Note: Descriptions are shown in the official language in which they were submitted.
CA 02252965 1998-12-O1
NOVEL SUPPRESSOR OF FUSED (Sufis) GENE
Field of the Invention
The present invention relates to a novel mammalian suppressor of fused (Sufis)
gene and
the regulatory protein it encodes.
Background of the Invention
Hedgehog (Hh) signaling is involved in many developmental processes and
disease
conditions, including cancer. Current knowledge of Hh signaling has been
derived from genetic
studies in Drosophila (Ingham, EMBO J (1998)17:3505-3511; Ming et al.,
Molecular Medicine
Today (1998) 4:343-349) in which two transmembrane proteins, Patched (Ptc) and
Smoothened
(Smo), have been identified as opposing partners in the Hh receptor (Chen &
Struhl, Cell (1996)
87:553-563). The current model suggests that Ptc functions as a negative
regulator of Hh
signaling by inhibiting the activity of Smo. When Hh binds to Ptc, repression
of Smo is released.
Derepressed Smo then signals through a Gli zinc forger protein, Cubitus
interruptus (Ci), to
activate the transcription of target genes. Ci is believed to form a mufti-
protein complex
including a serine-threonine protein kinase, Fused (Fu) (Therond et al.,
Genetics (1996) 142:
1181-1198); a kinesin-like molecule, Costal 2 (Cos2)(Sisson et al., Cell
(1997) 90:235-245); and
a novel PEST-domain containing protein, Suppressor of fused (Su(fu))(Pham et
al., Genetics
(1995) 140:587-598; Monnier et al., Current Biology (1998) 8:583-586). While
the role of
Su(fu) is still unclear, Fu and Cos2 function, respectively, as positive and
negative regulators of
Hh signaling.
In Drosophila, Su(fu) was identified using a genetic screen for suppressors of
fu
phenotypes (Preat, Genetics (1992) 132:725-736). It encodes a novel PEST-
domain containing
protein and is expressed both maternally and zygotically (Pham et a1.,1995).
Although loss-of
function Su(fu) mutations are viable and display no visible phenotype, they
can fully suppress all
the known phenotypes associated with loss of Fu function, including the
segmentation defects,
wing defects, ovarian tumors and pupal lethality (Preat, 1992). Su(fu)
antagonizes Fu in a dose-
dependent manner (Preat, 1992). Loss of one dose of Su(fu) can partially
suppress the Fu
phenotype and removal of both copies of Su(fu) fully rescues the Fu phenotype.
Conversely, the
Fu phenotype is enhanced by a gene duplication of Su(fu). While Su(fu)
mutations can suppress
CA 02252965 1998-12-O1
the phenotype of null alleles (Fu0) and Fu[I] mutants (mutations in the kinase
domain), double
mutants of Su(fu) and Fu[II] (mutations in the regulatory domain) display a
novel phenotype
which is similar to Cos2 mutants (Preat et al., Genetics (1993) 135:1047-1062;
Therond et al.,
1996). It has also been shown that both Fu[II] and Su(fu) mutations can
enhance the phenotype
of Cos2 mutants (Preat et al., 1993). Taken together, these observations
suggest that there is a
tight relationship between Su(fu), Fu and Cos2 throughout Drosophila
development and in Hh
signaling.
It has recently been demonstrated that Su(fu) can bind directly to the
regulatory domain
of Fu and the N-terminal region of Ci (Monnier et al., 1998). In yeasts, Fu
cannot bind Ci but
Su(fu) can link Fu to Ci. Immunoprecipitation experiments further show that
Su(fu) and Ci
interact in Drosophila embryos. These biochemical data indicate that Su(fu) is
part of the multi-
protein complex involved in the transduction of Hh signal and can serve as a
linker protein
bringing Fu and Ci together. It has been suggested that Fu activation triggers
the degradation of
Su(fu) through the phosphorylation of its PEST sequence (Monnier et al.,
1998). Therefore, the
level of Su(fu) might affect the phosphorylation of Ci by Fu. The proteolysis
of Ci is known to
be inhibited in Cos2-deficient Drosophila embryos (Sisson et al., 1997). Since
Su(fu) behaves
like Cos2 in the absence of Fu (Preat et al., 1993), it may also play an
indirect role in the control
of Ci proteolysis. These observations suggest that homologs of Su(fu) as well
as Fu and Cos2
are potentially important regulatory components in mammalian Hh signaling.
Mammalian Hh signaling is more complex and involves at least two Ptc genes
(Motoyama et al., Nature Genetics (1998) 18:104-106) and three Gli genes (Hui
et al.,
Developmental Biology (1994) 162:402-413). Through mutational analysis in
mice, the role of
mammalian Gli proteins has been investigated. These analyses indicated that
Gli2 functions as a
major mediator of Shh (Sonic hedgehog) signaling while Gli3 appears to
function as a repressor
of Shh signaling (Hui & Joyner, Nature Genetics (1993) 3:241-246; Ding et al.,
Development
(1998) 125:2533-2543). Furthermore, double mutant analysis demonstrates that
Gli2 and Gli3
possess overlapping developmental functions (Mo et al., Development (1997)
124:113-123;
Hardcastle et al., 1998; Motoyama et al., Nature Genetics (1998) 20:54-57). In
contrast, Glil has
been shown to be dispensable with regard to developmental functions
(unpublished
observations). A neuroepithelial cell line MNS-70 (Nakagawa et al.,
Development (1996)
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CA 02252965 1998-12-O1
122:2449-2464) has also been used to investigate the roles of the three Gli
transcription factors.
Using this cell line, both Gli 1 and Gli2 activate Gli binding site-dependent
transcription, whereas
Gli3 acts as a transcriptional repressor (Sasaki et al., 1997). Consistent
with genetic analysis,
Gli2 is seen to be primarily responsible for the transduction of Shh signal in
this cell line.
The human Gli 1 gene was initially identified as an amplified oncogene in
glioblastoma
and several mesenchymal tumors (Kinzler et al., Nature (1988) 332:371-374;
Ruppert et al., Mol.
Cell Biol (1991) 11:1724-1728). Recently, it was also shown to be
overexpressed in basal cell
carcinomas (BCCs) and medulloblastomas (MBs) (Dahmane et al., Nature (1997)
389:876-881;
Reifenberger et al., Cancer Research (1998) 58:1798-1803). Interestingly,
although forced Glil
expression in early frog embryos lead to skin tumors (Dahmane et al., 1997),
BCCs could not be
induced in transgenic mice with Gli 1 overexpression in the skin (Andrzej
Dlugosz, personal
communication). In transgenic mice, Glil overexpression itself is apparently
not sufficient for
BCC formation. While the roles of Gli2 and Gli3 in tumorigenesis remain
unclear, it has
recently been observed that Gli2+~- and Gli2+/-/Gli3+~- mutants exhibit a
higher incidence of
spontaneous tumors, including BCCs.
Through genetic manipulation, forced Shh signaling has been shown to lead to
BCC
formation. Furthermore, a putative activating mutation in Shh has also been
identified in one
BCC, one MB and one breast carcinoma although its role in tumorigenesis
remains unclear (Oro
et al., Science (1997) 276;817-821). It is worth noting that BCCs represent
the most common
cancer in humans with more than 750,000 new cases diagnosed annually and MBs
(malignant
primitive neuroectodermal tumors of the cerebellum) occur predominantly in
childhood with an
incidence of about five per million children. About one-third of sporadic BCCs
and 10-15% of
sporadic MBs are now estimated to have mutations in Ptc (Hahn et al., 1996;
Johnson et al.,
Science (1996) 272:1668-1671; Reifenberger et al., 1998). Recently, several
activating Smo
mutations have also been found in both BCCs and MBs (Reifenberger et al.,
1998; Xie et al.
Nature (1998) 391:90-92). Together, these observations indicate that Shh
signaling is an
oncogenic pathway in the developing skin and cerebellum, and that Ptc and Smo
are important
targets for genetic alterations in BCCs and MBs.
Aberrant Hh signaling is likely involved in other tumors as Ptc mutations have
been
found in a variety of tumors (Ming et al., 1988). In addition to MBs, Ptc+~-
mutant mice also
CA 02252965 1998-12-O1
exhibit a high incidence of spontaneous tumors such as rhabdomyosarcomas
(Goodrich et al.,
1997; Hahn et al., 1998). In Drosophila, the diffusion of Hh signal is
controlled by Tout-velu
(Bellaiche et al., Nature (1998) 394:85-88). Tout-velu was recently found to
be a homolog of the
multiple exotoses syndrome gene, EXT 1. This latter observation further
indicates that aberrant
Hh signaling is involved in other types of tumors and suggests that mutations
in other
components of Hh signaling, such as Fu, Cos2 and Su(fu) homologs, might cause
BCCs, MBs as
well as other tumors.
It is clear that Fu, Cos2 and Su(fu) are significant factors in the regulation
of Hh
signaling and that Hh signaling is involved in many developmental processes
and disease
conditions, particularly cancer. It would be desirable, thus, to isolate
mammalian homologs of
these factors in order to better understand their influence in mammalian
systems, and to provide
tools useful for screening compounds having potential as therapeutic agents.
Summary of the Invention
Accordingly, in one aspect of the present invention, an isolated
polynucleotide, consisting
of either DNA or RNA, that encodes a mammalian suppressor of fused protein is
provided, as
well as the novel suppressor of fused protein itself.
In other aspects of the present invention, there are provided cells that have
been
genetically engineered to produce mammalian suppressor of fused protein and
methods for
producing suppressor of fused protein from such cells. In related aspects of
the present
invention, recombinant DNA constructs are provided as well as antibodies to
suppressor of
fused.
Other aspects of the present invention will become apparent from the following
detailed
description, and from the accompanying drawings in which:
Brief Reference to the Drawings
Figure 1 is the nucleotide sequence of a Sufu-encoding polynucleotide in
accordance with
the present invention;
Figure 2 is the amino acid sequence of a Sufu protein encoded by the
polynucleotide of
Fig. l;
Figure 3A is a Northern blot analysis of mouse embryonic and adult RNA
indicating
localization of Sufu;
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CA 02252965 1998-12-O1
Figure 3B illustrates the results of RNA in situ hybridization to determine
Sufu
localization;
Figure 4 illustrates the results of a co-immunoprecipitation assay to
determine the
interaction between Sufu and Gli proteins;
Figure 5 is a bar graph indicating the effect of Sufu overexpression on Gli-
dependent
transactivation;
Figure 6A identifies amino- and carboxyl-terminal Gli2 mutants and the results
of a co-
immunoprecipitation assay to determine Sufu/Gli2 mutant binding;
Figure 6B depicts a yeast hybrid assay scheme to confirm the Sufu/Gli2 mutant
binding
set out in Fig. 6A;
Figure 7 is a Northern blot analysis of Hh signaling components in C3H10T1/2
cells;
Figure 8 is a bar graph illustrating the results of Gli-dependent
transactivation of BGlixB-
BS luciferase transcription in C3H10T1/2 cells;
Figure 9 is a bar graph illustrating the results of Shh-dependent
transactivation of BGlix8-
BS luciferase transcription in C3H10T1/2 cells; and
Figure 10 illustrates the subcellular localization of Sufu, Glil and Gli2 in
COS cells.
Detailed Description of the Invention
A polynucleotide encoding mammalian suppressor of fused (Sufu) has been
isolated.
Such a polynucleotide is useful for the screening and isolation of other Sufu
and Sufu-related
mammalian genes, including human Sufu genes, the proteins encoded by which are
implicated in
the suppression of tumor growth. Sufu is a regulatory protein, in particular,
a negative regulator
of hedgehog (Hh) signalling and is characterized by its ability to bind Gli
transcription factors as
determined in assays of conventional design, such as the assays herein
described.
As used herein, the term "Sufu" is meant to refer to the protein herein
referred to as
"suppressor of fused", and specifically to mammalian suppressor of fused.
"Mammalian" as it is
used with respect to Sufu is meant to encompass Sufu of any mammal, including
human.
The sequence of a particular isolated mammalian Sufu polynucleotide is set out
in SEQ
ID NO: 1 (Fig.l). The polynucleotide encodes mammalian Sufu consisting of 483
amino acid
residues in its mature form, as identified by three-letter code in SEQ ID NO:
2, and single-letter
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CA 02252965 1998-12-O1
code in Fig.2. The protein contains several PEST regions, regions which are
rich in proline,
glutamine, serine and threonine residues.
In order to make mammalian Sufu, techniques of genetic engineering may be
applied to
prepare a mammalian cell line that produces mammalian Sufu in functional form
as a
heterologous product. The construction of such cell lines is achieved by
introducing into a
selected host cell a recombinant DNA construct in which DNA coding for
mammalian Sufu is
associated with expression controlling elements that are functional in the
selected host to drive
expression of Sufu-encoding DNA, thus elaborating the desired Sufu protein.
The particular cell
type selected to serve as host for production of Sufu can be any of several
cell types currently
available in the art, including both prokaryotic and eukaryotic cell types.
Chinese hamster ovary
(CHO) cells for example of K1 lineage (ATCC CCL 61) including the Pros variant
(ATCC CRL
1281); the fibroblast-like cells derived from SV40-transformed African Green
monkey kidney of
the CV-1 lineage (ATCC CCL 70), of the COS-1 lineage (ATCC CRL 1650) and of
the COS-7
lineage (ATCC CRL 1651); murine L-cells, murine 3T3 cells (ATCC CRL 1658),
murine C127
cells, human embryonic kidney cells of the 293 lineage (ATCC CRL 1573), human
carcinoma
cells including those of the HeLa lineage (ATCC CCL 2), and neuroblastoma
cells of the lines
IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC HTB 11 ) all
represent examples of suitable cell types for the production of mammalian
Sufu.
A variety of gene expression systems have been adapted for use with these
hosts and are
now commercially available. Any one of these systems can be selected to drive
expression of
the Sufu-encoding DNA. These systems, available typically in the form of
plasmidic vectors,
incorporate expression cassettes the functional components of which include
DNA constituting
expression controlling sequences, which are host-recognized and enable
expression of Sufu-
encoding DNA when linked 5' thereof. Such Sufu-encoding DNA is referred to
herein as being
incorporated "expressibly" into the system, and incorporated "expressibly" in
a cell once
successful expression from a cell is achieved. These systems further
incorporate DNA sequences
which terminate expression when linked 3' of the receptor-encoding region.
Thus, for expression
in the selected mammalian cell host, there is generated a recombinant DNA
expression construct
in which the Sufu-encoding DNA is linked with expression controlling DNA
sequences
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CA 02252965 1998-12-O1
recognized by the host, and which include a region 5' of the Sufu-encoding DNA
to drive
expression, and a 3' region to terminate expression.
Included among the various recombinant DNA expression systems that can be used
to
achieve mammalian cell expression of the Sufu-encoding DNA are those that
exploit promoters
of viruses that infect mammalian cells, such as the promoter from
cytomegalovirus (CMV), the
Rous sarcoma virus (RSV), simian virus (SV40), murine mammary tumor virus
(MMTV) and
others. Also useful to drive expression are promoters such as the LTR of
retroviruses, insect cell
promoters, including those isolated from Drosophila which are regulated by
temperature, as well
as mammalian gene promoters such as those regulated by heavy metals, i.e. the
metallothionein
gene promoter, and other steroid-inducible promoters.
The plasmidic vector harbouring the expression construct typically
incorporates a marker
to enable selection of stably transformed recombinant cells. The marker
generally comprises a
gene conferring some survival advantage on the transformants allowing for the
selective growth
of successful transformants in a chosen medium. For example, common gene
markers include
genes which code for resistance to specific drugs, such as tetracycline,
ampicillin and neomycin.
Thus, transformants which have successfully taken up the plasmid DNA will
incorporate both
the gene of interest, i.e. the Sufu gene and the marker gene, e.g. gene for
drug resistance such as
neomycin, and will survive culturing in media containing the drug which they
could otherwise
not tolerate.
For incorporation into the recombinant DNA expression vector, DNA coding for
Sufu
can be obtained by applying selected techniques of gene isolation or gene
synthesis. As
described in more detail in the examples herein, Sufu can be obtained by
careful application of
conventional gene isolation and cloning techniques. This typically will entail
extraction of total
messenger RNA from a fresh source of mammalian tissue, followed by conversion
of message to
cDNA and formation of a cDNA library e.g. a bacteriophage cDNA library. Such
bacteriophage
harboured fragments of the human DNA are grown by plating on a lawn of
susceptible E. coli
bacteria such that individual phage plaques or colonies can be isolated. The
DNA carried by the
phage colony is then immobilized on a nitrocellulose or nylon-based
hybridization membrane,
and then hybridized, under carefully controlled conditions, to a labelled,
e.g. radioactively or
otherwise labelled, probe sequence to identify the particular phage colony
carrying the DNA
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CA 02252965 1998-12-O1
fragment of particular interest, in this case a mammalian Sufu gene. The phage
carrying the gene
of interest is then isolated from contaminating phages in order that the gene
may be more easily
characterized. For convenience, the gene or a portion thereof is generally
subcloned into a
plasmidic vector at this stage.
Having herein provided the nucleotide sequence of a mammalian Sufu protein and
a
Sufu-encoding gene, it will be appreciated that automated techniques of gene
synthesis and/or
amplification can be performed to generate Sufu-encoding DNA. In this case,
because of the
length of the Sufu-encoding DNA, application of automated synthesis may
require staged gene
construction in which regions of the gene up to about 300 nucleotides in
length are synthesized
individually and then ligated in correct succession via designed overlaps.
Individually
synthesized gene regions can then be amplified by PCR.
With appropriate template DNA in hand, the technique of PCR amplification may
be
used to directly generate all or part of the final gene. In this case, primers
are synthesized which
will prime the PCR amplification of the final product, either in one piece, or
in several pieces
that may subsequently be ligated together via step-wise ligation of blunt
ended, amplified DNA
fragments, or preferentially via step-wise ligation of fragments containing
naturally occurnng
restriction endonuclease sites. Both cDNA or genomic DNA are suitable as
templates for PCR
amplification. The former may be obtained from a number of sources including
commercially
available cDNA libraries, single- or double-stranded cDNA, or cDNA constructed
from isolated
messenger RNA from a suitable tissue sample. Human genomic DNA may also be
used as a
template for the PCR-based amplification of the gene; however, the gene
sequence of such
genomic DNA may contain unwanted intervening sequences.
Once obtained, the Sufu-encoding DNA is incorporated for expression into any
suitable
expression vector, and host cells are transfected therewith using conventional
procedures, such as
DNA-mediated transformation including calcium phosphate precipitation,
protoplast fusion,
microinjection, lipofection and electroporation. Expression vectors may be
selected to provide
transformed cell lines that express the Sufu-encoding DNA in a stable manner.
Suitable
expression vectors will typically harbour a gene coding for a product that
confers on the
transformants a survival advantage to enable their subsequent selection. Genes
coding for such
selectable markers include the _E. coli gpt gene which confers resistance to
mycophenolic acid,
CA 02252965 1998-12-O1
the neon gene from transposon Tn5 which confers resistance to neomycin and to
the neomycin
analog 6418, the dhfr sequence from murine cells or E. coli which changes the
phenotype of
DHFR- cells into DHFR+ cells, and the tk gene of herpes simplex virus, which
makes TK- cells
phenotypically TK+ cells. Other methods of selecting for transformants may of
course be used,
if desired, including selection by morphological parameters, or detection of
surface antigen or
receptor expression. The latter can be monitored using specifically labelled
antibodies and a
cell-sorter, e.g. fluorescent activated.
As one of skill in the art will appreciate, Sufu-encoding DNA may be modified
prior to
its incorporation into an expression vector to enhance protein expression.
Specifically,
modifications may be made to the 5' and 3' non-coding regions of Sufu-encoding
DNA in order
to increase the level of protein expression. For example, the 5' non-coding
end of Sufu-encoding
DNA may be modified to provide a 5' "translation-enhancing sequence" (TES).
Such
modifications include truncating the 5' end of the Sufu-encoding DNA preceding
the native
translation-enhancing sequence. Alternatively, the DNA is truncated and the
native translation-
1 S enhancing sequence is replaced with a heterologous translation-enhancing
sequence using
conventional methods of restriction enzyme digestion followed by ligation
techniques. By
"heterologous" is meant a sequence that is not native to Sufu-encoding DNA.
Further, by
"translation-enhancing sequence" is meant the 5' sequence which is required
for translation to
occur, and includes the translation initiation codon, i.e. ATG.
The present invention also provides, in another of its aspects, antibody to
mammalian
Sufu. To raise such antibodies, there may be used as immunogen either full-
length Sufu or an
immunogenic fragment thereof, produced in a microbial or mammalian cell host
as described
above or by standard peptide synthesis techniques. Regions of Sufu
particularly suitable for use
as immunogenic fragments include regions which are determined to have a high
degree of
antigenicity based on a number of factors, as would be appreciated by those of
skill in the art,
including for example, amino acid residue content,
hydrophobicity/hydrophilicity and secondary
structure. Specific examples of immunogenic fragments of Sufu suitable for
generating
antibodies include, but are not limited to, the region spanning residues 305
to residues 325, the
region spanning residues 452 to residues 468.
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CA 02252965 1998-12-O1
The raising of antibodies to mammalian Sufu or to desired immunogenic
fragments can
be achieved, for polyclonal antibody production, using immunization protocols
of conventional
design, and any of a variety of mammalian hosts, such as sheep, goats and
rabbits. Alternatively,
for monoclonal antibody production, immunocytes such as splenocytes can be
recovered from
the immunized animal and fused, using hybridoma technology, to myeloma cells.
The fusion
cell products, i.e. hybridoma cells, are then screened by culturing in a
selection medium, and
cells producing the desired antibody are recovered for continuous growth, and
antibody recovery.
Recovered antibody can then be coupled covalently to a reporter molecule, i.e.
a detectable label,
such as a radiolabel, enzyme label, luminescent label or the like, using
linker technology
established for this purpose, to form a specific probe for Sufu.
According to a further aspect of the present invention, DNA or RNA encoding
mammalian Sufu, and selected regions thereof, may also be used in detestably
labeled form, e.g.
radiolabeled form, as hybridization probes to identify sequence-related genes
existing in the
human or other mammalian genomes (or cDNA libraries) or to locate Sufu-
encoding DNA in
any other specimen. This can be done using the intact coding region, due to a
high level of
conservation expected between related genes, or by using a highly conserved
fragment thereof,
having radiolabeled nucleotides, for example, 32P nucleotides, incorporated
therein.
Embodiments and aspects of the present invention will now be described by
reference to
the following specific examples which are not to be construed as limiting.
Example 1-Cloning of mouse Su~pressor of fused (Msuful eene
In order to isolate a full length clone of the mouse homolog of suppressor of
fused
(Msufu), a partial cDNA clone (EST clone number 513730 containing sequences
homologous to
Drosophila suppressor of fused gene; Research Genetics) was radio-labeled and
used as a probe
to screen a lambda phage cDNA library constructed from E11.5 mouse embryo mRNA
using
procedures well-known in the art such as those set out in Current Protocols in
Molecular Biology
(Whey).
A cDNA insert encoding mouse Sufu (Msufu) was isolated from the cDNA library
and
cloned into the plasmid vector, pBluescriptII (Stratagene), and the nucleotide
sequence, as set out
in SEQ ID NO: 1, was determined by DNA sequencing.
CA 02252965 1998-12-O1
Example 2 - Expression of the Msufu eene
In order to express the isolated Msufu gene, an expression vector was prepared
as
follows. A SaII restriction enzyme site was generated at the N-terminal end of
the Sufu coding
region of the isolated gene by PCR using procedures as set out in Current
Protocols in Molecular
Biology. The gene was then subcloned into the eukaryotic expression vector,
pCMVS(3 (gift
from Dr. Jeff Wrana of the Hospital for Sick Children, Toronto, Ontario; see
P.A. Hoodless et
al., Cell (1996) 85:489-500), downstream of a Myc-tag driven by the
cytomegalovirus promoter.
The predicted open reading frame of Msufu encodes 483 amino acid residues. At
the
amino acid level, the overall sequence identity between the mouse and
Drosophila proteins is
36%. Northern blot analysis of embryonic and adult mouse RNA revealed a 4.5 kb
Msufu
transcript (Fig. 3A). During mouse embryogenesis, Msufu is expressed from E8.5
to term.
Msufu transcripts are found in many adult tissues, including heart, brain and
liver. RNA in situ
hybridization revealed that Msufu transcripts are ubiquitous from E8.5 to
E10.5 (Fig. 3B) and its
expression becomes more restricted during organogenesis (data not shown).
Example 3 - Interactions between Msufu and Gli proteins
To study potential interactions between Msufu and Gli proteins, eukaryotic
expression
vectors containing FLAG-, Myc- and HA-tagged versions of Msufu were generated
by PCR,
using the procedure described above in Example 2, for use in co-
immunoprecipitation
experiments. FLAG-tagged Gli 1 and GIi2 were generated by subcloning the
protein coding
region of Gli 1 and Gli2 downstream of a FLAG-tag in the eukaryotic expression
vector,
pCMVS(3 (gift of Hiroshi Sasaki, Osaka University, Japan; unpublished).
The co-immunoprecipation experiment generally used is as described in Current
Protocols in Molecular Biology. FLAG-tagged GIi2 and Myc-tagged Sufu were co-
transfected
into HeLa and COS cells by lipofectamine treatment. Forty eight hours post-
transfection, lysate
was prepared from the cells. GIi2 or Sufu was precipitated from the cell
lysate using FLAG- or
Myc-antibodies (Sigma) respectively, and the immunoprecipitates were subjected
to SDS-PAGE
analysis. The interactions between Gli2 and Sufu were then determined by
Western blot analysis
11
CA 02252965 1998-12-O1
of FLAG-immunoprecipitate using Myc-antibodies and Myc-immunoprecipitate using
FLAG-
antibodies.
Similar results were obtained in both cells. As shown in Fig.4, the results of
these
experiments revealed that Msufu forms a protein complex with Gli2 upon
transfection into COS
cells.
Similar co-immunoprecipitation experiments were conducted to determine if
Msufu also
interacted with Gli 1 and Gli3. The results of these experiments confirmed the
formation of
Msufu-Gli 1 and Msufu-Gli3 protein complexes.
Example 4 - Modulatorv Effect of Msufu on transcriptional activities of Gli
proteins
To determine the functional significance of the Msufu-Gli interactions, the
transcriptional
activities of the Gli proteins were assayed with and without Msufu.
Specifically, Glil and Gli2
are known to activate transcription in HeLa cells from a luciferase reporter
gene (Gli-luc) via the
Gli binding sites while Gli3 represses this transcription as previously
described (Sasaki et al.,
1997, Development 124, 1313-1322). Using this assay, the effect of Msufu
overexpression on
Gli-dependent transactivation was determined.
The assay was conducted as described (Sasaki, 1997). As shown in Fig. 5,
although
Msufu overexpression alone has no effect on the transcription of the
luciferase reporter gene, it
does affect Gli-dependent transactivation. In particular, Msufu overexpression
dramatically
decreases the transactivation by Gli2. In contrast, Msufu has very little or
no effect on the
transactivation by Glil . Since Gli2 is the primary target of Shh signaling,
the ability of Msufu to
differentially regulate the transcriptional activities of Glil and Gli2
suggests the regulatory role
that Msufu plays with respect to Shh signaling. Moreover, Msufu overexpression
converts the
activity of Gli3 from repressor to activator. Although the physiological
significance of this latter
observation is unclear, it is important to note that Gli3 acts as a repressor
of Shh signaling. In
additional control experiments, Msufu overexpression was found to have an
insignificant effect
on the transactivation of a GAL4 reporter by a GAL4-VP 16 activator (data not
shown) indicating
the specificity of Msufu overexpression on Gli-dependent transactivation.
Example 5 - Interaction of Msufu with amino- and carboxyl-termini of Gli2
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CA 02252965 1998-12-O1
To determine the regions of Gli 2 involved in Msufu-Gli2 interactions, a
series of amino-
terminal and carboxyl-terminal deletion mutants of Gli2 (identified in Fig.
6A) were prepared
and co-transfected with Msufu into COS cells. Msufu-Gli2 interaction was then
determined by
co-immunoprecipitation as described above in detail in Example 3. As set out
in Fig. 6A, the
results of the assay reveal that the N-terminal region (between amino acid
residues 1 and 389) of
Gli2 can form a stable complex with Msufu. Interestingly, Msufu can also
interact with a
carboxyl-terminal region (between residues 1184 and 1544) of Gli2.
To further examine these interactions, these truncated amino- and carboxyl-
terminal
regions of Gli2 were cloned in frame with the GAL4 activation domain in
pGAD424 (Clontech)
to generate, respectively, plasmids Gli2N-TA and Gli2C-TA. These were each
assayed for
protein-protein interactions in yeast with an Msufu-GAL4 DNA-binding domain
fusion protein
cloned in pAS2-1 (Clontech) identified as Msufu-DB. As shown in Fig. 6B, both
regions can
interact with Msufu in yeasts, indicating that Msufu forms a complex with Gli2
through two
distinct regions.
Example 6 -Inhibition of Shh signaling in C3H10T1/2 cells by Msufu
To examine the role of Msufu in Shh signaling, transcriptional assays in the
pluripotent
mouse mesenchymal cell lines, C3H10T1/2 (ATCC - CCL-226), were developed as
previously
described in Example 4. Since C3H10T1/2 cells are known to respond to Shh
(Nakamura et al.,
1997, Biochemical and Biophysical Research Communications 237, 465-469) and
express high
levels of Gli2 and Msufu as well as Ptch and Smo transcripts (see Fig.7), they
can serve as a
useful in vitro system for studying the molecular mechanism of Shh signaling,
and, in particular,
the interactions between Msufu and Gli2.
As shown in Fig.8, the activity of Gli-luc in C3H10T1/2 cells was found to be
3- to 4-
fold more active than a control luciferase reporter lacking the Gli binding
site suggesting that
Gli2 can act as an activator of the Gli-luc reporter in the absence of
exogenous Shh signal. In
this cell line, Gli2 activity seems to be rate-limiting because Gli2
overexpression can further
augment the activity of Gli-luc. Similar to the results observed in HeLa
cells, the activity of Gli-
luc, but not the control luciferase reporter, was activated by Glil
overexpression and repressed
by Gli3 overexpression. When C3H10T1/2 cells were treated with Shh, the
transcription of Gli-
13
CA 02252965 1998-12-O1
luc, but not the control reporter, is enhanced (Fig.9). Furthermore,
preliminary experiments
demonstrate that this Shh-dependent enhancement is inhibited by cotransfection
with Msufu (not
shown). Together, these results indicate that Msufu overexpression can inhibit
Shh signaling in
C3H10T1/2 cells.
Example 7 - Subcellular localization of Msufu. Glil, Gli2 and Gli3
To study the subcellular distribution of Msufu and the three Gli proteins,
FLAG-tagged
cDNA expression vectors were introduced into COS and HeLa cells by lipofection
and analyzed
by immunofluorescence using a monoclonal anti-FLAG antibody.
Similar staining results were observed in both cell lines. The results
obtained in COS
cells are illustrated in Fig.lO. Msufu was found to be uniformly distributed
in the cytoplasm of
transfected cells. Both Gli2 and Gli3 (data not shown) are distributed
primarily in the cytoplasm
of transfected cells although 10-20% of the transfected cells also show
nuclear staining. In
contrast, Gli 1 was found to be nuclear in the transfected cells. Preliminary
experiments indicate
a similar subcellular distribution of these proteins in C3H10T1/2 cells.
Example 8 - Generation of Sufu-specific Antibodies
Full length Sufu as well as various subfragments of the N- and C- terminal
regions of
mouse Sufu were subcloned into a His-tagged bacterial expression vector (such
as pQE30,
pQE31 and pQE32 available from Qiagen). Purified His-tagged Sufu was injected
into rabbits to
raise polyclonal antibodies by using standard procedures such as those
described in Harlow &
Lane (Antibodies: A laboratory manual. Cold Spring Harbor Laboratory, 1988).
The following
Sufu-specific peptides were synthesized:
1) the region spanning residues 305 to residues 325; and
2) the region spanning residues 452 to residues 468
and used for raising antibodies. The specificity of the antibodies was
determined using Western
blot analysis of C3H10T1/2 cell extracts and mouse embryo extracts. The size
of epitope-tagged
Sufu has been determined to be about 53 kDa by Western blot analysis (see
Fig.4). Specificity
was also confirmed by the ability of the antibodies to immunoprecipitate Sufu
as set out in
Example 3. The immunoprecipitate was analyzed by Western blot analysis using a
pan-Gli
14
CA 02252965 1998-12-O1
antibody (gift from Dr. David Markowitz, University of Michigan) which
recognizes all three
mouse Gli proteins. Interaction of endogenous Sufu and Gli2 proteins in
C3H10T1/2 cells is
determined by a 200 kDa band on the Western blot, while interactions between
Sufu and Gli 1 in
mouse embryos is determined by a 140 kDa band, Gli2 interaction is determined
by a 200 kDa
band and Gli3 interaction is determined by a 210-220 kDa band.
Example 9 - Mutagenesis of Sufu
Mutagenesis of Sufu is conducted to determine the domains required for binding
and
repressing Gli2. Sequence analysis of Drosophila and mouse Sufu proteins have
revealed three
highly conserved regions. Within these regions, there are eight
serine/threonine residues that can
serve as potential phosphorylation sites. Based on this information, a series
of amino-terminal,
carboxyl-terminal and internal deletion mutants of Sufu were generated by PCR
as follows:
Mutant Gli2 Interaction
Residues 1-105 -
Residues 1-211 ++
Residues 1-308 +++
Residues 1-408 +
Residues 99-211 -
Residues 99-308 +++
Residues 99-408 -
Residues 99-482 +
Residues 244-482 ++
The mutants were cloned into an epitope-tagged expression vector, Myc-tagged
mammalian expression vector, and the interactions of these mutant Sufu
proteins with Gli2 were
assayed. The interaction between each mutant and Gli2 is set out above.
Two different methods were used to determine the mutant Sufu/Gli2 interaction.
First, mutant
Sufu was co-transfected with Gli2 into COS cells and their interactions were
analyzed by co-
immunoprecipitation assays as set out in Example 3. Second, radio-labeled
mutant Sufu was
generated by in vitro transcription and translation, and examined for the
ability to bind GST
CA 02252965 1998-12-O1
(glutathione-S-transferase) columns containing GST fusion proteins with either
amino- or
carboxyl-terminal regions of Gli2 in a GST-pull down assay.
It is possible for the results of these two assays to be different. For
example, the GST-
pull down assay could reveal a Gli2 binding region in Sufu that is missed in
the co-
immunoprecipitation assay because a mutant form of Sufu might form a complex
with Gli2
through indirect interaction with a third protein in transfected cells.
Therefore, both regions of
Sufu required so that (1) the formation of a stable complex Gli2 (co-
immunoprecipitation assay)
and (2) direct interactions with Gli2 (GST-pull down assay) can be determined.
To fine map the Gli2 binding region(s), additional mutants (internal deletions
or smaller
subfragments of Sufu) are constructed and examined as set out above.
Various mutants are also transfected into C3H10T1/2 cells and assayed for
their abilities
to repress Gli2 to determine whether stable complex formation and/or direct
Gli2 binding are
required for the repression of Gli2 transactivation.
The above-described assays can also be conducted to determine the effect of
Sufu
mutants in modulating Gli 1 and Gli3 transcriptional activities and to
determine differential
abilities in modulating Gli proteins.
16
CA 02252965 1998-12-O1
SEQUENCE LISTING
(1) GENERAL
INFORMATION:
S
(i) APPLICANT:
(A) NAME: Chi-Chung Hui
(B) STREET: 1023 St. Clarens Avenue
(C) CITY: TORONTO
IO (D) STATE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE (ZIP): M6H 3X8
(A) NAME: Qi Ding
IS (B) STREET: 1117 McIntyre Drive
(C) CITY: Ann Arbor
(D) STATE: Michigan
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 48105
20
(ii) TITLE OF INVENTION: Novel Mammalian Suppressor
of Fused Gene
(iii) NUMBER OF SEQUENCES: 2
ZS (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(EPO)
30
(2) INFORMATION
FOR
SEQ
ID NO:
1:
(i) SEQUENCE CHARACTERISTICS:
3S (A) LENGTH: 1663 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
4O (ii) MOLECULE TYPE: cDNA
4S(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 1:
GAATTCAAGC CGGAGCCTCGTGCGCAGGCGCGGAGTTAGACCTCGCCGTAGCCCCCATCG 60
CCTCGGGGAG TCTCATCCACAGTGGGTCCGCTGGCCATAGGGGCGCTTTCCCTCCTCTAC 120
SO
TCCCGGGTTC TCGGCCCTACGGCCCCAATGGCGGAGCTGCGGCCTAGCGTCGCCCCCGGT 180
CCCGCCGCGC CCCGCTCTGGCCCTAGTGCCCCTCCGGCCTTTGCTTCACTCTTTCCCCCG 240
SSGGACTGCACG CCATCTACGGAGAGTGTCGCCGCCTCTACCCTGACCAGCCGAACCCGCTC 300
17
CA 02252965 1998-12-O1
CAGGTTACCGCTATCGTCAA GGTGGTCCGGACCCCTTGGACTATGTTAGC 360
GTACTGGTTG
ATGTACAGGAACATGGGGTGTCCTTCTGCCAACATCCCTGAGCACTGGCACTACATCAGC 420
$
TTTGGCCTGAGTGATCTCTATGGTGACAACAGAGTCCATGAGTTTACAGGAACAGACGGA 480
CCAAGTGGATTTGGCTTTGAGTTGACGTTTCGTCTGAAGAGAGAAACTGGGGAGTCTGCC 540
lO CCACCAACATGGCCAGCAGAGCTGATGCAGGGCCTAGCCCGATATGTCTTCCAGTCAGAG 600
AACACCTTCTGTAGCGGGGACCATGTGTCTTGGCACAGCCCTTTGGATAACAGTGAGTCA 660
AGAATTCAGCACATGCTGCTGACGGAGGACCCACAGATGCAGCCTGTGCGGACACCCTTT 720
1$
GGGGTAGTGACTTTCCTCCAGATTGTTGGTGTCTGCACTGAGGAGTTACATTCAGCCCAA 780
CAGTGGAACGGGCAGGGCATCCAGGAACTACTACGGACAGTGCCCATTGCTGGCGGTCCC 840
2O TGGCTGATAACTGACATGCGGCGGGGAGAAACCATATTTGAGATCGATCCGCACCTGCAA 900
CAGGAGAGAGTTGACAAAGGCATTGAGACAGACGGTTCTAACCTGAGCGGCGTCAGTGCC 960
AAGTGTGCCTGGGATGACCTCAGCCGGCTCCGGAGGATGAAGAGGATAGCCGGAGCATCT 1020
2$
GCTCGGCACACGCCTCGGAGGCTGTCTGGCAAAGACACAGAGCAGATCCGGGAGACCCTG 1080
AGGCGGGGACTGGAGATTAACAGCAAACCTGTCCTTCCACCAATCAATTCTCAGCGACAG 1140
3O AACGGCCTCACCCACGACAGGGCTCCGAGCCGCAAGGACAGTTTGGGCAGCGACAGCTCC 1200
ACGGCCATCATCCCCCACGAGCTGATCCGCACACGGCAGCTCGAGAGCGTGCATCTAAAA 1260
TTTAACCAAGAGTCGGGAGCCCTCATCCCTCTCTGCCTAAGGGGCAGACTCCTACATGGC 1320
3$
CGGCACTTCACCTACAAGAGTATCACAGGCGACATGGCCATCACGTTTGTGTCCACGGGA 1380
GTGGAAGGCGCCTTTGCCACTGAGGAACACCCGTATGCAGCTCACGGACCCTGGTTACAA 1440
4O ATTCTGTTGACAGAAGAGTTTGTAGAGAAGATGTTGGAGGACTTAGAAGATCTAACCTCT 1500
CCAGAGGAATTTAAACTTCCCAAAGAGTACAGCTGGCCTGAGAAGAAACTCAAAGTGTCC 1560
ATTCTCCCCGACGTGGTGTTCGACAGTCCACTGCACTAGCCTGGCTGTGCCTGCAGGGGC 1620
4$
CAAGAGGAGCCCAGCTGCTCCTGGTGACTTCCAGTGTGACAGG 1663
(2) INFORMATION
FOR SEQ
ID NO:
2:
SO (i) SEQUENCE :
CHARACTERISTICS
(A) LENGTH:483 aminocids
a
(B) TYPE:
amino
acid
(C) STRANDEDNESS: e
singl
(D) TOPOLOGY:
linear
$$
18
CA 02252965 1998-12-O1
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE
DESCRIPTION:
SEQ
ID
NO:
2:
Met AlaGlu LeuArgPro SerValAla ProGlyPro AlaAlaPro Arg
1 5 10 15
Ser GlyPro SerAlaPro ProAlaPhe AlaSerLeu PheProPro Gly
20 25 30
Leu HisAla IleTyrGly GluCysArg ArgLeuTyr ProAspGln Pro
1$ 35 40 45
Asn ProLeu GlnValThr AlaIleVal LysTyrTrp LeuGlyGly Pro
50 55 60
Asp ProLeu AspTyrVal SerMetTyr ArgAsnMet GlyCysPro Ser
65 70 75 80
Ala AsnIle ProGluHis TrpHisTyr IleSerPhe GlyLeuSer Asp
85 90 95
Leu TyrGly AspAsnArg ValHisGlu PheThrGly ThrAspGly Pro
100 105 110
Ser GlyPhe GlyPheGlu LeuThrPhe ArgLeuLys ArgGluThr Gly
115 120 125
Glu SerAla ProProThr TrpProAla GluLeuMet GlnGlyLeu Ala
130 135 140
3$ Arg TyrVal PheGlnSer GluAsnThr PheCysSer GlyAspHis Val
145 150 155 160
Ser TrpHis SerProLeu AspAsnSer GluSerArg IleGlnHis Met
165 170 175
Leu LeuThr GluAspPro GlnMetGln ProValArg ThrProPhe Gly
180 185 190
Val ValThr PheLeuGln IleValGly ValCysThr GluGluLeu His
195 200 205
Ser AlaGln GlnTrpAsn GlyGlnGly IleGlnGlu LeuLeuArg Thr
210 215 220
$0 Val Pro Ile Ala Gly Gly Pro Trp Leu Ile Thr Asp Met Arg Arg Gly
225 230 235 240
Glu Thr Ile Phe Glu Ile Asp Pro His Leu Gln Gln Glu Arg Val Asp
245 250 255
19
CA 02252965 1998-12-O1
Lys GlyIle GluThrAsp GlySerAsn LeuSerGly ValSerAla Lys
260 265 270
Cys AlaTrp AspAspLeu SerArgLeu ArgArgMet LysArgIle Ala
$ 275 280 285
Gly AlaSer AlaArgHis ThrProArg ArgLeuSer GlyLysAsp Thr
290 295 300
Glu GlnIle ArgGluThr LeuArgArg GlyLeuGlu IleAsnSer Lys
305 310 315 320
Pro ValLeu ProProIle AsnSerGln ArgGlnAsn GlyLeuThr His
325 330 335
Asp ArgAla ProSerArg LysAspSer LeuGlySer AspSerSer Thr
340 345 350
Ala IleIle ProHisGlu LeuIleArg ThrArgGln LeuGluSer Val
355 360 365
His LeuLys PheAsnGln GluSerGly AlaLeuIle ProLeuCys Leu
370 375 380
Arg GlyArg LeuLeuHis GlyArgHis PheThrTyr LysSerIle Thr
385 390 395 400
Gly AspMet AlaIleThr PheValSer ThrGlyVal GluGlyAla Phe
405 410 415
Ala ThrGlu GluHisPro TyrAlaAla HisGlyPro TrpLeuGln Ile
420 425 430
Leu LeuThr GluGluPhe ValGluLys MetLeuGlu AspLeuGlu Asp
435 440 445
Leu ThrSer ProGluGlu PheLysLeu ProLysGlu TyrSerTrp Pro
450 455 460
Glu LysLys LeuLysVal SerIleLeu ProAspVal ValPheAsp Ser
465 470 475 480
Pro Leu His
20
CA 02252965 1998-12-O1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
S
(i) APPLICANT:
(A) NAME: Chi-Chung Hui
(B) STREET: 1023 St. Clarens Avenue
(C) CITY: TORONTO
IO (D) STATE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE (ZIP): M6H 3X8
(A) NAME: Qi Ding
IS (B) STREET: 1117 McIntyre Drive
(C) CITY: Ann Arbor
(D) STATE: Michigan
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 48105
(ii) TITLE OF INVENTION: Novel Mammalian Suppressor of Fused Gene
(iii) NUMBER OF SEQUENCES: 2
2S (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
3S (A) LENGTH: 1663 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
4O (ii) MOLECULE TYPE: cDNA
4S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GAATTCAAGC CGGAGCCTCG TGCGCAGGCG CGGAGTTAGA CCTCGCCGTA GCCCCCATCG 60
CCTCGGGGAG TCTCATCCAC AGTGGGTCCG CTGGCCATAG GGGCGCTTTC CCTCCTCTAC 120
S0
TCCCGGGTTC TCGGCCCTAC GGCCCCAATG GCGGAGCTGC GGCCTAGCGT CGCCCCCGGT 180
CCCGCCGCGC CCCGCTCTGG CCCTAGTGCC CCTCCGGCCT TTGCTTCACT CTTTCCCCCG 240
SS GGACTGCACG CCATCTACGG AGAGTGTCGC CGCCTCTACC CTGACCAGCC GAACCCGCTC 300
CA 02252965 1998-12-O1
CAGGTTACCG CTATCGTCAA GTACTGGTTG GGTGGTCCGG ACCCCTTGGA 360
CTATGTTAGC
ATGTACAGGA ACATGGGGTG TCCTTCTGCC AACATCCCTG AGCACTGGCA 420
CTACATCAGC
TTTGGCCTGA GTGATCTCTA TGGTGACAAC AGAGTCCATG AGTTTACAGG 480
AACAGACGGA
CCAAGTGGAT TTGGCTTTGA GTTGACGTTT CGTCTGAAGA GAGAAACTGG 540
GGAGTCTGCC
IO CCACCAACAT GGCCAGCAGA GCTGATGCAG GGCCTAGCCC GATATGTCTT 600
CCAGTCAGAG
AACACCTTCT GTAGCGGGGA CCATGTGTCT TGGCACAGCC CTTTGGATAA 660
CAGTGAGTCA
AGAATTCAGC ACATGCTGCT GACGGAGGAC CCACAGATGC AGCCTGTGCG 720
1$ GACACCCTTT
GGGGTAGTGA CTTTCCTCCA GATTGTTGGT GTCTGCACTG AGGAGTTACA 7g0
TTCAGCCCAA
CAGTGGAACG GGCAGGGCAT CCAGGAACTA CTACGGACAG TGCCCATTGC 840
TGGCGGTCCC
2O TGGCTGATAA CTGACATGCG GCGGGGAGAA ACCATATTTG AGATCGATCC 900
GCACCTGCAA
CAGGAGAGAG TTGACAAAGG CATTGAGACA GACGGTTCTA ACCTGAGCGG 960
CGTCAGTGCC
AAGTGTGCCT GGGATGACCT CAGCCGGCTC CGGAGGATGA AGAGGATAGC 1020
25 CGGAGCATCT
GCTCGGCACA CGCCTCGGAG GCTGTCTGGC AAAGACACAG AGCAGATCCG 1080
GGAGACCCTG
AGGCGGGGAC TGGAGATTAA CAGCAAACCT GTCCTTCCAC CAATCAATTC 1140
TCAGCGACAG
3O AACGGCCTCA CCCACGACAG GGCTCCGAGC CGCAAGGACA GTTTGGGCAG 1200
CGACAGCTCC
ACGGCCATCA TCCCCCACGA GCTGATCCGC ACACGGCAGC TCGAGAGCGT 1260
GCATCTAAAA
TTTAACCAAG AGTCGGGAGC CCTCATCCCT CTCTGCCTAA GGGGCAGACT 1320
35 CCTACATGGC
CGGCACTTCA CCTACAAGAG TATCACAGGC GACATGGCCA TCACGTTTGT 1380
GTCCACGGGA
GTGGAAGGCG CCTTTGCCAC TGAGGAACAC CCGTATGCAG CTCACGGACC 1440
CTGGTTACAA
4O ATTCTGTTGA CAGAAGAGTT TGTAGAGAAG ATGTTGGAGG ACTTAGAAGA 1500
TCTAACCTCT
CCAGAGGAAT TTAAACTTCC CAAAGAGTAC AGCTGGCCTG AGAAGAAACT CAAAGTGTCC 1560
ATTCTCCCCG ACGTGGTGTT CGACAGTCCA CTGCACTAGC CTGGCTGTGC CTGCAGGGGC 1620
CAAGAGGAGC CCAGCTGCTC CTGGTGACTT CCAGTGTGAC AGG 1663
(2) INFORMATION FOR SEQ ID N0: 2:
SO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02252965 1998-12-O1
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
2:
Met Ala Glu Leu Arg Pro Ser Val Ala Pro
Gly Pro Ala Ala Pro Arg
1 5 10 15
Ser Gly Pro Ser Ala Pro Pro Ala Phe Ala Pro Pro
Ser Leu Phe Gly
25 30
Leu His Ala Ile Tyr Gly Glu Cys Arg Arg ProAsp Gln
1$ Leu Tyr Pro
35 40 45
Asn Pro Leu Gln Val Thr Ala Ile Val Lys Trp LeuGly Gly
Tyr Pro
50 55 60
20 Asp Pro Leu Asp Tyr Val Ser Met Tyr Arg Met GlyCys Pro
Asn Ser
65 70 75 80
Ala Asn Ile Pro Glu His Trp His Tyr Ile Phe GlyLeu Ser
Ser Asp
85 90 95
Leu Tyr Gly Asp Asn Arg Val His Glu Phe Gly ThrAsp Gly
Thr Pro
100 105 110
Ser Gly Phe Gly Phe Glu Leu Thr Phe Arg Lys ArgGlu Thr
Leu Gly
115 120 125
Glu Ser Ala Pro Pro Thr Trp Pro Ala Glu Met GlnGly Leu
Leu Ala
130 135 140
3$ Arg Tyr Val Phe Gln Ser Glu Asn Thr Phe Ser Gly
Cys Asp
His
Val
145 150 155
160
Ser Trp His Ser Pro Leu Asp Asn Ser Glu Ser Arg Ile Gln His Met
40 16s 170 17s
Leu Leu Thr Glu Asp Pro Gln Met Gln Pro Val Arg Thr Pro Phe Gly
180 185 190
Val Val Thr Phe Leu Gln Ile Val Gly Val Cys Thr Glu Glu Leu His
4$ 195 200 205
Ser Ala Gln Gln Trp Asn Gly Gln Gly Ile Gln Glu Leu Leu Arg Thr
210 215 220
$0 Val Pro Ile Ala Gly Gly Pro Trp Leu Ile Thr Asp Met Arg Arg Gly
225 230 235
240
Glu Thr Ile Phe Glu Ile Asp Pro His Leu Gln Gln Glu Arg Val Asp
$$ 245 250 255
