Note: Descriptions are shown in the official language in which they were submitted.
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ALS2 GENE AND AMYOTROPHIC LATERAL SCLEROSIS TYPE 2
Related Applications
This application claims priority from United States application no. 60/267,723
filed
S February 12, 2001; Japanese application no. 2001-116973 filed April 16,
2001; and, United
States application no. 60/318,352 filed September 12, 2001, which applications
are hereby
incorporated by reference.
Field of the Invention
This invention relates to genetic causes of amyotrophic lateral sclerosis of
type 2
("ALS2").
Backeround of the Invention
Amyotrophic lateral sclerosis ("ALS") is a progressive neurodegenerative
disease in
which distal and proximal motor neurons are selectively degenerated'. Its
cause is
ambiguous and its onset is mostly at middle age and thereafter. Its rate of
onset is about 2-6
per 100,000 persons and begins with lowering of muscular strength and
myoatrophy of wrist
muscle as a secondary neuron hindrance resulting in bulbar paralysis symptoms
such as
atrophy of muscle of limbs, atrophy of tongue, alalia, dysphagia and dyspnea.
No
therapeutic method has been established yet and most of the afflicted die
within five years
from onset.
Juvenile amyotrophic lateral sclerosis of type 2 ("ALS2"; OMIM2151002) is a
somatically recessive type hereditary disease. Although the frequency of its
onset is rare,
muscular convulsion of limbs, face and throat is gradually expressed in
persons of teens or
twenties and becomes chronic by bulbar paralysis as described above.
Amyotrophic lateral sclerosis of type 2 has been mapped to the 1.7 cM interval
flanked by D2SI16 and D2S2237 on human chromosome 2q333'4. Alterations in 391
exons
and their flanking regions derived from 43 non-overlapping transcripts have
been noted
within this intervals'6.
ALS is a very severe disease and there is a need for development of means for
its
early detection or diagnosis and for treatment.
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Summary of the Invention
We have now identified a gene associated with amyotrophic lateral sclerosis
type 2,
termed the ALS2 or ALS2CR6 gene. This gene is expressed in various human
tissues
including neurons in the brain and spinal cord, and encodes a protein with
homology to
RanGED and RhoGEF.
This invention now provides mammalian ALS2 genes and mutant versions thereof
as
well as peptides (including proteins) encoded by such genes. Also included are
fragments
and nucleic acids derived from these genes, corresponding peptides, and
oligonucleotides
suitable for use as amplification primers and/or probes. Antibodies to the
peptides of this
invention are also provided.
This invention also provides methods of diagnosis of ALS2 which may include
identifying in a patient at risk, an altered ALS2 gene or protein. The patient
may be tested to
characterize one or more mutations in the gene or protein produced. Such a
mutation may
comprise the A261de1 mutation or the AGI548de1 mutations described herein.
This invention also provides nucleic acids which correspond to a region of the
ALS2
gene, which nucleic acids typically hybridize to at least about 6, at least
about 10, at least
about 15, at least about 20, or at least about 25 consecutive nucleotides of
an ALS2 sequence
as described herein, or to complements of such sequences, or to naturally
occurring mutants
or allelic variants thereof. The probes or primers may be chosen to be capable
of
distinguishing (such as by amplification or hybridization) allelic variants,
including the
A261 del and AGI548de1 mutations described herein. Such probes or primers may
fiuther
include a label which is capable of being detected. This invention also
provides kits for
identifying ALS2 genes, including those comprising alleles associated with an
ALS2 disease
state, wherein the kits may comprise a probe or primer as described herein.
The kit may
further comprise instructions for using the probes or primers to distinguish
alleles as
described herein.
This invention also provides vectors containing nucleic acids of this
invention,
including vectors adapted for expression of such nucleic acids in a target
cell or organism.
Such vectors may comprise appropriate transcription regulatory elements for
directing
transcription of the nucleic acids in a target cell or organism. Nucleic acids
and peptides of
this invention may be expressed in bacterial as well as eukaryotic cells,
including
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mammalian cells. Such vectors may be adapted to express nucleic acids of this
invention in
a reverse direction so as to generate anti-sense transcription products.
This invention also provides non-human mammals comprising a genome in which an
ALS2 gene has been mutated, including by deletion. Such a mammal may be a
mouse and
S methods for altering the murine genome such as to produce an ALS2 "knock-
out" mouse,
are described herein and are known in the art.
This invention also provides the use of nucleic acids and peptides as
disclosed herein
for the preparation of medicaments for treatment of ALS2 or in the treatment
of ALS2.
This invention also provides methods of treating patients for ALS2, which
methods
may comprise testing the patient to diagnose or characterize an ALS2 disease
state. A
patient may be treated for ALS2, for example by administering to the patient
or by otherwise
providing a native form or functional fragment or derivative of the ALS2
peptide described
herein or such other therapeutic agent as which will restore function of the
protein in a
patient. Also included in this invention are vectors suitable for use in gene
therapy and gene
therapy methodologies whereby a patient is treated to restore the function of
ALS2 by
delivering or producing a functional gene for expression in the patient. Gene
therapy vectors
may, for example, be adeno-associated vector, such as those known in the art.
General
methods for gene therapy are also known in the art.
This invention includes a human ALS2 gene which is present in human second
chromosome q33 region and may code for a GTPase regulatory factor. The gene
may
encode an amino acid sequence of SEQ ID N0:2. cDNA synthesized from mRNA that
may
be transcribed by this gene has a base sequence of SEQ ID NO:1.
This invention includes a human ALS2 mutated gene which is related to
amyotrophic lateral sclerosis of type 2 and codes for a modified protein
having an amino
acid sequence of SEQ ID NO: 3 or SEQ ID N0:84, by a deficiency of one or two
bases of
the above human ALS2 gene.
This invention includes nucleic acids purified from genomic DNA, mRNA or cDNA
as well as synthesized nucleic acids.
This invention includes oligonucleotides which hybridize to ALS2 genes and
variants thereof, preferably under.stringent conditions.
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This invention includes kits comprising oligonucleotides or oligonucleotide
primer
sets which may be used to carry out amplification of ALS2 encoded nucleic
acids, for
example by the polymerase chain reaction (PCR).
This invention includes oligonucleotide probes which hybridize to the regions
containing base deficient sites (A261de1 and AGI548de1J in ALS2 under
stringent
conditions.
This invention includes oligonucleotide primer sets which carry out a PCR
amplification of the region containing a base deficient site in ALS2 as
described herein. A
specific example of this primer set is a pair of synthetic oligonucleotides
comprising the base
sequences of SEQ ID NO: 6 and NO: 7 or a pair of synthetic oligonucleotides
comprising
the base sequences of SEQ ID NO: 8 and NO: 9.
This invention includes recombinant vectors comprising the above nucleic acids
and
cells transcribed by said recombinant vectors.
This invention includes a GTPase regulatory factor or a GEF which is
characterized
in being an expression product of an ALS2 gene as described herein.
Embodiments of such GTPase regulatory factors of GEF's are recombinant
proteins
produced by the transformed cells transformed according to this invention..
This invention includes a peptide comprising an amino acid sequence having
continuous 5 or more acid amino residues in the first to the 46th amino acid
sequence in
SEQ ID NO: 2 and also a peptide comprising an amino acid sequence having
continuous 5
or more acid amino residues in the 47th to the 1657th amino acid sequence in
SEQ ID NO:
2. These peptides may be used for production of antibodies.
This invention also provides a modified protein which may be an expression
product
of a mutant human ALS2 gene and which comprises the amino acid sequence of SEQ
ID
NO: 3. An embodiment of this modified protein is a recombinant protein
produced by a
transformed cell.
This invention includes an antibody which recognizes peptides (including
proteins)
as disclosed herein. Embodiments of this antibody are an antibody which is
prepared using
a peptide according to this invention as an antigen, including a peptide
comprising an amino
acid sequence having continuous 5 or more acid amino residues in the first to
the 46th amino
acid sequence in SEQ ID NO: 2 and also an antibody which is prepared using a
peptide
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comprising an amino acid sequence having continuous S or more acid amino
residues in the
47th to the 1657th amino acid sequence in SEQ ID NO: 2 as an antigen.
This invention furthermore provides methods for the diagnosis of amyotrophic
lateral sclerosis of type 2 which is characterized in detecting ALS2 mutated
genes. An
5 embodiment of this method for the diagnosis it that genomic DNA of the cells
of a person to
be diagnosed is subjected to a PCR amplification using a primer set comprising
a pair of
synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 6 and
NO: 7 or a
pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO:
8 and NO:
9, the resulting DNA fragments are treated with a restriction enzyme NarI and
the said
person where each of the DNA fragments is divided into two fragments is judged
to be
suffering from amyotrophic lateral sclerosis of type 2.
This invention also provides a method for the diagnosis of amyotrophic lateral
sclerosis of type 2 which is characterized in that the transcribed product of
an ALS2 gene or
mutated gene is detected. In an embodiment of this diagnostic method, the
transcribed
product is cDNA or mRNA of the gene of an ALS2 mutated gene or the modified
protein
expressed by the said mutated gene. An embodiment of the case of detection of
the
modified protein is a method for the detection of the protein where the
antibody recognizing
the first to the 46th amino acid sequences in SEQ ID NO: 2 reacts but the
antibody
recognizing the 47th to the 1657th amino acid sequence region in SEQ ID NO: 2
does not
react.
Further, this invention provides a mouse ALS2 gene which may have an amino
acid
sequence of SEQ ID NO:S as well as nucleic acids derived therefrom including
nucleic acids
synthesized or purified from genomic DNA, mRNA or cDNA of the mouse gene or a
complementary sequence thereof.
This invention also provides a gene-defective non-human mammal such as a
rodent,
preferably a mouse, where function of an ALS2 gene is substantially deficient.
Also
provided are tissues of such a mouse.
The human ALS2 gene according to this invention is a genomic gene which has 33
introns and 34 exons, exists in a genomic DNA of 80.3 kb adjacent to a
polymorphic DNA
marker D2S2309 in human second chromosome q 33 region (refer to Fig. 1) and
codes for a
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human GTPase regulatory factor having an amino acid sequence of SEQ ID N0:2.
In this
ALS2 gene, its cDNA has a base sequence of SEQ ID NO: 1.
This invention provides an isolated nucleic acid that codes for a peptide
having at
least about 75, 80, 85, 90, 95, 97 or 100% identity to all of an amino acid
sequence selected
from the group consisting of SEQ ID N0:2; SEQ ID N0:3; SEQ ID N0:5; SEQ ID
N0:84; and, amino acids 372-1657 of SEQ ID N0:2. Also provided are the
peptides
encoded by these nucleic acids.
This invention also provides an isolated nucleic acid consisting essentially
of a
nucleotide sequence having at least about 75, 80, 85, 90, 95, 97 or 100%
identity to all of a
nucleotide sequence or a complementary sequence thereof, selected from the
group
consisting of SEQ ID NO:1; SEQ ID N0:4; nucleotides 124-5094 of SEQ ID NO:1;
nucleotides 1225-5094 of SEQ ID NO:1; and, nucleotides 124-5076 of SEQ ID
N0:4. Also
provided are the peptides encoded by these nucleic acids.
The nucleic acids of this invention may be joined to a second nucleic acid not
naturally associated with the nucleic acid of this invention. By not naturally
associated, it is
meant that the second nucleic acid is not part of an ALS2 gene and is not
directly joined to
an ALS2 gene in the genome of a mammal.
This invention also provides an oligonucleotide of 6 to 75 nucleotides,
wherein the
oligonucleotide hybridizes to a nucleic acid of this invention or a
complementary sequence
thereof, under stringent conditions. An oligonucleotide of this invention may
be joined to a
label, which is any moiety suitable for detectable labelling of the nucleic
acid or for binding
of the nucleic acid to a non-nucleic acid moiety.
This invention also provides a peptide consisting essentially of a sequence of
at least
5 contiguous amino acids from a sequence selected from the group consisting of
amino
acids 1-46 of SEQ ID N0:2; amino acids 47-1657 of SEQ ID N0:2; SEQ ID N0:3;
amino
acids 43-49 of SEQ ID N0:3; SEQ ID N0:84; and amino acids 476 to 545 of SEQ ID
N0:84. These peptides are useful, for example in raising antibodies of this
invention and for
investigating the function of the ALS2 protein.
This invention also provides a non-human mammal comprising a mutated gene,
wherein the gene but for the mutation would encode a protein having at least
about 75, 80,
85, 90, 95, 97 or 100% sequence identity to all of SEQ ID N0:2 or SEQ ID N0:5.
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This invention also provides a method for the diagnosis of amyotrophic lateral
sclerosis type 2 in a patient, comprising detecting the presence of a mutation
in a gene that
encodes a protein having at least about 75, 80, 85, 90, 95, 97 or 100%
sequence identity to
SEQ ID N0:2 in a patient or a biological sample from a patient.
This invention also provides a method for the diagnosis of amyotrophic lateral
sclerosis type 2, comprising detecting the presence or absence of a protein
having at least
about 75, 80, 85, 90, 95, 97 or 100% sequence identity to all of SEQ ID N0:2
in a patient or
a biological sample from a patient.
This invention also provides a method for the diagnosis of amyotrophic lateral
sclerosis type 2, comprising detecting the presence or absence of a protein
having at least
about 75, 80, 85, 90, 95, 97 or 100% sequence identity to all of SEQ ID N0:3
or SEQ ID
N0:84 in a patient or a biological sample from a patient.
In the diagnostic methods of this invention, sequences may be compared to
determine the presence of mutations; oligonucleotides may be used to detect
hybridization to
nucleic acids of the patient; amplification of nucleic acids of the patient
may be performed;
proteins of the patient may be contacted with antibodies of this invention; or
proteins
produced in the patient may be evaluated for the function of ALS2 protein.
This invention also provides a method of treatment of amyotrophic lateral
sclerosis
type 2, comprising administering a peptide, a nucleic acid, or a
pharmaceutical composition
comprising the peptide or nucleic acid to a patient in need thereof, wherein
the peptide
comprises an amino acid sequence having at least about 75, 80, 85, 90, 95, 97
or 100%
identity to SEQ ID N0:2 or a fragment thereof, and wherein the nucleic acid
codes for said
peptide.
This invention also provides a method of treatment of amyotrophic lateral
sclerosis
type 2, comprising administering a composition to a patient in need thereof,
wherein the
composition mimics the biological activity of the peptide of SEQ ID NO. 2 or a
fragment
thereof.
This invention also provides the use of a peptide or a nucleic acid for
preparation of
a medicament for treatment of amyotrophic lateral sclerosis type 2, wherein
the peptide
comprises an amino acid sequence having at least about 75, 80, 85, 90, 95, 97
or 100%
identity to SEQ ID N0:2 or a fragment thereof, and the nucleic acid codes for
said peptide.
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In this specification the term "isolated" with reference to a nucleic acid or
peptide
means that a nucleic acid is separate from the genome of a cell, a peptide is
separate from a
cell but does not mean that the subject matter has been obtained from a genome
or a cell. In
some instances, nucleic acids and peptides of this invention may be
synthesized using
conventional techniques.
Two nucleic acid or protein sequences are considered substantially identical
if, when
optimally aligned, they share at least about 70% sequence identity. In
alternative
embodiments, sequence identity may for example be at least 75%, at least 90%
or at least
95%. Optimal alignment of sequences for comparisons of identity may be
conducted using a
variety of algorithms, such as the local homology algorithm of Smith and
Waterman,1981,
Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and
Wunsch,
1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and
Lipman, 1988,
Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of
these
algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence
alignment
may also be carned out using the BLAST algorithm, described in Altschul et
al., 1990, J.
Mol. Biol. 215:403-10 (using the published default settings).
Nucleic acid sequences of the invention may in some embodiments be
substantially
identical, such as substantially identical gene targeting substrates and
target sequences. The
substantial identity of such sequences may be reflected in percentage of
identity when
optimally aligned that may for example be greater than 50%, 80% to 100%, at
least 80%, at
least 90% or at least 95%, which in the case of gene targeting substrates may
refer to the
identity of a portion of the gene targeting substrate with a portion of the
target sequence,
wherein the degree of identity may facilitate homologous pairing and
recombination and/or
repair. An alternative indication that two nucleic acid sequences are
substantially identical is
that the two sequences hybridize to each other under moderately stringent, or
preferably
stringent, conditions. Hybridization to filter-bound sequences under
moderately stringent
conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium dodecyl
sulfate
(SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% SDS at
42°C (see Ausubel, et
al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green
Publishing
Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
Alternatively,
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hybridization to filter-bound sequences under stringent conditions may, for
example, be
performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in
0.1 x
SSC/0.1% SDS at 68°C (see Ausubel, et al. (eds), 1989, supra).
Hybridization conditions
may be modified in accordance with known methods depending on the sequence of
interest
(see Tijssen, 1993, Laboratory Technigues in Biochemistry and Molecular
Biology --
Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of
principles of
hybridization and the strategy of nucleic acid probe assays", Elsevier, New
York).
Generally, stringent conditions are selected to be about 5°C lower than
the thermal melting
point for the specific sequence at a defined ionic strength and pH.
It is well known in the art that some modifications and changes can be made in
the
structure of a polypeptide without substantially altering the biological
function of that
peptide, to obtain a biologically equivalent polypeptide. In one aspect of the
invention, LPL
S447X therapeutics may include peptides that differ from a portion of the wild-
type LPL
sequence by conservative amino acid substitutions. As used herein, the term
"conserved
amino acid substitutions" refers to the substitution of one amino acid for
another at a given
location in the peptide, where the substitution can be made without loss of
function. In
making such changes, substitutions of like amino acid residues can be made,
for example, on
the basis of relative similarity of side-chain substituents, for example,
their size, charge,
hydrophobicity, hydrophilicity, and the like, and such substitutions may be
assayed for their
effect on the function of the peptide by routine testing.
In some embodiments, conserved amino acid substitutions may be made where an
amino acid residue is substituted for another having a similar hydrophilicity
value (e.g.,
within a value of plus or minus 2.0), where the following hydrophilicity
values are assigned
to amino acid residues (as detailed in United States Patent No. 4,554,101,
incorporated
herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser
(+0.3); Asn (+0.2);
Gln (+p.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-
1.0); Met (-1.3); Val
(-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).
In alternative embodiments, conserved amino acid substitutions may be made
where
an amino acid residue is substituted for another having a similar hydropathic
index (e.g.,
within a value of plus or minus 2.0). In such embodiments, each amino acid
residue may be
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assigned a hydropathic index on the basis of its hydrophobicity and charge
characteristics, as
follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met
(+1.9); Ala (+1.8);
Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-
3.2); Glu (-3.5); Gln
(-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
5
In alternative embodiments, conserved amino acid substitutions may be made
where
an amino acid residue is substituted for another in the same class, where the
amino acids are
divided into non-polar, acidic, basic and neutral classes, as follows: non-
polar: Ala, Val,
Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral:
Gly, Ser, Thr,
10 Cys, Asn, Gln, Tyr.
Brief DescriRtion of the Drawings
Figure 1 is a transcription map of 3Mb region of human chromosome 2q33
including
an ALS2 candidate region. The white open rectangle is between D2S116 to
D2S2237z'3.
Positions of 7 STS markers, 12 polymorphic DNA markers and 42 independent
transcription
units are shown. Polarity of 38 transcription units are shown by arrows. The
location of the
ALS2 gene is designated "ALS2CR6" which term may be used interchangeably for
ALS2
below.
Figure 2 shows a process for the detection of ALS2 associated mutations. "a"
is an
example of the Tunisian and Kuwaiti ALS2 families. Genotypes of the members
constituting a family is shown based on previously reported results3'4. "b"
shows the result
of sequence determination of mutation (A261 del) in genomic DNA of the
Tunisian ALS2
family. Patient 10797 is A261 del of a homozygotic type and the carrier 10784
is a
heterozygotic type. The sequence determination was carried out for PCR
products. "c"
shows the results of determination of mutation (AGI548de1) in the genomic DNA
in the
Kuwaiti ALS2 family. Sequence of the reverse strand of exon 5 in the region of
interest are
shown. Individual 18279 is a normal sibling, who is unaffected by ALS2 and
carries two
normal haplotypes. The box in this sequence indicates the position of the
bases deleted in
affected members. Individual 18281 is an unaffected parent .who carnes one
disease
haplotype. The overlapping normal and mutated sequences are shown. Individual
18275 is
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affected and the figure shows a homozygous CT deletion in the reverse strand
of exon 5:
The position of the deleted bases is indicated by the arrow. The corresponding
forward
sequence and coded normal amino acids and novel amino acids produced by
frameshifting
are indicated. "d" shows segregation of the A261de1 mutation in the Tunisian
ALS2 family.
The presence of the deletion was assayed by the digestion with NarI, which
only cuts
mutated gene product. For exon-PCR products, the 339 by fragment representing
the
normal allele was cleaved into two fragments (225 by and 113 bp) in the mutant
allele. For
RT-PCR product, the 302 by product which represents the normal allele was
cleaved into
two fragments (195 by and 106 bp) in the mutant allele.
Figure 3 shows northern blot analysis of the ALS2 (ALS2CR6) mRNA. In "a", a
northern blot containing 2 ~g of poly A+ mRNA of many adult human tissues is
hybridized
with exon 4 of ALS2 cDNA. In the lower drawings, the same blot is hybridized
with human
~3-actin cDNA for confirmation of the property and the comparative load of
RNA. In the
left, size of the ALS2 transcript is shown. In "b", northern blot containing
10 pg of total
RNA obtained normal whole brain and 20 ~g of total RNA obtained from
lymphocytes of
patients and healthy persons (10788 persons) was hybridized to exon 4 of the
ALS2 cDNA.
The right panel shows an agarose gel electrophoresis of an RNA sample.
Figure 4 is a comparison of amino acid sequences in human ALS2CR6 and mouse
homolog mALS2CR6. The same residues are shown by frames. There are shown the
position of the additional three amino acid residues of the Tunisian mutant
protein (starting
from the 47th amino acid residue), the position of the 25 amino acid residues
(starting from
the 372nd residue) of a short variant part of the ALS2 gene and the position
of the additional
70 amino acid residues of the Kuwaiti mutant protein (starting from the 476th
residue).
Figure S shows an expression of ALS2 mRNA in brain and spinal cord of adult
mouse. "a" is an arrow-like whole image of an RNA/RNA in situ hybridization
using an
antisense ALS2 riboprobe while "b" is a control image using a sense strand
probe.
Significant expression was noted in neurons of hippocampus and dentate gyrus
(c and g),
Purkinje cells of cerebellum (d and h), neurons of cerebral cortex (e and i)
and cinerea of
spinal cord including anterior horn cells (f and j). A scale bar shows a
length of 10 pm.
Figure 6 is a result of an amino acid sequence analysis. "a" is a schematic
chart of
domains and motifs in normal and mutated ALS2 protein. RCCl is a regulatory
factor for
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chromosome condensation, DH is a homologous domain to Dbl, PH is a pleckstrin-
homologous domain, MORN is membrane structure and recognition nexus and VPS9
is a
vacuole protein for discrimination of 9 domains. "b" is comparison of amino
acid sequences
of RCCl repeat-containing regions for human ALS2 (hALS2CR6), mouse ALS2
S (mALS2CR6), human (h) RCC I , human (h) RPGR and mouse (m) RPGR. The amino
acid
residues shown by open frames are the same. Conserved amino acid residues are
abundantly
contained as well. Positions of the seven blades corresponding to RCC 1 are
shown
according to the literature3o.
Figure 7 is a chart that compares the wild type human, mouse, and short human
variant of the ALS2 proteins and the coding products of the A261de1 (Tunisian)
and
AGI548de1 (Kuwaiti) mutations.
Detailed Description of the Invention
The locus of a 1.7 cM region specified by microsatellite markers D2S116 and
D2S2237 of a human second chromosome q 33 region has been mapped3'4. The
inventors
previously prepared a physical map on the basis of YACBAC/PAC of 3Mb genomic
region
covering the candidate region in Figure 15'6. Sequences of cDNA clones and
EST's have
now been analyzed and 42 non-duplicated transcription units including 10 new
genes
mapped. 411 pairs in of primers were designed depending upon genomic DNA of 14
persons of a family of ALS2 (Fig. 2a) and 6 normal control persons having no
kinship with
the former was amplified by PCR. Seventy-seven base sequence polymorphs of
introns or
exons were identified by determining the sequence for all of the PCR products.
Among
them, a gene having base deletions related to onset of ALS2 was identified.
The ALS2 gene also includes restriction regions and regulatory regions
(promoter/enhancer, suppressor, etc.) which function in expression of protein
which is coded
thereby. Such restriction and regulation regions are useful for clarifying the
functions of the
ALS2 gene product as a GEF or a GTPase regulatory factor.
This ALS2 gene may, for example, by isolated by screening a human genome
library using pure polynucleotide or oligonucleotide comprising a base
sequence of SEQ ID
NO: 1 or a partial sequence thereof as a probe. The resulting genomic gene may
be
amplified by commonly used genetic amplifying methods such as, for example, a
PCR
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(polymerase chain reaction) method, an NASBN (nucleic acid sequence based
amplification) method, a TMA (transcription-mediated amplification) method or
an SDA
(strand displacement amplification) method.
A pure polynucleotide (DNA fragments and RNA fragments) may also be prepared
from this ALS2 genomic gene, mRNA transcribed by this gene or cDNA synthesized
from
mRNA. For example, cDNA may be synthesized using poly(A) + RNA extracted from
human cells as a template. The human cells may be either those excised from
human body
by operation, etc. or incubated cells. cDNA may be synthesized by known
methods (Mol.
Cell Biol., 2, 161-170, 1982; J. Gene, 25, 263-269, 1983; Gene, 150, 243-250,
1994). One
may also synthesize cDNA by an RT-PCR method using an oligonucleotide as a
primer and
mRNA isolated from human cells as a template. Specifically, the cDNA prepared
as such
has a base sequence of SEQ ID NO: 1. Those polynucleotides may be used for
recombinant
expression of a human GTPase regulatory factor.
The oligonucleotides of this invention are DNA fragments or RNA fragments
which
1 S hybridize to the above-mentioned ALS2 or the above-mentioned nucleic acids
under
stringent conditions. For example it is a continuous DNA fragment of 10-100 by
in the base
sequence of SEQ ID NO: 1. Here, stringent conditions means a condition whereby
a
specific hybrid formation of target with a probe is made possible by salt
concentration,
concentration of organic solvent (such as formamide), or temperature condition
during
hybridization and washing steps. Methods are described in U. S. Patent No.
6,100,037.
One methodology for creating stringent hybridization conditions is [insert B &
K)
A primer set of this invention is typically a pair of oligonucleotides for
amplification
of ALS2 gene or related nucleic acids. Such a primer set may be designed on
the basis of
the base sequence of SEQ ID NO: 1, synthesized and subjected to purification
using known
methods. Size (base numbers) of the primer preferably is 15-40 bases or more
preferably,
15-30 bases which specificity anneal with a template DNA. However, when LA
(long
accurate) PCR is carried out, it is effective to use primers in excess of 30
bases. A pair (two)
primers comprising sense strand (5'-terminal side) and antisense strand (3'-
terminal side)
should not be complementary. In addition, a self complementary sequence is to
be avoided
in a primer to prevent the formation of a hairpin structure. Further, in order
to ensure a
stable bond to a template DNA, the GC content should be about SO% and
occurrence of GC-
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rich or AT-rich regions in a primer should be avoided. Since an annealing
temperature is
dependent upon Tm (melting temperature), primers having Tm of 55-65°C
are chosen so as
to prepare a PCR product having a high specificity. The final concentration of
the primer
used in PCR should be about 0.1 to about 1 ~M. It is possible to use
commercially available
software for designing a primer including the OligoTM software [manufactured
by National
Bioscience Inc. (U. S. A.)] and GenetyxT"" software [manufactured by Software
Development KK (Japan)}.
Mutated ALS2 genes may be obtained by a method where a DNA library prepared
from cells of a patient thought to be suffering from ALS2 is screened with a
probe which
hybridizes to a region containing mutant (e.g. a base deficient site) under a
stringent
condition. Pure polynucleotide (DNA fragment or RNA fragment) may be obtained
from
genomic DNA, mRNA or cDNA of an ALS2 mutated gene or a complementary sequence
thereof. For example, an ALS2 mutated gene comprises a nucleic acid where the
261 st base
a of SEQ ID NO: 1 is deficient. Such a polynucleotide may be used for
recombinant
1 S production of ALS2 modified protein or for diagnosis of ALS2.
A primer set for a PCR amplification of ALS2, including various regions having
base deficient sites in mutated ALS2 is (for example) a pair of synthetic
oligonucleotides
comprising base sequences of SEQ ID NO: 6 and NO: 7. This primer set is
capable of a
PCR amplification of the region (339 bp) including exon 3 and introns before
and after that
in the ALS2 gene. Another primer set may be composed of synthetic
oligonucleotides
comprising base sequences of SEQ ID NO: 8 and NO: 9 and is capable of PCR
amplification of exons 2-4 (302 bp) of the ALS2 gene using RNA as a template.
Any PCR
product not cleaved by the restriction enzyme NarI is derived from the normal
ALS2 gene
but PCR products derived from a mutated ALS2 gene may be cleaved by NarI to
give two
fragments (Fig. 2c).
A recombinant vector of this invention may be a cloning vector or an
expression
vector. Vectors will be constructed depending upon the type of the
polynucleotide as an
insert or upon the object for use. For example, when an ALS2 protein or a
modified protein
thereof is produced using cDNA or its ORF region as an insert, there may be
used an
expression vector for an in vitro transcription or an expression suitable for
each of
prokaryotic cells such as Escherichia coli and Bacillus subtilis and
eukaryotic cells such as
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yeast, insect cells and mammalian cells. When a genomic DNA of the ALS2 gene
or a
mutated gene thereof is used as an insert, it is also possible to use a BAC
(bacterial artificial
chromosome) vector or a cosmid vector. Such recombinant vectors are also
useful, for
example, as probes for diagnosis of chromosome abnormality by hybridization
including
5 fluorescent in situ hybridization (FISH). Further, a nucleic acid derived
from a normal
ALS2 gene may be recombined in a virus vector such as adenovirus or the like
and the
product may be used for genetic therapy.
In the manufacture of ALS2 peptide (including protein), a transformed cell of
this
invention may be a prokaryotic cell such as Escherichia coli and Bacillus
subtilis or an
10 eukaryotic cell such as from yeast, insects, and mammals. In addition,
cells (such as blood
stem cells) derived from a patient suffering from ALS2 which are transformed
by a virus
vector of this invention in which a nucleic acid derived from a normal ALS2
gene is
recombined, may be used for a genetic therapy of ALS2. Such transformed cells
may be
prepared by introducing a recombinant vector into cells by means of known
methods such as
15 electroporation, calcium phosphate method, liposome method and DEAE dextran
method.
A peptide of this invention may be an expression product of a normal ALS2 gene
or
an expression product of a mutated ALS2 gene. The normal gene product is a
GTPase
transcription factor or GEF having an amino acid sequence of SEQ ID NO: 2.
Peptides of
this invention are useful as immunogens for the preparation of an antibody, as
target
molecules for the development of therapeutic agents for ALS2, etc. These
peptides may be
prepared by methods involving isolating peptides from the cells of healthy
persons or
patients suffering from ALS2. Methods of chemical synthesis on the basis of a
desired
amino acid sequence from SEQ ID N0:2 or SEQ ID N0:3, etc. and (preferably) by
production and isolation or purification from the above-mentioned transformed
cells. Such
transformed cells are incubated and isolation and purification are carried out
for the culture
by, for example, means of treatment with a modifier such as urea or with a
surface-active
agent, ultrasonic wave treatment, enzymatic digestion, precipitation by
salting out or by
solvent, dialysis, centrifugal separation, ultrafiltration, gel filtration,
SDS-PAGE, isoelectric
electrophoresis, ion exchange chromatography, hydrophobic chromatography,
affinity
chromatography and reversed phase chromatography. Such proteins may include
fused
proteins with any other protein. For example, fused proteins with glutathione-
S-transferase
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(GST) or green fluorescent protein (GFP) may be exemplified. In addition, the
protein
expressed in cells may be subjected to various kinds of modifications in the
cells after being
translated. Accordingly, modified proteins are also included in the coverage
of the protein
of this invention. Examples of the modification after translation as such are
elimination of
N-terminal methionine, N-terminal acetylation, addition of sugar chain,
limited
decomposition by intracellular protease, myristoylation, isoprenylation and
phosphorylation.
An antibody of this invention is a polyclonal antibody or monoclonal antibody
which
recognizes a peptide of this invention. Examples include an antibody prepared
using a
peptide comprising an amino acid sequence of continuous 5 amino acid residues
or more of
the first to the 46th amino acid sequence in SEQ ID NO: 2 as an antigen and an
antibody
prepared using a peptide comprising an amino acid sequence of continuous 5
amino acid
residues or more of the 47th to the 1657th amino acid sequence in SEQ ID NO: 2
as an
antigen. When those two kinds of antibodies are used, it is possible to detect
and
differentiate normal and A261 del mutant proteins. The antibody of this
invention includes
all molecules which are able to bind to an epitope of an ALS2 protein or other
peptide of this
invention, and all of Fab, F(ab')2, Fv fragments, etc. thereof. Such an
antibody can be
obtained from serum after an animal is immunized using ALS2 derived protein or
peptide as
an antigen. Alternatively, the above expression vectors for eukaryotic cells
may be
introduced into muscle or skin of animals by injection or particle gun and
then serum is
collected therefrom. Examples of animals that may be used are mouse, rat,
rabbit, goat,
chicken, etc. When B cells collected from the spleen of an immunized animal
are fused with
myeloma cells to produce a hybridoma, it is possible to produce monoclonal
antibodies.
The diagnostic method of this invention is one in which an ALS2 mutated gene
or a
transcription product of an ALS2 mutated gene is detected whereby the risk of
onset of
ALS2 may be estimated. Particularly amenable are persons of known ALS2
families
although diagnosis is not limited thereto.
Genomic DNA of a person to be diagnosed may be subjected to a PCR
amplification
using any of the above-mentioned primer sets or other oligonucleotides of this
invention.
The resulting DNA fragment may be treated with one or more restriction enzymes
such as
NarI, and the person to be diagnosed where the DNA fragment is cleaved into
fragments
different from cleaving product produced from a person not suffering from ALS2
is
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indicative of a patient suffering from ALS2 or a person with some risk of ALS2
in view of
the presence of a mutation in the ALS2 gene.
It is also possible to detect the ALS2 mutated genes by (for example) an
allele
specific oligonucleotide probe method, an oligonucleotide ligation assay
method, a PCR
SSCP method, a PCR-CFLP method, a PCR-PHFA method, an invader method, an RCA
(rolling circle amplification) method and a primer oligo base extension
method.
In detecting transcription products of ALS2 mutated genes, diagnosis may be
carried
out by determining the sequence of mRNA of the person to be diagnosed or cDNA
thereof.
It is also possible to carry out the diagnosis in such a manner that an ALS2
gene of a person
to be diagnosed or cDNA thereof is recombined with an expression vector,
transfected to
cells and the expression product thereof measured.
Expression products of normal and mutant ALS2 genes may be assessed by
measurement of molecular weight. For example, the frame shift caused by
deletion of one
base in normal ALS2 gene whereupon the modified protein is changed to a low-
molecular
protein (SEQ ID NO: 3) comprising the first to the 46th amino acid residues of
SEQ ID NO:
2 and three amino acid residues (Pro-Ser-Glu) newly coded by the frame shift
results in a
product having a molecular weight easily comparable to naturally occurnng gene
products
of the normal ALS2 gene. Further, diagnosis may be also carned out by the
above antibody
provided by this invention in which the ALS2 modified protein reacts with an
antibody
recognizing (for example) the first to the 46th amino acid sequence in SEQ ID
NO: 2 or a
region comprising amino acids 43-49 of SEQ ID N0:3, but does not react with an
antibody
recognizing the 47th to the 1657th amino acid sequence region in SEQ ID NO: 2.
Antibodies specific for amino acids 476 to 545 of SEQ ID N0:84 as compared to
any of the
amino acids of SEQ ID N0:2 could be similarly used for diagnosis of the
AGI548de1.
Diagnosis using antibodies may, for example, be carried out with an ELIZA
method.
The-mouse ALS2 gene of this invention is a mouse genomic gene isolated as a
homolog of the human ALS2 gene and which codes for a mouse ALS2 protein
comprising
an amino acid sequence of SEQ ID NO: 5. Its cDNA has a base sequence of SEQ ID
NO: 4.
This gene may be used for the preparation of a "knock-out" mouse.
Such a "knock-out" mouse can be prepared by known gene targeting methods
(Science, 244: 1288-1292, 1989) or generally according to the following
exarriple.
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First, a DNA fragment of the mouse ALS2 gene including the initiation codon of
the
gene is modified whereupon a defective DNA fragment which deletes expression
of the
ALS2 gene is obtained. This defective DNA fragment is used for the preparation
of a
targeting vector for introduction of the modification into a mouse totipotent
cell (ES cell)
according to known methods (such as the method described in Science, 244: 1288-
1292,
1989). For example, genomic DNA comprising the ALS2 gene is substituted or
inserted
with a resistant gene to a cytotoxin to prepare a recombinant plasmid DNA
possessing the
defective gene having a sequence homologous to the genomic DNA of the ALS2
gene at
both terminals (the targeting vector). It is also possible for the resistant
gene to be connected
to a sequence such as PGKl promoter and PGKl polyadenylation signal for
controlling the
expression. It is preferred that the genomic DNA site of the ALS2CR6 gene
which is
substituted with or inserted by resistant gene be a genomic DNA region
containing an exon
region containing an initiation codon.
There are no particular limitations on such target vectors except that it will
have a
sequence which is homologous to genomic DNA of the ALS2 gene and a resistance
sequence or other sequence useful for cell sorting (such as diphtheria toxin A
gene and
thymidine kinase gene of herpes virus). A promoter and enhancer may be
appropriately
combined and used. The targeting vector is then introduced into an ES
(embryonic stem)
cell according to known methods (e.g. Nature, 292: 154-156, 1981). Such
methods include
electric pulse, a liposome and calcium phosphate. When recombination
efficiency of the
gene to be introduced is of concern, the electric pulse methods is preferred.
DNA in each of
the ES cells into which gene is introduced is extracted and, by means of a
southern blot
analysis or a PCR assay, cells are selected in which a homologous gene
recombination has
taken place between the wild type ALS2 gene existing on the chromosome and the
introduced defective ALS2 gene fragment resulting in placement of the
defective gene
fragment in the chromosome.
An ES cell having a defective gene prepared above may be injected into a
blastocyst
of a wild type animal and chimera-embryos obtained which are transplanted to
the uterus of
a preliminary parent. Resulting progeny are selected for the ALS2 defective
gene and bred.
Selection may be carned out by checking the difference in the color of hair or
by extraction
of DNA from a part of the body (such as the tail end) followed by conducting a
southern blot
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analysis, a PCR assay after extraction of DNA, etc. As to the offspring
obtained by a
crossbreeding of animal of a wild type with a chimera animal where the ALS2
defective
gene is in the generative cells, a southern blot analysis, a PCR assay or the
like may be
carned out using the DNA extracted from a part of the body (such as the tail
end) as a
material to identify a heterozygote into which the ALS2 defective gene is
introduced. A
heterozygote possessing the ALS2 defective gene which is stable in all
generative cells and
somatic cells may be bred to produce progeny in which the ALS2 gene is
completely
knocked-out".
An animal prepared as such may be used for analysis of function of ALS2 gene
in
onset of ALS2 and for screening of therapeutic drugs or development of
therapeutic methods
as an ALS2 model animal.
Methods and results of procedures carned out for cloning of the ALS2 gene and
for
functional analysis thereof are shown.
1. Methods
1-1. ALS2 family
Sixteen cases including 8 individuals suffering from the disease obtained from
a
Tunisian consanginous ALS2 family (literature 2) were analyzed. The
characteristic of
ALS2 is a progressive convulsion of muscles of the limbs and the face
accompanied by
distal myoatrophy of the hand and the foot. Age of onset is between 3 and 10
years age
(literature 2). According to biopsy of nerves and muscles and also to
electromyography test,
there was confirmed deletion of distal motor neuron (literature 2). When a
gene type of the '
polymorphic DNA markers was analyzed together with clinical test data, ALS2
was clearly
an autosomal recessive inheritance.
1-2. Transcription map
Genome Data Base (GDB) (http://www.gdbwww.gdb.org) and UniGene
(http://www.ncbi.nJm.nih.gov) of the Biotechnology Information Center (NCBI)
which were
open to the public for discriminating the sequence of transcribed DNA mapped
within an
objective region were retrieved. Sequence of genomic DNA. overlapped with the
objective
region of ALS2 was retrieved from the "nr" or "htgs" data base of GenBank and
utilized as
the object for the test when a BLAST retrieval to the dbEST data base is
conducted. In order
to isolate the transcript of a full length, there were carned out RT-PCR, 5'-
RACE and cDNA
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library screening. In addition, EST clone was purchased from Research Genetics
and
sequencing for DNA was carned out for measuring the insertion of the whole
clone.
Sequence of double stranded DNA was determined by conducting a dideoxy
sequencing
using a BigDye Terminator Cycle Sequencing Kit (ABI) and an AB 1377DNA
sequences.
5 All sequences of EST data, PCR products and DNA obtained from cDNA clone
were
determined and an estimated independent transcription unit was established.
Then each unit
was mapped on a physical map by a PCR method.
1-3. Identification of exon
In order to determine the constitution of intron and exon of the transcription
DNA,
10 genomic DNA sequence data open to the public in GenBank data base was
compared with
the sequence of cDNA using a Sequences Version 3.0 (Gene Codes Corporation)
program
according to the descriptions of BLAST (literature 28) and literatures (5 and
6).
1-4. PCR
Exon and intron/exon boundaries were subjected to a PCR amplification. ExTaq
15 polyrnerase (Takara) was used and a cycle of 95°C for 15 seconds,
60°C for 30 seconds and
72°C for 30 seconds was repeated for 35 times whereby about SO mg of
genomic DNA were
amplified by a PCR. In order to detect the deficient form of the transcription
DNA, an RT-
PCR was carried out. Total RNA from lymphocytes of four patients of a family
of ALS2
and two carriers was isolated. Total RNA extracted from a healthy human brain
was
20 purchased from Clontech. An RT-PCR was carried out using a Superscript pre-
amplification system (Gibco-BRL) according to the protocol of the
manufacturer. The
oligonucleotide primer for such a PCR was designed using Primer 3.0
(http://www-
genome.wi.mit.edu). Table 1 lists the primers used for amplification of ALS2
(ALS2CR6).
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1-5. Analysis of mutation
In order to detect the mutation of the DNA sequence at the exon or the
intron/exon
boundary, a DNA sequence of a PCR product of exon was determined. The DNA
sequence
of a PCR product of exon was analyzed using the same oligonucleotide as a
primer. The
S sequence in the data base open to the public was compared with the DNA
sequence obtained
from patients, carriers and healthy persons and changes in the nucleotide were
discriminated.
[0044]
It was also confirmed that a new NarI site was formed (A261 del) after the
treatment
with NarI by means of an RT-PCR amplification of exons 2-4 or a PCR
amplification of
exon 3. As to the primers for exon 3-PCR, there were used 5'
CCTAGTCATCCATGTGCTGG-3' (SEQ ID NO: 6) and 5'
TCCCATACCTGACCTTCCAC-3' (SEQ ID NO: 7). As to the primers for the RT-PCR of
exons 2-4, there were used 5'-CTTGATAGACTTTCTGTAAAGAAG-3' (SEQ ID NO: 8)
and 5'-GGCTACTTGGACAAATCTCCACTG-3' (SEQ ID NO: 9). Decomposed product
with NarI was separated by 1.5% agarose gel.
1-6. Northern blot analysis
Northern (MTN) blot (Clontech) of many human adult tissues was hybridized with
exon 4 labelled with 32P-dCTP of ALS2CR6 or human (3-actin cDNA in a Perfect
Hyb
hybridizing solution (Toyobo). The membrane was washed with 0.1 x SSC
containing 1%
of SDS and subjected to an X-ray film (Bio-MAX, Kodak).
1-7. mRNA in situ hybridization
Antisense and sense cRNA probes were prepared from two mouse cDNA clones m2-
as and m2-s. Those mouse cDNA clones covered a part of mouse mALS2CR6 cDNA
(from
the 1732nd to the 2685th bases of SEQ ID NO: 4; 954 bp) and inserted into
pCR2.1
(Invitrogen) in an opposite direction. The probes were prepared according to
the protocol of
the manufacturer (Roche Molecular Biochemicals) by an in vitro transcription
reaction
where digoxigenin-labelled UTP and T7 polymerase were mixed. Preparation of
the sample
and method for the in situ hybridization were in accordance with the
literature (29).
1-8. Retrieval of the data base
Each of DNA and amino acid sequences was compared with the data base of
sequences of nucleotide and protein which were not overlapped each other using
BLASTN
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and BLASTP. Domain and motive of protein were identified by MOTIF servers of
Genome
Net Japan (http://www.~enome.ad.jp), search launcher of BCM
(http://www.hgse.hem.tmc.edu/Search.launcher) and CD search of NCBI
(http://www.hcbi.nlm.nih.~ov).
S 2. Results
The inventors have prepared a physical map on the basis of YAC/BAC/PAC of
genomic region of 3 Mb covering a complete candidate region to ALS2
(literatures 5 and 6).
Sequences of EST and cDNA clone were analyzed within a broad area and, at the
same time,
this physical map was used for the mapping of 43 independent transcription
units including
previously analyzed 18 genes (KIAA0005, CLKl, PP1L3, ORC2L, NDUFB3, CFLAR,
CASP10, CASPB, FZD7, NOPS, UBL1, BMPR2, FLJ10881, LOC57404, AIP-1, CD28,
CTLA4 and AILIM) and new 10 full-length transcription products (ALS2CR1,
ALS2CR2,
ALS2CR3, ALS2CR4, ALS2CR5/MPP4, ALS2CR6, ALS2CR7, ALS2CR8, ALS2CR9
and ALS2CR12). Those genetic sequences were present in the locus of ALS2 (Fig.
1 ).
[0045]
Juvenile ALS2 is rare and has a sign that, in teens and twenties, muscular
convulsion
of limbs, face and throat gradually expresses. Since ALS2 is recessively
hereditary, it is
predicted that this ALS2 disease may take place by a loss of a functional
mutation. . Big
deletion or translocation in the ALS2 locus was investigated by a mapping of
STS/EST
content on the basis of a PCR and a southern blot analysis but that was not
detected. After
that, small deletion or base substitution in exon or intron-exon boundary was
investigated.
In order to detect those mutations, each gene was analyzed and an intron/exon
boundary
thereof was determined. Until now, 395 exons have been identified from 42
genes. In order
to amplify exon and flanking sequence thereof including consensus sequence to
splicing
donor and acceptor, 411 primers in total were designed and those primers were
used to
amplify the genomic DNA of 10 normal control persons who were not related to
14 persons
of the ALS2 family (Fig. 2a) by PCR. Sequence of each of those PCR products
was
determined whereby 77 sequence polymorphs in total of intron or exon were
identified.
Among those 77 polymorphs, 8 mutations contained in 4 different genes were
related to
ALS2 (Table 2).
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Table 2
Gene Region Normal ALS2
NOPS intron 2 tatctc(T~9aattct
NOPS intron 6 gttttg(TTG 2ttttta ~ (TTG)3
ALS2CR6 intron 2 ggtaaAtcattt
ALS2CR6 exon 3 gcaggcAgccctc -~ A261 deletion*
ALS2CR8 intron 6 gtcagtAttataa
ALS2CR9 exon 4 ctccagCatggac ~ T (3rd codon)
ALS2CR9 intron 7 ttgggaTtttttt
ALS2CR9 intron 8 aaaataCggatat .-~ T
Among those sequence mutations, one nucleotide deletion (A261 del) noted in
exon 3
of ALS2CR6 broke the reading frame and it is suggested that such a mutation
mutates the
protein. All of the suspicious hetero-conjugative carriers show a duplicated
sequence pattern
starting from the first nucleotide after the deficient part (Fig. 2b). This
deletion clearly
moves together with an ALS2 expression type (Fig. 2c) and is not noted in 533
normal
control individuals of various races (data not shown). In other mutations, one
base
substitution from C to T in exon 4 of ALS2CR9 gene is included (C873T).
However, this
mutation corresponds to the third codon and, therefore, it does not change the
amino acid
residue. In order to detect a splicing error which is made latent or manifest
by other
sequence mutation, an RT-PCR was carned out using total RNA extracted from
lymphocytes of patients and healthy control persons but no sequence mutation
of mRNA
was detected (data not shown). Accordingly, the mutation related to ALS2 of
intron or exon
region does not cause a splicing error. From those results, it has been
confirmed that
deletion of one base in exon 3 of ALS2CR6 (A261 del; Table 1 ) is mutation
concerning
ALS2.
ALS2CR6 gene contains 33 introns and 34 exons and is present in a genomic DNA
of 80.3 kb adjacent to a polymorphic DNA marker D2S2309 (Fig. 1).
Transcription polarity
of the ALS2CR6 gene is in the direction of central body from telomere. An
ALS2CR6
transcription product (mRNA) has a full length of 6394 by (SEQ ID NO: 1)
having a single
open reading frame (ORF) with a length of 4974 nucleotides (124-5,097 nt) and
codes for a
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protein of 184 KDa comprising 1,657 amino acid residues. Polyadenylated
estimated signal
(AATAAA: 6,375-6,380 nt) and poly(A) region are clear. A short ALS2CR6
transcription
product in a full length of 2,651 by having 1,191 by ORF coding for a 396
amino acid
sequence was identified as well. This short variant splices a 5'-donor site
after exon 4 and,
S as a result, a stop codon is formed after 25 amino acid residues of intron
4. Being
correspondent to those results, 2 transcription products of about 6.5 kb and
2.6 kb were
identified in many adult human tissues by a northern blot analysis (Fig. 3a).
Except the liver
where short transcription products are mostly expressed, both transcription
products showed
the similar expression pattern. It has been confirmed that a big transcription
product of 6.5
10 kb is expressed in a slightly higher level than a transcription product of
2.6 kb and is most
abundantly expressed in the cerebellum. This gene has been also confirmed to
be expressed
in cells of ALS2 patients (Fig. 3b).
Further, a mouse homolog of ALS2CR6 was isolated and named mALS2CR6. A
transcription product of mALS2CR6 is in a full length of 6,349 by (SEQ ID NO:
4) having
15 one ORF of 4,956 by (124-5,076 nt) and codes for a protein of 183 kDa
comprising 1651
amino acids (SEQ ID NO: 5). The ORF as a whole is well reserved in a DNA level
(87%
same) and a protein level (91 % same; 94% similar; Fig. 4) between human being
and mouse
and it is suggested that ALS2CR6 gene is a gene which is well reserved in
mammals.
In order to check the localization property of expression of mALS2CR6
transcription
20 product in the brain and the spinal cord of mouse, an in situ hybridization
using riboprobe
corresponding to a part of mALS2CR6 cDNA was carned out. The result was that,
as
shown in Fig. 5, the mALS2CR6 transcription products were expressed in various
levels in
nerve cells from the brain to the spinal cord, especially in neurons of
hippocampal and
dentate gyrus, cerebellar Purkinje cells, neurons of cerebral cortex and
spinal cinerea
25 including anterior horn cells. In addition, a significant expression was
noted in neurons of
olfactory bulb, basal nucleus and cranial nerve nucleus as well.
Human ALS2CR6 protein showed many interesting properties (Fig. 6a). The first
property is present in a region of A-terminal side of ALS2CR6 and it showed a
high
homology to RCC 1 (regulatory factor for concentrating the chromosome;
literature 7) and
RPGR (GTPase for pigmentary retinitis; literature 8)(Fig. 8). RCC1 and RPGR
protein acts
as a guanine nucleotide exchange factor (GEF) for GTPase like Ran. The second
property is
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that ALS2CR6 has a Dbl-homologous (DH) domain and a pleckstrin-homologous (PH)
domain and both domains are typical domains noted in RhoGEF protein
(literatures 9 and
10). In addition, VPS9 domain is noted in a C-terminal region as well. VPS9
domain is
noted in many GEF including Vps9 (literature 11 ) and Rabex-S (literature 12)
and each is
said to mediate the selection of vacuole protein and the phagocytic
transportation. Two
MORN motives comprising 14 amino acids (literature 13) were noted as well.
According to
the recent study for junctophilin containing an MORN motive, this motive is
shown to
contribute in bonding of plasma membrane (literature 13). It has been known
that GEF is
related to a GDP bonding form of GTPase and promotes the dissociation of GDP
and
bonding of GTP whereby GTPase is activated. Since it has been known
(literatures 18 and
19) that GEF plays an important role in many signal transmission cascades
(literature 14),
neuron formation (literature 15), membrane transportation (literature 16) and
formation of
actin cell skeleton (literature 17), it is likely that ALS2CR6 acts as a
regulatory
factor/activator of Ran-related GTPase, regulates the formation of membrane,
and acts in a
(membrane) transportation of cells including neurons.
According to an RT-PCR analysis, a transcription product of mutated ALS2CR6
gene is transcribed from chromosomes of the patient (Fig. 2c) and produces a
modified
protein comprising 49 amino acids having three new residues (Pro-Ser-Glu) at C-
terminal
(Fig. 6a). Since this modified protein has no functional domain corresponding
to ALS2CR6
protein, it seems to make the inherent function lost. Accordingly, the A261de1
mutation
noted in this ALS2CR6 is related to the fact that ALS2 is recessively
hereditary.
A recent finding that ALS is related to defect for the transportation of axon
and the
formation of cell skeleton (literatures 20, 21 and 22) induces a hypothesis
that ALS2CR6
gene corresponds to ALS2 and that ALS2 is generated by the defect of membrane
structure
due to lacking in a regulatory function of membrane structure Ran-related
GTPase.
ALS2CR6 gene is the second ALS gene succeeding to the determination of role of
copper-zinc superoxide desmutase (SDS-1) in~ ALS. Mutation of SOD-1 is related
to the
form of tardive autosomal dominance (literature 23).
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
readily apparent
to those of skill in the art in light of the teachings of this invention that
changes and
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modification may be made thereto without departing from the spirit or scope of
the
appended claims. All patents, patent applications and publications referred to
herein are
hereby incorporated by reference.
S
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SEQUENCE LISTING
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<151> 2001-02-12
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595 600 605
agt gaa aat gga gtc tgg agc ata get gca ggc agg gat tat tcc ctg 1992
Ser Glu Asn Gly Val Trp Ser Ile Ala Ala Gly Arg Asp Tyr Ser Leu
610 615 620
ttt tta gtg gat aca gaa gac ttc cag cct ggg tta tat tac agt ggc 2040
Phe Leu Val Asp Thr Glu Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly
625 630 635
cga cag gac cct aca gaa ggt gac aac ctt cca gag aat cac agt ggt 2088
Arg Gln Asp Pro Thr Glu Gly Asp Asn Leu Pro Glu Asn His Ser Gly
640 645 650 655
tct aag act cca gta ctt ctc tcc tgt agt aag ctt gga tat ata agc 2136
Ser Lys Thr Pro Val Leu Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser
660 665 670
aga gtg aca gca gga aaa gat agc tat tta gcc ttg gtg gat aaa aac 2184
Arg Val Thr Ala Gly Lys Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn
675 680 685
att atg ggg tat att gcc agt ctc cac gag tta get act aca gaa aga 2232
Ile Met Gly Tyr Ile Ala Ser Leu His Glu Leu Ala Thr Thr Glu Arg
690 695 700
cga ttc tat tca aaa cta agt gat atc aaa tct cag att ctc agg cct 2280
Arg Phe Tyr Ser Lys Leu Ser Asp Ile Lys Ser Gln Ile Leu Arg Pro
705 710 715
ctt ctc agt tta gaa aat ttg ggc act aca act aca gtc cag ctg ttg 2328
Leu Leu Ser Leu Glu Asn Leu Gly Thr Thr Thr Thr Val Gln Leu Leu
720 725 730 735
cag gag gtg get agc cga ttc agc aag ctg tgt tac ctc att ggt cag 2376
Gln Glu Val Ala Ser Arg Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln
740 745 750
cat gga gcc tca ttg agc agc ttc ctt cat ggg gta aag gaa gcc agg 2424
His Gly Ala Ser Leu Ser Ser Phe Leu His Gly Val Lys Glu Ala Arg
755 760 765
agt ttg gtc atc ctg aag cat tca agt ctc ttc ttg gat agt tat aca 2472
Ser Leu Val Ile Leu Lys His Ser Ser Leu Phe Leu Asp Ser Tyr Thr
770 775 780
gag tat tgc aca tct att aca aat ttc ctg gtt atg gga gga ttc cag 2520
Glu Tyr Cys Thr Ser Ile Thr Asn Phe Leu Val Met Gly Gly Phe Gln
785 790 795
ctt ctt get aag cct gcc att gat ttc cta aat aaa aac caa gag ctg 2568
Leu Leu Ala Lys Pro Ala Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu
800 805 810 815
ttg caa gat ttg tca gaa gtg aat gac gaa aac act cag ttg atg gaa 2616
Leu Gln Asp Leu Ser Glu Val Asn Asp Glu Asn Thr Gln Leu Met Glu
820 825 830
ata ctg aat act ttg ttt ttc ttg cca atc aga cga ctt cat aat tac 2664
4
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Ile Leu Asn Thr Leu Phe Phe Leu Pro Ile Arg Arg Leu His Asn Tyr
835 840 845
gca aaa gtt ttg cta aag ctt get act tgt ttt gaa gtg gca tct cca 2712
Ala Lys Val Leu Leu Lys Leu Ala Thr Cys Phe Glu Val Ala Ser Pro
850 855 860
gaa tat cag aaa ctg cag gat tcc agt tct tgt tat gag tgt ctt get 2760
Glu Tyr Gln Lys Leu Gln Asp Ser Ser Ser Cys Tyr Glu Cys Leu Ala
865 870 875
ctc cat ctc ggc agg aaa agg aag gaa gca gaa tac aca ctg ggc ttc 2808
Leu His Leu Gly Arg Lys Arg Lys Glu Ala Glu Tyr Thr Leu Gly Phe
880 885 890 895
tgg aag acc ttc ccc gga aaa atg acg gat tcc ttg agg aag cca gag 2856
Trp Lys Thr Phe Pro Gly Lys Met Thr Asp Ser Leu Arg Lys Pro Glu
900 905 910
cgt cga ctg ctg tgt gag agt agt aac cga gcc ctg tct ctg cag cat 2904
Arg Arg Leu Leu Cys Glu Ser Ser Asn Arg Ala Leu Ser Leu Gln His
915 920 925
get ggg agg ttt tcc gtg aat tgg ttc att ctc ttt aat gat gcc ctg 2952
Ala Gly Arg Phe Ser Val Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu
930 935 940
gtc cat gcc cag ttc tcc acg cac cat gtt ttc cct ctg gcc acg ctg 3000
Val His Ala Gln Phe Ser Thr His His Val Phe Pro Leu Ala Thr Leu
945 950 955
tgg gca gag cca ctg tct gaa gaa get ggt ggt gtg aat ggc tta aag 3048
Trp Ala Glu Pro Leu Ser Glu Glu Ala Gly Gly Val Asn Gly Leu Lys
960 965 970 975
ata act aca cct gag gag cag ttc act ctc att tca tct aca ccc cag 3096
Ile Thr Thr Pro Glu Glu Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln
980 985 990
gaa aag aca aag tgg cta cga get ata agc caa gcc gta gat cag get 3144
Glu Lys Thr Lys Trp Leu Arg Ala Ile Ser Gln Ala Val Asp Gln Ala
995 1000 1005
ttg aga ggg atg tct gat ctc ccc cct tat gga agt ggt agc agt gtt 3192
Leu Arg Gly Met Ser Asp Leu Pro Pro Tyr Gly Ser Gly Ser Ser Val
1010 1015 1020
cag aga cag gaa cca ccc att tca cgc agt gcc aaa tat act ttc tac 3240
Gln Arg Gln Glu Pro Pro Ile Ser Arg Ser Ala Lys Tyr Thr Phe Tyr
1025 1030 1035
aag gat cct cgc cta aag gat gcc acc tat gat gga cgc tgg ctt tca 3288
Lys Asp Pro Arg Leu Lys Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser
1040 1045 1050 1055
ggg aag cct cat ggc aga ggg gtt ttg aag tgg cct gat gga aag atg 3336
Gly Lys Pro His Gly Arg Gly Val Leu Lys Trp Pro Asp Gly Lys Met
1060 1065 1070
tat tct ggc atg ttc agg aat ggc ttg gaa gat ggg tat gga gaa tac 3384
Tyr Ser Gly Met Phe Arg Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr
1075 1080 1085
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
aga atc cca aac aag gca atg aac aaa gaa gac cat tat gtg ggc cat 3432
Arg Ile Pro Asn Lys Ala Met Asn Lys Glu Asp His Tyr Val Gly His
1090 1095 1100
tgg aaa gaa gga aaa atg tgc ggt caa gga gtc tac agc tat get tct 3480
Trp Lys Glu Gly Lys Met Cys Gly Gln Gly Val Tyr Ser Tyr Ala Ser
1105 1110 1115
ggt gaa gta ttt gag ggc tgt ttt caa gat aat atg cgt cat ggt cat 3528
Gly Glu Val Phe Glu Gly Cys Phe Gln Asp Asn Met Arg His Gly His
1120 1125 1130 1135
ggt ctt cta cga agt ggg aaa ttg acg tcc tct tct cct agt atg ttc 3576
Gly Leu Leu Arg Ser Gly Lys Leu Thr Ser Ser Ser Pro Ser Met Phe
1140 1145 1150
att ggc cag tgg gta atg gat aag aaa gca gga tat ggt gtc ttt gat 3624
Ile Gly Gln Trp Val Met Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp
1155 1160 1165
gat atc act agg ggg gaa aag tat atg gga atg tgg caa gat gat gtg 3672
Asp Ile Thr Arg Gly Glu Lys Tyr Met Gly Met Trp Gln Asp Asp Val
1170 1175 1180
tgt caa ggg aat ggt gtg gtg gtt acc cag ttt gga tta tac tac gag 3720
Cys Gln Gly Asn Gly Val Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu
1185 1190 1195
ggc aac ttt cac ctt aat aaa atg atg gga aat ggg gtt ttg ctt tcc 3768
Gly Asn Phe His Leu Asn Lys Met Met Gly Asn Gly Val Leu Leu Ser
1200 1205 1210 1215
gaa gat gat act atc tat gaa gga gaa ttt tca gat gac tgg act ctt 3816
Glu Asp Asp Thr Ile Tyr Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu
1220 1225 1230
agt gga aag gga aca ctg act atg cca aat gga gac tac att gaa ggt 3864
Ser Gly Lys Gly Thr Leu Thr Met Pro Asn Gly Asp Tyr Ile Glu Gly
1235 1240 1245
tat ttt agt gga gaa tgg gga tct ggg ata aaa atc act gga acc tac 3912
Tyr Phe Ser Gly Glu Trp Gly Ser Gly Ile Lys Ile Thr Gly Thr Tyr
1250 1255 1260
ttc aaa cct agt cta tat gag agt gat aaa gac aga cct aaa gtt ttc 3960
Phe Lys Pro Ser Leu Tyr Glu Ser Asp Lys Asp Arg Pro Lys Val Phe
1265 1270 1275
agg aag cta gga aac ctg gca gtg cca get gat gag aag tgg aaa gcg 4008
Arg Lys Leu Gly Asn Leu Ala Val Pro Ala Asp Glu Lys Trp Lys Ala
1280 1285 1290 1295
gtg ttt gac gaa tgt tgg cgc caa ctg ggc tgt gag ggc cca ggc caa 4056
Val Phe Asp Glu Cys Trp Arg Gln Leu Gly Cys Glu Gly Pro Gly Gln
1300 1305 1310
ggg gaa gtt tgg aaa gca tgg gac aat att get gtg gcc ttg acc acc 4104
Gly Glu Val Trp Lys Ala Trp Asp Asn Ile Ala Val Ala Leu Thr Thr
1315 1320 1325
agt cgg cgc cag cac aga gac agt cca gaa ata ttg agt cgt tca cag 4152
6
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Ser Arg Arg Gln His Arg Asp Ser Pro Glu Lle Leu Ser Arg Ser Gln
1330 1335 1340
act cag aca cta gag agt ttg gaa ttc att cca cag cat gtt ggt gcc 4200
Thr Gln Thr Leu Glu Ser Leu Glu Phe Ile Pro Gln His Val Gly Ala
1345 1350 1355
ttc tct gtg gag aaa tat gat gac atc agg aaa tat tta ata aag gcc 4248
Phe Ser Val Glu Lys Tyr Asp Asp Ile Arg Lys Tyr Leu Ile Lys Ala
1360 1365 1370 1375
tgt gac act cct ctg cac ccc ctg ggc agg ctt gtg gag aca ctg gtt 4296
Cys Asp Thr Pro Leu His Pro Leu Gly Arg Leu Val Glu Thr Leu Val
1380 1385 1390
gca gtg tat aga atg aca tac gtg ggc gta gga gcc aac cgc agg tta 4344
Ala Val Tyr Arg Met Thr Tyr Val Gly Val Gly Ala Asn Arg Arg Leu
1395 1400 1405
ttg cag gag get gta aag gag att aag tcc tat ctt aag cga att ttc 4392
Leu Gln Glu Ala Val Lys Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe
1410 1415 1420
cag ctg gtg agg ttc tta ttt cct gag ctg cct gaa gaa ggc agc aca 4440
Gln Leu Val Arg Phe Leu Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr
1425 1430 1435
att cct ctc tct get cct ctg cca acc gaa agg aag tct ttt tgc act 4488
Ile Pro Leu Ser Ala Pro Leu Pro Thr Glu Arg Lys Ser Phe Cys Thr
1440 1445 1450 1455
ggg aag tca gat tcc cga tct gaa tca cca gag cca ggt tat gta gta 4536
Gly Lys Ser Asp Ser Arg Ser Glu Ser Pro Glu Pro Gly Tyr Val Val
1460 1465 1470
acg agt tct gga tta ttg ctt cct gtg ctg cta cct cgg ctc tac cca 4584
Thr Ser Ser Gly Leu Leu Leu Pro Val Leu Leu Pro Arg Leu Tyr Pro
1475 1480 1485
ccg ctg ttt atg ctt tat get ttg gat aat gat cgc gag gaa gac att 4632
Pro Leu Phe Met Leu Tyr Ala Leu Asp Asn Asp Arg Glu Glu Asp Ile
1490 1495 1500
tac tgg gaa tgt gtc ctt cga cta aat aag cag cca gat att get ctc 4680
Tyr Trp Glu Cys Val Leu Arg Leu Asn Lys Gln Pro Asp Ile Ala Leu
1505 1510 1515
ctg ggc ttt ctt ggg gtg cag agg aaa ttt tgg cca gca acc ttg tca 4728
Leu Gly Phe Leu Gly Val Gln Arg Lys Phe Trp Pro Ala Thr Leu Ser
1520 1525 1530 1535
atc ctt gga gag agt aaa aag gtt ttg cca acc acg aaa gat get tgt 4776
Ile Leu Gly Glu Ser Lys Lys Val Leu Pro Thr Thr Lys Asp Ala Cys
1540 1545 1550
ttt gcc tca gca gta gaa tgt ctg cag cag atc agc aca aca ttt acc 4824
Phe Ala Ser Ala Val Glu Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr
1555 1560 1565
cca tca gac aaa ctt aag gtc atc cag cag act ttt gag gag atc tct 4872
Pro Ser Asp Lys Leu Lys Val Ile Gln Gln Thr Phe Glu Glu Ile Ser
1570 1575 1580
7
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
cag agt gtc ctg gcg tca ctc cac gaa gac ttc ttg tgg tcc atg gat 4920
Gln Ser Val Leu Ala Ser Leu His Glu Asp Phe Leu Trp Ser Met Asp
1585 1590 1595
gac ttg ttt cct gtt ttc tta tat gtg gtg cta cgg gcc agg att agg 4968
Asp Leu Phe Pro Val Phe Leu Tyr Val Val Leu Arg Ala Arg Ile Arg
1600 1605 1610 1615
aat tta ggc tct gag gta cac ctc att gag gat cta atg gac ccc tat 5016
Asn Leu Gly Ser Glu Val His Leu Ile Glu Asp Leu Met Asp Pro Tyr
1620 1625 1630
ctt cag cat ggg gaa cag ggt ata atg ttc acc acc ttg aag gca tgt 5064
Leu Gln His Gly Glu Gln Gly Ile Met Phe Thr Thr Leu Lys Ala Cys
1635 1640 1645
tac tac cag att cag cgt gag aag ctt aac tag gctgcataac agcttgaaaa 5117
Tyr Tyr Gln Ile Gln Arg Glu Lys Leu Asn
1650 1655
ctggattatc tactacagag tgttataaca ccatctggag tcttcctgta gtggcaaaaa 5177
agaacagtgt tgaaattgga aaggactttg tgttatttag gttgttagaa tgagccttac 5237
caataataag agccctgagc ccagaaaaaa ggactgtata gtttaaaggg aggattgaaa 5297
gggaggtaaa aaatcagatt agaccagttc ttggcctatg ataagttcca aaaataccat 5357
ttatctacta tttgaaaaaa gaagaggata tcccttccta cagtaaaggg tatgtcagct 5417
acatgaagtt gtaagaaaag cttccagtag agcttcttat attaaagaag ttgatggata 5477
tttttgaatt tctggtttgc ctgaatccac ctgcagttac cccgatccgt ttgcaagaac 5537
cagatcgtac ttgaaactat agtggccaca ctctgccttc ctgagtccct tccagtcatg 5597
tgtgcatcat gtctctttgc caagggaggg gagaaaggaa cttttaaact gcagttttaa 5657
ctttttctaa gctgtttctt gatgggagag gttctgtgca aaactaccac attctgtccc 5717
caaaatgtgg aatgcatcca aataggagtc ttctgcctct taacttaaaa gaacatagga 5777
attttgtttt tggtttcttt atcatgctac agagagtgaa tacactggaa ttcagacacc 5837
gactctgagc tgctaggaac ctcatttgtc catgtgcaaa cgctgtattc caaggcctgt 5897
gaatggcagc ctgaggaagt tttgcatgca ggctgtgttt tcgagcagga ctaacaactg 5957
ggaaataagc aaaaaactgc atcgatcccc agcctggtgt tgttcttccc tatacttcac 6017
actgaactca ggatgggaag aaaaaggaaa caagctttgg ctttttccat ctcaaaagta 6077
ttgtggcacc tcaacatttc agtgttttgc tttttaaaaa atgccctatt gtaagttgtt 6137
ggtttatact gtataagtaa cactagtagc tgttttgaat aacataggtg ctcttcctca 6197
tctcatctcc tacaccgtgg tgagcataca gagtgtcctg atttgtgtta agtgactgag 6257
aagatgttaa ttacttttga aaaaggatca tggtttttgc tctactttat aatcaagaca 6317
agtgtttatt aaaatactgt tttggaatgt tggctgtaat gtaacagcaa ttttcataat 6377
aaaaggcatt catcttt 6394
<210> 2
<211> 1657
<212> PRT
<213> Homo Sapiens
<400> 2
Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu
1 5 10 15
Arg Gly Leu Val His Ile Trp Gln Ala Gly Ser Phe Pro Ile Thr Pro
20 25 30
Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala Leu
35 40 45
Gly Val Lys His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr Ser
50 55 60
Phe Gly Thr Leu Pro Trp Arg Ser Gly Pro Val Glu Ile Cys Pro Ser
65 70 75 80
Ser Pro Ile Leu Glu Asn Ala Leu Val Gly Gln Tyr Val Ile Thr Val
85 90 95
8
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Asp Asn Gly Val Ala
100 105 110
Tyr Met Trp Gly Glu Asn Ser Ala Gly Gln Cys Ala Val Ala Asn Gln
115 120 125
Gln Tyr Val Pro Glu Pro Asn Pro Val Ser Ile Ala Asp Ser Glu Ala
130 135 140
Ser Pro Leu Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu Glu
145 150 155 160
His Thr Leu Ala Leu Ser Ile Ser Arg Glu Ile Trp Ala Trp Gly Thr
165 170 175
Gly Cys Gln Leu Gly Leu Ile Thr Thr Ala Phe Pro Val Thr Lys Pro
180 185 190
Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys
195 200 205
Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Ser Gln Asp
210 215 220
Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu Ile
225 230 235 240
Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His Cys
245 250 255
Cys Pro Leu Gly Val Thr Leu Thr Glu Ser Gln Ala Glu Asn His Ala
260 265 270
Ser Thr Ala Leu Ser Pro Ser Thr Glu Thr Leu Asp Arg Gln Glu Glu
275 280 285
Val Phe Glu Asn Thr Leu Val Ala Asn Asp Gln Ser Val Ala Thr Glu
290 295 300
Leu Asn Ala Val Ser Ala Gln Ile Thr Ser Ser Asp Ala Met Ser Ser
305 310 315 320
Gln Gln Asn Val Met Gly Thr Thr Glu Ile Ser Ser Ala Arg Asn Ile
325 330 335
Pro Ser Tyr Pro Asp Thr Gln Ala Val Asn Glu Tyr Leu Arg Lys Leu
340 345 350
Ser Asp His Ser Val Arg Glu Asp Ser Glu His Gly Glu Lys Pro Met
355 360 365
Pro Ser Gln Pro Leu Leu Glu Glu Ala Ile Pro Asn Leu His Ser Pro
370 375 380
Pro Thr Thr Ser Thr Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala
385 390 395 400
Ser Ala Val Gly Val Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu
405 410 415
Ser Leu Lys Lys Val Met Asn Phe Tyr Ser Thr Thr Pro Cys Glu Thr
420 425 430
Gly Ala Gln Ala Gly Ser Ser Ala Ile Gly Pro Glu Gly Leu Lys Asp
435 440 445
Ser Arg Glu Glu Gln Val Lys Gln Glu Ser Met Gln Gly Lys Lys Ser
450 455 460
Ser Ser Leu Val Asp Ile Arg Glu Glu Glu Thr Glu Gly Gly Ser Arg
465 470 475 480
Arg Leu Ser Leu Pro Gly Leu Leu Ser Gln Val Ser Pro Arg Leu Leu
485 490 495
Arg Lys Ala Ala Arg Val Lys Thr Arg Thr Val Val Leu Thr Pro Thr
500 505 510
Tyr Ser Gly Glu Ala Asp Ala Leu Leu Pro Ser Leu Arg Thr Glu Val
515 520 525
Trp Thr Trp Gly Lys Gly Lys Glu Gly Gln Leu Gly His Gly Asp Val
530 535 540
Leu Pro Arg Leu Gln Pro Leu Cys Val Lys Cys Leu Asp Gly Lys Glu
545 550 555 560
Val Ile His Leu Glu Ala Gly Gly Tyr His Ser Leu Ala Leu Thr Ala
565 570 575
Lys Ser Gln Val Tyr Ser Trp Gly Ser Asn Thr Phe Gly Gln Leu Gly
580 585 590
9
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
His Ser Asp Phe Pro Thr Thr Val Pro Arg Leu Ala Lys Ile Ser Ser
595 600 605
Glu Asn Gly Val Trp Ser Ile Ala Ala Gly Arg Asp Tyr Ser Leu Phe
610 615 620
Leu Val Asp Thr Glu Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg
625 630 635 640
Gln Asp Pro Thr Glu Gly Asp Asn Leu Pro Glu Asn His Ser Gly Ser
645 650 655
Lys Thr Pro Val Leu Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg
660 665 670
Val Thr Ala Gly Lys Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile
675 680 685
Met Gly Tyr Ile Ala Ser Leu His Glu Leu Ala Thr Thr Glu Arg Arg
690 695 700
Phe Tyr Ser Lys Leu Ser Asp Ile Lys Ser Gln Ile Leu Arg Pro Leu
705 710 715 720
Leu Ser Leu Glu Asn Leu Gly Thr Thr Thr Thr Val Gln Leu Leu Gln
725 730 735
Glu Val Ala Ser Arg Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln His
740 745 750
Gly Ala Ser Leu Ser Ser Phe Leu His Gly Val Lys Glu Ala Arg Ser
755 760 765
Leu Val Ile Leu Lys His Ser Ser Leu Phe Leu Asp Ser Tyr Thr Glu
770 775 780
Tyr Cys Thr Ser Ile Thr Asn Phe Leu Val Met Gly Gly Phe Gln Leu
785 790 795 800
Leu Ala Lys Pro Ala Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu Leu
805 810 815
Gln Asp Leu Ser Glu Val Asn Asp Glu Asn Thr Gln Leu Met Glu Ile
820 825 830
Leu Asn Thr Leu Phe Phe Leu Pro Ile Arg Arg Leu His Asn Tyr Ala
835 840 845
Lys Val Leu Leu Lys Leu Ala Thr Cys Phe Glu Val Ala Ser Pro Glu
850 855 860
Tyr Gln Lys Leu Gln Asp Ser Ser Ser Cys Tyr Glu Cys Leu Ala Leu
865 870 875 880
His Leu Gly Arg Lys Arg Lys Glu Ala Glu Tyr Thr Leu Gly Phe Trp
885 890 895
Lys Thr Phe Pro Gly Lys Met Thr Asp Ser Leu Arg Lys Pro Glu Arg
900 905 910
Arg Leu Leu Cys Glu Ser Ser Asn Arg Ala Leu Ser Leu Gln His Ala
915 920 925
Gly Arg Phe Ser Val Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu Val
930 935 940
His Ala Gln Phe Ser Thr His His Val Phe Pro Leu Ala Thr Leu Trp
945 950 955 960
Ala Glu Pro Leu Ser Glu Glu Ala Gly Gly Val Asn Gly Leu Lys Ile
965 970 975
Thr Thr Pro Glu Glu Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln Glu
980 985 990
Lys Thr Lys Trp Leu Arg Ala Ile Ser Gln Ala Val Asp Gln Ala Leu
995 1000 1005
Arg Gly Met Ser Asp Leu Pro Pro Tyr Gly Ser Gly Ser Ser Val Gln
1010 1015 1020
Arg Gln Glu Pro Pro Ile Ser Arg Ser Ala Lys Tyr Thr Phe Tyr Lys
1025 1030 1035 1040
Asp Pro Arg Leu Lys Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly
1045 1050 1055
Lys Pro His Gly Arg Gly Val Leu Lys Trp Pro Asp Gly Lys Met Tyr
1060 1065 1070
Ser Gly Met Phe Arg Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg
1075 1080 1085
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Ile Pro Asn Lys Ala Met Asn Lys Glu Asp His Tyr Val Gly His Trp
1090 1095 1100
Lys Glu Gly Lys Met Cys Gly Gln Gly Val Tyr Ser Tyr Ala Ser Gly
1105 1110 1115 1120
Glu Val Phe Glu Gly Cys Phe Gln Asp Asn Met Arg His Gly His Gly
1125 1130 1135
Leu Leu Arg Ser Gly Lys Leu Thr Ser Ser Ser Pro Ser Met Phe Ile
1140 1145 1150
Gly Gln Trp Val Met Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp Asp
1155 1160 1165
Ile Thr Arg Gly Glu Lys Tyr Met Gly Met Trp Gln Asp Asp Val Cys
1170 1175 1180
Gln Gly Asn Gly Val Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly
1185 1190 1195 1200
Asn Phe His Leu Asn Lys Met Met Gly Asn Gly Val Leu Leu Ser Glu
1205 1210 1215
Asp Asp Thr Ile Tyr Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu Ser
1220 1225 1230
Gly Lys Gly Thr Leu Thr Met Pro Asn Gly Asp Tyr Ile Glu Gly Tyr
1235 1240 1245
Phe Ser Gly Glu Trp Gly Ser Gly Ile Lys Ile Thr Gly Thr Tyr Phe
1250 1255 1260
Lys Pro Ser Leu Tyr Glu Ser Asp Lys Asp Arg Pro Lys Val Phe Arg
1265 1270 1275 1280
Lys Leu Gly Asn Leu Ala Val Pro Ala Asp Glu Lys Trp Lys Ala Val
1285 1290 1295
Phe Asp Glu Cys Trp Arg Gln Leu Gly Cys Glu Gly Pro Gly Gln Gly
1300 1305 1310
Glu Val Trp Lys Ala Trp Asp Asn Ile Ala Val Ala Leu Thr Thr Ser
1315 1320 1325
Arg Arg Gln His Arg Asp Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr
1330 1335 1340
Gln Thr Leu Glu Ser Leu Glu Phe Ile Pro Gln His Val Gly Ala Phe
1345 1350 1355 1360
Ser Val Glu Lys Tyr Asp Asp Ile Arg Lys Tyr Leu Ile Lys Ala Cys
1365 1370 1375
Asp Thr Pro Leu His Pro Leu Gly Arg Leu Val Glu Thr Leu Val Ala
1380 1385 1390
Val Tyr Arg Met Thr Tyr Val Gly Val Gly Ala Asn Arg Arg Leu Leu
1395 1400 1405
Gln Glu Ala Val Lys Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln
1410 1415 1420
Leu Val Arg Phe Leu Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile
1425 1430 1435 1440
Pro Leu Ser Ala Pro Leu Pro Thr Glu Arg Lys Ser Phe Cys Thr Gly
1445 1450 1455
Lys Ser Asp Ser Arg Ser Glu Ser Pro Glu Pro Gly Tyr Val Val Thr
1460 1465 1470
Ser Ser Gly Leu Leu Leu Pro Val Leu Leu Pro Arg Leu Tyr Pro Pro
1475 1480 1485
Leu Phe Met Leu Tyr Ala Leu Asp Asn Asp Arg Glu Glu Asp Ile Tyr
1490 1495 1500
Trp Glu Cys Val Leu Arg Leu Asn Lys Gln Pro Asp Ile Ala Leu Leu
1505 1510 1515 1520
Gly Phe Leu Gly Val Gln Arg Lys Phe Trp Pro Ala Thr Leu Ser Ile
1525 1530 1535
Leu Gly Glu Ser Lys Lys Val Leu Pro Thr Thr Lys Asp Ala Cys Phe
1540 1545 1550
Ala Ser Ala Val Glu Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro
1555 1560 1565
Ser Asp Lys Leu Lys Val Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln
1570 1575 1580
11
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Ser Val Leu Ala Ser Leu His Glu Asp Phe Leu Trp Ser Met Asp Asp
1585 1590 1595 1600
Leu Phe Pro Val Phe Leu Tyr Val Val Leu Arg Ala Arg Ile Arg Asn
1605 1610 1615
Leu Gly Ser Glu Val His Leu Ile Glu Asp Leu Met Asp Pro Tyr Leu
1620 1625 1630
Gln His Gly Glu Gln Gly Ile Met Phe Thr Thr Leu Lys Ala Cys Tyr
1635 1640 1645
Tyr Gln Ile Gln Arg Glu Lys Leu Asn
1650 1655
<210> 3
<211> 49
<212> PRT
<213> Homo Sapiens
<400> 3
Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu
1 5 10 15
Arg Gly Leu Val His Ile Trp Gln Ala Gly Ser Phe Pro Ile Thr Pro
20 25 30
Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Pro Ser
35 40 45
Glu
<210> 4
<211> 6349
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (124)..(5079)
<400> 4
ccacgcgtcc ggcggtgcag tcgggctcgc gccgggagaa gagcgcggag ctgcgggagc 60
gtcaggtctt gagagagctt ttgctaatgg gatggtttgg tgatggagta ctcctcctga 120
ccg atg gac tca aag aag aaa agc tca aca gag gca gaa gga tcc aaa 168
Met Asp Ser Lys Lys Lys Ser Ser Thr Glu Ala Glu Gly Ser Lys
1 5 10 15
gaa aga ggc cta gtc cat gtc tgg cag gca gga tcc ttt tct cta aca 216
Glu Arg Gly Leu Val His Val Trp Gln Ala Gly Ser Phe Ser Leu Thr
20 25 30
cca gag agg ttg cca ggc tgg ggt gga aag aca gtt ctt cag gca gcc 264
Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala
35 40 45
ctt ggt gtg agg cat gga gtt ctt ctg act gaa gat ggt gag gtc tac 312
Leu Gly Val Arg His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr
50 55 60
agc ttt ggg act ctt ccc tgg aaa agt gaa tca gca gaa att tgt cca 360
Ser Phe Gly Thr Leu Pro Trp Lys Ser Glu Ser Ala Glu Ile Cys Pro
65 70 75
agc agc ccc ctt cta gaa agt gcc ctg gtt ggg cat cat gtt att act 408
Ser Ser Pro Leu Leu Glu Ser Ala Leu Val Gly His His Val Ile Thr
80 85 90 95
12
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
gtg gca aca ggg agc ttc cac agt gga gca gtg aca gag agc ggg gtg 456
Val Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Glu Ser Gly Val
100 105 110
gtg tac atg tgg gga gag aat get gcc ggg cag tgt gcg gta get aac 504
Val Tyr Met Trp Gly Glu Asn Ala Ala Gly Gln Cys Ala Val Ala Asn
115 120 125
cag cag tat gtg ccg gag ccg agt cct gtc agc att tct gac tcg gag 552
Gln Gln Tyr Val Pro Glu Pro Ser Pro Val Ser Ile Ser Asp Ser Glu
130 135 140
acc agc ccg tca tta gca gtt agg att ctg caa ttg gca tgt ggc gag 600
Thr Ser Pro Ser Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu
145 150 155
gaa cac aca ctg gca ttg tca ctc agc aga gag atc tgg gca tgg ggc 648
Glu His Thr Leu Ala Leu Ser Leu Ser Arg Glu Ile Trp Ala Trp Gly
160 165 170 175
acc ggc tgt cag ctg ggc ctc atc acc acc act ttc cca gtg aca aag 696
Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr Thr Phe Pro Val Thr Lys
180 185 190
cca cag aag gtg gaa cac ctt get gga cga gtg gtg ctc cag gtg gcc 744
Pro Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala
195 200 205
tgc ggt gca ttc cac agc ctt gca ctt gtg cag tgc ctc cct cct cag 792
Cys Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Pro Gln
210 215 220
gat ctg aag cca gtc cca gag aga tgc aat cag tgc agc cag ctg ctc 840
Asp Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu
225 230 235
atc acc atg aca gac aaa gag gac cat gtg ata ata tcg gac agc cat 888
Ile Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His
240 245 250 255
tgc tgc cct tta ggt gtg aca ttg tcc gag tct caa gca gaa aag cat 936
Cys Cys Pro Leu Gly Val Thr Leu Ser Glu Ser Gln Ala Glu Lys His
260 265 270
gcc agc cct get ccc agc cct cac cca gag gca ctg gat gag cag gga 984
Ala Ser Pro Ala Pro Ser Pro His Pro Glu Ala Leu Asp Glu Gln Gly
275 280 285
gag gtg ttt gag aac acg gtg gta gaa get gaa ctg aac atg gga agc 1032
Glu Val Phe Glu Asn Thr Val Val Glu Ala Glu Leu Asn Met Gly Ser
290 295 300
agt cag acc aca agt ggc agt gcc att tcc acc cag cag aac atc gtg 1080
Ser Gln Thr Thr Ser Gly Ser Ala Ile Ser Thr Gln Gln Asn Ile Val
305 310 315
gga aca get gaa gtg tct tct gcc aga aca get ccg tca tac cca gac 1128
Gly Thr Ala Glu Val Ser Ser Ala Arg Thr Ala Pro Ser Tyr Pro Asp
320 325 330 335
acc cat gcg gta act gca tac ctg cag aag ctg tca gag cat tcg atg 1176
Thr His Ala Val Thr Ala Tyr Leu Gln Lys Leu Ser Glu His Ser Met
13
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
340 345 350
agg gag aac cat gag cct gga gaa aag cca ccc cag gtc cag cct ctt 1224
Arg Glu Asn His Glu Pro Gly Glu Lys Pro Pro Gln Val Gln Pro Leu
355 360 365
gta gaa gaa gca gtt cct gat ctt cac agt cca cca acc aca agc acc 1272
Val Glu Glu Ala Val Pro Asp Leu His Ser Pro Pro Thr Thr Ser Thr
370 375 380
tca gcc ctc aac agc ttg gtg gtc tcc tgt gca tct get gtt ggt gtc 1320
Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala Val Gly Val
385 390 395
aga gtg get gcc acc tat gaa get ggg gcc ttg tct ctc aag aaa gtt 1368
Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu Lys Lys Val
400 405 410 415
atg aac ttt tac agc act gcc ccc tgc gag acg gca get cag tcg ggc 1416
Met Asn Phe Tyr Ser Thr Ala Pro Cys Glu Thr Ala Ala Gln Ser Gly
420 425 430
agt gcc tcc aca ggc cca gaa agt ctg aaa gat ctc cga gaa gag cag 1464
Ser Ala Ser Thr Gly Pro Glu Ser Leu Lys Asp Leu Arg Glu Glu Gln
435 440 445
gtg aaa cag gag tca ctg caa ggg aag aaa agc tca agt ctc atg gac 1512
Val Lys Gln Glu Ser Leu Gln Gly Lys Lys Ser Ser Ser Leu Met Asp
450 455 460
atc aga gag gaa gag tcg gag gga ggg agt cga aga ctc tcc ctc cca 1560
Ile Arg Glu Glu Glu Ser Glu Gly Gly Ser Arg Arg Leu Ser Leu Pro
465 470 475
ggg ttg ttg tcg caa gtt tcc ccc agg ctc tta agg aag get gcg agg 1608
Gly Leu Leu Ser Gln Val Ser Pro Arg Leu Leu Arg Lys Ala Ala Arg
480 485 490 495
gtg aaa act cgg aca gtg gtt ctg act ccc aca tac agt gga gaa gca 1656
Val Lys Thr Arg Thr Val Val Leu Thr Pro Thr Tyr Ser Gly Glu Ala
500 505 510
gat gcc ctt ctg cct tcc ctg agg aca gag gtg tgg acc tgg ggg aaa 1704
Asp Ala Leu Leu Pro Ser Leu Arg Thr Glu Val Trp Thr Trp Gly Lys
515 520 525
ggc aag gaa ggg cag cta ggg cac ggc gac gtc ctg ccc agg ctt cag 1752
Gly Lys Glu Gly Gln Leu Gly His Gly Asp Val Leu Pro Arg Leu Gln
530 535 540
ccg ttg tgt gtc aag tgt ctg gat ggt aaa gag gta atc cac ctg gag 1800
Pro Leu Cys Val Lys Cys Leu Asp Gly Lys Glu Val Ile His Leu Glu
545 550 555
gcg ggc ggc tcc cac tcc ctc gca ctc act gcg aaa tct cag gtt tac 1848
Ala Gly Gly Ser His Ser Leu Ala Leu Thr Ala Lys Ser Gln Val Tyr
560 565 570 575
tca tgg ggc agt aat acc ttt ggt cag ctt ggg cat tct gag ttt cca 1896
Ser Trp Gly Ser Asn Thr Phe Gly Gln Leu Gly His Ser Glu Phe Pro
580 585 590
14
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
aca acg gtt cct cga ctc tca aag gtt agc agt gaa aat gga gtc tgg 1944
Thr Thr Val Pro Arg Leu Ser Lys Val Ser Ser Glu Asn Gly Val Trp
595 600 605
agt gta get gca ggc caa gat tat tcc ttg ttt tta gtg gac acg gaa 1992
Ser Val Ala Ala Gly Gln Asp Tyr Ser Leu Phe Leu Val Asp Thr Glu
610 615 620
gac ttc cag cct ggg ttg tat tac agt ggc cga cag gac cgt gca gaa 2040
Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg Gln Asp Arg Ala Glu
625 630 635
ggt gat acc ctg cca gag aat ccc agt ggt aca aag act cca gta ctt 2088
Gly Asp Thr Leu Pro Glu Asn Pro Ser Gly Thr Lys Thr Pro Val Leu
640 645 650 655
ctc tcc tgt agt aag ctt gga tac ata agc aga gta aca gca gga aaa 2136
Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg Val Thr Ala Gly Lys
660 665 670
gat agc tat cta gcc ttg gtg gat aag aac atc atg gga tac atc gcc 2184
Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile Met Gly Tyr Ile Ala
675 680 685
agt ctc cat gag ttg get tct aca gaa aga cgg ttt tac tca aaa ctg 2232
Ser Leu His Glu Leu Ala Ser Thr Glu Arg Arg Phe Tyr Ser Lys Leu
690 695 700
agc gaa atc aaa tca cag ata ctt agg cct ctt ctc agt tta gaa aat 2280
Ser Glu Ile Lys Ser Gln Ile Leu Arg Pro Leu Leu Ser Leu Glu Asn
705 710 715
ttg ggc aca gtg acc act gtc cag ctg ttg cag gaa gtt gcc agc cgg 2328
Leu Gly Thr Val Thr Thr Val Gln Leu Leu Gln Glu Val Ala Ser Arg
720 725 730 735
ttc agc aag ttg tgt tac ctc att ggg cag cat gga gcc tca cta agc 2376
Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln His Gly Ala Ser Leu Ser
740 745 750
agc tac cta cag ggt atg aag gaa gcc agc agc ctg gtc atc atg aag 2424
Ser Tyr Leu Gln Gly Met Lys Glu Ala Ser Ser Leu Val Ile Met Lys
755 760 765
cac tca agt ctt ttc ctg gac agc tac aca gag tac tgc aca tca gtt 2472
His Ser Ser Leu Phe Leu Asp Ser Tyr Thr Glu Tyr Cys Thr Ser Val
770 775 780
tca aat ttc ctg gtt atg gga gga ttc cag ctt ctt get aag cct gcc 2520
Ser Asn Phe Leu Val Met Gly Gly Phe Gln Leu Leu Ala Lys Pro Ala
785 790 795
att gat ttc cta aat aaa aac caa gaa ctc ttg caa gat ttg tca gaa 2568
Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu Leu Gln Asp Leu Ser Glu
800 805 810 815
gtg aat gat gag aac act cag ttg atg gaa atc ctg aac atg ctg ttt 2616
Val Asn Asp Glu Asn Thr Gln Leu Met Glu Ile Leu Asn Met Leu Phe
820 825 830
ttc ttg cca atc aga cga ctt cat aat tat gca aaa gtt ttg cta aag 2664
Phe Leu Pro Ile Arg Arg Leu His Asn Tyr Ala Lys Val Leu Leu Lys
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
835 840 845
ctt gcc act tgc ttt gaa gtg aca tct cca gag tat caa aag ctg cag 2712
Leu Ala Thr Cys Phe Glu Val Thr Ser Pro Glu Tyr Gln Lys Leu Gln
850 855 860
gat tcc agt tct tgc tat gag tct ctt get ctc cat ctt ggc aag aag 2760
Asp Ser Ser Ser Cys Tyr Glu Ser Leu Ala Leu His Leu Gly Lys Lys
865 870 875
agg aag gaa gca gag tac aca ctg agc ttc tgg aag acc ttt cct ggg 2808
Arg Lys Glu Ala Glu Tyr Thr Leu Ser Phe Trp Lys Thr Phe Pro Gly
880 885 890 895
aaa atg acg gat tcc ttg agg aag cca gag cgc cgg ctg ctg tgt gag 2856
Lys Met Thr Asp Ser Leu Arg Lys Pro Glu Arg Arg Leu Leu Cys Glu
900 905 910
agc agt aac cga gcc ctc tcc ctg cag cat gcc ggc agg ttt tct gtg 2904
Ser Ser Asn Arg Ala Leu Ser Leu Gln His Ala Gly Arg Phe Ser Val
915 920 925
aat tgg ttc att ctc ttc aat gat gcc ctg gtc cat get cag ttc tct 2952
Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu Val His Ala Gln Phe Ser
930 935 940
aca cac cac gtg ttc cct ttg gcc aca ctc tgg gca gag cca cta tct 3000
Thr His His Val Phe Pro Leu Ala Thr Leu Trp Ala Glu Pro Leu Ser
945 950 955
gaa gaa get ggt agc gtg aat ggc tta aag ata act aca cct gaa gaa 3048
Glu Glu Ala Gly Ser Val Asn Gly Leu Lys Ile Thr Thr Pro Glu Glu
960 965 970 975
caa ttc aca ctc att tct tca aca ccc cag gaa aag acc aag tgg ctt 3096
Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln Glu Lys Thr Lys Trp Leu
980 985 990
cgg get att agc caa get gtg gat cag get ttg agg ggg acg tcc gat 3144
Arg Ala Ile Ser Gln Ala Val Asp Gln Ala Leu Arg Gly Thr Ser Asp
995 1000 1005
ttc cca ctt tac gga ggc ggc agc agt gtt cag aga cag gaa cca ccc 3192
Phe Pro Leu Tyr Gly Gly Gly Ser Ser Val Gln Arg Gln Glu Pro Pro
1010 1015 1020
atc tca aga agt gcc aaa tac act ttc tac aag gat act cgc cta aag 3240
Ile Ser Arg Ser Ala Lys Tyr Thr Phe Tyr Lys Asp Thr Arg Leu Lys
1025 1030 1035
gat gcc act tac gat ggg cgc tgg ctt tca ggg aag cct cat ggc agg 3288
Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly Lys Pro His Gly Arg
1040 1045 1050 1055
ggt gtt ctg aag tgg cct gat gga aag atg tac tct ggc atg ttc agg 3336
Gly Val Leu Lys Trp Pro Asp Gly Lys Met Tyr Ser Gly Met Phe Arg
1060 1065 1070
aat ggc ttg gaa gat gga tat ggt gaa tac aga atc cct aac aag gcc 3384
Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg Ile Pro Asn Lys Ala
1075 1080 1085
16
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
ctg aac aaa gaa gac cat tat gta ggc cat tgg aaa gag ggg aaa atg 3432
Leu Asn Lys Glu Asp His Tyr Val Gly His Trp Lys Glu Gly Lys Met
1090 1095 1100
tgt ggg caa gga gtc tac agc tat gcc tct ggt gaa gtg ttt gaa ggc 3480
Cys Gly Gln Gly Val Tyr Ser Tyr Ala Ser Gly Glu Val Phe Glu Gly
1105 1110 1115
tgc ttt caa gat aac atg cgc cat ggg cat ggt ctg ctc cgg agt gga 3528
Cys Phe Gln Asp Asn Met Arg His Gly His Gly Leu Leu Arg Ser Gly
1120 1125 1130 1135
aaa ctg act tct tct tct cct agc atg ttc att ggc cag tgg gta atg 3576
Lys Leu Thr Ser Ser Ser Pro Ser Met Phe Ile Gly Gln Trp Val Met
1140 1145 1150
gat aag aaa gca gga tat ggc gtc ttt gat gat atc acc agg gga gaa 3624
Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp Asp Ile Thr Arg Gly Glu
1155 1160 1165
aag tac atg gga atg tgg cag gat gat gtg tgc caa ggg aat ggg gta 3672
Lys Tyr Met Gly Met Trp Gln Asp Asp Val Cys Gln Gly Asn Gly Val
1170 1175 1180
gta gtc acc cag ttt ggg tta tac tac gaa ggc aac ttc cac ctg aat 3720
Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly Asn Phe His Leu Asn
1185 1190 1195
aag atg atg gga aat ggg gtt ttg ctt tct gaa gat gat acc atc tat 3768
Lys Met Met Gly Asn Gly Val Leu Leu Ser Glu Asp Asp Thr Ile Tyr
1200 1205 1210 1215
gaa gga gaa ttt tcc gat gac tgg aca ctt agt gga aag gga acg ctg 3816
Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu Ser Gly Lys Gly Thr Leu
1220 1225 1230
act atg cca cat gga gat tat att gaa ggt tat ttt agt gga gaa tgg 3864
Thr Met Pro His Gly Asp Tyr Ile Glu Gly Tyr Phe Ser Gly Glu Trp
1235 1240 1245
gga tct ggg ata aaa atc act ggg acc tac ttc aaa cct agc ctg tat 3912
Gly Ser Gly Ile Lys Ile Thr Gly Thr Tyr Phe Lys Pro Ser Leu Tyr
1250 1255 1260
gag agc gat aag gac aag ccc aaa gcc ttc agg aag ctg ggg aac ctg 3960
Glu Ser Asp Lys Asp Lys Pro Lys Ala Phe Arg Lys Leu Gly Asn Leu
1265 1270 1275
gcc gtg gca gca gac gag aaa tgg aga gca gtg ttt gaa gaa tgc tgg 4008
Ala Val Ala Ala Asp Glu Lys Trp Arg Ala Val Phe Glu Glu Cys Trp
1280 1285 1290 1295
cac cag ctg ggc tgt gag agc cca ggc caa ggg gag gtt tgg aaa gca 4056
His Gln Leu Gly Cys Glu Ser Pro Gly Gln Gly Glu Val Trp Lys Ala
1300 1305 1310
tgg gat aat att get gtg gcc ttg acc acg aac cgt cgc cag cat aaa 4104
Trp Asp Asn Ile Ala Val Ala Leu Thr Thr Asn Arg Arg Gln His Lys
1315 1320 1325
gac agt cca gaa ata cta agc cgc tct cag act cag acc ctg gag agt 4152
Asp Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr Gln Thr Leu Glu Ser
17
SUBSTITUTE SHEET (RULE 26)
ct
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
1330 1335 1340
ttg gag tac att ccc cag cac att ggc gcc ttc tct gtg gag aaa tat 4200
Leu Glu Tyr Ile Pro Gln His Ile Gly Ala Phe Ser Val Glu Lys Tyr
1345 1350 1355
gat gac atc aag aag tat tta ata aag gcc tgt gat act cct ctg cac 4248
Asp Asp Ile Lys Lys Tyr Leu Ile Lys Ala Cys Asp Thr Pro Leu His
1360 1365 1370 1375
cca ctg ggc agg ctt gtg gag acc ctg gtt gcg gtg tat aga atg aca 4296
Pro Leu Gly Arg Leu Val Glu Thr Leu Val Ala Val Tyr Arg Met Thr
1380 1385 1390
tat gtg ggt gta ggg gcc aac cgc cgg tta ctg cag gaa get gtg aag 4344
Tyr Val Gly Val Gly Ala Asn Arg Arg Leu Leu Gln Glu Ala Val Lys
1395 1400 1405
gag att aaa tct tat ctc aag agg att ttc cag ctt gtg agg ttc ttg 4392
Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln Leu Val Arg Phe Leu
1410 1415 1420
ttt cct gag ctt cct gag gag ggc agc aca att cct ctt tct get cct 4440
Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile Pro Leu Ser Ala Pro
1425 1430 1435
ctg ccc act gga agg aga tcc ttc tgt act ggg aaa ttg gat tcc aga 4488
Leu Pro Thr Gly Arg Arg Ser Phe Cys Thr Gly Lys Leu Asp Ser Arg
1440 1445 1450 1455
tcc gag tca cca gaa cca ggt tat gta gta aca agt tct ggc tta ctg 4536
Ser Glu Ser Pro Glu Pro Gly Tyr Val Val Thr Ser Ser Gly Leu Leu
1460 1465 1470
ctt ccg gtg ctg ctg ccg cgg ctc tac cca cct ctc ttc atg ctc tat 4584
Leu Pro Val Leu Leu Pro Arg Leu Tyr Pro Pro Leu Phe Met Leu Tyr
1475 1480 1485
gcc ctg gat aat gac cga gag gaa gac att tac tgg gaa tgt gtg ctt 4632
Ala Leu Asp Asn Asp Arg Glu Glu Asp Ile Tyr Trp Glu Cys Val Leu
1490 1495 1500
cga cta aac aag cag cca gat att get ctc ttg ggc ttc ctt gga gta 4680
Arg Leu Asn Lys Gln Pro Asp Ile Ala Leu Leu Gly Phe Leu Gly Val
1505 1510 1515
cag aaa aaa ttc tgg cca gcc acc ttg tca atc ctt gga gag agt aaa 4728
Gln Lys Lys Phe Trp Pro Ala Thr Leu Ser Ile Leu Gly Glu Ser Lys
1520 1525 1530 1535
aag gtg ttg tca acc aca aag gat get tgc ttt gca tct gca gta gaa 4776
Lys Val Leu Ser Thr Thr Lys Asp Ala Cys Phe Ala Ser Ala Val Glu
1540 1545 1550
tgc ctg cag cag atc agc aca aca ttt act cca tca gac aag ctt aaa 4824
Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro Ser Asp Lys Leu Lys
1555 1560 1565
gtg atc cag cag acc ttt gaa gag atc tcc cag agt gtc ctt gca tcg 4872
Val Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln Ser Val Leu Ala Ser
1570 1575 1580
18
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
ctg cag gag gac ttc ctc tgg tcc atg gat gac ttg ttc ccc gtc ttc 4920
Leu Gln Glu Asp Phe Leu Trp Ser Met Asp Asp Leu Phe Pro Val Phe
1585 1590 1595
tta tac gtg gtg ctg cgg gcc agg att cgg aac ttg ggc tct gaa gtt 4968
Leu Tyr Val Val Leu Arg Ala Arg Ile Arg Asn Leu Gly Ser Glu Val
1600 1605 1610 1615
cac ctc att gag gat ctg atg gac ccc ttt ctc cag cat ggg gaa caa 5016
His Leu Ile Glu Asp Leu Met Asp Pro Phe Leu Gln His Gly Glu Gln
1620 1625 1630
ggc atc atg ttc acc acc ttg aag gcc tgt tac ttc cag att cag cgg 5064
Gly Ile Met Phe Thr Thr Leu Lys Ala Cys Tyr Phe Gln Ile Gln Arg
1635 1640 1645
gag aag ctt aac tag ggcgcctgac agcttgagga ccggattatc tgctgcggag 5119
Glu Lys Leu Asn
1650
gctacagcta tggcacaggc accgactgga ggctgatggg gcaaagaaca gtgttgaata 5179
cagaatggac ttttgtgcta ttttggttgt aatttctgag ccttactaat aattagagcc 5239
cagcatggaa aacatactgt atcattcaaa tggagactgg aaaaggagat agggatagag 5299
tagagtcttt ggcctgtgct gagatccaca cacctactta gaaaaggaaa ctggttaccc 5359
tttcctgtag tgaaagctct cagctccatg cagttccagg aaacctttcc aggaaagctg 5419
cttagatgaa aagaagttga tgactgtgtt taagctcctg gtttgtctaa ttccatttgc 5479
agttacccaa taccctttgg caaggagcag gttttacttg aaactgaagc agccatccct 5539
tgccttccta gacctctcgc tcccaggcac aagtgcagca tgctactttg ctaggggtgg 5599
gggtggggga gaagaagttt taaactgtag ttttaacctt ttgtaagccc ctttaccaag 5659
gcatttgtgg tcagagagct cccacggggt gactatgaca tcctggtccc ctcgtggaat 5719
gcatccacat aggatcttct gcctgctgac tgaaaagaac ataggaatac actggagtgc 5779
aaacactgcc gtgccaagct gctccaaacc tcactgatcc gaggcccact gcctacccag 5839
gaggcccact gcctacccag gaggcccgta agcttcttag cacaagcttt gtgtggagac 5899
tgaagatctg cacatgtgag gaagcaggga gctacagtgg ccctcagccc agtctgcggg 5959
tcttccctct acctcacact gaactcagaa gggaaggaag gagagacgca catgggattc 6019
tcccacctca gaagtattgt gacagcaccg cataaccacg gtttgctctt ttacaagcag 6079
cctcacaagt gtgggttgtg ggtgtgcgct ggagcagtgc cactcgtagc tgtttggata 6139
ccacaggtgc tcttccgtct catctgctgt actcggaggc gagcgcagtg gcctgactca 6199
tgggaaatga ctcagcaggc ggcaactact tttgaaaagg atcatgattt ccgagctact 6259
ttataatcaa gacaagcatt tgttaacata ctgttttgga atgttggctg taatgtaaca 6319
gcagttttca taataaatga cattcatctc 6349
<210> 5
<211> 1651
<212> PRT
<213> Mus musculus
<400> 5
Met Asp Ser Lys Lys Lys Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu
1 5 10 15
Arg Gly Leu Val His Val Trp Gln Ala Gly Ser Phe Ser Leu Thr Pro
20 25 30
Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala Leu
35 40 45
Gly Val Arg His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr Ser
50 55 60
Phe Gly Thr Leu Pro Trp Lys Ser Glu Ser Ala Glu Ile Cys Pro Ser
65 70 75 80
Ser Pro Leu Leu Glu Ser Ala Leu Val Gly His His Val Ile Thr Val
85 90 95
Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Glu Ser Gly Val Val
100 105 110
19
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Tyr Met Trp Gly Glu Asn Ala Ala Gly Gln Cys Ala Val Ala Asn Gln
115 120 125
Gln Tyr Val Pro Glu Pro Ser Pro Val Ser Ile Ser Asp Ser Glu Thr
130 135 140
Ser Pro Ser Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu Glu
145 150 155 160
His Thr Leu Ala Leu Ser Leu Ser Arg Glu Ile Trp Ala Trp Gly Thr
165 170 175
Gly Cys Gln Leu Gly Leu Ile Thr Thr Thr Phe Pro Val Thr Lys Pro
180 185 190
Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys
195 200 205
Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Pro Gln Asp
210 215 220
Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu Ile
225 230 235 240
Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His Cys
245 250 255
Cys Pro Leu Gly Val Thr Leu Ser Glu Ser Gln Ala Glu Lys His Ala
260 265 270
Ser Pro Ala Pro Ser Pro His Pro Glu Ala Leu Asp Glu Gln Gly Glu
275 280 285
Val Phe Glu Asn Thr Val Val Glu Ala Glu Leu Asn Met Gly Ser Ser
290 295 300
Gln Thr Thr Ser Gly Ser Ala Ile Ser Thr Gln Gln Asn Ile Val Gly
305 310 315 320
Thr Ala Glu Val Ser Ser Ala Arg Thr Ala Pro Ser Tyr Pro Asp Thr
325 330 335
His Ala Val Thr Ala Tyr Leu Gln Lys Leu Ser Glu His Ser Met Arg
340 345 350
Glu Asn His Glu Pro Gly Glu Lys Pro Pro Gln Val Gln Pro Leu Val
355 360 365
Glu Glu Ala Val Pro Asp Leu His Ser Pro Pro Thr Thr Ser Thr Ser
370 375 380
Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala Val Gly Val Arg
385 390 395 400
Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu Lys Lys Val Met
405 410 415
Asn Phe Tyr Ser Thr Ala Pro Cys Glu Thr Ala Ala Gln Ser Gly Ser
420 425 430
Ala Ser Thr Gly Pro Glu Ser Leu Lys Asp Leu Arg Glu Glu Gln Val
435 440 445
Lys Gln Glu Ser Leu Gln Gly Lys Lys Ser Ser Ser Leu Met Asp Ile
450 455 460
Arg Glu Glu Glu Ser Glu Gly Gly Ser Arg Arg Leu Ser Leu Pro Gly
465 470 475 480
Leu Leu Ser Gln Val Ser Pro Arg Leu Leu Arg Lys Ala Ala Arg Val
485 490 495
Lys Thr Arg Thr Val Val Leu Thr Pro Thr Tyr Ser Gly Glu Ala Asp
500 505 510
Ala Leu Leu Pro Ser Leu Arg Thr Glu Val Trp Thr Trp Gly Lys Gly
515 520 525
Lys Glu Gly Gln Leu Gly His Gly Asp Val Leu Pro Arg Leu Gln Pro
530 535 540
Leu Cys Val Lys Cys Leu Asp Gly Lys Glu Val Ile His Leu Glu Ala
545 550 555 560
Gly Gly Ser His Ser Leu Ala Leu Thr Ala Lys Ser Gln Val Tyr Ser
565 570 575
Trp Gly Ser Asn Thr Phe Gly Gln Leu Gly His Ser Glu Phe Pro Thr
580 585 590
Thr Val Pro Arg Leu Ser Lys Val Ser Ser Glu Asn Gly Val Trp Ser
595 600 605
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Val Ala Ala Gly Gln Asp Tyr Ser Leu Phe Leu Val Asp Thr Glu Asp
610 615 620
Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg Gln Asp Arg Ala Glu Gly
625 630 635 640
Asp Thr Leu Pro Glu Asn Pro Ser Gly Thr Lys Thr Pro Val Leu Leu
645 650 655
Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg Val Thr Ala Gly Lys Asp
660 665 670
Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile Met Gly Tyr Ile Ala Ser
675 680 685
Leu His Glu Leu Ala Ser Thr Glu Arg Arg Phe Tyr Ser Lys Leu Ser
690 695 700
Glu Ile Lys Ser Gln Ile Leu Arg Pro Leu Leu Ser Leu Glu Asn Leu
705 710 715 720
Gly Thr Val Thr Thr Val Gln Leu Leu Gln Glu Val Ala Ser Arg Phe
725 730 735
Ser Lys Leu Cys Tyr Leu Ile Gly Gln His Gly Ala Ser Leu Ser Ser
740 745 750
Tyr Leu Gln Gly Met Lys Glu Ala Ser Ser Leu Val Ile Met Lys His
755 760 765
Ser Ser Leu Phe Leu Asp Ser Tyr Thr Glu Tyr Cys Thr Ser Val Ser
770 775 780
Asn Phe Leu Val Met Gly Gly Phe Gln Leu Leu Ala Lys Pro Ala Ile
785 790 795 800
Asp Phe Leu Asn Lys Asn Gln Glu Leu Leu Gln Asp Leu Ser Glu Val
805 810 815
Asn Asp Glu Asn Thr Gln Leu Met Glu Ile Leu Asn Met Leu Phe Phe
820 825 830
Leu Pro Ile Arg Arg Leu His Asn Tyr Ala Lys Val Leu Leu Lys Leu
835 840 845
Ala Thr Cys Phe Glu Val Thr Ser Pro Glu Tyr Gln Lys Leu Gln Asp
850 855 860
Ser Ser Ser Cys Tyr Glu Ser Leu Ala Leu His Leu Gly Lys Lys Arg
865 870 875 880
Lys Glu Ala Glu Tyr Thr Leu Ser Phe Trp Lys Thr Phe Pro Gly Lys
885 890 895
Met Thr Asp Ser Leu Arg Lys Pro Glu Arg Arg Leu Leu Cys Glu Ser
900 905 910
Ser Asn Arg Ala Leu Ser Leu Gln His Ala Gly Arg Phe Ser Val Asn
915 920 925
Trp Phe Ile Leu Phe Asn Asp Ala Leu Val His Ala Gln Phe Ser Thr
930 935 940
His His Val Phe Pro Leu Ala Thr Leu Trp Ala Glu Pro Leu Ser Glu
945 950 955 960
Glu Ala Gly Ser Val Asn Gly Leu Lys Ile Thr Thr Pro Glu Glu Gln
965 970 975
Phe Thr Leu Ile Ser Ser Thr Pro Gln Glu Lys Thr Lys Trp Leu Arg
980 985 990
Ala Ile Ser Gln Ala Val Asp Gln Ala Leu Arg Gly Thr Ser Asp Phe
995 1000 1005
Pro Leu Tyr Gly Gly Gly Ser Ser Val Gln Arg Gln Glu Pro Pro Ile
1010 1015 1020
Ser Arg Ser Ala Lys Tyr Thr Phe Tyr Lys Asp Thr Arg Leu Lys Asp
1025 1030 1035 1040
Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly Lys Pro His Gly Arg Gly
1045 1050 1055
Val Leu Lys Trp Pro Asp Gly Lys Met Tyr Ser Gly Met Phe Arg Asn
1060 1065 1070
Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg Ile Pro Asn Lys Ala Leu
1075 1080 1085
Asn Lys Glu Asp His Tyr Val Gly His Trp Lys Glu Gly Lys Met Cys
1090 1095 1100
21
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Gly Gln Gly Val Tyr Ser Tyr Ala Ser Gly Glu Val Phe Glu Gly Cys
1105 1110 1115 1120
Phe Gln Asp Asn Met Arg His Gly His Gly Leu Leu Arg Ser Gly Lys
1125 1130 1135
Leu Thr Ser Ser Ser Pro Ser Met Phe Ile Gly Gln Trp Val Met Asp
1140 1145 1150
Lys Lys Ala Gly Tyr Gly Val Phe Asp Asp Ile Thr Arg Gly Glu Lys
1155 1160 1165
Tyr Met Gly Met Trp Gln Asp Asp Val Cys Gln Gly Asn Gly Val Val
1170 1175 1180
Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly Asn Phe His Leu Asn Lys
1185 1190 1195 1200
Met Met Gly Asn Gly Val Leu Leu Ser Glu Asp Asp Thr Ile Tyr Glu
1205 1210 1215
Gly Glu Phe Ser Asp Asp Trp Thr Leu Ser Gly Lys Gly Thr Leu Thr
1220 1225 1230
Met Pro His Gly Asp Tyr Ile Glu Gly Tyr Phe Ser Gly Glu Trp Gly
1235 1240 1245
Ser Gly Ile Lys Ile Thr Gly Thr Tyr Phe Lys Pro Ser Leu Tyr Glu
1250 1255 1260
Ser Asp Lys Asp Lys Pro Lys Ala Phe Arg Lys Leu Gly Asn Leu Ala
1265 1270 1275 1280
Val Ala Ala Asp Glu Lys Trp Arg Ala Val Phe Glu Glu Cys Trp His
1285 1290 1295
Gln Leu Gly Cys Glu Ser Pro Gly Gln Gly Glu Val Trp Lys Ala Trp
1300 1305 1310
Asp Asn Ile Ala Val Ala Leu Thr Thr Asn Arg Arg Gln His Lys Asp
1315 1320 1325
Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr Gln Thr Leu Glu Ser Leu
1330 1335 1340
Glu Tyr Ile Pro Gln His Ile Gly Ala Phe Ser Val Glu Lys Tyr Asp
1345 1350 1355 1360
Asp Ile Lys Lys Tyr Leu Ile Lys Ala Cys Asp Thr Pro Leu His Pro
1365 1370 1375
Leu Gly Arg Leu Val Glu Thr Leu Val Ala Val Tyr Arg Met Thr Tyr
1380 1385 1390
Val Gly Val Gly Ala Asn Arg Arg Leu Leu Gln Glu Ala Val Lys Glu
1395 1400 1405
Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln Leu Val Arg Phe Leu Phe
1410 1415 1420
Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile Pro Leu Ser Ala Pro Leu
1425 1430 1435 1440
Pro Thr Gly Arg Arg Ser Phe Cys Thr Gly Lys Leu Asp Ser Arg Ser
1445 1450 1455
Glu Ser Pro Glu Pro Gly Tyr Val Val Thr Ser Ser Gly Leu Leu Leu
1460 1465 1470
Pro Val Leu Leu Pro Arg Leu Tyr Pro Pro Leu Phe Met Leu Tyr Ala
1475 1480 1485
Leu Asp Asn Asp Arg Glu Glu Asp Ile Tyr Trp Glu Cys Val Leu Arg
1490 1495 1500
Leu Asn Lys Gln Pro Asp Ile Ala Leu Leu Gly Phe Leu Gly Val Gln
1505 1510 1515 1520
Lys Lys Phe Trp Pro Ala Thr Leu Ser Ile Leu Gly Glu Ser Lys Lys
1525 1530 1535
Val Leu Ser Thr Thr Lys Asp Ala Cys Phe Ala Ser Ala Val Glu Cys
1540 1545 1550
Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro Ser Asp Lys Leu Lys Val
1555 1560 1565
Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln Ser Val Leu Ala Ser Leu
1570 1575 1580
Gln Glu Asp Phe Leu Trp Ser Met Asp Asp Leu Phe Pro Val Phe Leu
1585 1590 1595 1600
22
SUBSTETUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO PCT/CA02/00147
02/072822
TyrValVal Leu Ala Arg Ile Arg Leu Gly Ser ValHis
Arg Asn Glu
1605 1610 1615
LeuIleGlu Asp Met Asp Pro Phe Gln His Gly GlnGly
Leu Leu Glu
1620 1625 1630
IleMetPhe Thr Leu Lys Ala Cys Phe Gln Ile ArgGlu
Thr Tyr Gln
1635 1640 1645
LysLeuAsn
1650
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence: Synthesized Oligonucleotide
<400> 6
cctagtcatc catgtgctgg 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence: Synthesized Oligonucleotide
<400> 7
tcccatacct gaccttccac 20
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence: Synthesized Oligonucleotide
<400> 8
cttgatagac tttctgtaaa gaag 24
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence: Synthesized Oligonucleotide
<400> 9
ggctacttgg acaaatctcc actg 24
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
23
Sk)BSTfTUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<400> 10
ggagagactg tgctcccaag 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 11
agccctcctt agccaatagc 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 12
taagcttagt gggcaggctc 20
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 13
ttcccactta acaaccatca ac 22
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 14
ccaatttggt taaatctata gggg 24
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 15
gacaatgcca gagtgtgctc 20
24
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 16
ccagcccttt gttagcagtc 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 17
cttcttcctg cctgtcaagg 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 18
ttgtacaatg cctcccttcc 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 19
agcccaacat gacacctttc 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 20
gattgcttgt tgcataaggg 20
<210> 21
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 21
atacagcatg cgatgtcagg 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 22
ctggactccc actccttcac 20
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 23
gctagaagag cccagatttc c 21
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 24
tgactttgtg tgcctgtgtg 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 25
ataccctgga aaatctgggg 20
<210> 26
<211> 20
<212> DNA
26
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 26
tttgcgcatt atctctggtc 20
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 27
gtacgtatga aattcccccg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 28
ttccgtctta ctcctgcacc 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 29
gccttaggat ccaattcctg 20
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 30
caatgatgta ctgatgaacc agc 23
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
27
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 31
cctgatggtt taatggtggg 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 32
gcacatggca acaggttaag 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 33
tccttggcag aataaccctg 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 34
cccctaccac tccctttacc 20
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 35
ccagtggcta atagtacctg tcc 23
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
28
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
oligonucleotide
<400> 36
tggatgcatg attcatttcc 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 37
tccttggctt tccaaatgtc 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 38
ctatcctggg gtctctgctg 20
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 39
tgctatcgaa atggttgctg 20
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 40
agctacgacc agcaaattcc 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
29
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<400> 41
~
ataggggtcc acctttcagg 20
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 42
aaggggatat gggcagagtc 20
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 43
aaatgcttgc ttggttttgg 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 44
aaagggcatc ttcattgcac 20
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 45
cacaagaggc agaaagagcc 20
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 46
aatgcttgat gaattgttgc c 21
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 47
atgatcatcc tcaccccagg 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 48
ttgaagattt atgcctgggg 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 49
tgaggtcaca cggctatcag 20
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 50
gtgtagtggg gctgatgtcc 20
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 51
tggctatgca aacattcagg 20
<210> 52
31
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 52
aatgcaaaat accacacatg g 21
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 53
tcattggctt aaactgtggg 20
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 54
caacctaggg ttgatgcctg 20
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 55
catcttcgga aagcaaaacc . 20
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 56
ctttggggat atgactgcgt 20
<210> 57
<211> 26
<212> DNA
32
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 57
gtaaaagaat ttattaggga gaaaaa 26
<210> 58
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 58
ttcctctaac cccacatttt attc 24
<210> 59
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 59
tgcttttaaa atattaacca gctttg 26
<210> 60
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 60
tcagtcttgg cagttttggt c 21
<210> 61
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 61
ctgctgtatg ttgagcaggt g 21
<210> 62
<211> 20
<212> DNA
<213> Artificial Sequence
33
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 62
tggatgctcc actttgactg 20
<210> 63
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 63
ttaagaaccc ccttgagtgc 20
<210> 64
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 64
ttcctggtcc caaaattgac 20
<210> 65
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 65
cagggtgaaa ctacccaagc 20
<210> 66
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 66
ttttatgctt ttcaaccccc 20
<210> 67
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
34
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
oligonucleotide
<400> 67
acacactttc tcgctgggac 20
<210> 68
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 68
tgatctgagc acaaaggctg 20
<210> 69
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 69
taaacagcgg tgggtagagc 20
<210> 70
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 70
aatgctcctt ttctcccact c 21
<210> 71
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 71
tgccaaattt ccaataatgc 20
<210> 72
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<400> 72
taatggggac aaggaagcc 19
<210> 73
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 73
gctgaggcaa aacaagcatc 20
<210> 74
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 74
ccaaagacct gcactctgac 20
<210> 75
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 75
ctggcttggc tctctcctac 20
<210> 76
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 76
aaaaagcacg atcaaatggc 20
<210> 77
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthesized
oligonucleotide
<400> 77
ggaagagcgt actcctgctg 20
36
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<210> 78
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 78
gcaggagtac gctcttccac 20
<210> 79
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 79
gaacaaaatg tgctctaaag gc 22
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 80
tctttttctc tctggggcag 20
<210> 81
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 81
tgccttctgt gttttaccct g 21
<210> 82
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 82
gaagggaaca gggaaaagtg 20
<210> 83
37
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthesized
oligonucleotide
<400> 83
ttacctccct ttcaatcctc c 21
<210> 84
<211> 545
<212> PRT
<213> Homo sapiens
<400> 84
Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu
1 5 10 15
Arg Gly Leu Val His Ile Trp Gln Ala Gly Ser Phe Pro Ile Thr Pro
20 25 30
Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala Leu
35 40 45
Gly Val Lys His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr Ser
50 55 60
Phe Gly Thr Leu Pro Trp Arg Ser Gly Pro Val Glu Ile Cys Pro Ser
65 70 75 80
Ser Pro Ile Leu Glu Asn Ala Leu Val Gly Gln Tyr Val Ile Thr Val
85 90 95
Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Asp Asn Gly Val Ala
100 105 110
Tyr Met Trp Gly Glu Asn Ser Ala Gly Gln Cys Ala Val Ala Asn Gln
115 120 125
Gln Tyr Val Pro Glu Pro Asn Pro Val Ser Ile Ala Asp Ser Glu Ala
130 135 140
Ser Pro Leu Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu Glu
145 . 150 155 160
His Thr Leu Ala Leu Ser Ile Ser Arg Glu Ile Trp Ala Trp Gly Thr
165 170 175
Gly Cys Gln Leu Gly Leu Ile Thr Thr Ala Phe Pro Val Thr Lys Pro
180 185 190
Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys
195 200 205
Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Ser Gln Asp
210 215 220
Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu Ile
225 230 235 240
Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His Cys
245 250 255
Cys Pro Leu Gly Val Thr Leu Thr Glu Ser Gln Ala Glu Asn His Ala
260 265 270
Ser Thr Ala Leu Ser Pro Ser Thr Glu Thr Leu Asp Arg Gln Glu Glu
275 280 285
Val Phe Glu Asn Thr Leu Val Ala Asn Asp Gln Ser Val Ala Thr Glu
290 295 300
Leu Asn Ala Val Ser Ala Gln Ile Thr Ser Ser Asp Ala Met Ser Ser
305 310 315 320
Gln Gln Asn Val Met Gly Thr Thr Glu Ile Ser Ser Ala Arg Asn Ile
325 330 335
Pro Ser Tyr Pro Asp Thr Gln Ala Val Asn Glu Tyr Leu Arg Lys Leu
340 345 350
38
SUBSTITUTE SHEET (RULE 26)
CA 02437960 2003-08-11
WO 02/072822 PCT/CA02/00147
Ser Asp His Ser Val Arg Glu Asp Ser Glu His Gly Glu Lys Pro Met
355 360 365
Pro Ser Gln Pro Leu Leu Glu Glu Ala Ile Pro Asn Leu His Ser Pro
370 375 380
Pro Thr Thr Ser Thr Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala
385 390 395 400
Ser Ala Val Gly Val Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu
405 410 415
Ser Leu Lys Lys Val Met Asn Phe Tyr Ser Thr Thr Pro Cys Glu Thr
420 425 430
Gly Ala Gln Ala Gly Ser Ser Ala Ile Gly Pro Glu Gly Leu Lys Asp
435 440 445
Ser Arg Glu Glu Gln Val Lys Gln Glu Ser Met Gln Gly Lys Lys Ser
450 455 460
Ser Ser Leu Val Asp Ile Arg Glu Glu Glu Thr Gly Arg Gln Ser Lys
465 470 475 480
Thr Leu Pro Pro Trp Ile Val Val Thr Ser Phe Pro Gln Ala Leu Lys
485 490 495
Lys Gly Cys Thr Gly Glu Asn Glu Asp Ser Gly Ser Asp Pro His Ile
500 505 510
Gln Trp Arg Ser Arg Cys Ala Pro Ala Phe Ser Glu Asn Arg Ser Val
515 520 525
Asp Leu Gly Glu Arg Glu Gly Arg Ala Ala Gly Ala Arg Arg Cys Ser
530 535 540
Ala
545
39
SUBSTITUTE SHEET (RULE 26)
