Gln Lys Ile Leu Lys305 310 315 320Val Thr Lys Glu Ile
Thr Pro Gly Lys Lys Val Trp Leu Gly Glu Thr325 330 335Ser Ser Ala
Tyr Gly Gly Gly Ala Pro Leu Leu Ser Asn Thr Phe Ala340 345 350Ala
Gly Phe Met Trp Leu Asp Lys Leu Gly Leu Ser Ala Gln Met Gly355 360
365Ile Glu Val Val Met Arg Gln Val Phe Phe Gly Ala Gly Asn Tyr
His370 375 380Leu Val Asp Glu Asn Phe Glu Pro Leu Pro Asp Tyr Trp
Leu Ser Leu385 390 395 400Leu Phe Lys Lys Leu Val Gly Pro Arg Val
Leu Leu Ser Arg Val Lys405 410 415Gly Pro Asp Arg Ser Lys Leu Arg
Val Tyr Leu His Cys Thr Asn Val420 425 430Tyr His Pro Arg Tyr Gln
Glu Gly Asp Leu Thr Leu Tyr Val Leu Asn435 440 445Leu His Asn Val
Thr Lys His Leu Lys Val Pro Pro Pro Leu Phe Arg450 455 460Lys Pro
Val Asp Thr Tyr Leu Leu Lys Pro Ser Gly Pro Asp Gly Leu465 470 475
480Leu Ser Lys Ser Val Gln Leu Asn Gly Gln Ile Leu Lys Met Val
Asp485 490 495Glu Gln Thr Leu Pro Ala Leu Thr Glu Lys Pro Leu Pro
Ala Gly Ser500 505 510Ala Leu Ser Leu Pro Ala Phe Ser Tyr Gly Phe
Phe Val Ile Arg Asn515 520 525Ala Lys Ile Ala Ala Cys Ile530
535452396DNAHomo sapiensCDS(594)..(2198) 45tttctagttg cttttagcca
atgtcggatc aggtttttca agcgacaaag agatactgag 60atcctgggca gaggacatcc
tagctcggtc agatttgggc aggctcaagt gaccagtgtc 120ttaaggcaga
agggagtcgg ggtagggtct ggctgaaccc tcaaccgggg cttttaactc
180agggtctagt cctggcgcca aatggatggg acctagaaaa ggtgacagag
tgcgcaggac 240accaggaagc tggtcccacc cctgcgcggc tcccgggcgc
tccctcccca ggcctccgag 300gatcttggat tctggccacc tccgcaccct
ttggatgggt gtggatgatt tcaaaagtgg 360acgtgaccgc ggcggagggg
aaagccagca cggaaatgaa agagagcgag gaggggaggg 420cggggagggg
agggcgctag ggagggactc ccgggagggg tgggagggat ggagcgctgt
480gggagggtac tgagtcctgg cgccagaggc gaagcaggac cggttgcagg
gggcttgagc 540cagcgcgccg gctgccccag ctctcccggc agcgggcggt
ccagccaggt ggg atg 596Met1ctg agg ctg ctg ctg ctg tgg ctc tgg ggg
ccg ctc ggt gcc ctg gcc 644Leu Arg Leu Leu Leu Leu Trp Leu Trp Gly
Pro Leu Gly Ala Leu Ala5 10 15cag ggc gcc ccc gcg ggg acc gcg ccg
acc gac gac gtg gta gac ttg 692Gln Gly Ala Pro Ala Gly Thr Ala Pro
Thr Asp Asp Val Val Asp Leu20 25 30gag ttt tac acc aag cgg ccg ctc
cga agc gtg agt ccc tcg ttc ctg 740Glu Phe Tyr Thr Lys Arg Pro Leu
Arg Ser Val Ser Pro Ser Phe Leu35 40 45tcc atc acc atc gac gcc agc
ctg gcc acc gac ccg cgc ttc ctc acc 788Ser Ile Thr Ile Asp Ala Ser
Leu Ala Thr Asp Pro Arg Phe Leu Thr50 55 60 65ttc ctg ggc tct cca
agg ctc cgt gct ctg gct aga ggc tta tct cct 836Phe Leu Gly Ser Pro
Arg Leu Arg Ala Leu Ala Arg Gly Leu Ser Pro70 75 80gca tac ttg aga
ttt ggc ggc aca aag act gac ttc ctt att ttt gat 884Ala Tyr Leu Arg
Phe Gly Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp85 90 95ccg gac aag
gaa ccg act tcc gaa gaa aga agt tac tgg aaa tct caa 932Pro Asp Lys
Glu Pro Thr Ser Glu Glu Arg Ser Tyr Trp Lys Ser Gln100 105 110gtc
aac cat gat att tgc agg tct gag ccg gtc tct gct gcg gtg ttg 980Val
Asn His Asp Ile Cys Arg Ser Glu Pro Val Ser Ala Ala Val Leu115 120
125agg aaa ctc cag gtg gaa tgg ccc ttc cag gag ctg ttg ctg ctc cga
1028Arg Lys Leu Gln Val Glu Trp Pro Phe Gln Glu Leu Leu Leu Leu
Arg130 135 140 145gag cag tac caa aag gag ttc aag aac agc acc tac
tca aga agc tca 1076Glu Gln Tyr Gln Lys Glu Phe Lys Asn Ser Thr Tyr
Ser Arg Ser Ser150 155 160gtg gac atg ctc tac agt ttt gcc aag tgc
tcg ggg tta gac ctg atc 1124Val Asp Met Leu Tyr Ser Phe Ala Lys Cys
Ser Gly Leu Asp Leu Ile165 170 175ttt ggt cta aat gcg tta cta cga
acc cca gac tta cgg tgg aac agc 1172Phe Gly Leu Asn Ala Leu Leu Arg
Thr Pro Asp Leu Arg Trp Asn Ser180 185 190tcc aac gcc cag ctt ctc
ctt gac tac tgc tct tcc aag ggt tat aac 1220Ser Asn Ala Gln Leu Leu
Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn195 200 205atc tcc tgg gaa
ctg ggc aat gag ccc aac agt ttc tgg aag aaa gct 1268Ile Ser Trp Glu
Leu Gly Asn Glu Pro Asn Ser Phe Trp Lys Lys Ala210 215 220 225cac
att ctc atc gat ggg ttg cag tta gga gaa gac ttt gtg gag ttg 1316His
Ile Leu Ile Asp Gly Leu Gln Leu Gly Glu Asp Phe Val Glu Leu230 235
240cat aaa ctt cta caa agg tca gct ttc caa aat gca aaa ctc tat ggt
1364His Lys Leu Leu Gln Arg Ser Ala Phe Gln Asn Ala Lys Leu Tyr
Gly245 250 255cct gac atc ggt cag cct cga ggg aag aca gtt aaa ctg
ctg agg agt 1412Pro Asp Ile Gly Gln Pro Arg Gly Lys Thr Val Lys Leu
Leu Arg Ser260 265 270ttc ctg aag gct ggc gga gaa gtg atc gac tct
ctt aca tgg cat cac 1460Phe Leu Lys Ala Gly Gly Glu Val Ile Asp Ser
Leu Thr Trp His His275 280 285tat tac ttg aat gga cgc atc gct acc
aaa gaa gat ttt ctg agc tct 1508Tyr Tyr Leu Asn Gly Arg Ile Ala Thr
Lys Glu Asp Phe Leu Ser Ser290 295 300 305gat gcg ctg gac act ttt
att ctc tct gtg caa aaa att ctg aag gtc 1556Asp Ala Leu Asp Thr Phe
Ile Leu Ser Val Gln Lys Ile Leu Lys Val310 315 320act aaa gag atc
aca cct ggc aag aag gtc tgg ttg gga gag acg agc 1604Thr Lys Glu Ile
Thr Pro Gly Lys Lys Val Trp Leu Gly Glu Thr Ser325 330 335tca gct
tac ggt ggc ggt gca ccc ttg ctg tcc aac acc ttt gca gct 1652Ser Ala
Tyr Gly Gly Gly Ala Pro Leu Leu Ser Asn Thr Phe Ala Ala340 345
350ggc ttt atg tgg ctg gat aaa ttg ggc ctg tca gcc cag atg ggc ata
1700Gly Phe Met Trp Leu Asp Lys Leu Gly Leu Ser Ala Gln Met Gly
Ile355 360 365gaa gtc gtg atg agg cag gtg ttc ttc gga gca ggc aac
tac cac tta 1748Glu Val Val Met Arg Gln Val Phe Phe Gly Ala Gly Asn
Tyr His Leu370 375 380 385gtg gat gaa aac ttt gag cct tta cct gat
tac tgg ctc tct ctt ctg 1796Val Asp Glu Asn Phe Glu Pro Leu Pro Asp
Tyr Trp Leu Ser Leu Leu390 395 400ttc aag aaa ctg gta ggt ccc agg
gtg tta ctg tca aga gtg aaa ggc 1844Phe Lys Lys Leu Val Gly Pro Arg
Val Leu Leu Ser Arg Val Lys Gly405 410 415cca gac agg agc aaa ctc
cga gtg tat ctc cac tgc act aac gtc tat 1892Pro Asp Arg Ser Lys Leu
Arg Val Tyr Leu His Cys Thr Asn Val Tyr420 425 430cac cca cga tat
cag gaa gga gat cta act ctg tat gtc ctg aac ctc 1940His Pro Arg Tyr
Gln Glu Gly Asp Leu Thr Leu Tyr Val Leu Asn Leu435 440 445cat aat
gtc acc aag cac ttg aag gta ccg cct ccg ttg ttc agg aaa 1988His Asn
Val Thr Lys His Leu Lys Val Pro Pro Pro Leu Phe Arg Lys450 455 460
465cca gtg gat acg tac ctt ctg aag cct tcg ggg ccg gat gga tta ctt
2036Pro Val Asp Thr Tyr Leu Leu Lys Pro Ser Gly Pro Asp Gly Leu
Leu470 475 480tcc aaa tct gtc caa ctg aac ggt caa att ctg aag atg
gtg gat gag 2084Ser Lys Ser Val Gln Leu Asn Gly Gln Ile Leu Lys Met
Val Asp Glu485 490 495cag acc ctg cca gct ttg aca gaa aaa cct ctc
ccc gca gga agt gca 2132Gln Thr Leu Pro Ala Leu Thr Glu Lys Pro Leu
Pro Ala Gly Ser Ala500 505 510cta agc ctg cct gcc ttt tcc tat ggt
ttt ttt gtc ata aga aat gcc 2180Leu Ser Leu Pro Ala Phe Ser Tyr Gly
Phe Phe Val Ile Arg Asn Ala515 520 525aaa atc gct gct tgt ata
tgaaaataaa aggcatacgg tacccctgag 2228Lys Ile Ala Ala Cys Ile530
535acaaaagccg aggggggtgt tattcataaa acaaaaccct agtttaggag
gccacctcct 2288tgccgagttc cagagcttcg ggagggtggg gtacacttca
gtattacatt cagtgtggtg 2348ttctctctaa gaagaatact gcaggtggtg
acagttaata gcactgtg 239646385DNARattus norvegicus 46cggccgctgc
tgctgctgtg gctctggggg cggctccgtg ccctgaccca aggcactccg 60gcggggaccg
cgccgaccaa agacgtggtg gacttggagt tttacaccaa gaggctattc
120caaagcgtga gtccctcgtt cctgtccatc accatcgacg ccagtctggc
caccgaccct 180cggttcctca ccttcctgag ctctccacgg cttcgagccc
tgtctagagg cttatctcct 240gcgtacttga gatttggcgg caccaagact
gacttcctta tttttgatcc caacaacgaa 300cccacctctg aagaaagaag
ttactggcaa tctcaagaca acaatgatat ttgcgggtct 360gaccgggtct
ccgctgacgt gttga 38547541DNARattus
norvegicusmisc_feature(507)..(507)Any nucleotide 47aaatcaggac
atatccttca cttatttgcc tcttggtcat attggaggca tttgtattca 60tttttaataa
ccctcaaaat agtgcatgca aagtgctaag cgtcatttgc cacatggtgc
120cattaactgt caccacctgc agtggtctac ttagagaaca ccgcactgga
tgttaacact 180gaagcgcgtg ccccgccctc ccgaggctct ggatccagcg
ttgaagcttg ccccgccctc 240ccgaggctct ggatccagca ctggagcatg
ccccgccctc ccgaggctct ggagcttgct 300aaggagtccg ctccctaccg
ctggggtttt gctttattct tatgaatgac acccctgacc 360gctttcgtct
caggggtact gtaatgcctt ttattttcat atacaagctg cgattttggc
420atttcttatg acaaaaaacc cataggaaaa ggcgggcacg cttagtgagc
ttcctgcggg 480gagaggtttt tctgttagag ctggcanggt ctgctcatcg
accatcttca ggcctcgtgc 540c 541
* * * * *