Note: Descriptions are shown in the official language in which they were submitted.
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A MEMBRANE-BOUND NETRIN
FTRLD OF THE INVENTION
The present invention relates to a novel member of the
Netrin protein family having nervous system axon guidance
function, which differs structurally and functionally from
known Netrins.
HACRGROUND OF THE INV.NmTnN
In the process of development of the nervous system,
growing axons are guided appropriately to correct targets to
form precise wiring of intricate networks. The molecular
mechanism of axon guidance is being clarified through the
identification of a number of gene families encoding the
keys to the guidance of axon growth cone and cell migration.
These genes include members of the immunoglobulin super
family, ephrins, semaphorin, slits and Netrins (Tessier-
Lavigne and Goodman, Science 274: 1123-1133, 1996; Chisholm
and Tessier-Lavigne, Curr. Opin. Neurobiol. 9: 603-615,
1999). A number of these are membrane proteins that have
been identified as contact-repulsive molecules or contact-
attractive molecules while others are secretion proteins
that have been identified as diffusible chemotropic signals.
Netrin-1 and Netrin-2 are chemo-attractive factors,
first identified through the biochemical formation of
molecules having commissural axon outgrowth-promoting
activity (Serafini et al., Cell 78: 409-424, 1994; Kennedy
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et al., Cell 78:425-435, 1994). UNC-6 is a homologue of
Netrin found in C.elegans. In vertebrate animals, Netrins
play a role in attracting the lamina alaris axons running
along the rostrocaudal axis, toward the tabula or the
ventromedian area (Kennedy et al., Cell 78: 425-435, 1994;
Shirasaki et al., Neuron 17:1079-1088, 1996), and in the
repulsion of the trochlear motor axons away from the midline
(Colamarino and Tessier-Lavigne, Cell 81: 621-629, 1995).
From gene analysis in nematodes and, in single cell analysis
in Xenopus laevis spinal neurons, it was concluded that
these dual effects (attraction and repulsion) were
transmitted by a single ligand via two types of receptor
sub-family belonging to the immunoglobulin super family (de
la Torre et al., Neuron 19: 1211-1224, 1997; Hong et al.,
Cell 97: 927-941, 1999). The attraction effect is
transmitted by means of UNC-40 in nematodes, frazzled
protein in Drosophila, DCC (Deleted in colorectal cancer) in
vertebrate animals, and a DCC sub-family receptor to which
neogenin belongs. (Chan et al., Cell 87: 187-195, 1996;
Keino-Masu et al., Cell 87: 175-185, 1996; Kolodziej et al.,
Cell 87: 197-214, 1996; de la Torre et al., Neuron 19:
1211-1224, 1997). In contrast, several of the repulsion
effects of Netrin require members of the UNC-5 family in
addition to the members of the UNC-40/DCC family. (Hedgecock,
et al., Neuron 2: 61-85, 1990; Hong et al., 1999).
In mammals, three types of UNC-5 receptor family member,
UNC5H1, UNC5H2 and UNC5H3/RCM (rostral cerebellar
malformation) have been isolated (Ackerman et al., Nature
386: 838-842, 1997; Leonardo et al., Nature 386: 833-838,
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1997). The members of both receptor sub-families all bind to
Netrin.
It had come to be thought that Netrin and UNC-6
constituted a phylogenically conserved small protein family.
(Chisholm and Tessier-Lavigne, Curr. Opin. Neurobiol. 9:
603-615, 1999). It differs from other axon guidance
molecules, in other words, semaphorin (Dodd and Schuchardt,
Cell 81: 471-474, 1995) and ephrin (Flanagan and
Vanderhaegen, Annu. Rev. Neurosci. 21: 309-345, 1998), and,
in the types that have been studied up until now, at most
only two types of UNC-6/Netrin family members have been
isolated. However, as is the case with other families, the
existence of further members is predicted. The high level of
variety within the semaphorin and ephrin families possibly
reflects their contribution to the formation of complex and
high level unified neuron networks in vertebrate animals.
Therefore, to further clarify the function of Netrin in
the brains of vertebrate animals, and to provide suitable
treatment for brain damage caused by disease, and further to
allow promotion of normal neural pathway formation during
transplant treatment and the like, there is a need to
identify various unknown members of the UNC-6/Netrin family
that are predicted to exist and to analyze their function in
organisms.
HREIF SUMMARY OF THE INVENTION
Therefore, the present inventors, as a result of focused
examination directed to discovering a novel member of the
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UNC-6/Netrin family in mice, discovered a membrane-bound
Netrin differing structurally and functionally from
previously known Netrins, thereby completing the present
invention. The novel Netrin of the present invention differs
from classical Netrins, is primarily bound to the plasma
membrane by means of a glycosylphospha-tidylinsitol (GPI)
lipid anchor, and has numerous isoforms most likely formed
by alternative splicing. Further, it has become apparent
that the Netrin of the present invention does not bind with
classical Netrin receptors, and exhibits no duplication of
function with Netrin-1.
In other words, the present invention provides the
following (1) to (18)
(1) A membrane-bound Netrin having a hydrophobic region at
its C-terminus, wherein the hydrophobic region is able
to bind to the cell membrane by means of
glycosylphosphatidylinositol (GPI).
(2) The membrane-bound Netrin according to (1) above which
comprises an amino acid sequence indicated by SEQ ID
NOS: 8, 10, 12, 14 or 16.
(3) A polynucleotide encoding the membrane-bound Netrin
according to (1) or (2) above.
(4) The polynucleotide according to (3) above comprising
the nucleotide sequence indicated by SEQ ID NO: 7, 9,
11, 13 or 15.
(5) A protein or fragment thereof, according to either (a)
or (b) below:
(a) A protein comprising an amino acid sequence indicated
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by SEQ ID NO: 8, 10, 12, 14, 16 or 18; or, a fragment
of said protein.
(b) A protein comprising an amino acid sequence derived
from an amino acid sequence indicated by SEQ ID NO: 8,
10, 12, 14, 16 or 18, by deletion, substitution or
insertion of one or several amino acids, or a fragment
of said protein, wherein said protein or fragment has
Netrin function.
(6) A polynucleotide according to either (a) or (b) below,
or a fragment thereof:
(a) A polynucleotide comprising a nucleotide sequence
indicated by SEQ ID NO: 7, 9, 11, 13, 15 or 17; or, a
fragment of said polynucleotide.
(b) A polynucleotide comprising a nucleotide sequence
derived from the nucleotide sequence indicated by SEQ ID
NO: 7, 9, il, 13, 15, or 17, by deletion, substitution or
insertion of one or several nucleotides, or a fragment of
this polynucleotide, encoding a protein having Netrin
function.
The term "several" in (5) or (6) above refers to a
number of mutations able to be induced by site specific
mutagenesis, for example, and means a number within a range
such that pre-mutation function is not lost.
Further, a reference to the term "fragment" herein
includes, but is not limited to, each of the domains of the
protein of the present invention as described herein and the
corresponding polynucleotides that encode them.
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(7) An expression vector comprising the polynucleotide
according to (3), (4) or (6) above, or a fragment of
this polynucleotide.
A vector able to comprise the above-mentioned
polynucleotide can be any usable vector known within the
relevant field, and if suitable for expression of the
protein encoded by the above-mentioned polynucleotide, is
not particularly limited. However, the following can be
used: pcDNA4Myc/His (Invitrogen), pGEX-2T (Amersham
Pharmacia), pET-32a (Novagen), Retro-X System (Clontech),
and the like. Further, as is well known in the relevant
field, a regulatory sequence such as promoter, enhancer or
the like, able to control expression of the above
polynucleotide can be included within the expression vector
where thought suitable.
Further, the protein to be expressed can be just the
protein of the present invention or a fragment thereof or,
as required depending on one's purpose, for example, fusion
proteins with glutathione S-transferase and green
fluorescent protein, or with tags such as (His)6or myc, etc.,
may be expressed.
(8) A host cell transfected with the expression vector of
( 7 ) above .
As a host cell, any cell which can express the above
polypeptide is acceptable including animal cells, plant
cells, bacteria and the like that are known to be usable in
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this field. In particular, the use of E. coli, HEK293 cell,
COS7 cell or the like is preferable.
(9) A method for producing the membrane-bound Netrin
according to (1) or (2) above, or the protein or fragment
thereof according to (5) above comprising culturing the
host cell according to (8) above.
(10) A primer used in the amplification of the
membrane-bound Netrin according to (1) or (2) above, or
the protein or fragment thereof, according to claim 5.
(11) The primer according to (10) above, which
corresponds to the nucleotides encoding amino acids 353
to 359, and amino acids 520 to 526 in the amino acid
sequence indicated by SEQ ID NO: 8, or a portion of said
nucleotides.
(12) The primer according to (10) or (11), wherein
said primer comprises the sequences indicated by SEQ ID
NOS: 19 and 20.
In (11) and (12) above, a primer including forward
and/or reverse primers) either individually or in
combination, is used.
(13) A probe specific to the membrane-bound Netrin
isoform according to (1) or (2) above, or the protein, or
fragment thereof, according to (5) above.
(14) The probe according to (13) above having a
sequence corresponding to nucleotides 1959 to 2261 of the
nucleotide sequence indicated by SEQ ID NO: 7,
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nucleotides 1959 to 2084 of the nucleotide sequence
indicated by SEQ ID NO: 13, or nucleotides 2262-2403 of
the nucleotide sequence indicated by SEQ ID NO: 7.
In (13) and (14) above, the probe may be either DNA or
RNA, and may include either a sense sequence or an antisense
sequence. Further, the probe can be labeled with radioactive
indicators, or non-radioactive indicators such as
fluorescent indicators.
(15) An antibody having specificity against the
membrane-bound Netrin according to (1) or (2) above or
the protein, or fragment thereof, according to (5) above.
(16) The antibody according to (15) above having
specificity against specific isoforms of the membrane-
binding Netrin according to (1) or (2) above.
The "antibody" of (15) and (16) above, may either be a
polyclonal antibody or a monoclonal antibody. A polyclonal
antibody can be obtained by immunizing an animal by a
typical method with the above-mentioned membrane-bound
Netrin or a polypeptide segment thereof as an antigen.
Further, a monoclonal antibody may be obtained from a
hybridoma obtained by fusion of an antibody producing cell
and a myeloma cell. Preparation of monoclonal and polyclonal
antibodies can be performed according to procedures known
within the relevant art.
(17) A transgenic animal into which is introduced a
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gene encoding the membrane-bound Netrin according to (1)
or (2) above or the protein according to (5) above.
(18) A homozygotic or heterozygotic knock-out animal
in which a gene encoding the membrane-bound Netrin
according to (1) or (2) above, or the protein according
to (5) above, has been inactivated.
This specification includes part or all of the contents
as disclosed in the specification and/or drawings of
Japanese Patent Application No.2000-148843, which is a
priority document of the present application.
BREIFDESCRIPTION OF THE DRAWINGS
Figure 1 indicates expression of 7D5 in the brain:
(A) Indicates the expression profile of 7D5 in
the development process of the cerebellum according to
northern blot analysis.
(B) Indicates tissue distribution of 7D5 in an
adult mouse . -actin was used as a control for RNA loading.
Arrows (A) and (B) indicate 7D5 signals.
Figure 2 indicates the alignment of the putative amino
acid sequence of Netrin-Bla (7D5) with vertebrate Netrin.
Amino acid residues identical as between Netrin-Bla and
other Netrins are shown in negative (white print on black
background). The arrows above the sequences indicate domain
structures of Netrin-B1a (domains VI, V1, V2, V3 and C'),
defined by homology with other Netrins. The * symbol
indicates the putative N-glycosylation site of Netrin-Bia.
Figure 3 is a schematic representation and
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hydrophobicity plot of Netrin-B1 and other UNC-6/Netrins.
(A) is a schematic representation of the domain
structures of members of the UNC-6/Netrin family.
Homology(%) between the amino acid sequences of homologous
domains is represented. Netrin-Bla is bound on the cell
membrane by a GPI-linkage whereas Netrin-1 and Netrin-3 are
secreted. The C domain of classical Netrins is highly
charged ( indicated by + -I- ) .
(B) indicates a Kyte-Doolittle hydrophobic plot
of the amino acid sequences of Netrin-Bla and mouse Netrin-1.
Figure 4 indicates the structure and expression of
Netrin-B1 isoforms.
(A) indicates the sequences in the vicinity of
the variable regions of six types of isoform, Netrin-Bia-Blf.
Netrin-Bia most resembles Netrin-1, Netrin-2, and Netrin-3
in respect of domain structure. Netrin-Blb and Blc has no
domain V2 (EGF2) and no domain V2 and V3 (EGF3),
respectively. Netrin-B1d has an unknown domain (Ukd) insert
between domain V1 (EGF1) and C' whose sequence is shown in
negative. Netrin-B1e has a shorter Ukd. All of these five
types of isoform (Netrin-Bla to B1e) have domains VI, V1 and
C'. Netrin-Bif lacks domains V2, V3 and C'. This isoform,
exceptionally, has no C-terminus hydrophobic stretch. Absent
sequences are indicated by "~" and the "-" at the ends of a
sequence indicates that the sequence continues.
(B) is a schematic representation of Netrin-B1
isoforms. The hydrophobic stretches of the N and C termini
are indicated by shading, and the Ukd domains of Netrin-Bld
and Ble are indicated with dots.
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(C) indicates an RT-PCR analysis of 5 types of
isoform in adult whole brain and various regions of PO brain.
RT-PCR is indicated by an arrow in Fig. B. RT-PCR was
performed with primer sets corresponding to the nucleotides
of EGF1 and domain C', respectively. Further southern blot
hybridization was performed using the isoform specific
probes) indicated in the lower portion of Figure B.
Isoforms are indicated with an arrow at the right.
Figure 5 indicates the PI-PLC sensitivity of recombinant
Netrin-B1a and Netrin-Bld.
Figure 6 indicates the regional distribution of Netrin-
B1 transcription products in mouse brain analyzed by whole
mount in situ hybridization.
PO mouse brain parasagittal sections (A, B, D, E, G and
H), and P2 mouse brain coronal vibratome slices (C and F)
were hybridized with digoxigenin labeled antisense cRNA
probes specific for Netrin-B1 (A-F excluding B) or
Unc5h3/RCM (G and H). The sense probe for Netrin-B1 show no
signal under the same conditions as those for the antisense
probe (B). As can be seen in the lateral view of the
cerebral hemisphere, the distribution of Netrin-B1 (E) and
Unc5h3/RCM (H) is complementary, and delineates a boundary
between the neocortex and allocortex as indicated by an
arrow. The bars in the figure indicate 1mm (A, B, E, and H),
0.85mm (C), 0.65mm (F), and 0.8mm (D and G), respectively.
Abbreviations are as follows: deep cerebellar nuclei
(DCN), external germinal layer (EGL), hippocampal formation
(HP), inferior colliculus (IC), inferior olive (IO),
mammillary body (MB), olfactory bulb (OB), piriform cortex
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(PR), red nucleus (RN), retrosplenial cortex(RC), superior
colliculus (SC), and thalamus (TH).
Figure 7 indicates an ontogenic analysis of Netrin-B1
expression.
Figure 8 indicates Netrin-B1's lack of affinity to
Netrin receptors.
Unc5h3/RCM whose cytoplasm domains were replaced by ECFP
were expressed in COS7 cells. Expression of the receptor
protein was detected as ECFP fluorescence (blue signal) (A,
C and E). DCC was expressed in 293 EBNA cells, and detected
using mouse monoclonal anti-DCC antibody and Alexa 488
conjugated anti-mouse IgG (green signal) (G and I). In the
same field, binding of myc-tagged chick Netrin-1
(Netrini/myc) on the cells was detected with
immunocytochemical methods using monoclonal anti-myc
antibody (9E10) and Alexa 546 conjugated anti-mouse IgG(B)
in the case of Unc5h3/RCM, and rabbit polyclonal anti-myc
antibody and Alexa 546 conjugated anti-rabbit IgG (H) in the
case of DCC (red signal). However, with the secreted forms
of myc-tagged Netrin-B1a (sNetrin-B1a/Myc) and myc-tagged
Netrin-Bld (sNetrin-B1a/Myc), there was no indication of
binding on the cells expressing both receptors in high
concentration (D, F and J). A similar result was obtained
with cells expressing Unc5h1 or Unc5h2 receptors. With both
sNetrin-B1c and sNetrin-Ble, there was no indication of
binding to cells expressing either of the receptors
investigated (data not indicated). Figure K shows the
relative concentration of the tested ligands used in the
binding experiment as clarified by anti-myc immunoblotting.
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The figure shows representative results obtained from one of
three independent experiments. The bars in the figure
represent l0,um.
Figure 9 indicates Netrin-B1's inability to complement
Netrin-1 activity in collagen gel cerebellar plate explant.
Cerebellar plate (CP) obtained from E12 mouse were co-
cultured in collagen gel with an aggregate of HEK293T cells
expressing Netrin-1 (A), secretion-type Netrin-Bla (sNetrin-
Bla)(B), secretion type Netrin-Bld (sNetrin-Bld), and mock
transfected cells (D), respectively. Cells expressing
Netrinl attracted axons from the CP (A). However, neither
sNetrin-Bla nor sNetrin-Bld could elicit CP axon growth (B
and C) (n~8). The bars in the figure are 100,u m (A-D).
Figure 10 indicates the phylogenetic relationship
between Netrin-Bl and other members of the Unc-6/Netrin
family.
A phylogenetic tree was constructed based on the amino
acid sequences of C.elegans Unc-6 (P34710), chick Netrin-1
(Q90922), chick Netrin-2 (Q90923), mouse Netrin-1 (AAC52971),
mouse Netrin-3 (AAD40063), human Netrin-1 (NP004813), human
NTN2L (NP006172), zebra fish Netrin-1 (AAB70266), zebra fish
Netrin-1a (AAC60252), Drosophila Netrin-A (Q24567),
Drosophila Netrin-B (Q24568) and Netrin-B1a (AB038667, SEQ
ID NO: 8) using CLUSTAL X program. The figure indicates that
Netrin-B1 has evolved at a great distance from other members
of the family, and that Netrin-B1 may become the prototype
of a novel sub-family.
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Divergence of UNC-6/Netrin Familv
Members of the UNC-6/Netrin family are laminin-related
small proteins. Laminin is a heterotrimeric extracellular
matrix protein of a , a and y chains, the N-terminal portion
of each chain is constructed from a single spherical domain
(domain VI) and several EGF-like repeats (domain V)
continuing therefrom. (Sasaki et al., J. Biol. Chem. 263:
16536-16544, 1988; Beck et al., FASEB J. 4: 148-160, 1990;
Timpl et al., Matrix Biol. 14: 275-281, 1994). Members of
the UNC-6/Netrin family have the characteristic that domain
structure is conserved from domain VI of laminin to V (the
third EGF-like repeat) (Ishii et al., Neuron 9:873-881,
1992; Serafini et al., Cell 78: 409-424, 1994; Engvall et
al., J. Cell Biochem. 61: 493-501, 1996).
We screened the cDNA fragment of secretion protein and
membrane protein predicted by signal sequence trap method,
and identified a novel member of the UNC-6/Netrin family.
The overall homology level of the Netrin (denoted
Netrin-B1) of the present invention to known UNC-6/Netrin
family members is low. However, its domain structure matches
the structure of classical members which provides the
molecular basis for including this novel gene in the UNC-
6/Netrin family. As with other UNC-6/Netrin (Ishii et al.,
Neuron 9: 873-881, 1992; Serafini et al., Cell 78: 409-424,
1994), Netrin-B1 is related in respect of the y -chain more
than the ~ -chain of laminin (with 32~ homology to mouse y
chain and, 27% homology to the ~ -chain), further supporting
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this conclusion. Since in all types that were examined,
chick (Serafini et al., Cell 78: 409-424, 1994), fly
(Mitchell et al., Neuron 17: 203-215, 1996; Harris et al.,
Neuron 17: 217-228, 1996), mouse (Puschel, Mech Dev 83:65-75,
1999; Wang et al., J. Neurosci. 19: 4938-4947, 1999), and
human (Van Raay et al., Genomics 41: 279-282, 1997), only
two types of gene were isolated, the UNC-6/Netrin family was
thought to be a small family. The Netrin-B1 of the present
invention, is a third member of this family in mouse. The
present inventors suggest the possibility that the UNC-
6/Netrin family has, like other axon guidance molecule
families, such as semaphorin (Dodd and Schuchardt, Cell
81:471-474, 1995) and ephrin (Flanagan and Vanderhaeghen,
Annu. Rev. Neurosci 21:309-345, 1998), diversified during
the course of evolution more greatly than had been predicted
up until now. From a phylogenetic analysis (Fig. 10) of the
gene family, it is suggested that Netrin-B1 evolved
independently from classical Netrins, and that Netrin-Bl may
form the prototype of a new sub-family.
Netrin-B1 has several characteristics that differ from
classical Netrins. One characteristic of Netrin-B1 can be
seen in its C-terminal sequence. Domain C of Netrin-1 is
rich in lysine which is a basic residue and functions as a
heparin-binding site (Serafini et al., Cell 78: 409-424,
1994). This basic C domain can bind to the cell surfaces via
negatively charged molecules such as proteoglycan. Therefore,
it is thought that the diffusion range of Netrin is
determined by the expression level of Netrin relative to the
binding site concentration in the environment. The C-
CA 02324167 2000-11-20
terminal sequence of Netrin-B1 (domain C') is not rich in
lysine, rather it encodes a signal for the GPI anchor (Fig.
3). From expression in HEK293T cell and PI-PLC processing,
it became clear that Netrin-B1a and Bid were bound by means
of a GPI linkage on cell surface. This characteristic of
Netrin-B1 contrasts with diffuse classical Netrin. Therefore,
it is thought that Netrin-B1 has specialized in order to
play a role in a narrower region. However, this does not
exclude the possibility that Netrin-B1, will under certain
conditions, disperse afar. It has been reported that GPI
anchor molecules such as axonin 1/Tag1 may be released from
the membrane, by internally expressed glycosyl PtdIns
specific phosphalipase D, and that this oxygen activity was
detected in posterior root ganglion neurons and in the brain
(Lierheimer et al., Eur. J. Biochem. 243:502-510, 1997).
From broad-ranging cDNA analysis, it became clear that
Netrin-B1 included at least six types of isoform (Fig. 4).
The longest isoform most resembling classical Netrin in
respect of domain structure was named Netrin-Bla. Netrin-B1b
and Blc are respectively absent 1 unit and 2 units of EGF-
like repeat. Since it is thought that the EGF-like motif
functions in relation to protein-protein interaction, there
is the possibility that these isoform differ in their
binding specificity for related molecules. In the nematode,
C.elegans, a mutant strain lacking domain V2 of UNC-6
exhibits selective loss of function. A null mutant exhibited
loss of both dorsal and anterior guidance in relation to
nearby axons (Hedgecock et al., Neuron 2: 61-85, 1990),
however a V2 deletion mutant strain exhibited loss of dorsal
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guidance only (Wadsworth, et al., Neuron 16: 35-46, 1996)
suggesting that the existence of this domain was important
in some types of expression forms. Netrin-Bld and Bie are
absent two EGF-like repeats but have an unknown domain
insert between domain V1 (EGF-1) and C'. It is possible that
through this insert, the binding specificity of these
isoforms for their partners has been further altered. Since
Netrin-Blf is absent a C' domain, this isoform diffuses and
like classical Netrins can influence distant targets.
From Southern blot hybridization of genomic DNA and
partial sequencing through genomic cloning, it was suggested
that these isoforms are derived from a single gene, and are
likely formed by alternative splicing (Data not indicated).
This data suggests that the exon-intron structure of Netrin-
B1 also resembles that of other UNC-6/Netrin family members
(Ishii et al., Neuron 9: 873-881, 1992; Harris et al.,
Neuron 17: 217-228, 1996). The Ukd domain found in Netrin-
Bld and B1e is thought to encode a novel extra exon that was
obtained during the process of evolution. Interestingly,
from semi-quantitative RT-PCR (Fig. 4C), it was suggested
that alternative splicing appeared to be regulated in a
regional and developmental stage specific manner. It is
likely that alternative splicing further diversifies the
function of Netrin-B1. It is not known whether classical
Netrins having slicing-mutant forms or not.
Netrin B1's Lack of affinity to Netrin receptor
The binding ability of isoforms of Netrin-B1 to Netrin
receptors was examined. To assess any functional duplication,
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the binding ability of Netrin-B1 isoforms to Netrin
receptors was examined. Unc5hl, Unc5h2, Unc5h3/RCM, DCC and
neogenin are receptors for classical Netrins in vertebrate
animals (Keino-Masu et al., Cell 87: 175-185, 1996; Leonardo
et al., Cold Spring Harb. Symp. Quant. Biol. LXII: 467-478,
1997; Ackerman et al., Nature 386: 838-842, 1997). Classical
Netrin can bind with all of these receptors.
The binding ability of Netrin-Bla-myc, B1b-myc, Bld-myc,
and B1e-myc to Unc5h3/RCM expressed in COS7 and HEK293T
cells, and other Netrin receptors was examined. Netrinl-myc
used as a positive control bonded to these test receptors,
and exhibited functional expression of the receptor
molecules but not any of the test ligands exhibited
detectable affinity for these receptors even at a
concentration which was 100 times higher compared to
Netrinl-myc. Detection of bound molecules, and determination
of their relative concentrations was conducted using anti-
myc antibody. It is possible to obtain a false negative
result due to contamination of an excessive amount of
degradation product lacking the myc epitope tag.
Consequently, the quality of the test ligand was evaluated
and this possibility was excluded by Western Blot analysis
using a polyclonal antibody for Netrin-B1 domain VI. Netrin-
Blc and Blf were not examined. However, it was not
considered possible that these isoforms having less
similarity to classical Netrin would exhibit affinity for
these receptors.
To further evaluate the possibility of interactions the
naturally expressing Netrin receptors of Netrin-B1, an in
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vitro explant co-culture experiment using a collagen gel
matrix was performed. It has been well clarified that
commissure axons and cerebellar efferent axons express
Netrini receptors, and that Netrini promotes the growth of
these axons (Serafini et al., Cell 78: 409-424, 1994;
Shirasaki et al., Neuron 14: 961-972, 1995). Unlike Netrin 1,
the soluble form of Netrin-B1a demonstrated no detectable
effect on these axons. The results of the explant co-culture
experiment and the results of the receptor-binding assay
suggested that Netrin-Bl may have a function differing from
that of classical Netrins. It can be contemplated that
Netrin-B1 possesses specificity for receptors differing from
known Netrin receptors. However, the possibility remains
that Netrin-B1 interacts with previously known Netrin
receptors in the presence of co-factor.
ession and sredicted function of Netrin-B1
As for the predicted function of Netrin-B1, there are
thought to be two possibilities. Firstly, based on the
structural similarity to classical Netrin, it can be
predicted that Netrin-B1 plays a chemical attraction and/or
chemical repulsion role in axon growth, and the movement of
cells that express the putative receptors for Netrin-B1.
Alternatively, there is the possibility that GPI anchor type
Netrin-Bl plays an independent role in transmits a signal
from the extracellular environment such as that seen in the
CNTF a receptor (Davis et al., Science 253: 59-63, 1991).
The ephrin/Eph receptor system is proposed to involve
directional signal transmission (Araujo et al., Development
19
CA 02324167 2000-11-20
125: 4195-4204, 1998; Bruckner et al., Science 275: 1640-
1643, 1997; Holder and Klein, Development 126: 2033-2044,
1999; Davy et al., Genes Dev. 13:3125-3135, 1999).
Expression of Netrin-B1 was detected in the mid- and
hind-brain regions up to E12 (No data indicated). Thereafter,
Netrin-B1 expressed in many isolated regions in the CNS.
Expression was limited to the CNS during the developmental
process. Therefore, it was thought that Netrin-B1 is a
specialized molecule having a role in the CNS. This is in
contrast to Netrin-3 in mice. Netrin-3 is suggested be a
factor specialized for the peripheral nervous system
(Puschel, Mech. Dev. 83: 65-75, 1999; Wang, J. Neurosci. 19:
4938-4947, 1999).
Expression of Netrin-B1 is most prominent in the
thalamus. Neurons of the thalamus include mammillary bodies
also expressing Netrin-B1 interrupting afferent signals from
various regions of the nervous system to the cerebellar
cortex. Conversely, neocortical neurons in the V layer
project axons toward the thamalic nuclei through the
cortico-thalamic pathway. This projection is thought to be
influenced by Netrin-1 extruded from the ganglionic eminence
which is an intermediate target (Metin et al., Development
124:5063-5074, 1997; Richards et al., J. Neurosci. 17: 2445-
2458, 1997). There is the possibility that Netrin-B1
performs an attraction and/or target selection influencing
this projection beyond the ganglionic eminence toward the
thamalic nuclei.
Expression of Netrin-B1 in the cerebellum is limited to
the deep cerebellar nuclei. The deep cerebellar nuclei
CA 02324167 2000-11-20
receive the projection of three types of afferent fibers,
i.e. mossy fibers, climbing fibers, and Purkinje cells, and
extend efferent fibers to the red nucleus and other brain
regions. Interestingly, Netrin-B1 is also expressed in the
red nucleus which is a target for efferent fibers from
regions about deep cerebellar nuclei such as the inferior
olive nuclei, which is the origin of climbing fibers and
from the deep cerebellum. There is the possibility that
Netrin-B1 plays an important role in the formation of
functional pathways for the regulation of movement activity.
Further, the deep cerebellum nucleus is thought to regulate
movement or development of granular cells and Purkinje cells.
The deep cerebellar nucleus is believed to be one site that
can be used in the future evaluation of the functional
importance of Netrin-B1.
Expression in the olfactory bulb was detected up to E14
and high level expression was maintained up to adulthood.
This expression appears to be restricted to Mitral cells and
Tufted cells (Fig. 7D and G). Olfactory receptor neurons
project to the mitral cells at around E14 and can regenerate
throughout the life of the organism. Further, granular cells
are continuously supplemented even in adults from the
subventricular even in adults. Therefore, olfactory bulb
needs to continuously renew the structural connections
between cells. It is possible that the continuous expression
of Netrin-B1 supports the maintenance of circuitry in the
olfactory system.
The complementary expression of Netrin-B1 and Unc5h3/RCM
in the cerebral cortex is interesting. These genes are
21
CA 02324167 2000-11-20
potentially useful as marker genes of the allocortex and
neocortex.
Examples:
~inle 1' A novel member of the UNC-6/Netrin familv:
Tso~at~on of Netrin-B1 (7D5)
The date of birth was designated 0 days post-natum (PO).
To analyze the molecules involved in intercellular
communication during development of the cerebellum, a signal
sequence trap cDNA library was constructed from a 21 days
post-natal (P21) mouse cerebellum. The signal sequence trap
method is conducted with invertase-defective yeast strain
(Jacobs et al., Gene 198: 289-296, 1997), however it enables
selective analysis of cDNA encoding secretion and membrane
proteins.
The total RNA of a P21 mouse cerebellum was extracted by
the guanidinium thiocyanate technique (Chomczynski and
Sacchi, Anal. Biochem. 162: 156-159, 1987). Poly(A)+ RNA was
selected on a QuickPrep mRNA purification kit (Pharmacia).
Following the Yabe et al. procedure (J. Biol. Chem. 272:
18232-18239, 1997), a DNA library was synthesized from 3,u g
of Poly(A)+ RNA. To explain simply, a first cDNA chain was
primed with 40pmo1 of XhoI unidirectional primer . 5'-
GAGACGGTAATACGATCGACAGTAGSTCGAGNNNNNNNNN-3' (SEQ ID NO: 1).
After dA tailing, a second cDNA chain was synthesized using
l0pmol of EcoRI linker primer . 5'-
CCGCGAATTCTGACTAACTGATTTTTTTTTTTTTTTTTNN-3' (SEQ ID NO: 2).
Duplex cDNA was size fractionated by agarose gel
22
CA 02324167 2000-11-20
electrophoresis (300-800bp), and amplified by polymerase
chain reaction (PCR) using external primers . 5'-
GACGGTAATACGATCGACAGTAGC-3' (SEQ ID N0: 3) and 5'-
CCGCGAATTCTGACTAACTGATT-3' (SEQ ID NO: 4). The PCR product
was digested with EcoRI and Xhol, re-fractionated by agarose
gel electrophoresis, and unidirectionally ligated to the
EcoRI and XhoI of a pSuc2t7F1 o n vector (Yabe et al., J.
Biol. Chem. 272: 18232-18239, 1997). cDNA was inserted into
this vector upstream of the invertase gene lacking a signal
sequence and, downstream of the yeast ADH1 promoter for
effective expression.
To screen cDNA having a signal sequence, this library
was inoculated into invertase-deficient yeast strainYT455
(Suc2D9, ade2-101, ura3-52) that is unable to reproduce on a
raffinose plate (Jacobs et al., Gene 198: 289-296, 1997) . A
transformant that was able to export to the exterior of the
cell the invertase for fusion having a cDNA-derived signal
sequence, was able to reproduce on this plate. The inserted
cDNA was amplified using the following primer set: 5'-
CAGGAAACAGCTATGACCCAAGCATACAATCAACTCCAAGCTC-3' (SEQ ID NO:
5) and 5'-TGTAAAACGACGGCCAGTACTCCTCTGAAATTAATACGACTCAC-3'
(SEQ ID NO: 6). The amplified DNA was spotted on nylon
membrane in duplicate. These membranes were hybridized with
3zP-labelled cDNA directly synthesized from PO or P21 mouse
cerebellum mRNA. The sequence of the clones indicating
differential hybridization signal was determined by an
automated sequencer (ABI Prism 377) employing BigDye Primer
Cycle Sequencing method (Applied Biosystems). From among
these, clone 7D5 which exhibits low homology to UNC-6/Netrin
23
CA 02324167 2000-11-20
was isolated.
To obtain a full-length cDNA clone, 2.5 X 106 phage
plaques of PO and adult mouse brain cDNA libraries
(Stratagene) were screened with 'ZP-labelled 7D5 cDNA
fragment isolated by the signal sequence trap method. The
phage insert was excised in vivo according to the
manufacturer's manual.
Fig. 1 indicates the expression profile of clone 7D5
according to Northern blot analysis. Poly(A)+ RNA of PO and
P21 mouse cerebellum was extracted as described above. 2 ,u g
Poly(A)+ RNA was subjected to electrophoresis in 1% agarose
gel (2,u g each lane), and transferred to a nylon membrane
(Hybond N Filter, Amersham). This filter was hybridized with
32P- labeled cDNA probe of clone 7D5. The filter was washed
twice with 2 x SSC and 1% SDS at 65 'C for 30 minutes, and
then washed twice with 0.2 x SSC and 0.1% SDS. The membrane
was then analyzed using Image Analyzer (BAS5000, Fuji Film).
As a result, a single stranded band approx. 4.5kb in
size was detected. 7D5 expressed strongly in cerebellum at
PO and was down-regulated by P21 (Fig. 1A). Further, when an
adult mouse multiple tissue Northern blot (Clontech) was
hybridized with the above probe, 7D5 specifically expressed
in the brain and no signal was detected in various mouse
tissues other than the brain (Fig. 1B).
To isolate the full-length cDNA clone encoding the 7D5
gene, a cDNA library prepared from an adult and PO mouse
brain and primed with oligo-dT was screened using the
trapped cDNA fragment as a probe. The 39 overlapping clones
were isolated, and their sequences determined. The longest
24
CA 02324167 2000-11-20
cDNA fragment was 4090 nucleotides long (SEQ ID NO: 7). The
predicted open reading frame (ORF) of this clone consisted
of 539 amino acids (Fig. 2, SEQ ID NO: 8). The sequence of
the trapped cDNA fragment (7D5), corresponded, as was
expected, with the sequence of 180 amino acids of the N-
terminus. Signal peptides are commonly situated at the N-
terminus.
2: Comparison of the sequences of Netrin-B1 and
known Netrins
The putative amino acid sequence of Netrin-Bl (SEQ ID
N0: 8) exhibited low homology to chick Netrinl, Netrin2, and
mouse Netrin-1 and Netrin-3 (31%, 30%, 29% and 29%,
respectively) (Fig. 2 and 3A). The predicted domain
structure of this protein resembled that of the UNC-6/Netrin
family, in other words, in respect of the laminin globular
domain (domain VI) and three epidermal growth factor (EGF)-
like repeats continuing therefrom (domains Vl to V3) (Figs.
2 and 3A). The Cys residue was conserved in these domains of
the 7D5 gene which is a residue that is phylogenically
conserved among the UNC-6/Netrin family, further evidencing
the structural similarity of 7D5 to UNC-6/Netrin. Two thirds
of the N-terminus side of UNC-6 exhibited homology to the N-
terminal domain (domain VI and domain V) of laminin ~ and
chains which constitute the extracellular matrix molecule of
the large hetero-trimer. For this reason, UNC-6 was firstly
identified as a laminin-related protein. The 7D5 gene also
possesses this characteristic. As with UNC-6 and Netrin, 7D5
within these domains, has greater homology in respect of the
CA 02324167 2000-11-20
Y chain more so than the ~ chain of laminin (homology of 32%
to mouse Y -chain, homology of 27% to the ~ -chain). From
these facts, it was concluded that 7D5 is a novel member of
the UNC-6/Netrin family and it was named Netrin-B1.
Characteristics of Netrin-B1 which differ from other
family members, could be found in the C-terminal sequence.
The C-terminal sequence (domain C) of Chick Netrin-1 and
Netrin-2 is rich in basic amino acids consisting of lysine
residues whereas the corresponding domain of Netrin-B1 is
not rich in lysine residues. When a Kyte-Doolittle
hydrophobic plot was conducted on the amino acid sequences
of Netrin-B1a and mouse Netrin-1 described in Example 3 (Fig.
3B), two hydrophobic stretches were observed in both the N-
terminus and C-terminus of Netrin-Bla. Among UNC-6/Netrin
family members, a C-terminal stretch is particular to
Netrin-Bla.
The hydrophobic stretches of Netrin-Bia are thought to
be a signal for the signal peptide and GPI linker predicted
from ~ and ~ +2 rules (Von Heijne, Nucleic Acids Res. 14:
4683-5690, 1986; Gerber et al., J. Biol. Chem. 267: 12168-
12173, 1992). Thus, this domain is referred to as domain C'.
It was predicted that this hydrophobic stretch of the C-
terminus plays a role as a signal for the GPI link to the
membrane, and further, it was considered that there was the
possibility that Netrin-B1, in contrast to diffuse classical
Netrins, was restricted to the membrane surface.
xamg~P 3' Netrin-B1 isoforms
From a comparison of the sequences of 39 cDNA clones, it
26
CA 02324167 2000-11-20
became clear that there existed six types of isoform
probably formed by alternative splicing. These isoforms are
schematically represented in Fig. 4B. The isoform having the
longest amino acid sequence (Netrin-Bia, SEQ ID NO: 8), most
greatly resembled the UNC-6/Netrin family in respect of its
domain structure. Of the 39 types of cDNA clones, seven
encode this isoform. Netrin-Blb (whose nucleotide sequence
is indicated by SEQ ID NO: 9, and whose amino acid sequence
by SEQ ID NO: 10) and Blc (whose nucleotide sequence is
indicated by SEQ ID NO: 11, and whose amino acid sequence by
SEQ ID NO: 12), do have deletions of one or two EGF-like
repeat units (EGF 2 or EGF 2 to 3), respectively (Fig. 4A
and B). From the 39 clones, 1 and 3 of these mutants,
respectively, were obtained. Netrin-B1d (whose nucleotide
sequence is represented by SEQ ID NO: 13, and whose amino
acid sequence by SEQ ID NO: 14) and Bie (whose nucleotide
sequence is represented by SEQ ID N015, and whose amino acid
sequence by SEQ ID NO: 16) possess an insert of 42 amino
acids and 22 amino acids, respectively, between domain EGF1
and domain C' (Fig. 4 A and B). These isoforms were
indicated in 8 and 5 clones respectively. Half of the N-
terminal side of the insert in Netrin-B1d and Ble was
identical, but Netrin-Bld was just 20 amino acids longer
than Netrin-Ble. In a GeneBank data base search, the
inserted domain did not exhibit homology with any known gene,
so it was denoted (Unknown) domain (Ukd). The shortest
isoform, Netrin-Blf (whose nucleotide sequence is indicated
by SEQ ID NO: 17; and whose amino acid sequence is indicated
by SEQ ID NO: 18) was found in only one clone, domains V2
27
CA 02324167 2000-11-20
(EGF2), V3 (EGF3) and C' were absent (Fig. 4A and B). The
remaining 14 clones partially possessed coding sequences,
and it was impossible to determine to which of the isoforms
these clones belonged.
The above results show that the isoforms of Netrin-B1
had variety in their C-terminal structures but all isoforms
comprised N-terminal domain VI and EGF1. The nucleotide
sequences of Netrin-Bla and other isoforms were registered
in the GenBank database (Accession Numbers: AB038667,
AB038666, AB038665, AB038664, AB038663, AB038662).
ale 4: RT-PCR analysis
To confirm the expression of the isoforms thought to be
formed by alternative splicing, and to examine their
regional and temporal distribution, RT-PCR was performed
using a primers set of nucleotides corresponding to EGF1
(amino acids 353-359 of Netrin-B1a) and domain C' (amino
acids 520 to 526 of Netrin-Bla)(Fig. 4B).
Total RNA from various regions of the mouse brain were
extracted. For RT-PCR, 5,u g of DnaseI processed total RNA
was reverse transcribed with Superscript II (Gibco), and
1/50 volume of the reaction product (total RNAl00ng) was
subjected to PCR. For confirmation of the isoform mutants,
the following primer set was used: 5'-CCTGTATCCCCAGCATTTCC-
3' (SEQ ID NO: 19) and 5'-AGCAGCAGTGCTGGGGAGCC-3' (SEQ ID
NO: 20). These primers correspond respectively to the
nucleotide sequences of EGF1 (+1058 to +1077 of Netrin-Bla)
and domain C' (+1558 to +1577 of Netrin-Bla), and amplified
bands of 520, 352, 217, 343 and 283bp respectively were
28
CA 02324167 2000-11-20
formed from Netrin-Bla, Bib, Blc, Bid and Ble. Reaction
conditions were 96~C for 3 minutes, followed by 96'C for 1
minute, and 60 ~C for 1 minute and 72 ~C for 1 minute for 33
cycles, and finally 72~C for 7 minutes.
To identify the products, the isoform specificity of the
amplified fragment was confirmed by southern hybridization
using an isoform specific internal probe. (Fig. 4B, C). For
Netrin-Bla and Blb, the nucleotide sequence of EGF-2 and 3
corresponding to (a+1087 to +1389nt of Netrin-B1) was used
as a probe. For Netrin-Bid and Ble, a fragment corresponding
to the Ukd of Netrin-B1d (+1087 to +1212nt of Netrin-B1d)
was used. For Netrin-Blc, the internal sequence of domain C'
(+1390~'+1531nt of Netrin-B1a) was used.
From the probe corresponding to EGF2 and EGF3 (amino
acids 363-463 of Netrin-B1a (SEQ ID NO: 8)), the existence
of Netrin-Bia and Bld was confirmed. From the probe of
Domain Ukd (amino acids 363-404 of Netrin-B1d (SEQ ID NO:
14)), Netrin-B1d and B1e were detected. The shortest
fragment as clarified by the C' probe (amino acids 464-511
of Netrin-Bla (SEQ ID NO: 8) was identified as a product of
Netrin-Blc. Since there is the possibility that the
amplification efficiency of these isoforms differed, it was
not possible to evaluate the relative amounts of these
isoforms precisely. However, these results suggested that
all isoforms were being formed at the perinatal stage and
that Netrin-Bla and B1d appeared to be the main isoforms in
the brains of adults.
29
CA 02324167 2000-11-20
5~ Construction of a mammalian exsression vector
The full coding sequence of Netrin-B1a and B1d was
ligated to pcDNA4Myc/HisA (Invitrogen) at the EcoRI and PmeI
sites and synthesis of an intact protein was conducted. In
these constructions, the synthesized protein is not fused to
the myc and His-tagged peptide. The obtained constructions
were used in the PI-PLC processing experiment.
To maximize extracellular secretion of Netrin-Bla, Blb,
Bld and B1e in the mammalian cell system, the coding
sequences of these isoforms excluding the C-terminal
sequence were also sub-cloned in pcDNA4Myc/HisA. In other
words, the sequence corresponding to the 26 amino acids of
the hydrophobic C terminus of Netrin-B1 was substituted by a
spacer sequences comprising an ApaI site, ligated to
EcoRI/ApaI site and fused to a Myc/His-tagged sequence at
the C-terminus. These vectors included the sequence from
80nt upstream of these isoforms' initial ATG to the 5'-
terminus of the C-terminal hydrophobic domain. The linking
amino acid sequence between Netrin-B1 and myc/His was Gly-
Gly- Pro- Phe.
P 6- Construction of a Netrin-B1a domain- protein
~~rP~~;nn vector and expression in a host cell
The sequence corresponding to domain VI (amino acids 44-
259) of Netrin-Bla (SEQ ID NO: 8) was sub-cloned in pGEX-
2T(Amersham Pharmacia) and fused with the glutathione S -
transferase (GST) gene. The fusion protein was allowed to
express in E.Coli by isopropyl-b-D-thiogalactopyranoside
induction.
CA 02324167 2000-11-20
Further, (His)6-tagged domain VI protein was synthesized
by cloning the corresponding sequence in pET-32a (Novagen)
and expressing in E. coli.
.xamnle 7: Preparation of an antibodv
The Netrin-Bla domain VI-GST fusion protein allowed to
express in Example 6 was purified using a glutathione column.
A rabbit was immunized several times by typical techniques
using purified antigen and Freund's complete and incomplete
adjuvant. Antiserum was affinity purified against (His)6-
tagged domain VI protein (amino acid 66-116) that was
synthesized in Example 6. Affinity purification was
conducted by purifying (His)6-tagged domain VI protein
expressed in E. coli with a Ni-NTA column (Qiagen). The
purified fusion protein was coupled to a HiTrap NHS-
activated affinity column (Amersham Pharmacia).
E~gIP R- PI-PLC Treatment and Western blot analysis
A hydrophobic plot of Netrin-Bla indicates that Netrin-
Bl differs from Netrinl and has two hydrophobic sequences at
both termini (Fig. 3). The sequence at the N-terminus
supports the possibility of operating as a signal sequence
in secretion of yeast invertase but this activity has not
been confirmed in mammal cells. This signal sequence is
observed in common between members of the UNC-6/Netrin
family. However, the C-terminus sequence is particular to
Netrin-B1, and is thought to be a GPI lipid anchor.
To confirm this possibility, recombinant Netrin-Bla and
B1d were expressed in HEK293 cells.
31
CA 02324167 2000-11-20
HEK293T cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) to which 2mM of L-glutamine and 10% f,
et al. " calf serum (FCS) has been added. The cells were
inoculated onto l0cm tissue culture plates at a cell
concentration of 4 x 106 cells per plate. After 24 hours,
cells at 80% confluence were transfected with 20 ~ g of
expression vector (with no epitope fusion) obtained in
example 5, using a CellPhect Transfection Kit (Amersham
Pharmacia). After 40 hours, the transfected cells were
incubated in 8m1 OptiMEMI(Gibco) at 37'C for 2 hours, both
together with and without 100mU/ml PI-PLC (Sigma). The media
from the PI-PLC processed and unprocessed cultures (1.5m1)
were centrifuged at 2,OOOg for 10 minutes then 60,000 for
100 minutes thereby effecting clarification. These
supernatants were TCA-precipitated, and pellets dissolved in
an SDS-sample buffer. The cells cultures without PI-PLC were
dissolved mRIPA buffer 750 ~ 1 (50mM Tris-HC1 p H7.5, 150mM
NaCl, 1% Triton X-100, 1% cholic acid, 0.1% SDS and 20mM
EDTA). These samples were fractionated with 10% SDS-PAGE,
and electrotransferred to a PVDF membrane (Millipore).
Recombinant Netrin-B1 was detected using the affinity
purification rabbit anti-Netrin-B1 antibody and as a
secondary antibody, HRP-conjugated anti-rabbit IgG (Zymed),
and visualized using an ECL System (Amersham Pharmacia).
60 hours after transfection, the transfected HEK293T
cells were incubated in Opti-MEM, both together with
200mU/ml PI-PLC, and without PI-PLC at 37 C for two hours.
The supernatant was clarified by centrifugation, and
precipitated with TCA. A cell lysate was obtained from the
32
CA 02324167 2000-11-20
cells untreated with PI-PLC. These samples were fractionate
by SDS-PAGE, then from immunoblot analysis with affinity
purified anti-Netrin-B1 polyclonal antibody for Netrin-B1
common domain (domain VI), each of the isoforms were
detected in the cell in the lysate (Fig. 5), indicating that
synthesis of the recombinant protein was successful. The
transfected cell was exposed for 2 hours to phosphatidyl
inositol-specific phosphalipase C (PI-PLC) which
specifically cleaves the GPI anchor linkage protein from the
membrane surface. As a result the recombinant protein was
released into the supernatant (Fig. 5). In contrast, there
was substantially no release of the recombinant protein with
the same processing conducted without PI-PLC. From these
results, it was indicated that at least in respect of these
isoforms, and in all likelihood all isoforms with the
exception of Netrin-Blf are primarily bound to the cell
membrane by means of a GPI anchor. However, when the
transfected cells were incubated for one day in a standard
growth medium, a detectable amount of recombinant protein
was release into the supernatant without any evidence of
degradation (Data not indicated).
The calculated values of the molecular weights of
nascent Netrin-B1a and Hld were 60.5kDa and 53.9kDa
respectively. The cleavage site for GPI anchoring was
predicted from ~J and c.~ +2 rules (Gerber, J. Biol . Chem. 267
12168-12173, 1992) (Fig. 2). The calculated values of the
molecular weights of these cleaved proteins were 58.0 and
51.4kDa, respectively. The size as evaluated from immunoblot
analysis was fractionally larger than the above-mentioned
33
CA 02324167 2000-11-20
calculated value (Fig. 5), suggesting that other post-
translational modifications were involved. The putative N-
glycosylation site is indicated in Fig. 2. These
modifications were confirmed by N-glycosidase F treatment
(Data not shown). In Fig. 5, the recombinant protein
released by PI-PLC processing, traveled more slowly that the
product derived from the cell lysate. There is the
possibility that these electrophoresis speeds were
influenced by lipid elements bound to the recombinant
protein.
P 9' Expression of Netrin-B1 in the Central Nervous
Svstem (CNS)
To examine the regional distribution of Netrin-B1 in
detail, the N-terminal coding sequence was used as a probe
common to all isoforms, and in situ hybridization in mouse
cerebella was conducted.
First, to prepare a Netrin-B1 probe, the signal sequence
trapped cDNA sequence of 7D5 clone was transferred into
EcoRI and XhoI sites of pSP72 (Promega). Antisense and sense
ribo-probe (corresponding to 867-1052nt of SEQ ID NO: 7)
were each labeled with digoxigenin dUTP from a linearized
template using SP6 and T7 RNA polymerase (Boehringer
Mannheim).
For whole mount in situ hybridization, mouse brain was
dissected in phosphate-buffered saline, and immobilized
overnight in 4~ paraformaldehyde (PFA) PBS solution. The
immobilized brain was rinsed three times with PBS, and after
dehydration, rehydrated with a MeOH gradient, and co-
34
CA 02324167 2000-11-20
incubated with 10 ,~.~ g/ml of proteinase K at room temperature
for 40 minutes. Next, the brain sample was once again
immobilized in 0.2% glutalaldehyde and 4%PFA PBS solution at
room temperature, rinsed with PBS, then hybridized with l,u
g/ml of riboprobe for 16 hours at 65~C. The hybridized brain
was washed with 2 x SSC/0.1% CHAPS three times, at 65~C for
30 minutes and 0.2 x SSC/0.1% CHAPS three times at 65~C for
30 minutes. This was blocked with a 10% heat-inactivated
sheep serum and 2%BSA TBT (50mM Tris-HC1 pH7.5, 150mM NaCl
and 0.1% Triton X-100) solution, followed by co-incubation
overnight at 4~C with alkaline phosphatase conjugated anti-
digoxigenin Fab fragment (1:2000 dilution; Boehringer
Mannheim). After washing thoroughly with TBT, the hybridized
probe was detected using BMPurple as a phosphatase substrate.
In the case of coronal vibratome slices, slices of a
thickness of 300,u m were prepared. The following procedures
are the same as those described above except that the
duration of incubation with proteinase K was ten minutes.
For in situ hybridization of the thin sections, the
embryos were fixed in the same manner as above. PO and P21
brain were perfused with 4% paraformaldehyde PBS solution,
post-fixed overnight in 4% PFA, then submerged in 30%
sucrose PBS until the brain settled to the bottom. The
sample was embedded in O.C.T. compounds, and sliced to a
thickness of 20,u m using a cryostat. The sliced were re-
immobilized with 4% PFA, co-incubated with 2 ,u g/ml
proteinase K for 10 minutes at 37 C, then after acetylation,
the slices were further co-incubated with the above-
mentioned riboprobe for 16 hours at 55~C, and then wased at
CA 02324167 2000-11-20
55~C with 50% formamide/2 x SSC/0.01% Tween-20 solution.
This was then processed with 10~ g/ml RNase A at 37~C for 30
minutes, and then at 55~C , washed once with 2 x SSC/0.01%
Tween-20, then washed twice with 0.1 x SSC/0.01% Tween-20.
The hybridization signal was detected as described above
using anti-digoxigenin Fab antibody and BMPurple.
PO mouse brain parasagittal slices (A, B, D, E, G and H),
and P2 mouse brain coronal vibratome slices (C and F) were
hybridized with Netrin-B1 (A-F excluding B) or Unc5h3/RCM (G
and H) specific digoxigenin labeled antisense cRNA probe
(corresponding to the antisense sequence of 867-1052nt of
SEQ ID NO: 7). As a result of this, it was indicated that
Netrin-B1 was regionally limited in its expression (Fig. 6A
- F). The strongest expression was detected in thalamus (Fig.
6A and D). In the olfactory bulb, inferior collicus and
superior collicus, red nuclei, mammillary bodies, deep
cerebellar nuclei and inferior olive nuclei a medium level
expression was detected (Fig. 6A and D). In P2 brain coronal
vibratome slices, strong expression was detected in the
thamalic nuclei (Fig. 6C), and intermediate expression
detected in the inferior collicus and cerebellar deep nuclei
(Fig. 6F). Further, weak but clearly discernible expression
was detected in the piriform cortex, the posterior area of
the splenium of the corpus callosum, the granular cortex,
and the hippocampal formation from CA1 to CA3 (Fig. 6C).
In several regions of the brain, expression of Netrin-B1
was in contrast with expression of Unc5h3 (Fig. 6G and H).
For example, Unc5h3 was strongly expressed in the external
embryonic cell layer of the cerebellum and the Purkinje
36
CA 02324167 2000-11-20
cell layer (Fig. 6G, and Ackerman et al., Nature 386: 838-
842, 1997) but Netrin-B1 strongly expressed in the deep
cerebellum nucleus (Fig. 6D). Like Unc5h3, other Netrin
receptors are all reported to express in the external
embryonic cellular layer and the internal granular cell
layer (Leonardo et al., Cold Spring Harb. Symp. Quant. Biol.
LXII: 467-478, 1997). Among Netrin receptors only DCC is
known to express in the deep cerebellum nucleus (Livesey and
Hunt, Mol. Cell Neurosci. 8: 417-429, 1997). Interestingly,
in cerebellar cortex surface, Netrin-B1 and Unc5h3 expressed
respectively in the alhocortex and the neocortex (Fig. 6E
and H), and this expression delineated the boundary between
the allocortex and the neocortex.
~X.~.m,Dle 10 ~ Exsression of Netrin-B1 in the ontocrenesis
process
Ontogenetic expression of Netrin-B1 in the
representative region was examined by in situ hybridization
(Fig. 7) .
With the exception of B, expression of Netrin-Bl during
ontogenesis was examined by in situ hybridization using
frozen parasagittal slices. Digoxigenin-labeled antisense
cRNA to Netrin-B1 was used as a probe (corresponding to the
anitisense sequence of 867-1052nt in SEQ ID NO: 7) . At E14,
expression of Netrin-B1 was initially detected in the
accessory olfactory bulb (AOB), restricted to the mitral
cell layer (ML) and tufted cell (TF) at PO and persisting to
P21. In the thalamus, expression at E14 was seen in the
ventral thalamus (VT), dorsal thalamic nuclei (DT), and
37
CA 02324167 2000-11-20
peretectal area (PT), reaching a peak at birth and being
down-regulated during postnatal development. In the
cerebellum, expression of Netrin-Bl at E14 was detected,
however this was restricted to the deep cerebellar nuclei
(DCN), and in contrast to the olfactory bulb, this
expression was down-regulated at post-natal P21.
In the deep cerebellar nuclei, expression of Netrin-B1
was detected at E14. (Fig. 7C), sustained until PO (Fig. 7F),
but was down-regulated up until P21 (Fig. 7I) . This matched
well with the results of Northern analysis (Fig. 1A). Even
in the inferior collicus, post-natal expression of Netrin-B1
followed a similar course over time (Fig. 7F and I).
At E14, expression of Netrin-B1 was limited to the
ventral thalamus and dorsal thalamic nuclei, and the
peretectal area of the mesencephalon (Fig. 7B).
Interestingly, this expression was regulated layer-
specifically in the superior collicus (Fig. 7E and Fig. 6A).
Expression of Netrin-B1 in the thalamic region is thought to
follow the same course over time as in the cerebellum (Fig.
7E and7H).
Expression of Netrin-B1 in the olfactory bulb was
detected at E14 (Fig. 7A), which increased until PO (Fig.
7D), and was maintained at a high level to P21 (Fig. 7G) and
into adulthood (data not indicated).
Expression of Netrin-B1 up to E12 in the mesencephalon
and rhombencephalon regions was detected (data not
indicated). From the whole-mount hybridization, a signal was
obtained from either region in E10 (data not indicated).
38
CA 02324167 2000-11-20
Example 11: Recegtor bindinaa exseriment
Until now, all of the members identified as belonging to
the UNC-6/Netrin family were all previously known Netrin
receptors, that is, they bind to the Unc5 family (Unc5hl,
Unc5h2, andUnc5h3/RCM in mammals) (Leonardo et al., Nature
386: 833-838, 1997) and the Unc40 family (mammalian DCC and
neogenin) (Keino-Masu et al., Cell 87: 175-185, 1996). To
examine whether Netrin-B1 is a functional enhancer for UNC-
6/Netrin, Netrin-B1's binding activity toward Netrin
receptors was investigated.
Myc-tagged Netrin-1, and myc-tagged soluble forms the
recombinant proteins, Netrin-Bla, Blc, Bld and Ble, were
yielded as the culture supernatant from stable transformants
cultured overnight. The mass and amounts of fusion proteins
in the culture supernatant were examined by Western Blot
analysis using anti-myc antibody (9E10) and HRP conjugated
mouse IgG. The relative concentration of the fusion protein
was measured with Image Analyzer (LAS100, Fuji). The minimum
concentration of Netrin-1 binding with the receptor under
experimental conditions was determined by, continuous
dilution of the culture supernatant. To concentrate the
soluble forms of Netrin-Bia, Blc, Bld and Ble, a Centriprep-
30 Concentrator (Amicon) was used. This processing triggered
no aggregation of secreted Netrin-Bl.
The cDNA of the Netrin receptor was obtained from Marc
Tessier-Lavigne (Unc5hl, Unc5h2 and DCC; pCEP4-DCC) and
Susan Ackermann (Unc5h3/RCM). The cDNA of chick Netrin-1
(pGNetlmY°) was also obtained from Marc Tessier-Lavigne. The
coding sequence of the cytoplasm domain of Unc5hl, Unc5h2
39
CA 02324167 2000-11-20
and Unc5h3/RCM was substituted in frame, by an ECFP coding
sequence, and a fusion protein comprising the receptor and
ECFP-derived extracellar and transmembrane domains, was
obtained. To construct an expression vector, a l.7kb NaeI
fragment of Unc5hl, a l.7kb Eco47III/SmaI fragment of Unc5h2,
and a 2.2kb EcoRI/BalI fragment of Unc5h3/RCM was subcloned
in pECFP (Clontech) at the SmaI site. An expression vector
of the Unc5 family was transfected into a COS7 cell, and
pCEP4-DCC was transfected into a 293EBNA cell (InVitrogen).
48 hours into transfection, myc-tagged recombinant protein
was added to a medium to which 10% FCS and 20 ,u g/ml heparin
(Sigma) had been added, and was incubated for 90min at room
temperature. After washing one or four times with PBS, the
cells were immobilized in MeOH for 5 minutes then in 4%
paraformaldehyde PBS solution for 15 minutes. In the case of
the Unc5 family, binding of the recombinant protein was
detected using monoclonal anti-myc antibody (9E10) and Alexa
546 conjugated anti-mouse IgG (Molecular Probes). Cells
transfected with the receptor were visualized by CFP
fluorescence. In the case of DCC double staining was
performed with monoclonal anti-DCC antibody (Oncogene
Science) and affinity purified rabbit polyclonal anti-myc
antibody. The binding of these primary antibodies were
visualized using Alexa 488 conjugated anti-mouse IgG
(Molecular Probes) and Alexa 546 conjugated anti-rabbit IgG
(Molecular Probes), respectively. Fluorescent images were
obtained with a CCD camera (Prinston Instruments) having
MetaMorph software (Roper).
CA 02324167 2000-11-20
The C-termini of extracellular and transmembrane domains
of Unc5h3/RCM, Unc5h1 and Unc5h2 cells were fused with ECFP
which is a mutant green fluorescence protein, and this was
expressed in COS7 cells. Expression of the receptor molecule
was monitored by the fluorescence of the membrane, These
cells were co-incubated with myc-tagged Netrin-B1 and myc-
tagged chick Netrin-1 protein collected as the supernatant
from stably expressing cell. To maximize secretion, the
hydrophobic C-termini of the Netrin-B1 isoforms were removed.
In a culture incubated with chick Netrin-1, Unc5h3/RCM
expressing cells able to be recognized by ECFP fluorescence
were labeled by anti-myc antibody on their cell surface (Fig.
8 A and B), and this was consistent with previous reported
results (Leonardo et al., Nature 386: 833-838, 1997). In
contrast, no signal was detected in the culture treated with
the series of test ligands (myc-tagged Netrin-Bla, Blb, B1d
and B1e) (Fig. 8 D, F, etc.). The relative amounts of
proteins used in these binding assays were evaluated by
immunoblotting using anti-myc antibody. A representative
result is indicated in Fig. 8. These test ligands exhibited
no binding toward Unc5h3/RCM even at concentrations of 100
times greater than with chick Netrin-1. Therefore, Netrin-B1
is apparently not a ligand of Unc5h3/RCM. Similarly, Netrin-
B1 did not exhibit binding to Unc5h1 and Unc5h (data not
indicated). DCC belonging to another receptor family was
also expressed in COS cells, and binding experiment with
Netrin-B1 was conducted. In this case, the expression of DCC
was detected by immunocytological techniques employing DCC-
specific monoclonal antibodies (Fig. 8, G and I). Cells
41
CA 02324167 2000-11-20
expressing DCC exhibited binding toward chick Netrinl,
However binding toward Netrin-B1 was not exhibited even at
concentrations of over 100 times greater (Fig. 8 H and J).
Example 12: Ex 1
To further confirm the functional differences between
Netrin-B1 and Netrin l, 3-dimensional collagen explant
simultaneous culture experiment using dorsal spine and
cerebellum plates was conducted.
The procedures followed for an explant in collagen gel
were in accordance with the procedures described in
Shirasaki et al. (Neuron 14: 961-972, 1995). An E12 mouse
embryo was dissected in a DME/F12 medium (Sigma) to which
glucose had been added. Cerebellar plate explants were
obtained from open-book preparations of the hindbrain. An
aggregate of the cell strain (293T cell) stably expressing
Netrin-1, and the secretion form of Netrin-Bla and Netrin-
B1d was prepared as described above (Kennedy et al., Cell
78: 425-435, 1994) . The explant and 293T cell aggregate was
buried within a collagen matrix prepared from rat tail
tendons. The medium that was used was DME/F12 to which was
added 3.85mg/ml glucose, lOmg/ml streptomycin, 100 ,1.~ g/ml
transferin, 5,u g/ml insulin, 5.29ng/ml sodium selenite, 16.4
,u g/ml putrescine dihydrochloride, 6.29ng/ml progesterone,
7.40ng/ml hydrocortizone, and 10~ fetal calf serum.
As described above (Serafini et al., Cell 78: 409-424,
1994; Kennedy et al., Cell 78: 425-435, 1994; Shirasaki et
al., Neuron 14: 961-972, 1995), Netrin-1 secreted from the
transformed cell exhibited growth promotion and attraction
42
CA 02324167 2000-11-20
activity toward commissure axons and cerebellar efferent
axons (Fig. 9A). However, Netrin-B1 did not elicit the
growth of these axons (Fig. 9B and C), consistent with the
results of the binding assay.
These results strongly suggested that Netrin-B1 was not
a ligand to any known Netrin receptor.
ple 13: Creation of a transgenic animal
Transgenic animals can be produced by known techniques
within the relevant field. For example, a transgenic animal
may be produced by injecting the fertilized ovum of an
animal with a vector comprising the Netrin-B1 gene of the
present invention and regulatory sequences such as a
promoters and an enhancer to control expression of the gene.
This is then transplanted to a surrogate parent, and the
desired transgenic animal can be obtained by analyzing with
PCR whether or not the progeny have the introduced gene. In
this case, it is also possible to produce a chimeric animal
by injection of pluripotent embryonic stem cell (ES cell)
into the animal blastocyst.
Example 14: Creation of a Knock-out Animal
A knock-out animal can be produced by using known
standard genetic recombination techniques (gene targeting
for example, Methods in Enzymology 225: 803-890, 1993), for
example, as follows.
First, a targeting vector is produced by substituting
the sequence of the isolated Netrin-B1 gene of the present
invention by, for example, marker genes such as neomycin-
43
CA 02324167 2000-11-20
resistance gene, and further, adding to the terminal portion
of this gene, for example an MC1 promoter controlled
diphtheria toxin fragment A gene without a PolyA signal.
This targeting vector is introduced into the ES cells of a
mouse or the like, cell are selected in which homologous
recombination of the target sequence or the mutant sequence
it contains, into the cellular genomic DNA of the same gene.
The selection of such a gene recombinant cell can, for
example, be conducted by adding 6418 to the cell culture,
thereby allowing for removal of non-recombinant cells not
having a marker gene. Random recombinant cells are excluded
by their expression of fragment A of diphtheria toxin. The
gene of the selected gene recombinant cell, is a .mutant
sequence into whose coding sequence a marker gene has been
introduced, and is unable to produce Netrin-B1.
Next, this gene recombinant ES cell is injected into a
mouse early embryo (blastocyst), and this embryo is allowed
to develop into an individual in the body of a female mouse,
and a chimera mouse is allowed to be born. Then, this
chimera mouse is mated with a wild type mouse and allowed to
birth mice progeny, and by selecting mouse individuals
having the mutant sequence in one or both allele from among
the mice progeny, it is possible to obtain a knock-out mouse
having no ability to produce Netrin-B1 or who produces an
amount of Netrin-B1 that is relatively low compared to the
wild type. A knock-out animal includes homozygote in which
both alleles are substituted by mutant sequences ( - / - ),
and heterozygote in which only one allele is substituted by
a mutant sequence (+/-) .
44
CA 02324167 2000-11-20
As described in detail above, UNC-6/Netrin family is
highly conserved in the process of evolution. However, in
mammals, these family members are thought to be more
branched than was expected. Molecules that are highly
branched from previously known UNC-6 such as Netrin-B1 may
play a particular important role in the particularly highly
organized cell structure of vertebrate animals. The present
invention makes possible the further clarification of the
function of Netrin in the cerebella of vertebrate animals,
provision of suitable treatment for brain damage caused by
disease, and the promotion of normal neural pathway
formation during transplant treatment and the like. A mouse
with increased or defective function is effective for
examining the role of Netrin-B1, and it is thought that
useful information regarding candidate receptors can be
obtained.
Further, the Netrin-B1 protein and fragment thereof, and
the polynucleotide encoding this protein or fragment, being
the subjects of the present invention, is a potentially
useful medicaments in the treatment of patients required an
increase in Netrin-B1 protein expression, particularly in
cases such as where regeneration of neural circuit formation
is required. On the other hand the antibody against Netrin-
B1 protein and a fragment thereof, is a potentially useful
medicament in the treatment of patients requiring a
reduction in the expression of Netrin-B1 protein. Further,
the primer and probe of the present invention, apart from
the above-described analysis of function, are expected to be
CA 02324167 2000-11-20
useful in the treatment of the above diseases, their
diagnosis and in the production of medicaments.
All publications, patents and patent applications cited
herein are incorporated herein by reference in their
entirety.
46
CA 02324167 2001-07-27
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: RIKEN
(ii) TITLE OF INVENTION: A MEMBRANE-BOUND NETRIDT
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O.BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONTARIO
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,324,167
(B) FILING DATE: 20-NOV-2000
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: JP 2000-148843
(B) FILING DATE: 19-MAY-2000
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(C) REFERENCE/DOCKET NUMBER: 72813-131
(ix) TELECOMMUNICATION INFORMATION
(A) TELEPHONE: (613)-232-2486
(B) TELEFAX: (613)-232-8440
(2) SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40
(B) TYPE: nucleic acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GAGACGGTAA TACGATCGAC AGTAGCTCGA GNNNNNNNNN 40
(2) SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40
(B) TYPE: nucleic acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CCGCGAATTC TGACTAACTG ATTTTTTTTT TTTTTTTTNN 40
(2) SEQ ID NO: 3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24
(B) TYPE: nucleic acid
47
CA 02324167 2001-07-27
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GACGGTAATA CGATCGACAG TALC 24
(2) SEQ ID NO: 4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: nucleic acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CCGCGAATTC TGACTAACTG ATT 23
(2) SEQ ID NO: 5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43
(B) TYPE: nucleic acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Syntheaic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
CAGGAAACAG CTATGACCCA AGCATACAAT CAACTCCAAG CTC 43
(2) SEQ ID NO: 6
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44
(B) TYPE: nucleic acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
TGTAAAACGA CGGCCAGTAC TCCTCTGAAA TTAATACGAC TCAC 44
(2) SEQ ID N0: 7
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4090
(B) TYPE: nucleic acid
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 873..2492
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GAATTCGGCA CGAGAAAAAA CCCACAGTAA AAGTTTAAGG CGAGAAGTGG TGGCGGCGGC 60
GGCGGCGGCG CGGGGAAGCT GCGAGCGGAG AAGGTTGCCG AGACCTCGGA AGGCGGCGAT 120
48
CA 02324167 2001-07-27
a
TTGGTTCCTA ATCCCACTGT ATTTTTGGAG GGAGAGGCAC CTTTCTCATC CTCCCTTCCT 180
CTCCGCCCAC CCCTCTCCCT CCCCCTCATC TACCTGTCAA AGTCACTGAT CTTTTGCATT 240
TTGGAAGAGG ACGTCAACGG GAAGGAATTC CCCCTGTCAG GGTCCCGGCT CCGAGAGGGG 300
GCGACGCGCG ACAAGGCTGC CCCAGGGGCA AGAGACCAAG GTTGCTGGTG CCAGAAGAGG 360
GAAGAGAAAA GATTGAAGGG AAACAGATAC AGGACGATAG ACACCATCCC TTTGCTTCTG 420
ATGCTGATAC TTCAGCTCAT CTGAGGAGCC CTGATGAACC CAGAACAGCA AGGATCACTC 480
AGGATTATTG GATGTACAAC GGGAGAGCCG TCACTTTGCT AAAT'CATTAT CTGCTCCTGG 540
ACATCCCTGG ACATCTTTCA CAAAAGTCAA ATAGATATGT TCTAC:GAGGA GAAATGGCTG 600
CAAGCTGTTA ATGCCTAACA GATAAGCATG TTAGGCTTCT ACCAAAGTCC TCAGCATACC 660
TGACCGCATA GAATATTTCA ATCTGTCACA TTTGGTTTTG GAATCTGCTT TGAGATCTCA 720
GCCTATTTTT TTATACATAT ACAGACCTAC CTACATAAAG ATACATATAG ACACGTGCAC 780
ACACACACAC ACACACACAT ATAAGATACT CATGTATATT TAAAAGAGAC ACTGATTGCA 840
TAGAAGACAC GAAGATTGCC AAGATTTTAG AG ATG TAT TTG 7.'CA AGA TTC CTG 893
Met Tyr Leu :3er Arg Phe Leu
1 5
TCG ATC CAT GCC CTG TGG GTG ACA GTG TCC TCT GTG ATG CAG CCC TAC 941
Ser Ile His Ala Leu Trp Val Thr Val Ser Ser Val Met Gln Pro Tyr
10 15 20
CTT TTC GTG TGG GGA CAT TAT GAT GTA TGT AAG AGC C;TG ATT TAC ACA 989
Leu Phe Val Trp Gly His Tyr Asp Val Cys Lys Ser Leu Ile Tyr Thr
30 35
GAA GAA GGC AAA GTT TGG GAT TAC ACA GCC TGC CAG C;CG GAA TCC ACG 1037
Glu Glu Gly Lys Val Trp Asp Tyr Thr Ala Cys Gln Pro Glu Ser Thr
40 45 50 55
GAC ATG ACC AAG TAT CTG AAA GTG AAA CTG GAC CCT C;CG GAT ATT ACC 1085
Asp Met Thr Lys Tyr Leu Lys Val Lys Leu Asp Pro Pro Asp Ile Thr
60 65 70
TGT GGA GAC CCT CCA GAG TCC TTC TGT GCA ATG GGC F~AC CCT TAC ATG 1133
Cys Gly Asp Pro Pro Glu Ser Phe Cys Ala Met Gly F,sn Pro Tyr Met
75 80 85
TGC AAT AAT GAG TGT GAT GCG AGT ACC CCT GAA CTG GCA CAC CCT CCT 1181
Cys Asn Asn Glu Cys Asp Ala Ser Thr Pro Glu Leu P,la His Pro Pro
90 95 1.00
GAG CTG ATG TTT GAT TTT GAA GGA AGA CAT CCC TCC A,CA TTT TGG CAG 1229
Glu Leu Met Phe Asp Phe Glu Gly Arg His Pro Ser Thr Phe Trp Gln
105 110 115
TCT GCT ACT TGG AAG GAG TAC CCC AAA CCT CTC CAG CITT AAC ATC ACT 1277
Ser Ala Thr Trp Lys Glu Tyr Pro Lys Pro Leu Gln V'al Asn Ile Thr
120 125 130 135
CTG TCT TGG AGC AAA ACC ATT GAA CTC ACA GAC AAC A.TA GTT ATT ACC 1325
Leu Ser Trp Ser Lys Thr Ile Glu Leu Thr Asp Asn Ile Val Ile Thr
140 145 150
TTT GAA TCG GGG CGT CCA GAC CAA ATG ATC CTA GAG AAA TCT CTC GAC 1373
Phe Glu Ser Gly Arg Pro Asp Gln Met Ile Leu Glu Lys Ser Leu Asp
155 160 165
TAC GGA CGA ACA TGG CAG CCC TAT CAG TAT TAT GCC A.CA GAC TGC CTC 1421
Tyr Gly Arg Thr Trp Gln Pro Tyr Gln Tyr Tyr Ala Thr Asp Cys Leu
170 175 180
CAT GCA TTC CAC ATG GAC CCG AAA TCC GTG AAG GAT TTA TCT CAG CAC 1469
His Ala Phe His Met Asp Pro Lys Ser Val Lys Asp Leu Ser Gln His
185 190 195
49
CA 02324167 2001-07-27
ACG GTC TTG GAA ATC ATT TGC ACG GAA GAG TAC TCC ACT GGG TAC TCC 1517
Thr Val Leu Glu Ile Ile Cys Thr Glu Glu Tyr Ser Thr Gly Tyr Ser
200 205 210 215
ACG AAT AGC AAA ATA ATC CAC TTC GAG ATC AAA GAC F,GG TTT GCG TTT 1565
Thr Asn Ser Lys Ile Ile His Phe Glu Ile Lys Asp F,rg Phe Ala Phe
220 225 230
TTC GCT GGA CCT CGG CTA CGA AAT ATG GCT TCC CTC TAT GGA CAG CTG 1613
Phe Ala Gly Pro Arg Leu Arg Asn Met Ala Ser Leu Tyr Gly Gln Leu
235 240 245
GAT ACA ACC AAG AAA CTC AGA GAT TTC TTC ACT GTC P.CA GAC CTG AGG 1661
Asp Thr Thr Lys Lys Leu Arg Asp Phe Phe Thr Val Thr Asp Leu Arg
250 255 260
ATC AGG CTG TTG AGA CCC GCC GTT GGG GAA ATA TTT GTA GAT GAA CTA 1709
Ile Arg Leu Leu Arg Pro Ala Val Gly Glu Ile Phe V'al Asp Glu Leu
265 270 275
CAT TTG GCA CGT TAC TTT TAT GCG ATC TCA GAC ATA AAG GTG CGA GGA 1757
His Leu Ala Arg Tyr Phe Tyr Ala Ile Ser Asp Ile Lys Val Arg Gly
280 285 290 295
AGG TGC AAG TGC AAC CTG CAT GCC ACT TCG TGT TTG T'AT GAC AAC AGC 1805
Arg Cys Lys Cys Asn Leu His Ala Thr Ser Cys Leu T'yr Asp Asn Ser
300 305 310
AAA CTG ACA TGT GAA TGT GAG CAC AAC ACT ACA GGT CCC GAC TGT GGG 1853
Lys Leu Thr Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly
315 320 325
AAA TGC AAG AAG AAC TAC CAG GGC CGA CCT TGG AGC CCC GGC TCA TAC 1901
Lys Cys Lys Lys Asn Tyr Gln Gly Arg Pro Trp Ser Pro Gly Ser Tyr
330 335 340
CTC CCC ATC CCC AAA GGC ACC GCA AAC ACC TGT ATC CCC AGC ATT TCC 1949
Leu Pro Ile Pro Lys Gly Thr Ala Asn Thr Cys Ile Pro Ser Ile Ser
345 350 355
AGT ATC GGT AAC TGT GAA TGC TTC GGC CAC TCC AAT CGG TGC AGT TAT 1997
Ser Ile Gly Asn Cys Glu Cys Phe Gly His Ser Asn Arg Cys Ser Tyr
360 365 370 375
ATC GAT CTG CTA AAC ACA GTC ATT TGC GTG AGC TGT AAA CAC AAC ACT 2045
Ile Asp Leu Leu Asn Thr Val Ile Cys Val Ser Cys Lys His Asn Thr
380 385 390
AGA GGG CAG CAC TGT GAG TTA TGC AGG CTG GGC TAC TTC AGA AAT GCT 2093
Arg Gly Gln His Cys Glu Leu Cys Arg Leu Gly Tyr Phe Arg Asn Ala
395 400 405
TCT GCA CAG CTG GAC GAT GAG AAT GTG TGC ATA GAG TGT TAT TGT AAC 2141
Ser Ala Gln Leu Asp Asp Glu Asn Val Cys Ile Glu Cys Tyr Cys Asn
410 415 420
CCT TTG GGC TCA ATC CAT GAT CGT TGT AAT GGC TCA GGA TTT TGT GAG 2189
Pro Leu Gly Ser Ile His Asp Arg Cys Asn Gly Ser Gly Phe Cys Glu
425 430 435
TGT AAG ACT GGA ACA ACA GGG CCT AAA TGT GAT GAG TGT CTG CCA GGA 2237
Cys Lys Thr Gly Thr Thr Gly Pro Lys Cys Asp Glu C-ys Leu Pro Gly
440 445 450 455
CA 02324167 2001-07-27
AAT TCC TGG TAC TAC GGC TGT CAA CCT AAT GTC TGC GAC AAT GAG CTC 2285
Asn Ser Trp Tyr Tyr Gly Cys Gln Pro Asn val Cys Asp Asn Glu Leu
460 465 470
CTG CAC TGC CAG AAT GGA GGG ACC TGC CAG AAC AAT C~TG CGC TGC GCG 2333
Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn ~Tal Arg Cys Ala
475 480 485
TGC CCA GAC GCC TAC ACC GGC ATC CTC TGT GAG AAG C:TA CGG TGC GAA 2381
Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu
490 495 500
GAG GCG GGC AGC TGT GGC TCC GAA TCC GGC CAG GGA CJCA CCC CCG CGG 2429
Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg
505 510 515
GGC TCC CCA GCA CTG CTG CTG CTG ACC ATG CTG CTG CTGG ACT GCC GGT 2477
Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Cily Thr Ala Gly
520 525 530 535
CCC CTG GTG TTC TAG GGGTCACACC CAGCCCTCCG ACAGGCC',TGT GCTGTGGGGA 2532
Pro Leu Val Phe
540
AGCAAACACA ACCCAAGCGA TTGCCACTGA CATAGAAAAC ACGCP,CACCC ACTCCAACAC 2592
AGTGTATAAA AGAAGAGGGC CTAACTGAAC TAAGCCATAT CTCTC'.AGAAC CGGACAGCAC 2652
ATCGCACATC GGAGTTGAGA CTGTTCATCA TTGACTCCAG AGGAA,TTGGC AGCTGTTGCT 2712
ATTCTCACTG CAAATCTCAT TGCCAGCTGC AGAGCTGATT GCGGATTGGA AAGGCTGTGA 2772
GAGCGCCCCC AAGAGGAAAG ACGGAAAACA AACTGATCAA CCAAC'.CTAAA AACATTCGCT 2832
ACTCTACCGT GGTGCACCCT AGTACCGCTC TGCTCAGTGT GTGGGCCAAC CAAATAAAAG 2892
CATTCTTCGC TGTCAGGTGC ATTGTGGGTA TAAGGAAATC TGTTp,CAAGC TGCCATATTG 2952
GCCTGCTTCA GTCCCCCCGA CCCCCAAATC CCTTCCAACC TGTGC'.TTTAG TGAACGTTGC 3012
TCTGTAACCC TTGTTGGTTG AAAGATTTCT TTGTCTGATG TTAGTGATAC ACACGTGTAA 3072
CAGCCCCCTC CAAAGCGCAA GCCAGTCATA CCCCTGTATA TTTTP.GCAGC ACTGCGGTCC 3132
CAGTGCCAGC TCACGTCCAC TTCACAAGAG TGGTTAGAGG AAAAG'AGAAA GTGTATCTAT 3192
CCTTTTGTAT TCAAATGAAG TTATTTTTCT TGAAATAATG TAATA.TGTAG ATTTTTTGTA 3252
TTATTGCCAA TTTATGTTAC CAGACAATCT GTTAATGTAT CTAATTCGAA TCAGCAAAGA 3312
CTGACTGATG TTTGAGTTTT TGGTCCTCTT TGGTTTTGTT TCGTTTCGTC ATGCAGAGAT 3372
TTCTCTGTAA GGGCAACGAG CGTGCTGGCA TCAAAGAATA TCGGT'TTACA TATAGCAAGT 3432
GTAATAAGAT TCCACCAAAG GACATTTTAA ATGTTTTTTC TTGTTGCTTT AACACTGGAA 3492
GATTTAAAGA ATAAAAATTC CTGCAGAAAT GTTATCAGGA AATTGTATGG CCGTTTCTTA 3552
AGACGAAAGG AGCAACCACC CAGCAGTTTC CCAGTCACGT CACTGATTTT TGTGTGGACT 3612
GAACACAGTC AGCTGACAAC TTTAATAACC AGGAAGACGG ATTGA.TGGTC ACTAGCTTGG 3672
ACAACGTCTG CAAAATATGA GACTATTTTC CACCTGGGAA AAAATTATAA CCACAAAAAC 3732
AGAGAGAAAG AAATATCTAA GTGATTGCCA AGATTATGCC AAAGCCTGTT GGCAGAGCAC 3792
TAAGAGACTT TTATTTTTAA GTCATGCTAT TTTCACAGAT TTATGGTGAT CATGTGACTC 3852
TAGGGATGCC GATCTATGTA TCCTTCCAAA TACAGTGTTT ACATGGAGTA TCATAAGAGG 3912
CCACCTGGGG AACCAGGACA GCAGCAGGGA AATTGAGTGA TTAGCAATTT GACTTTGAAT 3972
ATATTCTAAG TATTTAAATG AAATATCAAA ATATACAGCA GCAAGTAGAC ATAACTGCTG 4032
TTCCTGAAAA TAAAGTCTGT TTCAAGTACT GCCAAAAAAA AAAAAAAAAA AACTCGAG 4090
(2) SEQ ID NO: 8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 539
(B) TYPE: amino acid
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu T:rp Val Thr val
1 5 10 15
51
CA 02324167 2001-07-27
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
Cys LysSerLeu IleTyr ThrGluGlu GlyLysVal TrpAsp TyrThr
35 40 45
Ala CysGlnPro GluSer ThrAspMet ThrLysTyr LeuLys ValLys
50 55 60
10Leu AspProPro AspIle ThrCysGly AspProPro CTluSer PheCys
65 70 75 80
Ala MetGlyAsn ProTyr MetCysAsn AsnGluCys AspAla SerThr
85 90 95
Pro GluLeuAla HisPro ProGluLeu MetPheAsp PheGlu GlyArg
100 105 110
His ProSerThr PheTrp GlnSerAla ThrTrpLys CPluTyr ProLys
20 115 120 1.25
Pro LeuGlnVal AsnIle ThrLeuSer TrpSerLys ThrIle GluLeu
130 135 140
Thr AspAsnIle ValIle ThrPheGlu SerGlyArg FroAsp GlnMet
145 150 155 160
Ile LeuGluLys SerLeu AspTyrGly ArgThrTrp GlnPro TyrGln
165 170 175
30
Tyr TyrAlaThr AspCys LeuHisAla PheHisMet AspPro LysSer
180 185 190
Val LysAspLeu SerGln HisThrVal LeuGluIle IleCys ThrGlu
195 200 205
Glu TyrSerThr GlyTyr SerThrAsn SerLysIle IleHis PheGlu
210 215 220
40Ile LysAspArg PheAla PhePheAla GlyProArg LeuArg AsnMet
225 230 235 240
Ala SerLeuTyr GlyGln LeuAspThr ThrLysLys LeuArg AspPhe
245 250 255
Phe ThrValThr AspLeu ArgIleArg LeuLeuArg ProAla ValGly
260 265 270
Glu IlePheVal AspGlu LeuHisLeu AlaArgTyr PheTyr AlaIle
50 275 280 285
Ser AspIleLys ValArg GlyArgCys LysCysAsn LeuHis AlaThr
290 295 300
Ser CysLeuTyr AspAsn SerLysLeu ThrCysGlu CysGlu HisAsn
305 310 315 320
Thr ThrGlyPro AspCys GlyLysCys LysLysAsn TyrGln GlyArg
325 330 335
60
Pro TrpSerPro GlySer TyrLeuPro IleProLys GlyThr AlaAsn
340 345 350
52
CA 02324167 2001-07-27
a
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Asn Cys C~lu Cys Phe Gly
355 360 365
His Ser Asn Arg Cys Ser Tyr Ile Asp Leu Leu Asn Thr Val Ile Cys
370 375 380
Val Ser Cys Lys His Asn Thr Arg Gly Gln His Cys Glu Leu Cys Arg
385 390 395 400
Leu Gly Tyr Phe Arg Asn Ala Ser Ala Gln Leu Asp Asp Glu Asn Val
405 410 415
Cys Ile Glu Cys Tyr Cys Asn Pro Leu Gly Ser Ile His Asp Arg Cys
420 425 430
Asn Gly Ser Gly Phe Cys Glu Cys Lys Thr Gly Thr Thr Gly Pro Lys
435 440 9:45
Cys Asp Glu Cys Leu Pro Gly Asn Ser Trp Tyr Tyr Gly Cys Gln Pro
450 455 460
Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys
465 470 475 480
Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu
485 490 495
Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser
500 505 510
Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu L~eu Leu Leu Thr
515 520 525
Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe
530 535
(2) SEQ ID NO: 9
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1452
(B) TYPE: nucleic acid
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1452
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATG TAT TTG TCA AGA TTC CTG TCG ATC CAT GCC CTG TGG GTG ACA GTG 48
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA CAT TAT GAT GTA 96
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
TGT AAG AGC CTG ATT TAC ACA GAA GAA GGC AAA GTT TGG GAT TAC ACA 144
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
53
CA 02324167 2001-07-27
s o
GCC TGC CAG CCG GAA TCC ACG GAC ATG ACC AAG TAT C:TG AAA GTG AAA 192
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
50 55 60
CTG GAC CCT CCG GAT ATT ACC TGT GGA GAC CCT CCA GAG TCC TTC TGT 240
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Cilu Ser Phe Cys
65 70 75 80
GCA ATG GGC AAC CCT TAC ATG TGC AAT AAT GAG TGT GAT GCG AGT ACC 288
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr
85 90 95
CCT GAA CTG GCA CAC CCT CCT GAG CTG ATG TTT GAT TTT GAA GGA AGA 336
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg
100 105 110
CAT CCC TCC ACA TTT TGG CAG TCT GCT ACT TGG AAG CiAG TAC CCC AAA 384
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys C9lu Tyr Pro Lys
115 12 0 1.2 5
CCT CTC CAG GTT AAC ATC ACT CTG TCT TGG AGC AAA FCC ATT GAA CTC 432
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT C'.CA GAC CAA ATG 480
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Faro Asp Gln Met
145 150 155 160
ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C'.AG CCC TAT CAG 528
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG G'~AC CCG AAA TCC 576
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P.sp Pro Lys Ser
180 185 190
GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC A.TT TGC ACG GAA 624
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A.TC CAC TTC GAG 672
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG CTA CGA AAT ATG 720
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met
225 230 235 240
GCT TCC CTC TAT GGA CAG CTG GAT ACA ACC AAG AAA CTC AGA GAT TTC 768
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe
245 250 255
TTC ACT GTC ACA GAC CTG AGG ATC AGG CTG TTG AGA CCC GCC GTT GGG 816
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
GAA ATA TTT GTA GAT GAA CTA CAT TTG GCA CGT TAC TTT TAT GCG ATC 864
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
TCA GAC ATA AAG GTG CGA GGA AGG TGC AAG TGC AAC CTG CAT GCC ACT 912
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
54
CA 02324167 2001-07-27
TCG TGT TTG TAT GAC AAC AGC AAA CTG ACA TGT GAA 7.'GT GAG CAC AAC 960
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu C:ys Glu His Asn
305 310 315 320
ACT ACA GGT CCC GAC TGT GGG AAA TGC AAG AAG AAC 7.'AC CAG GGC CGA 1008
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn 7:'yr Gln Gly Arg
325 330 335
CCT TGG AGC CCC GGC TCA TAC CTC CCC ATC CCC AAA CTGC ACC GCA AAC 1056
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys CTly Thr Ala Asn
340 345 350
ACC TGT ATC CCC AGC ATT TCC AGT ATC GGT AAG TGT TAT TGT AAC CCT 1104
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Lys Cys Tyr Cys Asn Pro
355 360 3.65
TTG GGC TCA ATC CAT GAT CGT TGT AAT GGC TCA GGA TTT TGT GAG TGT 1152
Leu Gly Ser Ile His Asp Arg Cys Asn Gly Ser Gly Phe Cys Glu Cys
370 375 380
AAG ACT GGA ACA ACA GGG CCT AAA TGT GAT GAG TGT C'TG CCA GGA AAT 1200
Lys Thr Gly Thr Thr Gly Pro Lys Cys Asp Glu Cys heu Pro Gly Asn
385 390 395 400
TCC TGG TAC TAC GGC TGT CAA CCT AAT GTC TGC GAC P,AT GAG CTC CTG 1248
Ser Trp Tyr Tyr Gly Cys Gln Pro Asn Val Cys Asp P,sn Glu Leu Leu
405 410 415
CAC TGC CAG AAT GGA GGG ACC TGC CAG AAC AAT GTG C'GC TGC GCG TGC 1296
His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val A.rg Cys Ala Cys
420 425 430
CCA GAC GCC TAC ACC GGC ATC CTC TGT GAG AAG CTA C'GG TGC GAA GAG 1344
Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu A.rg Cys Glu Glu
435 440 445
GCG GGC AGC TGT GGC TCC GAA TCC GGC CAG GGA GCA C'CC CCG CGG GGC 1392
Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly
450 455 460
TCC CCA GCA CTG CTG CTG CTG ACC ATG CTG CTG GGG A.CT GCC GGT CCC 1440
Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro
465 470 475 480
CTG GTG TTC TAG 1452
Leu Val Phe
(2) SEQ ID N0: 10
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483
(B) TYPE: amino acid
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
CA 02324167 2001-07-27
a
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
50 55 60
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
65 70 75 80
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys P,sp Ala Ser Thr
85 90 95
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp F~he Glu Gly Arg
100 105 110
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys
115 120 125
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met
145 150 155 160
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met A.sp Pro Lys Ser
180 185 190
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met
225 230 235 240
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe
245 250 255
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn
305 310 315 320
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn
340 345 350
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Lys Cys Tyr Cys Asn Pro
355 360 365
56
CA 02324167 2001-07-27
Leu Gly Ser Ile His Asp Arg Cys Asn Gly Ser Gly Phe Cys Glu Cys
370 375 380
Lys Thr Gly Thr Thr Gly Pro Lys Cys Asp Glu Cys Leu Pro Gly Asn
385 390 395 400
Ser Trp Tyr Tyr Gly Cys Gln Pro Asn Val Cys Asp Asn Glu Leu Leu
405 410 415
His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala Cys
420 425 430
Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu Glu
435 440 4:45
Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Fro Pro Arg Gly
450 455 460
Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro
465 470 475 480
Leu Val Phe
(2) SEQ ID NO: 11
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1317
(B) TYPE: nucleic acid
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1317
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATG TAT TTG TCA AGA TTC CTG TCG ATC CAT GCC CTG TGG GTG ACA GTG 48
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA CAT TAT GAT GTA 96
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
TGT AAG AGC CTG ATT TAC ACA GAA GAA GGC AAA GTT TGG GAT TAC ACA 144
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
GCC TGC CAG CCG GAA TCC ACG GAC ATG ACC AAG TAT CTG AAA GTG AAA 192
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
50 55 60
CTG GAC CCT CCG GAT ATT ACC TGT GGA GAC CCT CCA G.AG TCC TTC TGT 240
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
65 70 75 80
GCA ATG GGC AAC CCT TAC ATG TGC AAT AAT GAG TGT G.AT GCG AGT ACC 288
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr
85 90 95
CCT GAA CTG GCA CAC CCT CCT GAG CTG ATG TTT GAT T'TT GAA GGA AGA 336
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp P:he Glu Gly Arg
100 105 110
57
CA 02324167 2001-07-27
CAT CCC TCC ACA TTT TGG CAG TCT GCT ACT TGG AAG CiAG TAC CCC AAA 384
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys C~lu Tyr Pro Lys
115 12 0 1.2 5
CCT CTC CAG GTT AAC ATC ACT CTG TCT TGG AGC AAA ACC ATT GAA CTC 432
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT C'.CA GAC CAA ATG 480
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met
145 150 155 160
ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C'AG CCC TAT CAG 528
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG GAC CCG AAA TCC 576
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P.sp Pro Lys Ser
180 185 190
GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC P.TT TGC ACG GAA 624
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A.TC CAC TTC GAG 672
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG C'TA CGA AAT ATG 720
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met
225 230 235 240
GCT TCC CTC TAT GGA CAG CTG GAT ACA ACC AAG AAA CTC AGA GAT TTC 768
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe
245 250 255
TTC ACT GTC ACA GAC CTG AGG ATC AGG CTG TTG AGA CCC GCC GTT GGG 816
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
GAA ATA TTT GTA GAT GAA CTA CAT TTG GCA CGT TAC TTT TAT GCG ATC 864
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
TCA GAC ATA AAG GTG CGA GGA AGG TGC AAG TGC AAC CTG CAT GCC ACT 912
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
TCG TGT TTG TAT GAC AAC AGC AAA CTG ACA TGT GAA TGT GAG CAC AAC 960
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn
305 310 315 320
ACT ACA GGT CCC GAC TGT GGG AAA TGC AAG AAG AAC TAC CAG GGC CGA 1008
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
CCT TGG AGC CCC GGC TCA TAC CTC CCC ATC CCC AAA GGC ACC GCA AAC 1056
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn
340 345 350
ACC TGT ATC CCC AGC ATT TCC AGT ATC GGT ACT AAT GTC TGC GAC AAT 1104
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Asn Val Cys Asp Asn
355 360 365
58
i
CA 02324167 2001-07-27
GAG CTC CTG CAC TGC CAG AAT GGA GGG ACC TGC CAG AAC AAT GTG CGC 1152
Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln F~sn Asn Val Arg
370 375 380
TGC GCG TGC CCA GAC GCC TAC ACC GGC ATC CTC TGT GAG AAG CTA CGG 1200
Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg
385 390 395 400
TGC GAA GAG GCG GGC AGC TGT GGC TCC GAA TCC GGC C'AG GGA GCA CCC 1248
Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro
405 410 415
CCG CGG GGC TCC CCA GCA CTG CTG CTG CTG ACC ATG C'.TG CTG GGG ACT 1296
Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Lieu Leu Gly Thr
420 425 430
GCC GGT CCC CTG GTG TTC TAG 1317
Ala Gly Pro Leu Val Phe
435
(2) SEQ ID NO: 12
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 438
(B) TYPE: amino acid
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
40 45
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
55 60
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
65 70 75 80
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr
85 90 95
50 Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg
100 105 110
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys
115 120 125
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys T:hr Ile Glu Leu
130 135 140
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg P:ro Asp Gln Met
145 150 155 160
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp G.ln Pro Tyr Gln
165 170 175
59
CA 02324167 2001-07-27
a a
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P,sp Pro Lys Ser
180 185 190
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile I:le Cys Thr Glu
195 200 205
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met
225 230 235 240
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys L~eu Arg Asp Phe
245 250 255
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn
305 310 315 320
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn
340 345 350
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Asn Val Cys Asp Asn
355 360 365
Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys Gln Asn Asn Val Arg
370 375 380
Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg
385 390 395 400
Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro
405 410 415
Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu Thr Met Leu Leu Gly Thr
420 425 430
Ala Gly Pro Leu Val Phe
435
(2) SEQ ID NO: 13
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1443
(B) TYPE: nucleic acid
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1443
CA 02324167 2001-07-27
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATG TAT TTG TCA AGA TTC CTG TCG ATC CAT GCC CTG T'GG GTG ACA GTG 48
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA C'.AT TAT GAT GTA 96
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly H:is Tyr Asp Val
20 25 30
TGT AAG AGC CTG ATT TAC ACA GAA GAA GGC AAA GTT TGG GAT TAC ACA 144
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
GCC TGC CAG CCG GAA TCC ACG GAC ATG ACC AAG TAT C'TG AAA GTG AAA 192
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr L~eu Lys Val Lys
50 55 60
CTG GAC CCT CCG GAT ATT ACC TGT GGA GAC CCT CCA GAG TCC TTC TGT 240
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
65 70 75 80
GCA ATG GGC AAC CCT TAC ATG TGC AAT AAT GAG TGT GAT GCG AGT ACC 288
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys A.sp Ala Ser Thr
85 90 95
CCT GAA CTG GCA CAC CCT CCT GAG CTG ATG TTT GAT TTT GAA GGA AGA 336
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg
100 105 110
CAT CCC TCC ACA TTT TGG CAG TCT GCT ACT TGG AAG GAG TAC CCC AAA 384
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys
115 120 125
CCT CTC CAG GTT AAC ATC ACT CTG TCT TGG AGC AAA ACC ATT GAA CTC 432
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT CCA GAC CAA ATG 480
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met
145 150 155 160
ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG CAG CCC TAT CAG 528
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG G.AC CCG AAA TCC 576
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met Asp Pro Lys Ser
180 185 190
GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC ATT TGC ACG GAA 624
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A'TC CAC TTC GAG 672
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG C'TA CGA AAT ATG 720
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met
225 230 235 240
GCT TCC CTC TAT GGA CAG CTG GAT ACA ACC AAG AAA C'TC AGA GAT TTC 768
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys L.°u Arg Asp Phe
245 250 255
61
CA 02324167 2001-07-27
TTC ACT GTC ACA GAC CTG AGG ATC AGG CTG TTG AGA C;CC GCC GTT GGG 816
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
GAA ATA TTT GTA GAT GAA CTA CAT TTG GCA CGT TAC TTT TAT GCG ATC 864
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
TCA GAC ATA AAG GTG CGA GGA AGG TGC AAG TGC AAC C".TG CAT GCC ACT 912
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
TCG TGT TTG TAT GAC AAC AGC AAA CTG ACA TGT GAA TGT GAG CAC AAC 960
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu C",ys Glu His Asn
305 310 315 320
ACT ACA GGT CCC GAC TGT GGG AAA TGC AAG AAG AAC TAC CAG GGC CGA 1008
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
CCT TGG AGC CCC GGC TCA TAC CTC CCC ATC CCC AAA GGC ACC GCA AAC 1056
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Lily Thr Ala Asn
340 345 350
ACC TGT ATC CCC AGC ATT TCC AGT ATC GGT ACT CCT C'.CA AAG TTT AAT 1104
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro E~ro Lys Phe Asn
355 360 365
AGG ATA TGG CCG AAT ATT TCT TCC CTT GAG GTT TCT p,AC CCA AAA CAA 1152
Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser Asn Pro Lys Gln
370 375 380
GTT GCT CCC AAA TTA GCT TTG TCA ACA GTT TCT TCT GTT CAA GTT GCA 1200
Val Ala Pro Lys Leu Ala Leu Ser Thr Val Ser Ser v'al Gln Val Ala
385 390 395 400
AAC CAC AAG CGA GCT AAT GTC TGC GAC AAT GAG CTC C'.TG CAC TGC CAG 1248
Asn His Lys Arg Ala Asn Val Cys Asp Asn Glu Leu L~eu His Cys Gln
405 410 415
AAT GGA GGG ACC TGC CAG AAC AAT GTG CGC TGC GCG T'GC CCA GAC GCC 1296
Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala C'ys Pro Asp Ala
420 425 430
TAC ACC GGC ATC CTC TGT GAG AAG CTA CGG TGC GAA G'~AG GCG GGC AGC 1344
Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu G'~lu Ala Gly Ser
435 440 445
TGT GGC TCC GAA TCC GGC CAG GGA GCA CCC CCG CGG GGC TCC CCA GCA 1392
Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala
450 455 460
CTG CTG CTG CTG ACC ATG CTG CTG GGG ACT GCC GGT CCC CTG GTG TTC 1440
Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe
465 470 475 480
TAG
1443
(2) SEQ ID NO: 14
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480
(B) TYPE: amino acid
(ii) MOLECULE TYPE: protein
62
CA 02324167 2001-07-27
(iii) HYPOTHETICAL: No
{iv) ANTI-SENSE: No
{vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
50 55 60
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
65 70 75 80
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys P,sp Ala Ser Thr
85 90 95
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp F~he Glu Gly Arg
100 105 110
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys
115 120 125
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met
145 150 155 160
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met A.sp Pro Lys Ser
180 185 190
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met
225 230 235 240
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe
245 250 255
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn
305 310 315 320
63
CA 02324167 2001-07-27
n
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys C~ly Thr Ala Asn
340 345 350
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro Pro Lys Phe Asn
355 360 _I65
Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser Asn Pro Lys Gln
370 375 380
Val Ala Pro Lys Leu Ala Leu Ser Thr Val Ser Ser Val Gln Val Ala
385 390 395 400
Asn His Lys Arg Ala Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln
405 410 415
Asn Gly Gly Thr Cys Gln Asn Asn Val Arg Cys Ala C;ys Pro Asp Ala
420 425 430
Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg Cys Glu G~lu Ala Gly Ser
435 440 9:45
Cys Gly Ser Glu Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala
450 455 460
Leu Leu Leu Leu Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe
465 470 475 480
(2) SEQ ID N0: 15
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1383
(B) TYPE: nucleic acid
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1383
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ATG TAT TTG TCA AGA TTC CTG TCG ATC CAT GCC CTG T'GG GTG ACA GTG 48
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu T'rp Val Thr Val
1 5 10 15
TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA C'AT TAT GAT GTA 96
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly H:is Tyr Asp Val
20 25 30
TGT AAG AGC CTG ATT TAC ACA GAA GAA GGC AAA GTT TGG GAT TAC ACA 144
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
GCC TGC CAG CCG GAA TCC ACG GAC ATG ACC AAG TAT CTG AAA GTG AAA 192
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
50 55 60
CTG GAC CCT CCG GAT ATT ACC TGT GGA GAC CCT CCA GAG TCC TTC TGT 240
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
70 75 80
64
CA 02324167 2001-07-27
m
a n ° a
GCA ATG GGC AAC CCT TAC ATG TGC AAT AAT GAG TGT GAT GCG AGT ACC 288
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr
85 90 95
CCT GAA CTG GCA CAC CCT CCT GAG CTG ATG TTT GAT TTT GAA GGA AGA 336
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp F~he Glu Gly Arg
100 105 110
CAT CCC TCC ACA TTT TGG CAG TCT GCT ACT TGG AAG GAG TAC CCC AAA 384
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys
115 12 0 1.2 5
CCT CTC CAG GTT AAC ATC ACT CTG TCT TGG AGC AAA P.CC ATT GAA CTC 432
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT C'.CA GAC CAA ATG 480
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met
145 150 155 160
ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C'.AG CCC TAT CAG 528
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG GAC CCG AAA TCC 576
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P.sp Pro Lys Ser
180 185 190
GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC P.TT TGC ACG GAA 624
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA A.TC CAC TTC GAG 672
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG C'.TA CGA AAT ATG 720
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met
225 230 235 240
GCT TCC CTC TAT GGA CAG CTG GAT ACA ACC AAG AAA C'TC AGA GAT TTC 768
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys L~eu Arg Asp Phe
245 250 255
TTC ACT GTC ACA GAC CTG AGG ATC AGG CTG TTG AGA C'CC GCC GTT GGG 816
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
GAA ATA TTT GTA GAT GAA CTA CAT TTG GCA CGT TAC TTT TAT GCG ATC 864
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
TCA GAC ATA AAG GTG CGA GGA AGG TGC AAG TGC AAC C'TG CAT GCC ACT 912
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
TCG TGT TTG TAT GAC AAC AGC AAA CTG ACA TGT GAA T'GT GAG CAC AAC 960
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn
305 310 315 320
ACT ACA GGT CCC GAC TGT GGG AAA TGC AAG AAG AAC TAC CAG GGC CGA 1008
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
CA 02324167 2001-07-27
a
i
CCT TGG AGC CCC GGC TCA TAC CTC CCC ATC CCC AAA GGC ACC GCA AAC 1056
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn
340 345 350
ACC TGT ATC CCC AGC ATT TCC AGT ATC GGT ACT CCT C'.CA AAG TTT AAT 1104
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro Faro Lys Phe Asn
355 360 365
AGG ATA TGG CCG AAT ATT TCT TCC CTT GAG GTT TCT F,AC CCA AAA CAA 1152
Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser P.sn Pro Lys Gln
370 375 380
GCT AAT GTC TGC GAC AAT GAG CTC CTG CAC TGC CAG P,AT GGA GGG ACC 1200
Ala Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln P.sn Gly Gly Thr
385 390 395 400
TGC CAG AAC AAT GTG CGC TGC GCG TGC CCA GAC GCC TAC ACC GGC ATC 1248
Cys Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala T'yr Thr Gly Ile
405 410 415
CTC TGT GAG AAG CTA CGG TGC GAA GAG GCG GGC AGC T'GT GGC TCC GAA 1296
Leu Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser C'ys Gly Ser Glu
420 425 430
TCC GGC CAG GGA GCA CCC CCG CGG GGC TCC CCA GCA C'TG CTG CTG CTG 1344
Ser Gly Gln G1y Ala Pro Pro Arg Gly Ser Pro Ala L~eu Leu Leu Leu
435 440 445
ACC ATG CTG CTG GGG ACT GCC GGT CCC CTG GTG TTC TAG 1383
Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe
450 455 460
(2) SEQ ID N0: 16
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460
(B) TYPE: amino acid
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu T'rp Val Thr Val
1 5 10 15
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
50 55 60
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
65 70 75 80
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr
85 90 95
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg
100 105 110
66
CA 02324167 2001-07-27
a
m ~ ~ a
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys
115 120 1.25
Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Fro Asp Gln Met
145 150 155 160
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met P,sp Pro Lys Ser
180 185 190
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg L~eu Arg Asn Met
225 230 235 240
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe
245 250 255
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn
305 310 315 320
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn
340 345 350
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Pro Pro Lys Phe Asn
355 360 365
Arg Ile Trp Pro Asn Ile Ser Ser Leu Glu Val Ser Asn Pro Lys Gln
370 375 380
Ala Asn Val Cys Asp Asn Glu Leu Leu His Cys Gln Asn Gly Gly Thr
385 390 395 400
Cys Gln Asn Asn Val Arg Cys Ala Cys Pro Asp Ala Tyr Thr Gly Ile
405 410 415
Leu Cys Glu Lys Leu Arg Cys Glu Glu Ala Gly Ser Cys Gly Ser Glu
420 425 430
Ser Gly Gln Gly Ala Pro Pro Arg Gly Ser Pro Ala Leu Leu Leu Leu
435 440 445
67
CA 02324167 2001-07-27
a
m ~ s
Thr Met Leu Leu Gly Thr Ala Gly Pro Leu Val Phe
450 455 460
(2) SEQ ID NO: 17
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1095
(B) TYPE: nucleic acid
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1095
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 17:
ATG TAT TTG TCA AGA TTC CTG TCG ATC CAT GCC CTG TGG GTG ACA GTG 48
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
TCC TCT GTG ATG CAG CCC TAC CTT TTC GTG TGG GGA C'AT TAT GAT GTA 96
Ser Ser val Met Gln Pro Tyr Leu Phe Val Trp Gly H:is Tyr Asp Val
20 25 30
TGT AAG AGC CTG ATT TAC ACA GAA GAA GGC AAA GTT TGG GAT TAC ACA 144
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val T'rp Asp Tyr Thr
35 40 45
GCC TGC CAG CCG GAA TCC ACG GAC ATG ACC AAG TAT CTG AAA GTG AAA 192
Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys
50 55 60
CTG GAC CCT CCG GAT ATT ACC TGT GGA GAC CCT CCA GAG TCC TTC TGT 240
Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Ser Phe Cys
65 70 75 80
GCA ATG GGC AAC CCT TAC ATG TGC AAT AAT GAG TGT GAT GCG AGT ACC 288
Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr
85 90 95
CCT GAA CTG GCA CAC CCT CCT GAG CTG ATG TTT GAT TTT GAA GGA AGA 336
Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg
100 105 110
CAT CCC TCC ACA TTT TGG CAG TCT GCT ACT TGG AAG GAG TAC CCC AAA 384
His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys
115 120 125
CCT CTC CAG GTT AAC ATC ACT CTG TCT TGG AGC AAA ACC ATT GAA CTC 432
Pro Leu Gln val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu
130 135 140
ACA GAC AAC ATA GTT ATT ACC TTT GAA TCG GGG CGT CCA GAC CAA ATG 480
Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met
145 150 155 160
ATC CTA GAG AAA TCT CTC GAC TAC GGA CGA ACA TGG C.AG CCC TAT CAG 528
Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln
165 170 175
TAT TAT GCC ACA GAC TGC CTC CAT GCA TTC CAC ATG G;~1C CCG AAA TCC 576
Tyr Tyr Ala Thr Asp Cys Leu His Ala Phe His Met A;sp Pro Lys Ser
180 185 190
68
CA 02324167 2001-07-27
m
s
GTG AAG GAT TTA TCT CAG CAC ACG GTC TTG GAA ATC ATT TGC ACG GAA 624
Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu
195 200 205
GAG TAC TCC ACT GGG TAC TCC ACG AAT AGC AAA ATA ATC CAC TTC GAG 672
Glu Tyr Ser Thr Gly Tyr Ser Thr Asn Ser Lys Ile Ile His Phe Glu
210 215 220
ATC AAA GAC AGG TTT GCG TTT TTC GCT GGA CCT CGG CTA CGA AAT ATG 720
Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met
225 230 235 240
GCT TCC CTC TAT GGA CAG CTG GAT ACA ACC AAG AAA CTC AGA GAT TTC 768
Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe
245 250 255
TTC ACT GTC ACA GAC CTG AGG ATC AGG CTG TTG AGA CCC GCC GTT GGG 816
Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly
260 265 270
GAA ATA TTT GTA GAT GAA CTA CAT TTG GCA CGT TAC TTT TAT GCG ATC 864
Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile
275 280 285
TCA GAC ATA AAG GTG CGA GGA AGG TGC AAG TGC AAC CTG CAT GCC ACT 912
Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr
290 295 300
TCG TGT TTG TAT GAC AAC AGC AAA CTG ACA TGT GAA TGT GAG CAC AAC 960
Ser Cys Leu Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn
305 310 315 320
ACT ACA GGT CCC GAC TGT GGG AAA TGC AAG AAG AAC TAC CAG GGC CGA 1008
Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg
325 330 335
CCT TGG AGC CCC GGC TCA TAC CTC CCC ATC CCC AAA GGC ACC GCA AAC 1056
Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn
340 345 350
ACC TGT ATC CCC AGC ATT TCC AGT ATC GGT AGT AAG TAA 1095
Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Ser Lys
355 360 365
(2) SEQ ID NO: 18
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 364
(B) TYPE: amino acid
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val
1 5 10 15
Ser Ser Val Met Gln Pro Tyr Leu Phe Val Trp Gly His Tyr Asp Val
20 25 30
Cys Lys Ser Leu Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Thr
35 40 45
69
CA 02324167 2001-07-27
m
Ala CysGlnPro GluSer ThrAspMet ThrLysTyr LeuLysVal Lys
50 55 60
Leu AspProPro AspIle ThrCysGly AspProPro GluSerPhe Cys
65 70 75 80
Ala MetGlyAsn ProTyr MetCysAsn AsnGluCys AspAlaSer Thr
85 90 95
Pro GluLeuAla HisPro ProGluLeu MetPheAsp PheGluGly Arg
100 105 110
His ProSerThr PheTrp GlnSerAla ThrTrpLys GluTyrPro Lys
115 120 125
Pro LeuGlnVal AsnIle ThrLeuSer TrpSerLys ThrIleGlu Leu
130 135 140
Thr AspAsnIle ValIle ThrPheGlu SerGlyArg ProAspGln Met
145 150 155 160
Ile LeuGluLys SerLeu AspTyrGly ArgThrTrp GlnProTyr Gln
165 170 175
Tyr TyrAlaThr AspCys LeuHisAla PheHisMet A.spProLys Ser
180 185 190
Val LysAspLeu SerGln HisThrVal LeuGluIle IleCysThr Glu
195 200 205
Glu TyrSerThr GlyTyr SerThrAsn SerLysIle IleHisPhe Glu
210 215 220
Ile LysAspArg PheAla PhePheAla GlyProArg LeuArgAsn Met
225 230 235 240
Ala SerLeuTyr GlyGln LeuAspThr ThrLysLys LeuArgAsp Phe
245 250 255
Phe ThrValThr AspLeu ArgIleArg LeuLeuArg ProAlaVal Gly
260 265 270
Glu IlePheVal AspGlu LeuHisLeu AlaArgTyr PheTyrAla Ile
275 280 285
Ser AspIleLys ValArg GlyArgCys LysCysAsn LeuHisAla Thr
290 295 300
Ser CysLeuTyr AspAsn SerLysLeu ThrCysGlu CysGluHis Asn
305 310 315 320
Thr ThrGlyPro AspCys GlyLysCys LysLysAsn TyrGlnGly Arg
325 330 335
Pro TrpSerPro GlySer TyrLeuPro IleProLys GlyThrAla Asn
340 345 350
Thr CysIlePro SerIle SerSerIle GlySerLys
355 360
(2) SEQ ID NO: 19
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
I'.
CA 02324167 2001-07-27
~e; a
(B) TYPE: nucleic acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 19:
CCTGTATCCC CAGCATTTCC 20
(2) SEQ ID NO: 20
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid Synthetic DNA
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 20:
AGCAGCAGTG CTGGGGAGCC 20
71
