Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Galectin 8, 9, IO and IOSV
Background of the Invention
Field of the Invention
The present invention relates to novel galectins. More specifically,
isolated nucleic acid molecules are provided encoding human galectin 8, 9, 10,
or IOSV. Galectin 8, 9, 10 and IOSV polypeptides are also provided, as are
vectors, host cells and recombinant methods for producing the same. The
invention further relates to screening methods for identifying agonists and
antagonists of galectin 8, 9, 10, or l OSV activity. Also provided are
diagnostic
methods for detecting cell growth disorders and therapeutic methods for cell
growth disorders, including autoimmune diseases, cancer, and inflammatory
diseases.
Related Art
Lectins are proteins that bind to specific carbohydrate structures and can
thus recognize particular glycoconjugates. Barondes et al.) J. Biol. Chem.
Z69(33):20807-20810 (1994). Galectins are members of a family of
(3-galactoside-binding lectins with related amino acid sequences (For review
see,
Barondes et al.) Cell 76.597-598 (1994); Barondes et al., J. Biol. Chem.
269(33):20807-20810 (August 1994)). Galectin 1 (aka. L-14-1, L-14, RL-14.5,
gaIaptin, MGBP, GBP, BHL, CHA, HBP, HPL, HLBP 14, rIML-1 ) is a
homodimer with a subunit molecular mass of 14,S00 which is abundant in
smooth and skeletal muscle, and is present in many other cell types (Couraud
et
al., J. Biol. Chem. 264:1310-1316 ( 1989)). Galectin 2 was originally found in
hepatoma and is a homodimer with a subunit molecular weight of l4,650 (Gift
et al., J. Biol. Chem. 267:10601-10606 ( 1992)). Galectin 3 (aka. Mac-2, EPB,
CBP-35, CBP-30, and L-29) is abundant in activated macrophages and epithelial
cells and is a monomer with an apparent molecular mass between 26,320 and
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30,300 (Cherayil et al.) Proc. Natl. Acad. Sci. USA 87: 7324-7326 (l990)).
Galectin 4 has a molecular mass of 36,300 and contains two carbohydrate-
binding
domains within a single polypeptide chain (Oda et al., J. Biol. Chem. 268:
5929-
5939 (l993)). Galectins 5 and 6 are mentioned in Barondes et al., Cell 76: 597-
598 (I994). Human galectin 7 has a molecular mass of 15,073 and is found
mainly in stratified squamous epithelium (Madsen et al., J. Biol. Chem.
270(1l):5823-5829 (199S)).
Animal lectins, in general, often function in modulating cell-cell and cell-
matrix interactions. Galectin 1 has been shown to either promote or inhibit
cell
adhesion depending upon the cell type in which it is present. Galectin I
inhibits
cell-matrix interactions in skeletal muscle {Cooper et al., J. Cell Biol. Il
S:1437
1448 ( I 991 )). In other cell types, galectin 1 promotes cell-matrix adhesion
possibly by cross-linking cell surface and substrate glycoconjugates {Zhou et
al.,
Arch. Biochem. Biophys. 300:6-17 (1993); Skrincosky et al., Cancer Res.
S3:2667-2675 (1993)).
Galectin 1 also participates in regulating cell proliferation (Wells et al.,
Cell 64:91-97 (1991)) and some immune functions (Offner et al., J.
Neuroimmunol. 28:177-184 (I990)). Galectin I has been shown to regulate the
immune response by mediating apoptosis of T cells (Perillo et al., Nature 378:
736-739 (1995)).
Galectin 3 promotes the growth of cells cultured under restrictive culture
conditions (Yang et al., Proc. Natl. Acad. Sci. USA 93: 6737-6742 (June
1996)).
Galectin 3 expression in cells confers resistance to apoptosis which indicates
that
Galectin 3 could be a cell death suppressor which interferes in a common
pathway of apoptosis. Id.
Accordingly, there is a need in the art for the identification of novel
galectins which can serve as useful tools in the development of therapeutics
and
diagnostics for regulating immune response.
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Summary of the Invention
The present invention provides isolated nucleic acid molecules comprising
a polynucleotide encoding the galectin 8, 9, 10, or I OSV polypeptide having
the
amino acid sequence is shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B,
respectively {SEQ ID NOs:2, 4, 6, and 8, respectively) or the amino acid
sequence
encoded by the cDNA clones deposited in bacterial hosts as ATCC Deposit
Numbers 97732, 97733 and 97734 on September 24, l996.
The present invention also relates to recombinant vectors, which include
the isolated nucleic acid molecules of the present invention, and to host
cells
containing the recombinant vectors, as well as to methods of making such
vectors
and host cells and for using them for production of galectin 8, 9, 10, or IOSV
polypeptides or peptides by recombinant techniques.
The invention further provides an isolated galectin 8, 9, 10, or IOSV
polypeptide having an amino acid sequence encoded by a polynucleotide
described herein.
The present invention also provides a screening method for identifying
compounds capable of enhancing or inhibiting a cellular response induced by
galectin 8, 9, 10, or l OSV, which involves contacting cells which express
galectin
8, 9, 10, or l OSV with the candidate compound, assaying a cellular response,
and
comparing the cellular response to a standard cellular response, the standard
being
assayed when contact is made in absence of the candidate compound; whereby,
an increased cellular response over the standard indicates that the compound
is
an agonist and a decreased cellular response over the standard indicates that
the
compound is an antagonist.
In another aspect, a screening assay for agonists and antagonists is
provided which involves determining the effect a candidate compound has on
galectin 8, 9, 10, or l OSV binding to the (3-galactosidase sugar. In
particular, the
method involves contacting the ~3-galactosidase sugar with a galectin 8, 9,
10, or
l OSV polypeptide and a candidate compound and determining whether galectin
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8, 9, 10, or lOSV binding to ~3-galactosidase sugar is increased or decreased
due
to the presence of the candidate compound.
The invention provides a diagnostic method useful during diagnosis
disorder.
An additional aspect of the invention is related to a method for treating an
individual in need of an increased level of galectin 8, 9, 10, or l OSV
activity in
the body comprising administering to such an individual a composition
comprising a therapeutically effective amount of an isolated galectin 8, 9,
10, or
lOSV polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an
individual in need of a decreased level of galectin 8, 9, 10, or 1 OSV
activity in the
body comprising, administering to such an individual a composition comprising
a therapeutically effective amount of a galectin 8, 9, 10, or 1 OSV
antagonist.
Brief Description of the Figures
FIG. 1 shows the nucleotide (SEQ ID NO:1 ) and deduced amino acid
(SEQ ID N0:2) sequences of galectin 8. The protein has a deduced molecular
weight of about 36 kDa.
FIG. 2A-2B shows the nucleotide (SEQ ID N0:3) and deduced amino
acid (SEQ ID N0:4) sequences of galectin 9. The protein has a deduced
molecular weight of about 34.7 kDa.
FIG. 3A-3B shows the nucleotide (SEQ ID NO:S) and deduced amino
acid (SEQ ID N0:6) sequences of full length galectin 10. The protein has a
deduced molecular weight of about 35.7 kDa.
FIG. 4A-4B shows the nucleotide (SEQ ID N0:7) and deduced amino
acid (SEQ ID N0:8) sequences of a galectin 10 splice variant (galectin 10SV).
The protein has a deduced molecular weight of about 22.4 kDa.
FIG. SA-SB shows the regions of similarity between the amino acid
sequences of the galectin 8, 9, and 10 proteins and human galectin 2 (SEQ ID
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N0:9), human galectin 3 (SEQ ID NO:10), rat galectin 4 (SEQ ID NO:11 ), rat
galectin 5 (SEQ ID N0:12), human galectin 7 (SEQ ID N0:13), rat galectin 3
(SEQ ID N0:14), rat galectin 8 (SEQ ID NO:15), and human galectin 1 (SEQ ID
N0:16).
FIG. 6 shows the regions of similarity between the amino acid sequences
of the galectin l OSV protein and the rat RL30 protein (SEQ ID N0:17).
FIG. 7 shows a homology comparison between the galectin 10 and
galectin IOSV proteins.
FIGS. 8, 9, 10, and 1 I show an analysis of the galectin 8, 9, 10, and l OSV
amino acid sequence, respectively. Alpha, beta, turn and coil regions;
hydrophilicity and hydrophobicity; amphipathic regions; flexible regions;
antigenic index and surface probability are shown. In the "Antigenic Index -
Jameson-Wolf' graph, amino acid residues 55-101, l37-162, 180-193, 216-266
in FIG. 1 (SEQ ID N0:2), 62-102, 226-259, I97-308 in FIG. 2A-2B (SEQ ID
N0:4), 25-77, 84-105, 129-140, 156-183, l95-215, and 241-257 in FIG. 3A-3B
(SEQ ID N0:6), and 25-77, 84-105, 129-140, and 156-183 in FIG. 4A-4B (SEQ
ID N0:8) correspond to the shown highly antigenic regions of the galectin 8,
9,
10, or IOSV protein, respectively.
Detailed Description
The present invention provides isolated nucleic acid molecules comprising
a polynucleotide encoding a galectin 8, 9, 10, or l OSV polypeptide having the
amino acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B, respectively
(SEQ ID NOs:2, 4, 6, and 8, respectively), which was determined by sequencing
a cloned cDNA. The galectin 8, 9,10, and 1 OSV proteins of the present
invention
share sequence homology with other galectins and the rat RL30 protein (FIGS.
SA-SB and 6) (SEQ ID NOs:9-17). The nucleotide sequences shown in FIGs. l,
2A-2B, and 4A-4B (SEQ ID NO:1, 3, and 7, respectively) were obtained by
sequencing the HSIAL77, HTPBR22, and HETAS87 clones, which were
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deposited on September 24, 1996 at the American Type Culture Collection,
l2301 Park Lawn Drive, Rockville, Maryland 20852, and given accession
numbers 97732, 97733 and 97734, respectively. The deposited clones are
contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).
The nucleotide sequence shown in FIG. 3A-3B (SEQ ID NO:S), which
encodes the full-length galectin 10 protein, was obtained by sequencing a
clone
cDNA obtained from a human endometrial tumor library.
~Yucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by
sequencing a DNA molecule herein were determined using an automated DNA
sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino
acid sequences of polypeptides encoded by DNA molecules determined herein
were predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined by this
automated approach, any nucleotide sequence determined herein may contain
some errors. Nucleotide sequences determined by automation are typically at
least about 90% identical, more typically at least about 95% to at least about
99.9% identical to the actual nucleotide sequence of the sequenced DNA
molecule. The actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in the art.
As is also known in the art, a single insertion or deletion in a determined
nucleotide sequence compared to the actual sequence will cause a frame shift
in
translation of the nucleotide sequence such that the predicted amino acid
sequence encoded by a determined nucleotide sequence will be completely
different from the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or deletion.
Using the information provided herein, such as the nucleotide sequences
in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B a nucleic acid molecule of the present
,.
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invention encoding a galectin 8, 9, 10, or l OSV, respectively, polypeptide
may be
obtained using standard cloning and screening procedures, such as those for
cloning cDNAs using mRNA as starting material. Illustrative of the invention,
the nucleic acid molecules described in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B (SEQ
ID NO:1, 3, 5, and 7, respectively) were discovered in cDNA libraries derived
from human adult small intestine, human pancreatic tumor, human endometrial
tumor and human endometrial tumor, respectively. These genes were also
identified in cDNA libraries from the following tissues pancreas, colon, small
intestine, brain, bone marrow, kidney, lung, spleen, and testes tissue.
Galectin 8
(SEQ ID NO:1 ) appears to be mainly expressed in cells of the human colon and
small intestine.
The determined nucleotide sequences of the galectin 8, 9, 10, and l OSV
cDNAs of FIGs. l, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:I, 3,
5, and 7) contain open reading frames encoding proteins of 323, 311, 317, and
200 amino acid residues, with an initiation codon at positions 52-54, 16-18,
118-
120, and 118-120 ofthe nucleotide sequences in FIGs. 1, 2A-2B, 3A-3B, and 4A-
4B, respectively (SEQ ID NOs: l , 3, 5, and 7), and a deduced molecular weight
of about 36, 34.7, 35.7, and 22.4 kDa, respectively. The galectin 8, 9, 10 and
IOSV proteins shown in FIGS. 1, 2A-2B, 3A-3B, and 4A-4B respectively (SEQ
ID NOs:2, 4, 6, and 8) share homology with other galectins {See, e.g., FIG. 5A-
5B).
As one of ordinary skill would appreciate, due to the possibilities of
sequencing errors discussed above, as well as the variability of processing
sites
for different known proteins, the predicted galectin 8 and 9 polypeptides
encoded
by the deposited cDNAs comprise about 323 and 311 amino acids, but may be
anywhere in the range of 300 - 333 amino acids. Similarly, the predicted
galectin
10 polypeptide comprises about 317 amino acids, but may be anywhere in the
range of 305 - 329 amino acids. Further, the predicted galectin IOSV
polypeptide
encoded by the deposited eDNA comprises about 200 amino acids, but may be
anywhere in the range of l90 - 210 amino acids.
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Galectin lOSV is believed to be a splice variant of galectin 10. As used
herein the phrase "splice variant" refers to cDNA molecules produced from RNA
molecules initially transcribed from the same genomic DNA sequence but which
have undergone alternative RNA splicing. Alternative RNA splicing occurs when
S a primary RNA transcript undergoes splicing, generally for the removal of
introns, which results in the production of more than one mRNA molecule each
of which may encode different amino acid sequences. The term "splice variant"
also refers to the proteins encoded by the above cDNA molecules.
As indicated, nucleic acid molecules of the present invention may be in
the form of RNA, such as mRNA, or in the form of DNA, including, for instance,
cDNA and genomic DNA obtained by cloning or produced synthetically. The
DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA
may be the coding strand, also known as the sense strand, or it may be the
non-coding strand, also referred to as the anti-sense strand.
IS By "isolated" nucleic acid molecules} is intended a nucleic acid
molecule, DNA or RNA, which has been removed from its native environment
For example, recombinant DNA molecules contained in a vector are considered
isolated for the purposes of the present invention. Further examples of
isolated
DNA molecules include recombinant DNA molecules maintained in heterologous
host cells or purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA
molecules of the present invention. Isolated nucleic acid molecules according
to
the present invention further include such molecules produced synthetically.
Isolated nucleic acid molecules of the present invention include DNA
molecules comprising an open reading frame (ORF) shown in FIGs. 1, 2A-2B,
3A-3B, and 4A-4B, respectively (SEQ ID NOs:I, 3, 5, and 7); and DNA
molecules which comprise a sequence substantially different from those
described
above but which, due to the degeneracy of the genetic code, still encode the
galectin 8, 9, 10, or IOSV protein. Of course, the genetic code is well known
in
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the art. Thus, it would be routine for one skilled in the art to generate such
degenerate variants.
In addition, the invention provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID NO:1 which have
been determined from the following related cDNA clones: HSIAL77R (SEQ ID
N0:18), HGBDKSSR (SEQ ID N0:19), HCNAH29R (SEQ ID N0:20),
HKCAA85R (SEQ ID N0:21), HCNAISSR (SEQ ID N0:22), HCNAI87R (SEQ
ID N0:23), HCNAS74R (SEQ ID N0:24) and HCNAF43R (SEQ ID N0:25).
In addition, the invention provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID N0:3 which have
been determined from the following related cDNA clones: HMSCP 11 R (SEQ ID
N0:26), HMSEU32R (SEQ ID N0:27), HTPA071R (SEQ ID N0:28),
HJAAV54R (SEQ ID N0:29), HMSEU43R (SEQ ID N0:30), HILBP03R (SEQ
ID N0:3I), HTPCG81R (SEQ ID N0:32), HTBAA21R (SEQ ID N0:33), and
HFXBU26R (SEQ ID N0:34).
In addition, the invention provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID NO:S which have
been determined from the following related cDNA clones: HTNBX92R (SEQ ID
N0:35), HLTAZ64RB (SEQ ID N0:36), HJBAI38R (SEQ ID N0:37),
HETAS87R (SEQ ID N0:38), and HETAR45R (SEQ ID N0:39).
In addition, the invention provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID N0:7 which have
been determined from the following related eDNA clones: HTNBX92R (SEQ ID
N0:35), HLTAZ64RB (SEQ ID N0:36), HBNAF37R (SEQ ID N0:40), and
HETAS87R (SEQ ID N0:38).
In another aspect, the invention provides isolated nucleic acid molecules
encoding the galectin 8, 9, 10 or 10SV polypeptide having an amino acid
sequence encoded by the cDNA clone contained in the plasmid deposited as
ATCC Deposit Nos. 97732, 97733 and 97734, respectively, on September 24,
1996. In a further embodiment, nucleic acid molecules are provided encoding
the
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full-length galectin 8, 9, 10, or IOSV polypeptide lacking the N-terminal
methionine. The invention further provides an isolated nucleic acid molecule
having the nucleotide sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ
ID NOs:l, 3, S, or 7) or the nucleotide sequence of the galectin 8, 9, or IOSV
cDNA contained in the above-described deposited clones, or a nucleic acid
molecule having a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, are useful as probes for gene
mapping, by in situ hybridization with chromosomes, and for detecting
expression of the galectin 8, 9, 10, or lOSV gene in human tissue, for
instance,
by Northern blot analysis.
The present invention is further directed to fragments of the isolated
nucleic acid molecules described herein. By a fragment of an isolated nucleic
acid molecule having the nucleotide sequence of the deposited cDNA or the
nucleotide sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NO:1,
3, 5, or 7) is intended fragments at least about 15 nt, and more preferably at
least
about 20 nt, still more preferably at least about 30 nt, and even more
preferably,
at least about 40 nt in length which are useful as diagnostic probes and
primers
as discussed herein. Of course larger DNA fragments 50, I00, 1 S0, 200, 250,
300, 3S0, 400, 4S0, 500, 5S0, 600, 650, 700, 750, 800, 850, 900, I00, 1050, 1
l00,
or 1115 nt in length of the sequence shown in SEQ ID NO:1 are also useful
according to the present invention as are fragments corresponding to most, if
not
all, of the nucleotide sequence of the cDNA clone contained in the plasmid
deposited as ATCC Deposit No. 97732 or as shown in SEQ ID NO:I. Similarly,
larger DNA fragments 50, l00, 150, 200, 250, 300, 3S0, 400, 4S0, S00, 550,
600,
6S0, 700, 7S0, 800, 850, 900, 100, 1050, 1100, I150, 1200, I250, 1300, 1350,
1400, 1450, 1500, or 1525 nt in length of the sequence shown in SEQ ID N0:3
are also useful according to the present invention as are fragments
corresponding
to most, if not all, of the nucleotide sequence of the cDNA clone contained in
the
plasmid deposited as ATCC Deposit No. 97733 or as shown in SEQ ID N0:3.
Similarly, larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450,
500,
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550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250,
1300,
1350, 1400, 1450, or 1464 nt in length of the sequence shown in SEQ ID N0:5
are also useful according to the present invention as are fragments
corresponding
to most, if not all, of the nucleotide sequence of the cDNA molecule as shown
in
SEQ ID N0:5. Further, larger DNA fragments 50, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, l600, 1650, l700, 1750, 1800,
I 8S0, 1900, and 1908 nt in length of the sequence shown in SEQ ID N0:7 are
also useful according to the present invention as are fragments corresponding
to
most, if not all, of the nucleotide sequence of the cDNA clone contained in
the
plasmid deposited as ATCC Deposit No. 97734 or as shown in SEQ ID N0:7.
By a fragment at least 20 nt in length, for example, is intended fragments
which
include 20 or more contiguous bases from the nucleotide sequence of the
deposited cDNA or the nucleotide sequence as shown in SEQ ID NOs:l, 3, 5, or
7.
Preferred nucleic acid fragments of the present invention include nucleic
acid molecules encoding epitope-bearing portions of the galectin 8, 9, 10, or
l OSV protein. In particular, such nucleic acid fragments of the present
invention
include nucleic acid molecules encoding: a polypeptide comprising amino acid
residues from about 55-l01, 137-162, l80-193, 2l6-266 in FIG. 1 (SEQ ID
N0:2), 62-102, 226-259, 197-308 in FIG. 2A-2B (SEQ ID N0:4), 25-77, 84-105,
129-l40, 156-183, 195-215, and 24l-257 in FIG. 3A-3B (SEQ ID N0:6), and 25-
77, 84-105, 129-140, and 156-183 in FIG. 4A-4B (SEQ ID N0:8). The inventors
have determined that the above polypeptide fragments are antigenic regions of
the
galectin 8, 9, 10, and lOSV proteins. Methods for determining other such
epitope-bearing portions of the galectin 8, 9, I0, and 1 OSV proteins are
described
in detail below.
In another aspect, the invention provides an isolated nucleic acid molecule
comprising a polynucleotide which hybridizes under stringent hybridization
conditions to a portion of the polynucleotide in a nucleic acid molecule of
the
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invention described above, for instance, a cDNA clone contained in ATCC
Deposit Nos. 97732, 97733 and 97734. By "stringent hybridization conditions"
is intended overnight incubation at 42~C in a solution comprising: 50%
formamide, 5x SSC {150 mM NaCI, l5mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml
denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x
SSC at about 65~C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide
is intended a polynucleotide (either DNA or RNA) hybridizing to at least about
15 nucleotides (nt), and more preferably at least about 20 nt, still more
preferably
at least about 30 nt, and even more preferably about 30-70 nt of the reference
polynucleotide. These are useful as diagnostic probes and primers as discussed
above and in more detail below.
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is intended 20 or more contiguous nucleotides from the nucleotide sequence of
the reference polynucleotide (e.g., the deposited cDNA or the nucleotide
sequence
as shown in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7)). Of
course, a polynucleotide which hybridizes only to a poly A sequence {such as
the
3' terminal poly(A) tract of the galectin 8, 9, 10, or 1 OS V cDNA shown in
FIGs.
1, 2A-2B, 3A-3B, or 4A-4B, respectively {SEQ ID NOs:l, 3, 5, or 7)), or to a
complementary stretch of T (or U) resides, would not be included in a
polynucleotide of the invention used to hybridize to a portion of a nucleic
acid of
the invention, since such a polynucleotide would hybridize to any nucleic acid
molecule containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
As indicated, nucleic acid molecules of the present invention which
encode a galectin 8, 9, 10, or IOSV polypeptide may include, but are not
limited
to those encoding the amino acid sequence of the polypeptide, by itself; the
coding sequence for the polypeptide and additional sequences, such as those
encoding an amino acid leader or secretory sequence, such as a pre-, or pro-
or
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prepro- protein sequence; the coding sequence of the polypeptide, with or
without
the aforementioned additional coding sequences, together with additional,
non-coding sequences, including for example, but not limited to introns and
non-coding 5' and 3' sequences, such as the transcribed, non-translated
sequences
that play a role in transcription, rnRNA processing, including splicing and
polyadenylation signals, for example - ribosome binding and stability of mRNA;
an additional coding sequence which codes for additional amino acids, such as
those which provide additional functionalities. Thus, the sequence encoding
the
polypeptide may be fused to a marker sequence, such as a sequence encoding a
peptide which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector
(Qiagen, Inc.), among others, many of which are commercially available. As
described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 ( 1989), for
instance, hexa-histidine provides for convenient purification of the fusion
protein.
'The "HA" tag is another peptide useful for purification which corresponds to
an
epitope derived from the influenza hemagglutinin protein, which has been
described by Wilson et al., Cell 3 7:767-778 ( 1984). As discussed below,
other
such fusion proteins include the galectin 8, 9, 10, or lOSV fused to Fc at the
N
or C-terminus.
The present invention further relates to variants of the nucleic acid
molecules of the present invention, which encode portions, analogs or
derivatives
of the galectin 8, 9, 10, or l OSV protein. Variants may occur naturally, such
as a
natural allelic variant. By an "allelic variant" is intended one of several
alternate
forms of a gene occupying a given locus on a chromosome of an organism.
Genes ll, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally
occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions,
deletions or additions which may involve one or more nucleotides. The variants
may be altered in coding regions, non-coding regions, or both. Alterations in
the
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coding regions may produce conservative or non-conservative amino acid
substitutions, deletions or additions. Especially preferred among these are
silent
substitutions, additions and deletions, which do not alter the properties and
activities of the galectin 8, 9, 10, or IOSV protein or portions thereof. Also
especially preferred in this regard are conservative substitutions.
Further embodiments of the invention include isolated nucleic acid
molecules comprising a polynucleotide having a nucleotide sequence at least
95%
identical, and more preferably at least 96%, 97%, 98% or 99% identical to (a)
a
nucleotide sequence encoding the galectin 8, 9, 10, or IOSV polypeptide having
the amino acid sequence in FIGS. I, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l,
3, 5, or 7); (b) a nucleotide sequence encoding the polypeptide having the
amino
acid sequence in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7),
but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the
amino acid sequence encoded by the eDNA clone contained in ATCC Deposit
Nos. 97732, 97733 or 97734 on September 24, I996; or (d) a nucleotide sequence
complementary to any of the nucleotide sequences in (a), (b), or (c).
By a polynucleotide having a nucleotide sequence at least, for example,
95% "identical" to a reference nucleotide sequence encoding a galectin 8, 9,
10,
or IOSV polypeptide is intended that the nucleotide sequence of the
polynucleotide is identical to the reference sequence except that the
polynucleotide sequence may include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence encoding the galectin 8, 9,
10,
or lOSV polypeptide. In other words, to obtain a polynucleotide having a
nucleotide sequence at least 95% identical to a reference nucleotide sequence,
up
to 5% of the nucleotides in the reference sequence may be deleted or
substituted
with another nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the reference
sequence.
These mutations of the reference sequence may occur at the 5' or 3' terminal
positions of the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among nucleotides in the
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reference sequence or in one or more contiguous groups within the reference
sequence.
As a practical matter, whether any particular nucleic acid molecule is at
least 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide
S sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:I, 3, 5, or
7) or to the nucleotides sequence of the deposited cDNA clone can be
determined
conventionally using known computer programs such as the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, WI 53711.
Bestfit uses the local homology algorithm of Smith and Waterman, Advances in
Applied Mathematics 2:482-489 ( 1981 )), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present invention, the
parameters are set, of course, such that the percentage of identity is
calculated
over the full length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of nucleotides in the reference
sequence are allowed.
The present application is directed to nucleic acid molecules at least 95%,
96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIGs. 1,
2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7) or to the nucleic acid
sequence of one of the deposited cDNAs, irrespective of whether they encode a
polypeptide having galectin 8, 9, 10, or IOSV activity. This is because even
where a particular nucleic acid molecule does not encode a polypeptide having
galectin 8, 9, 10, or l OSV activity, one of skill in the art would still know
how to
use the nucleic acid molecule, for instance, as a hybridization probe or a
polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of
the present invention that do not encode a polypeptide having galectin 8, 9,
10,
or 10SV activity include, inter alia, (1) isolating the galectin 8, 9, 10, or
lOSV
gene or allelic variants thereof in a cDNA library; (2) in situ hybridization
(e.g.,
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"FISH") to metaphase chromosomal spreads to provide precise chromosomal
location of the galectin 8, 9, I0, or IOSV gene, as described in Verma et al.,
Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New
York ( 1988); and (3) Northern Blot analysis for detecting galectin 8, 9, 10,
or
IOSV mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least
95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in
FIGs. l, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:I, 3, S, or 7) or to the nucleic
acid sequence of one of the deposited cDNAs which do, in fact, encode a
polypeptide having gaIectin 8, 9, 10, or l OSV protein activity. By "a
polypeptide
having galectin 8, 9, 10, or IOSV activity" is intended polypeptides
exhibiting
activity similar, but not necessarily identical, to an activity of the
galectin 8, 9, 10,
or I OSV protein of the invention, as measured, in a particular biological
assay.
For example, galectin 8, 9, 10, or 1 OS V protein activity can be measured
using
a lactose binding assay.
Lactose binding activity of the expressed galectin 8, 9, 10, or lOSV is
assayed by immunodetection of in situ binding activity to asialofetuin (Sigma)
immobilized on nitrocellulose (Amersham) (Madsen et al., J. Biol. Chem.
270(l1): S823-S829 (1995)). Thirty ~.g of asialofetuin dissolved in 3 pl of
water
is spotted on a 1-cmz strip of nitrocellulose. The nitrocellulose pieces are
then
placed in a 24-well tissue culture plate and incubated overnight in buffer B
(58
mM Na2HP04, 18 mM KHzP04, 75 mM NaCI, 2 mM EDTA, and 3% BSA,
pH7.2) with constant agitation at 22~C. Following incubation, the blocking
medium is aspirated and the nitrocellulose pieces are washed three times in
buffer
A (58 mM NaZHP04, 18 mM KH2P04, 75 mM NaCI, 2 mM EDTA, 4 mM ~3-
mercaptoethanol and 0.2% BSA, pH7.2). Cell extracts (preferably, COS cells)
are prepared containing 1 % BSA and either with or without 150 mM lactose (
105
~l of primary extract, 15 pl of 10% BSA in buffer A and either 30 pl of 0.75 M
lactose in buffer A or 30 ~l of buffer A). The immobilized asialofetuin is
incubated with the extracts for 2 h and washed 5 times in buffer A. The
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nitrocellulose pieces are then fixed in 2% formalin in PBS (58 mM NazHP04, 18
mM KHZP04, 75 mM NaCI, 2 mM EDTA pH'7.2) for 1 hour to prevent loss of
bound galectin. Following extensive washing in PBS the pieces were incubated
with rabbit anti-galectin 8, 9, 10, or l OSV polyclonal serum diluted 1:I00 in
PBS
for 2 h at 22~C. The pieces are then washed in PBS and incubated with
peroxidase-labeled goat anti-rabbit antibodies (DAKO). Following incubation
for 2 h at 22~C, the pieces are washed in PBS and the substrate is added.
Nitrocellulose pieces are incubated until the color develops and the reaction
is
stopped by washing in distilled water.
Of course, due to the degeneracy of the genetic code, one of ordinary skill
in the art will immediately recognize that a large number of the nucleic acid
molecules having a sequence at least 95%, 96%, 97%, 98%, or 99% identical to
the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence
shown in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7,
IS respectively) will encode "a polypeptide having galectin 8, 9, 10, or l OSV
protein
activity." In fact, since degenerate variants of these nucleotide sequences
all
encode the same polypeptide, this will be clear to the skilled artisan even
without
performing the above described comparison assay. It will be further recognized
in the art that, for such nucleic acid molecules that are not degenerate
variants, a
reasonable number will also encode a polypeptide having galectin 8, 9, 10, or
lOSV protein activity. This is because the skilled artisan is fully aware of
amino
acid substitutions that are either less likely or not likely to significantly
effect
protein function (e.g., replacing one aliphatic amino acid with a second
aliphatic
amino acid).
For example, guidance concerning how to make phenotypically silent
amino acid substitutions is provided in Bowie, J. U. et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science
247:1306-1310 (1990), wherein the authors indicate that proteins are
surprisingly
tolerant of amino acid substitutions.
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Vectors and Host Cells
The present invention also relates to vectors which include the isolated
DNA molecules of the present invention, host cells which are genetically
engineered with the recombinant vectors, and the production of galectin 8, 9,
10,
or lOSV polypeptides or fragments thereof by recombinant techniques.
The polynucleotides may be joined to a vector containing a selectable
marker for propagation in a host. Generally, a plasmid vector is introduced in
a
precipitate, such as a calcium phosphate precipitate, or in a complex with a
charged lipid. If the vector is a virus, it may be packaged in vitro using an
appropriate packaging cell line and then transdueed into host cells.
The DNA insert should be operatively linked to an appropriate promoter,
such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters,
the
SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
Other suitable promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and,
in the transcribed region, a ribosome binding site for translation. The coding
portion of the transcripts expressed by the constructs will preferably include
a
translation initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture and tetracycline or ampicillin
resistance
genes for culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast
cells;
insect cells such as Drosophila S2 and Spodoptera Sf~ cells; animal cells such
as
CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture
mediums and conditions for the above-described host cells are known in the
art.
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Among vectors preferred for use in bacteria include pQE70, pQE60 and
pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript
vectors, pNHBA, pNH 16a, pNH 18A, pNH46A, available from Stratagene; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia.
S Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Other suitable vectors will be readily apparent to
the
skilled artisan.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAF-dextran mediated transfection, cationic
lipid-mediated transfection, electroporation, transduction, infection or other
methods. Such methods are described in many standard laboratory manuals, such
as Davis et al.) Basic Methods In Molecular Biology (l986).
The polypeptide may be expressed in a modified form, such as a fusion
I S protein, and may include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of additional amino
acids,
particularly charged amino acids, may be added to the N-terminus of the
polypeptide to improve stability and persistence in the host cell, during
purification, or during subsequent handling and storage. Also, peptide
moieties
may be added to the polypeptide to facilitate purification. Such regions may
be
removed prior to final preparation of the polypeptide. The addition of peptide
moieties to polypeptides to engender secretion or excretion, to improve
stability
and to facilitate purification, among others, are familiar and routine
techniques
in the art. A preferred fusion protein comprises a heterologous region from
immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464
533 (Canadian counterpart 2045869) discloses fusion proteins comprising
various
portions of constant region of immunoglobin molecules together with another
human protein or part thereof. In many cases, the Fc part in a fusion protein
is
thoroughly advantageous for use in therapy and diagnosis and thus results, for
example, in improved pharmacokinetic properties (EP-A 0232 262). On the other
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hand, for some uses it would be desirable to be able to delete tlae Fc part
after the
fusion protein has been expressed, detected and purified in the advantageous
manner described. This is the case when Fc portion proves to be a hindrance to
use in therapy and diagnosis, for example when the fusion protein is to be
used
as antigen for immunizations. In drug discovery, for example, human proteins,
such as, hILS-receptor has been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-S. See, D.
Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K.
Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp
9459-947l (1995).
The galectin 8, 9, 10, or IOSV protein can he recovered and purified from
recombinant cell cultures by well-known methods including ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography and
lectin chromatography. Most preferably, high performance liquid
chromatography ("HPLC"} is employed for purification. Polypeptides of the
present invention include naturally purified products, products of chemical
synthetic procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial, yeast,
higher
plant, insect and mammalian cells. Depending upon the host employed in a
recombinant production procedure, the poiypeptides of the present invention
may
be glycosylated or may be non-glycosylated. In addition, polypeptides of the
invention may also include an initial modified methionine residue, in some
cases
as a result of host-mediated processes.
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Galectin 8, 9, and 10 Polypeptides and Fragments
The invention further provides an isolated galectin 8, 9, 10, or IOSV
polypeptide having ( 1 ) the amino acid sequence encoded by one of the
deposited
cDNAs, (2) the amino acid sequence in FIGS. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ
ID NOs:2, 4, 6, or 8, respectively), or (3) the amino acid sequence of a
peptide or
polypeptide comprising a portion of the above polypeptides.
It will be recognized in the art that some amino acid sequences of the
galectin 8, 9, 10, or 1 OSV polypeptide can be varied without significant
effect of
the structure or function of the protein. If such differences in sequence are
contemplated, it should be remembered that there will be critical areas on the
protein which determine activity.
Thus, the invention further includes variations of the galectin 8, 9, 10, or
l OSV polypeptide which show substantial galectin 8, 9, 10, or 10SV
polypeptide
activity or which include regions of galectin 8, 9, 10, or l OSV protein such
as the
protein portions discussed below. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions. As indicated above, guidance
concerning which amino acid changes are likely to be phenotypically silent can
be found in Bowie, J.U., et al., "Deciphering the Message in Protein
Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-l310 {1990).
Thus, the fragment, derivative or analog of the polypeptide of SEQ ID
NOs:2, 4, 6, or 8, or that encoded by one of the deposited cDNAs, may be {i)
one
in which one or more of the amino acid residues are substituted with a
conserved
or non-conserved amino acid residue (preferably a conserved amino acid
residue)
and such substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid residues
includes
a substituent group, or (iii) one in which the mature poiypeptide is fused
with
another compound, such as a compound to increase the half life of the
polypeptide (for example, polyethylene glycol), or (iv) one in which the
additional amino acids are fused to the mature polypeptide, such as an IgG Fc
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fusion region peptide or leader or secretory sequence or a sequence which is
employed for purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the scope of
those skilled in the art from the teachings herein.
Of particular interest are substitutions of charged amino acids with
another charged amino acid and with neutral or negatively charged amino acids.
The latter results in proteins with reduced positive charge to improve the
characteristics of a galectin 8, 9, 10, or IOSV protein. The prevention of
aggregation is highly desirable. Aggregation of proteins not only results in a
loss
of activity but can also be problematic when preparing pharmaceutical
formulations, because they can be immunogenic. (Pinckard et al., Clin. Exp.
Immunol. 2:331-340 (I967); Robbins et al., Diabetes 36:838-845 (198?); Cleland
et al., Crit. Rev. Therapeutic Drug Carrier Systems 1D:307-377 (I993)).
As indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly affect the
folding
or activity of the protein (see Table 1 ).
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TABLE I . Conservative Amino Acid Substitutions.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Valine
Polar ~ Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic ~ Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
Of course, the number of amino acid substitutions a skilled artisan would
make depends on many factors, including those described above and below.
Generally speaking, the number of substitutions for any given galectin 8, 9,
10,
or 1 OSV polypeptide or mutant thereof will not be more than 50, 40, 30, 20,
10,
5, or 3, depending on the objective.
Amino acids in a galectin 8, 9, 10, or IOSV protein of the present
invention that are essential for function can be identified by methods known
in
the art, such as site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, Science 244:I081-1085 {1989)). The latter procedure
introduces single alanine mutations at every residue in the molecule. Sites
that
are critical for ligand binding can also be determined by structural analysis
such
as crystallization, nuclear magnetic resonance or photoaffinity labeling
(Smith et
al., J. Mol. Biol. 224:899-904 ( I 992) and de Vos et al., Science 25S:306-312
( 1992)).
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The polypeptides of the present invention are preferably provided in an
isolated form. By "isolated polypeptide" is intended a polypeptide removed
from
its native environment. Thus, a polypeptide produced andlor contained within a
recombinant host cell is considered isolated for purposes of the present
invention.
Also intended as an "isolated polypeptide" are polypeptides that have been
purified, partially or substantially, from a recombinant host cell. For
example,
a recombinantly produced version of a galectin 8, 9, 10, or l OSV polypeptide
can
be substantially purified by the one-step method described in Smith and
Johnson,
Gene 67:31-40 (1988).
The polypeptides of the present invention include the polypeptides
encoded by the deposited cDNAs; a polypeptide comprising amino acids about
1 to about 323 in SEQ ID N0:2, about 1 to about 311 in SEQ ID N0:4, about 1
to about 317 in SEQ ID N0:6, and about 1 to about 200 in SEQ ID N0:8; a
polypeptide comprising amino acids about 2 to about 323 in SEQ ID N0:2, about
2 to about 311 in SEQ ID N0:4, about 2 to about 317 in SEQ ID N0:6 and about
2 to about 200 in SEQ ID N0:8; as well as polypeptides which are at least 95%
identical, still more preferably at least 96%, 97%, 98% or 99% identical to
the
polypeptides described above and also include portions of such polypeptides
with
at least 30 amino acids and more preferably at least 50 amino acids.
By a polypeptide having an amino acid sequence at Least, for example,
95% "identical" to a reference amino acid sequence of a galectin 8, 9, 10, or
l OSV polypeptide is intended that the amino acid sequence of the polypeptide
is
identical to the reference sequence except that the polypeptide sequence may
include up to five amino acid alterations per each 100 amino acids of the
reference amino acid of the galectin 8, 9, 10, or IOSV polypeptide. In other
words, to obtain a polypeptide having an amino acid sequence at least 95%
identical to a reference amino acid sequence, up to 5% of the amino acid
residues
in the reference sequence may be deleted or substituted with another amino
acid,
or a number of amino acids up to 5% of the total amino acid residues in the
reference sequence may be inserted into the reference sequence. These
alterations
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of the reference sequence may occur at the amino or carboxy terminal positions
of the reference amino acid sequence or anywhere between those terminal
positions, interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 95%,
96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown
in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:2, 4, 6, or 8, respectively) or
to the amino acid sequence encoded by one of the deposited cDNA clones (ATCC
Deposit Numbers 97732, 97733 and 97734) can be determined conventionally
IO using known computer programs such the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, S75 Science Drive, Madison, WI 537l 1 ). When using Bestfit or
any other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence according to
the
present invention, the parameters are set, of course, such that the percentage
of
identity is calculated over the full length of the reference amino acid
sequence and
that gaps in homology of up to 5% of the total number of amino acid residues
in
the reference sequence are allowed.
The polypeptide of the present invention could be used as a molecular
weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns
using methods well known to those of skill in the art.
In another aspect, the invention provides a peptide or polypeptide
comprising an epitope-bearing portion of a polypeptide of the invention. The
epitope of this polypeptide portion is an immunogenic or antigenic epitope
described herein. An "immunogenic epitope" is defined as a part of a protein
that
elicits an antibody response when the whole protein is the immunogen. On the
other hand, a region of a protein molecule to which an antibody can bind is
defined as an "antigenic epitope." The number of immunogenic epitopes of a
protein generally is less than the number of antigenic epitopes. See, for
instance,
Geysen et al., Proc. Natl. Acad. Sci. USA 8l:3998- 4002 (I983).
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As to the selection of peptides or poIypeptides bearing an antigenic
epitope (i. e., that contain a region of a protein molecule to which an
antibody can
bind), it is well known in that art that relatively short synthetic peptides
that
mimic part of a protein sequence are routinely capable of eliciting an
antiserum
S that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G.,
Shinnick, T. M., Green, N. and Learner, R.A. ( 1983) Antibodies that react
with
predetermined sites on proteins. Science 2l9:660-666. Peptides capable of
eliciting protein-reactive sera are frequently represented in the primary
sequence
of a protein, can be characterized by a set of simple chemical rules, and are
confined neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore useful to raise antibodies, including monoclonal antibodies, that
bind
specifically to a polypeptide of the invention. See, for instance, Wilson et
al.,
Cell 37:767-778 (1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention
preferably contain a sequence of at least seven, more preferably at least nine
and
most preferably between about at least 15 to about 30 amino acids contained
within the amino acid sequence of a polypeptide of the invention.
Non-limiting examples of antigenic polypeptides or peptides that can be
used to generate galectin 8, 9, 10, or lOSV-specific antibodies include: a
polypeptide comprising amino acid residues from about SS-101, l37-162, 180-
l93, 216-266 in FIG. 1 (SEQ ID N0:2), 62-102, 226-259, 197-308 in FIG. 2A-2B
(SEQ ID N0:4), 25-77, 84-105, 129-I40, 156-183, l95-215, and 241-257 in FIG.
3A-3B (SEQ ID N0:6), and 25-77, 84-105, 129-140, and 156-183 in FIG. 4A-4B
(SEQ ID N0:8), respectively. As indicated above, the inventors have determined
that the above polypeptide fragments are antigenic regions of the galectin 8,
9, 10,
or IOSV protein.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means Houghten, R. A. ( 1985) General method
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for the rapid solid-phase synthesis of large numbers of peptides: specificity
of
antigen-antibody interaction at the level of individual amino acids. Proc.
Natl.
Acad. Sci. USA 82;513l-5135. This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten
et al. (l986).
As one of skill in the art will appreciate, galectin 8, 9, 10, or 10SV
polypeptides of the present invention and the epitope-bearing fragments
thereof
described above can be combined with parts of the constant domain of
immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion
proteins facilitate purif cation and show an increased half life in vivo. This
has
been shown, e.g., for chimeric proteins consisting of the first two domains of
the
human CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker
et al.) Nature 331:84- 86 (l988)). Fusion proteins that have a disulfide-
linked
dimeric structure due to the IgG part can also be more efficient in binding
and
neutralizing other molecules than the monomeric galectin 8, 9, 10, or IOSV
protein or protein fragment alone {Fountoulakis et al., JBiochem 270:3958-3964
( 1995)).
Diagnosis and Prognosis
It is believed that certain tissues in mammals with certain diseases
(cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic
diseases) express significantly altered (enhanced or decreased) levels of the
galectin 8, 9, 10, or IOSV protein and mRNA encoding the galectin 8, 9, 10, or
1 OSV protein when compared to a corresponding "standard" mammal, i. e., a
mammal of the same species not having the disease. Further, it is believed
that
altered levels of the galectin 8, 9, 10, or l OSV protein can be detected in
certain
body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with
the
disease when compared to sera from mammals of the same species not having the
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disease. Thus, the invention provides a diagnostic method useful during
diagnosis, which involves assaying the expression level of the gene encoding
the
galectin 8, 9, 10, or IOSV protein in mammalian cells or body fluid and
comparing the gene expression level with a standard galectin 8, 9, 10, or IOSV
gene expression level, whereby an increase or decrease in the gene expression
level over the standard is indicative of the disease.
Where a diagnosis has already been made according to conventional
methods, the present invention is useful as a prognostic indicator, whereby
patients exhibiting altered galectin 8, 9, 10, or lOSV gene expression will
experience a worse clinical outcome relative to patients expressing the gene
at a
normal Ievel.
By "assaying the expression level of the gene encoding the galectin 8, 9,
10, or IOSV protein" is intended qualitatively or quantitatively measuring or
estimating the level of the galectin 8, 9, 10, or l OSV protein or the level
of the
mRNA encoding the galectin 8, 9, 10, or IOSV protein in a first biological
sample
either directly (e.g., by determining or estimating absolute protein level or
mRNA
level) or relatively (e.g., by comparing to the galectin 8, 9, 10, or l OSV
protein
level or mRNA level in a second biological sample).
Preferably, the galectin 8, 9, 10, or 10SV protein level or mRNA level in
the first biological sample is measured or estimated and compared to a
standard
galectin 8, 9, 10, or IOSV protein level or mRNA level, the standard being
taken
from a second biological sample obtained from an individual not having the
cancer. As will be appreciated in the art, once a standard galectin 8, 9, 10,
or
lOSV protein level or mRNA level is known, it can be used repeatedly as a
standard for comparison.
By "biological sample" is intended any biological sample obtained from
an individual, cell line, tissue culture, or other source which contains
galectin 8,
9, 10, or l OSV protein or mRNA. Biological samples include mammalian body
fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which
contain
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secreted galectin 8, 9, 10, or IOSV protein, and ovarian, prostate, heart,
placenta,
pancreas liver, spleen, lung, breast and umbilical tissue.
The present invention is useful for detecting diseases in mammals (for
example, cancer, autoimmune diseases, inflammatory diseases, asthma, and
allergic diseases). In particular the invention is useful during diagnosis of
the of
following types of cancers in mammals: melanoma, renal astrocytoma, Hodgkin
disease, breast, ovarian, prostate, bone, liver, lung, pancreatic, and
spleenic.
Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses,
rabbits
and humans. Particularly preferred are humans.
Total cellular RNA can be isolated from a biological sample using the
single-step guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 ( l 987). Levels of mRNA
encoding the galectin 8, 9, 10, or IOSV protein are then assayed using any
appropriate method. These include Northern blot analysis, (Harada et al., Cell
63:303-312 (1990) S1 nuclease mapping, (Fijita et al., Cell 49:357-367 (1987))
the polymerase chain reaction (PCR), reverse transcription in combination with
the polymerase chain reaction (RT-PCR) (Makino et al., Technique 2:29S-30l
( 1990), and reverse transcription in combination with the ligase chain
reaction
(RT-LCR).
Assaying galectin 8, 9, 10, or IOSV protein levels in a biological sample
can antibody-based techniques. For example, galectin 8, 9, 10, or l OSV
protein
expression in tissues can be studied with classical immunohistological
methods.
(Jalkanen, M., et al., J. Cell. Biol. l01:976-985 (l985); Jalkanen, M., et
al., J.
Cell . Biol. 10S:3087-3096 (1987)).
Other antibody-based methods useful for detecting galectin 8, 9, 10, or
l OSV protein gene expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable labels are known in the art and include enzyme labels, such as,
Glucose oxidase, and radioisotopes, such as iodine ('25I, '2'I), carbon ('4C),
sulfur
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(35s), tritium {3H), indium ("ZIn), and technetium {99mTc}, and fluorescent
labels,
such as fluorescein and rhodamine, and biotin.
Therapeutics
It is to be understood that although the following discussion is specifically
S directed to human patients, the teachings are also applicable to any animal
that
expresses galectin 8, 9, 10, or 10SV.
As noted above, galectin 8, 9, 10, and l OSV share significant homology
with other galectins. Galectin 1 induces apoptosis of T cells and T cell
leukemia
cell lines. Thus, it is believed by the inventors that galectin 8, 9, 10, and
IOSV
are active in modulating growth regulatory activities, immunomodulatory
activity,
cell-cell and cell-substrate interactions, and apoptosis.
The ability of galectin 8, 9, 10, or l OSV to modulate growth regulatory
activity may be therapeutically valuable in the treatment of clinical
manifestations
of such cell regulatory disorders. Disorders which can be treated include, but
IS should not be limited to, autoimmune disease, cancer (preferably, melanoma,
renal, astrocytoma, and Hodgkin disease}, inflammatory disease, wound healing,
arteriosclerosis, other heart diseases, microbe infection (virus, fungal,
bacterial,
and parasite), asthma, and allergic diseases.
Given the activities modulated by galectin 8, 9, 10, and 1 OSV, it is readily
apparent that a substantially altered (increased or decreased) level of
expression
of galectin 8, 9, 10, or IOSV in an individual compared to the standard or
"normal" level produces pathological conditions such as those described above.
It will also be appreciated by one of ordinary skill that the galectin 8, 9,
10, or
IOSV protein of the invention will exert its modulating activities on any of
its
target cells. Therefore, it will be appreciated that conditions caused by a
decrease
in the standard or normal level of galectin 8, 9, 10, or IOSV activity in an
individual, can be treated by administration of galectin 8, 9, 10, or l OSV
protein
or an agonist thereof. Thus, the invention further provides a method of
treating
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an individual in need of an increased level of galectin 8, 9, 10, or I OSV
activity
comprising administering to such an individual a pharmaceutical composition
comprising an amount of an isolated galectin 8, 9, 10, or l OSV polypeptide of
the
invention or an agonist thereof to increase the galectin 8, 9, 10, or l OSV
activity
level in such an individual.
A still further aspect of the invention is related to a method for treating an
individual in need of a decreased level of galectin 8, 9, 10, or l OSV
activity in the
body comprising, administering to such an individual a composition comprising
a therapeutically effective amount of a galectin 8, 9, 10, or IOSV antagonist.
Preferred antagonists for use in the present invention are gaIectin 8, 9, 10,
or
IOSV-specific antibodies.
Modes of administration
It will be appreciated that conditions caused by a decrease in the standard
or normal level of galectin 8, 9, 10, or IOSV activity in an individual, can
be
treated by administration of galectin 8, 9, 10, or 10SV protein or an agonist
thereof. Thus, the invention further provides a method of treating an
individual
in need of an increased level of galectin 8, 9, 10, or l OSV activity
comprising
administering to such an individual a pharmaceutical composition comprising an
effective amount of an isolated galectin 8, 9, 10, or l OSV polypeptide of the
invention, particularly a mature form of the galectin 8, 9, I 0, or i OSV,
effective
to increase the galectin 8, 9, 10, or IOSV activity level in such an
individual.
As a general proposition, the total pharmaceutically effective amount of
galectin 8, 9, 10, or IOSV polypeptide administered parenterally per dose will
be
in the range of about 1 p.g/kg/day to 10 mg/kg/day of patient body weight,
although, as noted above, this will be subject to therapeutic discretion. More
preferably, this dose is at least 0.01 mg/kg/day, and most preferably for
humans
between about 0.01 and 1 mglkg/day for the hormone. If given continuously, the
galectin 8, 9, 10, or l OSV polypeptide is typically administered at a dose
rate of
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about 1 ~.g/kg/hour to about 50 ~g/kg/hour, either by 1-4 injections per day
or by
continuous subcutaneous infusions, for example, using a mini-pump. An
intravenous bag solution may also be employed.
Pharmaceutical compositions containing the galectin 8, 9, 10, or IOSV of
the invention may be administered orally, rectally, parenterally,
intracistemally,
intravaginally, intraperitoneally, topically (as by powders, ointments, drops
or
transdermal patch), bucally, or as an oral or nasal spray. By
"pharmaceutically
acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler,
diluent,
encapsulating material or formulation auxiliary of any type. The term
"parenteral" as used herein refers to modes of administration which include
intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
Chromosome Assays
The nucleic acid molecules of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to and can
hybridize with a particular location on an individual human chromosome. The
mapping of DNAs to chromosomes according to the present invention is an
important first step in correlating those sequences with genes associated with
disease.
In certain preferred embodiments in this regard, the cDNA herein
disclosed is used to clone genomic DNA of a galectin 8, 9, 10, or I OSV
protein
gene. This can be accomplished using a variety of well known techniques and
libraries, which generally are available commercially. The genomic DNA then
is used for in situ chromosome mapping using well known techniques for this
purpose.
In addition, in some cases, sequences can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the 3' untranslated region of the gene is used to rapidly select primers
that do
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not span more than one exon in the genomic DNA, thus complicating the
amplification process. These primers are then used for PCR screening of
somatic
cell hybrids containing individual human chromosomes.
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a
metaphase chromosomal spread can be used to provide a precise chromosomal
location in one step. This technique can be used with probes from the cDNA as
short as 50 or 60 bp. For a review of this technique, see Verma et al., Human
Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York
( 1988).
Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic map data. Such data are found, for example, in V. McKusick, Mendelian
Inheritance In Man, available on-line through Johns Hopkins University, Welch
Medical Library. The relationship between genes and diseases that have been
mapped to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is
observed
in some or a11 of the affected individuals but not in any normal individuals,
then
the mutation is likely to be the causative agent of the disease.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of illustration and are not intended as limiting.
Examples
Example l: Expression and Puriftcation of Galectin 8, 9, 10 and IOSV in E.
coli
The DNA sequence encoding the galectin 9 protein in the deposited
cDNA clone was amplified using PCR oligonucleotide primers specific to the
amino terminal sequences of the galectin 9 protein and to vector sequences 3 '
to
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the gene. Additional nucleotides containing restriction sites to facilitate
cloning
are added to the 5' and 3' sequences.
The DNA sequence encoding the galectin 8 or IOSV protein in the
deposited cDNA clone is amplified using PCR oligonucleotide primers specific
to the nucleotide sequences encoding the amino terminal sequences of the
galectin 8 or IOSV protein and to vector sequences 3' to the gene. Additional
nucleotides containing restriction sites to facilitate cloning are added to
the S' and
3' sequences.
The cDNA sequence encoding the galectin 10 protein is amplified from
either a human endometrial tumor or human fetal heart cDNA library using PCR
oligonucleotide primers specific to the nucleotide sequences encoding the
amino
terminal sequences of the galectin 10 protein and to vector sequences 3' to
the
gene. Additional nucleotides containing restriction sites to facilitate
cloning are
added to the 5' and 3' sequences.
The 5' galectin 8 oligonucleotide primer has the sequence 5' cgc ccAT
CCTATGTCCCCGCACCG 3' (SEQ ID N0:41 ) containing the underlined NcoI
restriction site and nucleotides 56 to 72 of the galectin 8 protein coding
sequence
in FIG. 1 (SEQ ID NO:1 ).
The 3' galectin 8 primer has the sequence 5' cgc AAG CTT TTAGATC
TGGACATAGGAC 3' (SEQ ID N0:42) containing the underlined HindIII
restriction site followed by nucleotides complementary to position 1005 to
1023
of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:1).
The 5' galectin 9 oligonucleotide primer has the sequence 5'cgc ccAT
CCTT CAGCGGTTCCCAG 3' (SEQ ID N0:43) containing the underlined NcoI
restriction site and nucleotides 20 to 36 of the galectin 9 protein coding
sequence
in FIG. 2A-2B (SEQ ID N0:3).
The 3' galectin 9 primer has the sequence 5'cgc AAG CTT CAGGGTT
GGAAAGGCTG (SEQ ID N0:44) containing the underlined HindIII restriction
site followed by nucleotides complementary to position 1029 to 1045 of the
galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID N0:3).
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The S' galectin 10 and lOSV oligonucleotide primer has the sequence
'cgc CCATGc TGTTGTCCTTAAACAAC 3' (SEQ ID N0:45) containing the
underlined Sphl restriction site and nucleotides 122-138 of the galectin 10
protein
coding sequence in FIG. 3A-3B {SEQ ID NO:S).
5 The 3' galectin 10 primer has the sequence 5' cgc CTG CAG CACAGAA
GCCATTCTG 3' (SEQ ID N0:46) containing the underlined PstI restriction site
followed by nucleotides complementary to position 1105-1 l20 of the galectin
10
protein coding sequence in FIG. 3A-3B (SEQ ID NO:S).
The 3' galectin IOSV primer has the sequence 5' CGCCTGCAGCTA
TGCAACTTTATAAAATATTCC 3' (SEQ ID N0:47) containing the underlined
PstI restriction site followed by nucleotides complementary to 3' end of the
galectin l OSV protein coding sequence in FIG. 4A-4B (SEQ ID N0:7).
The restriction sites are convenient to restriction enzyme sites in the
bacterial expression vector pQE60 (galectin 8 and 9) or pQE6 (galectin 10),
which are used for bacterial expression in these examples. (Qiagen, Inc. 9259
Eton Avenue, Chatsworth, CA, 91311 ). pQE60 encodes ampicillin antibiotic
resistance ("Amp"') and contains a bacterial origin of replication ("ori"), an
IPTG
inducible promoter, a ribosome binding site ("RBS"), a 6-His tag and
restriction
enzyme sites.
The amplified galectin 8, 9, 10, or 1 OSV DNA and the vector pQE60 or
pQE6 both are digested with NcoI and HindIII (for galectin 8 and 9) or SphI
and
PstI (for galectin I O) and the digested DNAs are then ligated together.
Insertion
of the galectin 8, 9, 10, or l OSV protein DNA into the restricted pQE60 or
pQE6
vector placed the galectin 8, 9, 10, or l OSV protein coding region downstream
of
and operably linked to the vector's IPTG-inducible promoter and in-frame with
an initiating AUG appropriately positioned for translation of galectin 8, 9,
10, or
lOSV protein.
The Iigation mixture is transformed into competent E. coli cells using
standard procedures. Such procedures are described in Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring
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Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. call strain
M15/rep4, containing multiple copies of the plasmid pREP4, which expresses lac
repressor and confers kanamycin resistance ("Kan"'), is used in carrying out
the
example described herein. This strain, which is only one of many that are
suitable
for expressing galectin 8, 9, 10, or IOSV protein, is available commercially
from
Qiagen.
Transformants are identified by their ability to grow on LB plates in the
presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant
colonies and the identity of the cloned DNA confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight ("OiN") in
liquid culture in LB media supplemented with both ampicillin (100 ug/ml) and
kanamycin (25 p.g/ml).
The O/N culture is used to inoculate a large culture, at a dilution of
approximately 1: l00 to 1:250. The cells are grown to an optical density at
600nm
ZS ("0D600") of between 0.4 and 0.6. Isopropyl-B-D-thiogalactopyranoside
("IPTG") is then added to a final concentration of 1 mM to induce
transcription
from lac repressor sensitive promoters, by inactivating the lacl repressor.
Cells
subsequently are incubated further for 3 to 4 hours. Cells then are harvested
by
centrifugation and disrupted, by standard methods. Inclusion bodies are
purified
from the disrupted cells using routine collection techniques, and protein is
solubilized from the inclusion bodies into 8M urea. The 8M urea solution
containing the solubilized protein is passed over a PD-10 column in 2X
phosphate-buffered saline ("PBS"), thereby removing the urea, exchanging the
buffer and refolding the protein. The protein is purified by a further step of
chromatography to remove endotoxin. Then, it is sterile filtered. The sterile
filtered protein preparation is stored in 2X PBS at a concentration of 95
p./ml.
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Example 2: Cloning and Expression of Galectin $, 9, 10 and IOSY protein in
a Baculovirus Expression System
The cDNA sequence encoding the full length galectin 8, 9, 10, or l OSV
protein in the deposited clone is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene:
The 5' galectin 8 oligonucleotide primer has the sequence 5'cgc CCC
GGG GCCTATGTCCCCGCAC 3' (SEQ ID N0:48) containing the underlined
SmaI restriction site and nucleotides 55 to 70 of the galectin 8 protein
coding
sequence in FIG. 1 (SEQ ID NO:1).
The 3' galectin 8 primer has the sequence 5' cgc GGT ACC
TTAGATCTGG ACATAGGAC 3' (SEQ ID N0:49) containing the underlined
Asp718 restriction site followed by nucleotides complementary to position 1005
to 1023 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:1).
The 5' galectin 9 oligonucleotide primer has the sequence 5' cgc ~
GGG GCCTTCAGCGGTTCCCAG 3' (SEQ ID NO:50) containing the
underlined Smal restriction site and nucleotides 19 to 36 of the galectin 9
protein
coding sequence in FIG. 2A-2B (SEQ ID N0:3).
The 3' galectin 9 primer has the sequence 5' cgc GGT ACC
CAGGGTTGG AAAGGCTG 3' (SEQ ID NO:S 1 ) containing the underlined
Asp7I8 restriction site followed by nucleotides complementary to position I
029
to 1045 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID N0:3).
The 5' galectin 10 oligonucleotide primer has the sequence 5' cgc ~C_
GGG TTGTCCTTAAACAACCTAC 3' (SEQ ID N0:52) containing the
underlined SmaI restriction site and nucleotides l24-142 of the galectin 10
protein coding sequence in FIG. 3A-3B (SEQ ID NO:S).
The 3' galectin 10 primer has the sequence 5' cgc SGT ACC CACA
GAAGCCATTCTG 3' (SEQ ID N0:53) containing the underlined Asp718
restriction site followed by nucleotides complementary to position 1105-1120
of
the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:S).
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The 3' galectin 1 OSV primer has the sequence 5' CGC TACCCTA
TGCAACTTTATAAAATATTCC 3' (SEQ ID N0:54) containing the underlined
Asp718 restriction site followed by nucleotides complementary to the 3' end of
the galectin l OSV protein coding sequence in FIG. 4A-4B (SEQ ID N0:7).
An efficient signal for initiation of translation in eukaryotic cells, as
described by Kozak, M., J. Mol. Biol. l96:947-950 (1987) is appropriately
located in the vector portion of the construct.
The amplified fragment is isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 10I Inc., La Jolla, Ca.). The
fragment then is digested with XbaI and again is purified on a 1 % agarose
gel.
This fragment is designated herein F2.
The vector pA2-GP is used to express the galectin 8, 9, I0, or IOSV
protein in the baculovirus expression system, using standard methods, as
described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS
VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas
Agricultural Experimental Station Bulletin No. 1555 (l987). This expression
vector contains the strong polyhedrin promoter of the Autographa californica
nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
The signal peptide of AcMNPV gp67, including the N-terminal methionine, is
located just upstream of a BamHI site. The polyadenylation site of the simian
virus 40 ("SV40") is used for efficient polyadenylation. For an easy selection
of
recombinant virus the beta-galactosidase gene from E. coli is inserted in the
same
orientation as the polyhedrin promoter and is followed by the polyadenylation
signal of the polyhedrin gene. The polyhedrin sequences are flanked at both
sides
by viral sequences for cell-mediated homologous recombination with wild-type
viral DNA to generate viable virus that express the cloned polynucleotide.
Many other baculovirus vectors could be used in place of pA2-GP, such
as pAc373, pVL941 and pAcIMl provided, as those of skill readily will
appreciate, that construction provides appropriately located signals for
transcription, translation, trafficking and the like, such as an in-frame AUG
and
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a signal peptide, as required. Such vectors are described in Luckow et al.,
Virology 170:31-39, among others.
The plasmid is digested with the restriction enzyme SmaI and Asp718 and
then is dephosphorylated using calf intestinal phosphatase, using routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
This vector DNA is designated herein "V2".
Fragment F2 and the dephosphorylated plasmid V2 are Iigated together
with T4 DNA ligase. E. coli HB101 cells are transformed with ligation mix and
spread on culture plates. Bacteria are identified that contain the pIasmid
with the
human galectin 8, 9, 10, or IOSV gene by digesting DNA from individual
colonies using XbaI and then analyzing the digestion product by gel
electrophoresis. The sequence of the cloned fragment is confirmed by DNA
sequencing. This plasmid is designated herein pBacgalectin 8, 9, 10, or IOSV.
5 pg of the plasmid pBacgalectin 8, 9, 10, or l OSV is co-transfected with
1.0 ug of a commercially available linearized bacuIovirus DNA ("BaculoGoldTM
baculovirus DNA", Pharmingen, San Diego, CA.), using the lipofection method
described by Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-74l7 (1987).
1 ug of BaculoGoldTM virus DNA and 5 ~g of the plasmid pBacgalectin 8, 9, 10,
or lOSV are mixed in a sterile well of a microtiter plate containing 50 pl of
serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD).
Afterwards 10 pI Lipofectin plus 90 q.l Grace's medium are added, mixed and
incubated for 15 minutes at room temperature. Then the transfection mixture is
added drop-wise to Sf~ insect cells (ATCC CRL 1711 ) seeded in a 3 S mm tissue
culture plate with 1 ml Grace's medium without serum. The plate is rocked back
and forth to mix the newly added solution. The plate is then incubated for S
hours at 27~C. After S hours the transfection solution is removed from the
plate
and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is
added. The plate is put back into an incubator and cultivation is continued at
27~C for four days.
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After four days the supernatant is collected and a plaque assay is
performed, as described by Summers and Smith, cited above. An agarose gel
with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy
identification and isolation of gal-expressing clones, which produce blue-
stained
plaques. (A detailed description of a "plaque assay" of this type can also be
found
in the user's guide for insect cell culture and baculovirology distributed by
Life
Technologies Inc., Gaithersburg, page 9-10).
Four days after serial dilution, the virus is added to the cells. After
appropriate incubation, blue stained plaques are picked with the tip of an
Eppendorf pipette. The agar containing the recombinant viruses is then
resuspended in an Eppendorf tube containing 200 ul of Grace's medium. The agar
is removed by a brief centrifugation and the supernatant containing the
recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four
days later the supernatants of these culture dishes are harvested and then
they are
stored at 4~C. A clone containing properly inserted hESSB I, II and III is
identified by DNA analysis including restriction mapping and sequencing. This
is designated herein as V-galectin 8, 9, 10, or l OSV.
Sf9 cells are grown in Grace's medium supplemented with 10% heat-
inactivated FBS. The cells are infected with the recombinant baculovirus
V-galectin 8, 9, 10, or IOSV at a multiplicity of infection ("MOI") of about 2
(about 1 to about 3). Six hours later the medium is removed and is replaced
with
SF900 II medium minus methionine and cysteine (available from Life
Technologies Inc., Gaithersburg}. 42 hours later, 5 pCi of 35S-methionine and
5
pCi 35S-cysteine (available from Amersham) are added. The cells are further
incubated for 16 hours and then they are harvested by centrifugation, lysed
and
the labeled proteins are visualized by SDS-PAGE and autoradiography.
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Example 3: Cloning and Expression in Mammalian Cells
Most of the vectors used for the transient expression of the galectin 8, 9,
10, or IOSV protein gene sequence in mammalian cells should carry the SV40
origin of replication. This allows the replication of the vector to high copy
numbers in cells (e.g. COS cells) which express the T antigen required for the
initiation of viral DNA synthesis. Any other mammalian cell line can also be
utilized for this purpose.
A typical mammalian expression vector contains the promoter element,
which mediates the initiation of transcription of mRNA, the protein coding
sequence, and signals required for the termination of transcription and
polyadenylation of the transcript. Additional elements include enhancers,
Kozak
sequences and intervening sequences flanked by donor and acceptor sites for
RNA splicing. Highly efficient transcription can be achieved with the early
and
late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses,
e.g. RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
However, cellular signals can also be used (e.g., human actin promoter).
Suitable
expression vectors for use in practicing the present invention include, for
example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBCI2MI (ATCC
67109). Mammalian host cells that could be used include, human HeLa, 283, H9
and Jurkart cells, mouse NIH3T3 and C 127 cells, Cos 1, Cos 7 and CV 1,
African
green monkey cells, quail QC 1-3 cells, mouse L cells and Chinese hamster
ovary
cells.
Alternatively, the gene can be expressed in stable cell lines that contain
the gene integrated into a chromosome. The co-transfection with a selectable
marker such as dhfr, gpt, neomycin, hygromycin allows the identification and
isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts of the
encoded protein. The DHFR (dihydrofolate reductase) is a useful marker to
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develop cell lines that carry several hundred or even several thousand copies
of
the gene of interest. Another useful selection marker is the enzyme glutamine
synthase (GS) (Murphy et al.) Biochem. J. 227:277-279 (1991 ); Bebbington et
al.,
BiolTechnology 10:169-175 (l992)). Using these markers, the mammalian cells
S are grown in selective medium and the cells with the highest resistance are
selected. These cell lines contain the amplified genes) integrated into a
chromosome. Chinese hamster ovary (CHO) cells are often used for the
production of proteins.
The expression vectors pC 1 and pC4 contain the strong promoter (LTR)
of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,
438-4470 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al.)
Cell 4l:521-530 (198S)). Multiple cloning sites, e.g., with the restriction
enzyme
cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the polyadenylation
and
termination signal of the rat preproinsulin gene.
Example 3(a): Cloning and Expression in COS Cells
The expression plasmid, pgalectin 8, 9, 10, or lOSV HA, is made by
cloning a cDNA encoding galectin 8, 9, 10, or lOSV into the expression vector
pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
The expression vector pcDNAIlamp contains: (1) an E. coli origin of
replication effective for propagation in E. coli and other prokaryotic cells;
(2) an
ampicillin resistance gene for selection of plasmid-containing prokaryotic
cells;
(3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a
CMV
promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged
so
that a cDNA conveniently can be placed under expression control of the CMV
promoter and operably linked to the SV40 intron and the polyadenylation signal
by means of restriction sites in the polylinker.
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A DNA fragment encoding the galectin 8, 9, 10, or l OSV protein and an
HA tag fused in frame to its 3' end is cloned into the polylinker region of
the
vector so that recombinant protein expression is directed by the CMV promoter.
The HA tag corresponds to an epitope derived from the influenza hemagglutinin
protein described by Wilson et al., Cell 37:7b7-778 {1984). The fusion of the
HA
tag to the target protein allows easy detection of the recombinant protein
with an
antibody that recognizes the HA epitope.
The plasmid construction strategy is as follows. The gaIectin 8, 9, 10, or
lOSV cDNA of the deposited clone is amplified using primers that contain
convenient restriction sites, much as described above regarding the
construction
of expression vectors for expression of galectin 8, 9, 10, or l OSV in ~.
coli. To
facilitate detection, purification and characterization of the expressed
galectin 8,
9, 10, or l OSV, one of the primers contains a hemagglutinin tag ("HA tag") as
described above.
Suitable primers include the following, which are used in this example.
The 5' galectin 8 primer has the sequence 5'cgc CCC GGG gcc atc ATG
GCCTATGTCCCCG 3' (SEQ ID N0:55) containing the underlined SmaI
restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (SEQ
ID
NO:1 ).
The 3' galectin 8 primer has the sequence 5' cgc SGT AS'C TTAGAT
CTGGACATAGGAC 3' (SEQ ID N0:56) containing the Asp718 restriction
followed by nucleotides complementary to nucleotides 10Q5-t 023 of the
galectin 8 coding sequence set out in FIG. 1 (SEQ ID NO:1 ).
The 5' galectin 9 primer has the sequence 5' cgc CCC GGG gcc atc
ATGGCCTTCAGCGGTTC 3' (SEQ ID N0:57) containing the underlined SmaI
restriction enzyme site followed by the nucleotide sequence of bases 16-32 of
FIG. 2A-2B (SEQ ID N0:3).
The 3' galectin 9 primer has the sequence 5' cgc CGT ACC CAGGGTT
GGAAAGGCTG 3' (SEQ ID N0:58) containing the Asp718 restriction followed
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by nucleotides complementary to nucleotides 1029-1045 of the galectin 9 coding
sequence set out in FIG. 2A-2B (SEQ ID N0:3), including the stop codon.
The 5' galectin 10 and l OSV primer has the sequence 5' cgc SCC GGG
gcc atc ATGATGTTGTCCTTAAAC 3' (SEQ ID N0:59) containing the
underlined SmaI restriction enzyme site followed by nucleotide sequence 118-
13S
of FIG. 3A-3B (SEQ ID NO:S).
The 3' galectin 10 primer has the sequence 5' cgc GGT ACC CACAG
AAGCCATTCTG 3' (SEQ ID N0:60) containing the Asp718 restriction
followed by nucleotides complementary to nucleotides 110S-1 l20 set out in
FIG.
3A-3B (SEQ ID NO:S).
The 3' galectin IOSV primer has the sequence 5' CGCGGTACCCTA
TGCAACTTTATAAAATATTCC 3' (SEQ ID N0:54) containing the Asp718
restriction followed by nucleotides complementary to the 3' end of the
galectin
l OSV coding sequence set out in FIG. 4A-4B (SEQ ID N0:7).
The PCR amplified DNA fragment and the vector, pcDNAIIAmp, are
digested with HindIII and XhoI and then ligated. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene Cloning
Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the
transformed culture is plated on ampicilIin media plates which then are
incubated
to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis and gel sizing for the
presence of the galectin 8, 9, 10, or l OSV-encoding fragment.
For expression of recombinant galectin 8, 9, 10, or l OSV, COS cells are
transfected with an expression vector, as described above, using DEAE-
DEXTRAN, as described, for instance, in Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, Cold Spring Laboratory Press, Cold
Spring Harbor, New York (1989). Cells are incubated under conditions for
expression of galectin 8, 9, 10, or lOSV by the vector.
Expression of the galectin 8, 9, 10, or 1 OSV HA fusion protein is detected
by radiolabelling and immunoprecipitation, using methods described in, for
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example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (I988).
To this end, two days after transfection, the cells are labeled by incubation
in
media containing 35S-cysteine for 8 hours. The cells and the media are
collected,
and the cells are washed and the lysed with detergent-containing RIPA buffer:
150 mM NaCI, 1 % NP-40, 0.1 % SDS, 1 % NP-40, 0.5% DOC, 50 mM TRIS, pH
7.5, as described by Wilson et al. cited above. Proteins are precipitated from
the
cell lysate and from the culture media using an HA-specific monoclonal
antibody.
The precipitated proteins then are analyzed by SDS-PAGE gels and
autoradiography. An expression product of the expected size is seen in the
cell
lysate, which is not seen in negative controls.
Example 3(b): Cloning and Expression in CHO Cells
The vector pCl is used for the expression of galectin 8, 9, 10, or IOSV
protein. Plasmid pC 1 is a derivative of the plasmid pSV2-dhfr [ATCC Accession
No. 37I 46]. Both plasmids contain the mouse DHFR gene under control of the
SV40 early promoter. Chinese hamster ovary- or other cells lacking
dihydrofolate
activity that are transfected with these plasmids can be selected by growing
the
cells in a selective medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate. The amplification of the DHFR
genes in cells resistant to methotrexate (MTX) has been well documented (see,
e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J.
Biol.
Chem. 2S3:1357-1370, Hamlin, J.L. and Ma, C. 1990, Biochem. et Biophys. Acta,
1097:107-143, Page, M.J. and Sydenham, M.A., Biotechnology 9:64-68 (l991)).
Cells grown in increasing concentrations of MTX develop resistance to the drug
by overproducing the target enzyme, DHFR, as a result of amplification of the
DHFR gene. If a second gene is linked to the DHFR gene it is usually co-
amplified and over-expressed. It is state of the art to develop cell lines
carrying
more than 1,000 copies of the genes. Subsequently, when the methotrexate is
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withdrawn, cell lines contain the amplified gene integrated into the
chromosome(s).
Plasmid pC 1 contains for the expression of the gene of interest a strong
promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen,
et al., Molecular and Cellular Biology, March 1985:438-4470) plus a fragment
isolated from the enhancer of the immediate early gene of human
cytomegalovirus (CMV) (Boshart et al., Cell 41:52I-530, 1985). Downstream
of the promoter are the following single restriction enzyme cleavage sites
that
allow the integration of the genes: BamHI, PvuII, and NruI. Behind these
cloning
sites the plasmid contains translational stop codons in all three reading
frames
followed by the 3' intron and the polyadenylation site of the rat
preproinsulin
gene. Other high efficient promoters can also be used for the expression,
e.g., the
human ~i-actin promoter, the SV40 early or late promoters or the long terminal
repeats from other retroviruses, e.g., HIV and HTLVI. For the polyadenylation
of the mRNA other signals, e.g., from the human growth hormone or globin genes
can be used as well.
Stable cell lines carrying a gene of interest integrated into the
chromosomes can also be selected upon co-transfection with a selectable marker
such as gpt, G418 or hygromycin. It is advantageous to use more than one
selectable marker in the beginning, e.g., G418 plus methotrexate.
The plasmid pC 1 is digested with the restriction enzyme BamHI and then
dephosphorylated using calf intestinal phosphates by procedures known in the
art.
The vector is then isolated from a I % agarose gel.
The DNA sequence encoding galectin 8, 9, or l OSV, ATCC Deposit Nos.
97732, 97733 and 97734, respectively, is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene. The galectin 10
sequence is similarly amplified from a human endometrial tumor or human fetal
heart cDNA library.
The S' galectin 8 primer has the sequence 5' cgcCC G GgccatcATG
GCCTATGTCCCCG 3' (SEQ ID NO:55) containing the underlined SmaI
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restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (SEQ
ID
NO:1 ). Inserted into an expression vector, as described below, the 5' end of
the
amplified fragment encoding human galectin 8 provides an efficient signal
peptide. An efficient signal for initiation of translation in eukaryotic
cells, as
S described by Kozak, M., J. ~I~IoI. Biol. l96:947-950 (1987) is appropriately
located in the vector portion of the construct.
The 3' galectin 8 primer has the sequence 5' cgc GGT ACC TTAGAT
CTGGACATAGGAC 3' (SEQ ID N0:56) containing the Asp718 restriction
followed by nucleotides complementary to nucleotides 100S-l023 of the
gaIectin 8 coding sequence set out in FIG. 1 (SEQ ID NO:1 ).
The 5' galectin 9 primer has the sequence 5' cgc CCC GGG gcc atc
ATGGCCTTCAGCGGTTC 3' (SEQ ID N0:57) containing the underlined SmaI
restriction enzyme site followed by the nucleotide sequence of bases 16-32 of
FIG. 2A-2B (SEQ ID N0:3). Inserted into an expression vector, as described
below, the S' end of the amplified fragment encoding human galectin 9 provides
an efficient signal peptide. An efficient signal for initiation of translation
in
eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 ( 1987)
is
appropriately located in the vector portion of the construct.
The 3' galectin 9 primer has the sequence 5' cgc GGT ACC CAGGGTT
GGAAAGGCTG 3' (SEQ ID N0:58) containing the Asp718 restriction followed
by nucleotides complementary to nucleotides 1029-104S of the galectin 9 coding
sequence set out in FIG. 2A-2B (SEQ ID N0:3), including the stop codon.
The 5' galectin 10 and l OSV primer has the sequence 5' cgc ACC GGG
gcc atc ATGATGTTGTCCTTAAAC 3' (SEQ ID N0:59) containing the
underlined SmaI restriction enzyme site followed by nucleotide sequence 118-
135
of FIG. 3A-3B (SEQ ID NO:S). Inserted into an expression vector, as described
below, the 5' end of the amplified fragment encoding human galectin 10
provides
an eff cient signal peptide. An efficient signal for initiation of translation
in
eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-95Q (1987)
is
appropriately located in the vector portion of the construct.
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The 3' galectin IO primer has the sequence 5' cgcGGTACCCACAG
AAGCCATTCTG 3' (SEQ ID N0:60) containing the Asp718 restriction
followed by nucleotides complementary to nucleotides 1105-1120 set out in FIG.
3A-3B (SEQ ID NO:S).
The 3' galectin IOSV primer has the sequence 5' CGCG TA CTA
TGCAACTTTATAAA.ATATTCC 3' (SEQ ID N0:54) containing the Asp7I8
restriction followed by nucleotides complementary to the 3' end of the
galectin
l OSV coding sequence set out in FIG. 4A-4B (SEQ ID N0:7).
The amplified fragments are isolated from a 1% agarose gel as described
above and then digested with the endonucleases SmaI and Asp7l8 and then
purified again on a 1 % agarose gel.
The isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB 1 O 1 cells are then transformed and bacteria
identified that contained the plasmid pC I inserted in the correct orientation
using
the restriction enzyme SmaI. The sequence of the inserted gene is confirmed by
DNA sequencing.
Transfection of CHO-DHFR-cells
Chinese hamster ovary cells lacking an active DHFR enzyme are used for
transfection. Five ~g of the expression plasmid C 1 are cotransfected with 0.5
pg
of the plasmid pSVneo using the lipofecting method (Felgner et al., supra).
The
plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5
encoding an enzyme that confers resistance to a group of antibiotics including
G418. The cells are seeded in alpha minus MEM supplemented with 1 mglml
G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning
plates (Greiner, Germany) and cultivated from 10-14 days. After this period,
single clones are trypsinized and then seeded in 6-well petri dishes using
different
concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM).
Clones growing at the highest concentrations of methotrexate are then
transferred
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to new 6-well plates containing even higher concentrations of methotrexate
(500
nM, I ~M, 2 pM, S ~M). The same procedure is repeated until clones grow at
a concentration of l00 ~M.
The expression of the desired gene product is analyzed by Western blot
analysis and SDS-PAGE.
Example 4: Tissue distribution of protein expression
Northern blot analysis is earned out to examine galectin 8, 9, 10, or IOSV
gene expression in human tissues, using methods described by, among others,
Sambrook et al., cited above. A cDNA probe containing the entire nucleotide
sequence of the galectin 8, 9, 10, or IOSV protein (SEQ ID NO:1, 3, 5, or 7,
respectively) is labeled with '2P using the rediprimeTM DNA labeling system
(Amersham Life Science), according to manufacturer's instructions. After
labeling, the probe is purified using a CHROMA SPIN-100T"' column (Clontech
Laboratories, Ine.), according to manufacturer's protocol number PT1200-1. The
1 S purified labeled probe is then used to examine various human tissues for
galectin
8, 9, 10, or IOSV mRNA.
Multiple Tissue Northern (MTN) blots containing various human tissues
(H) or human immune system tissues (IM) are obtained from Clontech and are
examined with labeled probe using ExpressHybTM hybridization solution
(Clontech) according to manufacturer's protocol number PT1190-1. Following
hybridization and washing, the blots are mounted and exposed to film at -70~C
overnight, and films developed according to standard procedures.
It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are
possible in light of the above teachings and, therefore, are within the scope
of the
appended claims.
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The entire disclosure of a11 publications (including patents, patent
applications, journal articles, laboratory manuals, books, or other documents)
cited herein are hereby incorporated by reference.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Human Genome Sciences, Inc.
94l0 Key West Avenue
Rockville) MD 20850
United States of America
APPLICANTS/INVENTORS: Ni, Jian
Gentz, Refiner L.
Ruben, Steven M.
(ii} TITLE OF INVENTION: Galectin 8, 9, 10 and lOSV
(iii) NUMBER OF SEQUENCES: 60
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterne, Kessler, Goldstein, & Fox P.L.L.C.
(B) STREET: 1100 New York Ave., Suite 600
(C} CITY: Washington
(D) STATE: D.C.
(E} COUNTRY: USA
(F) ZIP: 20005-3934
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B} COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To be assigned
(B} FILING DATE: Herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/028,093
(B) FILING DATE: 09-OCT-1996
(viii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US96/16565
(B) FILING DATE: 09-OCT-1996
(ix) ATTORNEY/AGENT INFORMATION:
(A} NAME: Steffe) Eric K.
(B) REGISTRATION NUMBER: 36,68S
(C) REFERENCE/DOCKET NUMBER: 1488.056PC01/EKS/SGW
(x) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-37l-2600
(B) TELEFAX: 202-371-2540
(2) INFORMATION FOR SEQ ID NO: l:
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{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1138 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A} NAME/KEY: CDS
(B) LOCATION: 52..1020
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
TTCGGCACGA GAGCTCTTCT CACAGGACCA GCCACTAGCG CACCTCGAGC G ATG GCC 57
Met Ala
1
TAT GTC CCC GCA CCG GGC TAC CAG CCC ACC TAC AAC CCG ACG CTG CCT l05
Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr Leu Pro
10 15
TAC TAC CAG CCC ATC CCG GGC GGG CTC AAC GTG GGA ATG TCT GTT TAC 153
Tyr Tyr Gln Pro Ile Pro Gly Gly Leu Asn Val Gly Met Ser Val Tyr
20 25 30
ATC CAA GGA GTG GCC AGC GAG CAC ATG AAG CGG TTC TTC GTG AAC TTT 201
Ile Gln Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val Asn Phe
35 40 45 50
GTG GTT GGG CAG GAT CCG GGC TCA GAC GTC GCC TTC CAC TTC AAT CCG 249
Val Val Gly Gln Asp Pro Gly Ser Asp Val Ala Phe His Phe Asn Pro
55 60 65
CGG TTT GAC GGC TGG GAC AAG GTG GTC TTC AAC ACG TTG CAG GGC GGG 297
Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gln Gly Gly
70 75 80
AAG TGG GGC AGC GAG GAG AGG AAG AGG AGC ATG CCC TTC AAA AAG GGT 345
Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys Lys Gly
85 90 95
GCC GCC TTT GAG CTG GTC TTC ATA GTC CTG GCT GAG CAC TAC AAG GTG 393
Ala Ala Phe Glu Leu Val Phe Ile Val Leu Ala Glu His Tyr Lys Val
l00 105 110
GTG GTA AAT GGA AAT CCC TTC TAT GAG TAC GGG CAC CGG CTT CCC CTA 441
Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu Pro Leu
l15 120 125 130
CAG ATG GTC ACC CAC CTG CAA GTG GAT GGG GAT CTG CAA CTT CAA TCA 489
Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Gln Leu Gln Ser
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135 l40 l45
ATC AAC TTC ATC GGA GGC CAG CCC CTC CGG CCC CAG GGA CCC CCG ATG S37
Ile Asn Phe Ile Gly Gly Gln Pro Leu Arg Pro Gln Gly Pro Pro Met
I50 155 l60
ATG CCA CCT TAC CCT GGT CCC GGA CAT TGC CAT CAA CAG CTG AAC AGC 585
Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gln Gln Leu Asn Ser
165 170 175
CTG CCC ACC ATG GAA GGA CCC CCA ACC TTC AAC CCG CCT GTG CCA TAT 633
Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val Pro Tyr
180 185 190
TTC GGG AGG CTG CAA GGA GGG CTC ACA GCT CGA AGA ACC ATC ATC ATC 681
Phe Gly Arg Leu Gln Gly Gly Leu Thr Ala Arg Arg Thr Ile Ile Ile
195 200 205 210
AAG GGC TAT GTG CCT CCC ACA GGC AAG AGC TTT GCT ATC AAC TTC AAG 729
Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala Ile Asn Phe Lys
2l5 220 225
GTG GGC TCC TCA GGG GAC ATA GCT CTG CAC ATT AAT CCC CGC ATG GGC 777
Val Gly Ser Ser Gly Asp Ile Ala Leu His Ile Asn Pro Arg Met Gly
230 235 240
AAC GGT ACC GTG GTC CGG AAC AGC CTT CTG AAT GGC TCG TGG GGA TCC 825
Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp Gly Ser
245 250 255
GAG GAG AAG AAG ATC ACC CAC AAC CCA TTT GGT CCC GGA CAG TTC TTT 873
Glu Glu Lys Lys Ile Thr His Asn Pro Phe Gly Pro Gly Gln Phe Phe
260 265 270
GAT CTG TCC ATT CGC TGT GGC TTG GAT CGC TTC AAG GTT TAC GCC AAT 921
Asp Leu Ser Ile Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr Ala Asn
275 280 285 290
GGC CAG CAC CTC TTT GAC TTT GCC CAT CGC CTC TCG GCC TTC CAG AGG 969
Gly Gln His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe Gln Arg
295 300 30S
GTG GAC ACA TTG GAA ATC CAG GGT GAT GTC ACC TTG TCC TAT GTC CAG l017
Val Asp Thr Leu Glu Ile Gln Gly Asp Val Thr Leu Ser Tyr Val Gln
310 315 320
ATC TAATCTATTC CTGGGGCCAT AACTCATGGG AAAACAGAAT TATCCCCTAG 1070
Ile
GACTCCTTTC TAAGCCCCTA ATAAAATGTC TGAGGGTGTC TCATGAAAAA F~~;~1AAAAAAA 1l30
1138
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(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 323 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr
1 5 10 15
Leu Pro Tyr Tyr Gln Pro Ile Pro Gly Gly Leu Asn Val Gly Met Ser
20 25 30
Val Tyr Ile Gln Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val
35 40 45
Asn Phe Val Val Gly Gln Asp Pro Gly Ser Asp Val Ala Phe His Phe
50 55 60
Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gln
65 70 75 80
Gly Gly Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys
85 90 95
Lys Gly Ala Ala Phe Glu Leu Val Phe Ile Val Leu Ala Glu His Tyr
100 105 l10
Lys Val Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu
l15 l20 125
Pro Leu Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Gln Leu
130 l35 140
Gln Ser Ile Asn Phe Ile Gly Gly Gln Pro Leu Arg Pro Gln Gly Pro
145 1S0 155 l60
Pro Met Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gln Gln Leu
165 170 175
Asn Ser Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val
180 185 190
Pro Tyr Phe Gly Arg Leu Gln Gly Gly Leu Thr Ala Arg Arg Thr Ile
195 200 205
Ile Ile Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala Ile Asn
210 215 220
Phe Lys Val Gly Ser Ser Gly Asp Ile Ala Leu His Ile Asn Pro Arg
225 230 235 240
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Met Gly Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp
245 2S0 255
Gly Ser Glu Glu Lys Lys Ile Thr His Asn Pro Phe Gly Pro Gly Gln
260 265 270
Phe Phe Asp Leu Ser Ile Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr
275 280 285
Ala Asn Gly Gln His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe
290 295 300
Gln Arg Val Asp Thr Leu Glu Ile Gln Gly Asp Val Thr Leu Ser Tyr
305 310 315 320
Val Gln Ile
(2} INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1545 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 16..948
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
AGAGGCGGCG GAGAG ATG GCC TTC AGC GGT TCC CAG GCT CCC TAC CTG AGT 51
Met Ala Phe Ser Gly Ser Gln A1a Pro Tyr Leu Ser
1 5 10
CCA GCT GTC CCC TTT TCT GGG ACT ATT CAA GGA GGT CTC CAG GAC GGA 99
Pro Ala Val Pro Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly
15 20 25
CTT CAG ATC ACT GTC AAT GGG ACC GTT CTC AGC TCC AGT GGA ACC AGG 147
Leu Gln Ile Thr Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg
30 35 40
TTT GCT GTG AAC TTT CAG ACT GGC TTC AGT GGA AAT GAC ATT GCC TTC 195
Phe Ala Val Asn Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe
45 50 55 60
CAC TTC AAC CCT CGG TTT GAA GAT GGA GGG TAC GTG GTG TGC AAC ACG 243
His Phe Asn Pro Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr
65 70 75
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AGG CAG AAC GGA AGC TGG GGG CCC GAG GAG AGG AAG ACA CAC ATG CCT 291
Arg Gln Asn Gly Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro
80 85 90
TTC CAG AAG GGG ATG CCC TTT GAC CTC TGC TTC CTG GTG CAG AGC TCA 339
Phe Gln Lys Gly Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser
95 100 105
GAT TTC AAG GTG ATG GTG AAC GGG ATC CTC TTC GTG CAG TAC TTC CAC 387
Asp Phe Lys Val Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His
1l0 l15 120
CGC GTG CCC TTC CAC CGT GTG GAC ACC ATC TCC GTC AAT GGC TCT GTG 435
Arg Val Pro Phe His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val
l25 130 135 140
CAG CTG TCC TAC ATC AGC TTC CAG ACC CAG ACA GTC ATC CAC ACA GTG 483
Gln Leu Ser Tyr Ile Ser Phe Gln Thr Gln Thr Val Ile His Thr Val
l45 150 155
CAG AGC GCC CCT GGA CAG ATG TTC TCT ACT CCC GCC ATC CCA CCT ATG 531
Gln Ser Ala Pro Gly Gln Met Phe Ser Thr Pro Ala Ile Pro Pro Met
l60 165 170
ATG TAC CCC CAC CCC GCC TAT CCG ATG CCT TTC ATC ACC ACC ATT CTG 579
Met Tyr Pro His Pro Ala Tyr Pro Met Pro Phe Ile Thr Thr Ile Leu
175 180 185
GGA GGG CTG TAC CCA TCC AAG TCC ATC CTC CTG TCA GGC ACT GTC CTG 627
Gly Gly Leu Tyr Pro Ser Lys Ser Ile Leu Leu Ser Gly Thr Val Leu
190 l95 200
CCC AGT GCT CAG AGG TTC CAC ATC AAC CTG TGC TCT GGG AAC CAC ATC 675
Pro Ser Ala Gln Arg Phe His Ile Asn Leu Cys Ser Gly Asn His Ile
205 2l0 215 220
GCC TTC CAC CTG AAC CCC CGT TTT GAT GAG AAT GCT GTG GTC CGC AAC 723
Ala Phe His Leu Asn Pro Arg Phe Asp Glu Asn Ala Val Val Arg Asn
225 230 235
ACC CAG ATC GAC AAC TCC TGG GGG TCT GAG GAG CGA AGT CTG CCC CGA 771
Thr Gln Ile Asp Asn Ser Trp Gly Ser Glu Glu Arg Ser Leu Pro Arg
240 245 250
AAA ATG CCC TTC GTC CGT GGC CAG AGC TTC TCA GTG TGG ATC TTG TGT 819
Lys Met Pro Phe Val Arg Gly Gln Ser Phe Ser Val Trp Ile Leu Cys
255 260 265
GAA GCT CAC TGC CTC AAG GTG GCC GTG GAT GGT CAG CAC CTG TTT GAA 867
Glu Ala His Cys Leu Lys Val Ala Val Asp Gly Gln His Leu Phe Glu
270 275 280
TAC TAC CAT CGC CTG AGG AAC CTG CCC ACC ATC AAC AGA CTG GAA GTG 9l5
Tyr Tyr His Arg Leu Arg Asn Leu Pro Thr Ile Asn Arg Leu Glu Val
285 290 295 300
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GGG GGC ATC CAG TAGGCGGCTTCCTGGCCCTG968
GAC CTG ACC
CAT GTG
CAG ACA
Gly Gly Ile Gln
Asp Leu Thr
His Val
Gln Thr
305 310
GGGCCGGGGGCTGGGGTGTGGGGCAGTCTGGGTCCTCTCATCATCCCCACTTCCCAGGCCI028
CAGCCTTTCCAACCCTGCCTGGGATCTGGGCTTTAATGCAGAGGCCATGTCCTTGTCTGG1088
TCCTGCTTCTGGCTACAGCCACCCTGGAACGGAGAAGGCAGCTGACGGGGATTGCCTTCC1148
TCAGCCGCAGCAGCACCTGGGGCTCCAGCTGCTGGAAATCCTACCATCCCAGGAGGCAGG1208
CACAGCCAGGGAGAGGGGAGGAGTGGGCAGTGAAGATGAAGCCCCATGCTCAGTCCCCTC1268
CCATCCCCCACGCAGCTCCACCCCAGTCCCAAGCCACCAGCTGTCTGCTCCTGGTGGGAG1328
GTGGCCTCCTCAGCCCCTCCTCTCTGACCTTTAACCTCACTCTCACCTTGCACCGTGCAC1388
CAACCCTTCACCCCTCCTGGAAAGCAGGCCTGATGGCTTCCCACTGGCCTCCACCACCTG1448
ACCAGAGTGTTCTCTTCAGAGGACTGGCTCCTTTCCCAGTGTCCTTAAAATAAAGAAATG1508
AAAATGCTTGTTGGCAAAAA AAP.AA.AA 1545
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro
1 5 10 15
Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr
20 25 30
Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn
35 40 45
Phe Gln Thr Gly Phe Ser Gly Asn Asp I1e Ala Phe His Phe Asn Pro
50 55 60
Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly
65 70 75 80
Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly
85 90 95
Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val
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100 l05 l10
Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe
115 120 125
His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val Gln Leu Ser Tyr
130 135 140
Ile Ser Phe Gln Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro
145 150 155 160
Gly Gln Met Phe Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His
165 170 l75
Pro Ala Tyr Pro Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr
180 185 190
Pro Ser Lys Ser Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln
195 200 205
Arg Phe His Ile Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu
2l0 215 220
Asn Pro Arg Phe Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp
225 230 235 240
Asn Ser Trp Gly Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe
245 250 255
Val Arg Gly Gln Ser Phe Ser Val Trp Ile Leu Cys Glu Ala His Cys
26d 265 270
Leu Lys Val Ala Val Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg
275 280 285
Leu Arg Asn Leu Pro Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile
290 295 300
Gln Leu Thr His Val Gln Thr
305 3l0
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1479 base pairs
(B) TYPE: nucleic acid
{C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 118..l068
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
ACACCAGTCT TTGGGGCCAG TGCCTCAGTT TCAATCCAGG TAACCTTTAA ATGAAACTTG 60
CCTAAAATCT TAGGTCATAC ACAGAAGAGA CTCCAATCGA CAAGAAGCTG GAAAAGA 117
ATG ATG TTG TCC TTA AAC AAC CTA CAG AAT ATC ATC TAT AAC CCG GTA l65
Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val
1 5 10 15
ATC CCG TTT GTT GGC ACC ATT CCT GAT CAG CTG GAT CCT GGA ACT TTG 213
Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu
20 25 30
ATT GTG ATA CGT GGG CAT GTT CCT AGT GAC GCA GAC AGA TTC CAG GTG 261
Ile Val Ile Arg Gly His Val Pro Ser Asp AIa Asp Arg Phe Gln Val
35 40 45
GAT CTG CAG AAT GGC AGC AGT GTG AAA CCT CGA GCC GAT GTG GCC TTT 309
Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe
50 55 60
CAT TTC AAT CCT CGT TTC AAA AGG GCC GGC TGC ATT GTT TGC AAT ACT 357
His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr
65 70 75 80
TTG ATA AAT GAA AAA TGG GGA CGG GAA GAG ATC ACC TAT GAC ACG CCT 405
Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro
85 90 95
TTC AAA AGA GAA AAG TCT TTT GAG ATC GTG ATT ATG GTG CTA AAG GAC 453
Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp
l00 105 110
AAA TTC CAG GTG GCT GTA AAT GGA AAA CAT ACT CTG CTC TAT GGC CAC 50l
Lys Phe Gln Val Ala VaI Asn Gly Lys His Thr Leu Leu Tyr Gly His
115 120 l25
AGG ATC GGC CCA GAG AAA ATA GAC ACT CTG GGC ATT TAT GGC AAA GTG 549
Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val
130 135 140
AAT ATT CAC TCA ATT GGT TTT AGC TTC AGC TCG GAC TTA CAA AGT ACC 597
Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr
145 150 15S 160
CAA GCA TCT AGT CTG GAA CTG ACA GAG ATA GTT AGA GAA AAT GTT CCA 645
Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Val Arg Glu Asn Val Pro
165 170 175
AAG TCT GGC ACG CCC CAG CTT AGC CTG CCA TTC GCT GCA AGG TTG AAC 693
Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn
180 185 190
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ACC CCC ATG GGC CCT GGA CGA ACT GTC GTC GTT AAA GGA GAA GTG AAT 741
Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn
195 200 205
GCA AAT GCC AAA AGC TTT AAT GTT GAC CTA CTA GCA GGA AAA TCA AAG 789
Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys
2l0 2l5 220
GAT ATT GCT CTA CAC TTG AAC CCA CGC CTG AAT ATT AAA GCA TTT GTG 837
Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val
225 230 235 240
AGA AAT TCT TTT CTT CAA GAG TCC TGG GGA GAA GAA GAG AGA AAT ATT 885
Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile
245 250 255
ACC GCT TTC CCA TTT AGT CCT GGG ATG TAC TTT GAG ATG ATA ATT TAT 933
Thr Ala Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr
260 265 270
TGT GAT GTT AGA GAA TTC AAG GTT GCA GTA AAT GGC GTA CAC AGC CTG 981
Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu
275 280 285
GAG TAC AAA CAC AGA TTT AAA GAG CTC AGC AGT ATT GAC ACG CTG GAA 1029
Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu
290 29S 300
ATT AAT GGA GAC ATC CAC TTA CTG GAA GTA AGG AGC TGG TAGCCTACCT 1078
Ile Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp
305 3l0 315
ACACAGCTGC TACAAAAACC AAAATACAGA ATGGCTTCTG TGATACTGGC CTTGCTGAAA 1l38
CGCATCTCAC TGTCATTCTA TTGTTTATAT TGTTAAAATG AGCTTGTGCA CCATTAGGTC 1198
CTGCTGGGTG TTCTCAGTCC TTGCCATGAA GTATGGTGGT GTCTAGCACT GAATGGGGAA 1258
ACTGGGGGCA GCAACACTTA TAGCCAGTTA AAGCCACTCT GCCCTCTCTC CTACTTTGGC 1318
TGACTCTTCA AGAATGCCAT TCAACAAGTA TTTATGGAGT CCTACTATAT ACAGTAGCTA 1378
ACATGTATTG AGCACAGATT TTTTTGGTAA ACCTGTGAGG GCTAGGGTAT ATCCTTGGGA l438
ACAAACCAGA ATGTCCTGTC CCTTGAAAAA A 1479
(2) INFORMATION FOR SEQ ID H0:6:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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{xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val
1 5 10 15
Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu
20 25 30
Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val
35 40 45
Asp Leu G1n Asn Gly Sex Ser Val Lys Pro Arg Ala Asp Val Ala Phe
50 55 60
His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr
65 70 75 80
Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro
85 90 95
Phe Lys Arg Glu Lys Sex Phe Glu Ile Val Ile Met Val Leu Lys Asp
100 105 1l0
Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His
115 120 125
Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val
130 13S 140
Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr
145 150 155 160
Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Va1 Arg Glu Asn Val Pro
165 170 175
Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn
180 185 190
Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn
195 200 205
AIa Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys
210 215 220
Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val
225 230 235 240
Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile
245 250 255
Thr Ala Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr
260 265 270
Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu
275 280 285
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Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu
290 295 300
Ile Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp
305 310 315
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 1936 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAMEiKEY: CDS
{B) LOCATION: 1l8..717
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
ACACCAGTCT TTGGGGCCAG TGCCTCAGTT TCAATCCAGG TAACCTTTAA ATGAAACTTG 60
CCTAAAATCT TAGGTCATAC ACAGAAGAGA CTCCAATCGA CAAGAAGCTG GAAAAGA 117
ATG ATG TTG TCC TTA AAC AAC CTA CAG AAT ATC ATC TAT AAC CCG GTA 165
Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val
1 5 10 15
ATC CCG TTT GTT GGC ACC ATT CCT GAT CAG CTG GAT CCT GGA ACT TTG 213
Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu
20 25 30
ATT GTG ATA CGT GGG CAT GTT CCT AGT GAC GCA GAC AGA TTC CAG GTG 261
Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val
35 40 45
GAT CTG CAG AAT GGC AGC AGC ATG AAA CCT CGA GCC GAT GTG GCC TTT 309
Asp Leu Gln Asn Gly Ser Ser Met Lys Pro Arg Ala Asp Val Ala Phe
50 55 60
CAT TTC AAT CCT CGT TTC AAA AGG GCC GGC TGC ATT GTT TGC AAT ACT 357
His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr
65 70 75 80
TTG ATA AAT GAA AAA TGG GGA CGG GAA GAG ATC ACC TAT GAC ACG CCT 405
Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro
85 90 95
TTC AAA AGA GAA AAG TCT TTT GAG ATC GTG ATT ATG GTG CTG AAG GAC 453
Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp
l00 105 110
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AAA TTC GTG GCT GTA AAT GGA AAA CTG CTC TAT GGC CAC S01
CAG CAT ACT
Lys Phe Val AIa Val Asn Gly Lys Leu Leu Tyr Gly His
Gln His Thr
1l5 l20 125
AGG ATC CCA GAG AAA ATA GAC ACT ATT TAT GGC AAA GTG 549
GGC CTG GGC
Arg Ile Pro Glu Lys Ile Asp Thr Ile Tyr Gly Lys Val
Gly Leu Gly
130 135 l40
AAT ATT TCA ATT GGT TTT AGC TTC GAC TTA CAA AGT ACC 597
CAC AGC TCG
Asn Ile Ser Ile Gly Phe Ser Phe Asp Leu Gln Ser Thr
His Ser Ser
145 150 155 160
CAA GCA AGT CTG GAA CTG ACA GAG AGA GAA AAT GTT CCA 645
TCT ATA AGT
Gln Ala Ser Leu Glu Leu Thr Glu Arg Glu Asn Val Pro
Ser IIe Ser
16S 170 l75
AAG TCT ACG CCC CAG CTT GTG AGT GCC TGG GTT ATT TCA 693
GGC ATT TTT
Lys Ser Thr Pro Gln Leu Val Ser Ala Trp Val Ile Ser
Gly Ile Phe
180 185 190
TGT GGA TTT TAT AAA GTT GCA TAGAAAATGA 747
ATA ACAGTTTAAA CCGTGGAGGG
Cys Gly Phe Tyr Lys Val Ala
Ile
195 200
CAGCTTCATTCATTCCATTC CTTACTGTAG AACTGTTTCCCTACAGCCTA GTAATAGAGG807
AGGAGACATTTCTAAAATCG CACCCAGAAC TGTCTACACCAAGAGCAAAG ATTCGACTGT867
CAATCACACTTTGACTTGCA CCAAAATACC ACCTATGAACTATGTGTCAA AGGGTTTGAA927
GAGCACCAAATTTTCTTAAC TCTATATAAA AATTAAGTTGTAATGAGCTG TTACGAGTAA987
CCTGTATCCACAATAGAGGC CCAAAGCAGC CCCCTCTGCATTTGTGTGCC GTCCCTGGAC1047
GGATTCGAGAGTCAACCAGG CCTGCCTCTG AGCCATTTCTGTGTATTTCC TCAGCACCTCl107
CCTGCTTGGCTGCTTCCCCT TCAGGCAGAA CACAGTACTGCCTCAGACCC CAGGCACAGG1167
GGGCCTTCCTGGCGTGTTTC ACTCATACAG AGGGCATCGGGTCCCACCCT GTCACTCATT1227
TCATCGTCTA AGGGACAGTG CTGCTGCAGG1287
AAATGTAATC
ATGTGTGTTT
GCTTCGAGCC
GGACCCAGCTGGGACCAAGG CAGACTGTCT CTCCCCTCCTGGGATTTACA GGGTCATGGC1347
TCTGAAACATTCCGTAGTGT TCTTTGGACA CGAGTTTTCCCTGGAGATCG CTTTCTGCAG1407
GCTCTTGGTCCTGACTGTGG CTTCTTTTCA GAGGCTGCCATTTCGCTGCA AGGTTGAACA1467
CCCCCATGGGCCCTGGACGA ACTGTCGTCG TTAAAGGAGAAGTGAATGCA AATGCCAAAA1527
GCTTTAATGTTGACCTACTA GCAGGAAAAT CAAAGGATATTGCTCTACAC TTGAACCCACl587
GCCTGAATATTAAAGCATTT GTAAGAAATT CTTTTCTTCAGGAGTCCTGG GGAGAAGAAG1647
AGAGAAATATTACCTCTTTC CCATTTAGTC CTGGGATGTACTTTGAGATG ATAATTTATT1707
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GTGATGTTAG AGAATTCAAG GTTGCAGTAA ATGGCGTACA CAGCCTGGAG TACAAACACA 1767
GATTTAAAGA GCTCAGCAGT ATTGACACGC TGGAAATTAA TGGAGACATC CACTTACTGG 1827
AAGTAAGGAG CTGGTAGCCT ACCTACACAG CTGCTACAAA AACCAAAATA CAGAATGGCT 18B7
TCTGTGATAC TGGCCTTGCT GAAACGCAAA P,~~J~1AAAAAA AAAAAAAAA l936
(2} INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val
1 5 10 15
Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu
20 25 30
Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val
35 40 45
Asp Leu Gln Asn Gly Ser Ser Met Lys Pro Arg Ala Asp Val Ala Phe
50 55 60
His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr
65 70 75 80
Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro
85 90 95
Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp
100 105 110
Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His
115 120 l25
Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val
l30 135 140
Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr
l45 150 l55 160
Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro
l65 170 l75
Lys Ser Gly Thr Pro Gln Leu Val Ser Ile Phe Ala Trp Val Ile Ser
180 185 19d
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Cys Gly Ile Phe Tyr Lys Val Ala
195 200
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Met Thr Gly Glu Leu Glu Val Lys Asn Met Asp Met Lys Pro Gly Ser
1 5 10 15
Thr Leu Lys Ile Thr Gly Ser Ile Ala Asp Gly Thr Asp Gly Phe Val
20 25 30
Ile Asn Leu Gly Gln Gly Thr Asp Lys Leu Asn Leu His Phe Asn Pro
35 40 45
Arg Phe Ser Glu Ser Thr Ile Val Cys Asn Ser Leu Asp Gly Ser Asn
50 55 60
Trp Gly Gln Glu Gln Arg Glu Asp His Leu Cys Phe Ser Pro Gly Ser
65 70 75 80
Glu Val Lys Phe Thr Val Thr Phe Glu Ser Asp Lys Phe Lys Val Lys
85 90 95
Leu Pro Asp Gly His Glu Leu Thr Phe Pro Asn Arg Leu Gly His Ser
100 105 1l0
His Leu Ser Tyr Leu Ser Val Arg Gly Gly Phe Asn Met Ser Ser Phe
l15 l20 12S
Lys Leu Lys Glu
l30
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 250 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn
1 5 10 15
Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Ala Gly
20 25 30
Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln
35 40 45
Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr His
50 55 60
Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly Val Tyr Pro
65 70 75 BO
Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser Ser Gly Gln Pro Ser
85 90 95
Ala Pro Gly Ala Tyr Pro Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly
100 105 110
Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val Val Pro
l15 l20 125
Arg Met Leu Ile Thr Ile Leu Gly Thr Val Lys Pro Asn Ala Asn Arg
130 135 140
Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val Ala Phe His Phe Asn
l45 150 155 l60
Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys
165 170 175
Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln Ser Val Phe Pro Phe
180 185 l90
Glu Ser Gly Lys Pro Phe Lys Ile Gln Val Leu Val Glu Pro Asp His
195 200 205
Phe Lys Val Ala Val Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg
210 215 220
Val Lys Lys Leu Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile
225 230 235 240
Asp Leu Thr Ser Ala Ser Tyr Thr Met Ile
245 250
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 324 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Ala Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr
1 5 10 15
Leu Pro Tyr Lys Arg Pro Ile Pro Gly Gly Leu Ser Val Gly Met Ser
20 25 30
Ile Tyr Ile Gln Gly Ile Ala Lys Asp Asn Met Arg Arg Phe His Val
35 40 45
Asn Phe Ala Val Gly Gln Asp Glu Gly Ala Asp Ile Ala Phe His Phe
50 55 60
Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Met Gln
65 70 75 80
Ser Gly Gln Trp Gly Lys Glu Glu Lys Lys Lys Ser Met Pro Phe Gln
85 90 95
Lys Gly His His Phe Glu Leu Val Phe Met Val Met Ser Glu His Tyr
l00 105 l10
Lys Val Val Val Asn Gly Thr Pro Phe Tyr Glu Tyr Gly His Arg Leu
11S 120 125
Pro Leu Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Glu Leu
130 135 140
Gln Ser Ile Asn Phe Leu Gly Gly Gln Pro Ala Ala Ser Gln Tyr Pro
145 150 155 160
Gly Thr Met Thr Ile Pro Ala Tyr Pro Ser Ala Gly Tyr Asn Pro Pro
I65 170 175
Gln Met Asn Ser Leu Pro Val Met Ala Gly Pro Pro Ile Phe Asn Pro
l80 I85 190
Pro Val Pro Tyr Val Gly Thr Leu Gln Gly Gly Leu Thr Ala Arg Arg
195 200 205
Thr Ile Ile Ile Lys Gly Tyr Val Leu Pro Thr Ala Lys Asn Leu Ile
210 2l5 220
Ile Asn Phe Lys Val Gly Ser Thr Gly Asp Ile Ala Phe His Met Asn
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225 230 235 240
Pro Arg Ile GIy Asp Cys Val Val Arg Asn Ser Tyr Met Asn Gly Ser
245 250 255
Trp Gly Ser Glu Glu Arg Lys I1e Pro Tyr Asn Pro Phe Gly Ala Gly
260 265 270
Gln Phe Phe Asp Leu Ser Ile Arg Cys Gly Thr Asp Arg Phe Lys Val
27S 280 285
Phe Ala Asn Gly Gln His Leu Phe Asp Phe Ser His Arg Phe Gln Ala
290 295 300
Phe Gln Arg Val Asp Met Leu Glu Ile Lys Gly Asp Ile Thr Leu Ser
305 310 315 320
Tyr Val Gln Ile
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l45 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Ser Ser Phe Ser Thr Gln Thr Pro Tyr Pro Asn Leu Ala Val Pro
1 5 10 15
Phe Phe Thr Ser Ile Pro Asn Gly Leu Tyr Pro Ser Lys Ser Ile Val
20 25 30
Ile Ser Gly Val Val Leu Ser Asp Ala Lys Arg Phe Gln Ile Asn Leu
35 40 45
Arg Cys Gly Gly Asp Ile Ala Phe His Leu Asn Pro Arg Phe Asp Glu
50 55 60
Asn Ala VaI Val Arg Asn Thr Gln Ile Asn Asn Ser Trp Gly Pro Glu
65 70 75 80
Glu Arg Ser Leu Pro Gly Ser Met Pro Phe Ser Arg Gly Gln Arg Phe
85 90 95
Ser Val Trp IIe Leu Cys Glu Gly His Cys Phe Lys Val Ala Val Asp
100 1D5 l10
CA 02268022 1999-04-09
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-69-
Gly Gln His Ile Cys Glu Tyr Ser His Arg Leu Met Asn Leu Pro Asp
115 120 l25
Ile Asn Thr Leu Glu Val Ala Gly Asp Ile Gln Leu Thr His Val Glu
l30 l35 140
Thr
l45
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 136 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Met Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro
1 5 10 15
Gly Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg
20 25 30
Phe His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala
35 40 45
Leu His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser
50 55 60
Lys Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro
65 70 75 80
Phe Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp
85 90 9S
Gly Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His
100 l05 1l0
Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val
115 120 125
Gln Leu Asp Ser Val Arg Ile Phe
l30 135
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids
CA 02268022 1999-04-09
, WO 98I15624 PCT/US97/18261
-70-
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Ala Asp Gly Phe Ser Leu Asn Asp Ala Leu Ala Gly Ser Gly Asn
1 5 10 15
Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Gly Ala
20 25 30
Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln Ala
35 40 45
Pro Pro Gly Gly Tyr Pro Gly Gln Ala Pro Pro Ser Ala Tyr Pro Gly
50 55 60
Pro Thr Gly Pro Ser Ala Tyr Pro Gly Pro Thr Ala Pro Gly Ala Tyr
65 70 75 80
Pro Gly Pro Thr Ala Pro Gly Ala Phe Pro Gly Gln Pro Gly Gly Pro
85 90 95
Gly Ala Tyr Pro Ser Ala Pro Gly Ala Tyr Pro Ser Ala Pro Gly Ala
100 105 110
Tyr Pro Ala Thr Gly Pro Phe Gly Ala Pro Thr Gly Pro Leu Thr Val
115 120 125
Pro Tyr Asp Met Pro Leu Pro Gly Gly Val Met Pro Arg Met Leu Ile
Z30 135 140
Thr Ile Ile Gly Thr Val Lys Pro Asn Ala Asn Ser Ile Thr Leu Asn
l45 150 l55 l60
Phe Lys Lys Gly Asn Asp Ile Ala Phe His Phe Asn Pro Arg Phe Asn
165 170 l75
Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys Gln Asp Asn Asn
180 185 l90
Trp Gly Arg Glu Glu Arg Gln Ser Ala Phe Pro Phe Glu Ser Gly Lys
195 2Q0 205
Pro Phe Lys Ile Gln Val Leu Val Glu Ala Asp His Phe Lys Val Ala
210 215 220
Val Asn Asp Val His Leu Leu Gln Tyr Asn His Arg Met Lys Asn Leu
225 230 235 240
CA 02268022 1999-04-09
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-71-
Arg Glu Ile Ser Gln Leu Gly Ile Ile Gly Asp Ile Thr Leu Thr Ser
245 250 255
Ala Ser His Ala Met Ile
260
(2) INFORMATION FOR SEQ ID N0:15:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Leu Ser Leu Ser Asn Leu Gln Asn IIe Ile Tyr Asn Pro Thr Ile
1 5 10 15
Pro Tyr Val Ser Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile
20 25 30
Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp
35 40 45
Phe Gln His Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His
50 55 60
Phe Asn Pro Arg Phe Lys Arg Ser Asn Cys Ile Val Cys Asn Thr Leu
65 70 75 80
Thr Asn Glu Lys Trp Gly Trp Glu Glu Ile Thr His Asp Met Pro Phe
85 ~ 90 95
Arg Lys Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asn Lys
l00 105 1l0
Phe His Val Ala Val Asn Gly Lys His Ile Leu Leu Tyr Ala His Arg
115 120 125
Ile Asn Pro Glu Lys Ile Asp Thr Leu Gly Ile Phe Gly Lys Val Asn
130 135 l40
Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu
145 150 155 160
Thr Ser Thr Leu Gly Leu Thr Gln Ile Ser Lys Glu Asn Ile Gln Lys
165 l70 17S
Ser Gly Lys Leu His Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala
CA 02268022 1999-04-09
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-72-
180 185 190
Ser Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Thr
195 200 205
Asn Ala Thr Ser Phe Asn Val Asp Leu Val Ala Gly Arg Ser Arg Asp
210 215 220
Ile Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg
225 230 235 240
Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr
245 250 255
Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys
260 265 270
Asp Val Arg Glu Phe Lys Val Ala VaI Asn Gly Val His Ser Leu Glu
275 2B0 285
Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ala Val
290 295 300
Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp
305 310 315
(2} INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu
1 5 10 15
Cys Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Va1
20 25 30
Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro
35 40 45
Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys
50 55 60
Asp Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe
65 70 75 80
CA 02268022 1999-04-09
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-?3-
Gln Pro Gly Ser Val Ala Glu Val Cys I1e Thr Phe Asp Gln Ala Asn
85 90 95
Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg
l00 l05 l10
Leu Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys
115 120 125
Ile Lys Cys Val Ala Phe Asp
l30 135
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Leu Ser Leu Ser Asn Leu Gln Asn Ile Ile Tyr Asn Pro Thr Ile
1 5 10 15
Pro Tyr Val Ser Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile
20 25 30
Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp
35 40 45
Phe Gln His Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His
50 55 60
Phe Asn Pro Arg Phe Lys Arg Ser Asn Cys ile Val Cys Asn Thr Leu
65 70 75 80
Thr Asn Glu Lys Trp Gly Trp Glu Glu Ile Thr His Asp Met Pro Phe
85 90 95
Arg Lys Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asn Lys
100 105 110
Phe His Val Ala Val Asn Gly Lys His Ile Leu Leu Tyr Ala His Arg
115 120 125
Ile Asn Pro Glu Lys Ile Asp Thr Leu Gly Ile Phe Gly Lys Val Asn
l30 135 140
Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu
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145 150 155 160
Thr Ser Thr Leu Gly Leu Thr Gln Ile Ser Lys Glu Asn Ile Gln Lys
I65 l70 175
Ser Gly Lys Leu His Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala
l80 1S5 190
Ser Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Thr
195 200 205
Asn Ala Thr Ser Phe Asn Val Asp Leu Val Ala Gly Arg Ser Arg Asp
210 215 220
IIe Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg
225 230 235 240
Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr
245 25d 255
Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys
260 26S 270
Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu
275 280 285
Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ala Val
290 295 300
Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp
305 3l0 315
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 499 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
AATTCGGCAC GAGAGCTCTT NTCACAGGAC CAGCCACTAG CGCANCTCGA GCGATGGCCT 60
ATGTCCCCGC ACCGGGCTAC CAGCCCACCT ACAACCCGAC GCTGCCTTAC TACCAGCCCA 120
TCCCGGGCGG GCTCAACGTG GGAATGTCTG TTTACATCCA AGGAGTGGCC AGCGAGCACA 180
TGAAGCGGTT CTTCGTGAAC TTTGTGGTTG GGCAGGATCC GGGCTCAGAC GTCGCCTTCC 240
CA 02268022 1999-04-09
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-75-
ACTTCAATCC GCGGTTTGAC GGCTGGGACA AGGTGGTCTT CAACACGTTG CAGGGCGGGA 300
AGTGGGGCAG CGAGGAGAGG AAGAGGAGCA TGCCCTTCAA AAAGGGTGCC GCCTTTGAGC 360
TTGGTCTTCA TAGTCCTNGG TTGAGCACTA CAAGGTNGTN GTAAATGGAA TCCCTCTATG 420
ANTAGGGGAC CGNTTTCCCT ANAATTGTAA CCANCTNNAA TTGATGGGNN TCAATTAATN 480
ATCAATTATT GGNGGCANC 499
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 391 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:19:
AGTGGATGGG GATCTGCAAC TTCAATCAAT CAACTTCATCGGAGGCCAGC CCCTCCGGCC60
CCAGGGACCC CCGATGATGC CACCTTACCC TGGTCCCGGACATTGCCATC AACAGCTGAAl20
CAGCCTGCCC ACCATGGAAG GACCCCCAAC CTTCAACCCGCCTGTGCCAT ATTTNGGGAG180
GCTGCAAGGA GGGCTCACAG CTCGAAGAAC CATCATCATCAAGGGCTATG TGCCTCCCAC240
AGGCAAGAGC TTTGCTATCA ACTTCAAGGT GGGCTCCTCAGGGGACATAG CTCTGCACAT300
TAATCCCCGC ATGGGCAACG GTACCGTGGT CCGGAACAGCCTTCTTGAAT GGTTCGTGGG360
GTTNCGAGGA GAAGAAGNTC ACCCACAACC C 391
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
CCGGCCCCAG GGACCCCCGA TGATGCCACC TTACCCTGGT CCCGGACATT GCCATCAACA 60
CA 02268022 1999-04-09
, WO 98l15624 PCTlUS97118261 _
-76-
GCTGAACAGC CTGCCCACCA TGGAAGGACC CCCAACCTTC AACCCGCCTG TGCCATATTT 120
CGGGAGGCTG CAAGGAGGGC TCACAGCTCG AAGAACCATC ATCATCAAGG GCTATGTGCC 1B0
TCCCACAGGC AAGAGCTTTG CTATCAACTT CAAGGTGGGC TCCTCAGGGG ACATAGCTCT 240
GCACATTAAT CCCCGCATGG GCAACGGTAC CGTGGTCCGG AACAGNCTTC TGAATGGCTC 300
GTGGGGATNC GAGGAGAAGG AAGGTCANCC ACAANCCATT TTGTNCCGGA CANTTTTTTT 360
NATCTGTCCA NTTGGTTGTG GTTTGGATCG TTTCAAGGTT TAAGGCAATG GCCAGAACTT 420
TTT 423
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:21:
AATTCGGCAC GAGCACAGGC AAGAGCTTTG CTATCAACTTCAAGGTGGGC TCCTCAGGGG60
ACATAGCTCT GCACATTAAT CCCCGCATGG GCAACGGTACCGTGGTCCGG AACAGCCTTC120
TGAATGGCTC GTGGGGATCC GAGGAGAAGA AGATCACCCACAACCCATTT GGTCCCGGAC180
AGTTCTTTGA TCTGTCCATT CGCTGTGGCT TGGATCGCTTCAAGGTTTAC GGCAATGGCC240
AGCACCTCTT TGACTTTGCC CATCGNCTCT CGGCCTTCCAGAGGGTGGAC ANATTNGAAA300
TCCAGGGTGA TGTCAACTTG TCCTATGTCC AGATCTAATCTTATTCCTGG GGCCATAATT360
CATGGGAAAC AGATTATNCN CTAGGGTTCT TTTTTAGGCCCTAATAAAAT GTCTTAGGGG420
GGTAAAAAAA AAAA 434
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02268022 1999-04-09
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-??-
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
CTTCAATCCG CGGTTTGACG GCTGGGACAA GGTGGTCTTC AACACGTTGC AGGGCGGGAA 60
GTGGGGCAGC GAGGAGAGGA AGAGGAGCAT GCCCTTCAAA AAGGGTGCCG CCTTTAAGCT l20
GGTCTTCATA GTCCTGGCTG AGCACTACAA GGTGGTGGTA AATGGAAATC CCTTCTATGA l80
GTACGGGCAC CGGCTTCCCC TACAGATGGT CACCCACCTG CAAGTGGATG GGGATCTNCA 240
ACTTCAATCA ATCAACTTCA TCGGGAGGNC AGCCCNTCCG GCCCCAGGGA CCCCCGATGA 300
TGCCACCTTA CCCTGGTNCC GGACATTGGC CATCAGCAGT TGAACAGCTG TCCA 354
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 329 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GTGGTCCGGA ACAGCCTTCT GAATGGCTCG TGGGGATCCG AGGAGAAGAA GATCACCCAC 60
AACCCATTTG GTCCCGGACA GTTCTTTGAT CTGTCCATTC GCTGTGGCTT GGATCGCTTC l20
AAGGTTTACG CCAATGGCCA GCACCTCTTT GACTTTGCCC ATCGCCTCTC GGCCTTCCAG 180
AGGGTGGACA CATTGGAAAT CCAGGGTGAT GTCACCTTGT CCTATGTCCA GATCTAATCT 240
ATTNCTGGGG CCATAACTCA TGGGAAAACA GAATTATCCC CTAGGACTCC TTTCTAAAGC 300
CCNCTAATAA AAANGTCTGA GGGTGTCTC 329
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02268022 1999-04-09
WO 98I15624 PCTIUS97/18261
_78_
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GCGGGCTCAA CGTGGGAATG TCTGTTTACA TCCAAGGAGT GGCCAGCGAG CACATGAAGC 60
GGTTCTTCGT GAACTTTGTG GTTGGGCAGG ATCCGGGCTC AGACGTCGCC TTCCACTTCA 120
ATCCGCGGTT TGACGGCTGG GACAAGGTGG TCTTCAACAC GTTGCAGGGC GGGAAGTGGG 180
GCAGCNAGGA GAGGAAGAGG AGCATGCCCT TCAAAAAGGG TGCCGCCTT 229
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GGAAGAGGAG CATGCCCTTC AAAAAGGGTG CCGCCTTTAA CCTGGTNTTC ATAGTCCTGG 60
CTGAGCACTA CAAGGTGGTG GTAAATGGAA ATCCCTTCTA TNAGTACGGG CACCGGCTTC 120
CCCTACAGAT GGTCACCCAC CTGCAAGTGG ATGGGGATCT GCAACTTCAT TCATTCAACT 180
TCATCGGAGG CCAG 194
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 499 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:26:
AATTCCGTTCTCTACTCCCGCCATCCCACCTATAATGTACCCCCACCCCG CCTATCCAAT60
GCCTTTAATCACCACCATTCTGGGAGGGCTGTACCCATCCAAGTCCATCC TCCTGTAAGG120
CACTTGCCTGCCCAGTGCTCANAGGTTCCACATCAACCTGTGCTCTGGGA AACCACATCG180
CCTTCCACCTGNAACCCCCGTTTTGAATGAGAATGCTGTGGTCCGCAACA CCCAGATNGA240
CA 02268022 1999-04-09
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-79-
CAACTCCTGG GGGTCTGAGG AGCGAAGTGT GCCCCGAAAA ATGCCCTTGG TNCGTGGCCA 300
GAGGTTNTNA GGTGGATCTT GTGTGAAGTT CAATGNGTNC AAGTGGGCCT GGATGGTNAG 360
NANTGTTTGN ATNATTANNC TGGGNTTGNG GNAACTGNGC AANNTTNAAC AGATNGNAGT 420
TGGGGGGGNG ANANTCAGNT GNACCGTTTT GNAGNNATAG GGGGNTTTNT TGGCCTTGGG 480
GGGGGGGGTT GGGGTTTTG 499
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 376 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
CTTTTGCCAA CAAGCATTTT NATTTCTTTA TTTTAAGGAC ACTGGGAAAG GAGCCAGTCC 60
CCTGAAGAGA ACACTCTGGT CAGGTGGTGG AGGCCAGTGG GAAGCCATCA GGCCTGCTTT I20
CCAGGAGGGG TGAAGGGTTG GTGCACGGTG CAAGGTGAGA GTGAAGGTTA AAGGTCAGAG 180
AGGAGGGGCT GAGGAGGCCA CCTTCCACCA GGAGCAGACA GCTGGTGGCT TGGGAACTGG 240
GGTGGAGCTG CGTGGGGGAT GGGAAGGGGA CTGAGCATGG GGCTTCATCT TNCACTGCCC 300
ACTCCTGCCC TCTTCCCTGG CTGTGCCTGC CTNCCTGGGA TGGTAGGGTT TCCANCANTT 360
GGAGGCCCCA NGTGCT 376
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
TTCAGATCAC TGTCAATGGG ACCGTTCTCA GCTCCAGTGG AACCAGGTTT NCTGTGAACT 60
CA 02268022 1999-04-09
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_8Q_
TTCAGACTGG CTTCAGTGGA AATAACATTG CCTTCCACTT CAACCCTCGG TTTGAAGATG 120
GAGGGTACGT GGTGTGCACA GNAGGCAGAA CGGAAGCTGG GGGCCCGAGG AGAGGAAGAC l80
ACACATGCCT TTCCAGAAGG GGATGCCCTT TAACCTCTGC TTCCTGGTGC AGAGCTCAGA 240
TTTCAAGGTG ATGGTGAACG GGATCCTCTT CGTGCAGTAC TT 282
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 274 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GTGCAGAGCG CCCCTGGACA GATGTNCTCT ACTCCCGCCA TCCCACCTAT GATGTACCCC 60
CACCCCGCCT ATCCGATGCC TTTNAACACC ACCATTCTGG GAGGGCTGTA CCCATCCAAG 120
ATCCATCCTC CTGTCAGGCA CTGTCCTGCC CAGTGCTCAG AGGTTCCACA TCAACCTGTG 180
CTCTGGGAAC CACATCGCCT TCCACCTGAA CCCCCGTTTT GATGAGAATG CTGTGGTCCG 240
CAACACCCAG ATCGACAAAT TCCTGGGGGG TCTT 274
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi} SEQUENCE
DESCRIPTION:
SEQ ID
N0:30:
CTTTTGCCAACAAGCATTTTNATTTCTTTATTTTAAGGAC ACTGGGAAAG GAGCCAGTCC60
CCTGAAGAGAACACTCTGGTCAGGTGGTGGAGGCCAGTGG GAAGCCATCA GGCCTGCTTT120
CCAGGAGGGGTGAAGGGTTGGTGCACGGTGCAAGGTGAGA GTNAAGGTTA AAGGTCAGAG180
AGGAGGGGCTGAGGAGGCCACCTTCCACCAGGAGCAGACA GCTGGTGGCT TGGGAACTGG240
CA 02268022 1999-04-09
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-81-
GGTGGGAGCT GTCGTNGGGG GATGGNAAGG GGACTGAGCC ATGGGGGCTT TCATCTTNCA 300
CTGCCCACTC CTGCCCTTTT CCCTGGTTTG TGNCTGNCCT TC 342
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
CCTGCTTCTG GCTACAGCCA CCNTGGAACG GAGAAGGCAG CTGACGGGGA TTGCCTTCNT 60
CAGCCGCAGC AGCACCTGGG GCTCCAGCTG CTGGAATCNT ACCATCCCAG GAGGCAGGCA 120
CAGCCAGGGA GAGGGGAGGA GTGGGCAGTG AAGATNAAGC CCCATGCTCA GTCCCCTCCC 180
ATCCCCCACG CAGCTCCACC CCAGTTCCAA GNCACCAGCT GTCTGCTCCT GGTGGGAGGT 240
GGCCTC 246
(2} INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 228 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
GGCANAGCAG AGGTGTGGAT CTTNTNTAAA GCTCACTGCC TCAAGGTGGC CGTGGATGGT 60
CAGCACCTGT TTAAATACTA CCATCGCCTG AGGAACCTGC CCACCATCAA CAGACTGGGA 120
GTGGGGGGCG AACATCCAGC TGACCCATGT GCAGACATAG GCGGCTTCCT GGCCCTGGGG 180
CGGGGGCTNA GNTTTGGGGN AGTCTGGGTC CTNTAATNAT CCNCANTT 22B
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
CA 02268022 1999-04-09
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-82-
(A) LENGTH: l61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
TTCCCTCTAC AAAGGACTTC CTAGTGGGTG TNAAAGGCAG CGGTGGCCAC ANAGGCGGCG 60
GAGAGATGGC CTTCAGCGGT TCCCAGGCTC CCTACCTGAG TCCAGCTGTC CCCTTTTTTG 120
GGACTATTCA AGGAGGTCTC CAGGACGGAC TTCAGATCAC T 161
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 306 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
CTCTGTGCAG CTGTCCTACA TCAGCTTCCA GGNNAGACTG TCCACCTGGC ACCGGTNCCA 60
GGGGCGGGGA ATGCGGGGNG NAGCGTAGTT GATACTGAAG NCNCTGATGG GTGGGGCNNA 120
AGNCANATCT CCTNACCCAG GTCACTCTGG GGGACAACCT CTGGCTTCCC TGTCCCAGTA 180
CCTGGCTGNC NACTTCTCCT CTGTGAACTC TGANCCCTCC TTCTGTGTTT ACTGTCTCTG 240
TCCGGAACAA CTGCCTTGGT CTCCCAGANT GCTCAGGTGA CCCTTTNTTN TTTCNACCCT 300
TCAATT 306
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:35:
CTCATACAGA GGGCATCGGG TCCCACCCTG TCACTCATTTCATCGTCTAA AATGTAATCA60
TGAGTGTTTG CTTCGAGCCA GGGACAGTNC TGCTGCAGGGGACCCAGCTG GGACCAAGGC120
AGACTGTCTC TCCCCTCCTG GGATTTACAG GGTCATGGCTCTGAAACATT CTGTAGTGTTl80
CTTTGAACAC GAGTTTTCCC TGGAGATCGC TTTCTGCAGGCCTCTTGGTC CTGACTGTGG240
CTTCTTTTCA GAGCCTGCCA TTCGCTGCAA GGTTGAACANCCCCATGGGC CCTGGGACGA300
ACTGTCGTCG TTAAAAGGAG AAGTGAATGC AAATGNCCAAAAAGCTTTTA ATGTTTGACC360
TACTAGCAGG AAATCAAAGG GTATTGCNTC TTACAATTGNACCCAGGCTG AATATTAAAG420
CATTTTAAAG AATTCTTTTT CTTCAGGAG 449
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
TTCAATCCTC GTTTCAAAAG GGCCGGCTGC ATTGTTTGCA ATACTTTNAT AAATGAAA.AA 60
TGGGGACGGG AAGAGATCAC CTATGACACG CCTTTCAAAA GAGAAAAGTC TTTTNAGATC 120
GTAATTATGG TGCTGAAGGA CAAATTCCAG GTGGCTGTAA ATGGAAAACA TACTCTGCTC 1$0
TATGGCCACA GGATCGGCCC AGAGAAAATA GACACTCTGG GCATTTATGG CAAAGTGAAT 240
ATTCACTCAA TTGGTTTTAG CTTCA 265
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 353 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:37:
AAGCCACTCTGCCCTCTCTCCTACTTTGGCTGACTCTTCAAGAATGCCATTCAACAAGTA 60
TTTATGGAGTACCTACTATAATACAGTAGCTAACATGTATTGAGCACAGATTTTTTTTGG 120
TAAAACTGTGAGGAGCTAGGATATATACTTGGTGAAACAAACCAGTATGTTCCCTGTTCT 180
CTTGAGCTTCGACTCTTCTGTGCTCTATTGCTGCGCACTGCTTTTTCTACAGGCATTACA 240
TCAACTCCTAAGGGGTCCTCTGGGGATTAGTTAAGCAGCTATTTAAATCACCCGAAGGAC 300
ACTTAATTTACAGATGACACAANTCCTTTCCCCAGTGATTCAACTGTTCATAA 353
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
GAAACACCAG TNTTTGGGGC CAGTNCCTCA NTTTCAATCC AGGTAACCTT TAANTGAAAC 60
TTGCCTAAAA TNTTAGGTCA TACACAGAAG AGACTCCAAT CGACAAGAAG CTGGAAAAGA 120
ATGATGTTGT CCTTAAACAA CCTACAGANT ATCATCTATA ACCCGGTAAT CCCGTTTNTT 180
GGCACCATTC CTGATCAGCT GGATCCTGGA ACTTTGATTG TAATACGTGG GCAT 234
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 344 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
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ACACGCTGGA AATTAATGGA GACATCCACT TACTGGAAGT AAGGNGNTGG TAGCCTACCT 60
ACACAGCTGC TACAAAAACC AAAATACAGA ATGGCTTCTG TGATACTGGC CTTGCTGAAA 120
CGCATCTCAC TGTCATTCTA TTGTTTATAT TGTTAAAATG AGCTTGTGCA CCATTAGGTC 180
CTGCTGGGTG TTCTCAGTCC TTGCCATGAA GTATGGTGGT GTCTAGCACT GAATGGGGAA 240
ACTGGGGGCA GCAACACTTA TAGCCAGTTA AAGCCACTCT GCCCTCTCTC CTACTTTGGG 300
CTGACTCTTC AAGAATGCCA TTCAACAAGT ATTTATGGGG TACC 344
(2) INFORMATION FOR SEQ ID N0:40:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 502 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:40:
AATTCGGCAN AGCTTCAAAC CTTTGAGACA TAGTTCATAGGTGGTATTTT GGTGCAAGTC60
AAAGTGTGAT NGACAGTCGA ATNTTTGCTC TTGGTGTAGACAGTTCTGGG TGCGATTTTA120
GAAATGTCTG CTCCTCTATT ACTAGGCTGT NGGGAAACAGTTCTACAGTA AGGAATGGAA180
TGANATGAAG CTGCCCTCCA CGGTTTAAAC TGTTCATTTTCTATGCAACT TTATAAAATA240
TTCCACATGA ANTAACCCAG GCAAAAATAC TTCACAGGCTGGGGGGCGTG GCCAGANCTT300
TGGGAACCTA TTGGGAAAAG GAAACCAAAN CACANCAATGTTTAGAAGGG GGAAGGATTT360
TTAGTTTATN AATNTGAAGT NTTGGGNNGT TGCTGAGGCTGAGGCCTGGG CCGGNGGCTT420
GGGGATTGTT TCCNGGTTNC CACTCTGGTG NGGNNTTNCCNGGGCAGTTG GGTGNTTTTA480
TGACGGGATT GGTATTGTGT TG 502
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
CGCCCATGGC CTATGTCCCC GCACCG 26
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
CGCAAGCTTT TAGATCTGGA CATAGGAC 28
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
CGCCCATGGC CTTCAGCGGT TCCCAG 26
(2) INFORMATION FOR SEQ ID N0:44:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
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CGCAAGCTTC AGGGTTGGAA AGGCTG 26
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
CGCCCATGCT GTTGTCCTTA AACAAC 26
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
CGCCTGCAGC ACAGAAGCCA TTCTG 25
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
CGCCTGCAGC TATGCAACTT TATAAAATAT TCC 33
(2) INFORMATION FOR SEQ ID N0:48:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
CGCCCCGGGG CCTATGTCCC CGCAC 25
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
CGCGGTACCT TAGATCTGGA CATAGGAC 28
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
CGCCCCGGGG CCTTCAGCGG TTCCCAG 27
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
CGCGGTACCC AGGGTTGGAA AGGCTG 26
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:52:
CGCCCCGGGT TGTCCTTAAA CAACCTAC 28
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
CGCGGTACCC ACAGAAGCCA TTCTG 25
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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{xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
CGCGGTACCC TATGCAACTT TATAAAATAT TCC 33
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
CGCCCCGGGG CCATCATGGC CTATGTCCCC G 31
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
CGCGGTACCT TAGATCTGGA CATAGGAC 2g
(2) INFORMATION FOR SEQ ID N0:57:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
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CGCCCCGGGG CCATCATGGC CTTCAGCGGT TC 32
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
CGCGGTACCC AGGGTTGGAA AGGCTG 26
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
CGCCCCGGGG CCATCATGAT GTTGTCCTTA AAC 33
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
CGCGGTACCC ACAGAAGCCA TTCTG 25
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below relate
to the microorganism referred to
in the description
on page 3 , line 6
B. IDENTIFICATION OF DEPOSIT rurther
deposits are identified on an additional
sheet
Name of depositary institution
American Type Culture Collection
Address of depositary institution
(including postal code and country)
12301 Parklawn Drive
Rockville, Maryland 20852
United States of America
Date of deposit Accession Number
September 24, 1996 ATCC 97732
C. ADDITIONAL INDICATIONS (lrave
blank if not applicable) 'IMis information
is continued on an additional sheet
DNA Plasmid, 93442
D. DESIGNATED STATES FOR W RICH INDICATIONS
ARE MADE (i jtht indications art
not for a1r designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(lurve blank if not applicable)
T'he indications listed below will
be submitted to the International
Bureau later (specifythegeneralnatureofthtindicationse.g.,
Accession
Number o f Deposit ")
For receiving OCfice use only i=or International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authorized officer ~ ~ Authorized officer
~~'1.~~.,~,
t:c,rtn !'t-tnt(tOtl1 rtuly IVy2)
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INDICATIONS RELATING TO A DEPOSITCD MICROORGANISM
(PCT Rute 136is)
A. The indicatiocu made below relate
to the microorganism referred to
in the description
on page 3 , line 6
B. IDENTIFICATION OF DEPOSTT Putther
deposits are identified on an additional
sheet X
Name of depositary institution
American Type Cuittire Collection
Address of depositary institution
(including postal code and country)
12301 Parklawn Drive
Rockville, Maryland 20852
United States of America
Date of deposit Accession Number
September 24, 1996 ATCC 97733
C. ADDITIONAL INDICATIONS (leave
blank ijnot applicable) This inforsnation
is continued on an additional sheet
DNA P laslnid , 91715
D. DESIGNATED STATES FOR W13ICH
INDICATIONS ARE MADE (ijtlu. indications
arc not jar all rlesigmrttd Slalcs)
E. SEPARATE FiIRIYISHING OF INDICATIONS
(lraveblankijnol applicable)
The indications I fisted below will
be submitted to the International
Bureau la ter (specify the general
nature ojthe indications e.g.)
Accession
Number ojDeposit
For receiving Office use only For International Bureau use only
'Ibis sheet was received with the international application Q 'This sheet was
received by the International Bureau on:
Authorized oCticcr ~ ~ /~uthurirc~ officer
r,~r~~~ lo-fncon3a duly m~zl
CA 02268022 1999-04-09
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rufe l3bis)
A. The indications made bciow relate
to the miaaorganism referred to
in the description
on page 3 , line 6
Q. IDENTIFICATION OF DEPOSTT f urther
deposits are identified on an additional
sheet
Name of depositary institution
American Type Culture Collection
Address of depositary institution
(including postal code and country)
I2301 Parklawn Drive
Rockvile, Maryland 20852
United States of America
Date of deposit Accession Number
September 24, l996 ATCC 97734
C. ADDITIONAL INDICATIONS (leave
blank ijnot applicable) 'Ibis information
is continued on an additional sheet
DID Plastnid, 221441
D. DESIGNATED STATES FOR WIiICIi
INDICATIONS ARE MADE (i jthe indications
are not jor all designated Slates)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ijnoe applicable)
'Ilteindicatianslistedbelowwiilbesubmittedtothelnternational8ureaulater(specify
thegeneratnatureojtheindicationsag.,
'Accrssion
Number ojDeposit')
For receiving OCGce use only For International Bureau use only
This sheet was received with the international application Q ll~is sheet was
received by the International Bureau on:
/~W horiicd oCficcr ~ ~ nmhorircd otTiccr
Pf~~p~," ~~~C
l:"r~" m-rntcm ~a rmly m~ml
i
