Note: Descriptions are shown in the official language in which they were submitted.
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LYMPHOMA ASSOCIATED MOLECULES AND USES THEREFOR
_Government Funding
Work described herein was supported, at least in part, under grant
S 1PO1CA66996-OlAl awarded by the NIH. The U.S. government therefore may have
certain rights in this invention.
Background of the Invention
The incidence of non-Hodgkin's lymphoma in the United States has increased by
75.1% between 1973 and 1992 (Kosary et al., SEER Cancer Statistics Review,
1973-
1992: Tables and Graphs, National Cancer Institute, NIH Publication No 96-
2789,
Bethesda, MD: NIH 1995), a percentage increase exceeded only by that for
prostate
cancer, lung cancer in women, and melanoma.
Diffuse large B-cell lymphoma (DLB-CL) is the most common non-Hodgkin's
lymphoma in adults. Although DLB-CL is curable in approximately 40% of
patients,
the majority of patients progress and die of their disease (Shipp et al. Non-
Hodgkin's
Lymphomas. In DeVita (ed): Principles and Practice of Oncology, 5th Edition,
Philadelphia, J.B. Lippincott Company. pp. 2165-2220, 1997). Additional
advancements in the treatment of this aggressive but potentially curable non-
Hodgkin's
lymphoma are likely to require a more precise understanding of the disease's
cellular and
molecular bases.
Summary of the Invention
To identify genes which contribute to the observed differences in clinical
outcome in DLB-CLs, the technique of differential display (Liang P. et al.
(1992)
Science, 257:967) was used in panels of primary tumors from patients with
known
clinical prognostic characteristics and mature follow-up. A novel 3' cDNA,
termed
BAL, was found to be significantly more abundant in tumors from patients with
"high-
risk (HR)" (International Prognostic Index, IPI) fatal disease than in tumors
from cured
"low risk (LR [IPI])" patients (Shipp M. et al. (1993) N. Engl. J. Med.,
329:987-994).
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Accordingly, the present invention is based, at least in part, on the
discovery of
novel molecules which are differentially expressed in tumors from patients
with "high
risk" fatal DLB-CL disease or "low risk" cured DLB-CL. Their differentially
expressed
gene products are referred to herein as the "B-aggressive lymphoma" ("BAL")
nucleic
acid and protein. The BAL molecules of the present invention are useful as
modulating
agents for regulating a variety of cellular processes. Accordingly, in one
aspect, this
invention provides isolated nucleic acid molecules encoding BAL proteins or
biologically active portions thereof, as well as nucleic acid fragments
suitable as primers
or hybridization probes for the detection of BAL-encoding nucleic acids.
In one embodiment, a BAL nucleic acid molecule of the invention is at least
50%, 55%, 60%, 65%, 70%, 72%, 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, or 6.
In a preferred embodiment, the isolated nucleic acid molecule includes the
nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof. In
another
embodiment, the nucleic acid molecule includes SEQ ID N0:3 and nucleotides 1-
228 of
SEQ ID NO:1. In another embodiment, the nucleic acid molecule includes SEQ ID
N0:3 and nucleotides 2791-3243 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
least 607 nucleotides (e.g., 607 contiguous nucleotides) of the nucleotide
sequence of
SEQ ID NO:1 or 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-
170 of
SEQ ID N0:4. In another embodiment, the nucleic acid molecule includes SEQ ID
N0:6 and nucleotides 2649-3024 of SEQ ID N0:4. In another preferred
embodiment,
the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID
N0:4 or
6. In another preferred embodiment, the nucleic acid molecule includes a
fragment of at
least 452 nucleotides (e.g., 452 contiguous nucleotides) of the nucleotide
sequence of
SEQ ID N0:4, SEQ ID N0:6, or a complement thereof.
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In another embodiment, a BAL nucleic acid molecule includes a nucleotide
sequence encoding a protein having an amino acid sequence sufficiently
homologous to
the amino acid sequence of SEQ ID N0:2 or 5. In a preferred embodiment, a BAL
nucleic acid molecule includes a nucleotide sequence encoding a protein having
an
S amino acid sequence at least 50%, 55%, 60%, 62%, 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 5.
In another preferred embodiment, an isolated nucleic acid molecule encodes the
amino acid sequence of human BAL. In yet another preferred embodiment, the
nucleic
acid molecule includes a nucleotide sequence encoding a protein having the
amino acid
sequence of SEQ ID N0:2 or S. In yet another preferred embodiment, the nucleic
acid
molecule is at least 452 or 607 nucleotides in length. In a further preferred
embodiment,
the nucleic acid molecule is at least 452 or 607 nucleotides in length and
encodes a
protein having a BAL activity (as described herein).
Another embodiment of the invention features nucleic acid molecules,
preferably
BAL nucleic acid molecules, which specifically detect BAL nucleic acid
molecules
relative to nucleic acid molecules encoding non-BAI, proteins. For example, in
one
embodiment, such a nucleic acid molecule is at least 300-350, 350-400, 400-
450, 452,
452-500, 500-550, 550-600, 607 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 or 4 or a complement thereof.
In preferred embodiments, the nucleic acid molecules are at least 15 (e.g.,
. contiguous) nucleotides in length and hybridize under stringent conditions
to nucleotides
1-333, 351-385, 463-824, 931-1082, or 3232-3244 of SEQ ID NO:1. In other
preferred
embodiments, the nucleic acid molecules comprise nucleotides 1-333, 351-385,
463-
824, 931-1082, or 3232-3244 of SEQ ID NO:1.
In other preferred embodiments, the nucleic acid molecules are at least 15
(e.g.,
contiguous) nucleotides in length and hybridize under stringent conditions to
nucleotides
1-39 or 57-1841 of SEQ ID N0:4. In other preferred embodiments, the nucleic
acid
molecules comprise nucleotides 1-39 or 57-1841 of SEQ ID N0:4.
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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
N0:2 or 5, wherein the nucleic acid molecule hybridizes to a nucleic acid
molecule
comprising SEQ ID NO:1, 3, 4, or 6 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule
which is antisense to a BAL nucleic acid molecule, e.g., the coding strand of
a BAL
nucleic acid molecule.
Another aspect of the invention provides a vector comprising a BAL 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 BAL 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.
Another aspect of this invention features isolated or recombinant BAL proteins
and polypeptides. In one embodiment, the isolated protein, preferably a BAL
protein,
includes at least one proline rich domain. In a preferred embodiment, the
isolated
protein, preferably a BAL protein, includes at least one proline rich domain
and at least
one tyrosine phosphorylation site. In a preferred embodiment, the protein,
preferably a
BAL protein, includes at least one proline rich domain, at least one tyrosine
phosphorylation site, and has an amino acid sequence at least about 50%, 55%,
60%,
62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the amino
acid sequence of SEQ ID N0:2 or 5. In another preferred embodiment, the
protein,
preferably a BAL protein, includes at least one proline rich domain, at least
one tyrosine
phosphorylation site, and plays a role in the pathogenesis of non-Hodgkin's
lymphoma.
In yet another preferred embodiment, the protein, preferably a BAL protein,
includes at
least one proline rich domain, at least one tyrosine phosphorylation site, and
is encoded
by a nucleic acid molecule having 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, or 6.
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In another embodiment, the invention features BAL proteins which are produced
by recombinant DNA techniques. Alternative to recombinant expression, a BAL
protein
or polypeptide can be synthesized chemically using standard peptide synthesis
techniques, based on the amino acid sequence of SEQ ID N0:2 or 5.
In another embodiment, the invention features fragments of the protein having
the amino acid sequence of SEQ ID N0:2 or 5, wherein the fragment comprises at
least
amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ
ID
N0:2 or 5. In another embodiment, the protein, preferably a BAL protein, has
the amino
acid sequence of SEQ ID N0:2 or 5, respectively.
10 In another embodiment, the invention features an isolated protein,
preferably a
BAL protein, which is encoded by a nucleic acid molecule consisting of a
nucleotide
sequence at least about 50%, 55%, 60%, 65%, 70%, 72%, 75%, 80%, 85%, 90%, 95%,
98% or more homologous to a nucleotide sequence of SEQ ID NO:1, 3, 4, or 6, or
a
complement thereof. This invention further features an isolated protein,
preferably a
15 BAL 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, or 6, 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-BAL polypeptide (e.g.,
heterologous
amino acid sequences) to form fusion proteins. The invention further features
antibodies, such as monoclonal or polyclonal antibodies, that specifically
bind proteins
of the invention, preferably BAL proteins. In addition, the BAL 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 BAL nucleic acid molecule, protein or polypeptide in a
biological sample
by contacting the biological sample with an agent capable of detecting a BAL
nucleic
acid molecule, protein or polypeptide such that the presence of a BAL nucleic
acid
molecule, protein or polypeptide is detected in the biological sample.
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In another aspect, the present invention provides a method for detecting the
presence of BAL activity in a biological sample by contacting the biological
sample
with an agent capable of detecting an indicator of BAL activity such that the
presence of
BAL activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating BAL activity
comprising contacting a cell capable of expressing BAL with an agent that
modulates
BAL activity such that BAL activity in the cell is modulated. In one
embodiment, the
agent inhibits BAL activity. In another embodiment, the agent stimulates BAL
activity.
In one embodiment, the agent is an antibody that specifically binds to a BAL
protein. In
another embodiment, the agent modulates expression of BAL by modulating
transcription of a BAL gene or translation of a BAL 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 BAL mRNA or a BAL gene.
In one embodiment, the methods of the present invention are used to treat a
subject having a disorder characterized by aberrant BAL protein or nucleic
acid
expression or activity, e.g., non-Hodgkin's lymphoma, by administering an
agent which
is a BAL modulator to the subject. In one embodiment, the BAL modulator is a
BAL
protein. In another embodiment the BAL modulator is a BAL nucleic acid
molecule. In
yet another embodiment, the BAL modulator is a peptide, peptidomimetic, or
other
small molecule. In a preferred embodiment, the disorder characterized by
aberrant BAL
protein or nucleic acid expression is a proliferative disorder, e.g., non-
Hodgkin's
lymphoma.
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 BAI, protein; (ii) mis-
regulation of the
gene; and (iii) aberrant post-translational modification of a BAL protein,
wherein a wild-
type form of the gene encodes a protein with a BAL activity.
In another aspect the invention provides a method for producing or identifying
a
compound that binds to or modulates the activity of a BAL protein, by
providing an
indicator composition comprising a BAL protein having BAL activity, contacting
the
indicator composition with a test compound, and determining the effect of the
test
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compound on BAL activity in the indicator compositian to produce or identify a
compound that modulates the activity of a BAL protein.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
Brief Description of the Drawings
Figure 1 depicts the cDNA sequence and predicted amino acid sequence of
human BAL. The nucleotide sequence corresponds to nucleic acids 1 to 3243 of
SEQ
ID NO:1. The amino acid sequence corresponds to amino acids 1 to 826 of SEQ ID
NO:
2. The coding region without the S' and 3' untranslated regions of the human
BAL gene
is shown in SEQ ID N0:3.
Figure 2 depicts the cDNA sequence and predicted amino acid sequence of
marine BAL. The nucleotide sequence corresponds to nucleic acids 1 to 3024 of
SEQ
ID N0:4. The amino acid sequence corresponds to amino acids 1 to 826 of SEQ ID
NO:
5. The coding region without the 5' and 3' untranslated regions of the marine
BAL gene
is shown in SEQ ID N0:6.
Figure 3 depicts an alignment of the human BAL protein with the marine BAL
protein using the ALIGN program (version 2.0), a P.AM120 weight residue table,
a gap
length penalty of 12 and a gap penalty of 2.
Figure 4 depicts an alignment of the human BAL nucleic acid molecule with the
marine BAL nucleic acid molecule using the ALIGN program (version 2.0), a
PAM120
weight residue table, a gap length penalty of 16 and a gap penalty of 4.
Figure S depicts a northern blot analysis of total RNA from S DLB-CL cell
lines,
4 "high-risk" and 5 "low-risk" primary tumors, and 2 pairs of normal B-
splenocytes,
with or without Ig-activation. A band of ~3.2kb (arrow), corresponding to the
BAL
message, is detected at high levels in only one of the cell lines (DHL-7), in
the "high-
risk" tumors and Ig-activated splenocytes. BAL transcripts are less abundant
in the
"low-risk" primary tumors. Likewise, BAL is expressed at lower levels in the
non-
activated B-cells. The ~i-actin blot demonstrates that loading does not
account for the
differences in BAL expression.
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Figure 6 depicts a northern blot comparing BAL transcripts in a tumor derived
from a DLB-CL cell line grown in SCID mouse and the parental suspension cells.
BAL
is expressed at significantly higher levels in the tumor derived RNA than in
the parental
cell line. The (3-actin blot demonstrates that loading does not account for
the observed
differences.
Figure 7 depicts a diagrammatic representation of BAL cDNA and protein
structure. Two alternatively spliced BAL cDNAs of 3243 by (BALL, long) and
3138 by
(BALS, short) encode previously uncharacterized 854 as and 819 as proteins.
The
alternatively spliced region and the start and stop codons are indicated in
BAL eDNA.
The region of partial homology to the non-histone region of histone macroH2A
and the
potential CRK/CRK-1 binding sites (YHVL, YSVP) and proline-rich region are
also
highlighted.
Figure 8 depicts a northern blot analysis of BAL transcripts in multiple human
tissues. BAL transcripts are most abundant in analyzed lymphoid/hematopoietic
tissues
(spleen, lymph node, fetal liver and peripheral blood) and several non-
hematopoietic
organs (heart, skeletal muscle and colon).
Figure 9 depicts FISH analysis of a normal human metaphase with genomic
PAC clones encompassing the BAL locus. The top panel shows the Giemsa-banded
metaphase. In the FISH panel (lower), the green signals represent the BAL-PAC
clones
hybridized to the two normal chromosome 3q21.
Figure 10 depicts sensitivity of the BAL semi-quantitative duplex RT-PCR. The
abundance of BAL in a given sample was determined by comparing the intensity
of co-
amplified BAL and control (ABL) PCR products by scanning densitometry. The
sensitivity of the duplex PCR was determined by mixing fixed amounts of cDNAs
from
a BAL negative and a BAL positive DLB-CL cell line to mimic BAL losses of 10%-
100% (lower panel). When the ratio of BAL/ABL signals was plotted against the
percentage of BAL lost in a given sample, the data yield a straight line and a
r2 value of
0.967 (top panel).
Figure 11 depicts the results from the duplex. PCR experiments.
Figure 12 is a graph depicting BAL expression as determined by the duplex PCR
experiments (quantified with scanning densitometry).
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Figure 13 depicts the results from a western blot analysis of aggressive
lymphoma transfectants expressing pEGFP vector only or pEGFP-BALS.
Figure 14 is a graph depicting the cell migration of pEGFP vector only or
pEGFP-BALS transfectants.
Detailed Description of the Invention
The present invention is based, at least in part, on the discovery of novel
molecules, referred to herein as BAL nucleic acid and protein molecules which
are
differentially expressed in malignancies such as lymphoma, e.g., non-Hodgkin's
lymphoma. The newly identified BAL nucleic acid and protein molecules can be
used
to identify cells exhibiting or predisposed to a malignancy such as lymphoma,
e.g., non-
Hodgkin's lymphoma, thereby diagnosing subjects having, or prone to developing
such
disorders.
As used herein, a "malignancy" includes a cancerous uncontrolled growth of
cells in an area of the body. Malignant cancers are typically classified by
their
microscopic appearance and the type of tissue from which they arise. Examples
of
malignancies include carcinomas, sarcomas, myelomas, chondrosarcomas,
adenosarcomas, angiosarcomas, neuroblastomas, gliomas, medulloblastomas,
erythroleukemias, and myelogenous leukemias.
As used herein, a "lymphoma" includes a malignant neoplastic disorder of
lymphoreticular tissue which produces a distinct tumor mass. Lymphomas include
tumors derived from the lymphoid lineage. Lymphomas usually arise in lymph
nodes,
the spleen, or other areas rich in lymphoid tissue. Lymphomas are typically
subclassified as Hodgkin's disease and Non-Hodgkin's lymphomas, e.g.,
Burkitt's
lymphoma, large-cell lymphoma, and follicular lymphoma.
As used herein, "differential expression" or differentially expressed"
includes
both quantitative as well as qualitative differences in the temporal and/or
cellular
expression pattern of a gene, e.g., the BAL gene, among, for example, normal
cells and
cells from patients with "high risk" fatal DLB-CL disease or "low risk" cured
DLB-CL.
Genes which are differentially expressed can be used as part of a prognostic
or
diagnostic marker for the evaluation of subjects at risk for developing a
malignancy such
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as a lymphoma, e.g., non-Hodgkin's lymphoma. Depending on the expression level
of
the gene, the progression state or the aggressiveness of the disorder can be
evaluated.
Methods for detecting the differential expression of a gene are described
herein.
The BAL molecules comprise a family of molecules having certain conserved
structural and functional features. 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 or motif and having
sufficient amino acid or nucleotide sequence homology as defined herein. Such
family
members can be naturally or non-naturally occurring and can be from either the
same or
different species. For example, a family can contain a first protein of human
origin, as
well as other, distinct proteins of human origin or alternatively, can contain
homologues
of non-human origin. Members of a family may also have common functional
characteristics.
For example, the family of BAL proteins comprise at least one "proline rich
domain." As used herein, the term "proline rich domain" includes an amino acid
sequence of about 4-6 amino acid residues in length having the general
sequence X-Pro-
X-X-Pro-X (where X can be any amino acid). Proline rich domains are usually
located
in a helical structure and bind through hydrophobic interactions to SH3
domains. SH3
domains recognize proline rich domains in both forward and reverse
orientations.
Proline rich domains are described in, for example, Sattler M. et al.,
Leukemia ( 1998)
12:637-644, the contents of which are incorporated herein by reference. BAL
proteins
of the invention preferably include at least one proline rich domain, but may
contain two
or more. Amino acid residues 781-786 of the human BAL and amino acid residues
748-
753 of the marine BAL comprise proline rich domains.
In another embodiment, a BAL protein of the present invention is identified
based on the presence of at least one "tyrosine phosphorylation site" in the
protein or
corresponding nucleic acid molecule. As used herein, the term "tyrosine
phosphorylation site" includes an amino acid sequence of about 4 amino acid
residues in
length having the general sequence Tyr-X-X-X (where X can be any amino acid).
The
tyrosine in this domain is phosphorylated in response to a cellular stimulus,
for example,
in response to a hematopoietic growth factor (e.g., thrombopoietin,
erythropoietin, or
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steel factor) stimulation. Tyrosine phosphorylation of cellular proteins plays
a major
role in cell signaling, e.g., hematopoietic cell signaling. Tyrosine
phosphorylation sites
are described in, for example, Sattler M. et al., Leukemia (1998) 12:637-644,
the
contents of which are incorporated herein by reference. BAL proteins of the
invention
include at least one or two tyrosine phosphorylation sites, but may contain
three or more.
Amino acid residues 392-395 and 495-498 of the human BAL comprise tyrosine
phosphorylation sites.
In another embodiment, a BAL protein of the present invention is identified
based on the presence of at least one "rod domain" in the protein or
corresponding
nucleic acid molecule. As used herein, the term "rod domain" includes an a-
helical,
filament forming structure that is made up of smaller repeating structures.
The smallest
repeating structure may contain seven amino acids in which small, generally
hydrophobic amino acids are typically found in the first and fourth positions
of the
repeat. The seven amino acids form two turns of an a-helix and the first and
fourth
positions fall in the hydrophobic interior of the a-helix. Rod domains in
alpha and beta
cardiac myosin are described in, for example, Warrick et al. (1987) Annual
Rev. Cell
Biol. 3:379-421.
In another embodiment, a BAL protein of the present invention is identified
based on the presence of at least one "a-helical region" in the protein or
corresponding
nucleic acid molecule that is homologous to the a-helical region of moesin,
the most
abundant ezrin-radixin-moesin (ERM) protein in lymphocytes. As used herein,
the term
"a-helical region" includes an amino acid sequence of about 10 to about 20
amino acids
in length that forms an a-helix. The a-helical region of ezrin-radixin-moesin
(ERM)
proteins is described in, for example, Bretscher (1999) Current Biology
8(12):721-4.
Due to their homology to protein families such as myosin heavy chain and
cytoskeleton linkers ezrin-radixin-moesin, the human BAL molecules may also be
involved in cellular functions such as cell migration, motility, and shape, as
well as in
cell/cell and cell/extra-cellular matrix interactions through adhesion
molecules.
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Isolated proteins of the present invention, preferably BAL proteins, have an
amino
acid sequence sufficiently homologous to the amino acid sequence of SEQ ID
N0:2 or 5 or
are encoded by a nucleotide sequence sufficiently homologous to SEQ ID NO:1,
3, 4, or 6.
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 or motifs
and/or a
common functional activity. For example, amino acid or nucleotide sequences
which share
common structural domains have at least 30%, 40%, or 50% homology, preferably
60%
homology, more preferably 70%-80%, and even more preferably 90-95% homology
across
the amino acid sequences of the domains and contain at least one and
preferably two
structural domains or motifs, are defined herein as sufficiently homologous.
Furthermore,
amino acid or nucleotide sequences which share at least 30%, 40%, or 50%,
preferably
60%, more preferably 70-80%, or 90-95% homology and share a common functional
activity are defined herein as sufficiently homologous.
As used interchangeably herein, "BAL activity", "biological activity of BAL"
or
"functional activity of BAL", refers to an activity exerted by a BAL protein,
polypeptide or
nucleic acid molecule on a BAL responsive cell or on a BAL protein substrate,
as determined
in vivo, ex vivo, or in vitro, according to standard techniques. In one
embodiment, a BAL
activity is a direct activity, such as an association with a BAL-target
molecule. As used
herein, a "target molecule" or "binding partner" is a molecule with which a
BAL protein
binds or interacts in nature, such that BAL-mediated function is achieved. A
BAL target
molecule can be a non-BAL molecule or a BAL protein or polypeptide of the
present
invention. In an exemplary embodiment, a BAL target molecule is a BAL ligand.
Alternatively, a BAL activity is an indirect activity, such as a cellular
signaling activity
mediated by interaction of the BAL protein with a BAL ligand. BAL activities
include
modulation of cellular adhesion and modulation of the aggressiveness or
severity of a
malignancy such as DLB-CL. BAL activities are described herein.
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Accordingly, another embodiment of the invention features isolated BAL
proteins and polypeptides having a BAL activity. Preferred proteins are BAL
proteins
having at least one tyrosine phosphorylation site and, preferably, a BAL
activity. Other
preferred proteins are BAL proteins having at least one proline rich domain
and,
preferably, a BAL activity. Other preferred proteins are BAL proteins having
at least
one tyrosine phosphorylation site and/or at least one proline rich domain, and
are,
preferably, encoded by a nucleic acid molecule having 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, or 6.
The nucleotide sequence of the isolated human BAL cDNA and the predicted
amino acid sequence of the human BAL polypeptide are shown in Figure 1 and in
SEQ
ID NOs:I and 2, respectively.
The human BAL gene, which is approximately 3244 nucleotides in length,
encodes a protein having a molecular weight of approximately 95 kD and which
is
approximately 826 amino acid residues in length. On a multiple tissue northern
blot,
BAL transcripts were most abundant in lymphoid organs (spleen, lymph node,
fetal
liver, and peripheral blood) and several additional non-hematopoietic organs
(heart,
skeletal muscle and colon).
The nucleotide sequence of the isolated murine BAL cDNA and the predicted
amino acid sequence of the murine polypeptide are shown in Figure 2 and in SEQ
ID
NOs:4 and 5, respectively.
The murine BAL gene, which is approximately 3024 nucleotides in length,
encodes a protein having a molecular weight of approximately 95 kD and which
is
approximately 826 amino acid residues in length.
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 BAL proteins or biologically active portions thereof, as well as
nucleic acid
fragments sufficient for use as hybridization probes to identify BAL-encoding
nucleic
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acid molecules (e.g., BAL mRNA) and fragments for use as PCR primers for the
amplification or mutation of BAL 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 BAL 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, or 6 or a portion
thereof, can be
isolated using standard molecular biology techniques and the sequence
information
provided herein. Using all or portion of the nucleic acid sequence of SEQ ID
NO:I, 3,
4, or 6, as a hybridization probe, BAL 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).
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Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID
NO:1, 3, 4, or 6 can be isolated by the polymerase chain reaction (PCR) using
synthetic
oligonucleotide primers designed based upon the sequence of SEQ ID NO:1, 3, 4,
or 6.
A nucleic acid 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 BAL 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:1. The sequence of SEQ ID
NO:1 corresponds to the human BAL cDNA. This cDNA comprises sequences
encoding the human BAL protein (i. e. , "the coding region", from nucleotides
229-2790),
as well as S' untranslated sequences (nucleotides 1-228) and 3' untranslated
sequences
(nucleotides 2791-3243). Alternatively, the nucleic acid molecule can comprise
only the
coding region of SEQ ID NO:1 (e.g., nucleotides 229-2790, corresponding to SEQ
ID
N0:3).
In a 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 BAL cDNA. This cDNA comprises sequences
encoding the marine BAL protein (i.e., "the coding region", from nucleotides
171-2648),
as well as 5' untranslated sequences (nucleotides 1-170) and 3' untranslated
sequences
(nucleotides 2649-3024). Alternatively, the nucleic acid molecule can comprise
only the
coding region of SEQ ID N0:4 (e.g., nucleotides 171-2648, corresponding to SEQ
ID
N0:6).
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, or 6, or a portion of any of these
nucleotide
sequences. A nucleic acid molecule which is complementary to the nucleotide
sequence
shown in SEQ ID NO:1, 3, 4, or 6, is one which is sufficiently complementary
to the
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nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, such that it can
hybridize to the
nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6.
In still another preferred embodiment, an isolated nucleic acid molecule of
the
present invention comprises a nucleotide sequence which is at least about 50%,
55%,
60%, 65%, 70%, 72%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the
entire length of the nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6 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, or 6, for example, a
fragment which
can be used as a probe or primer or a fragment encoding a portion of a BAL
protein, e.g.,
a biologically active portion of a BAL protein. The nucleotide sequence
determined
from the cloning of the BAL gene allows for the generation of probes and
primers
designed for use in identifying and/or cloning other BAL family members, as
well as
BAL homologues from other species. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide typically
comprises a region
of nucleotide sequence that hybridizes under stringent conditions to at least
about 12 or
15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,
60, 65, or 75
consecutive nucleotides of a sense sequence of SEQ ID NO:1, 3, 4, or 6, of an
anti-sense
sequence of SEQ ID NO:1, 3, 4, or 6, or of a naturally occurring allelic
variant or
mutant of SEQ ID NO:1, 3, 4, or 6. In an exemplary embodiment, a nucleic acid
molecule of the present invention comprises a nucleotide sequence which is
greater than
300-350, 350-400, 400-450, 452, 452-500, 500-550, 550-600, 607, 607-650, 650-
700,
700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, or more nucleotides in
length
and hybridizes under stringent hybridization conditions to a nucleic acid
molecule of
SEQ ID NO:1, 3, 4, or 6.
Probes based on the BAL nucleotide sequences can be used to detect transcripts
or genomic sequences encoding the same or homologous proteins. In preferred
embodiments, the probe further comprises a label 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 BAL protein, such as by measuring a level of a BAL
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encoding nucleic acid in a sample of cells from a subject e.g., detecting BAL
mRNA
levels or determining whether a genomic BAL gene has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion of a BAL
protein" can be prepared by isolating a portion of the nucleotide sequence of
SEQ ID
NO:1, 3, 4, or 6, which encodes a polypeptide having a BAL biological activity
(the
biological activities of the BAL proteins are described herein), expressing
the encoded
portion of the BAL protein (e.g., by recombinant expression in vitro) and
assessing the
activity of the encoded portion of the BAL protein.
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, due to degeneracy of the
genetic
code and thus encode the same BAL proteins as those encoded by the nucleotide
sequence shown in SEQ ID NO:1, 3, 4, or 6. 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 or 5.
In addition to the BAL nucleotide sequences shown in SEQ ID NO:1, 3, 4, or 6,
it will be appreciated by those skilled in the art that DNA sequence
polymorphisms that
lead to changes in the amino acid sequences of the BAL proteins may exist
within a
population (e.g., the human population). Such genetic polymorphism in the BAL
genes
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 BAL protein, preferably a
mammalian
BAL protein, and can further include non-coding regulatory sequences, and
introns.
Allelic variants of human BAL include both functional and non-functional BAL
proteins. Functional allelic variants are naturally occurring amino acid
sequence
variants of the human BAL protein that maintain the ability to bind a BAL
ligand and/or
modulate the occurrence or severity of a malignancy such as a lymphoma, e.g.,
non-
Hodgkin's lymphoma. Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID N0:2 or 5 or
substitution, deletion or insertion of non-critical residues in non-critical
regions of the
protein.
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Non-functional allelic variants are naturally occurring amino acid sequence
variants of the human BAL protein that do not have the ability to either bind
a BAL
ligand and/or modulate occurrence or severity of a malignancy such as a
lymphoma,
e.g., non-Hodgkin's lymphoma. Non-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 or 5 or a substitution, insertion or
deletion in
critical residues or critical regions.
The present invention further provides non-human orthologues of the human
BAL protein. Orthologues of the human BAL protein are proteins that are
isolated from
non-human organisms and possess the same BAL ligand binding and/or modulation
of
the occurrence or severity of a malignancy such as a lymphoma, e.g., non-
Hodgkin's
lymphoma capabilities of the human BAL protein. Orthologues of the human BAL
protein can readily be identified as comprising an amino acid sequence that is
substantially homologous to SEQ ID N0:2 or 5.
Moreover, nucleic acid molecules encoding other BAL family members and,
thus, which have a nucleotide sequence which differs from the BAL sequences of
SEQ
ID NO:1, 3, 4, or 6 are intended to be within the scope of the invention. For
example,
another BAL cDNA can be identified based on the nucleotide sequence of human
BAL.
Moreover, nucleic acid molecules encoding BAL proteins from different species,
and
which, thus, have a nucleotide sequence which differs from the BAL sequences
of SEQ
ID NO:1, 3, 4, or 6 are intended to be within the scope of the invention. For
example, a
monkey BAL cDNA can be identified based on the nucleotide sequence of a human
or
marine BAL.
Nucleic acid molecules corresponding to natural allelic variants and
homologues
of the BAL cDNAs of the invention can be isolated based on their homology to
the BAL
nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion
thereof, as
a hybridization probe according to standard hybridization techniques. under
stringent
hybridization conditions. Nucleic acid molecules corresponding to natural
allelic
variants and homologues of the BAL cDNAs of the invention can further be
isolated by
mapping to the same chromosome or locus as the BAL gene.
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Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 15, 20, 25, 30 or more nucleotides in length and
hybridizes under
stringent conditions to the nucleic acid molecule comprising the nucleotide
sequence of
SEQ ID NO:1, 3, 4, or 6. In other embodiment, the nucleic acid is at least 30,
50, 100,
150, 200, 250, 300, 350, 400, 450, 452, 500, 550, 607, 600, 650, 700, 750,
800, 850,
900, or 950 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 70%, more preferably at least about 80%, even more preferably at least
about 85%
or 90% 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, or 6 corresponds to a naturally-occurring nucleic acid molecule. As used
herein, a
"naturally-occurring" 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 BAL sequences that
may
exist in the population, the skilled artisan will further appreciate that
changes can be
introduced by mutation into the nucleotide sequences of SEQ ID NO:1, 3, 4, or
6 thereby
leading to changes in the amino acid sequence of the encoded BAL proteins,
without
altering the functional ability of the BAL proteins. 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, or 6. A "non-essential" amino acid residue
is a
residue that can be altered from the wild-type sequence of BAL (e.g., the
sequence of
SEQ ID N0:2 or 5) without altering the biological activity, whereas an
"essential" amino
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acid residue is required for biological activity. For example, amino acid
residues that are
conserved among the BAL proteins of the present invention, are predicted to be
particularly unamenable to alteration. Furthermore, additional amino acid
residues that
are conserved between the BAL proteins of the present invention and other
members of
the BAL family are not likely to be amenable to alteration.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding BAL proteins that contain changes in amino acid residues that are not
essential
for activity. Such BAL proteins differ in amino acid sequence from SEQ ID N0:2
or 5,
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 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%,
85%,
90%, 95%, 98% or more homologous to SEQ ID N0:2 or 5.
An isolated nucleic acid molecule encoding a BAL protein homologous to the
protein of SEQ ID N0:2 or 5 can be created by introducing one or more
nucleotide
substitutions, additions or deletions into the nucleotide sequence of SEQ ID
NO:1, 3, 4,
or 6, 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:1, 3, 4,
or 6, 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 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 (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a
predicted nonessential amino acid residue in a BAL protein 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 BAL
coding
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sequence, such as by saturation mutagenesis, and the resultant mutants can be
screened
for BAL biological activity to identify mutants that retain activity.
Following
mutagenesis of SEQ ID NO:1, 3, 4, or 6, the encoded protein can be expressed
recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant BAL protein can be assayed for the ability
to
(1) interact with a non-BAL protein molecule, e.g., CRK, CRK-L, SHP-2, PBK, or
ZAP70; (2) activate a BAL-dependent signal transduction pathway; or (3)
modulate the
occurrence or severity of a lymphoma, e.g., non Hodgkin's lymphoma.
In addition to the nucleic acid molecules encoding BAL 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 BAL 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
BAL. 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 human and murine BAL corresponds to SEQ ID NO:3 and 6, respectively). In
another
embodiment, the antisense nucleic acid molecule is antisense to a "noncoding
region" of
the coding strand of a nucleotide sequence encoding BAL. The term "noncoding
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 BAL disclosed herein (e.g., SEQ ID
N0:3 and 6), 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 BAL mRNA, but more preferably is
an
oligonucleotide which is antisense to only a portion of the coding or
noncoding region of
BAL mRNA. For example, the antisense oligonucleotide can be complementary to
the
region surrounding the translation start site of BAL mRNA. An antisense
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oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45
or SO
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-galactosylqueosine, inosine, N6-isopentenyladenine, 1-
methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-
1 S methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine, 5-
methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-
mannosylqueosine, 5'-methoxycarboxymethyluracil, S-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-S-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 BAL 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
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specific interactions in the major groove of the double helix. An example of a
route of
administration of antisense nucleic acid molecules of the 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 antisense 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 (3-units, the strands run parallel to each other (Gaultier et al. (1987)
Nucleic Acids.
Res. 15:6625-6641 ). The antisense nucleic acid molecule can also comprise a
2'-0
methylribonucleotide (moue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a
chimeric RNA-DNA analogue (moue et al. (1987) FEBS Lett. 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
eatalytically cleave BAL mRNA transcripts to thereby inhibit translation of
BAL
mRNA. A ribozyme having specificity for a BAL-encoding nucleic acid can be
designed based upon the nucleotide sequence of a BAL cDNA disclosed herein
(i.e.,
SEQ ID NO:1, 3, 4, or 6). 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 BAL-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,
BAL mRNA can be used to select a catalytic RNA having a specific ribonuclease
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activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W.
(1993)
Science 261:1411-1418.
Alternatively, BAL gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the BAL (e.g., the BAL
promoter
and/or enhancers) to form triple helical structures that prevent transcription
of the BAL
gene in 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 yet another embodiment, the BAL nucleic acid molecules of the present
invention 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 acid molecules can be
modified to
generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic &
Medicinal
Chemistry 4 (1): S-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.
Proc. Natl.
Acad. Sci. 93: 14670-675.
PNAs of BAL nucleic acid molecules can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or antigene agents
for
sequence-specific modulation of gene expression by, for example, inducing
transcription
or translation arrest or inhibiting replication. PNAs of BAL nucleic acid
molecules can
also be used in the analysis of single base pair mutations in a gene, (e.g.,
by PNA-
directed PCR clamping); as 'artificial restriction enzymes' when used in
combination
with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes
or
primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra;
Perry-
O'Keefe supra).
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In another embodiment, PNAs of BAL 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 BAL nucleic
acid
molecules 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 Res. 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-
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. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA
84:648-652;
PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT
Publication
No. W089/10134). In addition, oligonucleotides can be modified with
hybridization-
triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 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, or hybridization-triggered
cleavage agent).
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II. Isolated BAL Proteins and Anti-BAL Antibodies
One aspect of the invention pertains to isolated BAL proteins, and
biologically
active portions thereof, as well as polypeptide fragments suitable for use as
immunogens
to raise anti-BAL antibodies. In one embodiment, native BAL proteins can be
isolated
from cells or tissue sources by an appropriate purification scheme using
standard protein
purification techniques. In another embodiment, BAL proteins are produced by
recombinant DNA techniques. Alternative to recombinant expression, a BAL
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 BAL 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 BAL 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 BAL protein having less than about
30% (by
dry weight) of non-BAL protein (also referred to herein as a "contaminating
protein"),
more preferably less than about 20% of non-BAL protein, still more preferably
less than
about 10% of non-BAL protein, and most preferably less than about S% non-BAL
protein. When the BAL 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 BAL protein in which the protein is separated from
chemical
precursors 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 BAL protein having less than about 30% (by dry
weight) of
chemical precursors or non-BAL chemicals, more preferably less than about 20%
chemical precursors or non-BAL chemicals, still more preferably less than
about I O%
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chemical precursors or non-BAL chemicals, and most preferably less than about
5%
chemical precursors or non-BAL chemicals.
As used herein, a "biologically active portion" of a BAL protein includes a
fragment of a BAL protein which participates in an interaction between a BAL
molecule
and a non-BAL molecule. Biologically active portions of a BAL protein include
peptides comprising amino acid sequences sufficiently homologous to or derived
from
the amino acid sequence of the BAL protein, e.g., the amino acid sequence
shown in
SEQ ID N0:2 or 5, which include less amino acids than the full length BAL
proteins,
and exhibit at least one activity of a BAL protein. Typically, biologically
active portions
comprise a domain or motif with at least one activity of the BAL protein,
e.g.,
modulating cellular adhesion. A biologically active portion of a BAL protein
can be a
polypeptide which is, for example, 10, 25, 50, 100, 20U or more amino acids in
length.
Biologically active portions of a BAL protein can be used as targets for
developing
agents which modulate a BAL mediated activity, e.g., the occurrence or
severity of a
lymphoma, e.g., non-Hodgkin's lymphoma.
In one embodiment, a biologically active portian of a BAL protein comprises at
least one proline rich domain and/or at least one tyrosine phosphorylation
site. It is to be
understood that a preferred biologically active portion of a BAL protein of
the present
invention may contain at least one of the above-identified structural domains.
A more
preferred biologically active portion of a BAL 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 BAL
protein.
In a preferred embodiment, the BAL protein has an amino acid sequence shown
in SEQ ID N0:2 or 5. In other embodiments, the BAI. protein is substantially
homologous to SEQ ID N0:2 or 5, and retains the functional activity of the
protein of
SEQ ID N0:2 or 5, 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 BAL protein is a protein which comprises an amino acid
sequence at
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least about 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more
homologous to SEQ ID N0:2 or 5.
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
aligned for comparison purposes is at least 30%, preferably at least 40%, more
preferably at least 50%, 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 BAL amino acid sequence of SEQ ID NO:2 or 5 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://www.gcg.com), using either a Blosum 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
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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 PAM120 weight
residue
table, a gap length penalty of I2 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)
,I.
Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the
NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences
homologous to BAL nucleic acid molecules of the invention. BLAST protein
searches
can be performed with the XBLAST program, score = S0, wordlength = 3 to obtain
amino acid sequences homologous to BAL 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 BAL chimeric or fusion proteins. As used herein, a
BAL "chimeric protein" or "fusion protein" comprises a BAL polypeptide
operatively
linked to a non-BAL polypeptide. An "BAL polypeptide" refers to a polypeptide
having
an amino acid sequence corresponding to BAL, whereas a "non-BAL polypeptide"
refers
to a polypeptide having an amino acid sequence corresponding to a protein
which is not
substantially homologous to the BAL protein, e.g., a protein which is
different from the
BAL protein and which is derived from the same or a different organism. Within
a BAL
fusion protein the BAL polypeptide can correspond to all or a portion of a BAL
protein.
In a preferred embodiment, a BAL fusion protein comprises at least one
biologically
active portion of a BAL protein. In another preferred embodiment, a BAL fusion
protein comprises at least two biologically active portions of a BAL protein.
Within the
fusion protein, the term "operatively linked" is intended to indicate that the
BAL
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polypeptide and the non-BAL polypeptide are fused in-frame to each other. The
non-
BAL polypeptide can be fused to the N-terminus or C-terminus of the BAL
polypeptide.
For example, in one embodiment, the fusion protein is a GST-BAL fusion
protein in which the BAL sequences are fused to the C-terminus of the GST
sequences.
Such fusion proteins can facilitate the purification of recombinant BAL.
In another embodiment, the fusion protein is a BAL protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian
host cells), expression and/or secretion of BAL can be increased through use
of a
heterologous signal sequence.
The BAL fusion proteins of the invention can be incorporated into
pharmaceutical compositions and administered to a subject in vivo. The BAL
fusion
proteins can be used to affect the bioavailability of a BAL substrate. Use of
BAL fusion
proteins may be useful therapeutically for the treatment of disorders caused
by, for
example, (i) aberrant modification or mutation of a gene encoding a BAL
protein; (ii)
1 S mis-regulation of the BAL gene; and (iii) aberrant post-translational
modification of a
BAL protein.
Moreover, the BAL-fusion proteins of the invention can be used as immunogens
to produce anti-BAL antibodies in a subject, to purify BAL ligands and in
screening
assays to identify molecules which inhibit the interaction of BAL with a BAL
substrate.
Preferably, a BAL 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 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
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et al. John Wiley & Sons: 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). An
BAL-
encoding nucleic acid can be cloned into such an expression vector such that
the fusion
moiety is linked in-frame to the BAL protein.
The present invention also pertains to variants of the BAL proteins which
function as either BAL agonists {mimetics) or as BAL antagonists. Variants of
the BAL
proteins can be generated by mutagenesis, e.g., discrete point mutation or
truncation of a
BAL protein. An agonist of the BAL proteins can retain substantially the same,
or a
subset, of the biological activities of the naturally occurring form of a BAL
protein. An
antagonist of a BAL protein can inhibit one or more of the activities of the
naturally
occurring form of the BAL protein by, for example, competitively modulating a
BAL-
mediated activity of a BAL protein. 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 BAL protein.
In one embodiment, variants of a BAL protein which function as either BAL
agonists (mimetics) or as BAL antagonists can be identified by screening
combinatorial
libraries of mutants, e.g., truncation mutants, of a BAL protein for BAL
protein agonist
or antagonist activity. In one embodiment, a variegated library of BAL
variants is
generated by combinatorial mutagenesis at the nucleic acid level and is
encoded by a
variegated gene library. A variegated library of BAL variants can be produced
by, for
example, enzymatically ligating a mixture of synthetic oligonucleotides into
gene
sequences such that a degenerate set of potential BAL sequences is expressible
as
individual polypeptides, or alternatively, as a set of larger fusion proteins
(e.g., for phage
display) containing the set of BAL sequences therein. There are a variety of
methods
which can be used to produce libraries of potential BAL variants from a
degenerate
oligonucleotide sequence. Chemical synthesis of a 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
BAL
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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 a BAL protein coding sequence can be
used
to generate a variegated population of BAL fragments for screening and
subsequent
selection of variants of a BAL protein. In one embodiment, a library of coding
sequence
fragments can be generated by treating a double stranded PCR fragment of a BAL
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 stranded DNA which can include sense/antisense pairs from different
nicked
products, removing single stranded portions from reformed duplexes by
treatment with
S 1 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 BAL 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 BAL
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 BAL variants (Arkin and Yourvan (1992) Proc.
Natl. Acad.
Sci. USA 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
BAL library. For example, a library of expression vectors can be transfected
into a cell
line which ordinarily responds to a particular ligand in a BAL-dependent
manner. The
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transfected cells are then contacted with the ligand and the effect of
expression of the
mutant on signaling by the ligand can be detected, e.g., by measuring cell
survival or the
activity of a BAL-regulated transcription factor. Plasmid DNA can then be
recovered
from the cells which score for inhibition, or alternatively, potentiation of
signaling by
the ligand, and the individual clones further characterized.
An isolated BAL protein, or a portion or fragment thereof, can be used as an
immunogen to generate antibodies that bind BAL using standard techniques for
polyclonal and monoclonal antibody preparation. A full-length BAL protein can
be
used or, alternatively, the invention provides antigenic peptide fragments of
BAL for use
as immunogens. The antigenic peptide of BAL comprises at least 8 amino acid
residues
of the amino acid sequence shown in SEQ ID N0:2 or S and encompasses an
epitope of
BAL such that an antibody raised against the peptide forms a specific immune
complex
with BAL. Preferably, the antigenic peptide comprises at least 10 amino acid
residues,
more preferably at least 1 S amino acid residues, even more preferably at
least 20 amino
1 S acid residues, and most preferably at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of BAL
that
are located on the surface of the protein, e.g., hydrophilic regions, as well
as regions
with high antigenicity.
A BAL 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, recombinantly
expressed BAL protein or a chemically synthesized BAL 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
immunogenic BAL preparation induces a polyclonal anti-BAL antibody response.
Accordingly, another aspect of the invention pertains to anti-BAL 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 BAL. Examples of immunologically active portions of immunoglobulin
molecules include Flab) and F(ab')2 fragments which can be generated by
treating the
".
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antibody with an enzyme such as pepsin. The invention provides polyclonal and
monoclonal antibodies that bind BAL. 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
S particular epitope of BAL. A monoclonal antibody composition thus typically
displays
a single binding affinity for a particular BAL protein with which it
immunoreacts.
Polyclonal anti-BAL antibodies can be prepared as described above by
immunizing a suitable subject with a BAL immunogen. The anti-BAL 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 BAL. If
desired,
the antibody molecules directed against BAL 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-BAL 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)
Proc.
Natl. Acad. Sci. USA 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 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 BAL 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 BAL.
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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-
BAL
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, murine 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
lines are mouse myeloma cell lines that are sensitive to 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/O-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 unproductively 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 BAL, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal anti-BAL antibody can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody phage
display
library) with BAL to thereby isolate immunoglobulin library members that bind
BAL.
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 SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents 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
92/18619;
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Dower et al. PCT International Publication No. WO 91/I7271; 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) BiolTechnology 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) Proc. Natl.
Acad.
Sci. USA 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 )
Proc.
Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-
554.
Additionally, recombinant anti-BAL 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 al. 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) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu
et al.
(1987) J. Immunol. 139:3521-3526; Sun et al. (1987) 1'roc. Natl. Acad. Sci.
USA
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) BioTechniques
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.
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An anti-BAL antibody (e.g., monoclonal antibody) can be used to isolate BAL
by standard techniques, such as affinity chromatography or
immunoprecipitation. An
anti-BAL antibody can facilitate the purification of natural BAL from cells
and of
recombinantly produced BAL expressed in host cells. Moreover, an anti-BAL
antibody
can be used to detect BAL protein (e.g., in a cellular lysate or cell
supernatant) in order
to evaluate the abundance and pattern of expression of the BAL protein. Anti-
BAL
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, -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 luminol; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin, and examples of suitable radioactive
material include
125I~ 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 a BAL protein (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 ligated. 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
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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 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 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 Enzymology 185,
Academic Press, San Diego, CA ( 1990). Regulatory sequences include those
which
direct constitutive expression of a nucleotide sequence in many types of host
cells and
those which direct expression of the nucleotide sequence only in certain host
cells (e.g.,
tissue-specific regulatory sequences). It will 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, and
the like. 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
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described herein (e.g., BAL proteins, mutant forms of BAL proteins, fusion
proteins,
and the like).
The recombinant expression vectors of the invention can be designed for
expression of BAL proteins in prokaryotic or eukaryotic cells. For example,
BAL
proteins 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 promoters 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;
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 and
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 Biolabs, 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.
Purified fusion proteins can be utilized in BAL activity assays, (e.g., direct
assays or competitive assays described in detail below), or to generate
antibodies
specific for BAL proteins, for example. In a preferred embodiment, a BAL
fusion
protein expressed in a retroviral expression vector of the present invention
can be
utilized to infect bone marrow cells which are subsequently transplanted into
irradiated
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recipients. The pathology of the subject recipient is then examined after
sufficient 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 1 ld (Studier et al., Gene
Expression Technology: Methods in Enzymology 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 l 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 HMS 174(DE3) from a
resident
prophage harboring a T7 gnl gene under the transcriptional control of the
lacUV 5
promoter.
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 (Wads 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 BAL 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, BAL proteins can be expressed in insect cells using baculovirus
expression vectors. Baculovirus vectors available for 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~Summers (1989) Virology 170:31-
39).
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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) EMBO J. 6:187-195). When used in mammalian cells, the
S 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-
1 S 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
Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983)
Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad.
Sci. USA
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;1.
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
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RNA molecule which is antisense to BAL mRNA. Regulatory sequences operatively
linked to a nucleic acid cloned in the antisense orientation can be chosen
which direct
the continuous expression of the antisense 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 BAL
nucleic
acid molecule of the invention is introduced, e.g., a BAL nucleic acid
molecule within a
recombinant expression vector or a BAL nucleic acid molecule containing
sequences
which allow it to homologously recombine into a specific site of the host
cell's genome.
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 team as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, a BAL
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.
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
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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 a BAL protein
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) a BAL protein. Accordingly,
the invention
further provides methods for producing a BAL 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 a BAL protein
has been
introduced) in a suitable medium such that a BAL protein is produced. In
another
embodiment, the method further comprises isolating a BAL protein from the
medium or
the host cell.
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized
oocyte or an embryonic stem cell into which BAL-coding sequences have been
introduced. Such host cells can then be used to create non-human transgenic
animals in
which exogenous BAL sequences have been introduced into their genome or
homologous recombinant animals in which endogenous BAL sequences have been
altered. Such animals are useful for studying the function and/or activity of
a BAL and
for identifying and/or evaluating modulators of BAL activity. As used herein,
a
"transgenic animal" is a non-human animal, preferably a mammal, more
preferably a
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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, and the like. 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 animal,
preferably
a mammal, more preferably a mouse, in which an endogenous BAL 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 a BAL-
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 BAL cDNA sequence of SEQ ID NO:1 or 4
can be introduced as a transgene into the genome of a non-human animal.
Alternatively,
a nonhuman homologue of a human BAL gene, such as a mouse or rat BAL gene, can
be
used as a transgene. Alternatively, a BAL gene homologue, can be isolated
based on
hybridization to the BAL cDNA sequences of SEQ ID NO:1, 3, 4, or 6 (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 a BAL transgene to direct expression of a BAL 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 a BAL
transgene
in its genome and/or expression of BAL mRNA in tissues or cells of the
animals. A
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transgenic founder animal can then be used to breed additional animals
carrying the
transgene. Moreover, transgenic animals carrying a transgene encoding a BAL
protein
can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at least a portion of a BAL gene into which a deletion, addition or
substitution has been
introduced to thereby alter, e.g., functionally disrupt, the BAL gene. The BAL
gene can
be a human gene (e.g., the cDNA of SEQ ID N0:3), but more preferably, is a non-
human homologue of a human BAL gene (e.g., the cDNA of SEQ ID N0:6). For
example, a mouse BAL gene can be used to construct a homologous recombination
nucleic acid molecule, e.g., a vector, suitable for altering an endogenous BAL
gene in
the mouse genome. In a preferred embodiment, the homologous recombination
nucleic
acid molecule is designed such that, upon homologous recombination, the
endogenous
BAL gene is functionally disrupted (i.e., no longer encodes a functional
protein; also
referred to as a "knock out" vector). Alternatively, the homologous
recombination
nucleic acid molecule can be designed such that, upon homologous
recombination, the
endogenous BAL 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 BAL protein). In the homologous recombination
nucleic
acid molecule, the altered portion of the BAL gene is flanked at its 5' and 3'
ends by
additional nucleic acid sequence of the BAL gene to allow for homologous
recombination to occur between the exogenous BAL gene carried by the
homologous
recombination nucleic acid molecule and an endogenous BAL gene in a cell,
e.g., an
embryonic stem cell. The additional flanking BAL nucleic acid sequence 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
homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and
Capecchi, M. R. (1987) Cell 51:503 for a description of homologous
recombination
vectors). The homologous recombination nucleic acid molecule is introduced
into a cell,
e.g., an embryonic stem cell line (e.g., by electroporation) and cells in
which the
introduced BAL gene has homologously recombined with the endogenous BAL gene
are
selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells can
then injected
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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 nucleic acid molecules, e.g., vectors, or 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 Mouellec et al.; WO 91/01140 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
example of such a system is the crelloxP recombinase system of bacteriophage
P1. For
a description of the crelloxP recombinase system, see, e.g., Lakso et al.
(1992) Proc.
Natl. Acad. Sci. USA 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 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In
brief,
a cell, e.g., a somatic cell, from the transgenic animal can be isolated and
induced 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 enucleated oocyte from an animal of the
same species
from which the quiescent cell is isolated. The recontructed oocyte is then
cultured such
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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 Compositions
The BAL nucleic acid molecules, fragments of BAL proteins, and anti-BAL
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 carrier. 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.
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 parenteral, 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.
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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
liqui.~
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper
fluidit~;
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
1 S 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 fragment of a BAL protein or an anti-BAL antibody) in the
required
amount in an appropriate solvent with one or a combination of 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.
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Oral compositions generally include an inert diluent or an edible carrier.
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 carrier 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 like 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
release formulation, including implants and microencapsulated delivery
systems.
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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. Liposomal suspensions (including liposomes targeted to
infected
cells with monoclonal antibodies to viral antigens) can also be used as
pharmaceutically
acceptable carriers. 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
for 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
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 50% of the population) and the EDSO
(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
LD50/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
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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
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) Proc. Natl. Acad. Sci.
USA 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: a)
screening
assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays,
monitoring
clinical trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and
prophylactic). As described herein, a BAL protein of the invention has one or
more of
the following activities: (1 ) it interacts with a non-BAL protein molecule,
e.g., CRK,
CRK-L, SHP-2, PI3K, or ZAP70; (2) it activates a BAL-dependent signal
transduction
pathway; (3) it modulates the occurrence and severity of a malignancy such as
a
lymphoma, e.g., non-Hodgkin's lymphoma; and (4) it modulates cell migration,
motility,
and shape, as well as cell/cell and cell/extra-cellular matrix interactions,
and, thus, can
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be used to, for example, ( 1 ) modulate the interaction with a non-BAL protein
molecule;
(2) activate a BAL-dependent signal transduction pathway; (3) modulate the
occurrence
and severity of a malignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma;
and
(4) modulate cell migration, motility, and shape, as well as cell/cell and
cell/extra-cellular matrix interactions.
The isolated nucleic acid molecules of the invention can be used, for example,
to
express BAL protein (e.g., via a recombinant expression vector in a host cell
in gene
therapy applications), to detect BAL mRNA (e.g., in a biological sample) or a
genetic
alteration in a BAL gene, and to modulate BAL activity, as described further
below.
The BAL proteins can be used to treat disorders characterized by insufficient
or
excessive production of a BAL substrate or production of BAL inhibitors. In
addition,
the BAL proteins can be used to screen for naturally occurring BAL substrates,
to screen
for drugs or compounds which modulate BAL activity, as well as to treat
disorders
characterized by insufficient or excessive production of BAL protein or
production of
BAL protein forms which have decreased or aberrant activity compared to BAL
wild
type protein (e.g., Non-Hodgkin's lymphoma). Moreover, the anti-BAL antibodies
of
the invention can be used to detect and isolate BAL proteins, regulate the
bioavailability
of BAL proteins, and modulate BAL activity.
A. Screening Assays:
The invention provides a method (also referred to herein as a "screening
assay")
for identifying and/or producing modulators, i.e., candidate or test compounds
or agents
(e.g., peptides, peptidomimetics, small molecules or other drugs) which bind
to BAL
proteins, have a stimulatory or inhibitory effect on, for example, BAL
expression or
BAL activity, or have a stimulatory or inhibitory effect on, for example, the
expression
or activity of a BAL substrate.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which are substrates of a BAL protein or polypeptide or biologically
active
portion thereof. In another embodiment, the invention provides assays for
screening
candidate or test compounds which bind to or modulate the activity of a BAL
protein or
polypeptide or biologically active portion thereof. T'he test compounds of the
present
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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.
Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips
(Fodor
(1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner
USP
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) 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. 87:6378-63 82); (Felici ( 1991 )
.l. Mol. Biol.
222:301-310}; (Ladner supra.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a BAL protein or biologically active portion thereof is contacted
with a test
compound and the ability of the test compound to modulate BAL activity is
determined.
Determining the ability of the test compound to modulate BAL activity can be
accomplished by monitoring, for example, the survival of a cell which
expresses BAL or
the activity of a BAL-regulated transcription factor. The cell, for example,
can be of
mammalian origin, e.g., a peripheral blood cell.
The ability of the test compound to modulate BAL binding to a substrate or to
bind to BAL can also be determined. Determining the ability of the test
compound to
modulate BAL binding to a substrate can be accomplished, for example, by
coupling the
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BAL substrate with a radioisotope or enzymatic label such that binding of the
BAL
substrate to BAL can be determined by detecting the labeled BAL substrate in a
complex. Determining the ability of the test compound to bind BAL can be
accomplished, for example, by coupling the compound with a radioisotope or
enzymatic
label such that binding of the compound to BAL can be determined by detecting
the
labeled BAL compound in a complex. For examplc, compounds (e.g., BAL
substrates)
can be labeled with 125I~ 355 14C~ or 3H, either directly or indirectly, and
the
radioisotope detected by direct counting of radioemmission or by scintillation
counting.
Alternatively, 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.
It is also within the scope of this invention to determine the ability of a
compound (e.g., a BAL substrate) to interact with BAL without the labeling of
any of
the interactants. For example, a microphysiometer can be used to detect the
interaction
of a compound with BAL without the labeling of either the compound or the BAL.
McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument that
measures the rate
at which a cell acidifies its environment using a light-addressable
potentiometric sensor
(LAPS). Changes in this acidification rate can be used as an indicator of the
interaction
between a compound and BAL.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell expressing a BAL target molecule (e.g., a BAL substrate) with a test
compound and
determining the ability of the test compound to modulate (e.g. stimulate or
inhibit) the
activity of the BAL target molecule. Determining the ability of the test
compound to
modulate the activity of a BAL target molecule can be accomplished, for
example, by
determining the ability of the BAL protein to bind to or interact with the BAL
target
molecule.
Determining the ability of the BAL protein or a biologically active fragment
thereof, to bind to or interact with a BAL 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 BAL protein to bind to or interact with a BAL
target
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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,
and the like), detecting catalytic/enzymatic activity of the target an
appropriate substrate,
detecting the induction of a reporter gene (comprising a target-responsive
regulatory
element operatively linked to a nucleic acid encoding a detectable marker,
e.g.,
luciferase), or detecting a target-regulated cellular response.
In yet another embodiment, an assay of the present invention is a cell-free
assay
in which a BAL protein or biologically active portion thereof is contacted
with a test
compound and the ability of the test compound to bind to the BAL protein or
biologically active portion thereof is determined. Preferred biologically
active portions
of the BAL proteins to be used in assays of the present invention include
fragments
which participate in interactions with non-BAL molecules, e.g., fragments with
high
surface probability scores. Binding of the test compound to the BAL protein
can be
determined either directly or indirectly as described above. In a preferred
embodiment,
the assay includes contacting the BAL protein or biologically active portion
thereof with
a known compound which binds BAL to form an assay mixture, contacting the
assay
mixture with a test compound, and determining the ability of the test compound
to
interact with a BAL protein, wherein determining the ability of the test
compound,to
interact with a BAL protein comprises determining the ability of the test
compound to
preferentially bind to BAL or biologically active portion thereof as compared
to the
known compound.
In another embodiment, the assay is a cell-free assay in which a BAL 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
BAL protein
or biologically active portion thereof is determined. Determining the ability
of the test
compound to modulate the activity of a BAL protein can be accomplished, for
example,
by determining the ability of the BAL protein to bind to a BAL target molecule
by one
of the methods described above for determining direct binding. Determining the
ability
of the BAL protein to bind to a BAL target molecule can also be accomplished
using a
technology such as real-time Biomolecular Interaction Analysis (BIA).
Sjolander, S.
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and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995}
Curr.
Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for
studying
biospecific interactions in real time, without labeling any of the
interactants (e.g.,
BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR)
can
be used as an indication of real-time reactions between biological molecules.
In an alternative embodiment, determining the ability of the test compound to
modulate the activity of a BAL protein can be accomplished by determining the
ability
of the BAL protein to further modulate the activity of a downstream effector
of a BAL
target molecule. For example, the activity of the effector molecule on an
appropriate
target can be determined or the binding of the effector to an appropriate
target can be
determined as previously described.
The cell-free assays of the present invention are amenable to use of both
soluble
and/or membrane-bound forms of isolated proteins (e.g., BAL proteins or
biologically
active portions thereof ). In the case of cell-free assays in which a membrane-
bound
form of an isolated protein is used it may be desirable to utilize a
solubilizing agent such
that the membrane-bound form of the isolated protein is maintained in
solution.
Examples of such solubilizing agents include non-ionic detergents such as n-
octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-
methylglucamide,
decanoyl-N-methylglucamide, Triton~ X-100, Triton~~ X-114, Thesit~,
Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-
1-
propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-
propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
In more than one embodiment of the above assay methods of the present
invention, it may be desirable to immobilize either BAL 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 a BAL protein, or interaction of a BAL protein with a target
molecule in
the presence and absence of a candidate compound, can be accomplished in any
vessel
suitable for containing the reactants. Examples of such vessels include
microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein
can be
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provided which adds a domain that allows one or both of the proteins to be
bound to a
matrix. For example, glutathione-S-transferase/ BAL 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 BAL 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 BAL 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 a BAL protein or a BAL
target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated BAL protein or target molecules can be prepared from biotin-NHS
(N-
hydroxy-succinimide) using techniques 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 BAL protein
or target
molecules but which do not interfere with binding of the BAL protein to its
target
molecule can be derivatized to the wells of the plate, and unbound target or
BAL protein
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 BAL protein or
target
molecule, as well as enzyme-linked assays which rely on detecting an enzymatic
activity
associated with the BAL protein or target molecule.
In another embodiment, modulators of BAL expression are produced or
identified in a method wherein a cell is contacted with a candidate compound
and the
expression of BAL mRNA or protein in the cell is determined. The level of
expression
of BAL mRNA or protein in the presence of the candidate compound is compared
to the
level of expression of BAL mRNA or protein in the absence of the candidate
compound.
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The candidate compound can then be produced or identified as a modulator of
BAL
expression based on this comparison. For example, when expression of BAL mRNA
or
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
BAL mRNA or protein expression (i. e. a stimulator of BAL mRNA or protein
expression is produced). Alternatively, when expression of BAL mRNA or protein
is
less (statistically significantly less) in the presence of the candidate
compound than in its
absence, the candidate compound is produced or identified as an inhibitor of
BAL
mRNA or protein expression (i. e. an inhibitor of BAL mRNA or protein
expression is
produced). The level of BAL mRNA or protein expression in the cells can be
determined by methods described herein for detecting BAL mRNA or protein.
In yet another aspect of the invention, the BAL proteins can be used as "bait
proteins" in a two-hybrid assay or 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) Biotechni9ues 14:920-924; Iwabuchi et
al.
(1993) 4ncogene 8:1693-1696; and Brent W094/10300), to identify other
proteins,
which bind to or interact with BAL ("BAL-binding proteins" or "BAL-by") and
are
involved in BAL activity. Such BAL-binding proteins are also likely to be
involved in
the propagation of signals by the BAL proteins or BAL targets as, for example,
downstream elements of a BAL-mediated signaling pathway. Alternatively, such
BAL-
binding proteins are likely to be BAL inhibitors.
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 a
BAL protein 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
BAL-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
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(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 the BAL protein.
This invention further pertains to novel agents identified by the above-
described
screening assays. Accordingly, it is within the scope of this invention to
further use an
agent identified and/or produced as described herein in an appropriate animal
model.
For example, an agent identified and/or produced as described herein (e.g., a
BAL
modulating agent, an antisense BAL nucleic acid molecule, a BAL-specific
antibody, or
a BAL-binding partner) can be used in an animal model to determine the
efficacy,
toxicity, or side effects of treatment with such an agent. Alternatively, an
agent
identified and/or produced as described herein can be used in an animal model
to
determine the mechanism of action of such an agent. Furthermore, this
invention
pertains to uses of novel agents identified and/or produced by the above-
described
screening assays for treatments as described herein.
B. Detection Assays
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: (i) map
their
respective genes on a chromosome; and, thus, locate gene regions associated
with
genetic disease; (ii) identify an individual from a minute biological sample
(tissue
typing); and (iii) aid in forensic identification of a biological sample.
These applications
are described in the subsections below.
1. Chromosome Mapping
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 BAL
nucleotide sequences, described herein, can be used to map the location of the
BAL
genes on a chromosome (further described in Example 1, below). The mapping of
the
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BAL sequences to chromosomes is an important first step in correlating these
sequences
with genes associated with disease.
Briefly, BAL genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the BAL nucleotide sequences. Computer
analysis
of the BAL sequences can be used to predict 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
BAL sequences 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
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 BAL nucleotide
sequences 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 BAL sequence to its chromosome include in situ hybridization
(described
in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-
screening with
labeled flow-sorted chromosomes, and pre-selection by hybridization to
chromosome
specific cDNA libraries.
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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
one step. Chromosome spreads can be made using cells whose division has been
blocked in metaphase by a chemical such as 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 Welch Medical
Library). The
relationship between a gene and a disease, mapped to the same chromosomal
region, can
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 BAL 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
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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.
2. Tissue Typing
The BAL sequences of the present invention can also be used to identify
individuals from minute biological samples. The United States military, 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 BAL nucleotide
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 BAL nucleotide 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
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
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for identification purposes. Because greater numbers of polymorphisms occur in
the
noncoding regions, fewer sequences are necessary to differentiate individuals.
The
noncoding sequences of SEQ ID NO:1 or 4 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 or 6 are used, a more appropriate number of primers for positive
individual
identification would be 500-2,000.
If a panel of reagents from BAL nucleotide 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.
3 Use of Partial BAL 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 mentioned 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:l or
4 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.
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Examples of polynucleotide reagents include the BAL nucleotide sequences or
portions
thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:1 or
4
having a length of at least 20 bases, preferably at least 30 bases.
The BAL nucleotide 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 BAL probes can be used to identify
tissue by
species and/or by organ type.
In a similar fashion, these reagents, e.g., BAL 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).
C. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in
which
diagnostic assays, prognostic assays, and monitoring clinical trials 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 BAL protein and/or nucleic acid expression as well as BAL
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 BAL expression or activity, e.g., a
malignancy such as
a lymphoma, e.g., non-Hodgkin's lymphoma. The invention also provides for
prognostic (or predictive) assays for determining whether an individual is at
risk of
developing a disorder associated with BAL protein, nucleic acid expression or
activity.
For example, mutations in a BAL 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 BAL
protein, nucleic acid expression or activity e.g., a malignancy such as a
lymphoma, e.g.,
non-Hodgkin's lymphoma.
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Another aspect of the invention pertains to monitoring the influence of agents
(e.g., drugs, compounds) on the expression or activity of BAL in clinical
trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of BAL protein or
nucleic acid 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 BAL protein or nucleic acid (e.g., mRNA or genomic DNA) that encodes
BAL
protein such that the presence of BAL protein or nucleic acid is detected in
the
biological sample. A preferred agent for detecting BAL mRNA or genomic DNA is
a
labeled nucleic acid probe capable of hybridizing to BAL mRNA or genomic DNA.
The
nucleic acid probe can be, for example, a full-length BAL nucleic acid, such
as the
nucleic acid of SEQ ID NO:1, 3, 4, or 6, or a portion thereof, such as an
oligonucleotide
of at least 15, 30, 50, 100, 250 or S00 nucleotides in length and sufficient
to specifically
hybridize under stringent conditions to BAL mRNA or genomic DNA. Other
suitable
probes for use in the diagnostic assays of the invention are described herein.
A preferred agent for detecting BAL protein is an antibody capable of binding
to
BAL 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 BAL mRNA, protein, or
genomic DNA in a biological sample in vitro as well as in vivo. For example,
in vitro
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techniques for detection of BAL mRNA include Northern hybridizations and in
situ
hybridizations. In vitro techniques for detection of BAL protein include
enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of BAL genomic DNA
include
Southern hybridizations. Furthermore, in vivo techniques for detection of BAL
protein
include introducing into a subject a labeled anti-BAL 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. Alternatively, 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 serum sample isolated by conventional means from a subject.
In another embodiment, 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 BAL protein, rnRNA, or genomic DNA,
such
that the presence of BAL protein, mRNA or genomic DNA is detected in the
biological
sample, and comparing the presence of BAL protein, mRNA or genomic DNA in the
control sample with the presence of BAL protein, mRNA or genomic DNA in the
test
sample.
The invention also encompasses kits for detecting the presence of BAL in a
biological sample. For example, the kit can comprise a labeled compound or
agent
capable of detecting BAL protein or mRNA in a biological sample; means for
determining the amount of BAL in the sample; and means for comparing the
amount of
BAL 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
BAL protein or nucleic acid.
2. Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant
BAL expression or activity e.g., a malignancy such as a lymphoma, e.g., non-
Hodgkin's
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lymphoma. As used herein, the term "aberrant" includes a BAL expression or
activity
which deviates from the wild type BAL expression or activity. Aberrant
expression or
activity includes increased or decreased expression or activity, as well as
expression or
activity which does not follow the wild type developmental pattern of
expression or the
subcellular pattern of expression. For example, aberrant BAL expression or
activity is
intended to include the cases in which a mutation in the BAL gene causes the
BAL gene
to be under-expressed or over-expressed and situations in which such mutations
result in
a non-functional BAL protein or a protein which does not function in a wild-
type
fashion, e.g., a protein which does not interact with a BAL ligand or one
which interacts
with a non-BAL ligand.
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 a misregulation in BAL protein activity or nucleic
acid
expression, e.g., a malignancy such as a lymphoma, e.g., non-Hodgkin's
lymphoma.
Alternatively, the prognostic assays can be utilized to identify a subject
having or at risk
for developing a disorder associated with a misregulation in BAL protein
activity or
nucleic acid expression, such as a e.g., a malignancy such as a lymphoma,
e.g., non-
Hodgkin's lymphoma. Thus, the present invention provides a method for
identifying a
disease or disorder associated with aberrant BAL expression or activity in
which a test
sample is obtained from a subject and BAL protein or nucleic acid (e.g., mRNA
or
genomic DNA) is detected, wherein the presence of BAL protein or nucleic acid
is
diagnostic for a subject having or at risk of developing a disease or disorder
associated
with aberrant BAL 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)
to treat a disease or disorder associated with aberrant BAL expression or
activity. For
30, example, such methods can be used to determine whether a subject can be
effectively
treated with an agent for a e.g., a malignancy such as a lymphoma, e.g., non-
Hodgkin's
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lymphoma. Thus, the present invention provides methods for determining whether
a
subject can be effectively treated with an agent for a disorder associated
with aberrant
BAL expression or activity in which a test sample is obtained and BAL protein
or
nucleic acid expression or activity is detected (e.g., wherein the abundance
of BAL
protein or nucleic acid expression or activity is diagnostic for a subject
that can be
administered the agent to treat a disorder associated with aberrant BAL
expression or
activity).
The methods of the invention can also be used to detect genetic alterations in
a
BAL gene, thereby determining if a subject with the altered gene is at risk
for a disorder
characterized by misregulation in BAL protein activity or nucleic acid
expression, such
as a e.g., a malignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma. In
preferred embodiments, the methods include detecting, in a sample of cells
from the
subject, the presence or absence of a genetic alteration characterized by at
least one of an
alteration affecting the integrity of a gene encoding a BAL-protein, or the
mis-
expression of the BAL gene. For example, such genetic alterations can be
detected by
ascertaining the existence of at least one of 1 ) a deletion of one or more
nucleotides from
a BAL gene; 2) an addition of one or more nucleotides to a BAL gene; 3) a
substitution
of one or more nucleotides of a BAL gene, 4) a chromosomal rearrangement of a
BAL
gene; 5) an alteration in the level of a messenger RNA transcript of a BAL
gene, 6)
aberrant modification of a BAL 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 BAL gene, 8) a non-wild type level of a BAL-protein, 9) allelic loss of a
BAL gene,
and 10) inappropriate post-translational modification of a BAL-protein. As
described
herein, there are a large number of assays known in the art which can be used
for
detecting alterations in a BAL gene. A preferred biological sample is a tissue
ox serum
sample isolated by conventional means from a subject.
In certain embodiments, detection of the alteration 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) Proc. Natl. Acad. Sci. USA 91:360-364), the latter
of which
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can be particularly useful for detecting point mutations in the BAL-gene (see
Abravaya
et al. (1995) Nucleic Acids Res .23:675-682). This method can include the
steps of
collecting a sample of cells from a subject, isolating nucleic acid (e.g.,
genomic DNA or
mRNA) from the cells of the sample, contacting the nucleic acid sample with
one or
more primers which specifically hybridize to a BAL gene under conditions such
that
hybridization and amplification of the BAL-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 al. (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 BAL 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 BAL 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 al. (1996) Nature Medicine 2:
753-
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759). For example, genetic mutations in BAL 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 overlapping 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 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 BAL gene and detect mutations by
comparing the sequence of the sample BAL with the corresponding wild-type
(control)
sequence. Examples of sequencing reactions include those based on techniques
developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or
Sanger
((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any
of a
variety of automated sequencing procedures can be used 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 BAL gene include methods in
which protection from cleavage agents is used to detect mismatched bases in
RNA/RNA
or RNA/DNA 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 BAL 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
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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 Enrymol. 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 BAL cDNAs obtained from samples of cells. For example, the mutt
enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase
from HeLa cells cleaves T at G/T mismatches (Hsu et al. ( 1994) Carcinogenesis
15:1657-1662). According to an exemplary embodiment, a probe based on a BAL
sequence, e.g., a wild-type BAL 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 BAL 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 BAL 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 be labeled 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 uses heteroduplex analysis to separate double stranded
heteroduplex
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molecules on the basis of changes in electrophoretic 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 gel 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
oligonucleotides are hybridized to PCR amplified target DNA or a number of
different
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
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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 the use of pre-
packaged diagnostic kits which include 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 BAL gene such as non-Hodgkin's lymphoma. Such kits can optionally include
instructions for use.
Furthermore, any cell type or tissue in which BAL is expressed may be used in
the prognostic assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs) on the expression or activity
of a
BAL protein can be applied not only in basic drug screening, but also in
clinical trials.
1 S For example, the effectiveness of an agent determined by a screening assay
as described
herein to increase BAL gene expression, protein levels, or upregulate BAL
activity, can
be monitored in clinical trials of subjects exhibiting decreased BAL gene
expression,
protein levels, or downregulated BAL activity. Alternatively, the
effectiveness of an
agent determined by a screening assay to decrease BAL gene expression, protein
levels,
or downregulate BAL activity, can be monitored in clinical trials of subjects
exhibiting
increased BAL gene expression, protein levels, or upregulated BAL activity. In
such
clinical trials, the expression or activity of a BAL gene, and preferably,
other genes that
have been implicated in, for example, a BAL-associated disorder can be used as
a "read
out" or markers of the phenotype of a particular cell.
For example, and not by way of limitation, genes, including BAL, that are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule)
which modulates BAL activity (e.g., identified in a screening assay as
described herein)
can be identified. Thus, to study the effect of agents on BAL-associated
disorders (e.g.,
malignancies such as non-Hodgkin's lymphoma), for example, in a clinical
trial, cells
can be isolated and RNA prepared and analyzed for the levels of expression of
BAL and
other genes implicated in the BAL-associated disorder, respectively. The
levels of gene
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expression (e.g., 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 described herein, or by measuring the levels
of activity
of BAL or other genes. In this way, the gene expression pattern can serve as a
marker,
indicative of the physiological response of the 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) including 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 BAL protein, mRNA, or
genomic DNA
in the preadministration sample; (iii) obtaining one or more post-
administration samples
from the subject; (iv) detecting the level of expression or activity of the
BAL protein,
mRNA, or genomic DNA in the post-administration samples; (v) comparing the
level of
expression or activity of the BAL protein, mRNA, or genomic DNA in the pre-
administration sample with the BAL 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 may be
desirable to increase expression or activity of BAL to lower levels than
detected, i. e. to
decrease the effectiveness of the agent. According to such an embodiment, BAL
expression or activity may be used as an indicator of the effectiveness of an
agent, even
in the absence of an observable phenotypic response.
D. Methods of Treatment:
The present invention provides for both prophylactic and therapeutic methods
of
treating a subject at risk of (or susceptible to) a disorder or having a
disorder associated
with aberrant BAL expression or activity e.g., a malignancy such as a
lymphoma, e.g.,
non-Hodgkin's lymphoma. With regards to both prophylactic and therapeutic
methods
of treatment, such treatments may be specifically tailored or modified, based
on
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knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as
used herein, refers to the application of genomics technologies such as gene
sequencing,
statistical genetics, and gene expression analysis to drugs in clinical
development and on
the market. More specifically, the term refers the study of how a patient's
genes
determine his or her response to a drug (e.g., a patient's "drug response
phenotype", or
"drug response genotype".) Thus, another aspect of the invention provides
methods for
tailoring an individual's prophylactic or therapeutic treatment with either
the BAL
molecules of the present invention or BAL modulators according to that
individual's
drug response genotype. Pharmacogenomics allows a clinician or physician to
target
prophylactic or therapeutic treatments to patients who will most benefit from
the
treatment and to avoid treatment of patients who will experience toxic drug-
related side
effects.
1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject, a
disease or condition associated with an aberrant BAL expression or activity,
by
administering to the subject a BAL molecule or an agent which modulates BAL
expression or at least one BAL activity. Subjects at risk for a disease which
is caused or
contributed to by aberrant BAL expression or activity can be identified by,
for example,
any or a combination of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the manifestation of
symptoms
characteristic of the BAL aberrancy, such that a disease or disorder is
prevented or,
alternatively, delayed in its progression. Depending on the type of BAL
aberrancy, for
example, a BAL molecule, BAL agonist, or BAL antagonist can be used to treat
the
subject. The appropriate agent can be determined based on, for example, the
screening
assays described herein.
2. Therapeutic Methods
Another aspect of the invention pertains to methods of modulating BAL
expression or activity for therapeutic purposes. Accordingly, in an exemplary
embodiment, the modulatory method of the invention involves contacting a cell
with a
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BAL or agent that modulates one or more of the activities of BAL protein
activity
associated with the cell. An agent that modulates BAL protein activity can be
an agent
as described herein, such as a nucleic acid or a protein, a naturally-
occurring target
molecule of a BAL protein (e.g., a BAL substrate), a BAL antibody, a BAL
agonist or
antagonist, a peptidomimetic of a BAL agonist or antagonist, or other small
molecule.
In one embodiment, the agent stimulates one or more BAL activities. Examples
of such
stimulatory agents include active BAL protein and a nucleic acid molecule
encoding
BAL that has been introduced into the cell. In another embodiment, the agent
inhibits
one or more BAL activities. Examples of such inhibitory agents include
antisense BAL
nucleic acid molecules, anti-BAL antibodies, and BAL inhibitors. 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 BAL 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
modulate (e.g., upregulate or downregulate) BAL expression or activity. In
another
embodiment, the method involves administering a BAI. protein or nucleic acid
molecule
as therapy to compensate for reduced or aberrant BAL expression or activity.
Stimulation of BAL activity is desirable in situations in which BAL is
abnormally downregulated and/or in which increased BAL activity is likely to
have a
beneficial effect. For example, stimulation of BAL activity is desirable in
situations in
which a BAL molecule is downregulated and/or in which increased BAL activity
is
likely to have a beneficial effect. Likewise, inhibition of BAL activity is
desirable in
situations in which BAL is abnormally upregulated and/or in which decreased
BAL
activity is likely to have a beneficial effect.
3. Pharmacogenomics
The BAL molecules of the present invention, as well as agents, or modulators
which have a stimulatory or inhibitory effect on BAL activity (e.g., BAL gene
expression) as identified by a screening assay described herein can be
administered to
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individuals to treat (prophylactically or therapeutically) BAL-associated
disorders (e.g.,
e.g., malignancies such as non-Hodgkin's lymphoma). In conjunction with such
treatment, pharmacogenomics (i.e., the study of the relationship between an
individual's
genotype and that individual's response to a foreign compound or drug) 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, a physician or clinician may consider
applying
knowledge obtained in relevant pharmacogenomics studies in determining whether
to
administer a BAL molecule or BAL modulator as well as tailoring the dosage
and/or
therapeutic regimen of treatment with a BAL molecule or BAL modulator.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected
persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol.
23(10-11) :983-985 and Linden M.W. et al. (1997) Clin. Chem. 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 genetic defects or as naturally-occurring 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.
One pharmacogenomics approach to identifying genes that predict drug
response, known as "a genome-wide association", relies primarily on a high-
resolution
map of the human genome consisting of already known gene-related markers
(e.g., a "bi-
allelic" gene marker map which consists of 60,000-100,000 polymorphic or
variable
sites on the human genome, each of which has two variants.) Such a high-
resolution
genetic map can be compared to a map of the genome of each of a statistically
significant number of patients taking part in a Phase II/III drug trial to
identify markers
associated with a particular observed drug response or side effect.
Alternatively, such a
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high resolution map can be generated from a combination of some ten-million
known
single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a
"SNP" is a common alteration that occurs in a single nucleotide base in a
stretch of
DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may
be involved in a disease process, however, the vast majority may not be
disease-
associated. Given a genetic map based on the occurrence of such SNPs,
individuals can
be grouped into genetic categories depending on a particular pattern of SNPs
in their
individual genome. In such a manner, treatment regimens can be tailored to
groups of
genetically similar individuals, taking into account traits that may be common
among
such genetically similar individuals.
Alternatively, a method termed the "candidate gene approach", can be utilized
to
identify genes that predict drug response. According to this method, if a gene
that
encodes a drugs target is known (e.g., a BAL protein of the present
invention), all
common variants of that gene can be fairly easily identified in the population
and it can
be determined if having one version of the gene versus another is associated
with a
particular drug response.
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 CYP2C 19) 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
metabolizer (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 CYP2C19 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
rnetabolizers who
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do not respond to standard doses. Recently, the molecular basis of ultra-rapid
metabolism has been identified to be due to CYP2D6 gene amplification.
Alternatively, a method termed the "gene expression profiling", can be
utilized to
identify genes that predict drug response. For example, the gene expression of
an
animal dosed with a drug (e.g., a BAL molecule or BAL modulator of the present
invention) can give an indication whether gene pathways related to toxicity
have been
turned on.
Information generated from more than one of the above pharmacogenomics
approaches can be used to determine appropriate dosage and treatment regimens
for
prophylactic or therapeutic treatment an individual. 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
BAL
molecule or BAL modulator, such as a modulator identified by one of the
exemplary
screening assays described herein.
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, as well as the Figures and the
Sequence
Listing are incorporated herein by reference.
EXAMPLES
The following Materials and Methods were used in the Examples Described
herein.
Cell lines and Primary tumor specimens
Human DLB-CL cell lines DHL-4, DHL-7, DHL-8, DHL-10, HT, and the
Burkitt's cell line Namalwa, were cultured in RPMI 1640 supplemented with 10%
heat-inactivated fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM
Herpes buffer and penicillin/streptomycin. Cryopreserved primary tumor
specimens
were obtained from DLB-CL patients with known clinical prognostic
characteristics and
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long-term follow-up. Total RNA was isolated from the cell lines and primary
tumors as
described in Aguiar (1999) Blood 94(7):2403-13.
Differential Display, cDNA cloning and sequencing
Differential display was performed as described in Liang (1992) Science
257(5072):967-71, and Aguiar, Blood (supra). The relevant differential display
product
was used as a probe to screen a size-selected anti-Ig activated normal B-cell
cDNA
library. BAL full length cloning was completed with 5' and 3' RACE PCR,
performed
as described in Aguiar, Blood (supra). All DNA sequencing was performed and
analyzed on an Applied Biosystems model 373A automated sequencer (Perkin-Elmer
Corporation, Norwalk, CT).
Northern blot Analysis
Total RNA from DLB-CL cell lines and primary tumors was isolated,
size-fractionated in 1 % Agarose/formaldehyde gels and transferred to nylon
membranes
as described in Aguiar, Blood (supra). These membranes and additional multiple
tissue
northern blots (Clontech, Palo Alto, CA) were hybridized according to standard
protocols with either a differential display fragment probe, an 800 by BAL
probe
(nucleotides 900 to 1700) or B-actin.
PAC Library Screening, FISH and somatic cell hybrid mapping
The human PAC library RPCI l (UK HGMP Resource Centre, Cambridge UK)
was screened with a BAL cDNA probe according to standard protocols. DNA from
positive clones was Southern blotted and re-probed with a distinct BAL probe
to
confirm the specificity of the clones. PAC DNAs were Biotin labeled, and
hybridization
of human normal metaphases performed as described in Fletcher ( 1998) Nature
Genetics 18(1):84-7. Image analysis was performed with a cooled CCD camera
(Photometrics) in conjunction with an image analysis system (Oncor). A human
monochromosomal somatic cell hybrid DNA panel (UK HGMP Resource Centre,
Cambridge UK) was screened by PCR according to the manufacturer's
instructions.
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Semi-quantitative duplex RT-PCR
cDNAs from DLB-CLs patients with known clinical prognostic characteristics
and long-term follow-up and from DLB-CL cell lines were synthesized as
described in
Aguiar (1996) British J. Haematol. 95(4):673-7. To control for the quantity
and quality
of input cDNA and the amplification efficiency in individual test tubes, BAL
cDNA was
co-amplified with the constitutively expressed ABL gene. Duplex RT-PCR
products
were electrophoresed in 2% agarose gels, blotted and hybridized to internal
BAL and
ABL oligonucleotide probes. The abundance of BAL in a given sample was
determined
by comparing the intensity of the co-amplified PCR products with scanning
densitometry. The sensitivity of the duplex RT-PCR was determined by
constructing
and analyzing a standard dilutional curve. In brief, fixed amounts of BAL
negative and
BAL positive cell line cDNAs were added to mimic BAL losses of 10% -100%. Upon
co-amplification, the ratio of the intensity of the two bands plotted against
the
percentage of BAL "loss" yields a straight line and r2 value of 0.967,
indicating the
power of this system in detecting reduced BAL expression in the patient
samples.
Transfections, western blot and fluorescence microscopy
Full length BAL cDNA was cloned into the green fluorescent protein (GFP)
expression vector pEGFP-C (Clontech, Polo Alto, CA) and into the untagged
expression
vector pRc/CMV (Invitrogen, Carlsbad, CA). Linearized DNA from BAL-GFP and
BAL-pRc/CMV constructs were transfected by electroporation into the Namalwa B-
cell
lymphoma line and selected with 6418 (Sigma, St Louis, MO). Thereafter, the
stable
BAL-GFP or GFP-only bulk transfectants were sorted to select high GFP
expressing
cells. The pRcCMV transfectants were cloned by limiting dilution as described
in
Aguiar, Blood (supra). Total cell lysates, membrane, cytoplasm, and nuclear
fractions
from multiple BAL-GFP or GFP-only transfectants were obtained and western
blots
performed as described in Aguiar, Blood (supra). Rabbit polyclonal anti-GFP
anti-sera
(Clontech) was used for immunological detection of B~AL fusion proteins. The
mouse
fibroblast NIH3T3 cells were seeded on glass coverslides and transfected with
BAL-GFP or GFP-alone constructs by using lipofectin (Gibco). Forty-eight hours
after
transfection, the slides were rinsed with ice-cold phosphate-buffered saline
(PBS)-0.1
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NaN3, cells were fixed with 3% paraformaldehyde and analyzed by fluorescence
microscopy.
Cell migration assays
S Multiple BAL expressing stable transfectants (BAL-GFP and BAL into
pRc-CMV, selected on the basis of protein expression) and vector-only controls
(pEGFP
and pRcCMV, respectively) were seeded overnight at 1 x 106 cells/ml in RPMI
10%
FBS. Then, 2 x 106 /ml cells were starved in serum-free AIM-V medium (Gibco
BRL,
Gaithersburg, MD), for 1 hour at 37°C in 5% C02. Migration assays were
performed
using 8 p. pore filters (6.Smm Transwell, polycarbonate membrane, Costar,
Cambridge
MA). Cell suspensions (2 x 105 in 100 ~L) were plated into the upper chamber,
whereas
600pL of medium with or without recombinant human SDFI-a, (100ng/mL} (R & D
Systems, Minneapolis, MN) was added to the lower compartment. The transwells
were
incubated for 4 hours at 37 °C in 5% C02. Thereafter, cells in the
lower chamber were
recovered and counted (Coulter automatic cell and particle counter). All
clones (with
and without SDF1-a) were analyzed in duplicate and the entire assay repeated
three
times.
EXAMPLE 1: IDENTIFICATION AND CHARACTERIZATION OF
BAL cDNA
In this example, the identification and characterization of the genes encoding
human and marine BAL is described. To identify genes which contribute to the
observed differences in clinical outcome in DLB-CLs, the technique of
differential
display (Liang P. et al. (1992) Science, 257:967) was used in panels of
primary tumors
from patients with known clinical prognostic characteristics and mature follow-
up. BAL
was found to be significantly more abundant in tumors from patients with "high-
risk
(HR)" (International Prognostic Index, IPI) fatal disease than in tumors from
cured "low
risk (LR [IPI])" patients (Shipp M. et al. (1993) N. Engl. J. Med., 329:987-
994).
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In confirmatory northern analyses, primary tumors from cured "LR" patients
consistently expressed low levels of BAL whereas tumors from "HR" patients
with fatal
disease consistently expressed high levels of BAL (see Figure 5). However,
only 1 of 5
DLB-CL cell lines (DHL-7) expressed high levels of BAL. This observation was
of
particular interest because DHL-7 grows as a semi-adherent monolayer whereas
BAL-
negative DLB-CL cell lines grow in suspension. These findings suggest that BAL
can
be upregulated when DLB-CL cells interact with other cellular or extracellular
components in vivo. Consistent with this hypothesis, tumors derived from a DLB-
CL
cell line grown in SCID mice express significantly higher levels of BAL than
the
parental suspension cells (see Figure 6).
In addition to being differentially expressed in high-risk and low-risk
primary
DLB-CLs, BAL was expressed at higher levels in normal anti-Ig-activated
splenic B-
cells than in non-activated splenic B-cells (see Figure 5). For this reason,
the full length
BAL cDNA was cloned by probing an Ig-activated B-cell cDNA library with the 3'
BAL
differential display product. Several overlapping cDNA clones were identified
and 5'
RACE PCR (described in, for example, Ishimaru F. et al. (1995) Blood 85:3199-
3207)
was used to complete the full length BAL cDNA sequence. Two alternatively
spliced
BAL cDNAs of 3243bp (BALL) and 3138bp (BALS) were identified. These cDNAs
encode previously uncharacterized 854 as and 819 as proteins. In vitro
translation
experiments confirmed that BAL cDNAs encode ~85-87kd proteins.
For the Bal human cDNA, two human cDNA libraries derived from anti-
immunoglobulin activated splenocytes and the Raji Burkitts lymphoma cell line
cloned
into pCDM8 were screened to obtain additional full length Bal cDNAs.
For the Bal murine cDNAs, the BAL human sequence was used to search the
mouse EST database. A 418 by clone (Accession Number AA475710, Soares mouse
mammary gland) homologous to the human sequence was identified. This sequence
was
used as "anchor" to several rounds of 5' and 3' RACE assays performed with
mouse
(Balb-c) spleen cDNA. (The sequences obtained from the EST database were used
as
primers).
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The nucleotide sequence encoding the human BAL protein is shown in Figure 1
and is set forth as SEQ ID NO:1. The full length protein encoded by this
nucleic acid
comprises about 866 amino acids and has the amino acid sequence shown in
Figure 1
and set forth as SEQ ID N0:2. The coding region (open reading frame) of SEQ ID
NO:l is set forth as SEQ ID N0:3.
The nucleotide sequence encoding the mouse BAL protein is shown in Figure 2
and is set forth as SEQ ID N0:4. The full length protein encoded by this
nucleic acid
comprises about 866 amino acids and has the amino acid sequence shown in
Figure 2
and set forth as SEQ ID NO:S. The coding region (open reading frame} of SEQ ID
N0:4 is set forth as SEQ ID N0:6.
Tissue Distribution of BAL
Although BAL transcripts were detected in the majority of organs on a multiple
tissue northern blot, BAL transcripts were most abundant in lymphoid organs
(spleen,
1 S lymph colon mucosa, node, fetal liver, and peripheral blood) and several
additional non-
hematopoietic organs (heart, skeletal muscle) (see Figure 8).
Subcellular Localization of BAL
To determine the subcellular location of the BAL protein, the longer and the
shorter BAL cDNAs (BALL and BALS) were cloned into the green fluorescent
protein
(GFP) expression vector (pEGFP, Clontech) and transiently transfected in
NIH3T3
fibroblasts. These fibroblasts were then examined by fluorescence microscopy.
Fibroblasts were chosen for BAL subcel l ular localization because the
lymphoma cell
lines have scant cytoplasm, precluding optimal microscopic detection of
subcellular
structures. BALS was found to localize to the nucleus (confirmed with western
blotting
of cellular subfractions) whereas BALL localized in both the nucleus and the
perinuclear
cytoplasm. These data indicate that in NIH 3T3 cells, BAL does not interact
directly
with the cytoskeletal network, demonstrating that BAL may either influence
cellular
migration in an indirect manner, or may traffic between the cytoplasm and the
nucleus
and directly modulate cell migration via cytoskeleton interactions following
specific
signals.
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BAL Promotes Homotypic Aggregation
In additional experiments, aggressive lymphoma cell lines were transfected
with
pRc-CMV-BALL constructs and evaluated for changes in morphology. pRc-CMV-
BALL transfectants exhibited markedly increased homotypic aggregation,
indicating that
BAL enhances the adhesion of these cells. These observations are of particular
interest
because BAL was upregulated in DLB-CL tumors in SCID mice (see Figure 6) and
was
significantly more abundant in primary DLB-CLs and the adherent DLB-CL cell
line
than in DLB-CL suspension cell lines (see Figure 5 ).
Mapping of the BAL Locus
To map the BAL locus, a BAL cDNA probe was used to screen a human
genomic DNA PAC library (RPCI1). Genomic BAL-positive PAC clones were used to
perform FISH (fluorescence in situ hybridization) on normal human metaphases.
In
complementary experiments, a somatic cell hybrid panel was analyzed for BAL
sequences. The BAL locus was mapped to chromosome 3q21 (see Figure 9), an area
of
known abnormalities in multiple hematologic malignancies, including DLB-CL and
other aggressive B-cell lymphomas (Cabanillas F et al. ( 1988) Cancer Res.,
48:5557-
5564; Schouten H. et al. (1990) Blood, 75:1841; Monni O. et al. (1998), Genes,
Chrom.
& Cancer, 21:298-307; and (Mitelman F. et al. (1997) Nat. Genet, 15:417-474).
Analysis of the Human BAL Molecules
A BLASTP 2Ø6 search using an e-value threshold for inclusion of 0.001 and a
word length of 854 letters (Altschul et al. (1990) J. Mol. Biol. 215:403) of
the protein
sequence of human BAL revealed that human BAL is similar to the histone macro-
H2A.1 protein (Accession Number Q02874). The human BAL protein is 26%
identical
to the histone macro-H2A.1 protein (Accession Number Q02874) over amino acid
residues 319-485 and 22% identical over amino acid residues 160-288.
A BLASTN 2Ø5 search using an e-value threshold for inclusion of 9e-09, score
61) and a word length of 3244 letters (Altschul et al. (1990) J. Mol. Biol.
215:403) of the
nucleotide sequence of human BAL revealed that BAL is similar to the Soares
pregnant
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uterus NbHPU Homo sapiens cDNA clone 502921 (Accession Number AA151346).
The human BAL nucleic acid molecule is 98% identical to the Soares pregnant
uterus
NbHPU Homo Sapiens cDNA clone 502921 (Accession Number AA151346) over
nucleotides 2528 to 3132.
A BLASTP 2Ø6 search using an e-value threshold for inclusion of 0.001 and a
word length of 866 letters (Altschul et al. (1990) J. Mol. Biol. 215:403) of
the protein
sequence of marine BAL revealed that the marine BAL is similar to the histone
macro-
H2A.1 protein (Accession Number Q02874). The marine BAL protein is 26%
identical
to the histvne macro-H2A.1 protein (Accession Number Q02874) over amino acid
residues 261-450 and 24% identical over amino acid residues 74-250.
A BLASTN 2Ø5 search using an e-value threshold for inclusion of l e-08,
score
60) (Altschul et al. (1990) J. Mol. Biol. 215:403) of the nucleotide sequence
of marine
BAL revealed that BAL is similar to the Soares 2NbMT Mus musculus cDNA clone
1446050 (Accession Number AI157103). The marine BAL nucleic acid molecule is
99% identical to the Soares 2NbMT Mus musculus cDNA clone 1446050 (Accession
Number AI157103) over nucleotides 2295 to 2739.
The human BAL protein was aligned with the marine BAL protein using the
ALIGN program (version 2.0), a PAM120 weight residue table, a gap length
penalty of
12 and a gap penalty of 2. The results showed a 61.5% identity between the two
sequences (see Figure 3).
The human BAL nucleic acid molecule was aligned with the marine BAL
nucleic acid molecule using the ALIGN program (version 2.0), a PAM120 weight
residue table, a gap length penalty of 16 and a gap penalty of 4. The results
showed a
71.7% identity between the two sequences (see Figure 4).
Analysis of the predicted 819 as human BAL protein indicates that specific
regions of human BAL have partial homology to two previously characterized
domains
in otherwise related proteins (Figure 7). The human BAL N-terminal region (aa
136-
256 and 335-447) contains a duplicated domain of unknown function (Pfam at
www.wustl.edu) which is found in the non-histone region of histone Macro H2A,
non-
structural polyproteins of ssRNA viruses or on its own in a family of proteins
from
bacteria to eukaryotes (Pehrson J.R. et al. (1999) Nucleic Acids Research
26:2837-
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2842). Taken together, these data indicate that this evolutionarily conserved,
as yet
uncharacterized, domain is involved in an important and ubiquitous cellular
process.
The human BAL C-terminal region (aa 508-709), is partially homologous to
protein families (myosin heavy chain and cytoskeleton linkers ezrin-radixin-
moesin
[ERM]) (see Figure 7) which primarily govern physically integrated cellular
functions
such as cell migration, motility, and shape, as well as cell/cell and
cell/extra-cellular
matrix interactions through adhesion molecules. Specifically, BAL is
homologous to
the rod domain (filament forming properties) of alpha and beta cardiac myosin
(Warrick
et al. (1987) Annual Rev. Cell Biol. 3:379-421) and to the alpha-helical
region of
moesin, the most abundant ERM protein in lymphocytes (Bretscher ( 1999)
Current
Biology 8(12):721-4).
EXAMPLE 2: IDENTIFICATION OF CO-ASSOCIATING MOLECULES
AND SIGNALING PATHWAYS RELEVANT TO
BAL BIOLOGICAL ACTIVITY
The following experiments are designed to confirm that: ( 1 ) BAL is
phosphorylated on tyrosine; (2) BAL co-associates with other tyrosine
phosphoproteins;
(3) BAL specifically co-associates with CRK-L, SHP-2, P13K or ZAP70; and (4)
BAL
is a major component of signaling pathways in lymphohematopietic cells.
Generation of BALHA and BAL~~ DLB-CL Transfectants
BALS and BALL cDNAs are cloned into the GFP- and HA-tagged expression
vectors (pEGFP, Clontech and pHM6, Boehringer) and transfected into pEGFP-BAL
and MH6-BAL into DLB-CL cell lines that express the protein (DHL-7) or lack
endogenous BAL expression (DHL-4). Both BALGFP and BALHA transfectants are
evaluated in order to confirm BAL nuclear and perinuclear cytoplasmic
localization in
DLB-CLs. To characterize BAL function, BALD is preferentially used because its
smaller tagged protein is more likely to retain the physiologically relevant
binding sites.
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Tvrosine Phosphorylation of BAL and Additional Co-associated Proteins
To confirm that BAL itself is tyrosine phosphorylated and that BAL co-
associates with additional tyrosine phosphoproteins, BAL is immunoprecipitated
from
untreated or anti-Ig-treated DHL-7 and DHL-4 pHM6-BAL transfectants using an
HA
monoclonal antibody. Thereafter, the BALHA immunoprecipitates are
immunoblotted
with a phosphotyrosine antibody, 4610 (Upstate Biotechnology, Inc.). The
molecular
weights of identified tyrosine phosphoproteins are compared to that of BAL and
additional candidate co-associated tyrosine phosphoproteins. In complementary
experiments, BALHA immunoprecipitates are blotted with specific antibodies to
potential co-associating tyrosine phosphoproteins such as CRK/CRK-L, SHP-2,
P13K,
ZAP70 and additional candidates with molecular weights that are similar to
those of
identified BAL co-associated phosphoproteins. Because it may be easier to
isolate co-
associated proteins with concentrated highly purified recombinant BAL, the
BAL~sT
fusion protein beads (described above) are also incubated with DHL-7 cell
lysates, the
BAL complexes are blotted, and resulting blots are probed with a
phosphotyrosine
antibody (4G10).
Candidate co-associating proteins can further be identified using the yeast
two-
hybrid system (Matchmaker Two-Hybrid System by Clontech) as described in
Frederickson, R. (1998) Curr. Opin. Biotechnol., 9:90-96).
Tumorigenicity of BAL Transfectants
In addition to evaluating the role of BAL in specific signaling cascades,
BAL's
potential effects on the local growth and distant metastasis of DLB-CL cell
lines are
further determined in an in vivo marine model. The above-mentioned pRc-CMV
brief,
parental, vector-only, pRc-CMV-BALs and BALL I)LB-CL transfectants are
injected
subcutaneously or via tail vein into cohorts of SCID mice. Antisense BAL
constructs
can also be used because, as described herein, endogenous BAL is upregulated
when the
suspension DLB-CL cell lines form local tumors in vivo (see Figure 6). Local
tumorigenicity and distant metastasis can be scored at periodic intervals as
described in
Yakushijin Y. et al. (1998) Blood, 91:4282-4291.
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EXAMPLE 3: ANALYSIS OF BAL EXPRESSION AT RNA AND PROTEIN
LEVELS IN AN EXPANDED SERIES OF AGGRESSIVE
NHLs FROM WELL CHARACTERIZED UNIFORMLY
TREATED PATIENTS
Semi-quantitative duplex RT-PCR
A semi-quantitative duplex RT-PCR assay (as described in Aguiar, R. et al.
(1997) Blood 90 (Suppl. 1):491a and Aguiar, R. et al. (1997) Leukemia, 11:233-
238)
was performed to access BAL expression in a large series of primary DLB-CLs.
Briefly, the "target sequence" (BAL) was co-amplified with the constitutively
expressed
"control" ABL gene. The PCR products were size-fractionated, blotted and
hybridized
with internal BAL and ABL oligonucleotides. Autoradiogram signals were
captured
using a CCD camera linked to a frame grabber and intensities were quantified
using the
program NIH Image 1.55 (NIH, Bethesda, MD) (as described in Aguiar, R. et al.
( 1997)
Leukemia, 11:233-238). To establish the sensitivity of the assay, a series of
dilutional
controls were constructed by mixing fixed amounts of cDNA from the BAL-
negative
and BAL-positive DLB-CL cell lines. These controls mimic BAL losses at each
10%
interval up to 100% (see Figure 10). When the ratio of BAL/ABL signal was
plotted
against the percentage of BAL present in each of these test samples, it
yielded a straight
line and r2 value of 0.967, confirming the sensitivity of the assay (see
Figure 10). The
abundance of BAL in a given sample is determined by comparing the ratio of
intensity
of co-amplified BAL and ABL signals and correlating this ratio with that in
the
dilutional controls (as described in Aguiar, R. et al. ( 1997) Blood 90
(Suppl. ~1 ):491 a and
Aguiar, R. et al. (1997) Leukemia, 11:233-238).
To extend the initial observation regarding the risk related expression of BAL
in
DLBCLs, a larger series of 28 additional primary DLB-CL with well-
characterized risk
profiles and long term followup were studied (see Figure 11 ). In these
tumors, BAL
expression, as determined by the ratio of the intensity of the two co-
amplified cDNAs
(quantified with scanning densitometry) correlated closely with the clinical
risk profile
(see Figure 12). BAL transcripts were significantly more abundant in high
intermediate/high risk primary DLB-CLs than in cured low and low intermediate
risk
tumors (p=0.0023, Figure 12).
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Development of a BAL Antibody
In order to generate a BAL antibody, the BAL cDNA was cloned into the pGEX
expression vector (GST Gene Fusion System, Pharmacia). After sequencing the
construct to ensure that the fusion was in frame and that no mutations had
been
introduced in the BAL sequence, bacterial cultures containing pGEX-BAL were
treated
with IPTG (isopropyl-1-Thio-b-D-Galactopyearanoside) and the GST-Bal fusion
protein
was induced and affinity-purified on gluthathione-S-agarose beads. Balb-c mice
are
immunized with the affinity-purified GST-BAL protein and their spleens
harvested for
generation of BAL monoclonal antibodies. Monoclonal antibodies are initially
screened
for reactivity with pGEX-BAL and not with pGEX alone using ELISA. Positive
hybridoma supernatants are then screened against immunoblotted parental and
BAL
DLB-CL transfectants for reactivity with the appropriate-sized BAL protein.
Immunoperixidase Staining of Primary DLB-CLs for BAL
Immunoperoxidase staining of primary tumor specimens from a large series of
well-characterized DLB-CL patients, can also be performed using the BAL
monoclonal
antibody produced as described above.
EXAMPLE 4: EVALUATION OF THE INTEGRITY OF THE BAL LOCUS
IN AGGRESSIVE NHLs WITH 3q21 ABNORMALITIES
BAL maps to chromosome band 3q21, a region which is frequently associated
with non-random abnormalities (gain, loss and translocations) in NHLs
(Cabanillas F et
al. (1988) Cancer Res., 48:5557-5564); (Schouten H. et al. (1990) Blood,
75:1841);
(Monni O. et al. (1998), Genes, Chrom. & Cancer, 21:298-307); and (Mitelman F.
et al.
( 1997) Nat. Genet, 15:417-474). To determine whether BAL is the target of the
described 3q21 abnormalities in aggressive NHLs, the integrity of the BAL
locus is
assessed in informative samples. The patient's metaphases are initially prabed
with
PAC clones encompassing the BAL locus using the FISH technique (Aguiar, R. et
al.
(1997) Blood, 90:3130-3135; Carapeti, M. et al. (1998) Blood, 91:3127-3133;
Aguiar,
R. et al. ( 1997) Genes, Chrom. & Cancer, 20:408-411 ). In the case a
translocation
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involving the BAL locus is detected, Southern blotting is used to map the
breakpoint in
BAL. Thereafter, strategies to clone the translocation partner are used
including RACE
PCR and, if necessary, construction and screening of patient tumor cDNA
libraries with
BAL probes (Aguiar, R. et al. (1997) Blood, 90:3130-3135 and Carapeti, M. et
al.
(1998) Blood, 91:3127-3133. If loss or gain of DNA material is found with the
FISH,
Southern blots are performed to confirm these findings and DNA markers
flanking the
BAL locus are used to delineate the smallest region of loss or gain in the
tumor
specimens. To define the size of the region of gain or loss of 3q21 in
informative
samples, candidate 3q21 YAC clones are identified from the Human Genomic
Mapping
Project databases (www.genome.wi.mit.edu, www.genethon.fr). Southern blot and
PCR
analysis of these clones' DNA maps BAL to a particular YAC (Aguiar, R: et al.
(1997)
Blood 90 (Suppl. 1):491a). Thereafter, STS markers already anchored to that
particular
YAC are identified in the relevant databases (www.genome.wi.mit.edu) and used
as
probes or PCR targets to determine their copy number in informative primary
tumors as
previously described (Aguiar, R. et al. (1997) Leukemia, 1 I :233-238). Using
this
strategy, the smallest region of loss or gain at 3q21 is defined, and whether
BAL is the
target for these structural abnormalities is determined.
EXAMPLE 5: ROLE OF BAL IN MODULATING CELLULAR
MOTILITY AND MIGRATION
To investigate the potential role of BAL in modulating cellular motility and
migration, BALS was cloned into a GFP vector (pEGFP) and an untagged
expression
vector (pRc-CMV) and pEGFP-BALS, pRc-CMV BAL, and vector only transfectants
were generated in an aggressive lymphoma cell line that constitutively
expresses low
levels of BAL (see Figure 13). The effects of BAL overexpression an the
migration of
these transfectants was investigated using a transwell system. In initial
experiments,
BALS-GFP or GFP-only transfectants were plated in the upper chamber and
analyzed for
migration to the lower chamber in the presence of the hematopoietic
chemoattractant
factor, stromal derived factor 1-a (SDF-la) (see Figure 14). In multiple
independent
assays, aggressive lymphoma BALS-GFP transfectants migrated at a 2-4 times
higher
rate than the GFP-alone transfectants (p<0.01 ) (see Figure 14). Similar
results were
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obtained with multiple BALSpRcCMV transfectants indicating that the GFP moiety
did
not affect BAL function or the observed results. Taken together, these data
indicate that
BAL overexpression increases the migration of an aggressive NHL cell line.
EXAMPLE 6: EXPRESSION OF RECOMBINANT BAL PROTEIN
IN BACTERIAL CELLS
In this example, BAL is expressed as a recombinant glutathione-S-transferase
(GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and
characterized. Specifically, BAL is fused to GST and this fusion polypeptide
is
expressed in E. coli, e.g., strain PEB 199. Expression of the GST-BAL 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 7: EXPRESSION OF RECOMBINANT BAL PROTEIN
IN COS CELLS
To express the BAL 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 BAL protein and an HA tag (Wilson et al. (1984)
Cell
37:767) or a FLAG tag fused in-frame 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 BAL 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 BAL 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
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20 nucleotides of the BAL 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 BAL
gene is
5 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 un 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 fragment.
COS cells are subsequently transfected with the BAL-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,
I S Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The
expression
of the BAL 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 specif c 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, 1 SO
mM NaCI,
1% NP-40, 0.1% SDS, 0.5% DOC, SO 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 BAL 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 described above,
and the
expression of the BAL polypeptide is detected by radiolabelling and
immunoprecipitation using a BAL specific monoclonal antibody.
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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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SEQUENCE LISTING
<110> DANA-FARBER CANCER INSTITUTE
<120> LYMPHOMA ASSOCIATED MOLECULES AND USES THEREFOR
<130> DFN-031PC
<140>
<191>
<150> USSN 60/106,383
<151> 1998-10-29
<150> USSN 60/106,448
<151> 1998-10-30
<160> 6
<170> PatentIn Ver. 2.0
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<222> (229)..(2790)
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gggcttcgtg ttcctgggtg ctgaccgtgc actccccgcc gcccgaggac ttagagctct 60
ggaagtagct ctccagcttc cttcgtactc gggggccgga cttgtacacc cgcacgagga 120
gcggggacgg cgggcgcaga agtgggccac catatctgga aactacagtc tatgctttga 180
agcgcaaaag ggaataaaca tttaaagact cccccgggga cctggagg atg gac ttt 237
Met Asp Phe
1
tcc atg gtg gcc gga gca gca get tac aat gaa aaa tca ggt agg att 285
Ser Met Val Ala Gly Ala Ala Ala Tyr Asn Glu Lys Ser Gly Arg Ile
10 15
acc tcg ctc tca ctc ttg ttt cag aaa gtc ttt get cag atc ttt cct 333
Thr Ser Leu Ser Leu Leu Phe Gln Lys Val Phe Ala Gln Ile Phe Pro
20 25 30 35
cag tgg aga aag ggg aat aca gaa gaa tgt ctc ccc tac aag tgc tca 381
Gln Trp Arg Lys Gly Asn Thr Glu Glu Cys Leu Pro Tyr Lys Cys Ser
40 45 50
gag act ggt get ctt gga gaa aac tat agt tgg caa att ccc att aac 929
Glu Thr Gly Ala Leu Gly Glu Asn Tyr Ser Trp Gln Ile Pro Ile Asn
55 60 65
cac aat gac ttc aaa att tta aaa aat aat gag cgt cag ctg tgt gaa 477
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His AspPhe LysIleLeu LysAsnAsn GluArgGln LeuCys Glu
Asn
70 75 80
gtc ctccagaat aagtttggc tgtatctct accctggtc tctcca gtt 525
Val LeuGlnAsn LysPheGly CysIleSer ThrLeuVal SerPro Val
g5 90 95
cag gaaggcaac agcaaatct ctgcaagtg ttcagaaaa atgctg act 573
Gln GluGlyAsn SerLysSer LeuGlnVal PheArgLys MetLeu Thr
100 105 110 115
ect aggatagag ttateagtc tggaaagat gacetcacc acacat get 621
Pro ArgIleGlu LeuSerVal TrpLysAsp AspLeuThr ThrHis Ala
120 125 130
gtt gatgetgtg gtgaatgea gccaatgaa gatcttctg catggg gga 669
Val AspAlaVal ValAsnAla AlaAsnGlu AspLeuLeu HisGly Gly
135 140 195
ggc ctggcectg gccctggta aaagetggt ggatttgaa ateeaa gaa 717
Gly LeuAlaLeu AlaLeuVal LysAlaGly GlyPheGlu IleGln Glu
150 155 160
gag agcaaacag tttgttgcc agatatggt aaagtgtca getggt gag 765
Glu SerLysGln PheValAla ArgTyrGly LysValSer AlaGly Glu
165 170 175
ata getgtcacg ggagcaggg aggcttccc tgcaaacag atcatc cat 813
Ile AlaValThr GlyAlaGly ArgLeuPro CysLysGln IleIle His
180 185 190 195
get gttgggcct cggtggatg gaatgggat aaacaggga tgtact gga 861
Ala ValGlyPro ArgTrpMet GluTrpAsp LysGlnGly CysThr Gly
200 205 210
aag ctgcagagg gccattgta agtattctg aattatgtc atctat aaa 909
Lys LeuGlnArg AlaIleVal SerIleLeu AsnTyrVal IleTyr Lys
215 220 225
aat actcacatt aagacagta gcaattcca gcctt=gagc tctggg att 957
Asn ThrHisIle LysThrVal AlaIlePro AlaLeuSer SerGly Ile
230 235 290
ttt cagttccct ctgaatttg tgtacaaag actattgta gagact atc
1005
Phe GlnPhePro LeuAsnLeu CysThrLys ThrI:LeVal GluThr Ile
245 250 255
cgg gttagtttg caagggaag ccaatgatg agtaatttg aaagaa att
105 3
Arg ValSerLeu GlnGlyLys ProMetMet SerAsnLeu LysGlu Ile
260 265 270 275
cae etggtgagc aatgaggac ectactgtt getgecttt aaaget get
110 1
His LeuValSer AsnGluAsp ProThrVal AlaAlaPhe LysAla Ala
280 285 290
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tca gaa ttc atc cta ggg aag agt gag ctg gga caa gaa acc acc cct
1149
Ser Glu Phe Ile Leu Gly Lys Ser Glu Leu Gly G1n Glu Thr Thr Pro
295 300 305
tct ttc aat gca atg gtc gtg aac aac ctg acc ctc cag att gtc cag
1197
Ser Phe Asn Ala Met Val Val Asn Asn Leu Thr Leu Gln Ile Val Gln
310 315 320
ggc cac att gaa tgg cag acg gca gat gta att gtt aat tct gta aac
1245
Gly His Ile Glu Trp Gln Thr Ala Asp Val Ile Val Asn Ser Val Asn
325 330 335
cca cat gat att aca gtt gga cct gtg gca aag tca att cta caa caa
1293
Pro His Asp Ile Thr Val Gly Pro Val Ala Lys Ser Ile Leu Gln Gln
340 345 350 355
gca gga gtt gaa atg aaa tcg gaa ttt ctt gcc aca aag get aaa cag
1341
Ala Gly Val Glu Met Lys Ser Glu Phe Leu Ala Thr Lys Ala Lys Gln
360 365 370
ttt caa cgg tcc cag ttg gta ctg gtc aca aaa gga ttt aac ttg ttc
1389
Phe Gln Arg Ser Gln Leu Val Leu Val Thr Lys Gly Phe Asn Leu Phe
375 380 385
tgt aaa tat ata tac cat gta ctg tgg cat tca gaa ttt cct aaa cct
1437
Cys Lys Tyr Ile Tyr His Val Leu Trp His Ser Glu Phe Pro Lys Pro
390 395 400
cag -ata tta aaa cat gca atg aag gag tgt ttg gaa aaa tgc att gag
1485
Gln Ile Leu Lys His Ala Met Lys Glu Cys Leu Glu Lys Cys Ile Glu
905 410 415
caa aat ata act tcc att tcc ttt cct gcc ctt ggg act gga aac atg
1533
Gln Asn Ile Thr Ser Ile Ser Phe Pro Ala Leu Gly Thr Gly Asn Met
420 425 430 435
gaa ata aag aag gaa aca gca gca gag att ttg ttt gat gaa gtt tta
1581
Glu Ile Lys Lys Glu Thr Ala Ala Glu Ile Leu Phe Asp Glu Val Leu
490 495 950
aca ttt gcc aaa gac cat gta aaa cac cag tta act gta aaa ttt gtg
1629
Thr Phe Ala Lys Asp His Val Lys His Gln Leu Thr Val Lys Phe Val
455 960 465
atc ttt cca aca gat ttg gag ata tat aag get ttc agt tct gaa atg
1677
Ile Phe Pro Thr Asp Leu Glu Ile Tyr Lys Ala Phe Ser Ser Glu Met
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970 475 480
gca aag agg tcc aag atg ctg agt ttg aac aat tac agt gtc ccc cag
1725
Ala Lys Arg Ser Lys Met Leu Ser Leu Asn Asn Tyr Ser Val Pro Gln
485 490 4!35
tca acc aga gag gag aaa aga gaa aat ggg ctt gaa get aga tct cct
1773
Ser Thr Arg Glu Glu Lys Arg Glu Asn Gly Leu G.Lu Ala Arg Ser Pro
500 505 510 515
gcc atc aat ctg atg gga ttc aac gtg gaa gag atg tat gag gcc cac
1821
Ala Ile Asn Leu Met Gly Phe Asn Val Glu Glu Met Tyr Glu Ala His
520 525 530
gca tgg atc caa aga atc ctg agt ctc cag aac cac cac atc att gag
1869
Ala Trp Ile Gln Arg Ile Leu Ser Leu Gln Asn His His Ile Ile Glu
535 540 545
aat aat cat att ctg tac ctt ggg aga aag gaa cat gac att ttg tct
1917
Asn Asn His Ile Leu Tyr Leu Gly Arg Lys Glu His Asp Ile Leu Ser
550 555 560
cag ctt cag aaa act tca agt gtc tcc atc aca gaa att atc agc cca
1965
Gln Leu Gln Lys Thr Ser Ser Val Ser Ile Thr Glu Ile Ile Ser Pro
565 570 575
gga agg aca gag tta gag att gaa gga gcc cgg get gac ctc att gag
2013
Gly Arg Thr Glu Leu Glu Ile Glu Gly Ala Arg Ala Asp Leu Ile Glu
580 585 590 595
gtg gtt atg aac att gaa gat atg ctt tgt aaa gta cag gag gaa atg
2061
Val Val Met Asn Ile Glu Asp Met Leu Cys Lys Val Gln Glu Glu Met
600 605 610
gca agg aaa aag gag cga ggc ctt tgg cgc tcg tta gga cag tgg act
2109
Ala Arg Lys Lys Glu Arg Gly Leu Trp Arg Ser I~eu Gly Gln Trp Thr
615 620 625
att cag caa caa aaa acc caa gac gaa atg aaa gaa aat atc ata ttt
2157
Ile Gln Gln Gln Lys Thr Gln Asp Glu Met Lys Glu Asn Ile Ile Phe
630 635 640
ctg aaa tgt cct gtg cct cca act caa gag ctt cta gat caa aag aaa
2205
Leu Lys Cys Pro Val Pro Pro Thr Gln Glu Leu :Leu Asp Gln Lys Lys
645 650 655
cag ttt gaa aaa tgt ggt ttg cag gtt cta aag gtg gag aag ata gac
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2253
Gl s s Leu Gln Leu Lys Glu LysIleAsp
L Gly Val V,al
C
Gln u y y 675
Phe
660
665 670
aat gtcctt atggetgcc tttcaaaga aagaagaaa atgatggaa
gag
2301
V Leu MetAlaAla PheGlnArg LysLysLys MetMetGlu
l
Asn a 685 690
Glu
680
a ct cac aggcaacct gtgagccat aggctgttt cagcaagtc
gaa g
aa
2349
L His ArgGlnPro ValSerHis ArgLeuPhe GlnGlnVal
Glu eu 705
Lys
695 700
t ca ttc tgcaatgtg gtatgcaga gttggcttt caaagaatg
ac g
cca
2397
T GlnPhe CysAsnVal ValCysArg ValGlyPhe GlnArgMet
yr 715 720
Pro
710
tcg acacct tgcgatcca aaatacgga getggcata tacttcacc
ta c
2445
ThrPro CysAspPro LysTyrGly AlaGlyIle TyrPheThr
Tyr '735
Ser
725 730
aag ctcaaa aacctggca gagaaggcc aagaaaatc tctgetgca
aac
2493
A Leus AsnLeuAla GluLysAla Lys:LysIle SerAlaAla
L
sn y 750 755
Lys
740
745
gat ctgatc gtgttt gaggetgaa gtactcaca ggcttcttc
aag tat
2541
u Ile ValPhe GluAlaGlu LeuThr PhePhe
L Tyr Val Gly
Asp e 765 770
Lys
760
tgc ggacat ccgttaaat attgttccc ccaccactg agtcctgga
cag
2589
Gln GlyHis ProLeuAsn IleValPro ProProLeu SerProGly
C
ys 780 785
775
get gatggt catgacagt gtggttgac aatgtctcc agccctgaa
ata
2637
Il A Gl HisAspSer ValValAsp AsnValSer SerProGlu
e sp y
Ala
790 795 800
ttt tt att tttagtggc atgcagget atacctcag tatttgtgg
acc g
2685
Ph ValIle PheSerGly MetGlnAla IleProGln TyrLeuTrp
e 810 815
Thr
805
aca acccag gaatatgta cagtcacaa gattactca tcaggacca
tgc
2733
Th Gln GluTyrVal GlnSerGln AspTyrSer SerGlyPro
Thr r 830 835
Cys
820
825
atg cccttt gcacagcat ccttggagg ggattcgca agtggcagc
aga
2781
P Phe AlaGlnHis ProTrpArg GlyPheAla SerGlySer
Met ro 845 850
Arg
840
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cct gtt gat taatctctac atcattttaa cagctggtat ggccttacct
2830
Pro Val Asp
tgggtgaact aaccaaataa tgaccatcga tggctcaaag agtggcttga atatatccca
2890
tgggttatct gtatggactg actgggttat tgaaaggact agccacatac tagcatctta
2950
gtgcctttat ctgtctttat gtcttggggt tggggtaggt agataccaaa tgaaacactt
3010
tcaggacctt ccttcctctt gcagttgttc tttaatctcc tttactagag gagataaata
3070
ttttgcatat aatgaagaaa tttttctagt atataacgca ggccttttat tttctaaaat
3130
gatgatagta taaaaatgtt aggataacag aatgatttta gattttccag agaatattat
3190
aaagtgcttt aggtatgaaa ataaatcatc tttgtctgat taaaaaaaaa aaa
3243
<210> 2
<211> 854
<212> PRT
<213> Homo sapiens
<900>
2 PheSer ValAlaGly AlaAlaAla 'PyrAsn LysSer
Met Met 10 Glu 15
Asp 5
1
GlyArg IleThrSer LeuSerLeu LeuPheGln LysValPhe AlaGln
20 25 30
IlePhe ProGlnTrp ArgLysGly AsnThrGlu GluCysLeu ProTyr
35 40 45
LysCys SerGluThr GlyAlaLeu GlyGluAsn TyrSerTrp GlnIle
50 55 60
ProIle AsnHisAsn AspPheLys IleLeuLys AsnAsnGlu ArgGln
65 70 75 80
LeuCys GluValLeu GlnAsnLys PheGlyCys IleSerThr LeuVal
85 90 95
SerPro ValGlnGlu GlyAsnSer LysSerLeu GlnValPhe ArgLys
100 105 110
MetLeu ThrProArg IleGluLeu SerValTrp LysAspAsp LeuThr
115 120 125
Thr AlaValAsp AlaValVal AsnAlaAla AsnGluAsp LeuLeu
His
CA 02348758 2001-04-27
WO 00/26231
135 140
130
PCT/US99/25439
His Gly Gly Gly Leu Ala Leu Ala Leu Val Lys Ala Gly Gly Phe Glu
150 155 160
145
Ile Gln Glu Glu Ser Lys Gln Phe Val Ala Arg Tyr Gly Lys Val Ser
165 170 175
Ala Gly Glu Ile Ala Val Thr Gly Ala Gly Arg Leu Pro Cys Lys Gln
185 190
180
Ile Ile His Ala Val Gly Pro Arg Trp Met Glu Trp Asp Lys Gln Gly
195 200 205
Cys Thr Gly Lys Leu Gln Arg Ala Ile Val Ser Ile Leu Asn Tyr Val
210 215 220
Ile Tyr Lys Asn Thr His Ile Lys Thr Val Ala I.Le Pro Ala Leu Ser
230 235 240
225
Ser Gly Ile Phe Gln Phe Pro Leu Asn Leu Cys Thr Lys Thr Ile Val
250 255
245
Glu Thr Ile Arg Val Ser Leu Gln Gly Lys Pro Met Met Ser Asn Leu
260 265 270
Lys Glu Ile His Leu Val Ser Asn Glu Asp Pro Thr Val Ala Ala Phe
275 280 285
Lys Ala Ala Ser Glu Phe Ile Leu Gly Lys Ser Glu Leu Gly Gln Glu
295 300
290
Thr Thr Pro Ser Phe Asn Ala Met Val Val Asn Asn Leu Thr Leu Gln
310 315 320
305
Ile Val Gln Gly His Ile Glu Trp Gln Thr Ala Asp Val Ile Val Asn
325 330 335
Ser Val Asn Pro His Asp Ile Thr Val Gly Pro Val Ala Lys Ser Ile
340 345 350
Leu Gln Gln Ala Gly Val Glu Met Lys Ser Glu Phe Leu Ala Thr Lys
355 360 365
AlalLys Gln Phe Gln Arg Ser Gln Leu Val Leu Val Thr Lys Gly Phe
370 375 380
Asn Leu Phe Cys Lys Tyr Ile Tyr His Val Leu Trp His Ser Glu Phe
390 395 400
385
Pro Lys Pro Gln Ile Leu Lys His Ala Met Lys Glu Cys Leu Glu Lys
405 410 915
Cys Ile Glu Gln Asn Ile Thr Ser Ile Ser Phe Pro Ala Leu Gly Thr
420 425 430
Gly Asn Met Glu Ile Lys Lys Glu Thr Ala Ala Glu Ile Leu Phe Asp
435 440 445
CA 02348758 2001-04-27
WO 00/26231
_g_
PCT/US99/25439
Glu Val Leu Thr Phe Ala Lys Asp His Val Lys His Gln Leu Thr Val
450 455 460
Lys Phe Val Ile Phe Pro Thr Asp Leu Glu Ile Tyr Lys Ala Phe Ser
470 475 480
4 65
Ser Glu Met Ala Lys Arg Ser Lys Met Leu Ser Leu Asn Asn Tyr Ser
485 490 495
Val Pro Gln Ser Thr Arg Glu Glu Lys Arg Glu Asn Gly Leu Glu Ala
500 505 510
Arg Ser Pro Ala Ile Asn Leu Met Gly Phe Asn Val Glu Glu Met Tyr
515 520 525
Glu Ala His Ala Trp Ile Gln Arg Ile Leu Ser Leu Gln Asn His His
530 535 590
Ile Ile Glu Asn Asn His Ile Leu Tyr Leu Gly Arg Lys Glu His Asp
550 555 560
545
Ile Leu Ser Gln Leu Gln Lys Thr Ser Ser Val Ser Ile Thr Glu Ile
565 570 575
Ile Ser Pro Gly Arg Thr Glu Leu Glu Ile Glu Gly Ala Arg Ala Asp
580 585 590
Leu Ile Glu Val Val Met Asn Ile Glu Asp Met Leu Cys Lys Val Gln
595 600 605
Glu Glu Met Ala Arg Lys Lys Glu Arg Gly Leu Trp Arg Ser Leu Gly
610 615 620
Gln Trp Thr Ile Gln Gln Gln Lys Thr Gln Asp Glu Met Lys Glu Asn
630 635 640
625
Ile Ile Phe Leu Lys Cys Pro Val Pro Pro Thr Gln Glu Leu Leu Asp
645 650 655
Gln Lys Lys Gln Phe Glu Lys Cys Gly Leu Gln Val Leu Lys Val Glu
660 665 670
Lys Ile Asp Asn Glu Val Leu Met Ala Ala Phe Gln Arg Lys Lys Lys
675 680 685
Met Met Glu Glu Lys Leu His Arg Gln Pro Val Ser His Arg Leu Phe
690 695 700
Gln Gln Val Pro Tyr Gln Phe Cys Asn Val Val Cys Arg Val Gly Phe
710 715 720
705
Gln Arg Met Tyr Ser Thr Pro Cys Asp Pro Lys Tyr Gly Ala Gly Ile
725 730 735
Tyr Phe Thr Lys Asn Leu Lys Asn Leu Ala Glu Lys Ala Lys Lys Ile
740 745 750
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-9-
SerAlaAlaAsp LysLeu IleTyrVal PheGluAla GluValLeu Thr
755 760 765
GlyPhePheCys GlnGly HisProLeu AsnIleVal ProProPro Leu
770 775 780
SerProGlyAla IleAsp GlyHisAsp SerValVal AspAsnVal Ser
785 790 795 800
SerProGluThr PheVal IlePheSer GlyMetGln AlaIlePro Gln
805 810 815
TyrLeuTrpThr CysThr GlnGluTyr ValGlnSer GlnAspTyr Ser
820 825 830
SerGlyProMet ArgPro PheAlaGln HisProTrp ArgGlyPhe Ala
835 840 895
SerGlySerPro ValAsp
850
<210> 3
<211> 2562 '
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1}..(2562)
<400> 3
atg gac ttt tcc atg gtg gcc gga gca gca get tac aat gaa aaa tca 48
Met Asp Phe Ser Met Val Ala Gly Ala Ala Ala Tyr Asn Glu Lys Ser
1 5 10 15
ggt agg att acc tcg ctc tca ctc ttg ttt cag aaa gtc ttt get cag 96
Gly Arg Ile Thr Ser Leu Ser Leu Leu Phe Gln Lys Val Phe Ala Gln
20 25 30
atc ttt cct cag tgg aga aag ggg aat aca gaa gaa tgt ctc ccc tac 199
Ile Phe Pro Gln Trp Arg Lys Gly Asn Thr Glu Glu Cys Leu Pro Tyr
35 40 45
aag tgc tca gag act ggt get ctt gga gaa aac tat agt tgg caa att 192
Lys Cys Ser Glu Thr Gly Ala Leu Gly Glu Asn Tyr Ser Trp Gl.n Ile
50 55 60
ccc att aac cac aat gac ttc aaa att tta aaa aat aat gag cgt cag 240
Pro Ile Asn His Asn Asp Phe Lys Ile Leu Lys Asn Asn Glu Arg Gln
65 70 75 80
ctg tgt gaa gtc ctc cag aat aag ttt ggc tgt atc tct acc ctg gtc 288
Leu Cys Glu Val Leu Gln Asn Lys Phe Gly Cys I:le Ser Thr Leu Val
85 90 95
tct cca gtt cag gaa ggc aac agc aaa tct ctg caa gtg ttc aga aaa 336
Ser Pro Val Gln Glu Gly Asn Ser Lys Ser Leu Gln Val Phe Arg Lys
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-10-
100 105 110
atg ctg act cct agg ata gag tta tca gtc tgg aaa gat gac ctc acc 389
Met Leu Thr Pro Arg Ile Glu Leu Ser Val Trp Lys Asp Asp Leu Thr
115 120 125
aca cat get gtt gat get gtg gtg aat gca gcc aat gaa gat ctt ctg 432
Thr His Ala Val Asp Ala Val Val Asn Ala Ala Asn Glu Asp Leu Leu
130 135 140
cat ggg gga ggc ctg gcc ctg gcc ctg gta aaa get ggt gga ttt gaa 480
His Gly Gly Gly Leu Ala Leu Ala Leu Val Lys Ala Gly Gly Phe Glu
195 150 155 160
atc caa gaa gag agc aaa cag ttt gtt gcc aga t:at ggt aaa gtg tca 528
Ile Gln Glu Glu Ser Lys Gln Phe Val Ala Arg 7.'yr Gly Lys Val Ser
165 170 175
get ggt gag ata get gtc acg gga gca ggg agg cat ccc tgc aaa cag 575
Ala Gly Glu Ile Ala Val Thr Gly Ala Gly Arg Leu Pro Cys Lys Gln
180 185 190
atc atc cat get gtt ggg cct cgg tgg atg gaa t:gg gat aaa cag gga 624
Ile Ile His Ala Val Gly Pro Arg Trp Met Glu Trp Asp Lys Gln Gly
195 200 205
tgt act gga aag ctg cag agg gcc att gta agt att ctg aat tat gtc 672
Cys Thr Gly Lys Leu Gln Arg Ala Ile Val Ser I:le Leu Asn Tyr Val
210 215 2.20
atc tat aaa aat act cac att aag aca gta gca att cca gcc ttg agc 720
Ile Tyr Lys Asn Thr His Ile Lys Thr Val Ala I:le Pro Ala Leu Ser
225 230 235 240
tct ggg att ttt cag ttc cct ctg aat ttg tgt aca aag act att gta 768
Ser Gly Ile Phe Gln Phe Pro Leu Asn Leu Cys Thr Lys Thr Ile Val
295 250 255
gag act atc cgg gtt agt ttg caa ggg aag cca atg atg agt aat ttg 816
Glu Thr Ile Arg Val Ser Leu Gln Gly Lys Pro Met Met Ser Asn Leu
260 265 270
aaa gaa att cac ctg gtg agc aat gag gac cct act gtt get gcc ttt 864
Lys Glu Ile His Leu Val Ser Asn Glu Asp Pro Thr Val Ala Ala Phe
275 280 285
aaa get get tca gaa ttc atc cta ggg aag agt gag ctg gga caa gaa 912
Lys Ala Ala Ser Glu Phe Ile Leu Gly Lys Ser Glu Leu Gly Gln Glu
290 295 300
acc acc cct tct ttc aat gca atg gtc gtg aac aac ctg acc ctc cag 960
Thr Thr Pro Ser Phe Asn Ala Met Val Val Asn Asn Leu Thr Leu Gln
305 310 315 320
att gtc cag ggc cac att gaa tgg cag acg gca gat gta att gtt aat
1008
Ile Val Gln Gly His Ile Glu Trp Gln Thr Ala Asp Val Ile Val Asn
325 330 335
Met Met Glu Glu Lys Leu His Arg Gln Pro V
CA 02348758 2001-04-27
WO OO/Z6231 PCT/US99/25439
-11-
tct gta aac cca cat gat att aca gtt gga cct gtg gca aag tca att
1056
Ser Val Asn Pro His Asp Ile Thr Val Gly Pro Val Ala Lys Ser Ile
390 345 350
cta caa caa gca gga gtt gaa atg aaa tcg gaa ttt ctt gcc aca aag
1104
Leu Gln Gln Ala Gly Val Glu Met Lys Ser Glu Phe Leu Ala Th.r Lys
355 360 365
get aaa cag ttt caa cgg tcc cag ttg gta ctg gtc aca aaa gga ttt
1152
Ala Lys Gln Phe Gln Arg Ser Gln Leu Val' Leu Val Thr Lys Gly Phe
370 375 380
aac ttg ttc tgt aaa tat ata tac cat gta ctg tgg cat tca gaa ttt
1200
Asn Leu Phe Cys Lys Tyr Ile Tyr His Val Leu Trp His Ser Glu Phe
385 390 395 400
cct aaa cct cag ata tta aaa cat gca atg aag gag tgt ttg gaa aaa
1248
Pro Lys Pro Gln Ile Leu Lys His Ala Met Lys Glu Cys Leu Glu Lys
405 410 415
tgc att gag caa aat ata act tcc att tcc ttt cct gcc ctt ggg act
1296
Cys Ile Glu Gln Asn Ile Thr Ser Ile Ser Phe Pro Ala Leu Gly Thr
420 425 430
gga aac atg gaa ata aag aag gaa aca gca gca gag att ttg ttt gat
1344
Gly Asn Met Glu Ile Lys Lys Glu Thr Ala Ala Glu Ile Leu Phe Asp
935 490 945
gaa gtt tta aca ttt gcc aaa gac cat gta aaa cac cag tta act gta
1392
Glu Val Leu Thr Phe Ala Lys Asp His Val Lys His Gln Leu Thr Val
950 955 460
aaa ttt gtg atc ttt cca aca gat ttg gag ata tat aag get ttc agt
1440
Lys Phe Val Ile Phe Pro Thr Asp Leu Glu Ile Tyr Lys Ala Phe Ser
965 470 975 480
tct gaa atg gca aag agg tcc aag atg ctg agt ttg aac aat tac agt
1488
Ser Glu Met Ala Lys Arg Ser Lys Met Leu Ser Leu Asn Asn Tyr Ser
485 490 495
gtc ccc cag tca acc aga gag gag aaa aga gaa aat ggg ctt gaa get
1536
Val Pro Gln Ser Thr Arg Glu Glu Lys Arg Glu Asn Gly Leu Glu Ala
500 505 510
aga tct cct gcc atc aat ctg atg gga ttc aac gtg gaa gag atg tat
CA 02348758 2001-04-27
WO 00/26231 PCTNS99/25439
-12-
1584
Arg Ser Pro Ala Ile Asn Leu Met Gly Phe Asn Val Glu Glu Met Tyr
515 520 525
gag gcc cac gca tgg atc caa aga atc ctg agt ca c cag aac cac cac
1632
Glu Ala His Ala Trp Ile Gln Arg Ile Leu Ser Leu Gln Asn His His
530 535 540
atc att gag aat aat cat att ctg tac ctt ggg aga aag gaa cat gac
1680
Ile Ile Glu Asn Asn His Ile Leu Tyr Leu Gly Arg Lys Glu His Asp
545 550 555 560
att ttg tct cag ctt cag aaa act tca agt gtc tcc atc aca gaa att
1728
Ile Leu Ser Gln Leu Gln Lys Thr Ser Ser Val Ser Ile Thr Glu Ile
565 570 575
atc agc cca gga agg aca gag tta gag att gaa gga gcc egg get gac
1776
Ile Ser Pro Gly Arg Thr Glu Leu Glu Ile Glu Clly Ala Arg Ala Asp
580 585 590
ctc att gag gtg gtt atg aac att gaa gat atg ctt tgt aaa gta cag
1824
Leu Ile Glu Val Val Met Asn Ile Glu Asp Met Leu Cys Lys Val Gln
595 600 605
gag gaa atg gca agg aaa aag gag cga ggc ctt tgg cgc tcg tta gga
1872
Glu Glu Met Ala Arg Lys Lys Glu Arg Gly Leu Trp Arg Ser Leu Gly
610 615 Ei20
cag tgg act att cag caa caa aaa acc caa gac gaa atg aaa gaa aat
1920
Gln Trp Thr Ile Gln Gln Gln Lys Thr Gln Asp Glu Met Lys Glu Asn
625 630 635 640
atc ata ttt ctg aaa tgt cct gtg cct cca act c;aa gag ctt cta gat
1968
Ile Ile Phe Leu Lys Cys Pro Val Pro Pro Thr C:ln Glu Leu Leu Asp
645 650 655
caa aag aaa cag ttt gaa aaa tgt ggt ttg cag gtt cta aag gtg gag
2016
Gln Lys Lys Gln Phe Glu Lys Cys Gly Leu Gln Val Leu Lys Val Glu
660 665 670
aag ata gac aat gag gtc ctt atg get gec ttt c;aa aga aag aag aaa
2064
Lys Ile Asp Asn Glu Val Leu Met Ala Ala Phe Gln Arg Lys Lys Lys
675 680 685
atg atg gaa gaa aaa ctg cac agg caa cct gtg agc cat agg ctg ttt
2112
Met Met Glu Glu Lys Leu His Arg Gln Pro Val Ser His Arg Leu Phe
690 695 700
CA 02348758 2001-04-27
WO 00/26231 PCTNS99/25439
-13-
cag caa gtc cca tac cag ttc tgc aat gtg gta tgc aga gtt ggc ttt
2160
Gln Gln Val Pro Tyr Gln Phe Cys Asn Val Val Cys Arg Val Gly Phe
705 710 715 720
caa aga atg tac tcg aca cct tgc gat cca aaa tac gga get ggc ata
2208
Gln Arg Met Tyr Ser Thr Pro Cys Asp Pro Lys Tyr Gly Ala Gly Ile
725 730 735
tac ttc acc aag aac ctc aaa aac ctg gca gag aag gcc aag aaa atc
2256
Tyr Phe Thr Lys Asn Leu Lys Asn Leu Ala Glu Lys Ala Lys Lys Ile
740 745 750
tct get gca gat aag ctg atc tat gtg ttt gag get gaa gta ctc aca
2304
Ser Ala Ala Asp Lys Leu Ile Tyr Val Phe Glu Ala Glu Val Leu Thr
755 760 765
ggc ttc ttc tgc cag gga cat ccg tta aat att gtt ccc cca cca ctg
2352
Gly Phe Phe Cys Gln Gly His Pro Leu Asn Ile Val Pro Pro Pro Leu
770 775 780
agt cct gga get ata gat ggt cat gac agt gtg gtt gac aat gtc tcc
2400
Ser Pro Gly Ala Ile Asp Gly His Asp Ser Val Val Asp Asn Val Ser
785 790 795 800
agc cct gaa acc ttt gtt att ttt agt ggc atg cag get ata cct cag
2448
Ser Pro Glu Thr Phe Val Ile Phe Ser Gly Met Gln Ala Ile Pro Gln
805 810 815
tat ttg tgg aca tgc acc cag gaa tat gta cag tca caa gat tac tca
2496
Tyr Leu Trp Thr Cys Thr Gln Glu Tyr Val Gln Ser Gln Asp Tyr Ser
820 825 830
tca gga cca atg aga ccc ttt gca cag cat cct tgg agg gga ttc gca
2544
Ser Gly Pro Met Arg Pro Phe Ala Gln His Pro Trp Arg Gly Phe Ala
835 840 845
agt ggc agc cct gtt gat
2562
Ser Gly Ser Pro Val Asp
850
<210> 9
<211> 3029
<212> DNA
<213> Murinae gen. sp.
<220>
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-14-
<221> CDS
<222> (171)..(2648)
<400> 4
aggaacggaa gtttggcggg aacccggatt cccaggttca ggcctctcaa gggtggagcg 60
gaatagaggg aaacaggcca ccatctcctc gatctacaga ctacacttgg aaacacaaac 120
aaatataaat atctgaagac ccacgtggga cctgaagaat ggcctattac atg gat 176
Met Asp
1
aca tgg gcg gca get ccc gcc gaa aga cca gcc aac aat tct ctt gaa 224
Thr Trp Ala Ala Ala Pro Ala Glu Arg Pro Ala Asn Asn Ser Leu Glu
10 15
gaa cat tat aga tgg caa att ccc att aaa cac aat gtc ttc gaa att 272
Glu His Tyr Arg Trp G1'n Ile Pro Ile Lys His Asn Val Phe Glu Ile
20 25 30
tta aag agc aat gag agt cag cta tgt gaa gtc ctc caa aat aag ttt 320
Leu Lys Ser Asn Glu Ser Gln Leu Cys Glu Val Leu Gln Asn Lys Phe
35 40 45 50
gga tgc atc tct acc ctg agc tgt cca act cta gca ggg agc agc tct 368
Gly Cys Ile Ser Thr Leu Ser Cys Pro Thr Leu Ala Gly Ser Ser Ser
55 60 65
cctget cagagagtc ttcagaagg accctgatc cctgggata gagtta 416
ProAla GlnArgVal PheArgArg ThrLeuIle ProGlyIle GluLeu
70 75 80
tctgtc tggaaggat gaccttacc agacacgtt gttgatget gtggtg 464
SerVal TrpLysAsp AspLeuThr ArgHisVal ValAspAla ValVal
85 90 95
aacgca gccaatgaa aaccttttg catggaagt ggcctggcc ggaagc 512
AsnAla AlaAsnGlu AsnLeuLeu HisGlySer GlyLeuAla GlySer
100 105 110
ttggtg aaaactggt ggctttgaa atccaagaa gagagcaaa agaatc 560
LeuVal LysThrGly GlyPheGlu IleGlnGlu GluSerLys ArgIle
115 120 125 130
attgcc aacgttggt aaaatctca gttggtgga atcgetatc accggt 608
IleAla AsnValGly LysIleSer ValGlyGly IleAlaIle ThrGly
135 190 145
gcgggg agacttcct tgccatttg attatccat gcggttgga cctcgg 656
AlaGly ArgLeuPro CysHisLeu IleIleHis AlaValGly ProArg
150 155 160
tggaca gttacgaac agccagaca getatcgaa ttactgaaa tttgcc 709
TrpThr ValThrAsn SerGlnThr AlaIleGlu LeuLeuLys PheAla
165 170 175
attagg aacattcta gattatgtc accaaatat gatctacgc attaag 752
IleArg AsnIleLeu AspTyrVal ThrLysTyr AspLeuArg IleLys
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-15-
180 185 190
aca gta gca att cca gcc ctg agc tct gga att ttc cag ttc cct ctg 800
Thr Val Ala Ile Pro Ala Leu Ser Ser Gly Ile Phe Gln Phe Pro Leu
195 200 205 210
gat ttg tgt aca agc ata att tta gaa act atc cgg ctt tat ttc caa 848
Asp Leu Cys Thr Ser Ile Ile Leu Glu Thr Ile Arg Leu Tyr Phe Gln
215 220 225
gac aag caa atg ttc ggt aat ttg aga gag att cat ctg gtg agc aat 896
Asp Lys Gln Met Phe Gly Asn Leu Arg Glu Ile H.is Leu Val Ser Asn
230 235 240
gag gac ccc act gtt gcg tcc ttt aaa tcc gcc tca gaa agc atc cta 944
Glu Asp Pro Thr Val Ala Ser Phe Lys Ser Ala Ser Glu Ser Ile Leu
245 250 255
ggg agg gac ctg agc tct tgg ggg ggt cca gaa act gac cct get tcc 992
Gly Arg Asp Leu Ser Ser Trp Gly Gly Pro Glu Thr Asp Pro Ala Ser
260 265 270
acc atg act ctt cgc atc ggc cgg ggc ctg act ctc cag att gtc caa
1040
Thr Met Thr Leu Arg Ile Gly Arg Gly Leu Thr L~eu Gln Ile Val Gln
275 280 285 290
ggc tgt att gaa atg caa aca aca gat gta att ggt aat tct gga tac
lobe
Gly Cys Ile Glu Met Gln Thr Thr Asp Val Ile Gly Asn Ser Gly Tyr
295 300 305
atg cag gat ttt aaa tca gga cga gtg gca cag tcg att ctt aga caa
1136
Met Gln Asp Phe Lys Ser Gly Arg Val Ala Gln Ser Ile Leu Arg Gln
310 315 320
gca ggg gtt gaa atg gaa aag gaa ctt gac aag gtt aac ctg tcc aca
1184
Ala Gly Val Glu Met Glu Lys Glu Leu Asp Lys Val Asn Leu Ser Thr
325 330 335
gat tat caa gag gtg tgg gtc aca aaa gga ttt aaa ttg tcc tgt cag
1232
Asp Tyr Gln Glu Val Trp Val Thr Lys Gly Phe Lys Leu Ser Cys Gln
340 345 3.50
tat gtc ttc cat gtg gca tgg cat tcc caa atc aac aaa tac cag ata
1280
Tyr Val Phe His Val Ala Trp His Ser Gln Ile Asn Lys Tyr Gln Ile
355 360 365 370
ttg aaa gat gca atg aag tcc tgt cta gaa aaa t.gc ctt aaa cca gat
1328
Leu Lys Asp Ala Met Lys Ser Cys Leu Glu Lys C.'ys Leu Lys Pro Asp
375 380 385
ata aat tcc att tcc ttt cct get ctc ggg aca gga ttg atg gat ttg
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-16-
1376
Ile Asn Ser Ile Ser Phe Pro Ala Leu Gly Thr Gly Leu Met Asp Leu
390 395 400
aag aag agt aca gca get cag ata atg ttt gag gaa gtt ttt gca ttt
1924
Lys Lys Ser Thr Ala Ala Gln Ile Met Phe Glu Glu Val Phe Al_a Phe
405 410 415
get aaa gag cac aag gaa aaa acg cta act gta aag att gtg atc ttt
1472
Ala Lys Glu His Lys Glu Lys Thr Leu Thr Val Lys Ile Val Ile Phe
420 425 430
cca gta gat gtg gag acg tac aag att ttt tat get gaa atg aca aaa
1520
Pro Val Asp Val Glu Thr Tyr Lys Ile Phe Tyr Ala Glu Met Thr Lys
435 490 445 450
agg tcc aac gag ctg aat ctc agc ggt aat agt ggt get tta gcc ctg
1568
Arg Ser Asn Glu Leu Asn Leu Ser Gly Asn Ser Gly Ala Leu Ala Leu
455 460 965
cag tgg tcc agt ggg gag caa aga aga ggc ggc ctt gaa get gga tct
1616
Gln Trp Ser Ser Gly Glu Gln Arg Arg Gly Gly Leu Glu Ala Gly Ser
470 475 480
cct gcc atc aat ctc atg ggt gta aaa gtg gga gag atg tgt gag gcc
1664
Pro Ala Ile Asn Leu Met Gly Val Lys Val Gly Glu Met Cys Glu Ala
485 490 495
cag gaa tgg att gaa agg ttg ctg gtc tcc ctg gac cac cac atc att
1712
Gln Glu Trp Ile Glu Arg Leu Leu Val Ser Leu Asp His His Ile Ile
500 505 510
gag aat aat cat att ctc tat ctt ggg aaa aaa gag cac gac gtg ctg
1760
Glu Asn Asn His Ile Leu Tyr Leu Gly Lys Lys Glu His Asp Val Leu
515 520 525 530
tct gag ctc cag acc agc aca aga gtc tcc att tca gag act gtc agt
1808
Ser Glu Leu Gln Thr Ser Thr Arg Val Ser Ile Ser Glu Thr Val Ser
535 540 545
cca aga acg gcc act ttg gag att aaa ggt ccc cag get gac ctc att
1856
Pro Arg Thr Ala Thr Leu Glu Ile Lys Gly Pro Gln Ala Asp Leu Ile
550 555 560
gac gca gtt atg agg att gaa tgt atg ctg tgt gac gtt cag gaa gaa
1904
Asp Ala Val Met Arg Ile Glu Cys Met Leu Cys Asp Val Gln Glu Glu
565 570 575
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
- 17-
gtg gca gga aaa agg gag aaa aat ctt tgg agc t.tg tca gga cag ggg
1952
Val Ala Gly Lys Arg Glu Lys Asn Leu Trp Ser heu Ser Gly Gln Gly
580 585 590
acc aac cag caa gaa aaa ctg gat aaa atg gaa gaa tcg tac aca ttt
2000
Thr Asn Gln Gln Glu Lys Leu Asp Lys Met Glu Glu Ser Tyr Thr Phe
595 600 605 610
caa cga tac cca gca tca tta act cag gaa ctt cag gac cga aag aaa
2098
Gln Arg Tyr Pro Ala Ser Leu Thr Gln Glu Leu Gln Asp Arg Lys Lys
615 620 625
cag ttt gaa aag tgt ggc ttg tgg gtt gtg cag gtg gag cag ata gac
2096
Gln Phe Glu Lys Cys Gly Leu Trp Val Val Gln Val Glu Gln Ile Asp
630 635 640
aat aag gtg ctg ctg get gcc ttc caa gag aag aag aaa atg atg gaa
2144
Asn Lys Val Leu Leu Ala Ala Phe Gln Glu Lys hys Lys Met Met Glu
645 650 655
gag agg acg cca aag gga tct ggg agc caa agg t.tg ttt cag cag gtc
2192
Glu Arg Thr Pro Lys Gly Ser Gly Ser Gln Arg Leu Phe Gln Gln Val
660 665 670
cca cat cag ttc tgc aat acg gtg tgc aga gtc ggc ttc cac aga atg
2240
Pro His Gln Phe Cys Asn Thr Val Cys Arg Val Gly Phe His Arg Met
675 680 685 690
tat tcg aca tcc tat aac cca gtt tat gga gcc ggc ata tat ttc acc
2288
Tyr Ser Thr Ser Tyr Asn Pro Val Tyr Gly Ala Gly Ile Tyr Phe Thr
695 700 705
aag agc ctc aaa aat cta gca gac aag gtc aag aaa acc tca agc aca
2336
Lys Ser Leu Lys Asn Leu Ala Asp Lys Val Lys Lys Thr Ser Ser Thr
710 715 720
gac aag cta atc tat gtg ttt gag gca gaa gta ctc aca ggg tcc ttc
2384
Asp Lys Leu Ile Tyr Val Phe Glu Ala Glu Val Leu Thr Gly Ser Phe
725 730 735
tgt cag ggt aat tcc tca aat atc atc cct cca c:ca ttg agt cct ggg
2932
Cys Gln Gly Asn Ser Ser Asn Ile Ile Pro Pro Pro Leu Ser Pro Gly
740 745 750
gcc tta gat gtc aat gac agc gta gtt gac aat gtt tcc agc cct gaa
2480
CA 02348758 2001-04-27
WO 00/Z6231 PCTNS99/25439
-18-
Ala Leu Asp Val Asn Asp Ser Val Val Asp Asn Val Ser Ser Pro Glu
755 760 765 770
acc att gtt gtt ttt aat ggc atg cag gcc atg ccc ctg tac ttg tgg
2528
Thr Ile Val Val Phe Asn Gly Met Gln Ala Met Pro Leu Tyr Leu Trp
775 780 785
act tgc aca cag gat agg aca ttc tca cag cat ccg atg tgg tca cag
2576
Thr Cys Thr Gln Asp Arg Thr Phe Ser Gln His Pro Met Trp Ser Gln
790 795 800
gac tac tca tca gga cca gga atg gtc tct tcg ctg cag tcc tgg gaa
2624
Asp Tyr Ser Ser Gly Pro Gly Met Val Ser Ser Leu Gln Ser Trp Glu
805 810 815
tgg gtc tta aat ggc agc tct gtt tagtgtctac atcagtttaa caagcagaag
2678
Trp Val Leu Asn Gly Ser Ser Val
820 825
gggttgagag aactgacaaa atgataaata acaggttacc tgttcagaat gatggggtca
2738
ctaaaggcac cgaccacaca ctagcatcat agtgcctttg tctttacctc tgggcttgac
2798
tgggcagatg ccagctaaaa cttcctcact gtcttttcta tttgacatct ttcatctcct
2858
ttcctatagg tgacagcaag aatactttat atagaacaag gatatttttt tcaagcctgt
2918
tattttctaa aatgatagca caaactagga caacaggatg atttcaggtt ttctatataa
2978
tttataaagt gctttggata tccaaataaa tcacctttgt ctgagt
3024
<210> 5
<211> 826
<212> PRT
<213> Murinae gen. sp.
<400> 5
Met Asp Thr Trp Ala Ala Ala Pro Ala Glu Arg Pro Ala Asn Asn Ser
1 5 10 15
Leu Glu Glu His Tyr Arg Trp Gln Ile Pro Ile Lys His Asn Val Phe
20 25 30
Glu Ile Leu Lys Ser Asn Glu Ser Gln Leu Cys Glu Val Leu Gln Asn
35 40 45
Lys Phe Gly Cys Ile Ser Thr Leu Ser Cys Pro Thr Leu Ala Gly Ser
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-19-
50 55 60
Ser Ser Pro Ala Gln Arg Val Phe Arg Arg Thr Leu Ile Pro Gly Ile
65 70 75 80
Glu Leu Ser Val Trp Lys Asp Asp Leu Thr Arg His Val Val Asp Ala
85 90 95
Val Val Asn Ala Ala Asn Glu Asn Leu Leu His Gly Ser Gly Leu Ala
100 105 110
Gly Ser Leu Val Lys Thr Gly Gly Phe Glu Ile Gln Glu Glu Ser Lys
115 120 125
Arg Ile Ile Ala Asn Val Gly Lys Ile Ser Val Gly Gly Ile Ala Ile
130 135 140
Thr Gly Ala Gly Arg Leu Pro Cys His Leu Ile Ile His Ala Val Gly
145 150 155 160
Pro Arg Trp Thr Val Thr Asn Ser Gln Thr Ala Ile Glu Leu Leu Lys
165 170 175
Phe Ala Ile Arg Asn Ile Leu Asp Tyr Val Thr Lys Tyr Asp Leu Arg
180 185 190
Ile Lys Thr Val Ala Ile Pro Ala Leu Ser Ser Gly Ile Phe Gln Phe
195 200 205
Pro Leu Asp Leu Cys Thr Ser Ile Ile Leu Glu Thr Ile Arg Leu Tyr
210 215 220
Phe Gln Asp Lys Gln Met Phe Gly Asn Leu Arg Glu Ile His Leu Val
225 230 235 240
Ser Asn Glu Asp Pro Thr Val Ala Ser Phe Lys Ser Ala Ser Glu Ser
295 250 255
Ile Leu Gly Arg Asp Leu Ser Ser Trp Gly Gly Pro Glu Thr Asp Pro
260 265 270
Ala Ser Thr Met Thr Leu Arg Ile Gly Arg Gly :Leu Thr Leu Gln Ile
275 280 285
Val Gln Gly Cys Ile Glu Met Gln Thr Thr Asp Val Ile Gly Asn Ser
290 295 300
Gly Tyr Met Gln Asp Phe Lys Ser Gly Arg Val Ala Gln Ser Ile Leu
305 310 315 320
Arg Gln Ala Gly Val Glu Met Glu Lys Glu Leu Asp Lys Val Asn Leu
325 330 335
Ser Thr Asp Tyr Gln Glu Val Trp Val Thr Lys Gly Phe Lys Leu Ser
340 345 350
Cys Gln Tyr Val Phe His Val Ala Trp His Ser Gln Ile Asn Lys Tyr
355 360 365
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-20-
Gln Ile Leu Lys Asp Ala Met Lys Ser Cys Leu Glu Lys Cys Leu Lys
370 375 380
Pro Asp Ile Asn Ser Ile Ser Phe Pro Ala Leu Gly Thr Gly Leu Met
385 390 395 900
Asp Leu Lys Lys Ser Thr Ala Ala Gln Ile Met F'he Glu Glu Val Phe
905 410 415
Ala Phe Ala Lys Glu His Lys Glu Lys Thr Leu Thr Val Lys Ile Val
420 425 430
Ile Phe Pro Val Asp Val Glu Thr Tyr Lys Ile Phe Tyr Ala Glu Met
435 490 445
Thr Lys Arg Ser Asn Glu Leu Asn Leu Ser Gly Asn Ser Gly Ala Leu
950 455 460
Ala Leu Gln Trp Ser Ser Gly Glu Gln Arg Arg Gly Gly Leu Glu Ala
465 470 475 980
Gly Ser Pro Ala Ile Asn Leu Met Gly Val Lys Val Gly Glu Met Cys
985 490 995
Glu Ala Gln Glu Trp Ile Glu Arg Leu Leu Val Ser Leu Asp His His
500 505 510
Ile Ile Glu Asn Asn His Ile Leu Tyr Leu Gly Lys Lys Glu His Asp
515 520 525
Val Leu Ser Glu Leu Gln Thr Ser Thr Arg Val Ser Ile Ser Glu Thr
530 535 590
Val Ser Pro Arg Thr Ala Thr Leu Glu Ile Lys Gly Pro Gln Ala Asp
545 550 555 560
Leu Ile Asp Ala Val Met Arg Ile Glu Cys Met Leu Cys Asp Val Gln
565 570 575
Glu Glu Val Ala Gly Lys Arg Glu Lys Asn Leu Trp Ser Leu Ser Gly
580 585 590
Gln Gly Thr Asn Gln Gln Glu Lys Leu Asp Lys Met Glu Glu Ser Tyr
595 600 605
Thr Phe Gln Arg Tyr Pro Ala Ser Leu Thr Gln Glu Leu Gln Asp Arg
610 615 620
Lys Lys Gln Phe Glu Lys Cys Gly Leu Trp Val Val Gln Val Glu Gln
625 630 635 640
Ile Asp Asn Lys Val Leu Leu Ala Ala Phe Gln Glu Lys Lys Lys Met
645 650 655
Met Glu Glu Arg Thr Pro Lys Gly Ser Gly Ser Gln Arg Leu Phe Gln
660 665 670
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-21
Gln Val Pro His Gln Phe Cys Asn Thr Val Cys Arg Val Gly Phe His
675 680 685
Arg Met Tyr Ser Thr Ser Tyr Asn Pro Val Tyr Gly Ala Gly Ile Tyr
690 695 700
Phe Thr Lys Ser Leu Lys Asn Leu Ala Asp Lys Val Lys Lys Thr Ser
705 710 715 720
Ser Thr Asp Lys Leu Ile Tyr Val Phe Glu Ala Glu Val Leu Thr Gly
725 730 735
Ser Phe Cys Gln Gly Asn Ser Ser Asn Ile Ile Pro Pro Pro Leu Ser
740 745 750
Pro Gly Ala Leu Asp Val Asn Asp Ser Val Val Asp Asn Val Ser Ser
755 760 765
Pro Glu Thr Ile Val Val Phe Asn Gly Met Gln Ala Met Pro Leu Tyr
770 775 780
Leu Trp Thr Cys Thr Gln Asp Arg Thr Phe Ser Gln His Pro Met Trp
785 790 795 800
Ser Gln Asp Tyr Ser Ser Gly Pro Gly Met Val Ser Ser Leu Gln Ser
805 810 815
Trp Glu Trp Val Leu Asn Gly Ser Sex Val
820 825
<210> 6
<211> 2478
<212> DNA
<213> Murinae gen. sp.
<220>
<221> CDS
<222> (1)..(2478)
<900> 6
atg gat aca tgg gcg gca get ccc gcc gaa aga cca gcc aac aat tct 98
Met Asp Thr Trp Ala Ala Ala Pro Ala Glu Arg Pro Ala Asn Asn Ser
1 5 10 15
ctt gaa gaa cat tat aga tgg caa att ccc att aaa cac aat gtc ttc 96
Leu Glu Glu His Tyr Arg Trp Gln Ile Pro Ile Lys His Asn Val Phe
20 25 30
gaa att tta aag agc aat gag agt cag cta tgt gaa gtc ctc caa aat 149
Glu Ile Leu Lys Ser Asn Glu Ser Gln Leu Cys Glu Val Leu Gln Asn
35 40 45
aag ttt gga tgc atc tct acc ctg agc tgt cca act cta gca ggg agc 192
Lys Phe Gly Cys Ile Ser Thr Leu Ser Cys Pro Thr Leu Ala Gly Ser
50 55 60
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
-22-
agc tctcctget cagagagtc ttcagaagg accctgatc cctgggata 240
Ser SerProAla GlnArgVal PheArgArg ThrL~euIle ProGlyIle
65 70 75 80
gag ttatctgtc tggaaggat gaccttacc agacacgtt gttgatget 288
Glu LeuSerVal TrpLysAsp AspLeuThr ArgHisVal ValAspAla
85 90 95
gtg gtgaacgca gccaatgaa aaccttttg catggaagt ggcctggcc 336
Val ValAsnAla AlaAsnGlu AsnLeuLeu HisGlySer GlyLeuAla
100 105 110
gga agcttggtg aaaactggt ggctttgaa atccaagaa gagagcaaa 384
Gly SerLeuVal LysThrGly GlyPheGlu IleG'lnGlu GluSerLys
115 120 125
aga atcattgcc aacgttggt aaaatctca gttggtgga atcgetatc 432
Arg IleIleAla AsnValGly LysIleSer ValGIyGly IleAlaIle
130 135 140
acc ggtgcgggg agacttcct tgccatttg attatccat gcggttgga 480
Thr GlyAlaGly ArgLeuPro CysHisLeu IleIleHis AlaValGly
145 150 155 160
cct cggtggaca gttacgaac agccagaca getatcgaa ttactgaaa 528
Pro ArgTrp.Thr ValThrAsn SerGlnThr AlaIleGlu LeuLeuLys
165 170 175
ttt gccattagg aacattcta gattatgtc accaaatat gatctacgc 576
Phe AlaIleArg AsnIleLeu AspTyrVal ThrLysTyr AspLeuArg
180 185 190
att aagacagta gcaattcca gccctgagc tctggaatt ttccagttc 624
Ile LysThrVal AlaIlePro AlaLeuSer SerGlyIle PheGlnPhe
195 200 205
cct ctggatttg tgtacaagc ataatttta gaaactatc cggctttat 672
Pro LeuAspLeu CysThrSer IleIleLeu GluThrIle ArgLeuTyr
210 215 220
ttc caagacaag caaatgttc ggtaatttg agagagatt catctggtg 720
Phe GlnAspLys GlnMetPhe GlyAsnLeu ArgGluIle HisLeuVal
225 230 235 240
agc aatgaggac cccactgtt gcgtccttt aaat:ccgcc tcagaaagc 768
Ser AsnGluAsp ProThrVal AlaSerPhe Lys:>erAla SerGluSer
245 250 255
atc ctagggagg gacctgagc tcttggggg ggtc:cagaa actgaccct 816
Ile LeuGlyArg AspLeuSer SerTrpGly GlyProGlu ThrAspPro
260 265 270
get tccaccatg actcttcgc atcggccgg ggcca act ctccagatt 864
g
Ala SerThrMet ThrLeuArg IleGlyArg GlyLeuThr LeuGlnIle
275 280 285
gtc caaggctgt attgaaatg caaacaaca gatgtaatt ggtaattct 912
Val GlnGlyCys IleGluMet GlnThrThr AspValIle GlyAsnSer
CA 02348758 2001-04-27
WO 00/26231 PCT/US99/25439
- 23 -
290 295 300
gga tac atg cag gat ttt aaa tca gga cga gtg gca cag tcg att ctt 960
Gly Tyr Met Gln Asp Phe Lys Ser Gly Arg Val Ala Gln Ser Ile Leu
305 310 315 320
aga caa gca ggg gtt gaa atg gaa aag gaa ctt gac aag gtt aac ctg
1008
Arg Gln Ala Gly Val Glu Met Glu Lys Glu Leu Asp Lys Val Asn Leu
325 330 335
tcc aca gat tat caa gag gtg tgg gtc aca aaa gga ttt aaa ttg tcc
1056
Ser Thr Asp Tyr Gln Glu Val Trp Val Thr Lys Gly Phe Lys Leu Ser
390 345 350
tgt cag tat gtc ttc cat gtg gca tgg cat tcc caa atc aac aaa tac
1104
Cys Gln Tyr Val Phe His Val Ala Trp His Ser Gln Ile Asn Lys Tyr
355 360 365
cag ata ttg aaa gat gca atg aag tcc tgt cta gaa aaa tgc ctt aaa
1152
Gln Ile Leu Lys Asp Ala Met Lys Ser Cys Leu Glu Lys Cys Leu Lys
370 375 380
cca gat ata aat tcc att tcc ttt cct get ctc ggg aca gga ttg atg
1200
Pro Asp Ile Asn Ser Ile Ser Phe Pro Ala Leu Gly Thr Gly Leu Met
385 390 395 400
gat ttg aag aag agt aca gca get cag ata atg ttt gag gaa gtt ttt
1248
Asp Leu Lys Lys Ser Thr Ala Ala Gln Ile Met Phe Glu Glu Val Phe
405 910 415
gca ttt get aaa gag cac aag gaa aaa acg cta act gta aag att gtg
1296
Ala Phe Ala Lys Glu His Lys Glu Lys Thr Leu Thr Val Lys Ile Val
420 425 ~ 430
atc ttt cca gta gat gtg gag acg tac aag att ttt tat get gaa atg
1349
Ile Phe Pro Val Asp Val Glu Thr Tyr Lys Ile Phe Tyr Ala Glu Met
435 940 445
aca aaa agg tcc aac gag ctg aat ctc agc ggt aat agt ggt get tta
1392
Thr Lys Arg Ser Asn Glu Leu Asn Leu Ser Gly Asn Ser Gly Ala Leu
450 455 460
gcc ctg cag tgg tcc agt ggg gag caa aga aga ggc ggc ctt gaa get
1440
Ala Leu Gln Trp Ser Ser Gly Glu Gln Arg Arg Gly Gly Leu Glu Ala
465 470 475 480
gga tct cct gcc atc aat ctc atg ggt gta aaa gtg gga gag atg tgt
1488
CA 02348758 2001-04-27
WO 00/Z6231 PCT/US99/25439
-24-
Gly Ser Pro Ala Ile Asn Leu Met Gly Val Lys Val Gly Glu Met Cys
485 990 495
gag gcc cag gaa tgg att gaa agg ttg ctg gtc tcc ctg gac cac cac
1536
Glu Ala Gln Glu Trp Ile Glu Arg Leu Leu Val Ser Leu Asp His His
500 505 510
atc att gag aat aat cat att ctc tat ctt ggg aaa aaa gag cac gac
1584
Ile Ile Glu Asn Asn His Ile Leu Tyr Leu Gly Lys Lys Glu His Asp
515 520 525
gtg ctg tct gag ctc cag acc agc aca aga gtc t:cc att tca gag act
1632
Val Leu Ser Glu Leu Gln Thr Ser Thr Arg Val :ier Ile Ser Glu Thr
530 535 'i40
gtc agt cca aga acg gcc act ttg gag att aaa ggt ccc cag get gac
1680
Val Ser Pro Arg Thr Ala Thr Leu Glu Ile Lys Gly Pro Gln Ala Asp
545 550 555 560
ctc att gac gca gtt atg agg att gaa tgt atg ctg tgt gac gtt cag
1728
Leu Ile Asp Ala Val Met Arg Ile Glu Cys Met Leu Cys Asp Val Gln
565 570 575
gaa gaa gtg gca gga aaa agg gag aaa aat ctt tgg agc ttg tca gga
1776
Glu Glu Val Ala Gly Lys Arg Glu Lys Asn Leu 'Prp Ser Leu Ser Gly
580 585 590
cag ggg acc aac cag caa gaa aaa ctg gat aaa atg gaa gaa tcg tac
1824
Gln Gly Thr Asn Gln Gln Glu Lys Leu Asp Lys Met Glu Glu Ser Tyr
595 600 605
aca ttt caa cga tac cca gca tca ttaact cag gaa ctt cag gac cga 1872
Thr Phe Gln Arg Tyr Pro Ala Ser Leu Thr Gln Glu Leu Gln Asp Arg
610 615 620
aag aaa cag ttt gaa aag tgt ggc ttg tgg gtt gtg cag gtg gag cag
1920
Lys Lys Gln Phe Glu Lys Cys Gly Leu Trp Val Val Gln Val Glu Gln
625 630 635 690
ata gae aat aag gtg ctg etg get gce ttc caa gag aag aag aaa atg
1968
Ile Asp Asn Lys Val Leu Leu Ala Ala Phe Gln Glu Lys Lys Lys Met
645 650 655
atg gaa gag agg acg cca aag gga tct ggg agc caa agg ttg ttt cag
2016
Met Glu Glu Arg Thr Pro Lys Gly Ser Gly Ser Gln Arg Leu Phe Gln
660 665 670
cag gtc cca cat cag ttc tgc aat acg gtg tgc aga gtc ggc ttc cac
CA 02348758 2001-04-27
WO 00/Z6231 PCT/US99/25439
-25-
2069
Gln Val Pro His Gln Phe Cys Asn Thr Val Cys Arg Val Gly Phe His
675 680 685
aga atg tat tcg aca tcc tat aac cca gtt tat gga gcc ggc ata tat
2112
Arg Met Tyr Ser Thr Ser Tyr Asn Pro Val Tyr Gly Ala Gly Ile Tyr
690 695 700
ttc acc aag agc ctc aaa aat cta gca gac aag gtc aag aaa acc tca
2160
Phe Thr Lys Ser Leu Lys Asn Leu Ala Asp Lys Val Lys Lys Thr Ser
705 710 715 720
agc aca gac aag cta atc tat gtg ttt gag gca gaa gta ctc aca ggg
2208
Ser Thr Asp Lys Leu Ile Tyr Val Phe Glu Ala Gl.u Val Leu Thr Gly
725 730 735
tcc ttc tgt cag ggt aat tcc tca aat atc atc cct cca cca ttg agt
2256
Ser Phe Cys Gln Gly Asn Ser Ser Asn Ile Ile Pro Pro Pro Leu Ser
740 745 750
cct ggg gcc tta gat gtc aat gac agc gta gtt gac aat gtt tcc agc
2304
Pro Gly Ala Leu Asp Val Asn Asp Ser Val Val Asp Asn Val Ser Ser
755 760 765
cct gaa acc att gtt gtt ttt aat ggc atg cag gcc atg ccc ctg tac
2352
Pro Glu Thr Ile Val Val Phe Asn Gly Met Gln Al.a Met Pro Leu Tyr
770 775 780
ttg tgg act tgc aca cag gat agg aca ttc tca cag cat ccg atg tgg
2900
Leu Trp Thr Cys Thr Gln Asp Arg Thr Phe Ser Gl.n His Pro Met Trp
785 790 795 800
tca cag gac tac tca tca gga cca gga atg gtc tct tcg ctg cag tcc
2498
Ser Gln Asp Tyr Ser Ser Gly Pro Gly Met Val Ser Ser Leu Gln Ser
805 810 815
tgg gaa tgg gtc tta aat ggc agc tct gtt
2478
Trp Glu Trp Val Leu Asn Gly Ser Ser Val
820 825
