Note: Descriptions are shown in the official language in which they were submitted.
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SIGNAL PEPTIDE CONTAINING PROTEINS AND USES THEREFOR
Background of the Invention
Cells of the immune response characteristically express a variety of cell-
surface
proteins which are crucial to proper functioning of the immune system. Such
proteins
include surface immunoglobulins, non-immunoglobulin cell surface antigen
receptors,
cellular adhesion molecules, as well as other selected phenotypic markers.
Many of
these cell surface proteins are members of the immunoglobulin (Ig)superfamily
of
proteins, characterized by the existence of at least one immunoglobulin (Ig)
domain.
Such proteins function in an variety of immune cell functions ranging from
immune cell
development and differentiation, antigen recognition, antibody production,
cellular
signal transduction, and cellular homing of immune responsive cells from the
circulation
to cites of increased antigen concentration.
In some instances, the diversified nature of immune cell function can be
attributed to the specific pattern of expression of such cell surface
proteins. For
example, cells expressing VCAM proteins of the Ig superfamily are known to be
involved primarily in cellular adhesion, whereas T lymphocytes
characteristically
express distinct patterns of the phenotypic markers, CD4, CD3, and CDB. Giver
importance of such cell surface proteins in the proper functioning of the
immune system,
there exists a need to identify novel cell-surface molecules which function to
regulate
the immune response and whose aberrant function can lead to immune response
disorders such as congenital or acquired immunodeficiency, and or inflammatory
disorders such as arthritis.
The placenta is the source of several peptide hormones that are homologous to
hormones synthesized in other endocrine tissues. These placental hormones,
which
belong to the prolactin-growth hormone superfamily, are believed to play
crucial roles in
normal fetal development. Members of the prolactin-growth hormone superfamily
include mouse placental lactogen I (mPL-I), mouse placental lactogen II (mPL-
II),
which bind to the prolactin receptor, and other proteins Iike mouse proliferin
(PLF)
(Linzer D.LH. et al., Proc. Natl. Acad. Sci. U.S.A. {1985) 82:4356; Lee S.J.
et al.,
Endocrinology (1988) 122:1761), mouse proliferin-related protein (PRP) (Linzer
D.LH.
and Nathans D., EMBO J. 4:1419; Colosi P. et al., Mol. Endocrinol. ( 1988)
2:579), rat
PL-I variant (Deb S. et al., J. Biol. Chem. (1991) 266:1605-1610), and rat
P1RL-like
proteins (PLP) A {Campbell W.J. et al., Endocrinology ( 1989) 125:1565-1574),
B
(Ogilvie S. et al., Endocrinology (1990) 126:2561-2566), and C (Deb S. et al.,
Endocrinology (1991) 128:3066-3072; Deb S. et al., J. Biol. Chem. (1991)
266:23027-
23032). PLF was discovered as a serum growth factor-inducible mRNA (Linzer
D.LH.
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and Nathans D., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:4271; ibid. (1984)
81:4255) and
protein (Nilsen-Hamilton M. et al., Cell (/ 980) 20:19) in mouse fibroblasts,
and
expression of PLF in muscle cells has been shown to inhibit muscle cell-
specific gene
expression and differentiation (Wilder E.L. and Linzer D.LH., Mol. Cell. Biol.
(1989)
9:430; Muscat G.E.O. et al., Mol. Endocrinol. (1991) 5:802). The PRP mRNA was
detected in placenta as a cDNA clone which cross-hybridized to the PLF cDNA
(Linzer
D.LH. and Nathans D., EMBO J. 4:1419). All of these proteins show significant
structural similarity (Southard J.N., Molecular and Cellular Endocrinoilogy
(1991), 79:
C 133-C 140) and are produced by the same trophoblast giant cells (Yamaguchi
M.,
Program of the 75th Annual Meeting of the Endocrine Society, Las Vegas (1993),
p.l 13
abstract), but their biological activities and gestationaI profiles in the
maternal blood
differ. The known biological activities of mPL-I and mPL-II are prolactin-
like. The
functions of PRP and PLF are not understood, but both have been postulated to
be
involved in regulating the initiation and then the cessation of placental
neovascularization (Jackson D. et al., Science (1994) 266:1581-1584).
The differentiation of hematopoietic stem cells (HSCs) involves a series of
lineage commitment steps accompanied by the acquisition of specific phenotypic
characteristics (Huang, S. and Terstappen, L. (1992) Nature 360: 745-49).
Cells gain or
lose antigenic features and responsiveness to specific cytokines and growth
factors based
on their lineage and stage of differentiation. As development proceeds, HSCs
become
committed to specific myeloid, lymphoid or erythroid lineages. These committed
"progenitor" stem cells ultimately differentiate into a wide variety of
specialized cell
types which include erythrocytes, neutrophils, basophils; eosinophils,
platelets, mast
cells, monocytes, tissue macrophages, osteoclasts, and the T and B
lymphocytes.
Red blood cells (erythrocytes), white blood cells (leucocytes) and platelets
(thrombocytes) are the predominant cell-types in the blood. Platelets are
derived from
detached fragments of larger cells called megakaryocytes which reside
predominantly in
the bone marrow. Megakaryopoiesis and platelet production are central to the
release of
cytokines, wound healing and blood coagulation. The failure of an organism to
maintain
adequate megakaryocyte numbers lends to thrombocytopenia and consequent
bleeding
disorders that can, in the extreme, result in death. Several humoral factors
have been
shown to promote megakaryocyte and platelet development, including interleukin-
1 (IL-
1) (Schmidt, J.A., J. Exp. Med 160:772-787, 1984; March, C.J. et al., Nature
315:641-
647, 1985); IL-3 (Yang, Y.C. et al., Cell 47:3-10, 1986; Ikebuchi, K. et al.,
Proc. Natl.
Acad. Sci. USA 84:9035-9039, 1987), IL-6 (Hirano, T., et al., Proc. Natl.
Acad. Sci. USA
82:5490-5494, 1985; Hirano, T., et al., Nature 324:73-76,1986; Ishibashi, T.
et al., Proc.
Natl. Acad Sci. USA 8b:5953-5957, 1989); IL-11 (Paul, S.R. et al., Proc. Natl.
Acad.
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Sci. USA 87:7512-7516, 1990; Teramura, M. et al., Blood 79:327-331, 1992),
leukemia
inhibitory factor (Metcalf, D. et al., Blood 77:2150-2153, 1991), granulocyte-
macrophage colony-stimulating factor (Wong, G., et al., Science 228:810-815,
1985),
erythropoietin (Miyake, T. et al., J. Biol. Chem. 252:5558-5564, 1977; Jacobs,
K. et al.,
Nature 313:806-815, 1985), and stem cell factor (Hendrie, P.C. et al., Exp.
Hematol.
19:1031-1037, 1991). However, most of these factors are pleiotropic and
consequently
their roles in physiological replication of thrombocyte poiesis are unclear.
Additional
activities implicated in megakaryopoiesis include megakaryocyte-potentiating
factor
(Yamaguchi, N. et al., J. Biol. Chem. 269:805-808, 1994), megakaryocyte
stimulatory
factor (Tayrien, G., and Rosenberg, R.D., J. Biol. Chem. 262:3262-3268, 1987;
Greenberg, S.M. et al., Exp. Hematol. 19:1031-1037, 1987), megakaryocyte
colony-
stimulating factor (Ogata, K. et al., Int. J. Cell Cloning 8:103-120, 1990;
Erikson-Miller,
C.L. et al., Blood Cell Growth Factors: Their Present and Future Use in
Hematology
and Oncology, Alpha Med Press, pp. 204-220, 1992; Erikson-Miller, C.L. et al.,
Br. J.
Haematol. 84:197-203, 1993), thrombopoiesis-stimulating factor (McDonald, T.P.
et al.,
J. Lab. Clin. Med. 85:59-66, 1975), and thrombopoietin (Hill, R. and Levin,
J., Exp.
Hematol. 14:752-759, 1986; Hill, R.J. et al., J. Exp. Hematol 20:354-360,
1992).
Sources for these activities have included the urine, serum or plasma from
aplastic
and/or thrombocytopenic humans (McDonald, T.P., Biochem. Med. 13:101-110,
1975;
Ogata, K. et al., Int. J. Cell Cloning 8:103-120, 1990), rats (Odell, T.T. et
al., Proc. Soc.
Exp. Biol. Med. 108:428-431, 1961), rabbits (Evatt, B.L. et al., J. Lab. Clin.
Med.
83:364-371, 1974; Hill, R.J. et al., Exp. Hematol. 20:354-360, 1992), and dogs
(Mazur,
E. and South, K., Exp. Hematol. 13:1164-1172, 1985).
Recent studies have implicated the ligand of the c-mpl cytokine receptor,
thrombopoietin (TPO), as a megakaryocyte lineage-specific factor (Bartley,
T.D. et al.
1994; Cell Vol. 77:1117-I 124; de Sauvage, F.J., et al. (1994) Vol. 369:533-
38; Gurney,
A.L. (1995) Blood Vol. 85 (4):981-88; Sohma, Y. et al. (1994) FEBS Letters
353:47-61).
The human TPO cDNA encodes a mature protein of approximately 332 amino acids
that
can be divided into two domains: an amino terminal domain of 153 amino acids
with
homology to erythropoietin and a unique C-terminal domain of 175 amino acids
containing multiple N-linked glycosylation sites. Both recombinant full-length
human
TPO and a truncated form consisting of the EPO-like domain (TPOI53) stimulate
[3H]-
thymidine incorporation in marine BaF3 cells transfected with human e-mpl,
demonstrating that he epo-like domain alone is sufficient for activation of c-
mpl
(deSauvage F.J. et al. (1994) Nature 369:533). Recombinant human TPO
stimulated
human megakaryocytopoiesis in vitro alone or in the presence of other
exogenously
added early-acting hematopoietic growth factors (deSauvage F.J. et al., Nature
369:533,
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1994; Bartley, T.D. et al., Cell 77:I 117, 1994; Kaushansky K. et al., Nature
369:568,
1994; Wendling F., Nature 369:571, 1994) Also, TPO stimulated platelet
production in
mice and dramatically increased the number of megakaryocytes in the spleen and
bone
marrow, indicating that TPO regulates thrombopoiesis in vivo (deSauvage F.J.
et al.,
Nature 369:533, 1994; Lok S. et al., Nature 369:565, 1994; Kaushansky K. et
al.,
Nature 369:568, 1994; Wendling F., Nature 369:571, 1994).
Summary of the Invention
The present invention is based, at least in part, on the discovery of novel
signal
peptide containing molecules referred to herein as Leukocyte-Specific Protein-
1 ("LSP-
1 ") , ProIiferin Analog I ("PA-I"), and "Thrombopoietin Analog Protein" ("TAP-
1 ")
nucleic acid and protein molecules. The LSP-1, PA-I, and TAP-1 molecules of
the
present invention are useful as modulating agents in regulating a variety of
cellular
processes. Accordingly, in one aspect, this invention provides isolated
nucleic acid
molecules encoding LSP-1, PA-I, and TAP-1 proteins or biologically active
portions
thereof, as well as nucleic acid fragments suitable as primers or
hybridization probes for
the detection of LSF-1, PA-I, and TAP-1-encoding nucleic acids. In one
embodiment,
the LSP-1, PA-I, and TAP-1 nucleic acid molecule is a naturally occurring
nucleotide
sequence.
In one embodiment, a LSP-1, PA-I, or TAP-1 nucleic acid molecule of the
invention is at least 46%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, or more identical to the nucleotide sequence (e.g., to the entire length
of the
nucleotide sequence) shown in SEQ ID NO:1, 3, 4, 6, 7, or 9 or the nucleotide
sequence
of the DNA insert of the plasmid deposited with ATCC as Accession Number 98554
or
, or a complement thereof. In a preferred embodiment, a LSP-1 nucleic acid
molecule of the invention is at least 46%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%,
90%, 95%, 98%, or more identical to the nucleotide sequence (e.g., to the
entire length
of the nucleotide sequence) shown in SEQ ID NO:1 or 3, or the nucleotide
sequence of
the DNA insert of the plasmid deposited with ATCC as Accession Number , or a
complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the
nucleotide sequence shown SEQ ID NO: l or 3, or a complement thereof. In
another
embodiment, the nucleic acid molecule includes SEQ ID N0:3 and nucleotides 1-
1331
of SEQ ID NO:1. In another embodiment, the nucleic acid molecule includes SEQ
ID
N0:3 and nucleotides 2010-2462 of SEQ ID NO:1. In another preferred
embodiment,
the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID
NO:1 or
3. In another preferred embodiment, the nucleic acid molecule includes a
fragment of at
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least 601 nucleotides of the nucleotide sequence of SEQ ID NO: I, 3, or a
complement
thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes
the
nucleotide sequence shown SEQ ID N0:4 or 6, or a complement thereof. In
another
embodiment, the nucleic acid molecule includes SEQ ID N0:6 and nucleotides 1-
54 of
SEQ ID N0:4. In another embodiment, the nucleic acid molecule includes SEQ ID
N0:6 and nucleotides 814-933 of SEQ ID N0:4. In another preferred embodiment,
the
nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:4
or 6.
In a preferred embodiment, the isolated nucleic acid molecule includes the
nucleotide sequence shown SEQ ID N0:7 or 9, or a complement thereof. In
another
embodiment, the nucleic acid molecule includes SEQ ID N0:9 and nucleotides 259-
523
of SEQ ID N0:7. In another preferred embodiment, the nucleic acid molecule
consists
of the nucleotide sequence shown in SEQ ID N0:7 or 9.
In another embodiment, a LSP-1; PA-I, and TAP-1 nucleic acid molecule
1 S includes a nucleotide sequence encoding a protein having an amino acid
sequence
sufficiently homologous to the amino acid sequence of SEQ ID N0:2, 5, or 8 or
an
amino acid sequence encoded by the DNA insert of the plasmid deposited with
ATCC as
Accession Nuunber 98554 or . In a preferred embodiment, a LSP-l, PA-I, and
TAP-1 nucleic acid molecule includes a nucleotide sequence encoding a protein
having
an amino acid sequence at least 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98% or more homologous to the entire length of the amino acid sequence of
SEQ
ID N0:2, 5, or 8 or the amino acid sequence encoded by the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98554 or In another preferred
embodiment, a LSP-1 nucleic acid molecule includes a nucleotide sequence
encoding a
protein having an amino acid sequence at least 52%, 55%, 60%, 65%, 70%, 75%,
80%,
85%, 90%, 95%, 98% or more homologous to the entire length of the amino acid
sequence of SEQ ID N0:2 or the amino acid sequence encoded by the DNA insert
of the
plasmid deposited with ATCC as Accession Number
In another preferred embodiment, an isolated nucleic acid molecule encodes the
amino acid sequence of human LSP-l, PA-I, and TAP-1. In yet another preferred
embodiment, the nucleic acid molecule includes a nucleotide sequence encoding
a
protein having the amino acid sequence of SEQ ID NO: 2, 5, or 8 or the amino
acid
sequence encoded by the DNA insert of the plasmid deposited with ATCC as
Accession
Number 98554 or . In yet another preferred embodiment, the nucleic acid
molecule is at least 601 nucleotides in length. In a further preferred
embodiment, the
nucleic acid molecule is at least 601 nucleotides in length and encodes a
protein having a
LSP-1, PA-I, and TAP-1 activity (as described herein).
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Another embodiment of the invention features nucleic acid molecules,
preferably
LSP-1, PA-I, and TAP-1 nucleic acid molecules, which specifically detect LSP-
l, PA-I,
and TAP-1 nucleic acid molecules relative to nucleic acid molecules encoding
non-LSP-
1, non-PA-I, and non-TAP-1 proteins. For example, in one embodiment, such a
nucleic
acid molecule is at least 300-350, 350-400, 400-450, 450-500, 500-550, 550-
600, 601 or
more nucleotides in length and hybridizes under stringent conditions to a
nucleic acid
molecule comprising the nucleotide sequence shown in SEQ ID NO:1, 4, or 7, the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98554 or , or a complement thereof.
In other preferred embodiments, the nucleic acid molecule encodes a naturally
occurring allelic variant of a polypeptide comprising the amino acid sequence
of SEQ ID
NO: 2, 5, or 8 or an amino acid sequence encoded by the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98554 or , wherein the nucleic acid
molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, 3, 4,
6, 7, or 9
under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule
which is antisense to a LSP-1, PA-I, and TAP-1 nucleic acid molecule, e.g.,
the coding
strand of a LSP-l, PA-I, and TAP-1 nucleic acid molecule.
Another aspect of the invention provides a vector comprising a LSP-l, PA-I,
and TAP-1 nucleic acid molecule. In certain embodiments, the vector is a
recombinant
expression vector. In another embodiment, the invention provides a host cell
containing
a vector of the invention. In yet another embodiment, the invention provides a
host cell
containing a nucleic acid molecule of the invention. The invention also
provides a
method for producing a protein, preferably a LSP-1, PA-I, and TAP-1 protein,
by
culturing in a suitable medium, a host cell, e.g., a mammalian host cell such
as a non-
human mammalian cell, of the invention containing a recombinant expression
vector,
such that the protein is produced.
In another embodiment, the invention features fragments of the protein having
the amino acid sequence of SEQ ID NO: 2, 5, or 8, wherein the fragment
comprises at
least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence
of SEQ
ID NO: 2, S, or 8 or an amino acid sequence encoded by the DNA insert of the
plasmid
deposited with the ATCC as Accession Number 98554 or . In another
embodiment, the protein, preferably a LSP-1, PA-I, or TAP-1 protein, has the
amino
acid sequence of SEQ ID NO: 2, 5, or 8, respectively.
In another embodiment, the invention features an isolated protein, preferably
a
LSP-1, PA-I, and TAP-1 protein, which is encoded by a nucleic acid molecule
consisting of a nucleotide sequence at Least about 46%, 50%, 55%, 60%, 65%,
70%,
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75%, 80%, 85%, 90%, 95%, 98% or more homologous to a nucleotide sequence of
SEQ
ID NO:1, 3, 4, 6, 7, or 9, or a complement thereof. This invention further
features an
isolated protein, preferably a LSP-l, PA-I, or TAP-1 protein, which is encoded
by a
nucleic acid molecule consisting of a nucleotide sequence which hybridizes
under
stringent hybridization conditions to a nucleic acid molecule comprising the
nucleotide
sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9, or a complement thereof.
The proteins of the present invention or portions thereof, e.g., biologically
active
portions thereof, can be operatively linked to a non-LSP-1, non-PA-I, or non-
TAP-1
polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
The
invention fiu-ther features antibodies, such as monoclonal or polyelonal
antibodies, that
specifically bind proteins of the invention, preferably LSP-1, PA-I, and TAP-1
proteins.
In addition, the LSP-l, PA-I, and TAP-1 proteins or biologically active
portions thereof
can be incorporated into pharmaceutical compositions, which optionally include
pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the
presence of a LSP-1, PA-I, and TAP-1 nucleic acid molecule, protein or
polypeptide in a
biological sample by contacting the biological sample with an agent capable of
detecting
a LSP-1, PA-I, and TAP-1 nucleic acid molecule, protein or polypept~de such
that the
presence of a LSP-1, PA-I, and TAP-1 nucleic acid molecule, protein or
polypeptide is
detected in the biological sample.
In another aspect, the present invention provides a method for detecting the
presence of LSP-l, PA-I, and TAP-1 activity in a biological sample by
contacting the
biological sample with an agent capable of detecting an indicator of LSP-1, PA-
I, and
TAP-1 activity such that the presence of LSP-1, PA-I, and TAP-1 activity is
detected in
the biological sample.
In another aspect, the invention provides a method for modulating LSP-1, PA-I,
and TAP-1 activity comprising contacting a cell capable of expressing LSP-1,
PA-I, and
TAP-1 with an agent that modulates LSP-l, PA-I, and TAP-1 activity such that
LSP-1,
PA-I, and TAP-1 activity in the cell is modulated. In one embodiment, the
agent
inhibits LSP-1, PA-I, and TAP-1 activity. In another embodiment, the agent
stimulates
LSP-1, PA-I, and TAP-I activity. In one embodiment, the agent is an antibody
that
specifically binds to a LSP-1, PA-I, and TAP-1 protein. In another embodiment,
the
agent modulates expression of LSP-1, PA-I, and TAP-1 by modulating
transcription of a
LSP-1, PA-I, and TAP-1 gene or translation of a LSP-1, PA-I, and TAP-1 mRNA.
In
yet another embodiment, the agent is a nucleic acid molecule having a
nucleotide
sequence that is antisense to the coding strand of a LSP-1, PA-I, and TAP-1
mRNA or a
LSP-1, PA-I, and TAP-1 gene.
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In one embodiment, the methods of the present invention are used to treat a
subject having a disorder characterized by aberrant LSP-1, PA-I, and TAP-1
protein or
nucleic acid expression or activity by administering an agent which is a LSP-
l, PA-I,
and TAP-1 modulator to the subject. In one embodiment, the LSP-1, PA-I, and
TAP-1
modulator is a LSP-1, PA-I, and TAP-1 protein. In another embodiment the LSP-
1, PA
I, and TAP-1 modulator is a LSP-1, PA-I, and TAP-1 nucleic acid molecule. In
yet
another embodiment, the LSP-1, PA-I, and TAP-1 modulator is a peptide,
peptidomirnetic, or other small molecule. In a preferred embodiment, the
disorder
characterized by aberrant LSP-1, PA-I, and TAP-1 protein or nucleic acid
expression is a
disorder characterized by deregulated angiogenesis, deregulated immune
response, or
deregulated hematopoiesis.
The present invention also provides a diagnostic assay for identifying the
presence or absence of a genetic alteration characterized by at least one of
(i) aberrant
modification or mutation of a gene encoding a LSP-I, PA-I, and TAP-1 protein;
(ii) mis-
regulation of the gene; and (iii) aberrant post-translational modification of
a LSP-1, PA
I, and TAP-1 protein, wherein a wild-type form of the gene encodes a protein
with a
LSP-1, PA-I, and TAP-1 activity.
In another aspect the invention provides a method for identifying a compound
that binds to or modulates the activity of a LSP-1, PA-I, and TAP-1 protein,
by
providing an indicator composition comprising a LSP-l, PA-I, and TAP-1 protein
having LSP-1, PA-I, and TAP-1 activity, contacting the indicator composition
with a
test compound, and determining the effect of the test compound,on LSP-1, PA-I,
and
TAP-1 activity in the indicator composition to identify a compound that
modulates the
activity of a LSP=1, PA-I, and TAP-1 protein.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
Brief Description of the DrawinEs
Figure 1 depicts the cDNA sequence and predicted amino acid sequence of
human LSP-1. The nucleotide sequence corresponds to nucleic acids 1 to 2462 of
SEQ
ID NO:1. The amino acid sequence corresponds to amino acids 1 to 22b of SEQ ID
N0:2, 5, or 8.
Figure 2 is a schematic drawing depicting selected clones which were isolated
and sequenced to derive the nucleotide sequence of the gene encoding human LSP-
1.
The figure details the relationship between the original, partial LSP-1 clone
isolated
from a bone marrow cDNA library, three additional clones for which partial
sequence
information was available, and the final composite sequence generated from
complete
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sequence analysis of the additional clones as well as the nucleotide sequence
of the
original partial clone.
Figure 3 is a schematic diagram depicting the biological and functional
domains
of human LSP-1. The LSP-1 protein comprises at least a signal peptide from
about
amino acids 1-20 of the amino acid sequence depicted (which corresponds to the
amino
acid sequence of SEQ ID N0:2, 5, or 8), an Ig-like domain from about amino
acids 46-
128 of the amino acid sequence depicted, and a transmembrane domain from about
amino acids I 92-213 of the amino acid sequence depicted.
Figure 4 depicts the cDNA sequence and predicted amino acid sequence of
marine Proliferin analog I. The nucleotide sequence corresponds to nucleic
acids 1 to
933 of SEQ ID N0:4. The amino acid sequence corresponds to amino acids 1 to
253 of
SEQ ID NO:S.
Figure 5 depicts an alignment of the amino acid sequences of marine Proliferin
analog I (corresponding to amino acids 1 to 247 of SEQ ID NO:S), and marine
proliferin
related protein (Swiss-ProtT"" Accession No. P04769).
Figure E depicts northern blots performed using clone aa014234 as a probe.
Figure 6A depicts a northern blot using human tissue from placenta, heart,
brain, lung,
liver, skeletal muscle, kidney and pancreas. Figure 6B depicts a northern blot
using
tissue from mouse embryos (day 7, 11, 15, and 17 embryos). Figure 6C depicts a
northern blot using human tissue from brain, lung, liver, and kidney.
Figure 7 depicts the cDNA sequence and predicted amino acid sequence of
human TAP-1. The nucleotide sequence corresponds to nucleic acids 1 to 528 of
SEQ
ID NO:7. The amino acid sequence corresponds to amino acids 1 to 86 of SEQ ID
N0:8.
Figure 8 depicts an alignment of the amino acid sequences of human TAP-1
(corresponding to amino acids 15 to 75 of SEQ ID N0:8) and human TPO (Swiss-
ProtT""
Accession Numbers P40225, 1401246, 939627). Identical amino acids are
indicated by
a single amino acid code in the row between the TAP-1 and TPO sequences,
conserved
amino acids are indicated as (+).
Figure 9 depicts an alignment of the LSP-1 nucleic acid molecule with
theFDF03 nucleic acid molecule (described in W0/24906) using the GAP program
in
the GCG software package (pam120 matrix) and a gap weight of 12 and a length
weight
of 4.
Figure 10 depicts an alignment of the LSP-1 protein with theFDF03 protein
(described in W0/24906) using the GAP program in the GCG software package
(pam120 matrix) and a gap weight of 12 and a length weight of 4.
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Detailed Description of the Invention
The present invention is based, at least in part, on the discovery of novel
molecules specific to peripheral blood leukocytes, referred to herein as LSP-1
protein
and nucleic acid molecules, which comprise a family of molecules having
certain
S conserved structural and functional features. The present invention is
further based, at
least in part, on the discovery of novel molecules of the prolactin-growth
hormone
superfamily, referred to herein as "ProIiferin analog I" or "PA-I" protein and
nucleic
acid molecules, which comprise a family.of molecules having certain conserved
structural and functional features. Moreover, the invention is based, at least
in part, on
the discovery of novel hematopoietic specific factors, referred to herein as
"Thrombopoietin Analog Protein" or "TAP-1" nucleic acid and protein molecules.
The term "family" when referring to the protein and nucleic acid molecules of
the invention is intended to mean two or more proteins or nucleic acid
molecules having
a common structural domain and having sufficient amino acid or nucleotide
sequence
homology as defined herein. Such family members can be naturally occurring and
can
be from eiiher the same or different species. For example, a family can
contain a first
protein of human origin and a homologue of that protein of marine origin, ~as
well as a
second, distinct protein of human origin and a inurine homologue of that
protein.
Members of a family may also have common functional characteristics.
One embodiment of the invention features LSP-l, PA-I, and TAP-1 nucleic acid
and protein molecules, preferably human LSP-1, PA-I, and TAP-1 nucleic acid
and
protein molecules. The LSP-1, PA-I, and TAP-1 nucleic acid and protein
molecules of
the invention are described in further detail in the following subsections.
A. The LSP-1 Nucleic Acid and Protein Molecules
In one embodiment, a LSP-1 family member is identified based on the presence
of "an immunoglobulin (Ig)-like domain" and a "transmembrane domain" in the
protein
or corresponding nucleic acid molecule. As used herein, the term
"immunoglobulin
(Ig)-like domain" refers to a protein domain having an amino acid sequence of
at least
about 50, preferably at least about 60, more preferably at least about 70
amino acid
residues, and even more preferably at least about 80-90 amino acids of which
at least
about 30%, preferably at least about 40%, more preferably at least about 50%,
60% or
70% of the amino acids are homologous to the amino acid sequence of an
immunoglobulin domain. The homologous amino acids between an Ig-like domain
and
an Ig domain can be positioned across the entire Ig-like domain, referred to
herein as a
"full" Ig-like domain. Alternatively, the homologous amino acids between an Ig-
like
domain and an Ig domain can be concentrated in regions of high homology
dispersed
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throughout the Ig-like domain, referred to as a "partial" Ig-like domain. In a
preferred
embodiment, an Ig-like domain is located in the N-terminal region of a LSP-1
protein.
For example, in one embodiment, a LSP-1 protein contains an Ig-like domain
containing
about amino acids 46-128 of SEQ ID N0:2, wherein amino acids homologous to an
Ig
S domain are concentrated between amino acids 46-78 and amino acids 109-128.
In
another embodiment an LSP-1 protein includes an Ig-like domain or a partial Ig-
like
domain which is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%,
75%, 80%, 85%, 90%, 95%, 98%, or more homologous to the Ig-like domain of SEQ
ID
N0:2.
Also as used herein, a "transmembrane domain" refers to a protein domain
having an amino acid sequence containing at least about 10, preferably about
13,
preferably about 16, more preferably about 19, and even more preferably about
21, 23,
25, 30, 35 or 40 amino acid residues, of which at least about 60-70%,
preferably about
80% and more preferably about 90% of the amino acid residues contain non-polar
side
chains, for example, alanine, valine, leucine, isoleucine, proline,
phenylalanine,
tryptophan, and methionine. A transmembrane domain is lipophillic in nature.
In a
preferred embodiment, a transmembrane domain is located in the C-terminal
region of a
LSP-1 protein. For example, in one embodiment, a LSP-1 protein contains a
transmembrane domain containing about amino acids 192-213 of SEQ ID N0:2.
In another preferred embodiment, a LSP-1 family member is identified further
based on the presence of an "N-terminal signal sequence". As used herein, a
"signal
sequence" refers to a peptide containing about 20 amino acids which occurs at
the
extreme N-terminal end of secretory and integral membrane proteins and which
contains
large numbers of hydrophobic amino acid residues. Such a "signal sequence",
also
referred to in the art as a "signal peptide", serves to direct a protein
containing such a
sequence to a lipid bilayer. For example, in one embodiment, an LSP-1 protein
contains
a signal sequence containing about amino acids 1-20 of SEQ ID N0:2.
Preferred LSP-1 molecules of the present have an amino acid sequence
suffciently homologous to the amino acid sequence of SEQ ID N0:2. As used
herein,
the term "sufficiently homologous" refers to a first amino acid or nucleotide
sequence
which contains a sufficient or minimum number of identical or equivalent
(e.g., an
amino acid residue which has a similar side chain) amino acid residues or
nucleotides to
a second amino acid or nucleotide sequence such that the first and second
amino acid or
nucleotide sequences share common structural domains andlor a common
functional
activity. For example, amino acid or nucleotide sequences which share common
structural domains have at least about 40% homology, preferably 50% homology,
more
preferably 60%-70% homology across the amino acid sequences of the domains and
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contain at least one, preferably two, more preferably three, and even more
preferably
four, five or six structural domains, are defined herein as sufficiently
homologous.
Furthermore, amino acid or nucleotide sequences which share at least 40%,
preferably
50%, more preferably 60, 70, or 80% homology and share a common functional
activity
are defined herein as sufficiently homologous.
As used interchangeably herein a "LSP-1 activity", "biological activity of LSP-
1" or "functional activity of LSP-1 ", refers to an activity exerted by a LSP-
1 protein,
polypeptide or nucleic acid molecule on a LSP-1 responsive cell as determined
in vivo,
or in vitro, according to standard techniques. In one embodiment, a LSP-1
activity is a
direct activity, such as an association with or an enzymatic activity on a
second protein.
In another embodiment, a LSP-1 activity is an indirect activity, such as a
cellular
signaling activity mediated by interaction of the LSP=1 protein with a second
protein. In
a preferred embodiment, a LSP-1 activity is at least one or more of the
following
activities: (i) interaction of a LSP-1 protein on the cell surface with a
second non-LSP-1
protein molecule on the surface of the same cell; (ii) interaction of a LSP-1
protein on
the cell surface with a second non-LSP-1 protein molecule on the surface of a
different
cell; (iii) complex formation between a soluble LSP-1 protein and a cognate
ligand; (iv)
complex formation between a membrane-bound LSP-1 protein and a cytokine; (v)
interaction of a LSP-1 protein with an intracellular protein via a second
protein on the
cell surface. In yet another preferred embodiment, a LSP-1 activity is at
least one or
more of the following activities: (i) modulation of cellular signal
transduction; (ii)
regulation of a cell involved in an inflammatory response; (iii) homing of a
cell having a
LSP-1 protein on its cell surface from a first to a second anatomical
location; and (iv)
modulation of a cell involved in the immune response.
Accordingly, another embodiment of the invention features isolated LSP-1
proteins and polypeptides having a LSP-1 activity. Preferred LSP-1 proteins
have at
least an N-terminal partial immunoglobulin (Ig) domain, a C-terminal
transmembrane
domain, and a LSP-1 activity. In another preferred embodiment, the LSP-1
protein has
at least at least an N-terminal partial immunoglobulin (Ig) domain, a C-
terminal
transmembrane domain, a LSP-1 activity, and an amino acid sequence
sufficiently
homologous to an amino acid sequence of SEQ ID N0:2.
In a particularly preferred embodiment, the LSP-1 protein and nucleic acid
molecules of the present invention are human LSP-1 molecules. A human LSP-1
cDNA
was identified by the Signal Peptide trAP methodology described herein. {See
Example
1). The nucleic acid sequence of a positive cDNA clone was used to search the
GenBankTM EST database and multiple ESTs having greater than 95% nucleotide
identity were found. Three clones containing published nucleotide sequences
were
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purchased from Research Genetics (Huntsville, AL) as part of the IMAGE
Consortium.
The full sequence of the human clone was assembled as depicted in Figure 2. A
nucleotide sequence of the isolated human LSP-1 cDNA and the predicted amino
acid
sequence of the human LSP-1 protein are shown in Figure 1 and in SEQ ID NOs: l
and
2, respectively. In addition, the nucleotide sequence corresponding to the
coding region
of the human LSP-1 cDNA (nucleotides 1332-2009) is represented as SEQ ID N0:3.
An approximately 1.5 kb LSP-1 mRNA transcript is expressed at very low levels
in most human tissues tested. Significant expression of LSP-1 mRNA was
detected only
in peripheral blood leukocytes. Chromosomal mapping indicates that the human
LSP-1
gene maps to chromosome 7q21-q22, at 111-112 cM.
The human LSP-1 cDNA, which is approximately 2462 nucleotides in length,
encodes a protein which is approximately 226 amino acid residues in length.
The
human LSP-1 protein contains an N-terminal signal sequence, an N-terminal
partial
immunoglobulin (Ig) domain, and a C-terminal transmembrane domain. A LSP-1 N-
terminal partial immunoglobulin (Ig) domain can be found at least, for
example, from
about amino acids 46-128 of SEQ ID N0:2. A LSP-1 C-terminal transmembrane can
be
found at least, for example, from about amino acids 192-213 of SEQ ID N0:2.
The
human LSP-1 protein is a membrane-bound protein which further contains a
signal
sequence at about amino acids 1-20 of SEQ ID N0:2. The prediction of such a
signal
peptide can be made, for example, utilizing the computer algorithm SIGNALP
(Henrik,
et al. ( 1997) Protein Engineering 10:1-6).
B. The PA-I Nucleic Acid and Protein Molecules
In another embodiment, a PA-I family is identified based on the presence of at
least one "cysteine-rich domain" in the protein or corresponding nucleic acid
molecule.
As used herein, the term "cysteine rich domain" includes a protein domain
having an
amino acid sequence of at least about 20 amino acids of which at least about 2
amino
acids are cysteine residues. Preferably, a cysteine rich domain includes at
least about 30,
more preferably at least about 35-40 amino acid residues, of which at least
about 2,
preferably at least about 3, more preferably at least about 4, 5 or 6 amino
acids are
cysteine residues. Cysteine-rich domains having lengths of 45-50 or 60 amino
acid
residues and having up to 7, 8, 9 or 10 cysteine residues are also within the
scope of this
invention. Cysteine rich domains are described in, for example, Lodish H. et
aI.
Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y.,
1995), the
contents of which are incorporated herein by reference. In one embodiment, a
PA-I
protein includes a cysteine rich domain having at least about 20%, preferably
at least
about 30%, and more preferably about 40% amino acid sequence homology to a
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proliferin-related protein cysteine-rich domain, such as the cysteine-rich
domain of SEQ
ID NO:10 (e.g., amino acid residues 198-243 of murine proliferin-related
protein, Swiss-
ProtT"" Accession No. P04769).
Preferred PA-I molecules of the invention have an amino acid sequence
sufficiently homologous to an amino acid sequence of SEQ ID NO:S. As used
herein,
the term "sufficiently homologous" refers to a first amino acid or nucleotide
sequence
which contains a sufficient or minimum number of identical or equivalent
(e.g., an
amino acid residue which has a similar side chain) amino acid residues or
nucleotides to
a second amino acid or nucleotide sequence such that the first and second
amino acid or
nucleotide sequences have a common structural domain and/or common functional
activity. For example, amino acid or nucleotide sequences which contain a
common
structural domain having about 40% homology, preferably 50% homology, more
preferably 60%-70% homology are defined herein as sufficiently homologous. In
one
embodiment, the PA-I protein contains a cysteine-rich domain and a PA-I
activity.
As used interchangeably herein a "PA-I activity", "biological activity of PA-
I" or
"functional activity of PA-I", refers to an activity exerted by a PA-I
protein, polypeptide
or nucleic acid molecule on a PA-I responsive cell as determined in vivo, or
in vitro,
according to standard techniques. In o~-~e embodiment, a PA-I activity is a
direct
activity, such as an association with or an enzymatic activity on a second
protein. In
another embodiment, a PA-I activity is an indirect activity, such as a
cellular signaling
activity mediated by interaction of the PA-I protein with a second protein. In
a preferred
embodiment, a PA-I activity is at least one or more of the following
activities: (i)
formation of a complex with a cell-surface proteins} or a ligand, e.g., a
lipid or
carbohydrate; (ii) formation of a complex with a prolactin and/or growth
hormone
receptor. In yet another preferred embodiment, a PA-I activity is at least one
or more of
the following activities: (i) regulation of cellular growth; (ii) regulation
of cellular
proliferation; (iii) regulation of angiogenesis; (iv} regulation of cellular
differentiation;
and (v) regulation of cell survival.
Accordingly, another embodiment of the invention features isolated PA-I
proteins and polypeptides having a PA-I activity. Preferred PA-I proteins have
at least
one cysteine-rich domain and a PA-I activity. In another preferred embodiment,
the PA-
I protein has at least one cysteine-rich domain, a PA-I activity and an amino
acid
sequence sufficiently homologous to an amino acid sequence of SEQ ID NO:S.
Yet another embodiment of the invention features PA-I molecules which contain
a signal sequence. As used herein, a "signal sequence" refers to a peptide
containing
about 20 amino acids which occurs at the extreme N-terminal end of secretory
and
integral membrane proteins and which contains large numbers of hydrophobic
amino
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acid residues. Such a "signal sequence", also referred to in the art as a
"signal peptide",
serves to direct a protein containing such a sequence to a lipid bilayer.
In a further embodiment, the invention features PA-I molecules which are
secreted. As used herein, "secreted" refers to protein molecules which have
the ability
to be directed to the cellular plasma membrane (usually through a signal
peptide) and
subsequently released into the extracellular space. Such secreted PA-I
molecules lack a
trasmembrane domain.
In a particularly preferred embodiment, the PA-I protein and nucleic acid
molecules of the present invention are human PA-I molecules. Using clone
aa014234 as
a probe for northern blots, a band of 1 Kb was detected in human placenta
tissue (see
Figure 6).
In another preferred embodiment, the PA-I protein and nucleic acid molecules
of
the present invention are marine PA-I molecules. A marine PA-I cDNA (also
refered to
as MOPAI or TANGO 95) was identified by homology with human growth hormone.
In particular, a human growth hormone cDNA sequence was used to search the
GenBankTM EST database and clone aa014234 was identified. This clone,
containing
the published nucleotide sequence, was purchased from Research Genetics
(Huntsville,
AL) as part of the IMAGE Consortium and subsequently fully sequenced. A
nucleotide
sequence of the isolated marine PA-I cDNA and the predicted amino acid
sequence of
the marine PA-I protein are shown in Figure 4 and in SEQ ID NOs:4 and 5,
respectively.
In addition, the nucleotide sequence corresponding to the coding region of the
marine
PA-I cDNA (nucleotides 55-816) is represented as SEQ ID N0:6.
A BlastP search (BLASTTM searching, utilizing an amino acid sequence against a
protein database), using the translation product (frame 1 ) of the cDNA
sequence
represented as SEQ ID N0:4, revealed homology tv proteins belonging to the
prolactin-
growth hormone superfamily. One example of such a protein is mouse proliferin-
related
protein, which is 243 amino acids in length and is 35% identical (see Figure
5) to amino
acids 1-247 of the marine PA-I amino acid sequence depicted in Figure 4 and
SEQ ID
NO:S.
A I kb PA-I mRNA transcript is expressed in marine tissues from day 7 embryos
(see Figure 6).
The marine PA-I cDNA, which is 933 nucleotides in length, encodes a protein
which is approximately 253 amino acid residues in length. The marine PA-I
protein
contains one cysteine-rich domain. A PA-I cysteine rich domain can be found at
least,
for example, from about amino acids 201-247 of SEQ ID NO:S (Lys201 to Lys247
of
the marine PA-I amino acid sequence). The marine PA-I protein is a secreted
protein
which lacks a transmembrane domain. The marine PA-I protein further contains a
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signal sequence at amino acids 1-30 of SEQ ID NO:S (Metl to Ser30 of the
marine PA-I
amino acid sequence. The prediction of such a signal peptide can be made
utilizing the
computer algorithm SIGNALP {Henrik, et al. (1997) Protein Engineering 10:1-6).
C. The TAP-1 Nucleic Acid and Protein Molecules
The carboxy-terminal domain of TAP-1 molecules has homology with the
carboxy-terminal domain of human thrombopoietin (TPO). TPO has been identified
as
the ligand of the c-mpl cytokine receptor which upon activation of the
receptor functions
as a megakaryocyte lineage-specific factor. The N-terminal domain of human TPO
shares homology to erythropoietin (EPO). Thus, TAP-1, TPO and EPO may comprise
a
family of structurally- and functionally related factors. TAP-1 molecules of
the present
invention may influence cell proliferation or differentiation, for example,
hematopoietic
cell proliferation or differentiation, e.g., the maturation or differentiation
of,
megakaryocytes into platelets, or erythroid progenitor cells into
erythrocytes.
In one embodiment, a TAP-1 family is identified based on the presence of at
least one "serine-proline-threonine rich" in the protein or corresponding
nucleic acid
molecule. As used herein, the term"serine-proline-threonine rich" refers to a
protein
domain of about 7, 10, 15, 20, 30, 40, 50, 60, 70, or 80 amino acids,
preferably 10 to 30
amino acids, and most preferably 18-22 amino acids having at least about 15%
serine,
proline and/or threonine residues, more preferably about 20 amino acids having
at least
about 20% serine, proline and/or threonine residues, and even more preferably
about 20
amino acids having at least about 30% serine, proline and/or threonine
residues.
In one embodiment, a TAP-1 protein includes a serine-proline-threonine rich
domain having at least about 20%, preferably at least about 30%, and more
preferably
about 40% amino acid sequence homology to a TAP-1 serine-proline-threonine
rich
domain, such as the domain of SEQ ID N0:8 (e.g., amino acid residues 1-20 or
40-60 of
SEQ ID N0:8).
Preferred TAP-1 molecules of the present have an amino acid sequence
sufficiently homologous to all or a portion of the amino acid sequence of SEQ
ID N0:8,
such as a serine-proline-threonine rich domain of the amino acid sequence of
SEQ ID
N0:8. As used herein, the term "sufficiently homologous" refers to a first
amino acid or
nucleotide sequence which contains a sufficient or minimum number of identical
or
equivalent (e.g., an amino acid residue which has a similar side chain) amino
acid
residues or nucleotides to a second amino acid or nucleotide sequence such
that the first
and second amino acid or nucleotide sequences have a common structural domain
and/or
common functional activity. For example, amino acid or nucleotide sequences
which
contain a common structural domain having about 40% homology, preferably 50%
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homology, more preferably 60%-70% homology are defined herein as sufficiently
homologous. In one embodiment, the a TAP-1 protein contains a serine-proline-
threonine rich domain and a TAP-1 activity.
In one embodiment, a TAP-1 family is identified by protein which include a
unique carboxyl terminal domain. The terms "C-terminal unique domain" or
"carboxy-
terminal domain" as used herein, refer to a protein domain of a TAP-1 protein
family
member which includes at least one serine-proline-threonine-rich domain and
shares
structural similarity to a human TPO C-terminal domain. A C-terminal unique
domain
is suff ciently homologous between TAP-1 protein family members such that the
domain is at least about 40%, preferably about 50%, more preferably about 60%,
even
more preferably about 70%, 80%, or 90% homologous. As defined herein, a C-
terminal
unique domain of a TAP-1 protein family member, however, is not sufficiently
homologous to a C-terminal unique domain of a member of another protein
family, such
as a TPO protein family.
As used interchangeably herein a "TAP-1 activity", "biological activity of TAP-
1" or "functional activity of TAP-1 ", refers to an activity exerted by a TAP-
1 protein,
polypeptide or nucleic acid molecule on a TAP-1 responsive cell as determined
in vivo,
or in vitro, according to standard techniques. In one emuodiment, a TAP-1
activity is a
direct activity, such as an association with, or an enzymatic activity, on a
second protein,
e.g., a cell-surface receptor. In another embodiment, a TAP-1 activity is an
indirect
activity, such as a cellular signaling activity mediated by interaction of the
TAP-1
protein with a second protein. In a preferred embodiment, a TAP-1 activity is
at least
one or more of the following activities: (i) interaction, e.g., binding to, a
cell-surface
receptor, e.g., a hematopoietic-cell surface receptor; (ii) modulation of,
e.g., activation or
inhibition of, a cell-surface receptor; (iii) modulation of cellular signal
transduction. In
yet another preferred embodiment, a TAP-1 activity is at least one or more of
the
following activities: (i) regulation of cellular proliferation; (ii)
regulation of cellular
differentiation; (iii) regulation of cell survival; (iv) modulation of a cell
involved in the
immune response (v) regulation of maturation and/or differentiation of a
hematopoietic
stem cell; (vi) modulation of megakaryocytopoiesis; (vii) modulation of
thrombopoiesis;
(viii) regulation of maturation andlor differentiation of a megalcaryocyte
into platelets;
and (ix) regulation of maturation and/or differentiation of erythroid
progenitor cells into
erythrocytes.
Accordingly, another embodiment of the invention features isolated TAP-1
proteins and polypeptides having a TAP-1 activity. Preferred TAP-1 proteins
have at
least one serine-proline-threonine rich domain and a TAP-1 activity. In
another
preferred embodiment, the TAP-1 protein has at Ieast one serine-proline-
threonine rich
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domain; a TAP-1 activity and an amino acid sequence sufficiently homologous to
an
amino acid sequence of SEQ ID N0:8.
In a particularly preferred embodiment, the TAP-1 protein and nucleic acid
molecules of the present invention are human TAP-1 molecules. A partial human
TAP-
S 1 cDNA, also referred to as TANGO-94, was identified by analysis of an EST
database
using mouse TPO sequence as a probe. A partial human clone (jthqb070d08) was
obtained from a human prostate cDNA library and was subsequently fully
sequenced.
Clone jthqb070d08 was deposited with the American Type Culture Collection on
October 2, 1997 and has ATCC Accession Number 98554. This clone contains a
nucleotide sequence of the isolated C-terminal domain of human TAP-1 cDNA
(nucleotides 1-528 corresponding to the C-terminus and 3' untranslated
sequence) and
the predicted amino acid sequence of the human TAP-1 protein (amino acids 1-
86) are
shown in Figure 7 and in SEQ ID NOs:7 and 8, respectively. The amino acid
sequences
showed 32% identitity to the C-terminal part of human TPO. The nucleotide
sequence
corresponding to the coding region of the human TAP-1 cDNA are nucleotides 1-
258 of
SEQ ID N0:7, nucleotides 259-528 correspond to the 3' untranslated region of
the gene.
Using jthqb070d08 cDNA as a probe, a 3 kb TAP-1 mRNA transcript is
expressed in human fetal liver tissues. In addition to the 3 kb band, several
bands were
detected in the Northern blots which may indicate splice variants of TAP-1.
Two other
less intense bands of approximately 5 and 2 kb were detected in all tissues
tested.
The partial human TAP-1 cDNA, which is approximately 528 nucleotides in
length, and which is approximately 86 amino acid residues in length. The human
TAP-1
protein contains four serine-proline-threonine-rich domains. A TAP-1 serine-
proline-
threonine-rich domain can be found at least, for example, from about amino
acids 1-20
of SEQ ID N0:8 (Glyl to GIy20 of SEQ ID N0:8); from about amino acids 21-40 of
SEQ ID N0:8 (I1e20 to A1a40 of SEQ ID N0:8); from about amino acids 41-60 of
SEQ
ID N0:8 (Va140 to G1y60 of SEQ ID N0:8); and from about amino acids 61-81 of
SEQ
ID N0:8 (Pro61 to Thr81 of SEQ ID N0:8). The human TAP-I C-terminal domain
appears to encode a secreted protein, e.g., growth factor a secreted protein.
The human TAP-1 amino acid sequence shares significant homology, about 32%
homology, with the C-terminal region of TPO. An alignment of the human TAP-1
amino acid sequences to human TPO sequences is presented in Figure 8. The
figure
depicts an alignment of the amino acid sequences of TAP-1 (corresponding to
amino
acids 15 to 75 of SEQ ID N0:8) and human TPO sequences (Swiss-ProtT""
Accession
Numbers P40225, 1401246, 939627). Identical residues are indicated in the row
between the TAP-1 and the TPO sequences by a single amino acid code; conserved
amino acid residues are indicated as (+).
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Using jthqb070d08 cDNA as a probe, 8 clones from a human fetal liver library
were isolated and submitted for sequencing. 3 out of the 8 clones contain an
insert of
approximately 3 kb.
Various aspects of the invention are described in further detail in the
following
subsections:
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that
encode LSP-l, PA-I, and TAP-1 proteins or biologically active portions
thereof, as well
as nucleic acid fragments sufficient for use as hybridization probes to
identify LSP-1,
PA-I, and TAP-1-encoding nucleic acids (e.g., LSP-1, PA-I, and TAP-1 mRNA) and
fragments for use as PCR primers for the amplification or mutation of LSP-1,
PA-I, and
TAP-1 nucleic acid molecules. As used herein, the term "nucleic acid molecule"
is
intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA
molecules
(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
The
nucleic acid molecule can be single-stranded or double-stranded, but
preferably is
double-stranded DNA.
The term "isolated nucleic acid molecule" includes nucleic acid molecules
which
are separated from other nucleic acid molecules which are present in the
natural source
of the nucleic acid. For example, with regards to genomic DNA, the term
"isolated"
includes nucleic acid molecules which are separated from the chromosome with
which
the genomic DNA is naturally associated. Preferably, an "isolated" nucleic
acid is free
of sequences which naturally flank the nucleic acid (i.e., sequences located
at the 5' and
3' ends of the nucleic acid) in the genomic DNA of the organism from which the
nucleic
acid is derived. For example, in various embodiments, the isolated LSP-1, PA-
I, and
TAP-1 nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1
kb, 0.5
kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid
molecule in
genomic DNA of the cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of
other cellular material, or culture medium when produced by recombinant
techniques, or
substantially free of chemical precursors or other chemicals when chemically
synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule
having the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9, or a portion
of these
nucleotide molecules, can be isolated using standard molecular biology
techniques and
the sequence information provided herein. Using all or portion of the nucleic
acid
sequences of SEQ ID NO:1, 3, 4, 6, 7, or 9 as a hybridization probe, LSP-1, PA-
I, and
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TAP-1 nucleic acid molecules can be isolated using standard hybridization and
cloning
techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis,
T. Molecular
Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), .
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID
NO:I, 3; 4, 6, 7, or 9 can be isolated by the polymerase chain reaction using
synthetic
oligonucleotide primers designed based upon the sequence of SEQ ID NO:I, 3, 4,
6, 7,
or 9.
A nucleic acid molecule of the invention can be amplified using cDNA, mRNA
or alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers
according to standard PCR amplification techniques. The nucleic acid so
amplified can
be cloned into an appropriate vector and characterized by DNA sequence
analysis.
Furthermore, oligonucleotides corresponding to LSP-1, PA-I, and TAP-1
nucleotide
sequences can be prepared by standard synthetic techniques, e.g., using an
automated
DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention
comprises the nucleotide sequence shown in SEQ ID NO:I . The sequence of SEQ
ID
NO:I corresponds to the human LSP-1 cDNA. This cDNA comprises sequences
encoding the human LSP-1 protein (i.e., "the coding region", from nucleotides
1332-
2009), as well as 5' untranslated sequences (nucleotides 1 to 1331 ) and 3'
untranslated
sequences (nucleotides 2010 to 2462). Alternatively, the nucleic acid molecule
can
comprise only the coding region of SEQ ID NO:I (e:g., nucleotides 1332 to
2009,
corresponding to SEQ ID N0:3).
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID N0:4. The sequence
of
SEQ ID N0:4 corresponds to the marine PA-I cDNA. This cDNA comprises sequences
encoding the marine PA-I protein (i.e., "the coding region", from nucleotides
55 to 816
of SEQ ID N0:4), as well as 5' untranslated sequences (nucleotides 1 to 54 of
SEQ ID
N0:4) and 3' untranslated sequences (nucleotides 817 to 933 of SEQ ID N0:4).
Alternatively, the nucleic acid molecule can comprise only the coding region
of SEQ ID
N0:4 (e.g., nucleotides 55 to 816 of SEQ ID N0:4, or SEQ ID N0:6).
In yet another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID N0:7. The sequence
of
SEQ ID N0:7 corresponds to the human TAP-1 cDNA. This cDNA comprises
sequences encoding the human TAP-1 protein (i.e., "the coding region", from
nucleotides 1 to 258 of SEQ ID N0:7, or SEQ ID N0:9), as well as 3'
untranslated
sequences (nucleotides 259 to 528 of SEQ ID N0:7). Alternatively, the nucleic
acid
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molecule can comprise only the coding region of SEQ ID N0:7 (e.g., nucleotides
1 to
258 of SEQ ID N0:7, or SEQ ID N0:9).
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleic acid molecule which is a complement of the
nucleotide
sequence shown in SEQ ID NO:1, 3, 4, 6, 7, or 9, or a portion of either of
these
nucleotide sequences. A nucleic acid molecule which is complementary to the
nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, or 9, is one which is
sufficiently
complementary to the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, or
9, such
that it can hybridize to the nucleotide sequence shown in SEQ ID NO:1, 3, 4,
6, 7, or 9,
thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule
of.the
present invention comprises a nucleotide sequence which is at least about 60-
65%,
preferably at least about 70-75%, more preferable at least about 80-85%, and
even more
preferably at least about 90-95% or more homologous to the nucleotide sequence
shown
in SEQ ID NO:1, 3, 4, 6, 7, or 9, or a portion of any of these nucleotide
sequences.
Moreover, the nucleic acid molecule of the invention can comprise only a
portion
of the nucleic acid sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9, for example a
fragment
which can be used as a probe or primer or a fragment encoding a biologically
active
portion of LSP-1, PA-I, and TAP-1. The nucleotide sequence determined from the
cloning of the LSP-1, PA-I, and TAP-1 gene allows for the generation of probes
and
primers designed for use in identifying and/or cloning LSP-1, PA-I, and TAP-1
homologues in other cell types, e.g. from other tissues, as well as LSP-1, PA-
I, and
TAP-1 homologues from other mammals. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide typically
comprises a region
of nucleotide sequence which hybridizes under stringent conditions to at least
about 12,
preferably about 25, more preferably about 40, 50 or 75 consecutive
nucleotides of SEQ
ID NO:1, 3, 4, 6, 7, or 9, of an anti-sense sequence of SEQ ID NO:1, 3, 4, 6,
7, or 9, or
of a naturally occurring mutant of either SEQ ID NO:1, 3, 4, 6, 7, or 9.
Probes based on the LSP-1, PA-I, and TAP-1 nucleotide sequence can be used to
detect transcripts or genomic sequences encoding the same or homologous
proteins. In
preferred embodiments, the probe further comprises a Iabel group attached
thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or
an enzyme
co-factor. Such probes can be used as a part of a diagnostic test kit for
identifying cells
or tissue which misexpress a LSP-1, PA-I, and TAP-1 protein, such as by
measuring a
level of a LSP-1, PA-I, and TAP-1-encoding nucleic acid in a sample of cells
from a
subject e.g., detecting LSP-1, PA-I, and TAP-1 mRNA levels or determining
whether a
genomic LSP-1, PA-I, and TAP-1 gene has been mutated or deleted.
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A nucleic acid fragment encoding a "biologically active portion of LSP-1, PA-
I,
and TAP-1" can be prepared by isolating a portion of SEQ ID NO:1, 3, 4, 6, 7,
or 9,
which encodes a polypeptide having a LSP-l, PA-I, and TAP-1 biological
activity (the
biological activities of the LSP-1, PA-I, and TAP-1 proteins are described
herein),
expressing the encoded portion of LSP-1, PA-I, and TAP-1 protein (e.g., by
recombinant
expression in vitro) and assessing the activity of the encoded portion of LSP-
1, PA-I,
and TAP-1. For example, a nucleic acid fragment encoding a biologically active
portion
of PA-I encompasses at least nucleic acids 654-795 of SEQ ID N0:4 (encoding a
marine
PA-I cysteine rich domain).
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, or 9 (and portions
thereof) due
to degeneracy of the genetic code and, thus, encode the same LSP-1, PA-I, and
TAP-1
protein as that encoded by the nucleotide sequence shown in SEQ ID NO:1, 3, 4,
6, 7, or
9. In another embodiment, an isolated nucleic acid molecule of the invention
has a
nucleotide sequence encoding a protein having an amino acid sequence shown in
SEQ
ID N0:2, 5, or 8.
In addition to~the LSP-1, PA-I, and TAP-1 nucleotide sequences shown in SEQ
ID NO:1, 3, 4, 6, 7, and 9, respectively, it will be appreciated by those
skilled in the art
that DNA sequence polymorphisms that lead to changes in the amino acid
sequences of
LSP-l, PA-I, and TAP-1 may exist within a population (e.g., the human
population).
Such genetic polymorphism in the LSP-1, PA-I, and TAP-1 gene may exist among
individuals within a population due to natural allelic variation.
As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid
molecules which include an open reading frame encoding a LSP-1, PA-I, and TAP-
1
protein, preferably a mammalian LSP-1, PA-I, and TAP-1 protein, and can
further
include non-coding regulatory sequences, and introns.
Allelic variants of LSP-1, PA-I, and TAP-1 include both functional and non-
functional LSP-1, PA-I, and TAP-1 proteins. Functional allelic variants are
naturally
occurring amino acid sequence variants of the LSP-1, PA-I, and TAP-1 protein
that
maintain the ability to bind a LSP-l, PA-I, and TAP-1 ligand and/or modulate a
LSP-1,
PA-I, and TAP-1 activity. Functional allelic variants will typically contain
only
conservative substitution of one or more amino acids of SEQ ID N0:2, 5, or 8,
or
substitution, deletion or insertion of non-critical residues in non-critical
regions of the
protein.
Non-functional allelic variants are naturally occurring amino acid sequence
variants of the human LSP-I, PA-I, and TAP-1 protein that do not have the
ability to
either bind an ARP ligand and/or modulate a LSP-1, PA-I, and TAP-1 activity.
Non-
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functional allelic variants will typically contain a non-conservative
substitution, a
deletion, or insertion or premature truncation of the amino acid sequence of
SEQ ID
N0:2, 5, or 8 or a substitution, insertion or deletion in critical residues or
critical
regions.
The present invention further provides non-human orthologues of the LSP-1,
PA-I, and TAP-1 protein. Orthologues of the LSP-1, PA-I, and TAP-1 protein are
proteins that are isolated from non-human organisms and possess the same LSP-
1, PA-I,
and TAP-1 ligand binding and/or modulation of LSP-1, PA-I, and TAP-1
activities of
the LSP-1, PA-I, and TAP-1 protein. Orthologues of the LSP-1, PA-I, and TAP-1
protein can readily be identified as including an amino acid sequence that is
substantially homologous to SEQ ID N0:2, 5, or 8, as defined herein.
Moreover, nucleic acid molecules encoding LSP-1, PA-I, and TAP-1 proteins
from other species, and which, thus, have a nucleotide sequence which differs
from the
sequence of SEQ ID NO:1, 3, 4, 6, 7, and 9 are intended to be within the scope
of the
invention. Nucleic acid molecules corresponding to natural allelic variants
and
homologues of the LSP-I, PA-I, and TAP-1 cDNAs of the invention can be
isolated
based on their homology to the LSP-1, PA-I, and TAP-1 nucleic acids disclosed
herein
using these cDNAs, or a portion thereof, as a hybridization probe according to
standard
hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 15 nucleotides in length and hybridizes under stringent
conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,
3, 4, 6,
7, or 9. In other embodiment, the nucleic acid is at least 30, 50, 100, 250 or
500
nucleotides in length. As used herein, the term "hybridizes under stringent
conditions"
is intended to describe conditions for hybridization and washing under which
nucleotide
sequences at least 60% homologous to each other typically remain hybridized to
each
other. Preferably, the conditions are such that sequences at least about 65%,
more
preferably at least about 70%, and even more preferably at least about 75%
homologous
to each other typically remain hybridized to each other. Such stringent
conditions are
known to those skilled in the art and can be found in Current Protocols in
Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-
limiting
example of stringent hybridization conditions are hybridization in 6X sodium
chloride/sodium citrate (SSC) at about 45°C, followed by one or more
washes in 0.2 X
SSC, 0.1% SDS at 50°C, preferably at 55°C, more preferably at
60°C, and even more
preferably at 65°C. Preferably, an isolated nucleic acid molecule of
the invention that
hybridizes under stringent conditions to the sequence of SEQ ID NO:1, 3, 4, 6,
7, or 9
corresponds to a naturally-occurring nucleic acid molecule. As used herein, a
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"naturally-occurnng" nucleic acid molecule refers to an RNA or DNA molecule
having
a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In addition to naturally-occurring allelic variants of the LSP-l, PA-I, and
TAP-I
sequence that may exist in the population, the skilled artisan will further
appreciate that
changes can be introduced by mutation into the nucleotide sequence of SEQ ID
NO:1, 3,
4, 6, 7, or 9, thereby leading to changes in the amino acid sequence of the
encoded LSP-
1, PA-I, and TAP-1 protein, without altering the functional ability of the LSP-
1, PA-I,
and TAP-1 protein. For example, nucleotide substitutions leading to amino acid
substitutions at "non-essential" amino acid residues can be made in the
sequence of SEQ
ID NO:1, 3, 4, 6, 7, or 9. A "non-essential" amino acid residue is a residue
that can be
altered from the wild-type sequence of LSP-l, PA-I, and TAP-I (e.g., the
sequence~of
SEQ ID N0:2, 5, or 8) without altering the biological activity, whereas an
"essential"
amino acid residue is required for biological activity. For example; amino
acid residues
of PA-I that are conserved among the proIactin-growth hormone family members
of this
invention (as indicated by the alignment and comparison of the amino acid
sequences of
SEQ ID NOs:S and 10 presented as Figure 5) are predicted to be essential in PA-
I and,
thus, are not likely to be amenable to alteration. For example, most proteins
of the
prolactin-growth hormone family, as well as the PA-I protein of the present
invention,
contain at least four cysteine residues among the cysteine rich domains
(residues 101,
218, 235 and 244 of SEQ ID NO:S). Furthermore, amino acid residues that are
conserved between LSP-1 protein and other proteins having Ig-like or Ig
domains are
not likely to be amenable to alteration. Moreover; amino acid residues of TAP-
1 that are
conserved among the family members of this invention (as indicated by an
alignment .
and comparison of the amino acid sequences of SEQ ID N0:8 with sequences of
TPO,
e.g., human TPO shown in Figure 8) are predicted to be essential in TAP-1 and,
thus, are
not likely to be amenable to alteration. Identical or conserved amino acid
sequences
between TAP-1 and human TPO are indicated in the middle row between these
sequences in Figure 8.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding LSP-1, PA-I, and TAP-1 proteins that contain changes in amino acid
residues
that are not essential for activity. Such LSP-1, PA-I, and TAP-1 proteins
differ in amino
acid sequence from SEQ ID N0:2, S, or 8 yet retain biological activity. In one
embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence
encoding a protein, wherein the protein comprises an amino acid sequence at
least about
60% homologous to the amino acid sequence of SEQ ID N0:2, 5, or 8. Preferably,
the
protein encoded by the nucleic acid molecule is at least about 70% homologous
to SEQ
ID N0:2, 5, or 8, more preferably at least about 80% homologous to SEQ ID
NO:2, 5, or
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8, even more preferably at least about 90% homologous to SEQ ID N0:2, 5, or 8,
and
most preferably at least about 95% homologous to SEQ ID N0:2, 5, or 8.
An isolated nucleic acid molecule encoding a LSP-1, PA-I, and TAP-I protein
homologous to the protein of ~SEQ ID N0:2, 5, or 8 can be created by
introducing one or
more nucleotide substitutions, additions or deletions into the nucleotide
sequence of
SEQ ID NO:1, 3, 4, 6, 7, or 9 such that one or more amino acid substitutions,
additions
or deletions are introduced into the encoded protein. Mutations can be
introduced into
SEQ ID NO: l, 3, 4, 6, 7, or 9 by standard techniques, such as site-directed
mutagenesis
and PCR-mediated mutagenesis. Preferably, conservative amino acid
substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative
amino acid substitution" is one in which the amino acid residue is replaced
with an
amino acid residue having a similar side chain. Families of amino acid
residues having
similar side chains have been defined in the art. These families include amino
acids
with basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic
1 S acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine,
serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine,
valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains {~.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino
acid residue
in LSP-1, PA-I, and TAP-1 is preferably replaced with another amino acid
residue from
the same side chain family. Alternatively, in another embodiment, mutations
can be
introduced randomly along all or part of a LSP-1, PA-I, and TAP-1 coding
sequence,
such as by saturation mutagenesis, and the resultant mutants can be screened
for LSP-1,
PA-I, and TAP-1 biological activity activity to identify mutants that retain
activity.
Following mutagenesis of SEQ ID NO:1, 3, 4, 6, 7, or 9, the encoded protein
can be
expressed recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant LSP-1 protein can be assayed for (1) the
ability to modulate cellular signal transduction; (2) regulation of a cell
involved in an
inflammatory response; (3) homing of a cell having a LSP-1 protein on its cell
surface
from a first to a second anatomical location; and {4) the ability to modulate
a cell
involved in the immune response.
In another preferred embodiment, a mutant PA-I protein can be assayed for ( 1
)
the ability to form a complex with a cell-surface proteins) or a ligand, e.g.,
a lipid or
carbohydrate; (2) the ability to form a complex with a prolactin and/or growth
hormone
receptor. In yet another preferred embodiment, a mutant LSP-I, PA-I, and TAP-I
can
be assayed for the ability to (1) regulate cellular growth; (2) regulate
cellular
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proliferation; (3) regulate angiogenesis; (4) regulate cellular
differentiation; and (5)
regulate cell survival.
In yet another preferred embodiment, a mutant TAP-1 protein can be assayed for
( 1 ) the ability to form protein:protein interactions with cell-surface
proteins, e.g., a TAP
S 1 receptor, or biologically active portions thereof; (2) modulation of,
e.g., activation or
inhibition of, a cell-surface receptor; (3) modulation of cellular signal
transduction.. In
yet another preferred embodiment, a mutant TAP-1 can be assayed for the
ability to (1)
modulate cellular signal transduction; (2) regulate cellular proliferation;
(3) regulate
cellular differentiation; (4) regulate cell survival; (5) modulate a cell
involved in the
immune response; (6) regulation of maturation and/or differentiation of a
hematopoietic
stem cell; (7) modulation of megakaryocytopoiesis; (8) modulation of
thrombopoiesis;
(9) regulation of maturation and/or differentiation of a megakaryocyte into
platelets; and
(10) regulation of maturation and/or differentiation of erythroid progenitor
cells into
erythrocytes.
In addition to the nucleic acid molecules encoding LSP-1, PA-I, and TAP-1
proteins described above, another aspect of the invention pertains to isolated
nucleic acid
molecules which are antisense thereto. An "antisense" nucleic acid comprises a
nucleotide sequence which is complementary to a "sense" nucleic acid encoding
a
protein, e.g., complementary to the coding strand of a double-stranded cDNA
molecule
or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid
can
hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire LSP-1, PA-I, and TAP-1 coding strand, or to only a
portion
thereof. In one embodiment, an antisense nucleic acid molecule is antisense to
a "coding
region" of the coding strand of a nucleotide sequence encoding LSP-1, PA-I,
and TAP-1.
The term "coding region" refers to the region of the nucleotide sequence
comprising
codons which are translated into amino acid residues (e.g., the coding region
of LSP-1,
PA-I, and TAP-1 corresponds to SEQ ID N0:3, 6, and 9, respectively). In
another
embodiment, the antisense nucleic. acid molecule is antisense to a "non-coding
region"
of the coding .strand of a nucleotide sequence encoding LSP-I, PA-I, and TAP-
1. The
term "non-coding region" refers to 5' and 3' sequences which flank the coding
region
that are not translated into amino acids (i.e., also referred to as 5' and 3'
untranslated
regions).
Given the coding strand sequences encoding LSP-1, PA-I, and TAP-1 disclosed
herein (e.g., SEQ ID N0:3, 6, and 9, respectively), antisense nucleic acids of
the
invention can be designed according to the rules of Watson and Crick base
pairing. The
antisense nucleic acid molecule can be complementary to the entire coding
region of
LSP-1, PA-I, and TAP-1 mRNA, but more preferably is an oIigonucieotide which
is
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antisense to only a portion of the coding or noncoding region of LSP-1, PA-I,
and TAP-
1 mRNA. For example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of LSP-1, PA-I, and TAP-1 mRNA.
An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45 or
S 50 nucleotides in length. An antisense nucleic acid of the invention can be
constructed
using chemical synthesis and enzymatic ligation reactions using procedures
known in
the art. For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can
be chemically synthesized using naturally occurring nucleotides or variously
modified
nucleotides designed to increase the biological stability of the molecules or
to increase
the physical stability of the duplex formed between the antisense and sense
nucleic
acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides
can be
used. Examples of modified nucleotides which can be used to generate the
antisense
nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-
iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil,
dihydrouracil, beta-D-galactosyiqueosine; inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-
thiouracil,
5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid
(v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced
biologically using an expression vector into which a nucleic acid has been
subcloned in
an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid
will be of
an antisense orientation to a target nucleic acid of interest, described
further in the
following subsection).
The antisense nucleic acid molecules of the invention are typically
administered
to a subject or generated in situ such that they hybridize with or bind to
cellular mRNA
and/or genomic DNA encoding a LSP-1, PA-I, and TAP-1 protein to thereby
inhibit
expression of the protein, e.g., by inhibiting transcription and/or
translation. The
hybridization can be by conventional nucleotide complementarity to form a
stable
duplex, or, for example, in the case of an antisense nucleic acid molecule
which binds to
DNA duplexes, through specific interactions in the major groove of the double
helix.
An example of a route of administration of antisense nucleic acid molecules of
the
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invention include direct injection at a tissue site. Alternatively, antisense
nucleic acid
molecules can be modified to target selected cells and then administered
systemically.
For example, for systemic administration, antisense molecules can be modified
such that
they specifically bind to receptors or antigens expressed on a selected cell
surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or antibodies
which bind to
cell surface receptors or antigens. The antisense nucleic acid molecules can
also be
delivered to cells using the vectors described herein. To achieve sufficient
intracellular
concentrations of the antisense molecules, vector constructs in which the
antisense
nucleic acid molecule is placed under the control of a strong pol II or pol
III promoter
are preferred.
In yet another embodiment, the andsense nucleic acid molecule of the invention
is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule
forms
specific double-stranded hybrids with complementary RNA in which, contrary to
the
usual ~i-units, the strands run parallel to each other (Gaultier et aI. (1987)
Nucleic Acids.
Res. 15:6625-6641 ). The antisense nucleic acid molecule can also comprise a
2'-0-
methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or
a
chimeric RNA-DNA analogue (moue et al. (1987) FEBSLett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity
which are
capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which
they
have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach ( 1988) Nature 334:585-591 )) can be used
to
catalytically cleave LSP-1, PA-I, and TAP-1 mRNA transcripts to thereby
inhibit
translation of LSP-1, PA-I, and TAP-1 mRNA. A ribozyme having specificity for
a
LSP-1, PA-I, and TAP-1-encoding nucleic acid can be designed based upon the
nucleotide sequence of a LSP-1, PA-I, and TAP-1 cDNA disclosed herein (i.e.,
SEQ ID
NO:1). For example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed
in which the nucleotide sequence of the active site is complementary to the
nucleotide
sequence to be cleaved in a LSP-1, PA-I, and TAP-1-encoding mRNA. See, e.g.,
Cech
et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
Alternatively, LSP-1, PA-I, and TAP-1 mRNA can be used to select a catalytic
RNA
having a specif c ribonuclease activity from a pool of RNA molecules. See,
e.g., Bartel,
D. and Szostak, J. W. ( 1993) Science 261:1411-1418.
Alternatively, LSP-1, PA-I, and TAP-1 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory region of the
LSP-1,
PA-I, and TAP-1 (e.g., the LSP-1, PA-I, and TAP-1 promoter and/or enhancers)
to form
triple helical structures that prevent transcription of the LSP-1, PA-I, and
TAP-1 gene in
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target cells. See generally, Helene, C. ( 1991 ) Anticancer Drug Des. 6(6):569-
84;
Helene, C. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher, L.J.
(1992)
Bioassays 14(12):807-15.
In preferred embodiments, the nucleic acids of LSP-1, PA-I, and TAP-1 can be
modified at the base moiety, sugar moiety or phosphate backbone to improve,
e.g., the
stability, hybridization, or solubility of the molecule. For example, the
deoxyribose
phosphate backbone of the nucleic acids can be modified to generate peptide
nucleic
acids (see Hyrup B. et al. ( 1996) Bioorganic & Medicinal Chemistry 4 ( 1 ): 5-
23). As
used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid
mimics,
e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are retained. The
neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA
under conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using standard solid phase peptide synthesis protocols as described
in Hyrup
B. et al. ( 1996) supra; Perry-O'Keefe et al. { 1996) PNAS 93: 14670-675.
PNAs of LSP-1, PA-I, and TAP-1 can be used therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or antigene agents
for
sequence-specific modulation of gene expression by, e.g., inducing
trascription o~
translation arrest or inhibiting replication. PNAs of LSP-1, PA-I, and TAP-1
can also be
used, e.g., in the analysis of single base pair mutations in a gene by, e.g.,
PNA directed
PCR clamping; as 'artificial restriction enzymes when used in combination with
other
enzymes, e.g., S 1 nucleases (Hyrup B. ( 1996) supra); or as probes or primers
for DNA
sequence and hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe
supra).
In another embodiment, PNAs of LSP-1, PA-I, and TAP-1 can be modified, e.g.,
to enhance their stability or cellular uptake, by attaching lipophilic or
other helper
groups to PNA, by the formation of PNA-DNA chimeras, or by the use of
liposomes or
other techniques of drug delivery known in the art. For example, PNA-DNA
chimeras
of LSP-1, PA-I, and TAP-1 can be generated which may combine the advantageous
properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g.,
RNAse H and DNA polymerases, to interact with the DNA portion while the PNA
portion would provide high binding affinity and specificity. PNA-DNA chimeras
can be
linked using linkers of appropriate lengths selected in terms of base
stacking, number of
bonds between the nucleobases, and orientation (Hyrup B. ( 1996) supra). The
synthesis
of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and
Finn P.J. et al. (1996) Nucleic Acids Research 24 (17): 3357-63. For example,
a DNA
chain can be synthesized on a solid support using standard phosphoramidite
coupling
chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-
deoxy-
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thymidine phosphoramidite, can be used as a between the PNA and the 5' end of
DNA
(Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA
segment
and a 3' DNA segment (Finn P.J. et al. (1996) supra). Alternatively, chimeric
molecules
can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H.
et al.
(1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
In other embodiments, the oligonucleotide may include other appended groups
such as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating
transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.
Natl. Acad.
Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-
652;
PCT Publication No. W088/09810, published December 15, 1988) or the blood-
brain
barrier (see, e.g., PCT Publication No. W089/10134, published April 25, 1988).
In
addition, oligonucleotides can be modified with hybridization-triggered
cleavage agents
(See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating
agents. (See,
e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may
be
conjugated to another molecule, e.g., a peptide, hybridization triggered cross-
linking
agent, transport agent, hybridization-triggered cleavage agent, etc.
II. Isolated LSP-1, PA-I, and TAP-1 Proteins and Anti-LSP-1, Anti-PA-I, and
Anti-TAP-1 Antibodies
One aspect of the invention pertains to isolated LSP-1, PA-I, and TAP-1
proteins, and biologically active portions thereof, as well as polypeptide
fragments
suitable for use as immunogens to raise anti-LSP-1, anti-PA-I, and anti-TAP-1
antibodies. In one embodiment, native LSP-1, PA-I, and TAP-1 proteins can be
isolated
from cells or tissue sources by an appropriate purification scheme using
standard protein
purification techniques. In another embodiment, LSP-1, PA-I, and TAP-1
proteins are
produced by recombinant DNA techniques. Alternative to recombinant expression,
a
LSP-1, PA-I, and TAP-1 protein or polypeptide can be synthesized chemically
using
standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is
substantially free of cellular material or other contaminating proteins from
the cell or
tissue source from which the LSP-l, PA-I, and TAP-1 protein is derived, or
substantially
free from chemical precursors or other chemicals when chemically synthesized.
The
language "substantially free of cellular material" includes preparations of
LSP-1, PA-I,
and TAP-1 protein in which the protein is separated from cellular components
of the
cells from which it is isolated or recombinantly produced. In one embodiment,
the
language "substantially free of cellular material" includes preparations of
LSP-1, PA-I,
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and TAP-1 protein having less than about 30% (by dry weight) of non-LSP-1, PA-
I, and
TAP-1 protein (also referred to herein as a "contaminating protein"), more
preferably
less than about 20% of non-LSP-1, PA-I, and TAP-1 protein, still more
preferably less
than about 10% of non-LSP-l, PA-I, and TAP-1 protein, and most preferably less
than
about 5% non-LSP-1, PA-I, and TAP-1 protein. When the LSP-l, PA-I, and TAP-1
protein or biologically active portion thereof is recombinantly produced, it
is also
preferably substantially free of culture medium, i.e., culture medium
represents less than
about 20%, more preferably less than about 10%, and most preferably less than
about
5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of LSP-1, PA-I, and TAP-1 protein in which the protein
is
separated from chemical precusors or other chemicals which are involved in the
synthesis of the protein. In one embodiment, the language "substantially free
of
chemical precursors or other chemicals" includes preparations of LSP-1, PA-I,
and TAP-
1 protein having less than about 30% (by dry weight) of chemical precursors or
non-
LSP-1, PA-I, and TAP-1 chemicals, more preferably less than about 20% chemical
precursors or non-LSP-1, PA-I, and TAP-1 chemicals, still more preferably less
than
about 10% chemical precursors or non-LSP-1, PA-I, and TAP-1 chemicals, and
most
preferably less than about 5% chemical precursors or non-LSP-1, PA-I, and TAP-
1
chemicals.
Biologically active portions of a LSP-1, PA-I, and TAP-1 protein include
peptides comprising amino acid sequences sufficiently homologous to or derived
from
the amino acid sequence of the LSP-1, PA-I, and TAP-1 protein, e.g., the amino
acid
sequence shown in SEQ ID N0:2, 5, or 8, which include less amino acids than
the full
length LSP-1, PA-I, and TAP-1 proteins, and exhibit at least one activity of a
LSP-1,
PA-I, and TAP-1 protein. Typically, biologically active portions comprise a
domain or
motif with at least one activity of the LSP-1, PA-I, and TAP-1 protein. A
biologically
active portion of a LSP-1, PA-I, and TAP-1 protein can be a polypeptide which
is, for
example, 10, 25, 50, 100 or more amino acids in length.
In one embodiment, a biologically active portion of a LSP-1 protein comprises
at
least a transmernbrane domain. In yet another embodiment, a biologically
active
portion of a LSP-1 protein comprises at least a signal sequence.
In an alternative embodiment, a biologically active portion of a LSP-1 protein
comprises a LSP-1 amino acid sequence lacking a transmembrane domain. In yet
another embodiment, a biologically active portion of a LSP-1 protein comprises
a LSP-1
amino acid sequence lacking a transmembrane domain and a signal sequence. Such
a
preferred LSP-1 molecules are referred to as a "LSP-1 extracellular domains".
For
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example, preferred LSP-1 extracellular domains contain at least about amino
acids 1-190
of SEQ ID N0:2.
In another embodiment, a biologically active portion of a PA-I protein
comprises
at least one cysteine rich domain, characteristic of the prolactin-growth
hormone
superfamily of proteins. In another embodiment, a biologically active portion
of a PA-I,
protein comprises at least a signal sequence.
In yet another embodiment, a biologically active portion of a TAP-1 protein
comprises at least one serine-proline-threonine rich region of TAP-1.
In an alternative embodiment, a biologically active portion of a TAP-1 protein
comprises at least a C-terminal unique domain of a TAP-1 protein.
It is to be understood that a preferred biologically active portion of a LSP-
l, PA-
I, and TAP-1 protein of the present invention may contain at least one of the
above-
identified structural domains. A more preferred biologically active portion of
a LSP-1,
PA-I, and TAP-1 protein may contain at least two of the above-identified
structural
domains. Moreover, other biologically active portions, in which other regions
of the
protein are deleted, can be prepared by recombinant techniques and evaluated
for one or
more of the functional activities of a native LSP-l, PA-I, and TAP-1 protein.
In a preferred embodiment, the LSP-1, PA-I, and TAP-1 protein has an amino
acid sequence shown in SEQ ID N0:2, 5, or 8. In other embodiments, the LSP-1,
PA-I,
and TAP-1 protein is substantially homologous to SEQ ID N0:2, 5, or 8 and
retains the
functional activity of the protein of SEQ ID N0:2, 5, or 8 yet differs in
amino acid
sequence due to natural allelic variation or mutagenesis, as described in
detail in
subsection I above. Accordingly, in another embodiment, the LSP-1, PA-I, and
TAP-1
protein is a protein which comprises an amino acid sequence at least about 60%
homologous to the amino acid sequence of SEQ ID N0:2, 5, or 8 and retains the
functional activity of the LSP-1, PA-I, and TAP-1 proteins of SEQ ID N0:2, 5,
or 8.
Preferably, the protein is at least about 70% homologous to SEQ ID N0:2, 5, or
8, more
preferably at least about 80% homologous to SEQ ID N0:2, 5, or 8, even more
preferably at least about 90% homologous to SEQ ID NO 2, and most preferably
at least
about 95% or more homologous to SEQ ID N0:2, 5, or 8. In a preferred
embodiment
the LSP-1 protein is at least 52%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%
or
more homologous to SEQ ID N0:2.
To determine the percent identity of two amino acid sequences or of two
nucleic
acid sequences, the sequences are aligned for optimal comparison purposes
(e.g., gaps
can be introduced in one or both of a first and a second amino acid or nucleic
acid
sequence for optimal alignment and non-homologous sequences can be disregarded
for
comparison purposes). In a preferred embodiment, the length of a reference
sequence
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aligned for comparison purposes is at least 30%, preferably at least 40%, more
preferably at least SO%, even more preferably at least 60%, and even more
preferably at
least 70%, 80%, or 90% of the length of the reference sequence (e.g., when
aligning a
second sequence to the LSP-l, PA-I, and TAP-1 amino acid sequence of SEQ ID
N0:2,
5, or 8 having 177 amino acid residues, at least 80, preferably at least 100,
more
preferably at least 120, even more preferably at least 140, and even more
preferably at
least 150, 160 or 170 amino acid residues are aligned). The amino acid
residues or
nucleotides at corresponding amino acid positions or nucleotide positions are
then
compared. When a position in the first sequence is occupied by the same amino
acid
residue or nucleotide as the corresponding position in the second sequence,
then the
molecules are identical at that position (as used herein amino acid or nucleic
acid
"identity" is equivalent to amino acid or nucleic acid "homology"). The
percent identity
between the two sequences is a function of the number of identical positions
shared by
the sequences, taking into account the number of gaps, and the length of each
gap, which
need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two
sequences can be accomplished using a mathematical algorithm. In a preferred
embodiment, the percent identity between two amino acid sequences is
determined using
the Needleman and Wunsch (J. Mol. Biol. {48):444-453 (1970)) algorithm which
has
been incorporated into the GAP program in the GCG software package (available
at
http:/lwww.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and
a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. In yet
another preferred embodiment, the percent identity between two nucleotide
sequences is
determined using the GAP program in the GCG software package (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,
60,
70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment,
the percent
identity between two amino acid or nucleotide sequences is determined using
the
algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM 120 weight
residue
table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be
used as a "query sequence" to perform a search against public databases to,
for example,
identify other family members or related sequences. Such searches can be
performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. ( 1990)
J.
Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the
NBLAST program, score = 100, wordIength = 12 to obtain nucleotide sequences
homologous to LSP-1, PA-I, and TAP-1 nucleic acid molecules of the invention.
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BLAST protein searches can be performed with the XBLAST program, score = S0,
wordlength = 3 to obtain amino acid sequences homologous to LSP-1, PA-I, and
TAP-1
protein molecules of the invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997)
Nucleic
Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and NBLAST)
can be
used. See http://www.ncbi.nlm.nih.gov.
The invention also provides LSP-1, PA-I, and TAP-1 chimeric or fusion
proteins.
As used herein, a LSP-1, PA-I, and TAP-1 "chimeric protein" or "fusion
protein"
comprises a LSP-1, PA-I, and TAP-1 polypeptide operatively linked to a non-LSP-
1,
non-PA-I, and non-TAP-1 polypeptide. A "LSP-1, PA-I, and TAP-1 polypeptide"
refers
to a polypeptide having an amino acid sequence corresponding to LSP-1, PA-I,
and
TAP-1, whereas a "non-LSP-1, non-PA-I, and non-TAP-1 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a protein which is
not
substantially homologous to the LSP-1, PA-I, and TAP-1 protein, e.g., a
protein which is
different from the LSP-1, PA-I, and TAP-1 protein and which is derived from
the same
or a different organism. Within a LSP-1, PA-I, and TAP-1 fusion protein the
LSP-1,
PA-I, and TAP-1 polypeptide can correspond to all or a portion of a LSP-1, PA-
I, and
TAP-1 protein. In a preferred embodiment, a LSP-I, PA-I, and TAP-1 fusion
protein
comprises at least one biologically active portion of a LSP-1, PA-I, and TAP-1
protein.
In another preferred embodiment, a LSP-1, PA-I, and TAP-1 fusion protein
comprises at
least two biologically active portions of a LSP-1, PA-I, and TAP-1 protein. In
another
preferred embodiment, a LSP-1, PA-I, and TAP-1 fusion protein comprises at
least three
biologically active portions of a LSP-1, PA-I, and TAP-1 protein. Within the
fusion
protein, the term "operatively linked" is intended to indicate that the LSP-1,
PA-I, and
TAP-1 polypeptide and the non-LSP-1, non-PA-I, and non-TAP-1 polypeptide are
fused
in-frame to each other. The non-LSP-1, non-PA-I, and non-TAP-1 polypeptide can
be
fused to the N-terminus or C-terminus of the LSP-1, PA-I, and TAP-1
polypeptide.
Such fusion proteins can be further utilized in screening assays for compounds
which
modulate LSP-1, PA-I, and TAP-1 activity (such assays are described in detail
below).
In yet another embodiment, the fusion protein is a GST-LSP-1, GST-PA-I, and
GST-TAP-1 fusion protein in which the LSP-1, PA-I, and TAP-1 sequences are
fused to
the C-terminus of the GST sequences. Such fusion proteins can facilitate the
purification of recombinant LSP-1, PA-I, and TAP-1.
In another embodiment, the fusion protein is a LSP-1, PA-I, and TAP-1 protein
containing a heterologous signal sequence at its N-terminus. For example, the
native
LSP-1, PA-I, and TAP-1 signal sequence (i.e, amino acids 1 to 20 of SEQ ID
N0:2, or
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amino acids 1 to 30 of SEQ ID NO:S) can be removed and replaced with a signal
sequence from another protein. In certain host cells (e.g., mammalian host
cells),
expression and/or secretion of LSP-1, PA-I, and TAP-1 can be increased through
use of
a heterologous signal sequence.
In yet another embodiment, the fusion protein is a LSP-1, PA-I, and TAP-1-
immunoglobulin fusion protein in which the LSP-1, PA-I, and TAP-1 sequences
comprising primarily the cysteine rich domain are fused to sequences derived
from a
member of the immunoglobulin protein family. Methods for preparing such fusion
proteins have been described in Capon, D.J. et al. (1989) Nature 337:525-531
and
Capon U.S. Patents 5,116,964 and 5,428,130 [CD4-IgGl constructs]; Linsley,
P.S. et al.
(1991) J. Exp. Med. 173:721-730 [a CD28-IgGl construct and a B7-1-IgGl
construct];
and Linsley, P.S. et al. (1991) J. Exp. Med. 174:561-569 and U.S. Patent
5,434,131 [a ,
CTLA4-IgG1 ]). Such fusion proteins have proven useful for modulating receptor-
ligand
interactions.
The LSP-1, PA-I, and TAP-1-immunoglobulin fusion proteins of the invention
can be incorporated into pharmaceutical compositions and administered to a
subject to
inhibit an interaction between a LSP-1, PA-I, and TAP-1 molecule and a protein
which
binds LSP-1, PA-I, and TAP-1 and exists on the surface of a cell (e.g., a LSP-
1, PA-I,
and TAP-1 receptor) to, thereby, suppress LSP-1, PA-I, and TAP-1-mediated
signal
transduction in vivo. The LSP-1, PA-I, and TAP-1-immunoglobulin fusion
proteins can
be used to affect the bioavailability of the LSP-1, PA-I, and TAP-1 molecule.
Inhibition
of the LSP-1, PA-I, and TAP-1 receptor/LSP-1, PA-I, and TAP-1 interaction may
be
useful therapeutically for both the treatment of LSP-1, PA-I, and TAP-1
associated
disorders, e.g., proliferative and differentiative disorders, as well as
modulating {e.g.,
promoting or inhibiting) angiogenesis. Moreover, the LSP-1, PA-I, and TAP-1-
immunoglobulin fusion proteins of the invention can be used as immunogens to
produce
anti-LSP-1, PA-I, and TAP-1 antibodies in a subject, to purify LSP-1, PA-I,
and TAP-1
receptors and in screening assays to identify molecules which inhibit the
interaction of
LSP-I, PA-I, and TAP-I with a LSP-1, PA-I, and TAP-1 receptor.
Preferably, a LSP-1, PA-I, and TAP-1 chimeric or fusion protein of the
invention
is produced by standard recombinant DNA techniques. For example, DNA fragments
coding for the different polypeptide sequences are ligated together in-frame
in
accordance with conventional techniques, for example by employing blunt-ended
or
stagger-ended termini for ligation, restriction enzyme digestion to provide
for
appropriate termini, filling-in of cohesive ends as appropriate, alkaline
phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. In another
embodiment,
the fusion gene can be synthesized by conventional techniques including
automated
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DNA synthesizers. Alternatively, PCR amplification of gene fragments can be
carried
out using anchor primers which give rise to complementary overhangs between
two
consecutive gene fragments which can subsequently be annealed and reamplified
to
generate a chimeric gene sequence (see, for example, Current Protocols in
Molecular
Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many
expression
vectors are commercially available that already encode a fusion moiety (e.g.,
a GST
polypeptide). A LSP-1, PA-I, and TAP-1-encoding nucleic acid can be cloned
into such
an expression vector such that the fusion moiety is linked in-frame to the LSP-
1, PA-I,
and TAP-1 protein.
The present invention also pertains to variants of the LSP-I, PA-I, and TAP-1
protein which function as either LSP-1, PA-I, and TAP-1 agonists (mimetics) or
as LSP-
1, PA-I, and TAP-1 antagonists. Variants of the LSP-1, PA-I, and TAP-1 protein
can be
generated by mutagenesis, e.g., discrete point mutation or truncation of the
LSP-1, PA-I,
and TAP-1 protein. An agonist of the LSP-1, PA-I, and TAP-1 protein can retain
substantially the same, or a subset, of the biological activities of the
naturally occurring
form of the LSP-1, PA-I, and TAP-1 protein. An antagonist of the LSP-1, PA-I,
and
TAP-1 protein can inhibit one or more of the activities of the naturally
occurring form of
the LSP-1, PA-I, and TAP-1 protein by, for example, competitively binding to a
LSP-1,
PA-I, and TAP-1 receptor. Thus, specific biological effects can be elicited by
treatment
with a variant of limited function. In one embodiment, treatment of a subject
with a
variant having a subset of the biological activities of the naturally
occurring form of the
protein has fewer side effects in a subject relative to treatment with the
naturally
occurring form of the LSP-1, PA-I, and TAP-1 proteins.
In one embodiment, variants of the LSP-1, PA-I, and TAP-1 protein which
function as either LSP-I, PA-I, and TAP-1 agonists (mimetics) or as LSP-1, PA-
I, and
TAP-I antagonists can be identified by screening combinatorial libraries of
mutants,
e.g., truncation mutants, of the LSP-1, PA-I, and TAP-1 protein for LSP-1, PA-
I, and
TAP-1 protein agonist or antagonist activity. In one embodiment, a variegated
library of
LSP-1, PA-I, and TAP-1 variants is generated by combinatorial mutagenesis at
the
nucleic acid level and is encoded by a variegated gene library. A variegated
library of
LSP-1, PA-I, and TAP-1 variants can be produced by, for example, enzymatically
ligating a mixture of synthetic oligonucleotides into gene sequences such that
a
degenerate set of potential LSP-1, PA-I, and TAP-1 sequences is expressible as
individual polypeptides or, alternatively, as a set of larger fusion proteins
(e.g., for phage
display) containing the set of LSP-1, PA-I, and TAP-1 sequences therein. There
are a
variety of methods which can be used to produce libraries of potential LSP-1,
PA-I, and
TAP-1 variants from a degenerate oligonucleotide sequence. Chemical synthesis
of a
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degenerate gene sequence can be performed in an automatic DNA synthesizer, and
the
synthetic gene then ligated into an appropriate expression vector. Use of a
degenerate
set of genes allows for the provision, in one mixture, of all of the sequences
encoding
the desired set of potential LSP-1, PA-I, and TAP-1 sequences. Methods for
synthesizing degenerate oligonucleotides are known in the art (see, e.g.,
Narang, S.A.
(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;
Itakura et al.
(1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the LSP-1, PA-I, and TAP-1 protein
coding
sequence can be used to generate a variegated population of LSP-1, PA-I, and
TAP-1
fragments for screening and subsequent selection of variants of a LSP-1, PA-I,
and TAP-
1 protein. In one embodiment, a library of coding sequence fragments can be
generated
by treating a double stranded PCR fragment of a LSP-1, PA-I, and TAP-1 coding
sequence with a nuclease under conditions wherein nicking occurs only about
once per
molecule, denaturing the double stranded DNA, renaturing the DNA to form
double
1 S stranded DNA which can include sense/antisense pairs from different nicked
products,
removing single stranded portions.from reformed duplexes by treatment with S1
nuclease, and ligating the resulting fragment library into an expression
vector. By this
method, an expression library can be derived which encodes N-terminal, C-
terminal and
internal fragments of various sizes of the LSP-1, PA-I, and TAP-1 protein.
Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of LSP-
1, PA-I, and TAP-1 proteins. The most widely used techniques, which are
amenable to
high through-put analysis, for screening large gene libraries typically
include cloning the
gene library into replicable expression vectors, transforming appropriate
cells with the
resulting library of vectors, and expressing the combinatorial genes under
conditions in
which detection of a desired activity facilitates isolation of the vector
encoding the gene
whose product was detected. Recrusive ensemble mutagenesis (REM), a new
technique
which enhances the frequency of functional mutants in the libraries, can be
used in
combination with the screening assays to identify LSP-1, PA-I, and TAP-1
variants
(Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993} Protein
Engineering 6(3):327-331 ).
In one embodiment, cell based assays can be exploited to analyze a variegated
LSP-1 library. For example, a library of expression vectors can be transfected
into a cell
line which ordinarily responds to a particular ligand in a LSP-1-dependent
manner. The
transfected cells are then contacted with the ligand and the effect of
expression of the
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mutant on signaling by the ligand can be detected, e.g., by measuring any of a
number of
immune cell responses. Plasmid DNA can then be recovered from the cells which
score
for inhibition, or alternatively, potentiation of ligand induction, and the
individual clones
further characterized.
In another embodiment, cell based assays can be exploited to analyze a
variegated PA-I library. For example, a library of expression vectors can be
transfected
into a cell line which ordinarily responds to a PA-I molecule, e.g., a cell
line derived
from placental tissue. The transfected cells are then contacted with the, PA-I
mutant and
the effect of expression of the mutant on cellular proliferation can be
detected, e.g., by
measuring a cellular proliferation-related parameter. Plasmid DNA can then be
recovered from the cells which score for inhibition, or alternatively,
potentiation of
cellular proliferation, and the individual clones further characterized.
In yet another embodiment, cell based assays can be exploited to analyze a
variegated TAP-1 library. For example, a library of expression vectors
containing the
appropriate secretory signals can be transfected into a cell line and
contacted with a
HARP-responsive cell line. The effect of expression of the mutant on a TAP-1-
responsive cell can be measured as, e.g., changes in signal transduction or by
measuring
cell proliferation, differentiation or survival. Plasmid DNA can then be
recovered from
the cells which score for modulation, e.g., inhibition, or potentiation, pf
TAP-1 activity,
and the individual clones further characterized.
An isolated LSP-1, PA-I, and TAP-1 protein, or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind LSP-1, PA-I, and
TAP-1
using standard techniques for polyclonal and monoclonal antibody preparation.
The
full-length LSP-1, PA-I, and TAP-1 protein can be used or, alternatively, the
invention
provides antigenic peptide fragments of LSP-1; PA-I, and TAP-1 for use as
immunogens. The antigenic peptide of LSP-1, PA-I, and TAP-1 comprises at least
8
amino acid residues of the amino acid sequence shown in SEQ ID N0:2, S, or 8
and
encompasses an epitope of LSP-1, PA-I, and TAP-1 such that an antibody raised
against
the peptide forms a specific immune complex with LSP-1, PA-I, and TAP-1.
Preferably, the antigenic peptide comprises at least 10 amino acid residues,
more
preferably at least 15 amino acid residues, even more preferably at least 20
amino acid
residues, and most preferably at least 30 amino acid residues. Preferred
epitopes
encompassed by the antigenic peptide are regions of LSP-1, PA-I, and TAP-1
that are
located on the surface of the protein, e.g., hydrophilic regions.
A LSP-1, PA-I, and TAP-1 immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal)
with the
immunogen. An appropriate immunogenic preparation can contain, for example,
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recombinantly expressed LSP-1, PA-I, and TAP-1 protein or a chemically
synthesized
LSP-1, PA-I, and TAP-1 polypeptide. The preparation can further include an
adjuvant,
such as Freund's complete or incomplete adjuvant, or similar immunostimulatory
agent.
Immunization of a suitable subject with an imrnunogenic LSP-1, PA-I, and TAP-1
preparation induces a polyclonal anti-LSP-I, PA-I, and TAP-1 antibody
response.
Accordingly, another aspect of the invention pertains to anti-LSP-1, PA-I, and
TAP-I antibodies. The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e.,
molecules that contain an antigen binding site which specifically binds
(immunoreacts
with) an antigen, such as LSP-1, PA-I, and TAP-1. Examples of immunologically
active
portions of immunoglobulin molecules include Flab) and F(ab')2 fragments which
can
be generated by treating the antibody with an enzyme such as pepsin. The
invention
provides polyclonal and monoclonal antibodies that bind LSP-1, PA-I, and TAP-
1. The
term "monoclonal antibody" or "monoclonal antibody composition", as used
herein,
refers to a population of antibody molecules that contain only one species of
an antigen
binding site capable of immunoreacting with a particular epitope of LSP-1, PA-
I, and
TAP-1. A monoclonal antibody composition thus typically displays a single
binding
. affinity for a particular LSP-1, PA-I, and TAP-1 protein with which it
imrnunoreacts.
Polyclonal anti-LSP-1, PA-I, and TAP-1 antibodies can be prepared as described
above by immunizing a suitable subject with a LSP-1, PA-I, and TAP-1
immunogen.
The anti-LSP-1, PA-I, and TAP-1 antibody titer in the immunized subject can be
monitored over time by standard techniques, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized LSP-1, PA-I, and TAP-1. If
desired,
the antibody molecules directed against LSP-I, PA-I, and TAP-1 can be isolated
from
the mammal (e.g., from the blood) and further purified by well known
techniques, such
as protein A chromatography to obtain the IgG fraction. At an appropriate time
after
immunization, e.g., when the anti-LSP-1, PA-I, and TAP-1 antibody titers are
highest,
antibody-producing cells can be obtained from the subject and used to prepare
monoclonal antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature 256:495-497) (see
also,
Brown et al. { 1981 ) J. Immunol. 127:539-46; Brown et al. ( 1980) J. Biol.
Chem
.255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J.
Cancer
29:269-75), the more recent human B cell hybridoma technique (Kozbor et al.
(1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. ( 1985),
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma
techniques.
The technology for producing monoclonal antibody hybridomas is well known (see
generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In
Biological
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Analyses, Plenum Publishing Corp., New York, New York ( 1980); E. A. Lerner (
1981 )
Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet.
3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to
lymphocytes
(typically splenocytes) from a mammal immunized with a LSP-l, PA-I, and TAP-1
immunogen as described above, and the culture supernatants of the resulting
hybridoma
cells are screened to identify a hybridoma producing a monoclonal antibody
that binds
LSP-1, PA-I, and TAP-1.
Any of the many well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied for the purpose of generating an anti-
LSP-1, PA-I,
and TAP-1 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature
266:55052;
Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol.~Med.,
cited supra;
Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled
worker
will appreciate that there are many variations of such methods which also
would be
useful. Typically, the immortal cell line (e.g., a myeloma cell line) is
derived from the
same mammalian species as the lymphocytes. For example, marine hybridomas can
be
made by fusing lymphocytes from a mouse immunized with an immunogenic
preparation of the present invention with an immortalized mouse cell line.
Preferred
immortal cell Iines are mouse myeloma cell lines that are sensitive ~io
culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a
number of myeloma cell lines can be used as a fusion partner according to
standard
techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma
lines.
These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse
myeloma cells are fused to mouse splenocytes using polyethylene glycol
("PEG").
Hybridoma cells resulting from the fusion are then selected using HAT medium,
which
kills unfused and unproducrively fused myeloma cells (unfused splenocytes die
after
several days because they are not transformed). Hybridoma cells producing a
monoclonal antibody of the invention are detected by screening the hybridoma
culture
supernatants for antibodies that bind LSP-1, PA-I, and TAP-1, e.g., using a
standard
ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal anti-LSP-1, PA-I, and TAP-1 antibody can be identified and isolated
by
screening a recombinant combinatorial immunoglobulin library (e.g., an
antibody phage
display library) with LSP-1, PA-I, and TAP-1 to thereby isolate immunoglobulin
library
members that bind LSP-1, PA-I, and TAP-1. Kits for generating and screening
phage
display libraries are commercially available (e.g., the Pharmacia Recombinant
Phage
Antibody System, Catalog No. 27-9400-O1; and the Stratagene SurflAPTMPhage
Display Kit, Catalog No. 240612). Additionally, examples of methods and
reagents
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particularly amenable for use in generating and screening antibody display
library can be
found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al.
PCT
International Publication No. WO 92118619; Dower et al. PCT International
Publication
No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791;
Markland
et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT
International
Publication WO 93/01288; McCafferty et al. PCT International Publication No.
WO
92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner
et al.
PCT International Publication No. WO 90/02809; Fuchs et al. (1991)
Biol~'echnology
9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al.
(1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et
al.
(1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et
al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) BiolTechnology 9:1373-1377;
Hoogenboom et al. ( 1991 ) Nuc. Acid Res. 19:4133-4137; Barbas et al. { 1991 )
PNAS
88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
Additionally, recombinant anti-LSP-1, PA-I, and TAP-1 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human and non-
human portions, which can be made using standard recombinant DNA techniques,
are
within the scope of the invention. Such chimeric and humanized monoclonal
antibodies
can be produced by recombinant DNA techniques known in the art, for example
using
methods described in Robinson et al. International Application No.
PCT/LTS86/02269;
Akira, et al. European Patent Application 184,187; Taniguchi, M., European
Patent
Application 171,496; Morrison et al. European Patent Application 173,494;
Neuberger
et aI. PCT International Publication No. WO 86/01533; Cabilly et al. U.S.
Patent No.
4,816,567; Cabilly et al. European Patent Application 125,023; Better et al.
(1988)
Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987)
J.
Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.
(1987)
Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al.
(1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science
229:1202-
1207; Oi et al. (1986) BioTechnigues 4:214; Winter U.S. Patent 5,225,539;
Jones et al.
(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and
Beidler et
al. (1988) J. Immunol. 141:4053-4060.
An anti-LSP-1, PA-I, and TAP-1 antibody (e.g., monoclonal antibody) can be
used to isolate LSP-1, PA-I, and TAP-1 by standard techniques, such as
affinity
chromatography or immunoprecipitation. An anti-LSP-1, PA-I, and TAP-1 antibody
can facilitate ttte purification of natural LSP-1, PA-I, and TAP-1 from cells
and of
recombinantly produced LSP-l, PA-I, and TAP-1 expressed in host cells.
Moreover, an
anti-LSP-1, PA-I, and TAP-1 antibody can be used to detect LSP-l, PA-I, and
TAP-1
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protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate
the abundance
and pattern of expression of the LSP-1, PA-I, and TAP-1 protein. Anti-LSP-1,
PA-I,
and TAP-1 antibodies can be used diagnostically to monitor protein levels in
tissue as
part of a clinical testing procedure, e.g., to, for example, determine the
efficacy of a
given treatment regimen. Detection can be facilitated by coupling (i.e.,
physically
linking) the antibody to a detectable substance. Examples of detectable
substances
include various enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials. Examples of
suitable
enzymes include horseradish peroxidase, alkaline phosphatase, (3-
galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes !amino!; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin, and examples of suitable radioactive
material include
125h 131h 35S or 3H.
III. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors, containing a nucleic acid encoding LSP-1, PA-I, and TAP-1 (or a
portion
thereof). As used herein, the term "vector" refers to a nucleic acid molecule
capable of
transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional
DNA segments can be Iigated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome. Certain vectors
are
capable of autonomous replication in a host cell into which they are
introduced (e.g.,
bacterial vectors having a bacterial origin of replication and episomal
mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the
genome of a host cell upon introduction into the host cell, and thereby are
replicated
along with the host genome. Moreover, certain vectors are capable of directing
the
expression of genes to which they are operatively linked. Such vectors are are
referred
to herein as "expression vectors". In general, expression vectors of utility
in
recombinant DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably as the
plasmid is the
most commonly used form of vector. However, the invention is intended to
include
such other forms of expression vectors, such as viral vectors (e.g.,
replication defective
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retroviruses, adenoviruses and adeno-associated viruses), which serve
equivalent
functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the invention in a form suitable for expression of the nucleic acid in a host
cell, which
means that the recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for expression,
which is
operatively linked to the nucleic acid sequence to be expressed. Within a
recombinant
expression vector, "operably linked" is intended to mean that the nucleotide
sequence of
interest is linked to the regulatory sequences) in a manner which allows for
expression
of the nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a
host cell when the vector is introduced into the host cell). The term
"regulatory
sequence" is intended to include promoters, enhancers and other expression
control
elements (e.g., polyadenylation signals). Such regulatory sequences are
described, for
example, in Goeddel; Gene Expression Technology: Methods in Enrymology 185,
1 S Academic Press, San Diego, CA ( 1990). Regulatory sequences include those
which
direct constitutive expression of a nucleotide sequence in many types of host
cell and
those which direct expression of the nucleotide sequence only in certain host
cells (e.g.,
tissue-specific .regulatory sequences). It wilt be appreciated by those
skilled in the art
that the design of the expression vector can depend on such factors as the
choice of the
host cell to be transformed, the level of expression of protein desired, etc.
The
expression vectors of the invention can be introduced into host cells to
thereby produce
proteins or peptides, including fusion proteins or peptides, encoded by
nucleic acids as
described herein (e.g., LSP-l, PA-I, and TAP-1 proteins, mutant forms of LSP-
1, PA-I,
and TAP-1, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of LSP-l, PA-I, and TAP-1 in prokaryotic or eukaryotic cells. For
example,
LSP-I, PA-I, and TAP-1 can be expressed in bacterial cells such as E. coli,
insect cells
(using baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host
cells are discussed further in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in vitro, for
example
using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli
with
vectors containing constitutive or inducible promotors directing the
expression of either
fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein
encoded therein, usually to the amino terminus of the recombinant protein.
Such fusion
vectors typically serve three purposes: 1 ) to increase expression of
recombinant protein;
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2) to increase the solubility of the recombinant protein; and 3) to aid in the
purification
of the recombinant protein by acting as a ligand in affinity purification.
Often, in fusion
expression vectors, a proteolytic cleavage site is introduced at the junction
of the fusion
moiety and the recombinant protein to enable separation of the recombinant
protein from
the fusion moiety subsequent to purification of the fusion protein. Such
enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin arid
enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith,
D.B.
and Johnson, K.S. {1988) Gene 67:31-40), pMAL (New England BioIabs, Beverly,
MA)
and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase
(GST),
maltose E binding protein, or protein A, respectively, to the target
recombinant protein.
In a preferred embodiment, the coding sequence of human LSP-l, PA-I, and
TAP-1 (i.e., encompassing amino acids 1 to 253) is cloned into a pCDS
expression
vector to create a vector encoding a LSP-1, PA-I, and TAP-1-Ig fusion protein.
In an
alternative preferred embodiment, the coding sequence of a for~rn of human LSP-
1, PA-I,
and TAP-1 lacking the signal sequence is cloned into a pPicZ expression vector
(InVitrogen) downstream and in frame with a yeast-derived signal sequence. In
yet
another preferred embodiment, the coding sequence of human LSP-1, PA-I, and
TAP-1
{e.g., the sequence shown in SEQ ID N0:3, 6, or 9) is cloned into a retroviral
expression
vector, pWZLBIastEC. The fusion proteins can be purified utilizing methods
well
known in the art of protein purification. Purified fusion proteins can be
utilized in LSP-
1, PA-I, and TAP-1 activity assays, in LSP-1, PA-I, and TAP-1 receptor binding
(e.g.
direct assays or competitive assays described in detail below), to generate
antibodies
specific for LSP-1, PA-I, and TAP-1 proteins, as examples. In a preferred
embodiment,
a LSP-1, PA-I, and TAP-1 fusion expressed in a retroviral expression vector of
the
present invention can be utilized to infect bone marrow cells which are
subsequently
transplanted into in adiated recipients. The pathology of the subject
recipient is then
examined after suffccient time has passed (e.g six (6) weeks).
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene
Expression Technolo~.~ Methods in Enrymology 185, Academic Press, San Diego,
California (1990) 60-89). Target gene expression from the pTrc vector relies
on host
RNA polymerise transcription from a hybrid trp-lac fusion promoter. Target
gene
expression from the pET 1 ld vector relies on transcription from a T7 gnl0-lac
fusion
promoter mediated by a coexpressed viral RNA polymerise {T7 gnl). This viral
polymerise is supplied by host strains BL21(DE3) or HMS174(DE3) from a
resident ~.
prophage harboring a T7 gn 1 gene under the transcriptional control of the
lacUV 5
promoter.
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One strategy to maximize recombinant protein expression in E. coli is to
express
the protein in a host bacteria with an impaired capacity to proteolytically
cleave the
recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an
expression vector so that the individual codons for each amino acid are those
preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res.
20:2111-2118).
Such alteration of nucleic acid sequences of the invention can be carried out
by standard
DNA synthesis techniques.
In another embodiment, the LSP-1, PA-I, and TAP-1 expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S. cerivisae
include
pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and
Herskowitz,
(1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123),
pYES2
(Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego,
CA).
Alternatively, LSP-1, PA-I, and TAP-1 can be expressed in insect cells using
baculovirus expression vectors. Baculovirus vectors available far expression
of proteins
in cultured insect cells (e.g., Sf 9 cells) include the pAc series {Smith et
al. (1983) Mol.
Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Sumrzers (1989)
Virology
170:31-3 9).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by.viral regulatory
elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable expression systems for
both
prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989.
In another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially in a
particular cell type
(e.g., tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-
specific regulatory elements are known in the art. Non-limiting examples of
suitable
tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al.
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto
and
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Baltimore ( 1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. ( I
983) Cell
33:729-740; Queen and Baltimore (1983) Cel133:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316
and
European Application Publication No. 264,166). Developmentally-regulated
promoters
are also encompassed, for example the murine hox promoters (Kessel and Gruss (
1990)
Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (
1989)
Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a
DNA molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in
a manner which allows for expression (by transcription of the DNA molecule) of
an
RNA molecule which is antisense to LSP-1, PA-I, and TAP-1 mRNA. Regulatory
1 S sequences operatively linked to a nucleic acid cloned in the antisense
orientation can be
chosen which direct the continuous expression of the aiitisense RNA molecule
in a
variety of cell types, for instance viral promoters and/or enhancers, or
regulatory
sequences can be chosen which direct constitutive, tissue specific or cell
type specific
expression of antisense RNA. The antisense expression vector can be in the
form of a
recombinant plasmid, phagemid or attenuated virus in which antisense nucleic
acids are
produced under the control of a high efficiency regulatory region, the
activity of which
can be determined by the cell type into which the vector is introduced. For a
discussion
of the regulation of gene expression using antisense genes see Weintraub, H.
et al.,
Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in
Genetics,
Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant .
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such
terms refer not only to the particular subject cell but to the progeny or
potential progeny
of such a cell. Because certain modifications may occur in succeeding
generations due
to either mutation or environmental influences, such progeny may not, in fact,
be
identical to the parent cell, but are still included within the scope of the
term as used
herein. _
A host cell can be any prokaryotic or eukaryotic cell. For example, LSP-1, PA-
I,
and TAP-1 protein can be expressed in bacterial cells such as E. coli, insect
cells, yeast
or mammalian cells, such as Chinese hamster ovary cells (CHO) or COS cells.
Other
suitable host cells are known to those skilled in the art.
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Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized
techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell,
including
calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Suitable methods for
transforming or
transfecting host cells can be found in Sambrook, et al. (Molecular Cloning. A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Preferred
selectable markers include those which confer resistance to drugs, such as
6418,
hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be
introduced into a host cell on the same vector as that encoding LSP-1, PA-I,
and TAP-1
or can be introduced on a separate vector. Cells stably transfected with the
introduced
nucleic acid can be identified by drug selection (e.g., cells that have
incorporated the
selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i.e., express) LSP-1, PA-I, and TAP-1
protein.
Accordingly, the invention further provides methods for producing LSP-l, PA-I,
and
TAP-1 protein using the host cells of the invention. In one embodiment, the
method
comprises culturing the host cell of invention (into which a recombinant
expression
vector encoding LSP-1, PA-I, and TAP-1 has been introduced) in a suitable
medium
such that LSP-1, PA-I, and TAP-1 protein is produced. In another embodiment,
the
method further comprises isolating LSP-1, PA-I, and TAP-1 from the medium or
the
host cell.
The host cells of the invention can also be used to produce nonhuman
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized
oocyte or an embryonic stem cell into which LSP-1, PA-I, and TAP-1-coding
sequences
have been introduced. Such host cells can then be used to create non-human
transgenic
animals in which exogenous LSP-1, PA-I, and TAP-1 sequences have been
introduced
into their genome or homologous recombinant animals in which endogenous LSP-1,
PA-I, and TAP-1 sequences have been altered. Such animals are useful for
studying the
function and/or activity of LSP-1, PA-I, and TAP-1 and for identifying andlor
evaluating
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modulators of LSP-1, PA-I, and TAP-1~ activity. As used herein, a "transgenic
animal"
is a non-human animal, preferably a mammal, more preferably a rodent such as a
rat or
mouse, in which one or more of the cells of the animal includes a transgene.
Other
examples of transgenic animals include non-human primates, sheep, dogs, cows,
goats,
chickens, amphibians, etc. A transgene is exogenous DNA which is integrated
into the
genome of a cell from which a transgenic animal develops and which remains in
the
genome of the mature animal, thereby directing the expression of an encoded
gene
product in one or more cell types or tissues of the transgenic animal. As used
herein, a
"homologous recombinant animal" is a non-human anirrial, preferably a mammal,
more
preferably a mouse, in which an endogenous LSP-1, PA-I, and TAP-1 gene has
been
altered by homologous recombination between the endogenous gene and an
exogenous
DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of
the
animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing LSP-1, PA-
I,
and TAP-I-encoding nucleic acid into the male pronuclei of a fertilized
oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. The marine LSP-1, PA-I, and TAP-1 cDNA
sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9 can be introduced as a transgene
into the
genome of a non-marine animal. Alternatively, a nonmurine homologue of the
marine
LSP-1, PA-I, and TAP-1 gene, such as a human LSP-1, PA-I, and TAP-1 gene, can
be
isolated based on hybridization to the marine LSP-1, PA-I, and TAP-1 cDNA
(described
further in subsection I above) and used as a transgene. Intronic sequences and
polyadenylation signals can also be included in the transgene to increase the
efficiency
of expression of the transgene. A tissue-specific regulatory sequences) can be
operably
linked to the LSP-1, PA-I, and TAP-1 transgene to direct expression of LSP-1,
PA-I, and
TAP-1 protein to particular cells. Methods for generating transgenic animals
via embryo
manipulation and microinjection, particularly animals such as mice, have
become
conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866 and
4,870,009, both by Leder et al., U.S. Patent No. 4,873,191 by Wagner et al.
and in
Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of
other
transgenic animals. A transgenic founder animal can be identified based upon
the
presence of the LSP-1, PA-I, and TAP-1 transgene in its genome and/or
expression of
LSP-l, PA-I, and TAP-1 mRNA in tissues or cells of the animals. A transgenic
founder
animal can then be used to breed additional animals carrying the transgene.
Moreover,
transgenic animals carrying a transgene encoding LSP-1, PA-I, and TAP-1 can
further
be bred to other transgenic animals carrying other transgenes.
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To create a homologous recombinant animal, a vector is prepared which contains
at least a portion of a LSP-1, PA-I, and TAP-1 gene into which a deletion,
addition or
substitution has been introduced to thereby alter, e.g., functionally disrupt,
the LSP-l,
PA-I, and TAP-1 gene. The LSP-1, PA-I, and TAP-1 gene can be a human gene, but
more preferably, is a non-human homologue of a human LSP-1, PA-I, and TAP-1
gene.
For example, a mouse LSP-1, PA-I, and TAP-1 gene of SEQ ID NO:1 can be used to
construct a homologous recombination vector suitable for altering an
endogenous LSP-
l, PA-I, and TAP-1 gene in the mouse genome. In a preferred embodiment, the
vector is
designed such that, upon homologous recombination, the endogenous LSP-1, PA-I,
and
TAP-1 gene is functionally disrupted (i.e., no longer encodes a functional
protein; also
referred to as a "knock out" vector). Alternatively, the vector can be
designed such that,
upon homologous recombination, the endogenous LSP-l, PA-I, and TAP-1 gene is
mutated or otherwise altered but still encodes functional protein (e.g., the
upstream
regulatory region can be altered to thereby alter the expression of the
endogenous LSP-
1, PA-I, and TAP-1 protein). In the homologous recombination vector, the
altered
portion of the LSP-1, PA-I, and TAP-1 gene is flanked at its 5' and 3' ends by
additional
nucleic acid ofthe LSP-1, PA-I, and TAP-1 gene to allow for homologous
recombination to occur between the exogenous LSP-l, PA-I, and TAP-1 gene
carried by
the vector and an endogenous LSP-1, PA-I, and TAP-1 gene in an embryonic stem
cell.
The additional flanking LSP-1, PA-I, and TAP-1 nucleic acid is of sufficient
length for
successful homologous recombination with the endogenous gene. Typically,
several
kilobases of flanking DNA (both at the 5' and 3' ends) are included in the
vector (see
e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of
homologous recombination vectors). The vector is introduced into an embryonic
stem
cell line (e.g., by electroporation) and cells in which the introduced LSP-1,
PA-I, and
TAP-1 gene has homologously recombined with the endogenous LSP-1, PA-I, and
TAP-
1 gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected
cells are then
injected into a blastocyst of an animal (e.g., a mouse) to form aggregation
chimeras (see
e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric
embryo
can then be implanted into a suitable pseudopregnant female foster animal and
the
embryo brought to term. Progeny harboring the homologously recombined DNA in
their germ cells can be used to breed animals in which all cells of the animal
contain the
homologously recombined DNA by germline transmission of the transgene. Methods
for constructing homologous recombination vectors and homologous recombinant
animals are described further in Bradley, A. ( 1991 ) Current Opinion in
Biotechnology
2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le
MoueIlec et
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al.; WO 91/01.140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO
93/04169
by Berns et al.
In another embodiment, transgenic non-humans animals can be produced which
contain selected systems which allow for regulated expression of the
transgene. One
S example of such a system is the crelloxP recombinase system of bacteriophage
Pl . For
a description of the crelloxP recombinase system, see, e.g., Lakso et al.
(1992) PNAS
89:6232-6236. Another example of a recombinase system is the FLP recombinase
system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-
1355. If
a crelloxP recombinase system is used to regulate expression of the transgene,
animals
containing transgenes encoding both the Cre recombinase and a selected protein
are
required. Such animals can be provided through the construction of "double"
transgenic
animals, e.g., by mating two transgenic animals, one containing a transgene
encoding a
selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced according to the methods described in Wilmut, I. et al. (1997) Nature
385:810-
813. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be
isolated and
induced to to exit the growth cycle and enter Go phase. The quiescent cell can
then be
fused, e.g., through the use of electrical pulses, to an enucIeated oocyte
from an animal
of the same species from which the quiescent cell is isolated. The
recontructed oocyte is
then cultured such that it develops to morula or blastocyte and then
transferred to
pseudopregnant female foster animal. The offspring borne of this female foster
animal
will be a clone of the animal from which the cell, e.g., the somatic cell, is
isolated.
IV. Pharmaceutical Comuositions
The LSP-1, PA-I, and TAP-1 nucleic acid molecules, LSP-l, PA-I, and TAP-1
proteins, and anti-LSP-1, anti-PA-I, and anti-TAP-1 antibodies (also referred
to herein
as "active compounds") of the invention can be incorporated into
pharmaceutical
compositions suitable for administration. Such compositions typically comprise
the
nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable
carnei.
As used herein the language "pharmaceutically acceptable carrier" is intended
to include
any and all solvents, dispersion media, coatings, antibacterial and antifungal
agents,
isotonic and absorption delaying agents, and the like, compatible with
pharmaceutical
administration. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any conventional media
or agent
is incompatible with the active compound, use thereof in the compositions is
contemplated. Supplementary active compounds can also be incorporated into the
compositions.
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A pharmaceutical composition of the invention is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation),
transdermal (topical), transmucosal, and rectal administration. Solutions or
suspensions
used for parenteraI, intradermal, or subcutaneous application can include the
following
components: a sterile diluent such as water for injection, saline solution,
fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants
such as
ascorbic acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents for the
adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or
bases,
such as hydrochloric acid or sodium hydroxide. The parenteral preparation can
be
enclosed in ampoules, disposable syringes or multiple dose vials made of glass
or
plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous administration, suitable carriers include physiological saline,
bacteriostatic
water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline
(PBS). In
all cases, the composition must be sterile and should be fluid to the extent
that easy
syringability exists. It must be stable under the conditions of manufacture
and storage
and must be preserved against the contaminating action of microorganisms such
as
bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity
can be maintained, for example, by the use of a coating such as lecithin, by
the
maintenance of the required particle size in the case of dispersion and by the
use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable
to include
isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol,
sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be
brought about by including in the composition an agent which delays
absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g., a LSP-1, PA-I, and TAP-1 protein or anti-LSP-1, PA-I, and TAP-
I
antibody) in the required amount in an appropriate solvent with one or a
combination of
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ingredients enumerated above, as required, followed by filtered sterilization.
Generally,
dispersions are prepared by incorporating the active compound into a sterile
vehicle
which contains a basic dispersion medium and the required other ingredients
from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, the preferred methods of preparation are vacuum drying and freeze-
drying
which yields a powder of the active ingredient plus any additional desired
ingredient
from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible can:ier.
They
can be enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral
therapeutic administration, the active compound can be incorporated with
excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also
be prepared
using a fluid Garner for use as a mouthwash, wherein the compound in the fluid
carrier is
applied orally and swished and expectorated or swallowed. Pharmaceutically ,
compatible binding agents, and/or adjuvant materials can be included as part
of the
composition. The tablets, pills, capsules, troches and the Iike can contain
any of the
following ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as
starch or
lactose, a disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant,
e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art,
and include, for example, for transmucosal administration, detergents, bile
salts, and
fusidic acid derivatives. Transmucosal administration can be accomplished
through the
use of nasal sprays or suppositories. For transdermal administration, the
active
compounds are formulated into ointments, salves, gels, or creams as generally
known in
the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention
enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will
protect the compound against rapid elimination from the body, such as a
controlled
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release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid.
Methods for preparation of such formulations will be apparent to those skilled
in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposom~l suspensions (including liposomes targeted to
infected
cells with monoclonal antibodies to viral antigens) can also be used as
pharmaceutically
acceptable Garners. These can be prepared according to methods known to those
skilled
in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form
as used herein refers to physically discrete units suited as unitary dosages
fox the subject
to be treated; each unit containing a predetermined quantity of active
compound
calculated to produce the desired therapeutic effect in association with the
required
1 S pharmaceutical carrier. The specification for the dosage unit forms of the
invention are
dictated by and directly dependent on the unique characteristics of the active
compound
and the particular therapeutic effect to be achieved, and the limitations
inherent in the art '
of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g., for
determining the LD50 (the dose lethal to SO% of the population) and the ED50
(the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio
LDSO/ED50. Compounds which exhibit Large therapeutic indices are preferred.
While
compounds that exhibit toxic side effects may be used, care should be taken to
design a
delivery system that targets such compounds to the site of affected tissue in
order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
The data obtained from the cell culture assays and animal studies can be used
in
formulating a range of dosage for use in humans. The dosage of such compounds
lies
preferably within a range of circulating concentrations that include the ED50
with little
or no toxicity. The dosage may vary within this range depending upon the
dosage form
employed and the route of administration utilized. For any compound used in
the
method of the invention, the therapeutically effective dose can be estimated
initially
from cell culture assays. A dose may be formulated in animal models to achieve
a
circulating plasma concentration range that includes the IC50 (i.e., the
concentration of
the test compound which achieves a half maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more accurately
determine
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useful doses in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
The nucleic acid molecules of the invention can be inserted into vectors and
used
as gene therapy vectors. Gene therapy vectors can be delivered to a subject
by, for
example, intravenous injection, local administration {see U.S. Patent
5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). The
pharmaceutical preparation of the gene therapy vector can include the gene
therapy
vector in an acceptable diluent, or can comprise a slow release matrix in
which the gene
delivery vehicle is imbedded. Alternatively, where the complete gene delivery
vector
can be produced intact from recombinant cells, e.g. retroviral vectors, the
pharmaceutical preparation can include one or more cells which produce the
gene
delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
V. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, and antibodies
described herein can be used in one or more of the following methods: 1 )
screening
assays; 2) predictive assays (e.g., diagnostic assays and pharrnacogenetics);
3)
prognostic assays; 3) monitoring clinical trials; and 4) methods of treatment
(e.g.,
therapeutic and prophylactic).
As described herein, a LSP-1 protein of the invention has one or more of the
following activities: (i) interaction of a LSP-1 protein on the cell surface
with a second
non-LSP-1 protein molecule on the surface of the same cell; (ii) interaction
of a LSP-1
protein on the cell surface with a second non-LSP-1 protein molecule on the
surface of a
different cell; (iii) complex formation between,a soluble LSP-1 protein and a
cognate
ligand; (iv) complex formation between a membrane-bound LSP-1 protein and a
cytokine; (v) interaction of a LSP-I protein with an intracellular protein via
a second
protein on the cell surface and can thus be used for (i) modulation of
cellular signal
transduction; (ii) regulation of a cell involved in an inflammatory response;
(iii) homing
of a cell having a LSP-1 protein on its cell surface from a first to a second
anatomical
location; and (iv) modulation of a cell involved in the immune response,
either in vitro
or in vivo. The isolated nucleic acid molecules of the invention can be used,
for
example, to express LSP-1 protein (e.g., via a recombinant expression vector
in a host
cell in gene therapy applications), to detect LSP-1 mRlVA (e.g., in a
biological sample)
or a genetic lesion in a LSP-I gene, and to modulate LSP-1 activity, as
described further
below. In addition, the LSP-I proteins can be used to screen drugs or
compounds which
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modulate the LSP-t activity as well as to treat disorders characterized by
insufficient or
excessive production of LSP-1 protein or production of LSP-1 protein forms
which have
decreased or aberrant activity compared to LSP-1 wild type protein (e.g.,
inflammatory
diseases such as arthritis and immune response disorders). Moreover, soluble
forms of
the LSP-1 protein can be used to bind ligands of membrane-bound LSP-1 and
influence
bioavailability. In addition, the anti-LSP-1 antibodies of the invention can
be used to
detect and isolate LSP-1 proteins and modulate LSP-1 activity.
As further described herein, a PA-I protein of the invention has one or more
of
the following activities: (i) formation of a complex with a cell-surface
proteins) or a
ligand, e.g., a lipid or carbohydrate; (ii) formation of a complex with a
prolactin and/or
growth hormone receptor; (iii) regulation of cellular growth; (iv) regulation
of cellular
proliferation; (v) regulation of angiogenesis; (vi) regulation of cellular
differentiation;
and (vii) regulation of cell survival, and can thus be used to (i) modulate
complex
formation with a cell-surface proteins) or a ligand, e.g., a lipid or
carbohydrate; (ii)
modulate complex formation with a prolactin and/or growth hormone receptor;
(iii)
regulate cellular growth; (iv) regulate cellular proliferation; (v) regulate
angiogenesis;
(vi) regulate cellular differentiation; and (vii) regulate cell survival,
either in vitro or in
vivo. The isolated nucleic acid molecules of the invention can be used, for
example, to
express PA-I protein (e.g., via a recombinant expression vector in a host cell
in gene
therapy applications), to detect PA-I mRNA (e.g., in a biological sample) or a
genetic
lesion in a PA-I gene, and to modulate PA-I activity, as described further
below. In
addition, the PA-I proteins can be used to screen drugs or compounds which
modulate
the PA-I activity as well as to treat disorders characterized by insufficient
or excessive
production of PA-I protein or production of PA-I protein forms which have
decreased or
aberrant activity compared to PA-I wild type protein (e.g., proliferative
disorders such as
cancer or angiogenesis related disorders). Moreover, soluble forms of PA-I
antagonists
can be used to bind membrane-bound PA-I receptors and influence
bioavailability. In
addition, the anti-PA-I antibodies of the invention can be used to detect and
isolate PA-I
proteins and modulate PA-I activity.
Furthermore, as described herein, a TAP-i protein of the invention has the
following activities: (i) interaction, e.g., binding to, a cell-surface
receptor, e.g., a
hematopoietic-cell surface receptor; (ii) modulation of, e.g., activation or
inhibition of, a
cell-surface receptor (iii) modulation of cellular signal transduction and can
thus be used
to (i) regulate cellular proliferation; (ii) regulate cellular
differentiation; (iii) regulate cell
survival; (iv) modulate a cell involved in the immune response (v) regulate
maturation
and/or differentiation of a hematopoietic stem cell; (vi) modulate
megakaryocytopoiesis;
(vii) modulate thrombopoiesis; (viii) regulate maturation andlor
differentiation of a
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megakaryocyte into platelets; and (ix) regulate maturation and/or
differentiation of
erythroid progenitor cells into erythrocytes, either in vitro or in vivo. The
isolated
nucleic acid molecules of the invention can be used to express TAP-1 protein
(e.g., via a
recombinant expression vector in a host cell in gene therapy applications), to
detect
TAP-1 mRNA (e.g., in a biological sample) or a genetic lesion in a TAP-1 gene,
and to
modulate TAP-1 activity, as described further below. In addition, the TAP-1
proteins
can be used to screen drugs or compounds which modulate the TAP-1 activity as
well as
to treat disorders characterized by insufficient or excessive production of
TAP-1 protein
or production of TAP-1 protein forms which have decreased or abherrent
activity
compared to TAP-1 wild type protein (e.g. hematopoietic disorders disorders
such as
thrombocytopenia or anemia). Moreover, soluble forms of the TAP-1 protein can
be
used to bind ligands of membrane-bound TAP-1 and influence bioavailability. In
addition, the anti-TAP-1 antibodies of the invention can be used to detect and
isolate
TAP-1 proteins and modulate TAP-1 activity.
A. Screening Assays:
The invention provides a method (also referred ;o herein as a "screening
assay")
for identifying modulators, i.e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) which have a LSP-1, PA-I, and
TAP-1
agonist or antagonist activity or have a stimulatory or inhibitory effect on,
for example,
LSP-1, PA-I, and TAP-1 expression or LSP-l, PA-I, and TAP-1 activity.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which have a LSP-1, PA-I, and TAP-1 agonist or antagonist activity.
The
test compounds of the present invention can be obtained using any of the
numerous
approaches in combinatorial library methods known in the art, including:
biological
libraries; spatially addressable parallel solid phase or solution phase
libraries; synthetic
library methods requiring deconvolution; the 'one-bead one-compound' library
method;
and synthetic library methods using affinity chromatography selection. The
biological
library approach is limited to peptide libraries, while the other four
approaches are
applicable to peptide, non-peptide oligomer or small molecule libraries of
compounds
(Lam, K.S. (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in
the
art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A.
90:6909; Erb et
al. ( 1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. ( 1994). J.
Med.
Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem.
Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.
33:2061; and in
Gallop et al. (1994) J. Med. Chem. 37:1233.
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Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques 13:412-421 ), or on beads (Lam { 1991 ) Nature 3 $4:82-84),
chips (Fodor
(1993) Nature 364:$$$-$$6), bacteria (Ladner USP $,223,409), spores (Ladner
USP
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:186$-1869) or on
phage
$ (Scott and Smith (1990) Science 249:386-390); (Devlin (1990)Science 249:404-
406);
(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici {199I) J.
Mol. Biol.
222:301-310); (Ladner supra.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses or responds to a LSP-1, PA-I, and TAP-1 protein, or a biologically
active
portion thereof, is contacted with a test compound and the ability of the test
compound
to inhibit or stimulate the biological activity of a LSP-1, PA-I, and TAP-1
protein is
determined. The cell, for example, can be of a mammalian origin or can be a
yeast cell.
Determining the ability of the test compound to inhibit or stimulate the
biological
activity of a LSP-1, PA-I, and TAP-1 protein can be accomplished, for example,
by
1$ coupling the test compound with a radioisotope or enzymatic label such that
binding of
the test compound to a LSP-1, PA-I, and TAP-1 receptor can be determined by
detecting
the labeled compound in a complex. For example, test compounds can be labeled
with
125h 35s~ i4C~ or 3H, either directly or indirectly, and the radioisotope
detected by direct
counting of radioemmission or by scintillation counting. Alternatively, test
compounds
can be enzymatically labeled with, for example, horseradish peroxidase,
alkaline
phosphatase, or luciferase, and the enzymatic label detected by determination
of
conversion of an appropriate substrate to product.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell which expresses or responds to a LSP-1, PA-I, and TAP-1 protein or
biologically
2$ active portion thereof with a test compound and determining the ability of
the test
compound to modulate {e.g. stimulate or inhibit) the activity of the LSP-l, PA-
I, and
TAP-1 protein or biologically active portion thereof. Determining the ability
of the test
compound to modulate the activity of LSP-1, PA-I, and TAP-1 or a biologically
active
portion thereof can be accomplished, for example, by determining the ability
of the LSP-
1, PA-I, and TAP-1 protein to bind to or interact with a LSP-l, PA-I, and TAP-
1 target
molecule. As used herein, a "target molecule" is a molecule with which a LSP-
I, PA-I,
and TAP-1 protein binds or interacts in nature, for example, a molecule on the
surface of
a cell which expresses a LSP-1, PA-I, and TAP-1 protein, a molecule on the
surface of a
second cell, a molecule in the extracellular milieu, a molecule associated
with the
3$ internal surface of a cell membrane or a cytoplasmic molecule. A LSP-1, PA-
I, and
TAP-1 target molecule can be a non-LSP-1, PA-I, and TAP-1 molecule. In one
embodiment, a LSP-1, PA-I, and TAP-1 target molecule is a component of a
signal
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transduction pathway which facilitates transduction of an extracellular signal
(e.g. a
signal generated by binding of a LSP-1, PA-I, and TAP-1 molecule to a membrane-
bound receptor) through the cell membrane and into the cell. The target, for
example,
can be a second intercellular protein which has catalytic activity or a
substrate for a
catalytic activity ofthe LSP-l, PA-I, and TAP-1 receptor.
Determining the ability of the LSP-1, PA-I, and TAP-1 protein to bind to or
interact with a LSP-1, PA-I, and TAP-1 target molecule can be accomplished by
one of
the methods described above for determining direct binding. In a preferred
embodiment,
determining the ability of the LSP-l, PA-I, and TAP-1 protein to bind to or
interact with
a LSP-1, PA-I, and TAP-1 target molecule can be accomplished by determining
the
activity of the target molecule. For example, the activity of the target
molecule can be
determined by detecting induction of a cellular second messenger of the target
(i.e.
intracellular Ca2~, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic
activity of the
target an appropriate substrate, detecting the induction of a reporter gene
(comprising a
LSP-1, PA-I, and TAP-1-responsive regulatory element operatively linked to a
nucleic
acid encoding a detectable marker, e.g. luciferase), or detecting a cellular
response, for
example, cellular differentiation, or cell proliferation.
In another embodiment, the assay is a cell-free assay in which a LSP-1, PA-I,
and TAP-I protein or biologically active portion thereof is contacted with a
test
compound and the ability of the test compound to modulate (e.g., stimulate or
inhibit)
the activity of the LSP-I, PA-I, and TAP-1 protein or biologically active
portion thereof
is determined. Determining the ability of the test compound to modulate the
activity of
LSP-l, PA-I, and TAP-1 can be accomplished, for example, by determining the
ability
of the LSP-1, PA-I, and TAP-1 protein to bind to a LSP-1, PA-I, and TAP-1
target
molecule by one of the methods described above for determining direct binding.
In an
alternative embodiment, determining the ability of the test compound to
modulate the
activity of LSP-l, PA-I, and TAP-1 can be accomplished by determining the
ability of
the LSP-1, PA-I, and TAP-I protein to further modulate a LSP-1, PA-I, and TAP-
1
target molecule. For example, the catalytic/enzymatic activity of the target
molecule on
an appropriate substrate can be determined as previously described.
In any of the above disclosed assay methods of the present invention, it may
be
desirable to immobilize either LSP-1, PA-I, and TAP-1 or its target molecule
to
facilitate separation of complexed from uncomplexed forms of one or both of
the
proteins, as well as to accommodate automation of the assay. Binding of a test
compound to LSP-I, PA-I, and TAP-1, or interaction of LSP-1, PA-I, and TAP-1
with a
target molecule in the presence and absence of a candidate compound, cari be
accomplished in any vessel suitable for containing the reactants. Examples of
such
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vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In
one
embodiment, a fusion protein can be provided which adds a domain that allows
one or
both of the proteins to be bound to a matrix. For example, glutathione-S-
transferase/
LSP-1, PA-I, and TAP-1 fusion proteins or glutathione-S-transferase/target
fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis,
MO) or glutathione derivatized microtitre plates, which are then combined with
the test
compound or the test compound and either the non-adsorbed target protein or
LSP-1,
PA-I, and TAP-1 protein, and the mixture incubated under conditions conducive
to
complex formation (e.g., at physiological conditions for salt and pH).
Following
incubation, the beads or microtitre plate wells are washed to remove any
unbound
components, the matrix immobilized in the case of beads, complex determined
either
directly or indirectly, for example, as described above. Alternatively, the
complexes can
be dissociated from the matrix, and the level of LSP-I, PA-I, and TAP-1
binding or
activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example; either LSP-1, PA-I, and TAP-1
or its
target molecule can be immobilized utilizing conjugation of biotin and
streptavidin.
Biotinylated LSP-1, PA-I, and TAP-I or target molecules can be prepared from
biotin-
NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the
wells of
streptavidin-coated 96 well plates (Pierce Chemical). Alternatively,
antibodies reactive
with LSP-I, PA-I, and TAP-1 or target molecules but which do not interfere
with
binding of the LSP-1, PA-I, and TAP-1 protein to its target molecule can be
derivatized
to the wells of the plate, and unbound target or LSP-1, PA-I, and TAP-1
trapped in the
wells by antibody conjugation. Methods for detecting such complexes, in
addition to
those described above for the GST-immobilized complexes, include
immunodetection of
complexes using antibodies reactive with the LSP-1, PA-I, and TAP-1 or target
molecule, as well as enzyme-linked assays which rely on detecting an enzymatic
activity
associated with the LSP-1, PA-I, and TAP-1 or target molecule.
In another embodiment, modulators of LSP-1, PA-I, and TAP-1 expression are
identified in a method wherein a cell is contacted with a candidate compound
and the
expression of LSP-1, PA-I, and TAP-1 mRNA or protein in the cell is
determined. The
level of expression of LSP-I, PA-I, and TAP-I mRNA or protein in the presence
of the
candidate compound is compared to the level of expression of LSP-1, PA-I, and
TAP-1
mRNA or protein in the absence of the candidate compound. The candidate
compound
can then be identified as a modulator of LSP-I, PA-I, and TAP-1 expression
based on
this comparison. For example, when expression of LSP-1, PA-I, and TAP-1 mRNA
or
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protein is greater (statistically significantly greater) in the presence of
the candidate
compound than in its absence, the candidate compound is identified as a
stimulator of
LSP-1, PA-I, and TAP-I mRNA or protein expression. Alternatively, when
expression
of LSP-I, PA-I, and TAP-1 mRNA or protein is less (statistically significantly
less) in
the presence of the candidate compound than in its absence, the candidate
compound is
identified as an inhibitor of LSP-1, PA-I, and TAP-1 mRNA or protein
expression. The
level of LSP-1, PA-I, and TAP-1 mRNA or protein expression in the cells can be
determined by methods described herein for detecting LSP-1, PA-I, and TAP-
l.mRNA
or protein.
In yet another aspect of the invention, the LSP-1, PA-I, and TAP-1 proteins
can
be used as "bait proteins" in a two-hybrid assay or in a three-hybrid assay
(see, e.g., U.S.
Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.
(1993) J. Biol.
Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi
et al.
(1993) Oncogene 8:1693-1696; and Brent W094/10300), to identify other
proteins,
which bind to or interact with LSP-1, PA-I, and TAP-1 ("LSP-1-binding
proteins", "PA-
I-binding proteins", and "TAP-1-binding proteins" or "LSP-1-by", "PA-I-by",
and
"TAP-1-by") and modulate LSP-1, PA-I, and TAP-1 activity. Such LSP-1-, PA-I-,
and
TAP-1-binding proteins are also likely to be involved in the propagation of
signals by
the LSP-1, PA-I, and TAP-1 proteins as, for example, upstream or downstream
elements
of the LSP-1, PA-I, and TAP-1 pathway, e.g., a LSP-1, PA-I, and TAP-1
receptor.
The two-hybrid system is based on the modular nature of most transcription
factors, which consist of separable DNA-binding and activation domains.
Briefly, the
assay utilizes two different DNA constructs. In one construct, the gene that
codes for
LSP-1, PA-I, and TAP-1 is fused to a gene encoding the DNA binding domain of a
_ known transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from
a library of DNA sequences, that encodes an unidentified protein ("prey" or
"sample") is
fused to a gene that codes for the activation domain of the known
transcription factor. If
the "bait" and the "prey" proteins are able to interact, in vivo, forming a
LSP-1, PA-I,
and TAP-1-dependent complex, the DNA-binding and activation domains of the
transcription factor are brought into close proximity. This proximity allows
transcription of a reporter gene (e.g., LacZ) which is operably linked to a
transcriptional
regulatory site responsive to the transcription factor. Expression of the
reporter gene can
be detected and cell colonies containing the functional transcription factor
can be
isolated and used to obtain the cloned gene which encodes the protein which
interacts
with LSP-I, PA-I, and TAP-1.
This invention further pertains to novel agents identified by the above-
described
screening assays and uses thereof for treatments as described herein.
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B. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in
which
diagnostic assays, prognostic assays, pharmacogenetics, and monitoring
clinical trails
are used for prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention relates to
diagnostic
assays for determining LSP-1, PA-I, and TAP-1 protein and/or nucleic acid
expression
as well as LSP-1, PA-I, and TAP-1 activity, in the context of a biological
sample (e.g.,
blood, serum, cells, tissue) to thereby determine whether an individual is
afflicted with a
disease or disorder, or is at risk of developing a disorder, associated with
aberrant LSP-
1, PA-I, and TAP-1 expression or activity. The invention also provides for
prognostic
(or predictive) assays for determining whether an individual is at risk of
developing a
disorder associated with LSP-1, PA-I, and TAP-1 protein, nucleic acid
expression or
activity. For example, mutations in a LSP-1, PA-I, and TAP-1 gene can be
assayed in a
biological sample. Such assays can be used for prognostic or predictive
purpose to
thereby phophylactically treat an individual prior to the onset of a disorder
characterized
by or associated with LSP-l, PA-I, and TAP-1 protein, nucleic acid expression
or
activity.
Another aspect of the invention provides methods for determining LSP-1, PA-I,
and TAP-1 protein, nucleic acid expression or LSP-1, PA-I, and TAP-1 activity
in an
individual to thereby select appropriate therapeutic or prophylactic agents
for that
individual (referred to herein as "pharmacogenetics). Pharmacogenetics allows
for the
selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of
an individual
based on the genotype of the individual (e.g., the genotype of the individual
examined to
determine the ability of the individual to respond to a particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of
agents
(e.g. drugs, compounds) on the expression or activity of LSP-1, PA-I, and TAP-
1 in
clinical trials. These and other agents are described in further detail in the
following
sections.
1. Diagnostic Assaxs
An exemplary method for detecting the presence or absence of LSP-1, PA-I, and
TAP-1 in a biological sample involves obtaining a biological sample from a
test subject
and contacting the biological sample with a compound or an agent capable of
detecting
LSP-1, PA-I, and TAP-1 protein or nucleic acid, (e.g., mRNA, genomic DNA) that
encodes LSP-I, PA-I, and TAP-1 protein such that the presence of LSP-1, PA-I,
and
TAP-1 is detected in the biological sample.
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A preferred agent for detecting LSP-1, PA-I, and TAP-1 mRNA is a labeled
nucleic acid probe capable of hybridizing to LSP-1, PA-I, and TAP-I mRNA or
genomic DNA. The nucleic acid probe can be, for example, a full-length LSP-1,
PA-I,
and TAP-I nucleic acid, such as the nucleic acid of SEQ ID NO:I, 3, 4, 6, 7,
or 9, or a
portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250
or 500
nucleotides in length and sufficient to specifically hybridize under stringent
conditions
to LSP-l, PA-I, and TAP-1 mRNA or genomic DNA. Other suitable probes for use
in
the diagnostic assays of the invention are described herein.
A preferred agent for detecting LSP-1, PA-I, and TAP-1 protein is an antibody
capable of binding to LSP-1, PA-I, and TAP-1 protein (preferably an antibody
with a
detectable label). Antibodies can be polyclonal, or more preferably,
monoclonal.. An
intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The
term
"labeled", with regard to the probe or antibody, is intended to encompass
direct labeling
of the probe or antibody by coupling (i.e., physically linking) a detectable
substance to
the probe or antibody, as well as indirect labeling of the probe or antibody
by reactivity
with another reagent that is directly labeled. Examples of indirect labeling
include
detection of a primary antibody using a fluorescently labeled secondary
antibody and
end-labeling of a DNA probe with biotin such that it can be detected with
fluorescently
labeled streptavidin. The term "biological sample" is intended to include
tissues, cells
and biological fluids isolated from a subject, as well as tissues, cells and
fluids present
within a subject. That is, the detection method of the invention can be used
to detect
LSP-1, PA-I, and TAP-I mIZNA, protein, or genomic DNA in a biological sample
in
vitro as well as in vivo. For example, in vitro techniques for detection of
LSP-1, PA-I,
and TAP-1 mRNA include Northern hybridizations and in situ hybridizations. In
vitro
techniques for detection of LSP-1, PA-I, and TAP-I protein include enzyme
linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of LSP-1, PA-I, and TAP-
1
genomic DNA include Southern hybridizations. Furthermore, in vivo techniques
for
detection of LSP-1, PA-I, and TAP-1 protein include introducing into a subject
a
labeled anti-LSP-1, PA-I, and TAP-1 antibody. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a subject can
be
detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the
test subject. Alternatiavely, the biological sample can contain mRNA molecules
from
the test subject or genomic DNA molecules from the test subject. A preferred
biological
sample is a peripheral blood lymphocyte sample isolated by conventional means
from a
subject.
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In another emodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
capable of detecting LSP-l, PA-I, and TAP-1 protein, mRNA, or genomic DNA,
such
that the presence of LSP-1, PA-I, and TAP-1 protein, mRNA or genornic DNA is
detected in the biological sample, and comparing the presence of LSP-1, PA-I.
and TAP
1 protein, mRNA or genomic DNA in the control sample with the presence of LSP-
1,
PA-I, and TAP-1 protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of LSP-1, PA-I,
and TAP-1 in a biological sample. For example, the kit can comprise a labeled
compound or agent capable of detecting LSP-1, PA-I, and TAP-1 protein or mIZNA
in a
biological sample; means for determining the amount of LSP-1, PA-I, and TAP-1
in the
sample; and means for comparing the amount of LSP-1, PA-I, and TAP-1 in the
sample
with a standard. The compound or agent can be packaged in a suitable
container. The
kit can further comprise instructions for using the kit to detect LSP-1, PA-I,
and TAP-1
protein or nucleic acid.
2. Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
idcntify
subjects having or at risk of developing a disease or disorder associated with
aberrant
LSP-1, PA-I, and TAP-1 expression or activity. For example, the assays
described
herein, such as the preceding diagnostic assays or the following assays, can
be utilized to
identify a subject having or at risk of developing a disorder associated with
LSP-1, PA-I,
and TAP-1 protein, nucleic acid expression or activity such as an inflammatory
or
immune disorder. Alternatively, the prognostic assays can be utilized to
identify a
subject having or at risk for developing an immune disease or disorder. Thus,
the
present invention provides a method for identifying a disease or disorder
associated with
aberrant LSP-1, PA-I, and TAP-1 expression or activity in which a test sample
is
obtained from a subject and LSP-1, PA-I, and TAP-1 protein or nucleic acid
(e.g.,
mRNA, genomic DNA) is detected, wherein the presence of LSP-1, PA-I, and TAP-1
protein or nucleic acid is diagnostic for a subject having or at risk of
developing a
disease or disorder associated with aberrant LSP-1, PA-I, and TAP-1 expression
or
activity. As used herein, a "test sample" refers to a biological sample
obtained from a
subject of interest. For example, a test sample can be a biological fluid
(e.g., serum),
cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug
candidate)
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to treat a disease or disorder associated with aberrant LSP-1, PA-I, and TAP-1
expression or activity. For example, such methods can be used to determine
whether a
subject can be effectively treated with an agent for a disorder, such as
inflammatory
disease or an immune system disease. Thus, the present invention provides
methods for
determining whether a subject can be effectively treated with an agent for a
disorder
associated with aberrant LSP-1, PA-I, and TAP-1 expression or activity in
which a test
sample is obtained and LSP-1, PA-I, and TAP-1 protein or nucleic acid is
detected (e.g.
wherein the presence of LSP-1, PA-I, and TAP-1 protein or nucleic acid is
diagnostic
for a subject that can be administered the agent to treat a disorder
associated with
aberrant LSP-1, PA-I, and TAP-1 expression or activity.)
The methods of the invention can also be used to detect genetic lesions in a
LSP-
1, PA-I, and TAP-1 gene, thereby determining if a subject with the gene, which
is
lesioned, is at risk for a disorder characterized by aberrant cell
proliferation and/or
differentiation. In preferred embodiments, the methods include detecting, in a
sample of
cells from the subject, the presence or absence of a genetic lesion
characterized by at
least one of an alteration affecting the integrity of a gene encoding a LSP-1,
PA-I, and
TAP-1-protein, or the mis-expression of the LSP-1, FA-I, and TAP-1 gene. For
example, such genetic lesions can be detected by ascertaining the existence of
at least
one of (1) a deletion of one or more nucleotides from a LSP-1, PA-I, and TAP-1
gene;
(2) an addition of one or more nucleotides to a LSP-l, PA-I, and TAP-1 gene;
(3) a
substitution of one or more nucleotides of a LSP-1, PA-I, and TAP-1 gene, (4)
a
chromosomal rearrangement of a LSP-1, PA-I, and TAP-1 gene; (5) an alteration
in the
level of a messenger RNA transcript of a LSP-I, PA-I, and TAP-1 gene, (6)
aberrant
modification of a LSP-1, PA-I, and TAP-1 gene, such as of the methylation
pattern of
the genomic DNA, (7) the presence of a non-wild type splicing pattern of a
messenger
RNA transcript of a LSP-1, PA-I, and TAP-1 gene, (8) a non-wild type level of
a LSP-l,
PA-I, and TAP-1-protein, (9) allelic loss of a LSP-1, PA-I, and TAP-1 gene,
and (10)
inappropriate post-translational modification of a LSP-1, PA-I, and TAP-1-
protein. As
described herein, there are a large number of assay techniques known in the
art which
can be used for detecting lesions in a LSP-1, PA-I, and TAP-1 gene.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos.
4,683,195
and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a
ligation chain
reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and
Nakazawa et al. ( 1994) PNAS 91:360-364), the latter of which can be
particularly useful
for detecting point mutations in the LSP-1, PA-I, and TAP-1-gene (see Abravaya
et al.
(1995) Nucleic Acids Res .23:675-682). This method can include the steps of
collecting
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a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA
or both)
from the cells of the sample, contacting the nucleic acid sample with one or
more
primers which specifically hybridize to a LSP-1, PA-I, and TAP-1 gene under
conditions
such that hybridization and amplification of the LSP-1, PA-I, and TAP-1-gene
(if
present) occurs, and detecting the presence or absence of an amplification
product, or
detecting the size of the amplification product and comparing the length to a
control
sample. It is anticipated that PCR and/or LCR may be desirable to use as a
preliminary
amplification step in conjunction with any of the techniques used for
detecting mutations
described herein.
Alternative amplification methods include: self sustained sequence replication
{Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),
transcriptional
amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-
1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6:1197),
or any
other nucleic acid amplification method, followed by the detection of the
amplified
molecules using techniques well known to those of skill in the art. These
detection
schemes are especially useful for the detection of nucleic acid molecules if
such
molecules are present in very low numbers.
In an alternative embodiment, mutations in a LSP-1, PA-I, and TAP-1 gene
from a sample cell can be identified by alterations in restriction enzyme
cleavage
patterns. For example, sample and control DNA is isolated, amplified
{optionally),
digested with one or more restriction endonucleases, and fragment length sizes
are
determined by gel electrophoresis and compared. Differences in fragment length
sizes
between sample and control DNA indicates mutations in the sample DNA.
Moreover,
the use of sequence specific ribozymes (see, for example, U.S. Patent No.
5,498,531)
can be used to score for the presence of specific mutations by development or
loss of a
ribozyme cleavage site.
In other embodiments, genetic mutations in LSP-1, PA-I, and TAP-1 can be
identified by hybridizing a sample and control nucleic acids, e.g., DNA or
RNA, to high
density arrays containing hundreds or thousands of oligonucleotides probes
(Cronin,
M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et a1 (1996) Nature
Medicine 2: 753-759). For example, genetic mutations in LSP-1, PA-I, and TAP-1
can
be identified in two dimensional arrays containing light-generated DNA probes
as
described in Cronin, M.T. et al. supra. Briefly, a first hybridization array
of probes can
be used to scan through long stretches of DNA in a sample and control to
identify base
changes between the sequences by making linear arrays of sequential ovelapping
probes.
This step allows the identification of point mutations. This step is followed
by a second
hybridization array that allows the characterization of specific mutations by
using
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smaller, specialized probe arrays complementary to all variants or mutations
detected.
Each mutation array is composed of parallel probe sets, one complementary to
the wild-
type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the art can be used to directly sequence the LSP-1, PA-I, and TAP-1 gene and
detect
mutations by comparing the sequence of the sample LSP-1, PA-I, and TAP-1 with
the
corresponding wild-type (control) sequence. Examples of sequencing reactions
include
those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74:560)
or
Sanger (( 1977) PNAS 74:5463). It is also contemplated that any of a variety
of
automated sequencing procedures can be utilized when performing the diagnostic
assays
((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see,
e.g.,
PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv.
Chromatogr.
36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
Other methods for detecting mutations in the LSP-1, PA-I, and TAP-1 gene
include methods in which protection from cleavage agents is used to detect
mismatched
bases in RNA/RNA or RNA/DNA duplexes or heteroduplexes (Myers et al. (1985)
Science 230:1242). In general, the art technique of "mismatch cleavage" starts
by
providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA
containing
the wild-type LSP-1, PA-I, and TAP-1 sequence with potentially mutant RNA or
DNA
obtained from a tissue sample. The double-stranded duplexes are treated with
an agent
which cleaves single-stranded regions of the duplex such as which will exist
due to
basepair mismatches between the control and sample strands. For instance,
RNA/DNA
duplexes can be treated with RNase and DNA/DNA hybrids treated with S 1
nuclease to
enzymatically digesting the mismatched regions. In other embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide and with piperidine in order to digest mismatched regions. After
digestion of
the mismatched regions, the resulting material is then separated by size on
denaturing
polyacrylamide gels to determine the site of mutation. See, for example,
Cotton et al
(1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol.
217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled
for
detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more proteins that recognize mismatched base pairs in double-stranded DNA (so
called
"DNA mismatch repair" enzymes) in defined systems for detecting and mapping
point
mutations in LSP-I, PA-I, and TAP-1 cDNAs obtained fram samples of cells. For
example, the mutt enzyme of E. coli cleaves A at GIA mismatches and the
thymidine
DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (
1994)
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Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe
based
on a LSP-1, PA-I, and TAP-1 sequence, e.g., a wild-type LSP-1, PA-I, and TAP-1
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex
is treated with a DNA mismatch repair enzyme, and the cleavage products, if
any, can be
detected from electrophoresis protocols or the like. See, for example, U.S.
Patent No.
5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify mutations in LSP-1, PA-I, and TAP-1 genes. For example, single strand
conformation polymorphism (SSCP) may be used to detect differences in
electrophoretic
mobility between mutant and wild type nucleic acids (Orita et al. ( 1989) Proc
Natl.
Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and
Hayashi
(1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample
and
control LSP-1, PA-I; and TAP-1 nucleic acids will be denatured and allowed to
renature.
The secondary structure of single-stranded nucleic acids varies according to
sequence,
the resulting alteration in electrophoretic mobility enables the detection of
even a single
base change. The DNA fragments may belabeled or detected with labeled probes.
The
sensitivity of the assay may be enhanced by using RNA (rather than DNA), in
which the
secondary structure is more sensitive to a change in sequence. In a preferred
embodiment, the subject method utilizes heteroduplex analysis to separate
double
stranded heteroduplex molecules on the basis of changes in eIectrophoretic
mobility
(Keen et al. ( 1991 ) Trends Genet 7:5).
In yet another embodiment the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing
gradient geI electrophoresis (DGGE) (Myers et al (1985) Nature 313:495). When
DGGE is used as the method of analysis, DNA will be modified to insure that it
does not
completely denature, for example by adding a GC clamp of approximately 40 by
of
high-melting GC-rich DNA by PCR. In a further embodiment, a temperature
gradient is
used in place of a denaturing gradient to identify differences in the mobility
of control
and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
Examples of other techniques for detecting point mutations include, but are
not
limited to, selective oligonucleotide hybridization, selective amplification,
or selective
primer extension. For example, oligonucleotide primers may be prepared in
which the
known mutation is placed centrally and then hybridized to target DNA under
conditions
which permit hybridization only if a perfect match is found (Saiki et al.
(1986) Nature
324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele
specific
oIigonucleotides are hybridized to PCR amplified target DNA or a number of
different
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mutations when the oligonucleotides are attached to the hybridizing membrane
and
hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on
selective PCR amplification may be used in conjunction with the instant
invention.
Oligonucleotides used as primers for specific amplification may carry the
mutation of
interest in the center of the molecule (so that amplification depends on
differential
hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the
extreme 3'
end of one primer where, under appropriate conditions, mismatch can prevent,
or reduce
polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be
desirable
to introduce a novel restriction site in the region of the mutation to create
cleavage-based
detection (Gasparini et al ( 1992) Mol. Cell Probes 6:1 ). It is anticipated
that in certain
embodiments amplification may also be performed using Taq ligase for
amplification
(Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will
occur
only if there is a perfect match at the 3' end of the 5' sequence making it
possible to
detect the presence of a known mutation at a specific site by looking for the
presence or
absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-
packaged diagnostic kits comprising at least one probe nucleic acid or
antibody reagent
described herein, which may be conveniently used, e.g., in clinical settings
to diagnose
patients exhibiting symptoms or family history of a disease or illness
involving a LSP-1,
PA-I, and TAP-1 gene.
Furthermore, any cell type or tissue, preferably peripheral blood lymphocytes,
in
which LSP-1, PA-I, and TAP-1 is expressed may be ulilized in the prognostic
assays
described herein.
3. PharmacoEenetics
Agents, or modulators which have a stimulatory or inhibitory effect on LSP-1,
PA-I, and TAP-I activity (e.g., LSP-1, PA-I, and TAP-1 gene expression) as
identified
by a screening assay described herein can be administered to individuals to
treat
(prophylactically or therapeutically) disorders associated with aberrant LSP-
1, PA-I, and
TAP-1 activity, e.g., proliferative disorders. In conjunction with such
treatment, the
pharmacogenetics (i.e., the study of the relationship between an individual's
genotype
and that individual's response to a foreign compound, e.g., a drug) of the
individual may
be considered. Differences in metabolism of therapeutics can lead to severe
toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the
pharmacologically active drug. Thus, the pharmacogenetics of the individual
permit the
selection of effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based
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on a consideration of the individual's genotype. Such pharmacogenetics can
further be
used to determine appropriate dosages and therapeutic regimens. Accordingly,
the
activity of LSP-1, PA-I, and TAP-1 protein, expression of LSP-1, PA-I, and TAP-
1
nucleic acid, or mutation content of LSP-I, PA-I, and TAP-1 genes in an
individual can
be determined to thereby select appropriate agents) for therapeutic or
prophylactic
treatment of the individual.
Pharmacogenetics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected
persons. See e.g., Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23(10-I
1) :983-
985 and Linden M.W., Clin Chem,1997, 43{2):254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic conditions
transmitted as a
single factor altering the way drugs act on the body (altered drug action) or
genetic
conditions transmitted as single factors altering the way the body acts on
drugs (altered
drug metabolism). These pharmacogenetic conditions can occur either as rare
defects or
as polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency
(G6PD) is a common inherited enzymopathy in which the main clinical
complication is
haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides,
analgesics,
nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major determinant of both the intensity and duration of drug action. The
discovery of
genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase
2 (NAT
2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation
as to why some patients do not obtain the expected drug effects or show
exaggerated
drug response and serious toxicity after taking the standard and safe dose of
a drug.
These polymorphisms are expressed in two phenotypes in the population, the
extensive
(EM) and poor metabolizer (PM). The prevalence of PM is different among
different
populations. For example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the absence of
functional
CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quite frequently experience
exaggerated drug response and side effects when they receive standard doses.
If a
metabolite is the active therapeutic moiety, PM show no therapeutic response,
as
demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed
metabolite morphine. The other extreme are the so called ultra-rapid
metabolizers who
do not respond to standard doses. Recently, the molecular basis of ultra-rapid
metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of LSP-1, PA-I, and TAP-1 protein, expression of LSP-1, PA-
I, and TAP-1 nucleic acid, or mutation content of LSP-I, PA-I, and TAP-1 genes
in an
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individual can be determined to thereby select appropriate agents) for
therapeutic or
prophylactic treatment of the individual. In addition, pharnzacogenetic
studies can be
used to apply genotyping of polymorphic alleles encoding drug-metabolizing
enzymes
to the identification of an individual's drug responsiveness phenotype. This
knowledge,
when applied to dosing or drug selection, can avoid adverse reactions or
therapeutic
failure and thus enhance therapeutic or prophylactic efficiency when treating
a subject
with a LSP-1, PA-I, and TAP-1 modulator, such as a modulator identified by one
of the
exemplary screening assays described herein.
4. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents {e.g., drugs, compounds) on the expression
or
activity of LSP-1, PA-I, and TAP-1 (e.g., the ability to modulate
inflammation, immune
responsiveness, or cellular homing) can be applied not only in basic drug
screening, but
also in clinical trials. For example, the effectiveness of an agent determined
by a
screening assay to increase LSP-1, PA-I, and TAP-1 gene expression, protein
levels, or
upregulate LSP-1, PA-I, and TAP-1 activity, can be monitored in clinical
trails of
subjectes exhibiting decreased LSP-1, PA-I, and TAP-1 gene expression, protein
levels,
or downregulated LS.P-1, PA-I, and TAP-1 activity. Alternatively, the
effectiveness of a
compounds determined by a screening assay to decrease LSP-1, PA-I, and TAP-1
gene
expression, protein levels, or downregulate LSP-1, PA-I, and TAP-1 activity,
can be
monitored in clinical trails of subjectes exhibiting increased LSP-1, PA-I,
and TAP-1
gene expression, protein levels, or upregulated LSP-1, PA-I, and TAP-1
activity. In
such clinical trials, the expression of LSP-l, PA-I, and TAP-1 and,
preferably, other
genes that have been implicated in a metabolic disorder as described herein,
can be used
as a "read out" or as a marker of the metabolic state of a particular cell.
For example, and not by way of limitation, genes, including LSP-l, PA-I, and
TAP-1, that are modulated in cells by treatment with an agent (e.g., compound,
drug or
small molecule) which modulates angiogenesis, cellular proliferation, or
immune
responses (e.g., identified in a screening assay as described herein) can be
identified.
Thus, to study the effect of agents on metabolic disorders, for example, in a
clinical trial,
cells can be isolated and RNA prepared and analyzed for the levels of
expression of
LSP-1, PA-I, and TAP-1 and other genes implicated in the metabolic disorder.
The
levels of gene expression (i.e., a gene expression pattern) can be quantified
by Northern
blot analysis or RT-PCR, as described herein, or alternatively by measuring
the amount
of protein produced, by one of the methods as described herein, or by
measuring the
levels of activity of LSP-1, PA-I, and TAP-1 or other gene. In this way, the
gene
expression pattern can serve as a marker, indicative of the physiological
response of the
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cells to the agent. Accordingly, this response state may be determined before,
and at
various points during, treatment of the individual with the agent.
In a preferred embodiment, the present invention provides a method for
monitoring the effectiveness of treatment of a subject with an agent (e.g. an
agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or
other drug
candidate identified by the screening assays described herein) comprising the
steps of (i)
obtaining a pre-administration sample from a subject prior to administration
of the
agent; (ii) detecting the level of expression of a LSP-1, PA-I, and TAP-1
protein,
mIZNA, or genomic DNA in the pre-administration sample; (iii) obtaining one or
more
post-administration samples from the subject; (iv).detecting the level of
expression or
activity of the LSP-1, PA-I, and TAP-1 protein, mRNA, or genomic DNA in the
post-
administration samples; (v) comparing the level of expression or activity of
the LSP-l,
PA-I, and TAP-1 protein, mRNA, or genomic DNA in the pre-administration sample
with the LSP-1, PA-I, and TAP-1 protein, mRNA, or genomic DNA in the post
administration sample or samples; and (vi) altering the administration of the
agent to the
subject accordingly. For example, increased administration of the agent maybe
desirable to increase the expression or activity of LSP-l, PA-I, and TAP-1 to
:~igher
levEls than detected, i.e,. to increase the effectiveness of the agent.
Alternati~ialy,
decreased administration of the agent may be desirable to decrease expression
or activity
of LSP-l, PA-I, and TAP-1 to lower levels than detected, i.e., to decrease the
effectiveness of the agent.
C. Methods of Treatment:
The present invention provides for both prophylactic and therapeutic methods
of
treating a subject at risk for (or succeptible to) a disorder or having a
disorder associated
with aberrant LSP-1, PA-I, and TAP-1 expression or activity.
1. Proehvlactic Methods
In one aspect, the invention provides a method for preventing in a subject, a
disease or condition associated with an aberrant LSP-1, PA-I, and TAP-1
expression or
activity, by administering to the subject an agent which modulates LSP-1, PA-
I, and
TAP-1 expression or at least one LSP-1, PA-I, and TAP-1 activity. Subjects at
risk for a
disease which is caused or contributed to by aberrant LSP-1, PA-I, and TAP-1
expression or activity can be identified by, for example, any or a combination
of the
diagnostic or prognostic assays as described herein. Administeration of a
phophylactic
agent can occur prior to the manifestation of symptoms characteristic of the
LSP-1, PA-
I, and TAP-1 aberrancy such that a disease or disorder is prevented or,
alternatively,
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delayed in its progression. Depending on the type of LSP-1, PA-I, and TAP-1
aberrancy, for example, a LSP-1, PA-I, and TAP-1 agonist or LSP-1, PA-I, and
TAP-1
antagonist agent can be used for treating the subject. The appropriate agent
can be
determined based on screening assays described herein. The prophylactic
methods of
the present invention are further described in the following subsection.
2. Therapeutic Methods
Another aspect of the invention pertains to methods of modulating LSP-1, PA-I,
and TAP-1 expression or activity for therapeutic purposes. Another aspect of
the
invention pertains to methods of modulating LSP-1, PA-I, and TAP-1 expression
or
activity associated with a cell for therapeutic purposes. LSP-1, PA-I, and TAP-
1
activity "associated with a cell" is intended to include one or more of the
activites of
LSP-1, PA-I, and TAP-1 protein within the cell, secreted by the cell and in
the
extracellular milieu surrounding the cell. The modulatory method of the
invention
involves contacting the cell with an agent that modulates one or more of the
activites of
LSP-l, PA-I, and TAP-1 protein activity associated with the cell. An agent
that
modulates LSP-1, PA-I, and TAP-1 protein activity can be an agent as described
herein
such as a nucleic acid or a protein, a naturally-occurring cogitate ligand of
a LSP-1, PA-
I, and TAP-1 protein, a peptide, a LSP-1, PA-I, and TAP-1 peptidomimetic, or
other
small molecule. In one embodiment, the agent stimulates one or more LSP-1, PA-
I, and
TAP-1 protein activity. Examples of such stimulatory agents include active LSP-
1, PA-
I, and TAP-1 protein and a nucleic acid molecule encoding LSP-1, PA-I, and TAP-
1 that
has been introduced into the cell. In another embodiment, the agent inhibits
one or more
LSP-1, PA-I, and TAP-1 protein activity. Examples of such inhibitory agents
include
antisense LSP-1, PA-I, and TAP-1 nucleic acid molecules and anti-LSP-1, PA-I,
and
TAP-1 antibodies. These modulatory methods can be performed in vitro (e.g., by
culturing the cell with the agent) or, alternatively, in vivo (e.g, by
administering the
agent to a subject). As such, the present invention provides methods of
treating an
individual afflicted with a disease or disorder characterized by aberrant
expression or
activity of a LSP-l, PA-I, and TAP-1 protein or nucleic acid molecule. In one
embodiment, the method involves administering an agent {e.g., an agent
identified by a
screening assay described herein), or combination of agents, that modulates
(e.g.,
upregulates or downregulates) LSP-1, PA-I, and TAP-1 expression or activity.
In
another embodiment, the method involves administering a LSP-1, PA-I, and TAP-1
protein or nucleic acid molecule as therapy to compensate for reduced or
aberrant LSP-
l, PA-I, and TAP-1 expression or activity.
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Stimulation of LSP-1, PA-I, and TAP-1 activity is desirable in situations in
which LSP-1, PA-I, and TAP-1 is abnormally downregulated and/or in which
increased
LSP-1, PA-I, and TAP-1 activity is likely to have a beneficial effect. One
example of
such a situation is where a subject has a disorder characterized by aberrant
cell
proliferation (e.g., cancer). Another example of such a situation is where the
subject has
an angiogenesis disorder. One example of such a situation is where a subject
has a
disorder characterized by aberrant immune responsiveness. Another example of
such a
situation is where the subject has a inflammatory disease (e.g., arthritis).
In one embodiment, a method for modulating, e.g., stimulating or inhibiting,
the
proliferation, maturation, differentiation or survival of a stem cell, e.g., a
hematopoietic
stem cell, is provided. The term "stem cell" refers to an undifferentiated
cell which is
capable of self renewal, i.e., proliferation to give rise to more stem cells,
and may give
rise to lineage committed progenitors which are capable of differentiation and
expansion
into a specific lineage. In a preferred embodiment, the term "stem cell"
refers to a
1 S generalized mother cell whose descendants (progeny) specialize, often in
different
directions, by differentiation, e.g., by acquiring completely individual
characters, as
occurs in progressive diversification of embryonic cells and tissues. As used
herein, the
term "stem cells" refers generally to both embryonic and hematopoietic stem
cells from
mammalian origin, e.g., human.
For example, the present invention provides a method of differentiating
hematopoietic cells can be differentiated into mature cell of erythroid,
lymphoid or
myeloid Iineages, e.g., by potentiating or disrupting the biological
activities of a TAP-1
protein. As used herein, the term "hematopoietic stem cell" (HSC) means a
population
of cells capable of both self renewal and differentiation into all defined
hematopoietic
lineages, i.e., myeloid, lymphoid or erythroid lineages. HSCs can ultimately
differentiate into hematopoietic cells, including without limitation, common
lymphoid
progenitor cells, T cells (e.g., helper, cytotoxic, and suppressor cells), B
cells, plasma
cells, natural killer cells, common myeloid progenitor cells, monocytes,
macrophages,
mast cells, leukocytes, basophils, neutrophils, eosinophils, megakaryocytes,
platelets,
and erythroids. Preferably, the hematopoietic cells are selected from
megakaryocytes,
platelets, and erythroids.
The the present invention further provides methods for treating hematopoietic
diseases. Examples of hematopoietic diseases include thrombocytopenia
associated with
bone marrow hypoplasia (e.g., aplastic anemia following radio- or chemotherapy
or
bone marrow transplant), immune thrombocytopenia (HIV and non-HIV induced
thrombocytopenia), disorders such as intravascular coagulation,
myeloproliferative
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thrombocytotic disorders, inflammatory thrombocytosis and iron deficiency,
among
others.
VI. Uses of Partial LSP-1. PA-I, and TAP-1 Seguences
Portions or fragments of the cDNA sequences identified herein (and the
corresponding complete gene sequences) can be used in numerous ways as
polynucleotide reagents. For example, these sequences can be used to: (a) map
their
respective genes on a chromosome; and, thus, locate gene regions associated
with
genetic disease; (b) identify an individual from a minute biological sample
(tissue
typing); and (c) aid in forensic identification of a biological sample. These
applications
are described in the subsections below.
a. Chromosome Ma~uins
Once the sequence (or a portion of the sequence) of a gene has been isolated,
this
sequence can be used, to map the location of the gene on a chromosome. This
process is
called chromosome mapping. Accordingly, portions or fragments of the LSP-1, PA-
I,
and TAP-1, sequences, described herein, can be used to map the location ofthe
LSP-1,
PA-I, and TAP-1 gene, respectively; on a chromosome. The mapping of the LSP-1,
PA-
I, and TAP-1 sequence to chromosomes is an important first step in correlating
these
sequence with genes associated with disease.
Briefly, the LSP-l, PA-I, and TAP-1 gene can be mapped to a chromosome by
preparing PCR primers (preferably 15-25 by in length) from the LSP-1, PA-I,
and TAP-
1 sequence. Computer analysis of the LSP-1, PA-I, and TAP-1, sequence can be
used to
rapidly select primers that do not span more than one exon in the genomic DNA,
thus
complicating the amplification process. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human chromosomes.
Only
those hybrids containing the human gene corresponding to the LSP-1, PA-I, and
TAP-1
sequence will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different
mammals (e.g., human and mouse cells). As hybrids of human and mouse cells
grow
and divide, they gradually lose human chromosomes in random order, but retain
the
mouse chromosomes. By using media in which mouse cells cannot grow, because
they
lack a particular enzyme, but human cells can, the one human chromosome that
contains
the gene encoding the needed enzyme, will be retained. By using various media,
panels
of hybrid cell lines can be established. Each cell line in a panel contains
either a single
human chromosome or a small number of human chromosomes, and a full set of
mouse
chromosomes, allowing easy mapping of individual genes to specific human
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chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell
hybrids
containing only fragments of human chromosomes can also be produced by using
human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular sequence to a particular chromosome. Three or more sequences can be
assigned per day using a single thermal cycler. Using the LSP-1, PA-I, and TAP-
1
sequence to design oligonucleotide primers, sublocalization can be achieved
with panels
of fragments from specific chromosomes. Other mapping strategies which can
similarly
be used to map a LSP-l, PA-I, and TAP-1 sequence to its chromosome include in
situ
hybridization (described in Fan, Y. et al. ( 1990) PNAS, 87:6223-27), pre-
screening with
labeled flow-sorted chromosomes, and pre-selection by hybridization to
chromosome
specific cDNA libraries.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise chromosomal
location in
1 S one step. Chromosome spreads can be made using cells whose division has
been
blocked in metaphase by a chemical Iike colcemid that disrupts the mitotic
spindle. The
chromosomes can be treated briefly with trypsin, and then stained with Giemsa.
A
pattern of light and dark bands develops on each chromosome, so that the
chromosomes
can be identified individually. The FISH technique can be used with a DNA
sequence
as short as 500 or 600 bases. However, clones larger than 1,000 bases have a
higher
likelihood of binding to a unique chromosomal location with sufficient signal
intensity
for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases
will
suffice to get good results at a reasonable amount of time. For a review of
this
technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques
(Pergamon Press, New York, 1988}.
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding regions of the genes actually are preferred for mapping purposes.
Coding
sequences are more likely to be conserved within gene families, thus
increasing the
chance of cross hybridizations during chromosomal mapping.
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 WeIch Medical Library). The
relationship between genes and disease, mapped to the same chromosomal region,
can
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then be identified through linkage analysis (co-inheritance of physically
adjacent genes),
described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the LSP-1, PA-I, and TAP-1 gene, can
be
determined. If a mutation is observed in some or all of the affected
individuals but not
in any unaffected individuals, then the mutation is likely to be the causative
agent of the
particular disease. Comparison of affected and unaffected individuals
generally involves
first looking for structural alterations in the chromosomes, such as deletions
or
translocations that are visible from chromosome spreads or detectable using
PCR based
on that DNA sequence. Ultimately, complete sequencing of genes from several
individuals can be performed to confirm the presence of a mutation and to
distinguish
mutations from polymorphisms.
b. Tissue TypinE
The LSP-I, PA-I, and TAP-1 sequences of the present invention can also be used
to identify individuals from minute biological samples. The United States
militar3~, for
example, is considering the use of restriction fragment length polymorphism
(RFLP) for
identification of its personnel. In this technique, an individual's genomic
DNA is
digested with one or more restriction enzymes, and probed on a Southern blot
to yield
unique bands for identification. This method does not suffer from the current
limitations
of "Dog Tags" which can be lost, switched, or stolen, making positive
identification
difficult. The sequences of the present invention are useful as additional DNA
markers
for RFLP (described in U.S. Patent 5,272,057).
Furthermore, the sequences of the present invention can be used to provide an
alternative technique which determines the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the LSP-1, PA-I, and TAP-1
sequences described herein can be used to prepare two PCR primers from the 5'
and 3'
ends of the sequences. These primers can then be used to amplify an
individual's DNA
and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner, can provide unique individual identifications, as each individual will
have a
unique set of such DNA sequences due to allelic differences. The sequences of
the
present invention can be used to obtain such identification sequences from
individuals
and from tissue. The LSP-1, PA-I, and TAP-1 sequences of the invention
uniquely
represent portions of the human genome. Allelic variation occurs to some
degree in the
coding regions of these sequences, and to a greater degree in the noncoding
regions. It
is estimated that allelic variation between individual humans occurs with a
frequency of
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about once per each 500 bases. Each of the sequences described herein can, to
some
degree, be used as a standard against which DNA from an individual can be
compared
for identification purposes. Because greater numbers of polymorphisms occur in
the
noncoding regions, fewer sequences are necessary to differentiate individuals.
The
S noncoding sequences of SEQ ID NO:1, 4, and 7, can comfortably provide
positive
individual identification with a panel of perhaps 10 to 1,000 primers which
each yield a
noncoding amplified sequence of 100 bases. If predicted coding sequences, such
as
those in SEQ ID N0:3, 6, and 9, are used, a more appropriate number of primers
for
positive individual identification would be S00-2,000.
If a panel of reagents from LSP-1, PA-I, and TAP-1 sequences described herein
is used to generate a unique identification database for an individual, those
same
reagents can later be used to identify tissue from that individual. Using the
unique
identification database, positive identification of the individual, living or
dead, can be
made from extremely small tissue samples.
c. Use of Partial LSP-1. PA-I, and TAP-1 Sequences in Forensic Biology
DNA-based identification techniques can also be used in forensic biology.
Forensic biology is a scientific field employing genetic typing of biological
evidence
found at a crime scene as a means for positively identifying, for example, a
perpetrator
of a crime. To make such an identification, PCR technology can be used to
amplify
DNA sequences taken from very small biological samples such as tissues, e.g.,
hair or
skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene.
The amplified
sequence can then be compared to a standard, thereby allowing identification
of the
origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide
reagents, e.g., PCR primers, targeted to specific loci in the human genome,
which can
enhance the reliability of DNA-based forensic identifications by, for example,
providing
another "identification marker" (i.e. another DNA sequence that is unique to a
particular
individual). As described above, actual base sequence information can be used
for
identification as an accurate alternative to patterns formed by restriction
enzyme
generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:1,
4, and
7 are particularly appropriate for this use as greater numbers of
polymorphisms occur in
the noncoding regions, making it easier to differentiate individuals using
this technique.
Examples of polynucleotide reagents include the LSP-1, PA-I, and TAP-1
sequences or
portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID
NO:1, 4,
and 7, having a length of at least 20 bases, preferably at least 30 bases.
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The LSP-l, PA-I, and TAP-1 sequences described herein can further be used to
provide polynucleotide reagents, e.g., labeled or labelable probes which can
be used in,
for example, an in situ hybridization technique, to identify a specific
tissue, e.g., brain
tissue. This can be very useful in cases where a forensic pathologist is
presented with a
tissue of unknown origin. Panels of such LSP-l, PA-I, and TAP-1 probes can be
used to
identify tissue by species and/or by organ type.
In a similar fashion, these reagents, e.g., LSP-1, PA-I, and TAP-1 primers or
probes can be used to screen tissue culture for contamination (i.e. screen for
the presence
of a mixture of different types of cells in a culture).
This invention is further illustrated by the following examples which should
not
be construed as limiting. The contents of all references, patents and
published patent
applications cited throughout this application (including the figures) are
hereby
incorporated by reference.
1 S EXAMPLES
Example 1: Isolation And Characterization of Human LSP-1 cDNAs
In this example, the isolation and characterization of .he gene encoding human
LSP-1 (also referred to as "HOMEDEPO" or "TANGO 111 ") is described.
Isolation of the human LSP-1 cDNA
The following methodology takes advantage of the fact that molecules such as
LSP-1 have an amino terminal signal sequence which directs certain secreted
and
membrane-bound proteins through the cellular secretory apparatus.
A partial LSP-1 mRIVA was identified by screening of a human bone marrow
cDNA library. This library was prepared using mRNA purchased from Clontech,
Palo
Alto (Cat. no, 6573-I ). A signal trap cDNA library was prepared by ligating
random
primed double stranded cDNA into the expression vector, ptrAPl, resulting in
fusions of
cDNAs to the reporter, alkaline phosphatase (AP). DNAs from individual clones
from
this library were prepared by standard techniques and transfected in to human
embryonic
kidney fibroblasts (293T cells). After 28 hours cell supernatants were
collected and
assayed for AP activity.
Clones giving rise to detectable AP activity in the supernatants of
transfected
cells were analyzed further by DNA sequencing and the novel clones subjected
to
further DNA sequencing.
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Once such clone, named LSP-1, was identified. The initial LSP-1 clone
contained an open reading frame of 169 amino acids, (fused in-frame to the AP
reporter). Blast searching of Genbank with this sequence indicated partial
homology to
certain immunoglobulin type proteins (illustrated more clearly in the sequence
analysis
of the full length clone below). A GenBankTM search of the dbEST database
utilizing
the sequence of this cDNA revealed several EST sequences with greater than 95%
nucleotide identity to the partial cDNA.
The LSP-1 nucleic acid molecule was aligned with the FDF03 molecule
(described in WO/24906) using the GAP program in the GCG software package
(pam120 matrix) and a gap weight of 12 and a length weight of 4. The results
showed a
45.3% identity between the two sequences (see Figure 9).
The LSP-1 protein was aligned with the FDF03 protein (described in W0/24906)
using the GAP program in the GCG software package (pam120 matrix) and a gap
weight of 12 and a length weight of 4. The results showed a 51.6% identity
between the
two protein sequences (see Figure 10).
Sequencing of Full Length LSP-1 cDNAs
The interesting chromosomal localization (see Example 3) of the LSP-1 genes
motivated a search for clones encoding a full Length LSP-1 cDNA. Searching of
the
Genbank database identified several EST sequences that showed a high degree of
identity to the partial LSP-1 cDNA identified by signal sequence trapping.
To obtain further sequence data, three ESTs present in the IMAGE clone
collection were located and subjected to further DNA sequencing. A single
sequence
was assembled which completed the LSP-1 open reading frame and extended a 3'
UTR
and poly A tail. Figure 2 shows the selected clones and details the
relationship between
the first LSP-1 clone identified by signal trapping, the IMAGE clones and the
final
composite sequence.
Structure of the LSP-1 protein
The domain structure of the full length LSP-1 proteins is depicted in Figure
3.
LSP-1 contains an N-terminal signal peptide (predicted by the signal Pa
algorithm), an
immunoglobulin-type domain, a transmembrane domain (predicted by MEMSAT,
Jones, D.T., Taylor, W.R., and Thornton, J.M. 1994 Biochemistry 33 3038-3049)
and a
short cytoplasmic domain. The predicted Ig domain is incomplete. However,
there is
precedent for Ig domains of this kind (Barclay et al., The Leucocyte Antigen
Factsbook,
Academic Press).
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Thus, the LSP-1 protein appears to be a Type I membrane protein composed of a
single extracellular immunoglobulin type domain, a transmembrane domain and a
short
cytoplasmic tail. This overall topological structure appears similar to that
of other
leukocyte membrane proteins, notable CD1, CD3 and CD28 (Barclay et al., The
S Leucocyte Antigen Factsbook, Academic Press). Also of note is the presence
within the
predicted LSP-1 membrane spanning domain of a lysine residue. The presence of
charged groups within this domain of cell surface proteins suggests that the
protein may
form homodimers in the cell membrane (Barclay et al., The Leucocyte Antigen
Factsbook, Academic Press). Thus, LSP-1 may exist at the cell surface as a
homodimer
and may exert its effects in this form.
Translation initiation
The translation of the LSP-1 sequence is shown starting from an ATG at
nucleotide 1332. This ATG is in a favorable context for translation initiation
(good
Kozak consensus). Although another in-frame ATG is present upstream at
nucleotide
1140, this is not in a favorable context for translation initiation and given
the overall
predicted topology of the mature protein translated from the second
methionine, it is
unlikely to be used.
Eaamule 2: Distribution of LSP-1 mRNA In Human Tissues
Probing of northern blots purchased from Clontech revealed a 1.5 kb transcript
for LSP-1 only in peripheral blood leukocytes (PBL) and not in any other
tissue. Blots
contained the following tissues: spleen, thymus, prostate, testis, ovary,
small intestine,
colon, peripheral blood leukocyte (human multiple tissue northern III, cat.
no. 7767-4)
spleen, lymph node, thymus, peripheral blood Leucocyte bone marrow and fetal
liver
(human immune system II cat. no. 7768-1). Also, a larger 4.5 kb transcript was
detectable in tissues of the endocrine system (pancreas, adrenal medulla,
thyroid, adrenal
cortex, testis, thymus, small intestine and stomach-human endocrine blot, cat.
no. 7751-
1 ).
PCR analysis of cDNA libraries from various sources was also performed using
primers and conditioned as described for chromosomal mapping of LSP-I . PCR
detects
lower levels of mRNA expression than northern blotting. LSP-1 mRNA was
detectable
in the following human cDNA libraries; placenta, fetal brain, fetal heart,
fetal liver, adult
heart, human umbilical vein endothelial cells, HeLa cells, fetal kidney, adult
adipose
tissue, adult prostate, colorectal adenocarcinoma. lymphocytes, adult lung,
adult spleen,
HL-60 cells (promyelocytic leukemia), human microvascular endothelial cells
and fetal
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spleen. LSP-1 mRNA was not detectable in prostrate epithelial cells,
megakaryocytes,
bronchial epithelial cells or primary osteoblasts.
Examule 3: Chromosomal Mapping Of Human LSP-1 Gene
This Example describes the chromosomal mapping of the human and marine
LSP-1 genes.
The LSP-1 gene maps to chromosome 7q21-q22, at 111- I 12 cM (using the
Genethon linkage map as reference).
The clone was mapped to two difference Radiation Hybrid (RH) panels, the
Stanford Human Genome Center G3 panel and the Genebridge G4 panel, using the
following primers (forward: TCACTCAACCAAAACACC (SEQ ID NO:11); reverse
CCAGTTCAGAAAGACC (SEQ ID N0:12)). LSP-1 was found to be linked to
Genebridge G4 framework marker D7S651, at a distance of 1.7 cR (3000) and a
lod
score >3; and to Stanford G3 framework marker WI-7004, at a distance of 0 cR {
10000)
(meaning without recombination between the clone and the marker) with a lod
score of
1000. Both Markers are also pan of an integrated gene map
(http://www.nci.bi.nlm.nih.gov/SCIENCE96n that serve as a general reference.
The cytogenetic location for LSP-1 was inferred from the map position of close-
by genes (Epo and Cytochrome P450 IIIA).
The results of the RH panel mapping place the LSP-1 gene very close to the
Erythropoietin (EPO) gene (precise distance unclear due to the lack of
resolution of RH
mapping). Possibly within SO-100 Kb.
Example 4: Isolation And Characterization Of Marine PA-I cDNAs
In this example, the isolation and characterization of the gene encoding
marine
Proliferin analog I (PA-I) is described.
The marine gene was discovered by analysis of an EST database (a GenBankTM
search of the dbEST database) using human growth hormone as a probe. dbEST
clone
aa014234 was identified and subsequently obtained from Research Genetics
(Huntsville,
AL). This EST represents a mouse placenta derived clone which contains an ATG
translation initiation codon and is annotated as mouse proliferin-related
protein. BlastP
searching (BLASTTM searching utilizing an amino acid sequence against a
protein
database), using the translation product (frame 1 ) of this sequence, revealed
homology to
proteins belonging to the prolactin-growth hormone superfamily. The mouse
clone was
fully sequenced (SEQ ID N0:4).
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Examule 5: Distribution of PA- I mRNA In Mouse And Human Tissues
Clone aa014234 was digested with a NotI and an EcoRI enzyme to excise the
fragment from the vector and this fragment was used as a probe for Northern
blots. The
fragment was labeled using the Prime It kit from Stratagene (La Jolla, CA) and
then
hybridized to mufti-tissue northern blots from Clontech (Palo Alto, CA) as
recommended by the manufacturer. Three blots were used: a human (7760-I ), a
human
immune system II (7768-1 ) and a mouse embryo (7763-1 ). A band of about 1 Kb
was
detected in tissue from a mouse day 7 embryo (see Figure 6B) under low
stringency
hybridization conditions (hybridization at 68 ° C, wash in 2X SSC,
0.05% SDS at 68' C
for 20 minutes). A band of the same size ( 1 Kb) was detected in human
placenta tissue
(see Figure 6A) and in human fetal liver (see Figure 6C) under similar low
stringency
hybridization conditions (hybridization at 50 ° C, wash in 2X SSC,
0.05% SDS at room
temperature for 20 minutes and at SO ° C for 20 more minutes).
I 5 Example 6: Screening for the human cDNA and genomic DNA
A human placenta library (Clonetech), as well as a human fetal liver Library
(Clonetechj and a human genomic library (Stratagene) are screened using the
same
probe that was used in the northern blot experiment described above.
Hybridization i~
performed under low stringency conditions. Briefly, the hybridization is
performed
overnight at 45 ° C, in Church buffer (7% SDS, 250 mM NaHP04, 2 P.M
EDTA), and is
followed by washing of the filters in 2X SSC, 1 % SDS. The blots are exposed
to film at
-80 ° C for 5 hours. Positive clones are isolated and sequenced using
art known
techniques.
Example 7: Isolation And Characterization Of Human TAP-1 cDNAs
In this example, the isolation and characterization of the genes encoding
human
TAP-1 is described.
Contraction of Libraries
Poly A+ RNA from human prostate tissue was used to construct a cDNA library.
The cDNA library was constructed by first and second strand synthesis as
recommended
by the manufacturer for the Gibco BRL kit Superscript Plasmid System for cDNA
Synthesis and Plasmid Cloning (Gibco/BRL; Bethesda MD).
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Isolation of a human TAP-1 cDNA
A partial human TAP-1 cDNA, also referred to as TANGO-94, was identified by
analysis of an EST database using mouse TPO sequence as a probe. A partial
human
clone (jthqb070d08) was obtained from a human prostate cDNA library and was
subsequently fully sequenced. Clone jthqb070d08 was deposited with the
American
Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-
2209, on October 2, 1997 and has ATCC Accession Number 98554.
A nucleotide sequence of the isolated C-terminal domain of human TAP-1
cDNA (nucleotides I-523 corresponding to the C-terminus and 3' untranslated
sequence)
and the predicted amino acid sequence of the human TAP-1 protein (amino acids
1-86)
are shown in Figure 7 and in SEQ ID NOs:7 and 8, respectively. The amino acid
sequences showed 32% identitity to the C-terminal part of human TPO. The
nucleotide
sequence corresponding to the coding region of the human TAP-1 cDNA are
nucleotides
1-258 of SEQ ID N0:7, nucleotides 259-528 correspond to the 3' untranslated
region of
the gene.
Isolation of Additional TAP-1 cDNAs
Using jthqb070d08 cDNA as a probe, additional TAP-1 clones were isolated
using standard protocols. In brief, clone (jthqb070d08 cDNA) was excised from
a
pMET vector using SaII and NotI restriction enzymes. The excised fragment was
labeled using the Prime It kit from Stratagene (La Jolla, CA) and then
hybridized under
high stringency conditions to a human fetal liver library. For high stringency
conditions,
hybridizations were carried out overnight at 65° C in Church buffer.
The filters were
washed the next day with 2 X SCC / 0.1% SDS. Eight clones were isolated from
human
fetal liver library and submitted for sequencing. 3 out of the 8 clones
contain an insert
of approximately 3 kb.
Example 8: Distribution of TAP-1 mRNA In Human Tissues
Northern blots using clone (jthqb070d08 cDNA) were perfomed using standard
protocols. In brief, clone (jthqb070d08 cDNA) was excised from a pMET vector
using
SaII and NotI restriction enzymes. The excised fragment was labeled using the
Prime It
kit from Stratagene (La Jolla, CA) and then hybridized to multi-tissue
northern blots
from Clontech (Palo Alto, CA) as recommended by the manufacturer. A strong
band
was detected at approxiamtely 3 kb in human fetal liver tissues. Additional
bands were
detected which may be indicative of alternate spliced variants. Two other less
intense
bands of approximately 5 and 2 kb were detected in all tissues tested.
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Example 9: Analysis of TAP-1 amino acid sequence
The partial human TAP-1 cDNA, which is approximately 523 nucleotides in
length, and which is approximately 86 amino acid residues in length. The human
TAP-1
protein contains four serine-proline-threonine-rich domains. A TAP-1 serine-
proline-
threonine-rich domain can be found at least, for example, from about amino
acids I-20
of SEQ ID N0:8 (Gly 1 to G1y20 of SEQ ID N0:8); from about amino acids 21-40
of
SEQ ID N0:8 (IIe20 to AIa40 of SEQ ID N0:8); from about amino acids 41-60 of
SEQ
ID N0:8 (Va140 to G1y60 of SEQ ID N0:8); and from about amino acids 61-81 of
SEQ
ID N0:8 (Pro61 to Thr81 of SEQ ID N0:8). The human TAP-1 C-terminal domain
appears to encode a secreted protein, e.g., growth factor a secreted protein
which shares
significant homology, about 32% identity, with the C-terminal region of human
TPO.
An alignment of the human TAP-1 amino acid sequences to human TPO
sequences is presented in Figure 8. The figure depicts an alignment of the
amino acid
sequences of TAP-1 (corresponding to amino acids 15 to 75 of SEQ ID N0:8) and
human TPO sequences (Swiss-ProtT"" Accession Numbers P40225, 1401246, 939627).
Identical residues are indicated in the row between the TAP-1 and the TPb
sequences by
a single amino acid code; conserved amino acid residues are indicated as (+).
Example 10: Expression of LSP-1. PA-I, and TAP-I in Bacterial Cells
In this example, LSP-1, PA-I, and TAP-1 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion
polypeptide
is isolated and characterized. Specifcally, LSP-1, PA-I, and TAP-1 is fused to
GST
and this fusion polypeptide is expressed in E. coli, e.g., strain PEB 199.
Expression of
the GST-LSP-1, PA-I, and TAP-1 fusion protein in PEB 199 is induced with IPTG.
The
recombinant fusion polypeptide is purified from crude bacterial lysates of the
induced
PEB 199 strain by affinity chromatography on glutathione beads. Using
polyacrylamide
gel electrophoretic analysis of the polypeptide purified from the bacterial
lysates, the
molecular weight of the resultant fusion polypeptide is determined.
Example 11: Expression of Recombinant LSP-1. PA-I, and TAP 1 Protein in
COS Cells
To express the LSP-1, PA-I, and TAP-1 gene in COS cells, the pcDNA/Amp
vector by Invitrogen Corporation (San Diego, CA) is used. This vector contains
an
SV40 origin of replication, an ampicillin resistance gene, an E. coli
replication origin, a
CMV promoter followed by a polylinker region, and an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire LSP-1, PA-I, and TAP-
1
protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused
in-frame
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to its 3' end of the fragment is cloned into the polylinker region of the
vector, thereby
placing the expression of the recombinant protein under the control of the CMV
promoter.
To construct the plasmid, the LSP-I, PA-I, and TAP-1 DNA sequence is
amplified by PCR using two primers. The 5' primer contains the restriction
site of
interest followed by approximately twenty nucleotides of the LSP-1, PA-I, and
TAP-1
coding sequence starting from the initiation codon; the 3' end sequence
contains
complementary sequences to the other restriction site of interest, a
translation stop
codon, the HA tag or FLAG tag and the last 20 nucleotides of the LSP-1, PA-I,
and
TAP-1 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are
digested with the appropriate restriction enzymes and the vector is
dephosphorylated
using the CIAP enzyme (New England Biolabs, Beverly, MA). Preferably the two
restriction sites chosen are different so that the LSP-1, PA-I, and TAP-1 gene
is inserted
in the correct orientation. The ligation mixture is transformed into E. coli
cells (strains
HB101, DHSa, SURE, available from Stratagene Cloning Systems, La Jolla, CA,
can be
used}, the transformed culture is plated on ampicillin media plates, and
resistant colonies
are selected. Plasmid DNA is isolated from transformants and examined by
restriction
analysis for the presence of the correct fragmen~.
COS cells are subsequently transfected with the LSP-1, PA-I, and TAP-1-
pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-
precipitation methods, DEAE-dextran-mediated transfection, lipofection, or
electroporation. Other suitable methods for transfecting host cells can be
found in
Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual.
2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, NY, 1989. The expression of the LSP-1, PA-I, and TAP-1
polypeptide
is detected by radiolabelling (35S-methionine or 35S-cysteine available from
NEN,
Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D.
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells
are
labelled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media
are then
collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCI,
1 % NP-
40, O.I% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the
culture
media are precipitated with an HA specific monoclonal antibody. Precipitated
polypeptides are then analyzed by SDS-PAGE.
Alternatively, DNA containing the LSP-I, PA-I, and TAP-I coding sequence is
cloned directly into the polylinker of the pCDNA/Amp vector using the
appropriate
restriction sites. The resulting plasmid is transfected into COS cells in the
manner
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described above, and the expression of the LSP-1, PA-I, and TAP-1 polypeptide
is
detected by radiolabelling and immunoprecipitation using a LSP-l, PA-I, and
TAP-1
specific monoclonal antibody.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
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(1) GENERAL INFORMATION:
SEQUENCE LISTING
(i) APPLICANT:
(A) NAME: MILLENNIUM BIOTHERAPEUTICS, INC.
(B) STREET: 640 MEMORIAL DRIVE
(C) CITY: CAMBRIDGE
(D) STATE: MASSACHUSETTS
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 02139
(G) TELEPHONE:
(H) TELEFAX:
(ii) TITLE OF INVENTION: SIGNAL PEPTIDE CONTAINING PROTEINS AND
USES THEREFOR
(iii) NUMBER OF SEQUENCES: 12
(ivy CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LAHIVE & COCKFIELD, LLP
(B) STREET: 28 STATE STREET
(C) CITY: BOSTON
(D) STATE: MASSACHUSETTS
(E) COUNTRY: US
(F) ZIP: 02109
(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 #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US98/
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/061,143
(B) FILING DATE: OCTOBER 6, 1997
(C) APPLICATION NUMBER: US 60/061,149
(D) FILING DATE: OCTOBER 6, 1997
(E) APPLICATION NUMBER: 60/061,159
(F) FILING DATE: OCTOBER 6, 1997
(G) APPLICATION NUMBER: 09/004,206
(H) FILING DATE: JANUARY 8, 1998
(I) APPLICATION NUMBER: 09/010,674
(J) FILING DATE: JANUARY 22, 1998
(K) APPLICATION NUMBER: 09/014,347
(L) FILING DATE: JANUARY 27, 1998
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MANDRAGOURAS, AMY E.
(B) REGISTRATION NUMBER: 36,207
(C) REFERENCE/DOCKET NUMBER: MEI-004CPPC
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(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617)227-7400
(B) TELEFAX: (617)742-4214
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2462 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
i.1 ) MOLECULE TYPE : cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1332..2009
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GAATTCGGCA CGAGCTGGGG CTCCCCTATG CCTGAGTTCC TGCAACTCAG AAGTTGAGGC 60
CCAGAGATCC AGGTAGAGGG GGCTCCAGTC CTGGGACTGC CCTGCAAGTC ATGTGGCCTG I20
GGACAGGGCT TCTCCAAGCT CTGTCCCCTC CTGTGTAGGG CAGGGAGGTC AGAAGGGACC 180
CTGCAGATCC AGTCTCATGT CTGGGGGTCA AGGGGTGGCC TCGAGAGGGA CCAGTCTCTG 240
TGTAGGGGAC CGTCAGCCCC CTCACCCCTT GAGCAAGACT GTGGTCCCTG CACCAAGGGA 300
GCAGGCCTGG GGTGGGAAGA GGCCAGCTGG GCTGTGGTGG TGCCTGGGGA CTGCATGGGA 360
GCCCCTGCCA GGGAGGGAGA GGGACAGAGG ACAACCTGGG GGCTCTGGTG CTTGGGCTGG 420
GGGCTGAGCG CCTGTGACCT CCACTGGCTT CCTCCTTCTC CTCTCTGAGG ACTGAATCTG 480
GGGTGCAGCA GAGCACAGAC TCAGGCCCGC CTTTCCTCTC CCTGAAAGAG CCTGCGCTGG 540
CCTTGGACAG AGAAAGGGAT GAGAAGTGAG GCTGAGTGCG GTGGGGTCTG CAGGGATCCA 600
GGTGGGAGGG GCCCAGCCAG CCAAGGTGAG GCCCAGCCCC TCAGCAGGAA GATGGGCACT 660
GGGGCCCTTG GGCAGGGCTG ACTTGACACT TTTGTGTGAC TTGGAGCCAC TGTGCCCAGC 720
CTGAACACCC TTTCCTGGTA AAACACTCCA CAAACCAGGA AGAGAAGGAA TATACTGCAA 780
CAAAATAAAG GCCAGTCATG CAAGGCCCAT GGCTGAAAGT CTTTCAGTCA TTTTAGATGA B40
AAGACTGAAA TCTTTGCCTC CAAGATCAGG AACAAGAGAA GGATGCCCGC TCTCACTACT 900
TCTATTCAAC ACAGGATTTG AAGTCAGGCC GGGCACAGTG GCTCACGCCT GTAATCCCAG 960
CACTTTTGGA GGCTGAGGCG GGCAGATTAC TTGAGCCTAT GAGTGTGAGA CCACCCTGGC 1020
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CAACATGGCA AAAAGGATTTGAAGTCCTGG1080
AAACCCCATC
TCTACTAAAA
AAAAAAAAAA
CCGGAGCAATTAGGCAAGGG ATAAAAAGGC ACTAAGGCCCTTTTGCAATAAGAAGCCAGA1140
TGGATAAAGGAAGTGCTGGT CACCCTGGAG GTGTACTGGTTTGGGGAAGGTCCCCGGCCC1200
CCACAGCCCTCTGGGGAGCC TCACCCTGGC TCTCCCCACTCACCTCAGCCCTCAGGCAGC1260
CCCTCCACAGGACCCCTCTC CTGCCTGGAC AGCTCTGCTGGTCTCCCCGTCCCCTGGAGA1320
AGAACAAGGCC ATG GGT CGG CCC CTG CTG C CTG CTG 1370
CTG CCC CTG CTG CT
Met Gly Arg Pro Leu Leu u Leu Leu
Leu Pro Leu Leu Le
1 5 10
CAG CCG GCA TTT CTG CAG CCT GGT ACA GGA GGT CCA 1418
CCA GGC TCC TCT
Gln Pro Ala Phe Leu Gln Pro Gly Thr Gly Gly Pro
Pro Gly Ser Ser
15 20 25
AGC TAC TAT GGG GTC ACT CAA CCA CTC TCA TCC ATG 1466
CTT AAA CAC GCC
Ser Tyr Tyr Gly Val Thr Gln Pro Leu Ser Ser Met
Leu Lys His Ala
30 35 40 45
GGT GGC GTG GAA ATC CCC TTC TCC TAC CCC GAG TTA 1514
TCT TTC TAT TGG
Gly Gly Val Glu Ile Pro Phe Ser Tyr Pro Glu Leu
Ser Phe Tyr Trp
50 55 60
GCC ACA CCC GAC GTG AGA ATA TCC CGG GGC TTC CAC 1562
GCT TGG AGA CAC
Ala Thr Pro Asp Val Arg Ile Ser Arg Gly Phe His
Ala Trp Arg His
65 70 75
GGGCAG TCCTTCTAC AGCACAAGG CCGCCTTCC ATTCAC AAGGATTAT 1610
GlyGln SerPheTyr SerThrArg ProProSer IleHis LysAspTyr
80 85 90
GTGAAC CGGCTCTTT CTGAACTGG ACAGAGGGT CAGGAG AGCGGCTTC 1658
ValAsn ArgLeuPhe LeuAsnTrp ThrGluGly GlnGlu SerGlyPhe
95 100 105
CTCAGG ATCTCAAAC CTGCGGAAG GAGGACCAG TCTGTG TATTTCTGC 1706
LeuArg IleSerAsn LeuArgLys GluAspGln SerVal TyrPheCys
110 115 120 125
CGAGTC GAGCTGGAC ACCCGGAGA TCAGGGAGG CAGCAG TTGCAGTCC 1754
ArgVal GluLeuAsp ThrArgArg SerGlyArg GlnGln LeuGlnSer
130 135 140
ATCAAG GGGACCAAA CTCACCATC ACCCAGGCT GTCACA ACCACCACC 1802
IleLys GlyThrLys LeuThrIle ThrGlnAla ValThr ThrThrThr
145 150 155
ACCTGG AGGCCCAGC AGCACAACC ACCATAGCC GGCCTC AGGGTCACA 1850
ThrTrp ArgProSer SerThrThr ThrIleAla GlyLeu ArgValThr
160 165 170
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GAA AAA GGGCACTCA TCATGG CAC CTA AGT CTG ACTGCC 1898
AGC GAA GAC
Glu Lys GlyHisSer SerTrp His Leu Ser Leu ThrAla
Ser Glu Asp
175 180 185
ATC GTT GCATTGGCT GCTGTG CTC AAA ACT GTC TTGGGA 1946
AGG GTC ATT
Ile Val AlaLeuAla AlaVal Leu Lys Thr Val LeuGly
Arg Val Ile
190 195 200 205
CTG TGC CTCCTCCTG TGGAGG AGA AGG AAA GGT AGGGCG 1994
CTG TGG AGC
Leu Cys LeuLeuLeu TrpArg Arg Arg Lys Gly ArgAla
Leu Trp Ser
210 215 220
CCA AGT GACTTCTGACCAACAG 2049
AGC AGTGTGGGGA
GAAGGGATGT
GTATTAGCCC
Pro Ser AspPhe ,
Ser
225
CGGAGGACGTGATGTGAGAC CCGCTTGTGA GTCCTCCACACTCGTTCCCC ATTGGCAAGA2109
TACATGGAGAGCACCCTGAG GACCTTTAAA AGGCAAAGCCGCAAGGCAGA AGGAGGCTGG2169
GTCCCTGAATCACCGACTGG AGGAGAGTTA CCTACAAGAGCCTTCATCCA GGAACATCCA2229
CACTGCAATGATATAGGAAT GAAGTCTGAA CTCCACTGAATTAAACCACT GGCATTTGGG2289
GGCTGTTCATTATAGCAGTG CAAAGAGTTC CTTTATCCTCCCCAAGGATG GAAAATACAA2349
TTTATTTTGCTTACCATACA CCCCTTTTCT CCTCGTCCACATTTTCCAAT CTGTATGGTG2409
GCTGTCTTCTATGGCAAAAG TTTTGGGGAA TAAATAACGTTAAATGCTGC TGA 2462
(2} INFORMATION
FOR SEQ
ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 226 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ :
ID N0:2
Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu Gln Pro Pro
1 5 10 15
Ala Phe Leu Gln Pro Gly Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu
20 25 30
Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser
35 40 45
Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala
50 55 60
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Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser
65 70 75 g0
Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg
85 90 95
Leu Phe Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile
100 105 120
Ser Asn Leu Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg Val Glu
115 120 125
Leu Asp Thr Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile Lys Gly
130 135 140
Thr Lys Leu Thr Ile Thr Gln Ala Val Thr Thr Thr Thr Thr Trp Arg
145 150 155 160
Pro Ser Ser Thr Thr Thr Ile Ala Gly Leu Arg Val Thr Glu Ser Lys
165 170 175
Gly His Ser Glu Ser Trp His Leu Ser Leu Asp Thr Ala IIe Arg Val
180 185 190
Ala Leu Ala Val Ala Val Leu Lys Thr Val Ile Leu Gly Leu Leu Cys
195 200 205
Leu Leu Leu Trp Trp Arg Arg Arg Lys Gly Ser Arg Ala Pro Ser Ser
210 215 220
Asp Phe
225
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 678 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..678
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
ATG GGT CGG CCC CTG CTG CTG CCC CTG CTG CTC CTG CTG CAG CCG CCA 48
Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu Gln Pro Pro
1 5 10 15
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GCA CTG GGT TCC GGT 96
TTT CAG GGC ACA CCA
CCT GGA AGC
TCT TAC
CTT
Ala Leu Gly Ser Gly Gly Ser Tyr Leu
Phe Gln Gly Thr Ser Pro
Pro
20 25 30
TAT GTCACT CCA CAC TCA TCCATG GGT GGC TCT 144
GGG CAA AAA CTC GCC
TyrGly ValThr ProLys HisLeuSer AlaSerMet Gly Gly Ser
Gln
35 40 45
GTGGAA ATCCCC TCCTTC TATTACCCC TGGGAGTTA GCC ACA GCT 192
TTC
ValGlu IlePro SerPhe TyrTyrPro TrpGluLeu Ala Thr Ala
Phe
50 55 60
CCCGAC GTGAGA TCCTGG AGACGGGGC CACTTCCAC GGG CAG TCC 240
ATA
ProAsp ValArg SerTrp ArgArgGly HisPheHis Gly Gln Ser
Ile
65 70 75 80
TTCTAC AGCACA CCGCCT TCCATTCAC AAGGATTAT GTG AAC CGG 288
AGG
PheTyr SerThr ProPro SerIleHis LysAspTyr Val Asn Arg
Arg
85 90 95
CTCTTT CTGAAC ACAGAG GGTCAGGAG AGCGGCTTC CTC AGG ATC 336
TGG
LeuPhe LeuAsn ThrGlu GlyGlnGlu SerGlyPhe Leu Arg Ile
Trp
100 105 110
TCAAAC CTGCGG GAGGAC CAGTCTGTG TATTTCTGC CGA GTC GAG 384
AAG
SerAsn LeuArg GluAsp GlnSerVal TyrPheCys Arg Val Glu
Lys
115 120 125
CTGGAC ACCCGG TCAGGG AGGCAGCAG TTGCAGTCC ATC AAG GGG 432
AGA
LeuAsp ThrArg SerGly ArgGlnGln LeuGlnSer Ile Lys Gly
Arg
130 135 140
ACCAAA CTCACC ACCCAG GCTGTCACA ACCACCACC ACC TGG AGG 480
ATC
ThrLys LeuThr ThrGln AlaValThr ThrThrThr Thr Trp Arg
Ile
145 150 155 160
CCCAGC AGCACA ACCATA GCCGGCCTC AGGGTCACA GAA AGC AAA 528
ACC
ProSer SerThr ThrIle AlaGlyLeu ArgValThr Glu Ser Lys
Thr
165 170 175
GGGCAC TCAGAA TGGCAC CTAAGTCTG GACACTGCC ATC AGG GTT 576
TCA
GlyHis SerGlu TrpHis LeuSerLeu AspThrAla Ile Arg Val
Ser
180 185 190
GCATTG GCTGTC GTGCTC ACTGTC ATTTTGGGA CTG CTG TGC 624
GCT AAA
AlaLeu Val ValLeu LysThrVal IleLeuGly Leu Leu Cys
Ala Ala
195 200 205
CTCCTC CTGTGG AGA AAA AGC GCG CCA AGC AGT 672
TGG AGG GGT AGG
AGG
LeuLeu Trp Arg Lys Ser Ala Pro Ser Ser
Leu Trp Arg Gly Arg
Arg
210 215 22.0
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GAC TTC 678
Asp Phe
225
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 933 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 55..813
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
TCGACGCGCC AGAG 57
TATTCTTGCA ATG
CGAGGCAGAC
AGGCTGTGCC
AGAACTCTTC
Met
1
TCA TTTTCT TTC CAA TGC CCC GGG CTT CTG GTG 105
TCT CCA TCA GCA CTG
Ser PheSer Phe Gln Cys Pro Gly Leu Leu Val
Ser Pro Ser Ala Leu
5 10 15
GTG GTGTCA AGC CTT TGG GAG GTG TCT CCT TTG 153
CTC TTA AAT GCC GTA
Val ValSer Ser Leu Trp Glu Val Ser Pro Leu
Leu Leu Asn Ala Val
20 25 30
AGT AGCAAT GAG GAT TAT CCA TCC AAT CTG TTT 201
ACT GGT TTA ATC GGG
Ser SerAsn Glu Asp Tyr Pro Ser Asn Leu Phe
Thr Gly Leu Ile Gly
35 40 45
CAT AATGCC ATG CTA TGG AAT AAA CTC ATG GAA 249
AGA ACT ATC AAC AAC
His AsnAla Met Leu Trp Asn Lys Leu Met Glu
Arg Thr Ile Asn Asn
50 55 60 65
CTG CGCAAG ACA ACA AAT CAA TCT AAA TAC GAG 297
TAT GTC GTC GAA TTA
Leu ArgLys Thr Thr Asn Gln Ser Lys Tyr Glu
Tyr Val Val Glu Leu
70 75 8.0
AAC TATATG CTT TTT GAG GAC GAG CTG AAG GCT 345
GAC ATT ATG TAT GTC
Asn TyrMet Leu Phe Glu Asp Glu Leu Lys Ala
Asp Ile Met Tyr Val
85 90 95
CTC ACCTGC TGC AAT TCC ATC ACT GAA CTG GAC 393
CAC TAT AAA CCA AAC
Leu ThrCys Cys Asn Ser Ile Thr Glu Leu Asp
His Tyr Lys Pro Asn
100 105 110
GAA GCTCAA CAG CCT AAC GAA CCA CTG CTC AGT 441
ATT TTT TTT AAG ATC
Glu AlaGln Gln Pro Asn Glu Pro Leu Leu Ser
Ile Phe Phe Lys Ile
115 120 125
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AGA CTC 489
ATG
TGG
GCT
TGG
AAT
GAA
ACT
TCT
AAA
GTT
CTA
CTG
ACC
ACA
Arg Met Trp Ala Trp Asn Glu Lys Val Leu Leu Thr Leu
Thr Ser Thr
130 135 140 145
AGA AGT ATT CCA GGA ATG CAT GTC ATT TCA TTA GCC AAC 537
GAT GAT AAA
Arg Ser Ile Pro Gly Met His Val Ile Ser Leu Ala Asn
Asp Asp Lys
150 155 160
ATT GAA ACA AAA CTT GCA GAG GAG TAC ACC CAG AGT CTC 585
CTT TTT ATA
Ile Glu Thr Lys Leu Ala Glu Glu Tyr Thr Gln Ser Leu
Leu Phe Ile
165 170 175
AAC TCG ATT TAT GGA ACA ACA GGA AAT GTG GAA TAC GTC 633
ACA ACA ACC
Asn Ser Ile Tyr Gly Thr Thr Gly Asn Val Glu Tyr Val
Thr Thr Thr
180 185 190
TTT TCT GGT CTT GAA GAC TTA TCT GAT GAA GAA TTT CTT 681
AAA TCA AGT
Phe Ser Gly Leu Glu Asp Leu Ser Asp Glu Glu Phe Leu
Lys Sex Ser
195 200 205
TTT GAC CTT TGT AAA TTT TCC TTA CGT GTA GAT ATA ATG 729
TAT TGC CAT
Phe Asp Leu Cys Lys Phe Ser Leu Arg Val Asp Ile Met
Tyr Cys His
210 215 220 225
GTT GAA CTT TAT CTC AAG CTA TGT GTG GTA TAT GTT AGT 77?
TTA GAG AGT
Val Glu Leu Tyr Leu Lys Leu Cys Val Val Tyr Val Ser
Leu Glu Ser
230 235 240
GAT GTT TGT TTA TCC AAA AAT GAT GCT TCA TGATGCTGAA 823
ATT AGA
Asp Val Cys Leu Ser Lys Asn Asp Ala Ser
Ile Arg
245 250
TCTTTTTAAA 883
TAATCTTAAT
TTTATAATTG
TGAAAGTATA
ATTGAGTATA
ACGAGTGTCT
TTTAAAATAA AAAAp,AA FI~F~AAAAAp~Ap 9
AAATAAACTA 3
TATATATAAA 3
1?~F~A
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A).LENGTH: 253 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: ID N0:5:
SEQ
Met
Ser
Phe
Ser
Phe
Ser
Gln
Pro
Cys
Pro
Ser
Gly
Ala
Leu
Leu
Leu
1 5 10 15
Val
Val
Val
Ser
Ser
Leu
Leu
Leu
Trp
Glu
Asn
Val
Ala
Ser
Val
Pro
20 25 30
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Leu Ser Ser Asn Glu Thr Asp Gly Tyr Pro Leu Ser Ile Asn Gly Leu
35 40 45
Phe His Asn Ala Met Arg Leu Thr Trp Asn Ile Lys Asn Leu Asn Met
50 55 60
Glu Leu Arg Lys Thr Tyr Thr Val Asn Gln Val Ser Glu Lys Leu Tyr
65 70 75 80
Glu Asn Tyr Met Leu Asp Phe Ile Glu Asp Met Glu Tyr Leu Val Lys
85 90 95
Ala Leu Thr Cys Cys His Asn Tyr Ser Ile Lys Thr Pro Glu Asn Leu
100 105 110
Asp Glu Ala Gln Gln Ile Pro Phe Asn Glu Phe Pro Lys Leu Ile Leu
115 120 125
Ser Arg Met Trp Ala Trp Asn Glu Thr Ser Lys Val Leu Leu Thr Thr
130 135 140
Leu Arg Ser Ile Pro Gly Met His Asp Asp Val Ile Ser Leu Ala Lys
145 150 155 160
Asn Ile Glu Thr Lys Leu Ala Glu Leu Phe Glu Tyr Thr Gln Ser Ile
' 165 170 175
Leu Asn Ser Ile Tyr Gly Thr Thr Thr Thr Gly Asn Val Glu Tyr Thr
180 185 190
Val Phe Ser Gly Leu Glu Asp Leu Lys Ser Ser Asp Glu Glu Phe Ser
195 200 205
Leu Phe Asp Leu Cys Lys Phe Ser Tyr Cys Leu Arg Val Asp Ile His
210 215 220
Met Val Glu Leu Tyr Leu Lys Leu Leu Glu Cys Val Val Tyr Val Ser
225 230 235 240
Ser Asp Val Cys Leu Ser Lys Asn Ile Arg Asp Ala Ser
245 250
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 762 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..759
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
ATG TCA TTT TCT TTC TCT CAA CCA TGC CCC TCA GGG GCA CTT 48
CTG CTG
Met Ser Phe Ser Phe Ser Gln Pro Cys Pro Ser Gly Ala Leu
Leu Leu
1 5 10 15
GTG GTG GTG TCA AGC CTC CTT TTA TGG GAG AAT GTG GCC TCT 96
GTA CCT
Val Val Val Ser Ser Leu Leu Leu Trp Glu Asn Val Ala Ser
Val Pro
20 25 30
TTG AGT AGC AAT GAG ACT GAT GGT TAT CCA TTA TCC ATC AAT 144
GGG CTG
Leu Ser Ser Asn Glu Thr Asp Gly Tyr Pro Leu Ser Ile Asn
Gly Leu
35 40 45
TTT CAT AAT GCC ATG AGA CTA ACT TGG AAT ATC AAA AAC CTC 192
AAC ATG
Phe His Asn Ala Met Arg Leu Thr Trp Asn Ile Lys Asn Leu
Asn Met
50 55 60
GAA CTG CGC AAG ACA TAT ACA GTC AAT CAA GTC TCT GAA AAA 240
TTA TAC
Glu Leu Arg Lys Thr Tyr Thr Val Asn Gln Val Ser Glu Lys
Leu Tyr
65 70 75 80
GAG AAC TAT ATG CTT GAC TTT ATT GAG GAC ATG GAG TAT CTG 28B
GTC AAG
Glu Asn Tyr Met Leu Asp Phe Ile Glu Asp Met Glu Tyr Leu
Val Lys
85 90 95
GCT CTC ACC TGC TGC CAC AAT TAT TCC ATC AAA ACT CCA GAA 336
AAC CTG
Ala Leu Thr Cys Cys His Asn Tyr Ser Ile Lys Thr Pro Glu
Asn Leu
100 105 110
GAC GAA GCT CAA CAG ATT CCT TTT AAC GAA TTT CCA AAG CTG 384
ATC CTC
Asp Glu Ala Gln Gln Ile Pro Phe Asn Glu Phe Pro Lys Leu
Ile Leu
115 120 125
AGT AGA ATG TGG GCT TGG AAT GAA ACT TCT AAA GTT CTA CTG 432
ACC ACA
Ser Arg Met Trp Ala Trp Asn Glu Thr Ser Lys Val Leu Leu
Thr Thr
130 135 140
CTC AGA AGT ATT CCA GGA ATG CAT GAT GAT GTC ATT TCA TTA 480
GCC AAA
Leu Arg Ser Ile Pro Gly Met His Asp Asp Val Ile Ser Leu
Ala Lys
145 150 155 160
AAC ATT GAA ACA AAA CTT GCA GAG CTT TTT GAG TAC ACC CAG 528
AGT ATA
Asn Ile Glu Thr Lys Leu Ala Glu Leu Phe Glu Tyr Thr Gln
Ser Ile
165 170 175
CTC AAC TCG ATT TAT GGA ACA ACA ACA ACA GGA AAT GTG GAA 576
TAC ACC
Leu Asn Ser Ile Tyr Gly Thr Thr Thr Thr Gly Asn Val Glu
Tyr Thr
180 185 190
GTC TTT TCT GGT CTT GAA GAC TTA AAA TCA TCT GAT GAA GAA 624
TTT AGT
Val Phe Ser Gly Leu Glu Asp Leu Lys Ser Ser Asp Glu Glu
Phe Ser
195 200 205
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CTT TTT GAC CTT TGT AAA TTT TCC TAT TGC TTA CGT GTA GAT ATA CAT 672
Leu Phe Asp Leu Cys Lys Phe Ser Tyr Cys Leu Arg Val Asp Ile His
210 215 220
ATG GTT GAA CTT TAT CTC AAG CTA TTA GAG TGT GTG GTA TAT GTT AGT 7
Met Val Glu Leu Tyr Leu Lys Leu Leu Glu Cys Val Val Tyr Val Ser
225 230 235 240
AGT GAT GTT TGT TTA TCC AAA AAT ATT AGA GAT GCT TCA 759
Ser Asp Val Cys Leu Ser Lys Asn Ile Arg Asp Ala Ser
245 250
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 523 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..261
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GGA TCG ACC CCC GCG TCC GCG GTT TCT GCG CGG TGC GCG ACC TCG TCC 48
Gly Ser Thr Pro Ala Ser Ala Val Ser Ala Arg Cys Ala Thr Ser Ser
1 5 10 15
CGA AGC CTG GGG ATA CAC CCT CTC GAG AGC CCG CTG TCG CCC TCC GTT 96
Arg Ser Leu Gly Ile His Pro Leu Glu Ser Pro Leu Ser Pro Ser Val
20 25 30
AAG GTC GAA CCC CTC ACA GTT GCT GTG GGC AAC TCC AGC CCA ACA TTC 144
Lys Val Glu Pro Leu Thr Val Ala Val Gly Asn Ser Ser Pro Thr Phe
35 40 45
CCT CGC TCT GGT TCT CGC CCC ATT GGG AAA CTC GGC CCC ACG CTT CCC 192
Pro Arg Ser Gly Ser Arg Pro Ile GIy Lys Leu Gly Pro Thr Leu Pro
50 55 60
ACT TTT CTG GAT GAG GTG TCC CCT TTC TCC CCA CTA AAA TGT CAA ATA 240
Thr Phe Leu Asp Glu Val Ser Pro Phe Ser Pro Leu Lys Cys Gln Ile
65 70 75 80
ACC TAC GGA GGG TCT TCC TGAAACCCGC AGAGGAAAAT TCAGGCAACG 288
Thr Tyr Gly Gly Ser Ser
85
CCTCGCGTTG TGGTTTCAGG CTGCATGTAC CAAGTAGTTC AGACGTTTGG CTCGGATGGA 348
i'.
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AAAAAATCTT CTGCAATTAT TTCCAATTCC TAAGTCTTCT TGGAAATCTT ATACCACTAG 408
TTCAATCTTC AAGTCATGTC TGATGCTTTG AAAGGGATTA CAGGAAACCA GTTCAAGTTA 468
TTTTCAGACC AGATTTCCAG CTCTTCCACA AGTGCATCAT TTCAATTGCC CATTT 523
(2) INFORMATION FOR SEQ ID N0:8:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 86 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Gly Ser Thr Pro Ala Ser Ala Val Ser Ala Arg Cys Ala Thr Ser Ser
1 5 10 15
Arg Ser Leu Gly Ile His Pro Leu Glu Ser Pro Leu Ser Pro Ser Val
20 25 30
Lys Val Glu Pro Leu Thr Val Ala Val Gly Asn Ser Ser Pro Thr Phe
35 40 45
Pro Arg Ser Gly Ser Arg Pro Ile Gly Lys Leu Gly Pro Thr Leu Pro
50 55 60
Thr Phe Leu Asp Glu Val Ser Pro Phe Ser Pro Leu Lys Cys Gln Ile
65 70 75 80
Thr Tyr Gly Gly Ser Ser
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 258 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GGATCGACCC CCGCGTCCGC GGTTTCTGCG CGGTGCGCGA CCTCGTCCCG AAGCCTGGGG 60
ATACACCCTC TCGAGAGCCC GCTGTCGCCC TCCGTTAAGG TCGAACCCCT CACAGTTGCT 120
GTGGGCAACT CCAGCCCAAC ATTCCCTCGC TCTGGTTCTC GCCCCATTGG GAAACTCGGC 180
CCCACGCTTC CCACTTTTCT GGATGAGGTG TCCCCTTTCT CCCCACTAAA ATGTCAAATA 240
CA 02306246 2000-04-OS
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-13-
ACCTACGGAG GGTCTTCC 258
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Leu Pro Ser Leu Ile Gln Pro Cys Ser Ser Gly Thr Leu Leu Met
1 5 10 15
Leu Leu Met Ser Asn Leu Phe Leu Trp Glu Lys Val Ser Ser Ala Pro
20 25 30
Ile Asn Ala Ser Glu Ala Val Leu Ser Aap Leu Lys Asp Leu Phe Asp
35 40 45
Asn Ala Thr Val Leu Ser Gly Glu Met Ser Lys Leu Gly Val Ile Met
50 55 60
Arg Lys Glu Phe Phe Met Asn Ser Phe Ser Ser Glu Thr Phe Asn Lys
65 70 75 80
Ile Ile Leu Asp Leu His Lys Ser Thr Glu Asn Ile Thr Lys Ala Phe
85 90 95
Asn Ser Cys His Thr Val Pro Ile Asn Val Pro Glu Thr Val Glu Asp
100 105 110
Val Arg Lys Thr Ser Phe Glu Glu Phe Leu Lys Met Val Leu His Met
115 I20 125
Leu Leu Ala Trp Lys Glu Pro Leu Lys His Leu Val Thr Glu Leu Ser
130 135 140
Ala Leu Pro Glu Cys Pro Tyr Arg Ile Leu Ser Lys Ala Glu Ala Ile
145 150 155 160
Glu Ala Lys Asn Lys Asp Leu Leu Glu Tyr Ile Ile Arg Ile Ile Ser
165 170 175
Lys Val Asn Pro Ala Ile Lys Glu Asn Glu Asp Tyr Pro Thr Trp Ser
180 185 190
Asp Leu Asp Ser Leu Lys Ser Ala Asp Lys Glu Thr Gln Phe Phe Ala
195 200 205
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-14-
Leu Tyr Met Phe Ser Phe Cys Leu Arg Ile Asp Leu Glu Thr Val Asp
210 215 220
Phe Leu Val Asn Phe Leu Lys Cys Leu Leu Leu Tyr Asp Asp Val Cys
225 230 235 240
Tyr Ser Glu
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1-18
(D) OTHER INFORMATION: /note= "forward PCR primer~~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TCACTCAACC AAAACACC lg
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix} FEATURE:
(A) NAME/REY: misc_feature
(B) LOCATION: 1-15
(D) OTHER INFORMATION: /note= ~~reverse PCR primer~~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
CAGTTCAGAA AGACC 15
