Note: Descriptions are shown in the official language in which they were submitted.
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FRINGE PROTEINS AND NOTCH SIGNALLING
Field of the Invention
This invention relates to control of the
interaction between Notch receptors and their ligands.
Background of the Invention
Various journal articles referred to herein are
identified by authors and date in parentheses and are
listed, with full citations, at the end of the
specification. The contents thereof are incorporated
herein by reference.
Genetic analysis of Drosophila melanogaster wing
development has elucidated many fundamental concepts of
tissue induction and specification. These studies have
highlighted the importance of compartment. boundaries in
organizing pattern (Basler and Struhl, 1994;
Garcia-Bellido et al., 1973; Lawrence and Morata, 1976;
Tabata and Kornberg, 1994).
The wing imaginal disc is divided into
anterior/posterior (A/P) compartments and dorso/ventral
(D/V) compartments, which are specified during
embryogenesis and second larval instar, respectively.
The posterior compartment cells express the secreted
protein Hedgehog (HH) but do not express the Zn-finger
protein Cubitus interruptus (CI) which is required for
cells to respond to HH (Dominguez et al., 1996). In
contrast, the anterior compartment cells express Ci but
no HH. Because the posterior cells express HH but cannot
respond to it, whereas the Ci-expressing anterior cells
can respond to HH but do not make it, HH response only
occurs at the boundary between posterior and anterior
compartments (Dominguez et al., 1996) and the HH
signalling system is activated at the A/P boundary.
The Notch signalling system is activated by the
juxtaposition of the dorsal and ventral compartments (de
Celis et al., 1996; Diaz-Benjumea and Cohen, 1993).
Establishment of the dorso/ventral compartment boundary
is also initiated through the restricted expression of a
transcription factor. Dorsal cells express the LIM
mi
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domain/homeodomain-containing protein, Apterous
(Diaz-Benjumea and Cohen, 1993). Apterous induces
expression of the secreted protein Fringe (Irvine and
Wieschaus, 1994). Consequently, Fringe is expressed
dorsally, whereas the secreted protein Wingless is
expressed ventrally (Ng et al., 1996).
Two Notch ligands are also expressed in the wing
imaginal disc. Serrate is most strongly expressed in
dorsal cells (Kim et al., 1995), and Delta in ventral
cells (Doherty et al., 1996). The juxtaposition of
dorsal cells expressing Fringe and Serrate with ventral
cells which express Wingless and Delta results in the
localized activation of Notch on either side of the D/V
compartment boundary (de Celis et al., 1996; Irvine and
Wieschaus, 1994; Kim et al., 1995). The activated Notch
receptor then signals the induction of wing margin tissue
at this boundary (de Celis et al., 1996; Diaz-Benjumea
and Cohen, 1995; Doherty et al., 1996; Rulifson and
Blair, 1995).
It is not understood how Fringe, Wingless, Serrate
and Delta proteins induce the activation of Notch at the
D/V boundary but not elsewhere in the wing pouch. It is,
however, known that Serrate can only activate Notch in
ventral cells (Kim et al., 1995) whereas Delta can only
activate Notch in dorsal cells (Doherty et al., 1996).
In addition, ectopic expression of Fringe in ventral
cells causes the local activation of Notch with resulting
induction of margin tissue and wing outgrowth (Irvine and
Wieschaus, 1994; Kim et al., 1995). Removal of Fringe
from dorsal cells has a similar effect (Irvine and
Wieschaus, 1994). Both of these phenomena have
implicated Fringe in creating boundaries and in
controlling Notch activation (Irvine and Wieschaus,
1994).
The precise mechanism by which Fringe regulates
Notch activation at the D/V boundary in developing wing
discs remains to be elucidated. It has been demonstrated
that Fringe boundaries can upregulate Serrate protein
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expression in the wing disc (Kim et al., 1995). How this
occurs, as well as the potential participation of Delta
(Doherty et al., 1996) and Wingless (Couso and Martinez
Arias, 1994) in ectopic margin induction by Fringe
remains to be investigated. In addition, the roles of
Fringe or Fringe related proteins (Wu et al., 1996) in
other developmental contexts are also unknown.
Summary of Drawings
Certain embodiments of the invention are described,
reference being made to the accompanying drawings,
wherein:
Figures lA and 1B show ectopic expression of Manic
and Radical Fringe in Drosophila. Figure lA shows panels
(A) wild type wing; D) eye from GAL4P" fly (similar to
wild type); (B) and (E) wing and eye from flies crossed
to UAS- Manic Fringe and GAL4 driver; (C) and (F) wing
and eye from flies crossed to UAS-Radical Fringe and GAL4
drivers. B, C, E, F . ectopic expression with GAL4P"
driver. H, I . ectopic expression with GAL 4~5 driver.
Figure 1B shows panels (G), (H) wings from flies crossed
to VAS-Manic Fringe and GAL4 drivers (ectopic expression
with GAL4 c96 and GAL4 ~5 drivers respectively.
Figure 2 shows wings ectopically expressing Radical
Fringe with GAL4P" driver in different genetic
backgrounds (A) GAL4p"/+; UAS-Radical Fringe/+ wing (B, C)
fng52/+ and GAL4P''/+; UAS-Radical Fringe/fng52 respectively;
(D, H) D1''/+ and GAL4P'°/+; UAS-Radical Fringe/D1''
respectively; (E, I) SerR'''°6/+ and GAL4P"/+; UAS-Radical
Fringe/SerR''lo' respectively; ( F, J) Df ( 1 ) 1Va/+ and
GAL4p"/+; UAS-Radical Fringe/Df(1) 11~ respectively;
(G, K) wg~?/+ and GAL4P''/+; UAS-Radical Fringe/wg~"2
respectively;
Figure 3 shows Northern blot analysis of Fringe gene
expression in mice. Probe detects polyA+ RNA from whole
mouse embryos at the indicated stages of development (eg.
E7=7 day embryo) hybridized with each of the three mouse
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Fringe genes.
- 4 -
Figure 4 shows Northern blot analysis of Fringe gene
expression in mice. Probe detects RNA from adult mouse
tissues hybridized with each of the three mouse Fringe
genes.
Figures 5A and 5B show expression of mouse Fringe
genes in embryonic and selected adult tissues. Figure 5A
shows panels (A and B) Whole mount in situ hybridization
with Lunatic Fringe antisense riboprobes in E8.5 and E9.5
day embryos respectively; (C and E) Dark field section in
situ hybridization with Lunatic Fringe antisense
riboprobes in E11.5 and E12.5 day embryos respectively;
(D) Bright field of E12.5 section shown in panel E
(F) Dark field section in situ hybridization with Radical
Fringe antisense riboprobes in E12.5 day embryo
Figure 5B shows panels (G) Bright field of section
in situ hybridization with Lunatic Fringe antisense
riboprobes in E13.5 day embryo with close up of grains on
S-shaped bodies in kidney; (H and J) Dark field section
in situ hybridization with Lunatic Fringe antisense
riboprobes in adult thymus and spleen respectively
(I) Bright field of spleen section shown in panel J
(K and L) Dark field and bright field of section in situ
hybridization with Manic Fringe antisense riboprobes in
adult spleen, with close up of grains in megakaryocytes
shown in panel L.
Figure 6 shows Fringe gene switch in differentiation
in the mouse. (A, B, C) Dark field section in situ
hybridization with antisense probes to Lunatic (A), Manic
(B) and Radical (C) Fringe genes in E10.5 mouse embryo
neural tubes. vz is the ventricular zone and mz is the
marginal zone of the neural tube. (D, E, F) Dark field
section in situ hybridization with antisense probes to
Lunatic (D) , Manic (E) and Radical (F) Fringe genes in
adult tongue. be is the basal epithelium and sbe is the
suprabasal
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epithelium.
Figure 7 shows expression of mouse. Notch ligands
and Lunatic Fringe during somitogenesis and neural tube
patterning.
(A, B, C) Whole mount in situ hybridization with
antisense probes to Deltal(A), Lunatic Fringe(B) and
Serratel(C) genes in E8.5 mouse embryo posterior
mesoderm. arrowhead points to a forming somite.
(D, E, F) Dark field section in situ hybridization with
antisense probes to Deltal(A), Lunatic Fringe(B) and
Serratel(C) genes in E10.5 mouse embryo neural tube.
Figure 8 shows a schematic diagram of the proposed
model for Fringe proteins as regulators of Notch
specificity and sensitivity for its ligands.
Figure 9 shows a schematic diagram for the model of
Figure 8 applied to the development of wing margin in
Drosophila.
Detailed Description of Invention
The interaction of Notch receptors with Notch
ligands plays an important role in development in mammals
and in insects. Activation of a Notch receptor by a
Notch ligand initiates signal transduction, the signal
being communicated to the cell via the cytosolic domain
of the Notch receptor protein.
Notch ligands which activate the Notch receptor and
initiate signal transduction include the DSL group of
ligands, for example, Delta protein, Serrate protein and
Lag-2 protein.
The inventors have cloned and characterized three novel
mammalian genes which are related to Drosophila Fringe,
as described in the Examples herein. These mammalian
genes are expressed in tissues which are undergoing
Notch-dependent development and differentiation.
Experiments in Drosophila with these mammalian fringe
genes revealed that the Fringe proteins control or
modulate activation of the Notch receptor by Notch
ligands. The Fringe system of proteins can be used to
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induce new cell fates at tissue boundaries, to reinforce
predetermined tissue boundaries and to block Notch
signalling in differentiating cells.
Mammals have at least four Notch receptors which can
interact with Notch ligands (Egan et al. (1997), Current
Topics in Microbiology and Immunology, 228, 273 - 324).
The three mammalian Fringe proteins, Lunatic Fringe,
Manic Fringe and Radical Fringe, act to promote or
inhibit the interaction of Notch receptors with Notch
ligands.
The cDNA sequences of murine Lunatic Fringe
(Sequence ID:NO:I), Manic Fringe (Sequence ID N0:3) and
Radical Fringe (Sequence ID N0:5) are shown in Tables lA,
2A and 3A respectively. The corresponding amino acid
sequences for Lunatic Fringe protein (Sequence ID N0:2),
Manic Fringe protein (Sequence ID N0:4) and Radical
Fringe protein (Sequence ID N0:6) are shown in Tables 1B,
2B and 3B respectively.
Undifferentiated mammalian cells appear to express
Lunatic Fringe but not Manic Fringe or Radical Fringe.
During differentiation, there is a switch over to
expression of Manic and Radical and a cessation of
expression of Lunatic.
The present invention demonstrates that the three
mammalian Fringe proteins may be used to facilitate or
block the Notch signal transduction pathway and Notch-
dependent processes by regulating the sensitivity of
Notch receptors for their specific ligands.
Isolated Nucleic Acids
In accordance with one series of embodiments, this
invention provides isolated nucleic acids corresponding
to or related to the nucleic acid sequences disclosed
herein which encode the murine Fringe proteins, Lunatic
Fringe, Radical Fringe and Manic Fringe.
One of ordinary skill in the art is now enabled to
identify and to isolate mammalian Frinae genes or cDNAs
which are allelic variants of the disclosed Mammalian
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Fringe sequences or are homologues thereof, in other
species, including humans, using standard hybridization
screening or PCR techniques.
In one embodiment, the invention provides cDNA
sequences encoding the murine Lunatic Fringe, Manic
Fringe and Radical Fringe proteins (Sequence ID NOS: i, 3
and 5 respectively) comprising the nucleotide sectuences
of Sequence ID NOS: 2, 4 and 6 respectively.
Also provided are portions of the Fringe gene
sequences useful as probes in PCR primers or for encoding
fragments, functional domains or antigenic determinants
of Fringe proteins.
The invention also provides portions of the
disclosed nucleic acid sequences comprising about 10
l5 consecutive nucleotides (eg. for use as PCR primers) to
nearly the complete disclosed nucleic acid sequences.
The invention provides isolated nucleic acid sequences
comprising sequences corresponding to at least 10,
preferably 15 and more preferably at least 20 consecutive
nucleotides of the Fringe genes as disclosed or enabled
herein or their complements.
In addition, the isolated nucleic acids of the
invention include any of the above described nucleotide
sequences included in a vector.
Substantially Pure Proteins
In accordance with a further series of embodiments,
this invention provides substantially pure mammalian
Fringe proteins, fragments of these proteins and fusion
proteins including these proteins and fragments.
The proteins, fragments and fusion proteins have
utility, as described herein, for the preparation of
polyclonal and monoclonal antibodies to mammalian Fringe
proteins, for the identification of binding partners of
the mammalian Fringe proteins and for diagnostic and
therapeutic methods, as described herein. For these
uses, the present invention provides substantially pure
proteins, polypeptides or derivatives of polypeptides
m i
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which comprise portions of the mammalian. Fringe amino
acid sequences disclosed or enabled herein and which may
vary from about 4 to 5 amino acids (e.g. for use as
immunogens? to the complete amino acid sequence of the
proteins. The invention provides substantially pure
proteins or polypeptides comprising sequences
corresponding to at least 5, preferably at least l0 and
more preferably 50 or 100 consecutive amino acids of the
mammalian Fringe proteins disclosed or enabled herein.
The proteins of the invention may be isolated and
purified by any conventional method suitable in relation
to the properties revealed by the amino acid sequences of
these proteins.
Alternatively, cell lines may be produced which
l5 overexpress the Fringe gene products, allowing
purification of the proteins for biochemical
characterization, large-scale production, antibody
production and patient therapy.
For protein expression, eukaryotic and prokaryotic
expression systems may be generated in which a Fringe
gene sequence is introduced into a plasmid or other
vector which is then introduced into living cells.
Constructs in which the Fringe cDNA sequence containing
the entire open reading frame is inserted in the correct
orientation into an expression plasmid may be used for
protein expression. Alternatively, portions of the
sequence may be inserted. Prokaryotic and eukaryotic
expression systems allow various important functional
domains of the protein to be recovered as fusion proteins
and used for binding, structural and functional studies
and also for the generation of appropriate antibodies.
Typical expression vectors contain promoters that
direct the synthesis of large amounts of mRNA
corresponding to the gene. They may also include
sequences allowing for their autonomous replication
within the host organism, sequences that encode genetic
traits that allow cells containing the vectors to be
selected, and sequences that increase the efficiency with
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which the mRNA is translated. Stable long-term vectors
may be maintained as freely replicating entities by using
regulatory elements of viruses. Cell lines may also be
produced which have integrated the vector into the
genomic DNA and in this manner the gene product is
produced on a continuous basis.
Expression of foreign sequences in bacteria such as
E. coli require the insertion of the sequence into an
expression vector, usually a plasmid which contains
several elements such as sequences encoding a selectable
marker that assures maintenance of the vector in the
cell, a controllable transcriptional promoter which upon
induction can produce large amounts of mRNA from the
cloned gene, translational control sequences and a
polylinker to simplify insertion of the gene in the
correct orientation within the vector. A relatively
simple E. coli expression system utilizes the lac
promoter and a neighboring lacZ gene which is cut out of
the expression vector with restriction enzymes and
replaced by the Fringe gene sequence. In vitro
expression of proteins encoded by cloned DNA is also
possible using the T7 late-promoter expression system.
Plasmid vectors containing late promoters and the
corresponding RNA polymerases from related bacteriophages
such as T3, T5 and SP6 may also be used for in vitro
production of proteins from cloned DNA. E. coli can also
be used for expression by infection with M13 Phage mGPI-
2. E. coli vectors can also be used with phage Lambda
regulatory sequences, by fusion protein vectors, by
maltose-binding protein fusions, and by glutathione-S-
transferase fusion proteins.
Eukaryotic expression systems permit appropriate
post-translational modifications to expressed proteins.
This allows for studies of the fringe genes and gene
products including determination of proper expression and
post-translational modifications for biological activity,
identifying regulatory elements in thel5~ region of the
gene and their role in tissue regulation of protein
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expression. It also permits the production of large
amounts of normal proteins for isolation and
purification, to test the effectiveness of
pharmacological agents or as a component of a signal
transduction system to study the function of the normal
complete protein, specific portions of the protein, or of
naturally occurring polymorphisms and artificially
produced mutated proteins.
The Fringe DNA sequences can be altered using
procedures such as restriction enzyme digestion, DNA
polymerase fill-in, exonuclease deletion, terminal
deoxynucleotide transferase extension, ligation of
synthetic or cloned DNA sequences and site-directed in
vitro mutagenesis, including site-directed sequence
alteration using specific oligonucleotides together with
PCR.
Once the appropriate expression vector containing
the selected gene is constructed, it is introduced into
an appropriate host cell by transformation techniques
including calcium phosphate transfection, DEAE-dextran
transfection, electroporation, microinjection, protoplast
fusion and liposome-mediated transfection.
The host cell which may be transfected with the
vector of this invention may be selected from the group
consisting of E. Coli, Pseudomonas, Bacillus subtilis, or
other bacilli, other bacteria, yeast, fungi, insect
(using baculoviral vectors for expression), mouse or
other animal or human tissue cells. Mammalian cells can
also be used to express the Fringe proteins using a
vaccinia virus expression system.
Methods for producing appropriate vectors, for
transforming cells with those vectors and for identifying
transformants are described in the scientific literature,
for example in Sambrook et al. (1989), Molecular Cloning:
A Laboratorv Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. or latest edition
thereof .
The cellular distribution of Fringe proteins in
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tissues can be analyzed by reverse transcriptase PCR
analysis. Antibodies can also be generated for several
applications including both immunocytochemistry and
immunofluorescence techniques to visualize the proteins
directly in cells and tissues in order to establish the
cellular location of the proteins.
The present invention includes effective fragments
or analogues of the Fringe proteins described herein.
"Effective" fragments or analogues retain the activity of
the described Fringe proteins to modulate Notch -
receptor/Notch ligand interactions.
The term °analogue "extends to any functional and/or
chemical equivalent of a mammalian Fringe protein and
includes proteins having one or more conservative amino
acid substitutions, proteins incorporating unnatural
amino acids and proteins having modified side chains.
Examples of side chain modifications contemplated by
the present invention include modification of amino
groups such as by reductive alkylation by reaction with
an aldehyde followed by reduction with NaBH4; amidation
with methylacetimidate; acetylation with acetic
anhydride; carbamylation of amino groups with cyanate;
trinitrobenzylation of amino groups with 2, 4, 6,
trinitrobenzene sulfonic acid (TNBS); alkylation of amino
groups with succinic anhydride and tetrahydrophthalic
anhydride; and pyridoxylation of lysine with pyridoxal-
5'-phosphate followed by reduction with NaBH4.
The guanidino group of arginine residues may be
modified by the formation of heterocyclic condensation
products with reagents such as 2, 3-butanedione,
phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide
activation via -acylisourea formation followed by
subsequent derivatisation, for example, to a
corresponding amide.
Sulfhydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide;
performic acid oxidation to cysteic acid; formation of
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mixed disulphides with other thiol compounds; reaction
with maleimide, malefic anhydride or other substituted
maleimide; formation of mercurial derivatives using 4-
chloromercuribenzoate, 4-chloromercuriphenylsulfonic
acid, phenylmercury chloride, 2-chloromercuric-4-
nitrophenol and other mercurials; carbamylation with
cyanate at alkaline pH.
Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the
indole ring with 2-hydroxy-5-nitrobenzyl bromide or
sulphonyl halides. Tyrosine residues may be altered by
nitration with tetranitromethane to form a 3-
nitrotyrosine derivative.
Modification of the imidazole ring of a histidine
residue may be accomplished by alkylation with iodacetic
acid derivatives of N-carbethoxylation with
diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not
limited to, use of norleucine, 4-amino butyric acid, 4-
amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic
acid-, t-butylglycine, norvaline, phenylglycine,
ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic
acid, 2-thienyl alanine and/or D-isomers or amino acids.
Examples of conservative amino acid substitutions
are substitutions within the following five groups of
amino acids (amino acids are identified by the
conventional single letter code): Group 1: F Y W; Group
2: V L I; Group 3: H K R; Group 4: M S T P A G; Group 5:
D E .
Fragments or analogues of the mammalian Fringe
proteins of the invention may be conveniently screened
for their effectiveness by a variety of methods.
For example, a Drosophila-based assay can be
employed. In Drosophila, the mammalian Manic and Radical
Fringe proteins interfere with specific Notch-dependent
developmental events (eg. Manic Fringe blocks wing margin
formation, and causes small eyes and fusion of ocelli in
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specific transgenic lines whereas Radical Fringe blocks
Margin induction and causes extra bristles to form and
can induce wing vein deltas in specific transgenic lines)
(Cohen et al. (1997), Nature Genetics, I6, 283-288 and as
described herein). Transgenic Drosophila may be used to
screen for Fringe proteins, analogues and fragments which
enhance or suppress these phenotypes. In addition, drugs
which enhance or suppress these phenotypes could be
identified which would be useful therapeutically in
humans to alter Fringe function and Notch signalling.
Alternatively, a cell culture assay could be used as
a screen. It has been reported that differentiation of
C2C12 myoblast cells can be blocked in culture by
activation of Notch expressed on the cell surface:
(Lindsell et al. (1995), Cell, 80, 909-917; Luo et al.,
(1997), Mol. Cell. Biol., 17, 6057-6067). This
activation can occur as a result of presenting DSL
ligands to the C2C12 cells. This is achieved by
coculturing cells expressing Notch ligands with the C2C12
cells. This assay can be easily adapted to screen for
the effect of Fringe proteins, analogues and fragments to
regulate the activation of mammalian Notch receptors by
their ligands. Similarly, any cell culture system which
shows in vitro differentiation dependent on Notch
activation may form the basis of a screening assay.
Antibodies
In order to prepare polyclonal antibodies, fusion
proteins containing defined portions or all of the Fringe
proteins can be synthesized in bacteria by expression of
corresponding DNA sequences in a suitable cloning
vehicle. Fusion proteins are commonly used as a source
of antigen for producing antibodies. Two widely used
expression systems for E. coli are glutathione-S-
tranferase or maltose binding protein fusions using the
pUR series of vectors and trpE fusions using the pATH
vectors. The protein can then be purified, coupled to a
carrier protein if desired, and mixed with Freund~s
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adjuvant (to help stimulate the antigenic response of the
animal) and injected into rabbits or other appropriate
laboratory animals. Alternatively, the protein can be
isolated from Fringe protein-expressing cultured cells.
Following booster injections at weekly intervals, the
rabbits or other laboratory animals are then bled and the
sera isolated. The sera can be used directly or purified
prior to use by various methods including affinity
chromatography employing Protein A-Sepharose, antigen
Sepharose or Anti-mouse-Ig-Sepharose. The sera can then
be used to probe protein extracts from cells and tissues
run on a polyacrylamide gel to identify the Fringe
protein. Alternatively, synthetic peptides can be made
to the antigenic portions of the proteins and used to
IS inoculate the animals.
The most common practice is to choose a 10 to 15
residue peptide corresponding to the carboxyl or amino
terminal sequence of a protein antigen and to chemically
cross-link it to a carrier molecule such as keyhole
limpet haemocyanin or BSA. However, if an internal
sequence peptide is desired, selection of the peptide is
based on the use of algorithms that predict potential
antigenic sites. These predictive methods are, in turn,
based on predictions of hydrophilicity (Kyte and
Doolittle (29), Hopp and Woods (30) or secondary
structure (Chou and Fasman (31)). The objective is to
choose a region of the protein that is either surface
exposed such a hydrophilic region or a region
conformationally flexible relative to the rest of the
structure, such as a loop region or a region predicted to
form a [3-turn. The selection process is also limited by
constraints imposed by the chemistry of the coupling
procedures used to attach peptide to carrier protein. A
carboxyl-terminal peptide is chosen because they are
often more mobile than the rest of the molecule and the
peptide can be coupled to a carrier in a straightforward
manner using glutaraldehyde. The amino-terminal peptide
has the disadvantage that it may be modified post-
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translationally by acetylation or by the removal of a
leader sequence. A comparison of the protein amino acid
sequence between species can yield important information.
Those regions with sequence differences are likely tc be
immunogenic. Synthetic peptides can also be synthesized
as immunogens as long as they mimic the native antigen as
closely as possible.
It is understood by those skilled in the art that
monoclonal anti-Fringe antibodies may also be produced
using Fringe protein obtained from cells actively
expressing the protein or by isolation from tissues. The
cell extracts, or recombinant protein extracts,
containing the Fringe protein, are injected in Freund's
adjuvant into mice. After being injected 9 times over a
three week period, the mice spleens are removed and
resuspended in phosphate buffered saline (PBS). The
spleen cells serve as a source of lymphocytes, some of
which are producing antibody of the appropriate
specificity. These are then fused with a permanently
growing myeloma partner cell, and the products of the
fusion are plated into a number of tissue culture wells
in the presence of a selective agent such as HAT. The
wells are then screened by ELISA to identify those
containing cells making binding antibody. These are then
plated and after a period of growth, these wells are
again screened to identify antibody-producing cells.
Several cloning procedures are carried out until over 900
of the wells contain single clones which are positive for
antibody production. From this procedure a stable line
of clones which produce the antibody is established. The
monoclonal antibody can then be purified by affinity
chromatography using Protein A Sepharose, ion-exchange
chromatography, as well as variants and combinations of
these techniques.
Truncated versions of monoclonal antibodies may also
be produced by recombinant techniques in which,plasmids
are generated which express the desired monoclonal
antibody fragments) in a suitable host. Antibodies
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specific for mutagenic epitopes can also be generated.
The mammalian proteins, Fringe analogues and
fragments thereof and/or peptides of the invention are
also useful as antigens in immunoassays including enzyme-
s linked immunosorbent assays (ELISA>, radioimmunoassays
(RIA) and other non-enzyme linked antibody binding assays
or procedures known in the art for the detection of the
protein.
Pharmaceutical Compositions
In a further embodiment, this invention provides
pharmaceutical compositions for the treatment of
mammalian disorders which involve inappropriate Fringe
function and/or Notch signalling, comprising a
therapeutic amount of a Fringe protein, an analog or an
effective derivative thereof in association with a
pharmaceutical carrier.
Administration of a therapeutically active amount of
a pharmaceutical composition of the present invention
means an amount effective, at dosages and for periods of
time necessary to achieve the desired result. This may
also vary according to factors such as the disease state,
age, sex, and weight of the subject, and the ability of
the mammalian Fringe protein to elicit a desired response
in the subject. Dosage regima may be adjusted to provide
the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may
be proportionally reduced as indicated by the exigencies
of the therapeutic situation.
By pharmaceutically acceptable carrier as used
herein is meant one or more compatible solid or liquid
delivery systems. Some examples of pharmaceutically
acceptable carriers are sugars, starches, cellulose and
its derivatives, powdered tragacanth, malt, gelatin,
collagen, talc, stearic acids, magnesium stearate,
calcium sulfate, vegetable oils, polyols, agar, alginic
acids, pyrogen-free water, isotonic saline, phosphate
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buffer, and other suitable non-toxic substances used in
pharmaceutical formulations. Other excipients such as
wetting agents and lubricants, tableting agents,
stabilizers, anti-oxidants and preservatives are alsc
contemplated.
The compositions described herein can be preparea by
known methods for the preparation of pharmaceutically
acceptable compositions which can be administered to
subjects, such that an effective quantity of the active
substance is combined in a mixture with a
pharmaceutically acceptable carrier. Suitable carriers
and formulations adapted for particular modes of
administration are described, for example, in Remington~s
Pharmaceutical Sciences (Remington's Pharmaceutical
Sciences, Mack Publishing Company, Easton, Pa., USA
1985). On this basis the compositions include, albeit
not exclusively, solutions of the substance in
association with one or more pharmaceutically acceptable
vehicles or diluents, and contained in buffered solutions
with a suitable pH and iso-osmotic with the physiological
fluids.
The pharmaceutical compositions of the invention may
be administered therapeutically by various routes such as
by injection or by oral, nasal, buccal, rectal, vaginal,
transdermal or ocular routes in a variety of
formulations, as is known to those skilled in the art.
Binding t~artners
The mammalian Fringe proteins, expressed as fusion
proteins, can be utilized to identify small peptides that
bind to these proteins. In one approach, termed phage
display, random peptides (up to 20 amino acids long) are
expressed with coat proteins (geneIII or geneVIII) of
filamentous phage such that they are expressed on the
surface of the phage thus generating a library of phage
that express random sequences. A library of these random
sequences is then selected by incubating the library with
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the mammalian Fringe protein or fragments thereof and
phage that bind to the protein are then eluted either by
cleavage of Fringe from the support matrix or by elution
using an excess concentration of soluble Fringe protein
S or fragments. The eluted phage are then repropagated and
the selection repeated many times to enrich for higher
affinity interactions. The random peptides can either be
completely random or constrained at certain positions
through the introduction of specific residues. After
several rounds of selection, the final positive phage are
sequenced to determine the sequence of the peptide.
An alternate but related approach uses affinity
purification techniques. Fringe proteins are immobilised
on a suitable solid support. Preparations such as cell
extracts which may contain Fringe protein binding
partners are passed over the affinity matrix and any
bound material is eluted and microsequenced. Suitable
methods are available in the scientific literature, for
example in Bartley et al., Nature (1994), 368, 558-560.
Expression cloning, for example through expression
of cDNA libraries in Cos or other cells followed by
binding of labelled Fringe protein to the transfected
cells, may also be used to screen for Fringe protein
binding partners, for example as described in Matthews et
al., Cell (1991) 65, 973-982.
The identification of proteins or peptides that
interact with Fringe Proteins can provide the basis for
the design of peptide antagonists or agonists of Fringe
protein function. Further, the structure of these
peptides determined by standard techniques such as
protein NMR or X-ray crystallography can provide the
structural basis for the design of small molecule drugs.
Animal Models
The present invention also provides for the
production of transgenic non-human animal models for the
study of mammalian Fringe gene function, for the
screening of candidate pharmaceutical compounds, for the
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creation of explanted mammalian cell cultures which
express the Fringe proteins or in which a Fringe gene has
been inactivated by knock-out deletion, and for the
evaluation of potential therapeutic interventions.
The invention enables a transgenic animal, including
a transgenic insect, wherein a genome of the animal or o~
an ancestor of the animal has been modified by
introduction of a transgene comprising a mammalian fringe
gene under the transcriptional control of tissue
l0 restricted regulatory elements including the mouse
mammary-tumour virus long term repeat sequences.
Transgenic fruit flies which express mammalian
Frincre genes may be made as described in the Examples
herein. Such transgenic flies may be used to screen for
compounds which can repair developmental defects observed
in these transgenic flies.
Transgenic animals may also be made and used
similarly. Further, transgenic animals with
inappropriate expression of Fringe proteins may be
examined for phenotypic changes, for example tumour
development, and may be used to screen for compounds with
potential as pharmaceuticals. Compounds which provide
reversal of the phenotypic changes are candidates for
development as pharmaceuticals.
Transgenic animal models in accordance with the
invention can be created by introducing a DNA sequence
encoding a selected mammalian Fringe protein either into
embryonic stem (ES) cells of a suitable animal, for
example a mouse, by transfection or microinjection, or
into a germ line or stem cell by a standard technique of
oocyte microinjection.
The ES cells are inserted into a young embryo and
this embryo or an injected oocyte are implanted into a
pseudo-pregnant foster mother to grow to term.
The techniques for generating transgenic animals are
now widely known and are described in detail, for
example, in Hogan et al., (1986), and M. Capecchi (1989).
i
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Methods of Treatment
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In accordance with one embodiment, the present
invention enables a method for preventing or treating a
disorder in a mammal characterised by an abnormality in a
signal transduction pathway which involves an interaction.
between a Notch protein and a Notch ligand, by modulating
the Notch protein/Notch ligand interaction.
The Notch protein/Notch ligand interaction is
modulated, in one embodiment, by administration of a
mammalian Fringe protein or an effective fragment or
analogue therof.
A further embodiment is a method for treating or
preventing such a disorder by promoting or inhibiting the
interaction of Notch with its ligands Serrate and Delta
by administration of an effective amount of Lunatic
Fringe protein, Manic Fringe protein or Radical Fringe
protein or of a derivative thereof.
In a further embodiment, the invention enables a
method for promoting differentiation of a mammalian cell
by suppressing expression of Lunatic Fringe protein in
the cell and/or promoting expression of Radical Fringe
protein and/or Manic Fringe protein in the cell.
In a further embodiment, the invention enables a
method for suppressing differentiation of a cell by
suppressing expression of Radical Fringe protein and/or
Manic Fringe protein in the cell and/or promoting
expression of Lunatic Fringe protein in the cell.
It has been recently demonstrated that the Notch4
receptor is highly expressed in endothelial cellsl. In
addition, the Jaggedl protein is induced by fibrin in
human endothelial cells2. Notch signalling may therefore
be an important regulator of endothelial cell migration,
proliferation and cell fate specification. In humans,
vasculature and cardiovascular system malfunction
accounts for a very large number of deaths.
One important application of the Fringe proteins,
and analogues is regulation of the response of Notch in
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mammalian blood vessels. For example, during
angioplasty, application of Fringe proteins, Fringe anti-
sense oligonucleotides3 or other reagents to modify
fringe function locally may be used to alter Notch
activation and therefore the migration and proliferation
of cells within vessels. These reagents may also be used
to regulate or treat symptoms related to atherosclerosis,
cardiovascular disease or diseases related to
angiogenesis, including cancer.
Screening Methods
In a further embodiment, the invention enables a
method for identifying compounds which can modulate the
expression of mammalian a Fringe gene comprising
contacting a cell with a candidate compound wherein
the cell includes a regulator of a Fringe gene operably
joined to a coding region; and
detecting a change in expression of the coding
region.
In a further embodiment, the invention enables a
method for identifying compounds which can selectively
bind to a mammalian Fringe protein comprising
providing a preparation including at least one
mammalian Fringe protein;
contacting the preparation with a candidate
compound; and
determining binding of the Fringe protein to the
compound.
Suitable methods for such screening include affinity
chromatography, co-immunoprecipitation, biomolecular
interaction assay.
In a further embodiment, the invention enables a
method for identifying compounds which can modulate the
activity of a Fringe protein to promote or inhibit the
interaction of a Notch receptor and a Notch ligand.
Methods are also enabled to identify compounds which
can modulate the interaction of a Fringe protein with a
Notch receptor signal transduction pathway.
1 I 1 i
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As an example, wing development in Drosophila
melanogaster can be used as a screening tool for
evaluating fringe/notch interactions.
Cell culture assays may be developed to measure
S fringe function in vitro. Inhibition of the specific
fringe response including an alteration in notch function
could be used to assay for chemicals which inhibit or
enhance fringe function.
In a further embodiment, the invention enables a
method for identifying a compound useful for preventing
or treating a disorder in a mammal characterised by an
abnormality in a signal transduction pathway which
involves an interaction between a Notch receptor and
Notch ligand, the method comprising screening candidate
compounds for their ability to promote or inhibit the
interaction of a Fringe protein with the Notch signal
transduction pathway.
In a further embodiment, the invention enables a
method for promoting or inhibiting an interaction between
a Notch receptor and a Notch ligand comprising
administering an effective amount of a Fringe protein or
of a fragment, analogue or derivative thereof.
In a further embodiment, the invention enables a
method for diagnosing in a subject a disorder
characterised by abnormal expression of a Fringe protein
comprising
obtaining a tissue sample from the subject;
determining Fringe protein expression in the tissue
sample.
Tissue samples could be used for isolation of RNA
which would then be subjected to RT-PCR analysis using
specific primers for fringe genes in order to amplify the
cDNA for sequencing. Control tissues could be used for
comparison of sequence.
With the identification of the mammalian Fringe gene
sequences and gene products, nucleotide probes and
antibodies raised to the gene products can be used in a
variety of hybridisation and immunological assays to
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screen for and detect the presence of either a normal or
mutated gene or gene product.
Patient therapy through removal or blocking of a
mutant gene product, as well as supplementation with a
normal gene product by amplification, by genetic and
recombinant techniques or by immunotherapy car. now be
achieved.
Correction or modification of the defective gene
product by protein treatment immunotherapy (using
antibodies to the defective protein) or knock-out of the
mutated gene together with wild-type supplementation is
now also possible. Suitable methods are described or
referenced for example, in Crystal, R.G. (1995), Science,
270, 404-410.
Fringe proteins as regulators of Notch responsiveness
Three mammalian homologues of Drosophila fringe have
been isolated. The mammalian proteins share extensive
sequence homology with each other as well as with Xenopus
and Drosophila Fringe proteins in the C-terminal region,
which is predicted to encode the mature Fringe
polypeptide in each case.
Severe loss of function mutants in Drosophila Fringe
are lethal as homozygotes, and therefore this gene must
be essential for development (Irvine and Wieschaus,
1994). D-fringe function has thus far only been
characterized in wing margin specification (Irvine and
Wieschaus, 1994; Kim et al., 1995). D-Fringe is required
in dorsal cells and must not be expressed in ventral
cells of the wing pouch in order for margin tissue to be
induced at the D/V boundary. Destruction of this D/V
Fringe+/Fringe- expression boundary through ectopic
expression of D-Fringe in ventral cells at the D/V
boundary, or through loss of D-Fringe expression in
dorsal cells at the D/V boundary both result in loss of
margin tissue. The similarity of phenotype caused by
ectopic ventral expression and loss of dorsal expression
has led to the suggestion that Fringe is a
m
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boundary-organizing molecule (Irvine and Wieschaus,
1994). The presence of a Fringe+/Fringe- expression
boundary is therefore thought to be important, rather
than simply the presence or absence of Fringe in any
particular cell.
Analysis of Fringe function in Drosophi~a wing
development must be considered in the context of other
molecules which are required for margin induction at the
D/V boundary. These include Serrate which is expressed
dorsally as well as Delta and Wingless which are
expressed ventrally. These four ligands all cooperate to
activate Notch exclusively at the D/V boundary. The
generation of an ectopic Fringe boundary in the ventral
wing pouch must, therefore, be considered in the context
of Delta and Wingless which are expressed in the ventral
compartment. Similarly, the generation of a novel Fringe
boundary in the dorsal wing at the intersection of
Fringe- clones with Fringe expressing dorsal cells must
be viewed in the context of the dorsal compartment which
expresses Serrate.
Expression of either Manic Fringe or Radical Fringe
in Drosophila, using the GAL4pt~ driver, results in loss
of margin tissue. Disruption of margin formation by
these two Fringe proteins is not associated with creation
of ectopic margins, indicating that margin destruction
and margin induction are genetically separable functions.
Other phenotypes induced by these two mammalian fringe
genes suggest that Manic Fringe and Radical Fringe
interfere with Notch-mediated processes in several
tissues. Loss of function of either the Serrate or the
Delta ligands for Notch results in loss of margin
formation in the wing (Doherty et al., 1996; Kim et al.,
1995), and both Manic and Radical give this same
phenotype at the margin. Analysis of margin alone cannot
therefore be used to distinguish between activities which
directly inhibit Serrate, Delta or Notch. Examination of
fly tissues which are dependent on either Serrate or
Delta (but not both) indicates that Manic Fringe and
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Radical Fringe misexpression yielded distinct
Serrate-like or Delta-Like phenotypes. If these genes
inhibited Notch via all Notch ligands, then both genes
should give the same phenotypes in each tissue examined.
In every analysis of Lunatic Fringe expression
during mouse development, it was found that Lunatic
Fringe was expressed in an undifferentiated cell
compartment. Interestingly, in two cases examined in
detail, somitogenesis and neurogenesis, it was found that
l0 Lunatic Fringe expression was localized to cells which
were responding to Delta expression in neighbouring
cells. It is predicted that Lunatic Fringe may be a
co-agonist for Delta by facilitating the Delta-mediated
activation of Notch. Separating agonist functions on to
two polypeptides, Deltal and Lunatic Fringe, could allow
for precise control of Notch activation (Figure 8).
Expression analysis also revealed that Lunatic
Fringe is shut off as cells differentiate.
Differentiation is then accompanied by a Fringe
expression switch as Manic and/or Radical Fringe genes
are turned on. This Fringe switch could function to
reinforce the differentiation decision and regulate the
ratio of undifferentiated cells to their committed
progeny.
Fringe regulates Notch activation at boundaries
The data obtained by the inventors on the function
of Fringe proteins analyzed in Drosophila and expression
in the mouse lead to the model shown in schematic form in
Figures 8 and 9. The loss of function Drosophila fringe
allele fng$2 produces wing vein deltas (Figure 2B).
Ectopic expression in Drosophila of Radical Fringe, which
appears to antagonize some Delta functions, also enhanced
the phenotype of fng52 /+. This suggests that a rate
limiting function of Drosophila Fringe may be to
facilitate the activation of Notch by Delta. This
hypothesis would explain the observation that ectopic
Delta can only induce novel wing margins on the dorsal
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wing surface (Doherty et al., 1996)
which expresses D-Fringe, but not on the ventral wing
surface which does not express D-Fringe. In addition, if
D-Fringe facilitates the activation of Notch by Delta,
then ectopic Fringe would be expected to induce novel
ventral margin by cooperating with ventrally expressed
Delta to activate Notch. This is also the case, and
therefore, all data are consistent with D-Fringe
facilitating Delta activation of Notch during wing
development.
Such synergy between D-Fringe and Delta to activate
Notch, however, does not explain why deletion of Fringe
in dorsal clones can induce an ectopic margin (Irvine and
Wieschaus, 1994). Perhaps Drosophila Fringe, like Manic
I5 Fringe, inhibits activation of Notch by Serrate in the
wing disc. Such inhibition would explain why ectopic
Serrate can only induce novel wing margins on the ventral
wing surface which does not normally express D-Fringe
(Kim et al., 1995), but not on the dorsal wing surface
which does express D-Fringe. In addition, if D-Fringe
inhibits the activation of Notch by Serrate, then
induction of a novel margin in dorsal tissue would occur
at the intersection of fringe- clones with Fringe
expressing cells. This induction occurs because dorsal
Serrate could activate Notch in the cells of the fringe-
clones. Thus a dual mechanism is proposed for regulation
of Notch by D-Fringe: (i) Fringe synergizes with Delta to
activate Notch at the D/V boundary and (ii) Fringe
antagonizes Serrate in the dorsal compartment (Figures 8
and 9 ) .
The question arises as to how dorsally expressed
D-Fringe and Serrate and ventrally expressed Wingless and
Delta cooperate to induce Notch activation only in a
single row of cells on either side of the D/V boundary.
It is proposed that Fringe blocks Serrate from
functioning everywhere in the dorsal compartment so that
the latter can only activate Notch in the ventral cells
which abut the dorsal compartment. (Serrate may even
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require Wingless to activate Notch in these cells, in
which case Wingless and Fringe may both be modifiers of
Notch specificity.) In addition, Wingless may block
Delta from functioning in the ventral compartment
(Axelrod et al., 1996). Delta would only activate Notci:
in the single row of Fringe-expressing dorsal cells wr.ich
abut the ventral compartment. Thus, specific
co-agonists/antagonists (D-Fringe and perhaps Wingless)
are localized in such a way that the membrane bound Notch
ligands only activate Notch at the D/V boundary (Figure
9) .
Vertebrates may use Lunatic Fringe protein to
localize Notchl activation during somitogenesis, since
Deltal is highly expressed in the forming somite and
Lunatic Fringe is highly expressed in the surrounding
mesoderm. Deltal may only activate Notchl at the
boundary of these expression domains. This hypothesis
is consistent with the requirement for Notchl in blocking
somite differentiation between the forming somites
(Conlon et al., 1995). Similarly, Lunatic Fringe
expression in the ventricular zone of the neural tube may
render cells responsive to Deltal, which inhibits
differentiation of ventricular neuroblasts (Chitnis et
al., 1995). The three mammalian fringe proteins may be
used either to facilitate or to block Notch-dependent
processes throughout development and adult life by
regulating the sensitivity of Notch for specific
membrane-bound ligands.
It is not yet clear from Drosophila studies whether
D-Fringe is required for Delta signalling throughout
development or just during specific cell fate decisions.
It is interesting to note, however, that GAL4ptC driven
expression of Radical Fringe inhibited only a small
fraction of Delta-dependent processes which occur in
tissue where ptc is expressed (Muskavitch, 1994). For
example, ptc is expressed in the eye and yet no
phenotypes) has been observed which would indicate that
Delta signalling had been compromised in this tissue of
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GAL4~'t~/Radical Fringe flies. D-Fringe expression has not
been detected in the S2 cell line, which when transfected
with Notch, can respond to Delta in vitro (not shown)
(Fortini and Artavanis-Tsakonas, 1994). Therefore Delta
does not always require D-Fringe to activate Notch. The
two tissues where Radical Fringe inhibited Delta
functions, wing and scutellum, are two locations which
express Wingless. It is proposed that D-Fringe, and
perhaps Lunatic Fringe in mammals, are required
coagonists for Delta only in the presence of Wingless or
other Wnt family proteins. Somitogenesis and
neurogenesis in mammals, like wing margin formation in
Drosophila, also require the function of Wnt proteins
(Dickinson et al., 1994; Gavin et al., 1990; McMahon et
i5 al., 1992; Takada et al., 1994). Fringe proteins may
modify the function of membrane-bound Notch ligands only
in the presence (for Delta) or absence (for Serrate) of
Wnt proteins. Biochemical studies are required to define
the precise site of interaction between the Fringes and
the Notch receptor system. Candidate interacting
proteins through which the Fringe proteins may regulate
the sensitivity and specificity of Notch include Notch
itself, as well as Delta, Serrate and Wnts.
It is postulated that Lunatic Fringe protein
facilitates the local activation of Notch during
somitogenesis, neurogenesis and other developmental
processes. The mammalian Fringe proteins described
herein may potentially be used to block cancer by
altering Notch function. They may also be used to
regulate skin growth and differentiation when applied
topically. It is expected that all developing organ
systems will have the potential to respond to these
proteins. Any normal process which is regulated by
signalling through the Notch receptor may be modulated by
administration of the Fringe proteins. Further, any
pathological condition or disorder which may be
ameliorated by inhibition or promotion of signalling
through the Notch receptor may be treated by
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administration of the Fringe proteins described herein.
The present invention is not limited to the features
of the embodiments described herein, but includes all
variations and modifications within the scope of the
claims.
EXAMPLES
The examples are described for the purposes of
illustration and are not intended to limit the scope of
the invention.
Methods of molecular genetics, protein and peptide
biochemistry and immunology referred to but not
explicitly described in this disclosure and examples are
reported in the scientific literature and are well known
to those skilled in the art.
RT-PCR
Mouse tissues were homogenized in TRIZOL (Gibco
BRL), total RNA extracted, and poly(A)+ RNA prepared
using Oligotex (Qiagen). mRNA was heated to 95°C for 5
minutes prior to cDNA synthesis and reverse transcription
was carried out at 37°C for 1-2 hours in lx First Strand
Buffer (Gibco BRL), 10 mM DTT, 1 mM dNTPs (Pharmacies),
10 U RNasin (Pharmacies), 0.5 mg pd(N)6 (Pharmacies), and
200 U of M-MLV Reverse Transcriptase. cDNA was then used
in Taq Polymerase PCR reactions containing 1X PCR Buffer
(Perkin Elmer), 1 mM MgCl2, 0.2 mM dNTPs, 0.01%
gelatin, and 1 mg of forward and reverse primers.
Degenerate primers were as follows: Fringe upstream 5'
GCC GAA TTC TGG TT(T/C) TG(T/C) CA(T/C) (G/T)TN GA(C/T)
GA (C/T) GA (C/T) AA (C/T) TA (C/T) GT (codes for amino acids
WFCH(V/F)DDDNYV with 5' EcoRI site); Fringe downstream 5'
GCC TCT AGA CA (G/A)AA NCC NGC NCC NCC NGT NGC (G/A)AA
CCA (G/A)AA (codes for anti-sense of amino acids
FWFATGGAGFC with 5' XbaI site). PCR reaction conditions
were as follows: initial denaturation at 96°C for 7 min.,
followed by 2 cycles of 94°C for 50 s, 50°C for 2 min,
72°C for 2 min, 35 cycles of 94°C for 50 s, 55°C for 2
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min, 72°C for 1.5 min, and a final incubation of 72°C for
min. PCR products (expected size 216 by based on the
human EST) were run out on 3o Nusieve agarose (Mandel)
gels, purified using Qiaex II (Qiagen), digested with
S EcoRI and XbaI and subcloned into Bluescript (Stratagene)
for dideoxy sequencing using Sequenase v2.0 (US
Biochemicals). DNA and amino acid sequences were
analyzed using MacDNASIS software (Hitachi) and searches
for related sequences were done through the BLAST network
10 service (Altschul et al., 1990) provided by the National
Center for Biotechnology Information.
Examt~le 1: Isolation of Murine Frincre cDNA Clones
Approximately 1 x 106 plaques of a mouse embryonic
(day 14) cDNA library (Stratagene) were transferred and
ultraviolet light cross-linked to uncharged nylon
membranes (Qiabrane, Qiagen), and screened with a mixture
of 32P-labeled inserts from PCR clones of mouse Lunatic,
Manic, and Radical Fringe. Hybridization was performed
at 48°C for 24 hours in 1M NaCl, to SDS, loo Dextran
Sulphate, 50 mM Tris pH 7.5, 1X Denhardt's, and 100 mg/ml
denatured salmon sperm DNA. Filters were washed twice
with 2X SSC, 0.5% SDS, once with 1X SSC, 0.5o SDS, and
once with 1X SSC, 0.5o SDS. All washes were at 48°C for
30 min. and filters were exposed to Kodak BioMax film for
48 hours. Twenty-two positively hybridizing plaques
were identified, purified, and cycle sequencing was
performed on 11 excised clones using an ABI Biotechnology
Automatic DNA sequences. Of these 11 mouse clones, 8
were Radical, 1 was Manic, and 2 were Lunatic Fringe.
The 5' ends of Lunatic and Manic Fringe were cloned by 5'
Race using 5'-AmpliFINDERTM Race Kit (Clontech) following
manufacturer's specifications. The 3' specific primer
used for Race PCR synthesis of Lunatic Fringe was 5'ATC
AGT GAA GAT GAA CGT CAT CTC CTT and the 3' specific
primer used for Race PCR synthesis of Manic Fringe was
5'CTG CAG AAC AGT TGG TGA.
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The cDNA nucleotide sequences of mouse lunatic
fringe, mouse manic fringe and mouse radical fringe are
shown in Tables lA, 2A and 3A respectively and the
corresponding predicted amino acid sequences are shown in
Tables 1B, 2B and 3B.
Table 4 shows a comparison of the predicted mouse
Fringe amino acid sequences (m), with the Drosophila
fringe (D) (Irvine & Wieschaus, 1994) and Xenopus Radical
fringe (X) (Wu et al., 1996) amino acid sequences.
Red bar indicates the predicted cleavage site for
Xenopus and mouse Lunatic Fringe proteins. The blue
arrows correspond to the amino acid sequences on which
degenerate oligonuceotide primers were designed for
RT-PCR cloning of the mammalian Fringe gene family and
red asterisk denotes conserved cysteine residues.
Identical residues are boxed in yellow highlight.
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Mammalian Fringe family
The public data bases were searched for mammalian
(human and mouse) sequences with homology to the
Drosophila fringe gene. One such sequence, which had
been obtained from a three month human brain cDNA
library, was identified in the expressed sequence tag
database (Accession number F13368). Comparison of the
potential translated products from this EST with
Drosophila Fringe revealed that one reading frame encoded
two stretches of almost perfect match with D-Fringe (12
of 13 amino acids and 11 of 11 amino acids respectively).
Degenerate oligonucleotide primers to these two regions
were designed (Table 4) and PCR was performed with cDNA
from several developing mouse tissues. PCR products were
cloned, sequenced and found to contain a mixture of
sequences from three genes, including a mouse orthologue
of the human EST noted above. A mouse embryo cDNA
library was then screened with these three PCR derived
probes to isolate the corresponding full length cDNA
clone for each gene. These three genes have been named
Lunatic fringe, Manic fringe and Radical fringe (the
original EST was a fragment of human Radical fringe).
Multiple overlapping clones were isolated for
Radical Fringe and the sequence of the full coding region
obtained from these cDNAs. This sequence is shown in
Table 3A and its predicted amino acid sequence in Table
3B. In contrast, the 5' ends of Lunatic and Manic fringe
genes were not obtained in this screen. 5' Rapid
Amplification of cDNA Ends, or 5' RACE, was used on mouse
brain cDNA to obtain the 5' regions of both genes. The
full coding regions for Lunatic fringe and Manic fringe
were derived from overlapping sequences obtained from
5'RACE clones and the cDNA clones isolated above. The
full coding regions of Lunatic fringe and Manic fringe
are shown in Tables lA and 2A respectively, with their
predicted amino acid sequences in Table 1B and 2B.
Analysis of the predicted amino acid sequence of the
three murine fringe genes reveals that in each case an
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N-terminal signal sequence is present to target these
proteins to the secretory pathway (Table 4). The red bar
in Table 4 indicates the predicted cleavage site for
Xenopus and mouse lunatic fringe proteins (Wu et al.,
1996). The N-terminus of each protein is variable in
both length and sequence. Notably, the Drosophila Fringe
protein N-terminus is significantly longer than all
vertebrate Fringe proteins (Irvine and Wieschaus, 1994;
Wu et al., 1996). In contrast, the C-terminal 270 amino
acids of all Fringe proteins are very highly conserved
(starting at residue 152 in Table 4 multiple sequence
alignment). In this region, mouse and Xenopus Lunatic
Fringe proteins are 77o identical; mouse and Xenopus
Radical Fringe proteins are 59o identical; mouse Lunatic,
Manic and Radical Fringe proteins are greater than 50a
identical to each other and all vertebrate Fringes are
approximately 30o identical to the fruit fly protein
(Irvine and Wieschaus, 1994; Wu et al., 1996).
Drosophila fringe encodes seven cysteine residues which
are thought to form disulfide bonds in the native protein
(Irvine and Wieschaus, 1994). All vertebrate Fringe
proteins (including the three described herein and the
two Xenopus published fringe proteins (lunatic, radical)
(Wu et al., 1996) contain six of these cysteines at
identical positions suggesting that they may form an
essential scaffold for this protein family. In addition,
the spacing of all conserved residues in the Fringe
C-terminal region is nearly identical, with only two
single amino acid gaps being necessary to line up all
vertebrate proteins with each other and with the
Drosophila protein.
The Xenopus Lunatic fringe gene contains a poorly
conserved N-terminal region between the leader peptide
and a basic motif which is predicted to be the target of
proteolytic processing required for maturation of a
functional ligand (Wu et al., 1996). The mouse Lunatic
fringe gene also encodes a poorly conserved N-terminal
putative "pro region" followed by a basic motif, and
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therefore, is likely produced as an inactive precursor.
In contrast, the Manic and Radical Fringe predicted
proteins contain only a few amino acid residues between
the leader sequence and the conserved C-terminal region
common to all Fringe proteins. Manic Fringe does not
contain a basic cleavage sequence and encodes only 29
amino acids from the start codon to the region where
Lunatic Fringe is predicted to be cleaved. These 29
amino acids code for little more than the leader
sequence, thus Manic Fringe may be secreted in an active
form which does not require proteolytic cleavage. The
mouse Radical Fringe protein also lacks a tetrabasic
cleavage site and contains a shorter N-terminus than the
Xenopus Radical Fringe gene. From start codon to the
IS location of predicted cleavage in Lunatic fringe genes,
the mouse Radical fringe cDNA only encodes forty four
amino acids including the leader sequence (Xenopus
Radical fringe encodes seventy one amino acids in the
corresponding region). Like mouse Manic Fringe, mouse
Radical Fringe may not require regulated proteolytic
activation.
Example 2: Expression of Fringe in mice
(a) Northern Blot Analysis
Total RNA from adult mouse brain, thymus, heart,
lung, liver, kidney, spleen, skeletal muscle, and ES
cells was prepared using Trizol (Gibco, BRL). RNA
samples (10 ug) were electrophoretically separated on a
1.2% agarose/formaldehyde gel, transferred and
ultraviolet light cross-linked to Genescreen (Dupont).
Hybridization was performed at 65°C in 1M NaCl, l00
Dextran Sulphate, to SDS and 100 mg/ml denatured salmon
sperm DNA. Blots were washed twice for 5 min. at RT in
2X SSC, O.lo SDS, twice for 5 min. at RT in 0.2X SSC,
O.lo SDS, twice for 15 min. at 42°C in 0.2X SSC, 0.1o SDS,
and twice for 15 min. at 68°C in O.1X SSC, 0.1% SDS.
Blots were exposed for 2 - 4 days to Kodak BioMax film in
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the presence of an intensifying screen. A mouse embryo
multiple tissue northern blot (Clontech) was probed using
the manufacturer's specifications. The probe for Manic
Fringe was a 159 by EcoRI-PvuII fragment which starts 383
by downstream of the last amino acid in the coding
sequence. The probe for Lunatic Fringe was 2kb EcoR
insert from pBK-phagemid vector (clone 24), and the probe
for Radical Fringe was a l.5kb EcoRI insert from
pBK-phagemid vector (clone 89). All probes were random
primed-labeled with [a-32P]dCTP and 2 x 106 cpm/ml were
used for hybridization.
The results are shown in Figures 3 and 4.
(b) Tissue section in situ hybridization
t5 In situ hybridization experiments were performed
using 8-~ paraffin or frozen sections from
developmentally-staged C.B.-17 mouse embryos. Midday of
the time of appearance of vaginal plugs was considered as
0.5 dpc to time pregnancies. For paraffin sections,
embryos were fixed overnight in 4a paraformaldehyde,
dehydrated in ethanol and embedded in paraffin. For
cryosections, embryos were protected by embedding in OCT
compound (Miles) prior to freezing in liquid N2.
Pretreatments of frozen sections included fixing in 40
paraformaldehyde for 1 h., followed by proteinase K
digestion (20 mg/ml, 7.5 min, 25°C) and acetylation (0.1 M
triethanolamine pH 8.0, 0.25a acetic anhydride, 10 min,
25°C). Subsequently, the sections were dehydrated with
ethanol and air-dried prior to addition of hybridization
solution.
Riboprobes were synthesized using T7 RNA polymerase
(Pharmacies, Boehringer Mannheim), T3 RNA polymerase
(Pharmacies), and SP6 RNA polymerase (Pharmacies) according
to the protocol of the manufacturer.
pBK-RadicaldKpnI(clone 16) was used to synthesize sense
(T3) and antisense (T7) probes of 709 by which span from
nt 418 of Radical fringe to 118 nt downstream of coding
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sequence. A probe for Lunatic Fringe was generated by
PCR using the following primers: 5' GAATTC CTG CTG TTC
GAG ACC TGG ATC (contains EcoRI site) and 5' AGATCT ACC
AGG ATT GTA GAA GAT CGC (contains BglII site) and
pBK-Lunatic (clone 24) as template. The 756 by PCR
product which spans nt 273 to nt 1030 of the Lunatic
coding sequence was subcloned into pGemT (Prornega) and
sense and antisense riboprobes synthesized with SP6 and
T7 polymerase respectively. pBK-Manic (clone 30) was
digested with EcoRI and a 426 by EcoRI fragment from 3'
untranslated region of Manic fringe (begins 284 by
downstream of last coding nt) was subcloned into
phosphatase-treated EcoRI-digested pBluescript vector
(Stratagene). Sense and antisense Manic riboprobes were
synthesized from this plasmid using T3 and T7 polymerases
respectively. A probe for mouse Serrate-1 was generated
using the following primers: 5' TCC AGC TGA CAG AGG TTT
CC and 5' GAC CAG AAT GGC AAC AAA ACC TGC. The 937 by
PCR product, which covers nt 641-1578 of the rat sequence
20.was designed by searching for stretches of DNA identity
between rat and chicken Serrate-1, which was predicted to
be identical in mouse Serrate-1. This PCR product was
subcloned into pGemT (Promega) and antisense riboprobes
generated by transcribing with T7 RNA polymerase. A 777
by ScaI/PstI fragment of mouse Delta spanning nt 669-1446
was subcloned in pBK (Stratagene) and antisense
riboprobes generated using T3 RNA polymerase.
Pretreatment of paraffin sections and hybridization
to [a33P]UTP-labeled sense and antisense probes
(15,000-40,000 cpm/ml) were conducted as described by Hui
and Joyner (Hui and Joyner, 1993), with the following
modifications. The hybridization and washing steps
omitted use of DTT. Following RNase treatment, the
sections were washed sequentially with 2x SSC, lx SSC and
0.5x SSC at 37°C, 10 min each, and with O.lx SSC at 65°C
for 30 min. Exposure of slides to emulsion was allowed
to proceed for 1-3 weeks and, after development, the
tissues were stained lightly with hematoxylin and eosin.
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(c) Whole Mount In Situ Hybridization
Embryos were dissected into PBS and extraembrvonic
tissues removed. Embryos were fixed overnight at 4°C with
4% paraformaldehyde (PFA) in PBS, rinsed once with cold
PBT (PBS with O.lo Tween 20) and dehydrated through an
ascending methanol series (250, 500, 75%) in PBT and then
stored in 1000 methanol at -20C until further use.
Antisense riboprobes were synthesized from the same DNA
templates as described previously for section in situ.,
using a digoxygenin RNA labeling kit (Boehringer
Mannheim). Embryos were rehydrated through a descending
methanol series rinsed twice in PBT, and then bleached
for 1 hour at RT in 6% hydrogen peroxide in PBT. After
three rinses with PBT, embryos were permeabilized with l0
ug/ml proteinase K (5 min. for E9.5 embryo and 2 min. for
E8.5 embryo), rinsed twice with PBT and then fixed with
Glutaraldehyde 0.2%/PFA 4%/PBT for 20 min at RT. After
fixation, embryos were washed 4X with PBT, washed once
with hybridization buffer (50a formamide, 5X SSC [pH
4.5], 50 ug/ml yeast tRNA, to SDS, 50 ug/ml heparin), and
incubated with 1.5 ml of fresh hybridization buffer for 1
hr at 70°C. Digoxygenin-labeled riboprobe (1.5 mg) was
added directly and embryos were incubated overnight at
70°C.
Following hybridization, embryos were washed twice
for 30 min at 70°C with solution 1 (50o formamide, 5X SSC
[pH 4.5], 1% SDS), washed once for 10 min at 70°C with
50/50 solution 1/solution 2 (0.5 M NaCl, 0.01 M Tris [pH
7.5], 0.1% Tween-20), rinsed 3X with solution 2 at RT,
rinsed once at RT with solution 3 (50o formamide, 2X SSC
[pH 4.5]), and twice for 30 min at 65°C with solution 3.
Embryos were then rinsed 3X at RT with TBS-TL (137 mM
NaCl, 2.7 mM KC1, 25 mM Tris [pH 7.5] plus 2 mM
Levamisole and O.la Tween 20) and then incubated for 1 hr
at RT with TBS-TL containing loo heat-inactivated (65C
for 30 min) goat serum to prevent non-specific binding of
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antibody. Anti-digoxygenin Fab alkaline phosphatase
conjugate (1/5000, Boehringer Mannheim) was preabsorbed
in TBS-TL with to heat-inactivated goat serum and
approximately 3 mg heat-inactivated embryo powder per ml
antibody. After an overnight incubation at 4°C with the
preabsorbed antibody, embryos were rinsed 3X with TBS-TL,
washed 4X for 1 h with TBS-TL at RT, and then left
overnight at 4°C in fresh TBS-TL. The buffer was
exchanged by washing 3X for 10 min with NTMT (0.1 M
NaCl, 0.1 M Tris [pH 9.5], 0.05 M MgCl2 , O.lo Tween-20,
2mM levamisole), and the antibody detection reaction was
performed by incubating embryos with detection solution
(hTTMT with 0.25 mg/ml nitroblue tetrazolium and 0.13
mg/ml 5-bromo-4-chloro-3-indolulphosphate toluidinium).
Detection reactions were complete within 15 min - 1 hour
and then embryos were washed twice in PBT. Color was
intensified by dehydration/rehydration through ascending
and descending methanol/PBT rinses. Embryos were then
cleared through 50o and 80% glycerol in CMFET (137 mM
NaCl, 3 mM KC1, 8 mM Na2HP04, l.5mM KH2POq , 0.7 mM EDTA,
0.1% EDTA, 0.1% Tween-20) and whole embryos were
photographed under transmitted Light using a Leica MZ12
microscope with Kodak Tungsten 160 ASA film.
Results of the in situ hybridisation studies are
shown in Figures 5 to 7
Mammalian Fringe gene expression in development
To identify tissues which express mammalian fringe
homologues, Northern blot analysis was performed on RNA
derived from mouse embryos and adult tissues. The three
genes were expressed at all stages of mouse embryonic
development analyzed, from day seven of gestation to day
seventeen (Figure 3). Two transcripts were detected for
Manic and Radical Fringe genes in mouse embryos. In
addition, the three fringe genes were widely expressed in
adult tissues, with Lunatic fringe having a more
restricted expression pattern than either Manic or
Radical (Figure 4). Liver RNA samples were underloaded
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and each of the three fringe genes can be detected in
this tissue on longer exposures of these northern blots.
In situ hybridization analysis revealed that ir. many
cases the expression of fringe genes in both embryos and
adults was localized to tissues undergoing development or
differentiation. Lunatic Fringe is highly expressed
during neurogenesis and somitogenesis. Mouse embryos at
8.5 and 9.5 days of gestation (E8.5 and E9.5) express
Lunatic Fringe in two stripes which surround the forming
somite (Figure 5A and 5B). These two stripes move with
time towards the posterior of the embryo as new somites
are generated, suggesting that the Lunatic Fringe gene is
involved in the segmentation of mesoderm into somites.
In addition, Lunatic Fringe is expressed throughout the
developing central nervous system in the undifferentiated
neuroblast layers of the neural tube, brain, and otic
vesicle. This expression continues in uncommitted
neuroblasts as mice continue to develop (Figure 5C, 5E
and data not shown). In day 11.5 embryos (Figure 5C),
Lunatic Fringe is also expressed in the myotome which
contains undifferentiated myoblasts and in the
intervertebral mesenchyme (data not shown). Throughout
development, this fringe gene continues to be expressed
in proliferating cells in the ventricular zones of the
nervous system, in uncommitted neuroblasts in the retina
(C. C. and B.G. unpublished) and in the perichondrium
which contains proliferating chondroblasts (Figure 5D and
data not shown). Lunatic Fringe is also expressed in
developing organs and continuously developing systems in
adult mice. For example, it is expressed in the fetal
heart and hematopoetic cells in the fetal liver at E12.5
(not shown), in S-shaped bodies of the developing kidney
(Figure 5G), in the thymic medulla (Figure 5H), in a
subset of splenic lymphocytes (Figure 5J), in the basal
epithelium of skin (not shown) and similarly in the basal
epithelium of the tongue (Figure 6E). It is concluded
that Lunatic Fringe is expressed in cells which have yet
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to complete their developmental program and remain
competent both to proliferate and to differentiate. The
fact that lunatic fringe is expressed in the basal
epithelium of the skin which is continuously undergoing
cellular regeneration and differentiation suggests that
this protein could be applied topically to the skin as a
therapeutic agent to alter abnormal skin growth seen in
various skin diseases.
Manic Fringe and Radical Fringe, on the other hand,
are often expressed in cells of a more committed cell
fate. Both of these genes are expressed in the marginal
zones of the neural tube and brain throughout development
(Figure 5F and data not shown). Both Manic and Radical
Fringe are expressed from day E11.5 to E13.5 in the
dorsal root ganglia. These genes are also expressed in
E12.5 fetal liver, and in the suprabasal epithelium of
both tongue and skin. Manic Fringe also appears to be
very highly expressed in megakaryocytes present in the
adult spleen (Figure 5K and 5L).
Differentiation induced Fringe gene switch
In many developing tissues, as described above, the
"stem cell" population which is undifferentiated and
proliferating expressed high levels of Lunatic Fringe.
In contrast, Manic and Radical Fringe genes do not seem
to be expressed in uncommitted cell compartments.
Sections of two tissues where stem cells and their
differentiated progeny are physically separated have been
analysed. Sections through the neural tube in day 10.5
embryos reveal that Lunatic Fringe is expressed in the
ventricular zone which corresponds to the neuroblastic
population but not in the marginal zone, which contains
differentiated neurons (Figure 6A). Adjacent sections
probed with Manic or Radical Fringe riboprobes reveal a
striking complementary expression profile for these two
genes. As neurons are born, they turn off Lunatic -
Fringe, leave the ventricular zone and turn on both Manic
and Radical Fringe genes (Figure 6B and 6C). Similarly,
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tongue epithelium is continuously regenerated through
division of basally located stem cells which express
Lunatic Fringe (Figure 6D). As cells differentiate, they
move apically, turn off Lunatic Fringe and turn on both
Manic and Radical Fringe genes (Figure 6E and 6F).
Lunatic Fringe and Deltal expression domains intersect
The process of differentiation is often regulated by
Notch and its ligands (Artavanis-Tsakonas et al., 1995;.
The importance of Notch in differentiation and
development of both somites and neural tube has been
demonstrated genetically in vertebrates (Chitnis et al.,
1995; Chitnis and Kintner, 1996; Coffman et al., 1993;
Conlon et al., 1995; Swiatek et al., 1994). During
somitogenesis, Notchl is required for proper segmentation
of presomitic mesoderm into somites (Conlon et al.,
1995). Notchl is not, however, required to form
somite-like tissue (Conlon et al., 1995). This
observation suggests that activation of Notch may block
differentiation of mesoderm into somite tissue at the
boundary between adjacent somites. Delta family Notch
ligands are typically turned on as cells commit to
differentiate (Muskavitch, 1994). Analysis of mouse
Deltal expression in mesoderm undergoing somitogenesis in
E8.5 embryos reveals that Deltal is most strongly
expressed in the forming somite (Figure 7A) (Bettenhausen
et al., 1995). Lunatic Fringe is expressed at this stage
in two bands which surround the forming somite (Figure
7B). Mouse Serrate3/Jagged is also weakly expressed in
two bands which surround the forming somite (Figure 7C).
Lunatic Fringe may control the sensitivity or
selectivity of Notch for its ligands, perhaps by ensuring
that Deltal and Serratel only activate Notchl in cells
between the forming somites.
In contrast to the developing somites, Deltal and
Lunatic Fringe genes are expressed in overlapping domains
within the developing neural tube (Figure 7D and 7E).
Deltal is turned on as cells differentiate into the three
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major types of neurons (sensory neurons, interneurons and
motor neurons) (Chitnis et al., 1995; Henrique et al.,
1995) and activates Notchl in the remaining neuroblastic
layer to prevent differentiation. Serrate3/Jagged is
expressed in complementary stripes in the neural tube
which express neither Deltal nor Lunatic Fringe (Figure
7F) (Lindsell et al., 1995; Myat et al., 1996). The
function of Serratel in regulating neural tube
development is unknown, although by analogy to the role
of Drosophila Serrate in imaginal disc proliferation
(Speicher et al., 1994), this mammalian Serrate gene may
regulate proliferation rather than differentiation of
neuroblasts. Thus, in two tissues which are known to
undergo Notch dependent patterning (somites and neural
tube), Lunatic Fringe appears to be expressed in cells
which are responding to Delta.
Example 3: Ectopic Expression of Mouse Fringe in
Drosophila
An EcoRI fragment containing the entire mouse Radical
fringe open reading frame was purified from pBK-phagemid
vector (clone 89) and ligated with phosphatase-treated
EcoRI digested transformation vector pUAST, which
contains several GAL4 upstream activator sequences and a
minimal promoter (Brand and Perrimon, 1993). The 5' end
of Manic Fringe was PCR-modified to contain Kozak
consensus sequence (5' GAT CTA CCA ATG G) and an ApaI
site was introduced by PCR at nt 304-309 to allow
ligation with pBK-phagemid vector Manic fringe cDNA
(clone 8). The entire Manic fringe cDNA was then
subcloned as a BglII fragment into phosphatase-treated
BglII-digested transformation vector pUAST (Brand and
Perrimon, supra). The recombinant plasmids,
pUAST-Radical and pUAST-Manic, with the open reading
frames in the correct orientation relative to the
promoter, were used to transform Drosophila embryos using
standard microinjection procedures (Spradling, 1986).
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For analysis of ectopic expression, transgenic flies
carrying pUAST-Manic and pUAST-Radical were crossed to
GAL4 enhancer trap lines. The GAL4 drivers used were
GAL4pt~ (Hinz et al., 1994), GAL4c5 which is expressed
throughout the wing disc pouch (Yeh et al., 1995), and
GAL4C96 which is expressed only along the D/V boundary
(Gustafson and Boulianne, 1996). These crosses were
repeated with several independent transgenic lines for
pUAST-Manic and pUAST-Radical. Progeny of such crosses
were scored for defects. In this way the mammalian
fringe genes were expressed in cells where the patched
gene is expressed (Hinz et al., ?994) which includes
specific locations in the eye, wing, ocelli, and most if
not all other imaginal discs. In the wing imaginal disc,
patched is expressed in a stripe of cells on the anterior
side of the A/P boundary (Hinz et al., 1994; Kim et al.,
1995). Wings were dissected from adult flies, mounted
in GMM (Lawrence et al., 1986) and photographed using a
Zeiss Axioskop. Pictures of adult fly eyes were obtained
by Scanning Electron Microscopy using standard
proceedures (Tomlinson and Ready, 1987).
Results are shown in Figures 2 and 3.
Loss of endogenous wing margin
Ectopic expression of D-fringe using the GAL4pt~ line
leads to a loss of wing margin tissue at the A/P
boundary. This phenotype is believed to occur because
ectopic expression of D-Fringe in ventral cells destroys
the natural D/V Fringe boundary at this location (Kim et
al., 1995). In addition, expression of D-fringe in the
ventral compartment causes the creation of a new Fringe
boundary in this compartment, and therefore, an ectopic
wing margin is generated on the ventral surface of the
wing (Kim et al., 1995). In contrast, expression of
either Manic or Radical Fringe causes the loss of
endogenous margin tissue without the generation of an
ectopic margin on the ventral surface (Figure 1B and 1C).
Notably the loss of wing margin is more dramatic in
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Manic Fringe-expressing flies than in Radical
Fringe-expressing flies. Radical Fringe usually only
induces loss of margin tissue when expressed at high
levels either by an extra copy of the GAL4pt~ driver,
extra copies of the pUAST transgene or when present in
sensitized genetic backgrounds (see below).
These results demonstrate that the loss of wing
margin tissue induced by ectopically expressed fringe
genes is a genetically distinct function from the
creation of novel ectopic margins. Loss of margin tissue
is normally associated with a loss of Notch activation at
the D/V boundary (Couso and Martinez Arias, 1994).
Induction of novel margins is associated with
inappropriate activation of Notch (Doherty et al., 1996;
Kim et al., 1995; Rulifson and Blair, 1995).
GAL4pt~-driven expression of either of these mammalian
fringes, like D-fringe, causes the disruption of normal
margin; but unlike D-fringe, they do not encode the
functions) necessary for induction of an ectopic ventral
margin. Manic Fringe and Radical Fringe, therefore,
appear to inhibit Notch activation by its ligands,
Serrate and/or Delta, at the D/V boundary, but seem
unable to induce Notch activation in either the dorsal or
ventral compartments. These two mammalian Fringes do not
mimic Drosophila Fringe because they fail to induce a new
margin in the ventral compartment, and do not inhibit
Drosophila Fringe; loss of Drosophila Fringe function in
the dorsal compartment induces an ectopic dorsal margin
(Irvine and Wieschaus, 1994).
Manic and Radical Fringe proteins inhibit distinct Notch
ligand dependent processes.
GAL4pt°-driven expression of Manic and Radical fringe
genes induces phenotypic effects in other tissues. Manic
Fringe induces a dramatic reduction in size of the eye
(Figure lE as compared to 1D and 1F) and fused ocelli
(not shown). Wing scalloping (loss of margin) and
dramatic reduction of eye size are both phenotypes
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associated with loss of Serrate function (Speicher et
al., 1994). Radical Fringe flies on the other hand had
normal eyes but an extra pair of scutellar setae within
the normal proneural region (not shown) (Simpson, 1996).
Extramacrochaetae/setae within the proneural region and
wing scalloping both represent a failure of processes
which depend on Delta signalling (Artavanis-Tsakonas et
al., 1995; Muskavitch, 1994).
In order to characterize in greater detail the
effect of Manic Fringe and Radical Fringe on wing
development, the UAS mammalian-Fringe transgenic lines
were crossed to other enhancer trap lines which express
GAL4 in distinct wing compartments. The GAL4C96 line
expresses GAL4 at the D/V boundary in the future wing
margin (Gustafson and Boulianne, 1996). Crossing Manic
Fringe lines to GAL4~96 lines produced a dramatic loss of
margin tissue and reduction of wing size (Figure 1G). In
contrast, crosses between Radical Fringe lines and GAL4~9s
produced a small loss of margin tissue in some but not
most flies (not shown). The GAL4~5 enhancer trap line
expresses GAL4 in all cells which will become the wing
blade (Yeh et al., 1995). Crosses between this line and
Manic Fringe flies also produced a dramatic loss of
margin and wing blade tissue (Figure 1H). In contrast,
crosses between GAL4~5 and the Radical Fringe flies
produced wings with small vein deltas, or vein splitting
(Figure lI, see insert). These distinct phenotypes are
characteristic of loss of Serrate function in the case of
Manic Fringe, and loss of Delta function in Radical
Fringe flies. Expression of either mammalian fringe gene
at the D/V boundary (as in ptcGAL or GAL4~96 crosses) may
induce a similar wing phenotype because both Serrate and
Delta are required for induction of normal margin tissue
and wing growth. Thus, examination of Manic and Radical
Fringe effects in other tissues of ptcGAL crosses (eyes
and bristles) and in other regions of the wing (as in
GAL4~5 crosses) has identified distinct properties of
these two mammalian Fringe proteins.
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Additional crosses were performed to test for
genetic interactions between ectopically expressed
Radical Fringe and the endogenous Drosophila fringe gene
as well as with other signalling molecules involved in
wing margin induction at the D/V boundary. As mentioned
above, GAL4pt~/UAS-Radical Fringe did not produce a
phenotype when both GAL4ptC and Radical Fringe were
present at single dose (Figure 2A). The Drosophila
fringe hypomorphic allele fng52 shows a weak wing vein
splitting or "delta" phenotype and normal margin in
heterozygotes (Figure 3B) (Irvine and Wieschaus, 1994).
When GAL4pt~/UAS-Radical Fringe flies were also
heterozygous for the fng52 D-Fringe allele, significant
wing scalloping was observed (Figure 2C). This genetic
interaction between Radical Fringe and D-Fringe suggests
that Radical Fringe interferes with an essential function
of D-Fringe. The mild wing vein deltas observed in fng52
flies suggests that D-Fringe is required for Delta to
stimulate Notch. Indeed, the wing vein deltas observed
in GAL4~5/UAS-Radical Fringe flies, described above,
indicates that Radical Fringe interferes with Delta
stimulation of Notch.
It has been demonstrated previously that both Delta
and Serrate function through Notch to induce margin
tissue on either side of the D/V boundary. Loss of
either of these Notch ligands or of Notch itself results
in a loss of margin (Doherty et al., 1996; Kim et al.,
1995). In addition, ventral expression of the secreted
protein Wingless is required early in wing development
for margin induction (Couso and Martinez Arias, 1994).
Flies heterozygous for a loss of function Delta allele
have widened wing veins as well as "deltas" where the
veins intersect the wing margin (Figure 2D) (Doherty et
al., 1996; Muskavitch, 1994). The margin in these flies
however is normal. Flies which are heterozygous for
del to and also express GAL4pt~/UAS-Radical Fringe show a
loss of margin tissue (Figure 2H). Expression of the
GAL4pt°/UAS-Radical Fringe transgene combination also
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enhances or reveals a phenotype in combination with
heterozygous Serrate, Notch and wingless loss of function
alleles (Figure 2F through 2K).
Thus, Radical Fringe inhibits the normal margin
specification which depends on D-Fringe, Delta, Serrate,
Wingless and the Notch receptor. These dosage sensitive
interactions may be a result of all four ligands
functioning together to activate Notch only at the D/V
boundary. Radical Fringe may, therefore, interfere with
wing margin induction by inhibiting only one of the four
essential ligands, Delta. It remains formally possible
that Drosophila and mouse Fringe genes studied here
function through some novel receptor and signal
transduction system, however, the simplest interpretation
IS of our data involve a very proximal regulation of
Notch-ligand function by the Fringe proteins.
Example 4:
Dimerisation of Frinae Proteins: Generation of Dominant
Inhibitory mutants:
Murine Lunatic fringe, Manic Fringe and Radical
Fringe were each tagged using PCR-mediated mutagenesis on
their C-termini with the Flag epitope. These chimeric
cDNAs were then cloned into the eukaryotic expression
vector pCDNA3. Each Fringe expression construct was
transfected into Cos cells and lysates were analyzed for
production of tagged Fringe protein by Western blot with
anti-Flag antibodies. Independently, we tagged the three
mammalian fringe genes with C-terminal myc-epitope tags
in the pCDNA3 vector. These proteins were expressed in
transfected Cos cells and the myc-tagged Fringe proteins
detected on Western blots from cell lysates using anti-
myc antibodies.
In order to test for dimerization of the Fringe
proteins, we transfected Flag-epitope tagged Lunatic
Fringe with myc-tagged Lunatic Fringe, myc-tagged Manic
Fringe or myc-tagged Radical Fringe. Cell lysates were
prepared and immunoprecipitated with anti Flag
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antibodies. In each case, precipitated Flag-tagged
Lunatic Fringe protein coprecipitated the myc-tagged
Fringe protein as detected on western blot analysis using
anti-myc antibodies. Similarly, Flag-tagged Manic Fringe
could associate with myc-tagged Lunatic, Manic and
Radical Fringe in cells. Finally the Flag-tagged Radical
Fringe protein also dimerized with myc-tagged Lunatic,
Manic and Radical Fringes indicating that all dimeric
combinations of the three Fringe polypeptides are capable
of forming in cells.
These dimeric interactions suggest that six distinct
Fringe dimeric complexes exist in vivo. In addition,
this result suggests that the Fringe proteins may
function as dimers to regulate the sensitivity of Notch
IS Receptors for their ligands. The dimeric nature of the
Fringes can be used to identify or generate dominant
inhibitory alleles or mutants of each Fringe. It is
expected that mutant Fringes can be made which can
either; (i) still dimerize with wild type proteins but
cannot form productive interactions with other Fringe
partners or (ii) still interact with Fringe-binding
partners but are unable to dimerize with wild type Fringe
proteins. Such mutants can be used to block endogenous
Fringe function.
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TAHLE lA
ATGCTCCAGCGGTGCGGCCGGCGCCTGCTGCTGGCGCTGGTGGGCGCGCTGTTGGCT
TGTCTCCTGGTGCTCACGGCCGACCCGCCACCGACTCCGATGCCCGCTGAGCGCGGA
CGGCGCGCGCTGCGTAGCCTGGCGGGCTCCTCTGGAGGAGCTCCGGCTTCAGGGTCC
AGGGCGGCTGTGGATCCCGGAGTCCTCACCCGCGAGGTGCATAGCCTCTCCGAGTAC
TTCAGTCTACTCACCCGCGCGCGCAGAGACGCGGATCCACCGCCCGGGGTCGCTTCT
CGCCAGGGCGACGGCCATCCGCGTCCCCCCGCCGAAGTTCTGTCCCCTCGCGACGTC
TTCATCGCCGTCAAGACCACCAGAAAGTTTCACCGCGCGCGGCTCGATCTGCTGTTC
GAGACCTGGATCTCGCGCCACAAGGAGATGACGTTCATCTTCACTGATGGGGAGGAC
GAAGCTCTGGCCAAGCTCACAGGCAATGTGGTGCTCACCAACTGCTCCTCGGCCCAC
AGCCGCCAGGCTCTGTCCTGCAAGATGGCTGTGGAGTATGACCGATTCATTGAGTCT
GGGAAGAAGTGGTTCTGCCACGTGGATGATGACAACTACGTCAACCTCCGGGCGCTG
CTGCGGCTCCTGGCCAGCTATCCCCACACCCAAGACGTGTACATCGGCAAGCCCAGC
CTGGACAGGCCCATCCAGGCCACAGAACGGATCAGCGAGCACAA.AGTGAGACCTGTC
CACTTTTGGTTTGCCACCGGAGGAGCTGGCTTCTGCATCAGCCGAGGGCTGGCCCTA
AAGATGGGCCCATGGGCCAGTGGAGGACACTTCATGAGCACGGCAGAGCGCATCCGG
CTCCCCGATGACTGCACCATTGGCTACATTGTAGAGGCTCTGCTGGGTGTACCCCTC
ATCCGGAGCGGCCTCTTCCACTCCCACCTAGAGAACCTGCAGCAGGTGCCCACCACC
GAGCTTCATGAGCAGGTGACCCTGAGCTATGGCATGTTTGAGAACAAGCGGAACGCA
GTGCACATCAAGGGACCATTCTCTGTGGAAGCTGACCCATCCAGGTTCCGCTCTGTC
CATTGCCACCTGTACCCAGACACACCCTGGTGTCCTCGCTCCGCCATCTTCTAGCAG
TCGTGGTTGA
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TABLE 1B
MLQRCGRRLLLALVGALLACLLVLTADPPPTPMPAERGRRALRTLAGSSGGAPASGS
RAAVDPGVLTREVHSLSEYFSLLTRARRDADPPPGVASRQGDGHPRPPAEVLSPRDV
FIAVKTTRKFHRARLDLLFETWISRHKEMTFIFTDGEDEALAKLTGNWLTNCSSAH
SRQALSCKMAVEYDRFIESGKKWFCHVDDDNYVNLRALLRLLASYPHTQDVYIGKPS
LDRPIQATERISEHKVRPVHFWFATGGAGFCISRGLALKMGPWASGGHFMSTAERIR
LPDDCTIGYIVEALLGVPLIRSGLFHSHLENLQQVPTTELHEQVTLSYGMFENKRNA
VHIKGPFSVEADPSRFRSVHCHLYPDTPWCPRSAIF
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TAHLE 2A
ATGCACTGCCGACTTTTTCGGGGCATGGCGGGAGCCCTCTTTACCCTCCTGTGCGTG
GGGCTCCTGTCTCTACGATACCACTCAAGTTTGTCCCAGAGGATGATACAGGGCGCG
CTCAGGCTGAACCAACGGAACCCAGGACCCCTGGAGCTGCAGCTAGGCGACATCTTC
ATCGCAGTCAAGACTACCTGGGCCTTCCATCGCTCCCGCCTGGACCTGCTACTAGAC
ACGTGGGTCTCCAGGATCAGGCAACAGACATTCATCTTCACTGACAGCCCAGATGAA
CGCCTCCAGGAGAGACTAGGCCCGCACCTCGTGGTCACCAACTGTTCTGCAGAGCAC
AGTCATCCTGCTCTGTCCTGCAAGATGGCTGCAGAGTTCGATGCCTTCTTGGTCAGT
GGCCTCAGGTGGTTCTGCCACGTGGATGATGACAACTATGTGAACCCCAAGGCTCTG
CTGCAGCTGTTGAAAACATTCCCGCAGGACCGTGATGTCTATGTGGGCAAGCCCAGC
CTGAACCGGCCCATCCACGCCTCTGAGCTGCAGTCAAA.AAACCGCACGAAGCTGGTG
CGGTTCTGGTTTGCCACAGGGGGTGCTGGTTTCTGCATCAACCGCCAACTGGCTTTG
AAGATGGTGCCATGGGCCAGCGGCTCCCACTTTGTGGACACTTCTGCTCTCATCCGG
CTCCCCGATGACTGCACTGTGGGCTACATCATCGAGTGCAAGCTGGGGGGTCGCCTG
CAGCCCAGCCCCCTCTTCCACTCACACCTGGAAACCCTGCAGCTGCTGGGGGCCGCC
CAGCTTCCGGAGCAGGTCACCCTCAGCTACGGTGTCTTTGAGGGGAAACTGAATGTC
ATCAAGCTACCGGGCCCCTTCTCCCATGAAGAGGACCCCTCCAGATTCCGCTCCCTC
CATTGTCTCCTCTACCCAGACACACCCTGGTGTCCGCTGCTGGCAGCGCCCTGA
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TABLE 2B
MHCRLFRGMAGALFTLLCVGLLSLRYHSSLSQRMIQGALRLNQRNPGPLELQLGDIF
IAVKTTWAFHRSRLDLLLDTWVSRIRQQTFIFTDSPDERLQERLGPHLWTNCSAEH
SHPALSCKMAAEFDAFLVSGLRWFCHVDDDNYVNPKALLQLLKTFPQDRDVYVGKPS
LNRPIHASELQSKNRTKLVRFWFATGGAGFCINRQLALKMVPWASGSHFVDTSALIR
LPDDCTVGYIIECKLGGRLQPSPLFHSHLETLQLLGAAQLPEQVTLSYGVFEGKLNV
IKLPGPFSHEEDPSRFRSLHCLLYPDTPWCPLLAAP
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TAHLE 3A
ATGAGCCGTGCGCGGCGGGTGTTGTGCCGGGCCTGCCTCGCGCTGGCCGCGGTCCTG
GCTGTGTTGCTGCTACTGCCGCTGCCGCTACCGCTGCCGCTGCCTCGCGCGCCCGCA
CCGGACCCCGATCGGGTCCCGACCCGGAGCCTGACCCTCGAGGGAGACCGCCTGCAA
CCCGACGACGTCTTCATTGCAGTCAAGACCACTCGGAAGAACCACGGCCCGCGCCTG
CGGCTGCTGCTGCGTACCTGGATCTCACGAGCCCCACGGCAGACGTTCATTTTCACC
GATGGAGACGACCCTGAGCTCCAGATGCTGGCAGGCGGCCGCATGATCAACACCAAT
TGCTCTGCTGTGCGCACCCGCCAAGCACTGTGCTGCAAAATGTCGGTGGAATATGAT
AAGTTCCTAGAATCTGGACGAAAATGGTTCTGCCACGTGGATGATGACAACTACGTG
AACCCCAAAAGCCTGCTGCACCTGCTTTCCACCTTCTCTTCCAACCAGGACATCTAC
CTGGGGCGACCTAGCCTGGACCACCCCATCGAAGCCACAGAGAGGGTCCAAGGCGGT
GGCACCTCAAACACAGTGAAATTCTGGTTTGCTACTGGTGGGGCTGGGTTCTGCCTG
AGCAGGGGCCTTGCCCTCAAAATGAGCCCGTGGGCCAGCCTTGGCAGTTTCATGAGC
ACAGCAGAGCGGGTTCGGCTGCCTGATGACTGCACTGTGGGATACATCGTGGAAGGA
CTTCTGGGCGCCCGTCTGCTCCATAGCCCCCTGTTCCACTCGCACCTGGAAAACCTG
CAGAGGCTGCCGTCTGGTGCTATTTTGCAGCAGGTTACCTTGAGCTATGGGGGTCCT
GAGAACCCACATAATGTGGTGAATGTAGCTGGCAGTTTCAACATACAGCAGGACCCT
ACACGGTTTCAGTCTGTGCACTGCCTTCTCTACCCAGACACCCACTGGTGTCCTATG
AAGAACAGGGTTGAGGGAGCTTTCCAGTAA
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TAHLE 3B
MSRARRVLCRACLALAAVLAVLLLLPLPLPLPLPRAPAPDPDRVPTRSLTLEGDRLQ
PDDVFIAVKTTRKNHGPRLRLLLRTWISRAPRQTFIFTDGDDPELQMLAGGRMINTN
S CSAVRTRQALCCKMSVEYDKFLESGRKWFCHVDDDNYVNPKSLLHLLSTFSSNQDIY
LGRPSLDHPIEATERVQGGGTSNTVKFWFATGGAGFCLSRGLALKMSPWASLGSFMS
TAERVRLPDDCTVGYIVEGLLGARLLHSPLFHSHLENLQRLPSGAILQQVTLSYGGP
ENPHNV'VNVAGSFNIQQDPTRFQSVHCLLYPDTHWCPMKNRVEGAFQ
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TABLE 4
10 20 3G 90 5G
D-Fringe 1 MlJSLTVLSPP QRFKRILQAM MLAVAVVYMT 1' PGI'~VPHS~~'~'SG
LLLYQS~.)'G
X-Lunatic 1 MLF:------- NtGKFLLLSI ---VGATLTC - --L-~~VDLQSR50
LLV------
m-Lunatic 1 MLQ------- RCGRRLLLAL ---VGALLAC - --LT-DPPPTSO
LLV------
m-Manic 1 M--------- HC--RLFRGM ---AGALFT- - --VG~-----5C
LLC------
m-Radical 1 M--_______ __-Sg,ARRVL ---CR-__AC - __A~.V-LAVL5C
LAL-_____
X-Radical i M--------- ---KZTYVGL ---IF:---VC 5C'
FLV------- --FLL-LCa.T
60 70 8D oD
1DD
D-Fringe 51 DALASEAVTT HRDQLLQDYV QSSTPTQPGA 10G
GAPAASPTTV IIRF;DIRSFIJ
X-Lunatic 51 HMLETQSDHE PCSAAAVHLR ADLDPAIJpGD lOG
G---GDP~.-- 1JSAQDSGTFS
m-Lunatic 51 PM-----PAE RGRRALRTLA GSSGGAPASG 100
SRAAt'DPG-- VLTREVHSLS
m-Manic 51 _____-____ ______LSLR __________
_____
_____ __________lOD
m-kadical 51 LLLP------ ---- -LP-- --------
-- _-_-______ _Lp___LPLP 1D0
X-kadical 51 VLLIJ------ ------IS::R QRDSSQSLQH
CNSTCSAh-- YLE--
-T>;LF: lOD
110 120 13G 190 15D
D-Fringe 101 FSDIEVSERP TATLLTELAR RSRNGELLRD 150
LSQR-AVTAT PQPPVTEL--
X-Lunatic 101 ---------- --AYFNKLTR~DVEQVFA PSF:------D 150
SAF.PEDITA
m-Lunatic 1D1 ---------- --EYFSLLTR ARRDADPFPG
VASRQ-GDGH PRPp~
EVLSP
. 25D
m-Manic 101 ---------- ---yHSSLS- -----qRl9IQ PGPLELQLG-15G
G~.LRL-IJQRN
m-Radical 101 ---------- --RAPAPDPD ------R'.'PT-----DRLQP15G
RSLTLEG---
X-Radical 1D1 ---------- --EAHLTGRH hF:::ETYRLDAHFFF;EPLQI150
>;PTSATGQGH
160 170 18D I9G 2GG
D-Fringe 151 DDIFZSVKTT fiNYHDTRLAL III:TWFQLARDHYYQEKTF:G200
DQTF:FFTDTD
X-Lunatic 151 IJDVFIAVKTT KKFHRSRMDL LMDTWZSRtJKDEELQ-KI;TG200
EQTFIFTDGE
m-Lunatic 151 RDVFIAVRTT RKFHRARLDL LFETWISRHK DE~.LA-KLTG2G0
19TFIFTDGE
m-Manic 151 -DIFIAVKTT riAFHRSRLDL LLDTWVSRIR DRLQERLGP200
QQTFIFTDSP
m-Radical 151 DDVFIAVKTT RKNHGPRLRL LLRTWISRAP DPELQIJLAGG200
RQTFIFTDGD
X-Radical 151 F:DLFIAVRTT fiKYHGNRL1JL LMQTWISRAKDQELRQKAGD200
EQTFIFTD'.:E
21G 22G 23D 24G ~ 250
D-Fringe 201 HLINTI:CSQG HFRKALCCKM SAELDVFLES DNYVNVPRLV250
GKKWFCHFDD
X-Lunatic 201 NVISTNCSAA HSRQALSCKM AVYDKFIES DNYVNVRTLV250
DKKWFCHVDD
m-Lunatic 201 NWLTNCSSA HSRQALSCKM AVYDRFIS GKKWFCHVDDDNYVNLRALL25D
m-1fanic 201 HLVVTNCSAE HSHPALSCKM AAEFDAFLVS DNYVNPF:ALL25D
CLRWFCHVDD
m-Radical 201 RMINTNCSAV RTRQALCCKM SVYDRFLES DNYVNPF;SLL250
GRKWFCHVDD
X-kadical 201 QI1VNTNCSAV HTRQALCCKM AVEYDKFVLS DNYLNLHALL250
DKKWFCHLDD
260 270 280 290 3DD
D-Fringe 251 KLLDEYSPSV DtaYLGKPSIS SPLEIHLDSK WFATGGAGFC3DG
NTTTNKF:ITF
X-Lunatic 251 KLLSRYSHTN DIYIGKPSLD RPIQATERI- WFATCGAGFC300
SESNMRPV1JF
m-Lunatic 251 RLLASYPHTQ DVYICKPSLD RPIQATERI- WFATGGAGFC300
SEHKVRPVHF
m-t9anic 251 QLLF;TFPQDR DVYVCKPSLN APIHASELQ- WFATGGAGFC3DD
ShtJRTfiLVRF
m-Radical 251 HLLSTFSSNQ DIYLGRPSLD HPIEATERVQ WFATGCACFC3D0
GGGTS1JTVKF
X-Radical 251 DLLSTFSHST DVYVGRPSLD HPVETVDRD1K WFATGGAGFC3D0
GDCSGS-LKF
t
31D 320 330 340 350
D-Fringe 301 LSRALTLKML PIAGGGKFIS ICDKIRFPDD LKVPLTVVDN350
VTMGFIIEHL
X-Lunatic 301 ISRGLALKMS PWASGGHFMN TAKIRLPDD LGVhLIRSNL350
CTIGYIISV
m-Lunatic 301 ZSRGLALKMG PWASGGHFMS TAERIRLPDD LGVPLIRSGL350
CTIGYIVEAL
m-Manic 301 I1JRQLALRMV PWASGSHFVD TSALIRLPDD LGGRLQPSPL350
CTVGYIIECF:
m-Radical 3G1 LSRGLALKMS PWASLGSFMS TAERVRLPDD LGARLLHSPL350
CTVGYIVEGL
X-Radical 301 ISRGLALKMS PWASMGNFIS TAEKVRLPDD LDVKt1QHSNL350
CTIGYIIEGM
360 370 380 390 9G0
D-Fringe 351 FHSHLEPMEF IRQDTFQDQV SFSYAHMhNQ DT1KTDPKRFY400
t;NVIKVDG-F
X-Lunatic 351 FHSHLENLHQ VPQSEIHNQV TLSYCMFENK SVEEDPSRFR4GG
RNAIL21KGAF
m-Lunatic 351 FHSHLNLQQ VPTTELHQV TLSYCMFENK SVEADPSRFR900
RNAVHIKGPF
m-Manic 351 FHSHLETLQL LGAAQLPQV TLSYGVFEGK SHEDPSRFR900
LNVIKLFGFF
m-Radical 351 FHSHLENLQR LPSGAILQQV TLSYGGPNP NIQQDPTRFQ40G
HNWIJVnCSF
X-Radical 351 FHSHLHLQR LPTESLLF;QV TLSYGGPDNR SLF,EDPTRFfi9D0
.'NVVRVFJGAF
910 42G 430 44~ 4c, G.
D-Fringe 9D1 SLHCQLFPYF SFCPPR---- -
, __., .__...._,. ... . 45G
.''_-Lunatic4G1 SVHCLLYPDT PWCP:;K---- -AAY
...... ...,._., 45D
m-Lunatic 901 SVHCHLYPDT PWCPRS-- - -AIF
...... .,_,__ ,. _ SG
m-Manic q
9D1 SLHCLLYPDT PWCpLL---- -AAP
...... .,_ _ 9c0
m-kadical 4G1 SVHCLLYPDT HWCPMKNR:'E GAFQ......
....,_ _ _ 4SG
-P.adical 9G1 SVHCLLYSDT DWCP--NHF;H IJPTT .
. . _ . . . 45G
SUBSTITUTE SHEET(RULE 26)
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CA 02268751 1999-04-19
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62
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: HSC RESEARCH AND DEVELOPMENT
LIMITED
PARTNERSHIP
(B) STREET: 555 UNIVERSITY AVENUE, SUITE 5270
(C) CITY: TORONTO
(D) STATE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE (ZIP): M5G 1X8
(G) TELEPHONE: 916 813 5982
(H) TELEFAX: 416 8137163
(ii) TITLE OF INVENTION: FRINGE PROTEINSAND NOTCH SIGNALLING
(iii) NUMBER OF SEQUENCES: 6
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0,
Version #1.25 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA PCT/CA97/0 0775
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1150 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID
NO: 1:
ATGCTCCAGC GGTGCGGCCG GCGCCTGCTG TGGGCGCGCT GTTGGCTTGT60
CTGGCGCTGG
CTCCTGGTGC TCACGGCCGA CCCGCCACCG CCGCTGAGCG CGGACGGCGC120
ACTCCGATGC
GCGCTGCGTA GCCTGGCGGG CTCCTCTGGA CTTCAGGGTC CAGGGCGGCT180
GGAGCTCCGG
GTGGATCCCG GAGTCCTCAC CCGCGAGGTG CCGAGTACTT CAGTCTACTC290
CATAGCCTCT
ACCCGCGCGC GCAGAGACGC GGATCCACCG CTTCTCGCCA GGGCGACGGC300
CCCGGGGTCG
CATCCGCGTC CCCCCGCCGA AGTTCTGTCC TCTTCATCGC CGTCAAGACC360
CCTCGCGACG
ACCAGAAAGT TTCACCGCGC GCGGCTCGAT AGACCTGGAT CTCGCGCCAC920
CTGCTGTTCG
AAGGAGATGA CGTTCATCTT CACTGATGGG CTCTGGCCAA GCTCACAGGC980
GAGGACGAAG
AATGTGGTGC TCACCAACTG CTCCTCGGCC AGGCTCTGTC CTGCAAGATG590
CACAGCCGCC
GCTGTGGAGT ATGACCGATT CATTGAGTCT GGTTCTGCCA CGTGGATGAT600
GGGAAGAAGT
SUBSTITUTE SHEET (RULE 26)
CA 02268751 1999-04-19
WO 98117793 PCTlCA97100775
63
GACAACTACGTCAACCTCCGGGCGCTGCTGCGGCTCCTGGCCAGCTATCC CCACACCCAA660
GACGTGTACATCGGCAAGCCCAGCCTGGACAGGCCCATCCAGGCCACAGA ACGGATCAGC720
GAGCACAAAGTGAGACCTGTCCACTTTTGGTTTGCCACCGGAGGAGCTGG CTTCTGCATC780
AGCCGAGGGCTGGCCCTAAAGATGGGCCCATGGGCCAGTGGAGGACACTT CATGAGCACG840
GCAGAGCGCATCCGGCTCCCCGATGACTGCACCATTGGCTACATTGTAGA GGCTCTGCTG900
,
GGTGTACCCCTCATCCGGAGCGGCCTCTTCCACTCCCACCTAGAGAACCT GCAGCAGGTG960
CCCACCACCGAGCTTCATGAGCAGGTGACCCTGAGCTATGGCATGTTTGA GAACAAGCGG1020
AACGCAGTGCACATCAAGGGACCATTCTCTGTGGAAGCTGACCCATCCAG GTTCCGCTCT1080
GTCCATTGCCACCTGTACCCAGACACACCCTGGTGTCCTCGCTCCGCCAT CTTCTAGCAG1190
TCGTGGTTGA 1150
(2) INFORMATION
FOR SEQ
ID NO:
2:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:378 amino
acids
(B) TYPE:
amino
acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:
linear
(xi)SEQUENCE DESCRIPTION: NO:2:
SEQ
ID
Met LeuGln ArgCysGlyArg ArgLeuLeu LeuAlaLeu ValGlyAla
1 5 10 15
Leu LeuAla CysLeuLeuVal LeuThrAla AspProPro ProThrPro
20 25 30
Met ProAla GluArgGlyArg ArgAlaLeu ArgThrLeu AlaGlySer
35 90 45
Ser GlyGly AlaProAlaSer GlySerArg AlaAlaVal AspProGly
50 55 60
Val LeuThr ArgGluValHis SerLeuSer GluTyrPhe SerLeuLeu
65 70 75 80
Thr ArgAla ArgArgAspAla AspProPro ProGlyVal AlaSerArg
B5 90 95
Gln GlyAsp GlyHisProArg ProProAla GluValLeu SerProArg
100 105 110
Asp ValPhe IleAlaValLys ThrThrArg LysPheHis ArgAlaArg
115 120 125
Leu AspLeu LeuPheGluThr TrpIleSer ArgHisLys GluMetThr
130 135 140
Phe IlePhe ThrAspGlyGlu AspGluAla LeuAlaLys LeuThrGly
145 150 155 160
SUBSTITUTE SHEET (RULE 28)
i
CA 02268751 1999-04-19
WO 98/I7793 PCT/CA97/00775
64
Asn Val Val Leu Thr Asn Cys Ser Ser Ala His Ser Arg Gln Ala Leu
165 170 175
Ser Cys Lys Met Ala Val Glu Tyr Asp Arg Phe Ile Glu Ser Gly Lys
180 185 190
Lys Trp Phe Cys His Val Asp Asp Asp Asn Tyr Val Asn Leu Arg Ala
195 200 205
Leu Leu Arg Leu Leu Ala Ser Tyr Pro His Thr Gln Asp Val Tyr Ile
210 215 220
Gly Lys Pro Ser Leu Asp Arg Pro Ile G1I1 Ala Thr Glu Arg Ile Ser
225 230 235 240
Glu His Lys Val Arg Pro Val His Phe Trp Phe Ala Thr Gly Gly Ala
295 250 255
Gly Phe Cys Ile Ser Arg Gly Leu Ala Leu Lys Met Gly Pro Trp Ala
260 265 270
Ser Gly Gly His Phe Met Ser Thr Ala Glu Arg Ile Arg Leu Pro Asp
275 280 285
Asp Cys Thr Ile Gly Tyr Ile Val Glu Ala Leu Leu Gly Val Pro Leu
290 295 300
Ile Arg Ser Gly Leu Phe His Ser His Leu Glu Asn Leu Gln Gln Val
305 310 315 320
Pro Thr Thr Glu Leu His Glu Gln Val Thr Leu Ser Tyr Gly Met Phe
325 330 335
Glu Asn Lys Arg Asn Ala Val His Ile Lys Gly Pro Phe Ser Val Glu
390 395 350
Ala Asp Pro Ser Arg Phe Arg Ser Val His Cys His Leu Tyr Pro Asp
355 360 365
Thr Pro Trp Cys Pro Arg Ser Ala Ile Phe
370 375
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 966 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ATGCACTGCC GACTTTTTCG GGGCATGGCG GGAGCCCTCT TTACCCTCCT GTGCGTGGGG 60
CTCC'FGTCTC TACGATACCA CTCAAGTTTG TCCCAGAGGA TGATACAGGG CGCGCTCAGG 120
CTGAACCAAC GGAACCCAGG ACCCCTGGAG CTGCAGCTAG GCGACATCTT CATCGCAGTC 1B0
SUBSTITUTE SHEET (RULE 26)
CA 02268751 1999-04-19
WO 98/17793
PCTlCA97/00775
65
AAGACTACCTGGGCCTTCCATCGCTCCCGCCTGGACCTGCTACTAGACACGTGGGTCTCC290
AGGATCAGGCAACAGACATTCATCTTCACTGACAGCCCAGATGAACGCCTCCAGGAGAGA300
CTAGGCCCGCACCTCGTGGTCACCAACTGTTCTGCAGAGCACAGTCATCCTGCTCTGTCC360
TGCAAGATGGCTGCAGAGTTCGATGCCTTCTTGGTCAGTGGCCTCAGGTGGTTCTGCCAC920
GTGGATGATGACAACTATGTGAACCCCAAGGCTCTGCTGCAGCTGTTGAAAACATTCCCG980
CAGGACCGTGATGTCTATGTGGGCAAGCCCAGCCTGAACCGGCCCATCCACGCCTCTGAG590
CTGCAGTCAAAAAACCGCACGAAGCTGGTGCGGTTCTGGTTTGCCACAGGGGGTGCTGGT600
TTCTGCATCAACCGCCAACTGGCTTTGAAGATGGTGCCATGGGCCAGCGGCTCCCACTTT660
GTGGACACTTCTGCTCTCATCCGGCTCCCCGATGACTGCACTGTGGGCTACATCATCGAG720
TGCAAGCTGGGGGGTCGCCTGCAGCCCAGCCCCCTCTTCCACTCACACCTGGAAACCCTG780
CAGCTGCTGGGGGCCGCCCAGCTTCCGGAGCAGGTCACCCTCAGCTACGGTGTCTTTGAG890
GGGAAACTGAATGTCATCAAGCTACCGGGCCCCTTCTCCCATGAAGAGGACCCCTCCAGA900
TTCCGCTCCCTCCATTGTCTCCTCTACCCAGACACACCCTGGTGTCCGCTGCTGGCAGCG960
CCCTGA 966
(2) INFORMATION
FOR
SEQ
ID NO:
4:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:321 amino
acids
(B) TYPE:
amino
acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:
linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Met His Cys Arg Leu Phe Arg Gly Met Ala Gly Ala Leu Phe Thr Leu
1 5 10 15
Leu Cys Val Gly Leu Leu Ser Leu Arg Tyr His Ser Ser Leu Ser Gln
20 25 30
Arg Met Ile Gln Gly Ala Leu Arg Leu Asn Gln Arg Asn Pro Gly Pro
35 90 45
Leu Glu Leu Gln Leu Gly Asp Ile Phe Ile Ala Val Lys Thr Thr Trp
50 55 60
Ala Phe His Arg Ser Arg Leu Asp Leu Leu Leu Asp Thr Trp Val Ser
65 70 75 80
Arg Ile Arg Gin Gln Thr Phe Ile Phe Thr Asp Ser Pro Asp Glu Arg
85 90 95
Leu Gln Glu Arg Leu Gly Pro His Leu Val Val Thr Asn Cys Ser Ala
100 105 110
SUBSTITUTE SHEET (RULE 26)
m i
CA 02268751 1999-04-19
WO 98/17793 PCT/CA97100775
66
Glu HisSerHis ProAlaLeu SerCysLys MetAlaAlaGlu PheAsp
115 120 125
Ala PheLeuVal SerGlyLeu ArgTrpPhe CysHisValAsp AspAsp
130 135 19C
Asn TyrValAsn ProLysAla LeuLeuGln LeuLeuLysThr PhePro
i45 150 155 160
Gln AspArgAsp ValTyrVal GlyLysPro SerLeuAsnArg ProIIe
165 170 175
Elis AlaSerGlu LeuGlnSer LysAsnArg ThrLysLeuVal ArgPhe
180 185 190
Trp PheAlaThr GlyGlyAla GlyPheCys IleAsnArgGln LeuAla
195 200 205
Leu LysMetVal ProTrpAla SerGlySer HisPheValAsp ThrSer
210 215 220
Ala LeuIleArg LeuProAsp AspCysThr ValGlyTyrIle IleGlu
225 230 235 290
Cys LysLeuGly GlyArgLeu GlnProSer ProLeuPheHis SerHis
295 250 255
Leu GluThrLeu GlnLeuLeu GlyAlaAla GlnLeuProGlu GlnVal
260 265 270
Thr LeuSerTyr GlyValPhe GluGlyLys LeuAsnValIle LysLeu
275 280 2B5
Pro GlyProPhe SerHisGlu GluAspPro 5erArgPheArg SerLeu
290 295 300
His CysLeuLeu TyrProAsp ThrProTrp CysProLeuLeu AlaAla
305 310 315 320
Pxo
(2) INFORMATION FOR SEQ ID N0: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 999 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ATGAGCCGTG CGCGGCGGGT GTTGTGCCGG GCCTGCCTCG CGCTGGCCGC GGTCCTGGCT 60
GTGTTGCTGC TACTGCCGCT GCCGCTACCG CTGCCGCTGC CTCGCGCGCC CGCACCGGAC 120
CCCGATeGGG TCCCGACCCG GAGCCTGACC CTCGAGGGAG ACCGCCTGCA ACCCGACGAC 180
GTCTTCATTG CAGTCAAGAC CACTCGGAAG AACCACGGCC CGCGCCTGCG GCTGCTGCTG 240
SUBSTITUTE SHEET (RULE 26)
CA 02268751 1999-04-19
WO 98!17793 PCTICA97I00775
67
CGTACCTGGATCTCACGAGCCCCACGGCAGACGTTCATTTTCACCGATGG AGACGACCCT300
GAGCTCCAGATGCTGGCAGGCGGCCGCATGATCAACACCAATTGCTCTGC TGTGCGCACC360
CGCCAAGCACTGTGCTGCAAAATGTCGGTGGAATATGATAAGTTCCTAGA ATCTGGACGF920
AAATGGTTCTGCCACGTGGATGATGACAACTACGTGAACCCCAAAAGCCT GCTGCACCTG980
CTTTCCACCTTCTCTTCCAACCAGGACATCTACCTGGGGCGACCTAGCCT GGACCACCCC59C
ATCGAAGCCACAGAGAGGGTCCAAGGCGGTGGCACCTCAAACACAGTGAA ATTCTGGTTT600
GCTACTGGTGGGGCTGGGTTCTGCCTGAGCAGGGGCCTTGCCCTCAAAAT GAGCCCGTGG660
GCCAGCCTTGGCAGTTTCATGAGCACAGCAGAGCGGGTTCGGCTGCCTGA TGACTGCACT720
GTGGGATACATCGTGGAAGGACTTCTGGGCGCCCGTCTGCTCCATAGCCC CCTGTTCCAC780
TCGCACCTGGAAAACCTGCAGAGGCTGCCGTCTGGTGCTATTTTGCAGCA GGTTACCTTG890
AGCTATGGGGGTCCTGAGAACCCACATAATGTGGTGAATGTAGCTGGCAG TTTCAACATA900
CAGCAGGACCCTACACGGTTTCAGTCTGTGCACTGCCTTCTCTACCCAGA CACCCACTGG960
TGTCCTATGAAGAACAGGGTTGAGGGAGCTTTCCAGTAA 999
(2) INFORMATION
FOR
SEQ
ID NO:
6:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:332 amino
acids
(B) TYPE:
amino
acid
(C) STRANDEDNESS: e
singl
(D) TOPOLOGY:
linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Ser Arg Ala Arg Arg Val Leu Cys Arg Ala Cys Leu Ala Leu Ala
1 5 10 15
Ala Val Leu Ala Val Leu Leu Leu Leu Pro Leu Pro Leu Pro Leu Pro
20 25 30
Leu Pro Arg Ala Pro Ala Pro Asp Pro Asp Arg Val Pro Thr Arg Ser
35 90 95
Leu Thr Leu Glu Gly Asp Arg Leu Gln Pro Asp Asp Val Phe Ile Ala
50 55 60
Val Lys Thr Thr Arg Lys Asn His Gly Pro Arg Leu Arg Leu Leu Leu
65 70 75 80
Arg Thr Trp Ile Ser Arg Ala Pro Arg Gln Thr Phe Ile Phe Thr Asp
85 90 95
Gly Asp Asp Pro Glu Leu Gln Met Leu Ala Gly Gly Arg Met Ile Asn
100 105 110
Thr Asn Cys Ser Ala Val Arg Thr Arg Gln Ala Leu Cys Cys Lys Met
SUBSTITUTE SHEET (RULE 26)
m
CA 02268751 1999-04-19
WO 98/17793 PCT/CA97100775
68
115 120 125
Ser Val Glu Tyr Asp Lys Phe Leu Glu Ser Gly Arg Lys Trp Phe Cys
130 135 190
His Val Asp Asp Asp Asn Tyr Val Asn Pro Lys Ser Leu Leu His Leu
145 150 155 160
Leu Ser Thr Phe Ser Ser Asn Gln Asp Ile Tyr Leu Gly Arg Pro Ser
165 170 175
Leu Asp His Pro Ile Glu Ala Thr Glu Arg Val Gln Gly Gly Gly Thr
180 lay 190
Ser Asn Thr Val Lys Phe Trp Phe Ala Thr Gly Gly Ala Gly Phe Cys
195 200 205
Leu Ser Arg Gly Leu Ala Leu Lys Met Ser Pro Trp Ala Ser Leu Gly
210 215 220
Ser Phe Met Ser Thr Ala Glu Arg Val Arg Leu Pro Asp Asp Cys Thr
225 230 235 240
Val Gly Tyr Ile Val Glu Gly Leu Leu Gly Ala Arg Leu Leu His Ser
245 250 255
Pro Leu Phe His Ser His Leu Glu Asn Leu Gln Arg Leu Pro Ser Gly
260 265 270
Ala Ile Leu Gln Gln Val Thr Leu Ser Tyr Gly Gly Pro Glu Asn Pro
275 280 285
His Asn Val Val Asn Val Ala Gly Ser Phe Asn Ile Gln Gln Asp Pro
290 295 300
Thr Arg Phe Gln Ser Val His Cys Leu Leu Tyr Pro Asp Thr His Trp
305 310 315 320
Cys Pro Met Lys Asn Arg Val Glu Gly Ala Phe Gln
325 330
SUBSTITUTE SHEET (RUES 26)
