Note: Descriptions are shown in the official language in which they were submitted.
CA 02398924 2002-07-19
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ISOLATED HUMAN PHOSPHATASE PROTEINS, NUCLEIC ACID MOLECULES
ENCODING HUMAN PHOSPHATASE PROTEINS, AND USES THEREOF
RELATED APPLICATIONS
The present application is a Continuation-In-Part of U.S. Serial No.
09/715,177, filed
November 20, 2000 and Continuation-In-Part of U.S. Serial No. 09/761,640.
FIELD OF THE INVENTION
The present invention is in the field of phosphatase proteins that are related
to the
mitogen-activated protein (MAP) kinase phosphatase subfamily, recombinant DNA
molecules
and protein production. The present invention provides novel phosphatase
peptides and proteins
and nucleic acid molecules encoding such peptide and protein molecules, all of
which are useful
in the development of human therapeutics and diagnostic compositions and
methods.
Specifically, the present invention provides three novel MAP kinase
phosphatase splice forms.
BACKGROUND OF THE INVENTION
Phosphatase proteins, particularly members of the MAP kinase phosphatase
subfamily, are a
major target for drug action and development. Accordingly, it is valuable to
the field of
pharmaceutical development to identify and characterize previously unknown
members of this
subfamily of phosphatase proteins. The present invention advances the state of
the art by providing
a previously unidentified human phosphatase proteins that have homology to
members of the MAP
kinase phosphatase subfamily.
Protein Phos hap tales
Cellular signal transduction is a fundamental mechanism whereby external
stimuli that
regulate diverse cellular processes are relayed to the interior of cells. The
biochemical pathways
through which signals are transmitted within cells comprise a circuitry of
directly or functionally
connected interactive proteins. One of the key biochemical mechanisms of
signal transduction
involves the reversible phosphorylation of certain residues on proteins. The
phosphorylation
state of a protein may affect its conformation and/or enzymic activity as well
as its cellular
location. The phosphorylation state of a protein is modified through the
reciprocal actions of
protein phosphatases (PKs) and protein phosphatases (PPs) at various specific
amino acid
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residues.
Protein phosphorylation is the ubiquitous strategy used to control the
activities of
eukaryotic cells. It is estimated that 10% of the proteins active in a typical
mammalian cell are
phosphorylated. The high-energy phosphate that confers activation and is
transferred from
adenosine triphosphate molecules to protein-by-protein phosphatases is
subsequently removed
from the protein-by-protein phosphatases. In this way, the phosphatases
control most cellular
signaling events that regulate cell growth and differentiation, cell-to-cell
contacts, the cell cycle,
and oncogenesis.
The protein phosphorylation/dephosphorylation cycle is one of the major
regulatory
mechanisms employed by eukaryotic cells to control cellular activities. It is
estimated that more
than 10% of the active proteins in a typical mammalian cell are
phosphorylated. During protein
phosphorylation/dephosphorylation, phosphate groups are transferred from
adenosine
triphosphate molecules to protein-by-protein phosphatases and are removed from
the protein-by-
protein phosphatases.
Protein phosphatases function in cellular signaling events that regulate cell
growth and
differentiation, cell-to-cell contacts, the cell cycle, and oncogenesis. Three
protein phosphatase
families have been identified as evolutionarily distinct. These include the
serine/threonine
phosphatases, the protein tyrosine phosphatases, and the acid/alkaline
phosphatases (Carbonneau
H. and Tonks N. K. (1992) Annu. Rev. Cell Biol. x:463-93).
The serinelthreonine phosphatases are either cytosolic or associated with a
receptor. ~n
the basis of their sensitivity to two thermostable proteins, inhibitors 1 and
2, and their divalent
cation requirements, the serine/threonine phosphatases can be separated into
four distinct groups,
PP-I, PP-IIA, PP-IIB, and PP-IIC.
PP-I dephosphorylates many of the proteins phosphorylated by cylic AMP-
dependent
protein phosphatase and is therefore an important regulator of many cyclic AMP
mediated,
hormone responses in cells. PP-IIA has broad specificity for control of cell
cycle, growth and
proliferation, and DNA replication and is the main phosphatase responsible for
reversing the
phosphorylations of serine/threonine phosphatases. PP-IIB, or calcineurin
(Cn), is a Ca+2 -
activated phosphatase; it is involved in the regulation of such diverse
cellular functions as ion
channel regulation, neuronal transmission, gene transcription, muscle glycogen
metabolism, and
lymphocyte activation.
PP-IIC is a Mg++ -dependent phosphatase which participates in a wide
variety of
functions including regulating cyclic AMP-activated protein-phosphatase
activity, Ca-H- -
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dependent signal transduction, tRNA splicing, and signal transmission related
to heat shock
responses. PP-IIC is a monomeric protein with a molecular mass of about 40-45
kDa. One
.alpha. and several .beta. isoforms of PP-IIC have been identified (Wenk, J.
et al. (1992) FEBS
Lett. 297: 135-138; Terasawa, T. et al. (1993) Arch. Biochem. Biophys. 307:
342-349; and Kato,
S. et al. (1995) Arch. Biochem. Biophys. 318: 387-393).
The levels of protein phosphorylation required for normal cell growth and
differentiation
at any time are achieved through the coordinated action of PKs and PPS.
Depending on the
cellular context, these two types of enzymes may either antagonize or
cooperate with each other
during signal transduction. An imbalance between these enzymes may impair
normal cell
functions leading to metabolic disorders and cellular transformation.
For example, insulin binding'to the insulin receptor, which is a PTK, triggers
a variety of
metabolic and growth promoting effects such as glucose transports biosynthesis
of glycogen and
fats, DNA synthesis, cell division and differentiation. Diabetes mellitus,
which is characterized
by insufficient or a lack of insulin signal transduction, can be caused by any
abnormality at any
step along the insulin signaling pathway. (Olefsky, 1988, in "Cecil Textbook
of Medicine," 18th
Ed., 2:1360-81).
It is also well known, for example, that the overexpression of PTKs, such as
HER2, can
play a decisive role in the development of cancer (Slamon et al., 1987,
Science 235:77-82) and
that antibodies capable of blocking the activity of this enzyme can abrogate
tumor growth
(Drebin et al., 1988, Oncogene 2:387-394). Blocking the signal transduction
capability of
tyrosine phosphatases such as Flk-1 and the PDGF receptor have been shown to
block tumor
growth in animal models (Millauer et al., 1994, Nature 367:577; Ueno et al.,
Science, 252:844-
848).
Relatively less is known with respect to the direct role of phosphatases in
signal
transduction; PPs may play a role in human diseases. For example, ectopic
expression of
RPTP.alpha. produces a transformed phenotype in embryonic fibroblasts (Zheng
et al., Nature
359:336-339), and overexpression of RPTP.alpha. in embryonal carcinoma cells
causes the cells
to differentiate into a cell type with neuronal phenotype (den Hertog et al.,
EMBO J 12:3789-
3798). The gene for human RPTP.gamma. has been localized to chromosome 3p21
which is a
segment frequently altered in renal and small lung carcinoma. Mutations may
occur in the
extracellular segment of RPTP.gamma. which renders a RPTP that no longer
respond to external
signals (LaForgia et aL, Wary et aL, 1993, Cancer Res 52:478-482). Mutations
in the gene
encoding PTP 1 C (also known as HCP, SHP) are the cause of the moth-eaten
phenotype in mice
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that suffer severe immunodeficiency, and systemic autoimmune disease
accompanied by
hyperproliferation of macrophages (Schultz et al., 1993, Cell 73:1445-1454).
PTP1D (also
known as Syp or PTP2C) has been shown to bind through SH2 domains to sites of
phosphorylation in PDGFR, EGFR and insulin receptor substrate 1 (IRS-1).
Reducing the
activity of PTP1D by microinjection of anti-PTP1D antibody has been shown to
block insulin or
EGF-induced mitogenesis (Xiao et al., 1994, J Biol Chem 269:21244-21248).
MAP I~inase Phosphatases
The present invention provides three novel alternative splice forms of mitogen-
activated
protein (MAP) kinase phosphatase. The alternative splice forms are herein
referred to as splice
forms 1, 2, and 3. Specifically, as indicated in Figure 3, splice form 2
includes exons 13 and 14,
which are absent in splice forms 1 and 3, and splice form 3 is missing exon 7,
which is present in
splice forms 1 and 2. cDNA clones from all three isoforms have been mapped to
the same region
of human chromsome 11. Splice form 1 has been previously disclosed by
applicant in LT.S.
application 09/715,177, filed November 20, 2000.
MAP kinase phosphatases are dual-specificity protein phosphatases involved in
numerous critical biological processes. In Drosophila, MAP kinase phosphatases
have been
found to be essential for viability. Furthermore, loss-of function mutations
cause kinked and/or
branched bristles and wing hairs. In plants, M~1P kinase phosphatases play a
critical role in
responses to stress and pathogens (Gupta et al., Plat J 1998 Dec;l6(5):581-9).
MAP kinase
phosphatases play a critical role in neuronal survival and neuronal cell death
following injury
and degenerative stimuli (Winter et al., Brain Res 1998 Aug 10;801(1-2):198-
205). MAP kinase
phosphatase may also play a role in diabetes and other insulin-related
conditions; insulin
regulates map kinase phosphatase-1 (MI~f-1) and it has been suggested that MKP-
1 acts as a
negative regulator of insulin signaling (Kusari et al., Mol Endoc~inol 1997
Sep; l l (10):1532-43).
Furthermore, MAP kinase phosphatases may be critical for the negative
regulation of cellular
proliferation (Emslie et al., Hum Genet 1994 May;93(5):513-6) and therefore,
novel human
MAP kinase phosphatase genes are useful as candidate tumor-suppressor genes.
Additionally,
MAP kinase phosphatases may specifically be involved in pancreatic cancer
(Furukawa et al.,
Cytogenet Cell Genet 1998;82(3-4):156-9).
For a further review of MAP kinase phosphatases, see Scimeca et al., Oncogene
1997
Aug 7;15(6):717-25. See Wang et al., Genomics 57 (2), 310-315 (1999) for
supporting
information on alternative splice variants.
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The discovery of new human protein phosphatases and the polynucleotides
encoding
them satisfies a need in the art by providing new compositions that are useful
in the diagnosis,
prevention and treatment of biological processes associated with abnormal or
unwanted protein
phosphorylation.
SUMMARY OF THE INVENTION
The present invention is based in part on the identification of amino acid
sequences of
human phosphatase peptides and proteins that are related to the MAP kinase
phosphatase
subfamily, as well as allelic variants and other mammalian orthologs thereof.
Specifically, the
present invention provides three novel MAP kinase phosphatase splice forms.
These unique
peptide sequences, and nucleic acid sequences that encode these peptides, can
be used as models
for the development of human therapeutic targets, aid in the identification of
therapeutic
proteins, and serve as targets for the development of human therapeutic agents
that modulate
phosphatase activity in cells and tissues that express the phosphatase.
Experimental data as
provided in Figure 1 indicates that splice form 1 of the present invention is
expressed in humans
in the pancreas, colon and pancreas adenocarcinomas, pancreas epitheliod
carcinomas, lung large
cell carcinomas, renal cell carcinomas, placenta choriocarcinomas, ovary tumor
tissue, brain
(including fetal), heart (including fetal), kidney (including fetal), uterus,
and thyroid.
Additionally, splice form 3 is expressed in fetal brain.
DESCRIPTION OF THE FIGURE SHEETS
FIGURE 1 provides the nucleotide sequence of three cDNA molecules that encode
each
of the phosphatase splice forms of the present invention. (splice form 1 = SEQ
ID NO:1, splice
form 2 = SEQ ID N0:4, splice form 3 = SEQ ID N0:6) In addition, structure and
functional
information is provided, such as ATG start, stop and tissue distribution,
where available, that
allows one to readily determine specific uses of inventions based on this
molecular sequence.
Experimental data as provided in Figure 1 indicates that splice form 1 of the
present invention is
expressed in humans in the pancreas, colon and pancreas adenocarcinomas,
pancreas epitheliod
carcinomas, lung large cell carcinomas, renal cell carcinomas, placenta
choriocarcinomas, ovary
tumor tissue, brain (including fetal), heart (including fetal), kidney
(including fetal), uterus, and
thyroid. Additionally, splice form 3 is expressed in fetal brain.
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FIGURE 2 provides the predicted amino acid sequence of the three phosphatase
splice
forms of the present invention. (splice form 1 = SEQ ID N0:2, splice form 2 =
SEQ ID NO:S,
splice form 3 = SEQ ID N0:7) In addition structure and functional information
such as protein
family, function, and modification sites is provided where available, allowing
one to readily
determine specific uses of inventions based on this molecular sequence.
FIGURE 3 provides genomic sequences that span the gene encoding the three
phosphatase splice forms of the present invention. (SEQ ID N0:3) In addition
structure and
functional information, such as intron/exon structure, promoter location,
etc., is provided where
available, allowing one to readily determine specific uses of inventions based
on this molecular
sequence. Figure 3 also indicates that the map position of the genomic
sequence encoding the
three splice forms is human chromosome 11. As illustrated in Figure 3, the
following SNP
variations were identified: G577A, G1451A, and G2641A. Figure 3 further
provides a gene
structure model and multiple alignments of the cDNA and peptide sequences of
the three splice
forms to illustrate the structure/sequence variations. Specifically, as
indicated in Figure 3, splice
form 2 includes exons 13 and 14, which are absent in splice forms 1 and 3, and
splice form 3 is
missing exon 7, which is present in splice forms 1 and 2.
DETAILED DESCRIPTION OF THE INVENTION
General Description
The present invention is based on the sequencing of the human genome. During
the
sequencing and assembly of the human genome, analysis of the sequence
information revealed
previously unidentified fragments of the human genome that encode peptides
that share
structural and/or sequence homology to protein/peptideldomains identified and
characterized
within the art as being a phosphatase protein or part of a phosphatase protein
and are related to
the MAP kinase phosphatase subfamily. Utilizing these sequences, additional
genomic
sequences were assembled and transcript and/or cDNA sequences were isolated
and
characterized. Based on this analysis, the present invention provides amino
acid sequences of
three novel human MAP kinase phosphatase splice forms, nucleic acid sequences
in the form of
cDNA sequences and genomic sequences that encode these phosphatase splice
forms, nucleic
acid variation (allelic information), tissue distribution of expression, and
information about the
closest art known protein/peptide/domain that has structural or sequence
homology to the
phosphatase proteins of the present invention.
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In addition to being previously unknown, the peptides that are provided in the
present
invention are selected based on their ability to be used for the development
of commercially
important products and services. Specifically, the present peptides are
selected based on
homology and/or structural relatedness to known phosphatase proteins of the
MAP kinase
phosphatase subfamily and the expression pattern observed. Experimental data
as provided in
Figure 1 indicates that splice form 1 of the present invention is expressed in
humans in the
pancreas, colon and pancreas adenocarcinomas, pancreas epitheliod carcinomas,
lung large cell
carcinomas, renal cell carcinomas, placenta choriocarcinomas, ovary tumor
tissue, brain
(including fetal), heart (including fetal), kidney (including fetal), uterus,
and thyroid.
Additionally, splice form 3 is expressed in fetal brain. The art has clearly
established the
commercial importance of members of this family of proteins and proteins that
have expression
patterns similar to that of the present gene. Some of the more specific
features of the peptides of
the present invention, and the uses thereof, are described herein,
particularly in the Background
of the Invention and in the annotation provided in the Figures, and/or are
known within the art
for each of the known MAP kinase phosphatase family or subfamily of
phosphatase proteins.
Specific Embodiments
Peptide Molecules
The present invention provides nucleic acid sequences that encode protein
molecules that
have been identified as being members of the phosphatase family of proteins
and are related to
the MAP kinase phosphatase subfamily (protein sequences are provided in Figure
2,
transcript/cDNA sequences are provided in Figure l and genomic sequences are
provided in
Figure 3). Specifically, the present invention provides three novel MAP kinase
phosphatase
splice forms. The peptide sequences provided in Figure 2, as well as the
obvious variants
described herein, particularly allelic variants as identified herein and using
the information in
Figure 3, will be referred herein as the phosphatase peptides of the present
invention,
phosphatase peptides, phosphatase splice forms, or peptides/proteins/splice
forms of the present
invention.
The present invention provides isolated peptide and protein molecules that
consist of,
consist essentially of, or comprise the amino acid sequences of the
phosphatase peptides
disclosed in the Figure 2, (encoded by the nucleic acid molecule shown in
Figure 1,
transcript/cDNA or Figure 3, genomic sequence), as well as all obvious
variants of these
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peptides that are within the art to make and use. Some of these variants axe
described in detail
below.
As used herein, a peptide is said to be "isolated" or "purified" when it is
substantially free
of cellular material or free of chemical precursors or other chemicals. The
peptides of the present
invention can be purified to homogeneity or other degrees of purity. The level
of purification will
be based on the intended use. The critical feature is that the preparation
allows for the desired
function of the peptide, even if in the presence of considerable amounts of
other components (the
features of an isolated nucleic acid molecule is discussed below).
In some uses, "substantially free of cellular material" includes preparations
of the peptide
having less than about 30% (by dry weight) other proteins (i.e., contaminating
protein), less than
about 20% other proteins, less than about 10% other proteins, or less than
about 5% other proteins.
When the peptide is recombinantly produced, it can also be substantially free
of culture medium,
i.e., culture medium represents less than about 20% of the volume of the
protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of the peptide in which it is separated from chemical precursors
or other chemicals that
are involved in its synthesis. In one embodiment, the language "substantially
free of chemical
precursors or other chemicals" includes preparations of the phosphatase
peptide having less than
about 30% (by dry weight) chemical precursors or other chemicals, less than
about 20% chemical
precursors or other chemicals, less than about 10% chemical precursors or
other chemicals, or less
than about 5% chemical precursors or other chemicals.
The isolated phosphatase peptide can be purified from cells that naturally
express it, purified
from cells that have been altered to express it (recombinant), or synthesized
using known protein
synthesis methods. Experimental data as provided in Figure 1 indicates that
splice form 1 of the
present invention is expressed in humans in the pancreas, colon and pancreas
adenocarcinomas,
pancreas epitheliod carcinomas, lung large cell carcinomas, renal cell
carcinomas, placenta
choriocarcinomas, ovary tumor tissue, brain (including fetal), heart
(including fetal), kidney
(including fetal), uterus, and thyroid. Additionally, splice form 3 is
expressed in fetal brain. For
example, a nucleic acid molecule encoding the phosphatase peptide is cloned
into an expression
vector, the expression vector introduced into a host cell and the protein
expressed in the host cell.
The protein can then be isolated from the cells by an appropriate purification
scheme using standard
protein purification techniques. Many of these techniques are described in
detail below.
Accordingly, the present invention provides proteins that consist of the amino
acid
sequences provided in Figure 2 (SEQ ID NOS:2,5,7), for example, proteins
encoded by the cDNA
S
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nucleic acid sequences shown in Figure 1 (SEQ ID NOS:1,4,6) and the genomic
sequences
provided in Figure 3 (SEQ ID N0:3). The amino acid sequence of such a protein
is provided in
Figure 2. A protein consists of an amino acid sequence when the amino acid
sequence is the final
amino acid sequence of the protein.
The present invention further provides proteins that consist essentially of
the amino acid
sequences provided in Figure 2 (SEQ ID NOS:2,5,?), for example, proteins
encoded by the cDNA
nucleic acid sequences shown in Figure 1 (SEQ ID NOS:1,4,6) and the genomic
sequences
provided in Figure 3 (SEQ ID N0:3). A protein consists essentially of an amino
acid sequence
when such an amino acid sequence is present with only a few additional amino
acid residues, for
example from about 1 to about 100 or so additional residues, typically from 1
to about 20 additional
residues in the final protein.
The present invention further provides proteins that comprise the amino acid
sequences
provided in Figure 2 (SEQ ID NOS:2,5,?), for example, proteins encoded by the
cDNA nucleic acid
sequences shown in Figure 1 (SEQ ID NOS:1,4,6) and the genomic sequences
provided in Figure 3
I S (SEQ ID N0:3). A protein comprises an amino acid sequence when the amino
acid sequence is at
least part of the final amino acid sequence of the protein. In such a fashion,
the protein can be only
the peptide or have additional amino acid molecules, such as amino acid
residues (contiguous
encoded sequence) that are naturally associated with it or heterologous amino
acid residues/peptide
sequences. Such a protein can have a few additional amino acid residues or can
comprise several
hundred or more additional amino acids. The preferred classes of proteins that
are comprised of the
phosphatase peptides of the present invention are the naturally occurring
mature proteins. A brief
description of how various types of these proteins can be made/isolated is
provided below.
The phosphatase peptides of the present invention can be attached to
heterologous
sequences to form chimeric or fusion proteins. Such chimeric and fusion
proteins comprise a
phosphatase peptide operatively linked to a heterologous protein having an
amino acid sequence not
substantially homologous to the phosphatase peptide. "Operatively linked"
indicates that the
phosphatase peptide and the heterologous protein are fused in-frame. The
heterologous protein can
be fused to the N-ternlinus or C-terminus of the phosphatase peptide.
In some uses, the fusion protein does not affect the activity of the
phosphatase peptide per
se. For example, the fusion protein can include, but is not limited to,
enzymatic fusion proteins, for
example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His
fusions, MYC-tagged,
HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions,
can facilitate the
purification of recombinant phosphatase peptide. In certain host cells (e.g.,
mammalian host cells),
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expression and/or secretion of a protein can be increased by using a
heterologous signal sequence.
A chimeric or fusion protein can be produced by standard recombinant DNA
techniques.
For example, DNA fragments coding for the different protein sequences are
ligated together in-
frame in accordance with conventional techniques. 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 re-amplified to generate a chimeric gene sequence (see Ausubel et
al., Current
Protocols ih Molecular Biology, 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST protein). A
phosphatase peptide-
encoding nucleic acid can be cloned into such an expression vector such that
the fusion moiety is
linked in-frame to the phosphatase peptide.
As mentioned above, the present invention also provides and enables obvious
variants of the
amino acid sequence of the proteins of the present invention, such as
naturally occurring mature
forms of the peptide, allelic/sequence variants of the peptides, non-naturally
occurring
recombinantly derived variants of the peptides, and orthologs and paralogs of
the peptides. Such
variants can readily be generated using art-known techniques in the fields of
recombinant nucleic
acid technology and protein biochemistry. It is understood, however, that
variants exclude any
amino acid sequences disclosed prior to the invention.
Such variants can readily be identifiedlmade using molecular techniques and
the sequence
information disclosed herein. Further, such variants can readily be
distinguished from other
peptides based on sequence and/or structural homology to the phosphatase
peptides of the present
invention. The degree of homologylidentity present will be based primarily on
whether the peptide
is a functional variant or non-functional variant, the amount of divergence
present in the paralog
family and the evolutionary distance between the orthologs.
To determine the percent identity of two amino acid sequences or 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, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of
the length
of a reference sequence is aligned for comparison purposes. 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
CA 02398924 2002-07-19
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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 and
similarity
between two sequences can be accomplished using a mathematical algorithm.
(Computational
Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Biocomputing:
Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York,
1993; Computer
Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds.,
Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; and
Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York,
1991). 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 Blossom 62 matrix or a PAM250 matrix, and
a gap weight
of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred
embodiment, the percent identity between two nucleotide sequences is
determined using the
GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids
Res. 12(1):387
(1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a
gap weight of
40, 50, 60, 70, or 80 and a length weight of I, 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. Myers 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 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be
used as a
"query sequence" to perform a search against sequence 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. (J. Mol. Biol. 215:403-10
(1990)). BLAST
nucleotide searches can be performed with the NBLAST program, score = 100,
wordlength = 12
to obtain nucleotide sequences homologous to the nucleic acid molecules of the
invention.
BLAST protein searches can be performed with the XBLAST program, score = 50,
wordlength =
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3 to obtain amino acid sequences homologous to the proteins of the invention.
To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et
al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and
gapped BLAST
programs, the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can
be used.
Full-length pre-processed forms, as well as mature processed forms, of
proteins that
comprise one of the peptides of the present invention can readily be
identified as having complete
sequence identity to one of the phosphatase peptides of the present invention
as well as being
encoded by the same genetic locus as the phosphatase peptide provided herein.
The gene encoding
the novel phosphatase proteins of the present invention is located on public
BAC AP001885, which
is known to be mapped to chromosome 11 (as indicated in Figure 3).
Allelic variants of a phosphatase peptide can readily~be identified as being a
human protein
having a high degree (significant) of sequence homology/identity to at least a
portion of the
phosphatase peptide as well as being encoded by the same genetic locus as the
phosphatase peptide
provided herein. Genetic locus can readily be determined based on the genomic
information
provided in Figure 3, such as the genomic sequence mapped to the reference
human. 'The gene
encoding the novel phosphatase proteins of the present invention is located on
public BAC
AP001885, which is known to be mapped to chromosome 11 (as indicated in Figure
3). As used
herein, two proteins (or a region of the proteins) have significant homology
when the amino acid
sequences are typically at least about 70-80%, 80-90%, and more typically at
least about 90-95%
or more homologous. A significantly homologous amino acid sequence, according
to the present
invention, will be encoded by a nucleic acid sequence that will hybridize to a
phosphatase
peptide encoding nucleic acid molecule under stringent conditions as more
fully described
below.
Figure 3 provides information on SNPs that have been found in the gene
encoding the
phosphatase proteins of the present invention. The following SNPs were
identified: G577A,
G1451A, and G2641A. G577A and G1451A are non-synonymous coding SNPs. Changes
in the
amino acid sequence caused by these SNPs is indicated in Figure 3 and can
readily be
determined using the universal genetic code and the protein sequence provided
in Figure 2 as a
reference. G2641A is 3' of the ORF and may affect control/regulatory elements.
Paralogs of a phosphatase peptide can readily be identified as having some
degree of
significant sequence homology/identity to at least a portion of the
phosphatase peptide, as being
encoded by a gene from humans, and as having similar activity or function. Two
proteins will
12
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
typically be considered paralogs when the amino acid sequences are typically
at Ieast about 60%
or greater, and more typically at least about 70% or greater homology through
a given region or
domain. Such paralogs will be encoded by a nucleic acid sequence that will
hybridize to a
phosphatase peptide encoding nucleic acid molecule under moderate to stringent
conditions as
more fully described below.
Orthologs of a phosphatase peptide can readily be identified as having some
degree of
significant sequence homology/identity to at least a portion of the
phosphatase peptide as well as
being encoded by a gene from another organism. Preferred orthologs will be
isolated from
mammals, preferably primates, for the development of human therapeutic targets
and agents. Such
orthologs will be encoded by a nucleic acid sequence that will hybridize to a
phosphatase peptide
encoding nucleic acid molecule under moderate to stringent conditions, as more
fully described
below, depending on the degree of relatedness of the two organisms yielding
the proteins.
Non-naturally occurring variants of the phosphatase peptides of the present
invention can
readily be generated using recombinant techniques. Suchwariants include, but
are not limited to
deletions, additions and substitutions in the amino acid sequence of the
phosphatase peptide. For
example, one class of substitutions are conserved amino acid substitution.
Such substitutions are
those that substitute a given amino acid in a phosphatase peptide by another
amino acid of like
characteristics. Typically seen as conservative substitutions are the
replacements, one for another,
among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser
and Thr; exchange of the acidic residues Asp and Glu; substitution between the
amide residues Asn
and Gln; exchange of the basic residues Lys and Arg; and replacements among
the aromatic
residues Phe and Tyr. Guidance concerning which amino acid changes are likely
to be
phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).
Variant phosphatase peptides can be fully functional or can lack function in
one or more
activities, e.g. ability to bind substrate, ability to dephosphorylate
substrate, ability to mediate
signaling, etc. Fully functional variants typically contain only conservative
variation or variation in
non-critical residues or in non-critical regions. Figure 2 provides the result
of protein analysis and
can be used to identify critical domains/regions. Functional variants can also
contain substitution of
similar amino acids that result in no change or an insignificant change in
function. Alternatively,
such substitutions may positively or negatively affect function to some
degree.
Non-functional variants typically contain one or more non-conservative amino
acid
substitutions, deletions, insertions, inversions, or truncation or a
substitution, insertion, inversion, or
deletion in a critical residue or critical region.
13
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
Amino acids that are essential for function can be identified by methods known
in the art,
such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham
et al., Science
244:1081-1085 (1989)), particularly using the results provided in Figure 2.
The latter procedure
introduces single alanine mutations at every residue in the molecule. The
resulting mutant
molecules are then tested for biological activity such as phosphatase activity
or in assays such as an
in vitro proliferative activity. Sites that are critical for binding
partner/substrate binding can also be
determined by structural analysis such as crystallization, nuclear magnetic
resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos
et al. Science
255:306-312 (1992)).
The present invention further provides fragments of the phosphatase peptides,
in addition to
proteins and peptides that comprise and consist of such fragments,
particularly those comprising the
residues identified in Figure 2. The fragments to which the invention
pertains; however, are not to
be construed as encompassing fragments that may be disclosed publicly prior to
the present
invention.
As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more
contiguous amino
acid residues from a phosphatase peptide. Such fragments can be chosen based
on the ability to
retain one or more of the biological activities of the phosphatase peptide or
could be chosen for the
ability to perform a function, e.g. bind a substrate or act as an immunogen.
Particularly important
fragments are biologically active fragments, peptides that are, for example,
about 8 or more amino
acids in length. Such fragments will typically comprise a domain or motif of
the phosphatase
peptide, e.g., active site, a transmembrane domain or a substrate-binding
domain. Further, possible
fragments include, but are not limited to, domain or motif containing
fragments, soluble peptide
fragments, and fragments containing immunogenic structures. Predicted domains
and functional
sites are readily identifiable by computer programs well known and readily
available to those of
skill in the art (e.g., PROSITE analysis). The results of one such analysis
are provided in Figure 2.
Polypeptides often contain amino acids other than the 20 amino acids commonly
referred to
as the 20 naturally occurring amino acids. Further, many amino acids,
including the terminal amino
acids, may be modified by natural processes, such as processing and other post-
translational
modifications, or by chemical modification techniques well known in the art.
Common
modifications that occur naturally in phosphatase peptides are described in
basic texts, detailed
monographs, and the research literature, and they are well known to those of
skill in the art (some of
these features are identified in Figure 2).
Known modifications include, but are not limited to, acetylation, acylation,
ADP-
14
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
ribosylation, amidation, covalent attachment of flavin, covalent attachment of
a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative, covalent
attachment of a lipid or lipid
derivative, covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond
formation, demethylation, formation of covalent crosslinks, formation of
cystine, formation of
pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor
formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic
processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
RNA mediated
addition of amino acids to proteins such as arginylation, and ubiquitination.
Such modifications are well known to those of skill in the art and have been
described in
great detail in the scientific literature. Several particularly common
modifications, glycosylation,
lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and
ADP-ribosylation, for instance, are described in most basic texts, such as
Proteins - Structure arcd
Molecular Properties, 2nd Ed., T.E. Creighton, W. H. Freeman and Company, New
York (1993).
Many detailed reviews are available on this subject, such as by Wold, F.,
Posttrauslational Covalent
Modification ofProteihs, B.C. Johnson, Ed., Academic Press, New York 1-12
(1983); Seifter et al.
(Meth. Ermymol. 182: 626-646 (1990)) and Rattan et al. (A~v~. N. Y. Acad. Sci.
663:48-62 (1992)).
Accordingly, the phosphatase peptides of the present invention also encompass
derivatives
or analogs in which a substituted amino acid residue is not one encoded by the
genetic code, in
which a substituent group is included, in which the mature phosphatase peptide
is fused with
another compound, such as a compound to increase the half life of the
phosphatase peptide, or in
which the additional amino acids are fused to the mature phosphatase peptide,
such as a leader or
secretory sequence or a sequence for purification of the mature phosphatase
peptide or a pro-protein
sequence.
ProteinlPeptide Uses
The proteins of the present invention can be used in substantial and specific
assays
related to the functional information provided in the Figures; to raise
antibodies or to elicit
another immune response; as a reagent (including the labeled reagent) in
assays designed to
quantitatively determine levels of the protein (or its binding partner or
ligand) in biological
fluids; and as markers for tissues in which the corresponding protein is
preferentially expressed
(either constitutively or at a particular stage of tissue differentiation or
development or in a
disease state). Where the protein binds or potentially binds to another
protein or ligand (such as,
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
for example, in a phosphatase-effector protein interaction or phosphatase-
ligand interaction), the
protein can be used to identify the binding partner/ligand so as to develop a
system to identify
inhibitors of the binding interaction. Any or all of these uses are capable of
being developed into
reagent grade or kit format for commercialization as commercial products.
Methods for performing the uses listed above are well known to those skilled
in the art.
References disclosing such methods include "Molecular Cloning: A Laboratory
Manual", 2d ed.,
Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989,
and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic
Press,
Berger, S. L. and A. R. Kimmel eds., 1987.
The potential uses of the peptides of the present invention are based
primarily on the
source of the protein as well as the class/action of the protein. For example,
phosphatases
isolated from humans and their humanlmammalian orthologs serve as targets for
identifying
agents for use in mammalian therapeutic applications, e.g. a human drug,
particularly in
modulating a biological or pathological response in a cell or tissue that
expresses the
phosphatase. Experimental data as provided in Figure 1 indicates that splice
form 1 of the
phosphatase protein of the present invention is expressed in humans in the
pancreas, colon and
pancreas adenocarcinomas, pancreas epitheliod carcinomas, lung large cell
carcinomas, renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid.
Additionally, splice form 3 is
expressed in fetal brain. Specifically, a virtual northern blot shows
expression in pancreas, colon
and pancreas adenocaxcinomas, pancreas epitheliod carcinomas, Lung large cell
carcinomas, renal
cell carcinomas, placenta choriocarcinomas, and ovary tumor tissue. In
addition, PCR-based
tissue screening panels indicate expression in the brain (including fetal),
heart (including fetal),
kidney (including fetal), uterus, and thyroid. A large percentage of
pharmaceutical agents are
being developed that modulate the activity of phosphatase proteins,
particularly members of the
MAP kinase phosphatase subfamily (see Background of the Invention). The
structural and
functional information provided in the Background and Figures provide specific
and substantial
uses for the molecules of the present invention, particularly in combination
with the expression
information provided in Figure 1. Experimental data as provided in Figure 1
indicates that splice
form 1 of the present invention is expressed in humans in the pancreas, colon
and pancreas
adenocarcinomas, pancreas epitheliod carcinomas, lung large cell carcinomas,
renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid.
Additionally, splice form 3 is
16
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
expressed in fetal brain. Such uses can readily be determined using the
information provided
herein, that which is known in the art, and routine experimentation.
The proteins of the present invention (including variants and fragments that
may have been
disclosed prior to the present invention) are useful for biological assays
related to phosphatases that
are related to members of the MAP kinase phosphatase subfamily. Such assays
involve any of the
known phosphatase functions or activities or properties useful for diagnosis
and treatment of
phosphatase-related conditions that are specific for the subfamily of
phosphatases that the one of the
present invention belongs to, particularly in cells and tissues that express
the phosphatase.
Experimental data as provided in Figure 1 indicates that splice form 1 of the
phosphatase protein of
the present invention is expressed in humans in the pancreas, colon and
pancreas adenocarcinomas,
pancreas epitheliod, carcinomas, lung large cell carcinomas, renal cell
carcinomas, placenta
choriocarcinomas, ovary tumor tissue, brain (including fetal), heart
(including fetal), kidney
(including fetal), uterus, and thyroid. Additionally, splice form 3 is
expressed in fetal brain.
Specifically, a virtual northern blot shows expression in pancreas, colon and
pancreas
adenocarcinomas, pancreas epitheliod carcinomas, lung large cell carcinomas,
renal cell
carcinomas, placenta choriocarcinomas, and ovary tumor tissue. In addition,
PCR-based tissue
screening panels indicate expression in the brain (including fetal), heart
(including fetal), kidney
(including fetal), uterus, and thyroid.
The proteins of the present invention are also useful in drug screening
assays, in cell-based
or cell-free systems. Cell-based systems can be native, i.e., cells that
normally express the
phosphatase, as a biopsy or expanded in cell culture. Experimental data as
provided in Figure 1
indicates that splice form 1 of the present invention is expressed in humans
in the pancreas, colon
and pancreas adenocarcinomas, pancreas epitheliod carcinomas, lung large cell
carcinomas, renal
cell carcinomas, placenta choriocarcinomas, ovary tumor tissue,' brain
(including fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid.
Additionally, splice form 3 is
expressed in fetal brain. In an alternate embodiment, cell-based assays
involve recombinant host
cells expressing the phosphatase protein.
The polypeptides can be used to identify compounds that modulate phosphatase
activity of
the protein in its natural state or an altered form that causes a specific
disease or pathology
associated with the phosphatase. Both the phosphatases of the present
invention and appropriate
variants and fragments can be used in high-throughput screens to assay
candidate compounds for
the ability to bind to the phosphatase. These compounds can be fiufiher
screened against a
functional phosphatase to deterniine the effect of the compound on the
phosphatase activity.
17
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
Further, these compounds can be tested in animal or invertebrate systems to
determine
activity/effectiveness. Compounds can be identified that activate (agonist) or
inactivate (antagonist)
the phosphatase to a desired degree.
Further, the proteins of the present invention can be used to screen a
compound for the
ability to stimulate or inhibit interaction between the phosphatase protein
and a molecule that
normally interacts with the phosphatase protein, e.g. a substrate or a
component of the signal
pathway that the phosphatase protein normally interacts (for example, another
phosphatase). Such
assays typically include the steps of combining the phosphatase protein with a
candidate compound
under conditions that allow the phosphatase protein, or fragment, to interact
with the target
molecule, and to detect the formation of a complex between the protein and the
target or to detect
the biochemical consequence of the interaction with the phosphatase protein
and the target, such as
any of the associated effects of signal transduction such as protein
phosphorylation, cAMP turnover,
and adenylate cyclase activation, etc.
Candidate compounds include, for example, 1) peptides such as soluble
peptides, including
Ig-tailed fusion peptides and members of random peptide libraries (see, e.g.,
Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial
chemistry-derived
molecular libraries made of D- and/or L- configuration amino acids; 2)
phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide libraries,
see, e.g., Songyang
et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal,
humanized, anti-
idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')a, Fab
expression library
fragments, and epitope-binding fragments of antibodies); and 4) small organic
and inorganic
molecules (e.g., molecules obtained from combinatorial and natural product
libraries).
One candidate compound is a soluble fragment of the receptor that competes for
substrate
binding. Other candidate compounds include mutant phosphatases or appropriate
fragments
containing mutations that affect phosphatase function and thus compete for
substrate. Accordingly,
a fragment that competes for substrate, for example with a higher afFmity, or
a fragment that binds
substrate but does not allow release, is encompassed by the invention.
The invention further includes other end point assays to identify compounds
that modulate
(stimulate or inhibit) phosphatase activity. The assays typically involve an
assay of events in the
signal transduction pathway that indicate phosphatase activity. Thus, the
dephosphorylation of a
substrate, activation of a protein, a change in the expression of genes that
are up- or down-regulated
in response to the phosphatase protein dependent signal cascade can be
assayed.
Any of the biological or biochemical functions mediated by the phosphatase can
be used as
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CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
an endpoint assay. These include all of the biochemical or
biochemical/biological events described
herein, in the references cited herein, incorporated by reference for these
endpoint assay targets, and
other functions known to those of ordinary skill in the art or that can be
readily identified using the
information provided in the Figures, particularly Figure 2. Specifically, a
biological function of a
cell or tissues that expresses the phosphatase can be assayed. Experimental
data as provided in
Figure 1 indicates that splice form 1 of the phosphatase protein of the
present invention is expressed
in humans in the pancreas, colon and pancreas adenocarcinomas, pancreas
epitheliod carcinomas,
lung large cell carcinomas, renal cell carcinomas, placenta choriocarcinomas,
ovary tumor tissue,
brain (including fetal), heart (including fetal), kidney (including fetal),
uterus, and thyroid.
Additionally, splice form 3 is expressed in fetal brain. Specifically, a
virtual northern blot shows
expression in pancreas, colon and pancreas adenocarcinomas, pancreas
epitheliod carcinomas, lung
large cell carcinomas, renal cell carcinomas, placenta choriocarcinomas, and
ovary tumor tissue. In
addition, PCR-based tissue screening panels indicate expression in the brain
(including fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid.
Binding and/or activating compounds can also be screened by using chimeric
phosphatase
proteins in which the amino terminal extracellular domain, or parts thereof,
the entire
transmembrane domain or subregions, such as any of the seven transmembrane
segments or any of
the intracellular or extracellular loops and the carboxy terminal
intracellular domain, or parts
thereof, can be replaced by heterologous domains or subregions. For example, a
substrate-binding
region can be used that interacts with a different substrate then that which
is recognized by the
native phosphatase. Accordingly, a different set of signal transduction
components is available as
an end-point assay for activation. This allows for assays to be performed in
other than the specific
host cell from which the phosphatase is derived.
The proteins of the present invention are also useful in competition binding
assays in
methods designed to discover compounds that interact with the phosphatase
(e.g. binding partners
and/or ligands). Thus, a compound is exposed to a phosphatase polypeptide
under conditions that
allow the compound to bind or to otherwise interact with the polypeptide.
Soluble phosphatase
polypeptide is also added to the mixture. If the test compound interacts with
the soluble
phosphatase polypeptide, it decreases the amount of complex formed or activity
from the
phosphatase target. This type of assay is particularly useful in cases in
which compounds are sought
that interact with specific regions of the phosphatase. Thus, the soluble
polypeptide that competes
with the target phosphatase region is designed to contain peptide sequences
corresponding to the
region of interest.
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WO 02/42436 PCT/USO1/42995
To perform cell free drug screening assays, it is sometimes desirable to
immobilize either
the phosphatase protein, or fragment, or its target molecule to facilitate
separation of complexes
from uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of
the assay.
Techniques for immobilizing proteins on matrices can be used in the drug
screening assays.
In one embodiment, a fusion protein can be provided which adds a domain that
allows the protein to
be bound to a matrix. For example, glutathione-S-transferase 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 cell lysates (e.g., 35S-labeled) and
the candidate
compound, and the mixture incubated under conditions conducive to complex
formation (e.g., at
physiological conditions for salt and pH). Following incubation, the beads are
washed to remove
any unbound label, and the matrix immobilized and radiolabel determined
directly, or in the
supernatant after the complexes are dissociated. Alternatively, the complexes
can be dissociated
from the matrix, separated by SDS-PAGE, and the level of phosphatase-binding
protein found in
the bead fraction quantitated from the gel using standard electrophoretic
techniques. For example,
either the polypeptide or its target molecule can be immobilized utilizing
conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively, antibodies
reactive with the
protein but which do not interfere with binding of the protein to its target
molecule can be
derivatized to the wells of the plate, and the protein trapped in the wells by
antibody conjugation.
Preparations of a phosphatase-binding protein and a candidate compound are
incubated in the
phosphatase protein-presenting wells and the amount of complex trapped in the
well can be
quantitated.. 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 phosphatase protein target molecule, or which are reactive with
phosphatase protein and
compete with the target molecule, as well as enzyme-linked assays which rely
on detecting an
enzymatic activity associated with the target molecule.
Agents that modulate one of the phosphatases of the present invention can be
identified
using one or more of the above assays, alone or in combination. It is
generally preferable to use a
cell-based or cell free system first and then confirm activity in an animal or
other model system.
Such model systems are well known in the art and can readily be employed in
this context.
Modulators of phosphatase protein activity identified according to these drug
screening
assays can be used to treat a subject with a disorder mediated by the kinase
pathway, by treating
cells or tissues that express the phosphatase. Experimental data as provided
in Figure 1 indicates
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
that splice form 1 of the present invention is expressed in humans in the
pancreas, colon and
pancreas adenocarcinomas, pancreas epitheliod carcinomas, lung large cell
carcinomas, renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart (including
fetal), kidney (including fetal), uterus, and thyroid. Additionally, splice
form 3 is expressed in fetal
brain. These methods of treatment include the steps of administering a
modulator of phosphatase
activity in a pharmaceutical composition to a subject in need of such
treatment, the modulator being
identified as described herein.
In yet another aspect of the invention, the phosphatase 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) Biotech~iques 14:920-924; Iwabuchi et al. (1993) Oncogehe
8:1693-1696;
and Brent W094/10300), to identify other proteins, which bind to or interact
with the
phosphatase and are involved in phosphatase activity. Such phosphatase-binding
proteins are
also likely to be involved in the propagation of signals by the phosphatase
proteins or
phosphatase targets as, for example, downstream elements of a kinase-mediated
signaling
pathway. Alternatively, such phosphatase-binding proteins are likely to be
phosphatase
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
phosphatase 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 phosphatase-dependent complex, the DNA-binding and activation
domains of the
transcription factor are brought into close proximity. This proximity allows
transcription of a
reporter gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive
to the transcription factor. Expression of the reporter gene can be detected
and cell colonies
containing the functional transcription factor can be isolated and used to
obtain the cloned gene
which encodes the protein which interacts with the phosphatase 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 as described herein in an appropriate animal model. For example, an
agent identified
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CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
as described herein (e.g., a phosphatase-modulating agent, an antisense
phosphatase nucleic acid
molecule, a phosphatase-specific antibody, or a phosphatase-binding partner)
can be used in an
animal or other model to determine the efficacy, toxicity, or side effects of
treatment with such
an agent. Alternatively, an agent identified as described herein can be used
in an animal or other
model to determine the mechanism of action of such an agent. Furthermore, this
invention
pertains to uses of novel agents identified by the above-described screening
assays for treatments
as described herein.
The phosphatase proteins of the present invention are also useful to provide a
target for
diagnosing a disease or predisposition to disease mediated by the peptide.
Accordingly, the
invention provides methods for detecting the presence, or levels of, the
protein (or encoding
mRNA) in a cell, tissue, or organism. Experimental data as provided in Figure
1 indicates that
splice form 1 of the present invention is expressed in humans in the pancreas,
colon and pancreas
adenocarcinomas, pancreas epitheliod carcinomas, lung large cell carcinomas,
renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart (including
fetal), kidney (including fetal), uterus, and thyroid. Additionally, splice
form 3 is expressed in fetal
brain. The method involves contacting a biological sample with a compound
capable of interacting.
with the phosphatase protein such that the interaction can be detected. Such
an assay can be
provided in a single detection format or a mufti-detection format such as an
antibody chip array.
One agent for detecting a protein in a sample is an antibody capable of
selectively binding to
protein. A biological sample includes tissues, cells and biological fluids
isolated from a subject, as
well as tissues, cells and fluids present within a subject.
The peptides of the present invention also provide targets for diagnosing
active protein
activity, disease, or predisposition to disease, in a patient having a variant
peptide, particularly
activities and conditions that are known for other members of the family of
proteins to which the
present one belongs. Thus, the peptide can be isolated from a biological
sample and assayed for the
presence of a genetic mutation that results in aberrant peptide. This includes
amino acid
substitution, deletion, insertion, rearrangement, (as the result of aberrant
splicing events), and
inappropriate post-translational modification. Analytic methods include
altered electrophoretic
mobility, altered tryptic peptide digest, altered phosphatase activity in cell-
based or cell-free assay,
alteration in substrate or antibody-binding pattern, altered isoelectric
point, direct amino acid
sequencing, and any other of the known assay techniques useful for detecting
mutations in a protein.
Such an assay can be provided in a single detection format or a mufti-
detection format such as an
antibody chip array.
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WO 02/42436 PCT/USO1/42995
Ih vitro techniques for detection of peptide include enzyme linked
immunosorbent assays
(ELISAs), Western blots, immunoprecipitations and immunofluorescence using a
detection reagent,
such as an antibody or protein binding agent. Alternatively, the peptide can
be detected ih vivo in a
subject by introducing into the subject a labeled anti-peptide antibody or
other types of detection
agent. 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.
Particularly useful are
methods that detect the allelic variant of a peptide expressed in a subject
and methods which detect
fragments of a peptide in a sample.
The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics
deal with
clinically significant hereditary variations in the response to drugs due to
altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clip. Exp.
Pharmacol. Physiol.
. 23(10-11):983-985 (1996)), and Linder, M.W. (Cli~z. Chem. 43(2):254-266
(1997)). The clinical
outcomes of these variations result in severe toxicity of therapeutic drugs in
certain individuals or
therapeutic failure of drugs in certain individuals as a result of individual
variation in metabolism.
Thus, the genotype of the individual can determine the way a therapeutic
compound acts on the
body or the way the body metabolizes the compound. Further, the activity of
drug metabolizing
enzymes effects both the intensity and duration of drug action. Thus, the
pharmacogenomics of the
individual permit the selection of effective compounds and effective dosages
of such compounds for
prophylactic or therapeutic treatment based on the individual's genotype. The
discovery of genetic
polymorphisms in some drug metabolizing enzymes has explained why some
patients do not obtain
the expected drug effects, show an exaggerated drug effect, or experience
serious toxicity from
standard drug dosages. Polymorphisms can be expressed in the phenotype of the
extensive
metabolizes and the phenotype of the poor metabolizes. Accordingly, genetic
polymorphism may
lead to allelic protein variants of the phosphatase protein in which one or
more of the phosphatase
functions in one population is different from those in another population. The
peptides thus allow a
target to ascertain a genetic predisposition that can affect treatment
modality. Thus, in a ligand-
based treatment, polymorphism may give rise to amino terminal extracellular
domains and/or other
substrate-binding regions that are more or less active in substrate binding,
and phosphatase
activation. Accordingly, substrate dosage would necessarily be modified to
maximize the
therapeutic effect within a given population containing a polymorphism. As an
alternative to
genotyping, specific polymorphic peptides could be identified.
The peptides are also useful for treating a disorder characterized by an
absence of,
inappropriate, or unwanted expression of the protein. Experimental data as
provided in Figure 1
23
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WO 02/42436 PCT/USO1/42995
indicates that splice form 1 of the present invention is expressed in humans
in the pancreas, colon
and pancreas adenocarcinomas, pancreas epitheliod carcinomas, lung large cell
carcinomas, renal
cell carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain
(including fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid.
Additionally, splice form 3 is
expressed in fetal brain. Accordingly, methods for treatment include the use
of the phosphatase
protein or fragments.
Antibodies
The invention also provides antibodies that selectively bind to one of the
peptides of the
present invention, a protein comprising such a peptide, as well as variants
and fragments thereof.
As used herein, an antibody selectively binds a target peptide when it binds
the target peptide and
does not significantly bind to unrelated proteins. An antibody is still
considered to selectively bind
a peptide even if it also binds to other proteins that are not substantially
homologous with the target
peptide so long as such proteins share homology with a fragment or domain of
the peptide target of
the antibody. In this case, it would be understood that antibody binding to
the peptide is still
selective despite some degree of cross-reactivity.
As used herein, an antibody is defined in terms consistent with that
recognized within the
art: they are mufti-subunit proteins produced by a mammalian organism in
response to an antigen
challenge. The antibodies of the present invention include polyclonal
antibodies and monoclonal
antibodies, as well as fragments of such antibodies, including, but not
limited to, Fab or Flab°)Z, end
Fv fragments.
Many methods are known for generating and/or identifying antibodies to a given
target
peptide. Several such methods are described by Harlow, Antibodies, Cold Spring
Harbor Press,
(1989).
In general, to generate antibodies, an isolated peptide is used as an
immunogen and is
administered to a mammalian organism, such as a rat, rabbit or mouse. The full-
length protein, an
antigenic peptide fragment or a fusion protein can be used. Particularly
important fragments are
those covering functional domains, such as the domains identified in Figure 2,
and domain of
sequence homology or divergence amongst the family, such as those that can
readily be identified
using protein alignment methods and as presented in the Figures.
Antibodies are preferably prepared from regions or discrete fragments of the
phosphatase
proteins. Antibodies can be prepared from any region of the peptide as
described herein.
24
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WO 02/42436 PCT/USO1/42995
However, preferred regions will include those involved in function/activity
and/or
phosphatase/binding partner interaction. Figure 2 can be used to identify
particularly important
regions while sequence alignment can be used to identify conserved and unique
sequence
fragments.
An antigenic fragment will typically comprise at least 8 contiguous amino acid
residues.
The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more
amino acid residues.
Such fragments can be selected on a physical property, such as fragments
correspond to regions that
are located on the surface of the protein, e.g., hydrophilic regions or can be
selected based on
sequence uniqueness (see Figure 2).
Detection on an antibody of the present invention can be facilitated by
coupling (i.e.,
physically linking) the antibody to a detectable substance. Examples of
detectable substances
include various enzymes, prosthetic groups, fluorescent materials, luminescent
materials,
bioluminescent materials, and radioactive materials. Examples of suitable
enzymes include
horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or
acetylcholinesterase; examples of
suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent materials
include luciferase,
luciferin, and aequorin, and examples of suitable radioactive material include
lash i3lI, ssS or 3H.
Antibod,
The antibodies can be used to isolate one of the proteins of the present
invention by standard
techniques, such as amity chromatography or immunoprecipitation. The
antibodies can facilitate
the purification of the natural protein from cells and recombinantly produced
protein expressed in
host cells. In addition, such antibodies are useful to detect the presence of
one of the proteins of the
present invention in cells or tissues to determine the pattern of expression
of the protein among
various tissues in an organism and over the course of normal development.
Experimental data as
provided in Figure 1 indicates that splice form 1 of the phosphatase protein
of the present invention
is expressed in humans in the pancreas, colon and pancreas adenocarcinomas,
pancreas epitheliod
carcinomas, lung large cell carcinomas, renal cell carcinomas, placenta
choriocarcinomas, ovary
tumor tissue, brain (including fetal), heart (including fetal), kidney
(including fetal), uterus, and
thyroid. Additionally, splice form 3 is expressed in fetal brain.
Specifically, a virtual northern blot
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shows expression in pancreas, colon and pancreas adenocarcinomas, pancreas
epitheliod
carcinomas, lung large cell carcinomas, renal cell carcinomas, placenta
choriocarcinomas, and
ovary tumor tissue. In addition, PCR-based tissue screening panels indicate
expression in the brain
(including fetal), heart (including fetal), kidney (including fetal), uterus,
and thyroid. Further, such
antibodies can be used to detect protein ih situ, in vitro, or in a cell
lysate or supernatant in order to
evaluate the abundance and pattern of expression. Also, such antibodies can be
used to assess
abnormal tissue distribution or abnormal expression during development or
progression of a
biological condition. Antibody detection of circulating fragments of the full
length protein can be
used to identify turnover.
Further, the antibodies can be used to assess expression in disease states
such as in active
stages of the disease or in an individual with a predisposition toward disease
related to the protein's
function. When a disorder is caused by an inappropriate tissue distribution,
developmental
expression, level of expression of the protein, or expressedlprocessed form,
the antibody can be
prepared against the normal protein. Experimental data as provided in Figure 1
indicates that splice
form 1 of the present invention is expressed in humans in the pancreas, colon
and pancreas
adenocarcinomas, pancreas epitheliod carcinomas, lung large cell carcinomas,
renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart (including
fetal), kidney (including fetal), uterus, and thyroid. Additionally, splice
form 3 is expressed in fetal
brain. If a disorder is characterized by a specific mutation in the protein,
antibodies specific for this
mutant protein can be used to assay for the presence of the specific mutant
protein.
The antibodies can also be used to assess normal and aberrant subcellular
localization of
cells in the various tissues in an organism. Experimental data as provided in
Figure 1 indicates that
splice form 1 of the present invention is expressed in humans in the pancreas,
colon and pancreas
adenocarcinomas, pancreas epitheliod carcinomas, lung large cell carcinomas,
renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart (including
fetal), kidney (including fetal), uterus, and thyroid. Additionally, splice
form 3 is expressed in fetal
brain. The diagnostic uses can be applied, not only in genetic testing, but
also in monitoring a
treatment modality. Accordingly, where treatment is ultimately aimed at
correcting expression level
or the presence of aberrant sequence and aberrant tissue distribution or
developmental expression,
antibodies directed against the protein or relevant fragments can be used to
monitor therapeutic
efficacy.
Additionally, antibodies are useful in pharmacogenomic analysis. Thus,
antibodies prepared
against polymorphic proteins can be used to identify individuals that require
modified treatment
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WO 02/42436 PCT/USO1/42995
modalities. The antibodies are also useful as diagnostic tools as an
immunological marker for
aberrant protein analyzed by electrophoretic mobility, isoelectric point,
tryptic peptide digest, and
other physical assays known to those in the art.
The antibodies are also useful for tissue typing. Experimental data as
provided in Figure 1
indicates that splice form 1 of the present invention is expressed in humans
in the pancreas, colon
and pancreas adenocarcinomas, pancreas epitheliod carcinomas, lung large cell
carcinomas, renal
cell carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain
(including fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid.
Additionally, splice form 3 is
expressed in fetal brain. Thus, where a specific protein has been correlated
with expression in a
specific tissue, antibodies that are specific for this protein can be used to
identify a tissue type.
The antibodies are also useful for inhibiting protein function, for example,
blocking the
binding of the phosphatase peptide to a binding partner such as a substrate.
These uses can also be
applied in a therapeutic context in which treatment involves inhibiting the
protein's function. An
antibody can be used, for example, to block binding, thus modulating
(agonizing or antagonizing)
the peptides activity. Antibodies can be prepared against specific fragments
containing sites
required for function or against intact protein that is associated with a cell
or cell membrane. See
Figure 2 for structural information relating to the proteins of the present
invention.
The invention also encompasses kits for using antibodies to detect the
presence of a protein
in a biological sample. The kit can comprise antibodies such as a labeled or
labelable antibody and
a compound or agent for detecting protein in a biological sample; means for
determining the amount
of protein in the sample; means for comparing the amount of protein in the
sample with a standard;
and instructions for use. Such a kit can be supplied to detect a single
protein or epitope or can be
configured to detect one of a multitude of epitopes, such as in an antibody
detection array. Arrays
are described in detail below for nuleic acid arrays and similar methods have
been developed for
antibody arrays.
Nucleic Acid Molecules
The present invention further provides isolated nucleic acid molecules that
encode a
phosphatase peptide or protein of the present invention (cDNA, transcript and
genomic sequence).
Such nucleic acid molecules will consist of, consist essentially of, or
comprise a nucleotide
sequence that encodes one of the phosphatase peptides of the present
invention, an allelic variant
thereof, or an ortholog or paralog thereof.
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WO 02/42436 PCT/USO1/42995
As used herein, an "isolated" nucleic acid molecule is one that is separated
from other
nucleic acid present in the natural source of the nucleic acid. 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. However, there can be some flanking nucleotide sequences, for example
up to about SKB,
4KB, 3I~B, 2KB, or lI~B or less, particularly contiguous peptide encoding
sequences and peptide
encoding sequences within the same gene but separated by introns in the
genomic sequence. The
important point is that the nucleic acid is isolated from remote and
unimportant flanking sequences
such that it can be subjected to the specific manipulations described herein
such as recombinant
expression, preparation of probes and primers, and other uses specific to the
nucleic acid sequences.
Moreover, an "isolated" nucleic acid molecule, such as a transcript/cDNA
molecule, can be
substantially free of other cellular material, or culture medium when produced
by recombinant
techniques, or chemical precursors or other chemicals when chemically
synthesized. However, the
nucleic acid molecule can be fused to other coding or regulatory sequences and
still be considered
isolated.
For example, recombinant DNA molecules contained in a vector are considered
isolated.
Further examples of isolated DNA molecules include recombinant DNA molecules
maintained in
heterologous host cells or purified (partially or substantially) DNA molecules
in solution. Isolated
RNA molecules include in vivo or ih vitro RNA transcripts of the isolated DNA
molecules of the
present invention. Isolated nucleic acid molecules according to the present
invention further include
such molecules produced synthetically.
Accordingly, the present invention provides nucleic acid molecules that
consist of the
nucleotide sequence shown in Figure 1 or 3 [SEQ ID NOS:1,4,6 (cDNA sequences)
and SEQ ID
N0:3 (genomic sequence)], or any nucleic acid molecule that encodes the
~protein provided in
Figure 2, SEQ ID NOS:2,5,7. A nucleic acid molecule consists of a nucleotide
sequence when the
nucleotide sequence is the complete nucleotide sequence of the nucleic acid
molecule.
The present invention further provides nucleic acid molecules that consist
essentially of the
nucleotide sequence shown in Figure 1 or 3 [SEQ ID NOS:1,4,6 (cDNA sequences)
and SEQ ID
NO:3 (genomic sequence)], or any nucleic acid molecule that encodes the
protein provided in
Figure 2, SEQ ID NOS:2,5,7. A nucleic acid molecule consists essentially of a
nucleotide sequence
when such a nucleotide sequence is present with only a few additional nucleic
acid residues in the
final nucleic acid molecule.
The present invention further provides nucleic acid molecules that comprise
the nucleotide
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WO 02/42436 PCT/USO1/42995
sequences shown in Figure 1 or 3 [SEQ ID NOS:1,4,6 (cDNA sequences) and SEQ ID
N0:3
(genomic sequence)], or any nucleic acid molecule that encodes the protein
provided in Figure 2,
SEQ ID NOS:2,5,7. A nucleic acid molecule comprises a nucleotide sequence when
the nucleotide
sequence is at least part of the final nucleotide sequence of the nucleic acid
molecule. In such a
fashion, the nucleic acid molecule can be only the nucleotide sequence or have
additional nucleic
acid residues, such as nucleic acid residues that are naturally associated
with it or heterologous
nucleotide sequences. Such a nucleic acid molecule can have a few additional
nucleotides or can
comprises several hundred or more additional nucleotides. A brief description
of how various types
of these nucleic acid molecules can be readily made/isolated is provided
below.
In Figures 1 and 3, both coding and non-coding sequences are provided. Because
of the
source of the present invention, humans genomic sequence (Figure 3) and
cDNA/transcript
sequences (Figure 1), the nucleic acid molecules in the Figures will contain
genomic intronic
sequences, 5' and 3' non-coding sequences, gene regulatory regions and non-
coding intergenic
sequences. In general such sequence features are either noted in Figures 1 and
3 or can readily
be identified using computational tools known in the art. As discussed below,
some of the non-
coding regions, particularly gene regulatory elements such as promoters, are
useful for a variety
of purposes, e.g. control of heterologous gene expression, target for
identifying gene activity
modulating compounds, and are particularly claimed as fragments of the genomic
sequence
provided herein.
The isolated nucleic acid molecules can encode the mature protein plus
additional amino or
carboxyl-terminal amino acids, or amino acids interior to the mature peptide
(when the mature form
has more than one peptide chain, for instance). Such sequences may play a role
in processing of a
protein from precursor to a mature form, facilitate protein trafficking,
prolong or shorten protein
half life or facilitate manipulation of a protein for assay or production,
among other things. As
generally is the case in situ, the additional amino acids may be processed
away from the mature
protein by cellular enzymes.
As mentioned above, the isolated nucleic acid molecules include, but are not
limited to, the
sequence encoding the phosphatase peptide alone, the sequence encoding the
mature peptide and
additional coding sequences, such as a leader or secretory sequence (e.g., a
pre-pro or pro-protein
sequence), the sequence encoding the mature peptide, with or without the
additional coding
sequences, plus additional non-coding sequences, for example introns and non-
coding 5' and 3'
sequences such as transcribed but non-translated sequences that play a role in
transcription, mRNA
processing (including splicing and polyadenylation signals), ribosome binding
and stability of
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WO 02/42436 PCT/USO1/42995
mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence
encoding, for
example, a peptide that facilitates purification.
Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in
the form
DNA, including cDNA and genomic DNA obtained by cloning or produced by
chemical synthetic
techniques or by a combination thereof. The nucleic acid, especially' DNA, can
be double-stranded
or single-stranded. Single-stranded nucleic acid can be the coding strand
(sense strand) or the non-
coding strand (anti-sense strand).
The invention furfiher provides nucleic acid molecules that encode fragments
of the peptides
of the present invention as well as nucleic acid molecules that encode obvious
variants of the
phosphatase proteins of the present invention that are described above. Such
nucleic acid molecules
may be naturally occurring, such as allelic variants (same locus), paralogs
(different locus), and
orthologs (different organism), or may be constructed by recombinant DNA
methods or by
chemical synthesis. Such non-naturally occurring variants may be made by
mutagenesis
techniques, including those applied to nucleic acid molecules, cells, or
organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions, deletions,
inversions and
insertions. Variation can occur in either or both the coding and non-coding
regions. The variations
can produce both conservative and non-conservative amino acid substitutions.
The present invention further provides non-coding fragments of the nucleic
acid molecules
provided in Figures 1 and 3. Preferred non-coding fragments include, but are
not limited to,
promoter sequences, enhancer sequences, gene modulating sequences and gene
termination
sequences. Such fragments are useful in controlling heterologous gene
expression and in
developing screens to identify gene-modulating agents. A promoter can readily
be identified as
being 5' to the ATG start site in the genomic sequence provided in Figure 3.
A fragment comprises a contiguous nucleotide sequence greater than 12 or more
nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500
nucleotides in length.
The length of the fragment will be based on its intended use. For example, the
fragment can encode
epitope bearing regions of the peptide, or can be useful as DNA probes and
primers. Such
fragments can be isolated using the known nucleotide sequence to synthesize an
oligonucleotide
probe. A labeled probe can then be used to screen a cDNA library, genomic DNA
library, or
mRNA to isolate nucleic acid corresponding to the coding region. Further,
primers can be used in
PCR reactions to clone specific regions of gene.
A probelprimer typically comprises substantially a purified oligonucleotide or
oligonucleotide pair. The oligonucleotide typically comprises a region of
nucleotide sequence that
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive
nucleotides.
Orthologs, homologs, and allelic variants can be identified using methods well
known in the
art. As described in the Peptide Section, these variants comprise a nucleotide
sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least
about 90-95% or
more homologous to the nucleotide sequence shown in the Figure sheets or a
fragment of this
sequence. Such nucleic acid molecules can readily be identified as being able
to hybridize under
moderate to stringent conditions, to the nucleotide sequence shown in the
Figure sheets or a
fragment of the sequence. Allelic variants can readily be determined by
genetic locus of the
encoding gene. The gene encoding the novel phosphatase proteins of the present
invention is
located on public BAC AP001885, which is known to be mapped to chromosome 11
(as indicated
in Figure 3).
Figure 3 provides information on SNPs that have been found in the gene
encoding the
phosphatase proteins of the present invention. The following SNPs were
identified: G577A,
G1451A, and G2641A. G577A and G1451A are non-synonymous coding SNPs. Changes
in the
amino acid sequence caused by these SNPs is indicated in Figure 3 and can
readily be determined
using the universal genetic code and the protein sequence provided in Figure 2
as a reference.
G2641A is 3' of the ORF and may affect control/regulatory elements.
As used herein, the term "hybridizes under stringent conditions" is intended
to describe
conditions for hybridization and washing under which nucleotide sequences
encoding a peptide at
least 60-70% homologous to each other typically remain hybridized to each
other. The conditions
can be such that sequences at least about 60%, at least about 70%, or at least
about 80% or more
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. One example of stringent hybridization
conditions are
hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C,
followed by one or more
washes in 0.2 X SSC, 0.1% SDS at 50-65C. Examples of moderate to low
stringency hybridization
conditions are well known in the art.
Nucleic Acid Molecule Uses
The nucleic acid molecules of the present invention are useful for probes,
primers, chemical
intermediates, and in biological assays. The nucleic acid molecules are useful
as a hybridization
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probe for messenger RNA, transcriptlcDNA and genomic DNA to isolate full-
length cDNA and
genomic clones encoding the peptide described in Figure 2 and to isolate cDNA
and genomic
clones that correspond to variants (alleles, orthologs, etc.) producing the
same or related peptides
shown in Figure 2. As illustrated in Figure 3, the following SNP variations
were identified: G577A,
G1451A, and G2641A.
The probe can correspond to any sequence along the entire length of the
nucleic acid
molecules provided in the Figures. Accordingly, it could be derived from 5'
noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed, fragments are
not to be construed
as encompassing fragments disclosed prior to the present invention.
The nucleic acid molecules are also useful as primers for PCR to amplify any
given region
of a nucleic acid molecule and are useful to synthesize antisense molecules of
desired length and
sequence.
The nucleic acid molecules are also useful for constructing recombinant
vectors. Such
vectors include expression vectors that express a portion of, or all of, the
peptide sequences.
Vectors also include insertion vectors, used to integrate into another nucleic
acid molecule
sequence, such as into the cellular genome, to alter in situ expression of a
gene and/or gene product.
For example, an endogenous coding sequence can be replaced via homologous
recombination with
all or part of the coding region containing one or more specifically
introduced mutations.
The nucleic acid molecules are also useful for expressing antigenic portions
of the proteins.
The nucleic acid molecules are also useful as probes for determining the
chromosomal
positions of the nucleic acid molecules by means of ih situ hybridization
methods. The gene
encoding the novel phosphatase proteins of the present invention is located on
public BAC
AP001885, which is known to be mapped to chromosome 11 (as indicated in Figure
3).
The nucleic acid molecules are also useful in making vectors containing the
gene regulatory
regions of the nucleic acid molecules of the present invention.
The nucleic acid molecules are also useful for designing ribozymes
corresponding to all, or
a part, of the mRNA produced from the nucleic acid molecules described herein.
The nucleic acid molecules are also useful for making vectors that express
part, or all, of the
peptides.
The nucleic acid molecules are also useful for constructing host cells
expressing a part, or
all, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful for constructing transgenic animals
expressing
all, or a part, of the nucleic acid molecules and peptides.
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The nucleic acid molecules are also useful as hybridization probes for
determining the
presence, level, form and distribution of nucleic acid expression.
Experimental data as provided in
Figure 1 indicates that splice form 1 of the phosphatase protein of the
present invention is expressed
in humans in the pancreas, colon and pancreas adenocarcinomas, pancreas
epitheliod carcinomas,
lung large cell carcinomas, renal cell carcinomas, placenta choriocarcinomas,
ovary tumor tissue,
brain (including fetal), heart (including fetal), kidney (including fetal),
uterus, and thyroid.
Additionally, splice form 3 is expressed in fetal brain. Specifically, a
virtual northern blot shows
expression in pancreas, colon and pancreas adenocarcinomas, pancreas
epitheliod carcinomas, lung
large cell carcinomas, renal cell carcinomas, placenta choriocarcinomas, and
ovary tumor tissue. In
addition, PCR-based tissue screening panels indicate expression in the brain
(including fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid. Accordingly,
the probes can be used
to detect the presence of, or to determine levels of, a specific nucleic acid
molecule in cells, tissues,
and in organisms. The nucleic acid whose level is determined can be DNA or
RNA. Accordingly,
probes corresponding to the peptides described herein can be used to assess
expression and/or gene
copy number in a given cell, tissue, or organism. These uses are relevant for
diagnosis of disorders
involving an increase or decrease in phosphatase protein expression relative
to normal results.
In vitro techniques for detection of mRNA include Northern hybridizations and
in situ
hybridizations. 1h vitro techniques for detecting DNA includes Southern
hybridizations and i~ situ
hybridization.
Probes can be used as a part of a diagnostic test kit for identifying cells or
tissues that
express a phosphatase protein, such as by measuring a level of a phosphatase-
encoding nucleic acid
in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining
if a phosphatase
gene has been mutated. Experimental data as provided in Figure 1 indicates
that splice form 1 of
the phosphatase protein of the present invention is expressed in humans in the
pancreas, colon and
pancreas adenocarcinomas, pancreas epitheliod carcinomas, lung large cell
carcinomas, renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart (including
fetal), kidney (including fetal), uterus, and thyroid. Additionally, splice
form 3 is expressed in fetal
brain. Specifically, a virtual northern blot shows expression in pancreas,
colon and pancreas
adenocarcinomas, pancreas epitheliod carcinomas, lung large cell carcinomas,
renal cell
carcinomas, placenta choriocarcinomas, and ovary tumor tissue. In addition,
PCR-based tissue
screening panels indicate expression in the brain (including fetal), heart
(including fetal), kidney
(including fetal), uterus, and thyroid.
Nucleic acid expression assays are useful for drug screening to identify
compounds that
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modulate phosphatase nucleic acid expression.
The invention thus provides a method for identifying a compound that can be
used to treat a
disorder associated with nucleic acid expression of the phosphatase gene,
particularly biological and
pathological processes that are mediated by the phosphatase in cells and
tissues that express it.
Experimental data as provided in Figure 1 indicates that splice form 1 of the
present invention is
expressed in humans in the pancreas, colon and pancreas adenocarcinomas,
pancreas epitheliod
carcinomas, lung large cell carcinomas, renal cell carcinomas, placenta
choriocarcinomas, ovary
tumor tissue, brain (including fetal), heart (including fetal), kidney
(including fetal), uterus, and
thyroid. Additionally, splice form 3 is expressed in fetal brain. The method
typically includes
assaying the ability of the compound to modulate the expression of the
phosphatase nucleic acid and
thus identifying a compound that can be used to treat a disorder characterized
by undesired
phosphatase nucleic acid expression. The assays can be performed in cell-based
and cell-free
systems. Cell-based assays include cells naturally expressing the phosphatase
nucleic acid or
recombinant cells genetically engineered to express specific nucleic acid
sequences.
The assay for phosphatase nucleic acid expression can involve direct assay of
nucleic acid
levels, such as mRNA levels, or on collateral compounds involved in the signal
pathway. Further,
the expression of genes that are up- or down-regulated in response to the
phosphatase protein signal
pathway can also be assayed. In this embodiment the regulatory regions of
these genes can be
operably linked to a reporter gene such as luciferase.
Thus, modulators of phosphatase gene expression can be identified in a method
wherein a
cell is contacted with a candidate compound and the expression of mRNA
determined. The level of
expression of phosphatase mRNA in the presence of the candidate compound is
compared to the
level of expression of phosphatase mRNA in the absence of the candidate
compound. The
candidate compound can then be identified as a modulator of nucleic acid
expression based on this
comparison and be used, for example to treat a disorder characterized by
aberrant nucleic acid
expression. When expression of mRNA is statistically significantly greater in
the presence of the
candidate compound than in its absence, the candidate compound is identified
as a stimulator of
nucleic acid expression. When nucleic acid expression is statistically
significantly less in. the
presence of the candidate compound than in its absence, the candidate compound
is identified as an
inhibitor of nucleic acid expression.
The invention further provides methods of treatment, with the nucleic acid as
a target, using
a compound identified through drug screening as a gene modulator to modulate
phosphatase nucleic
acid expression in cells and tissues that express the phosphatase.
Experimental .data as provided in
34
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
Figure 1 indicates that splice form 1 of the phosphatase protein of the
present invention is expressed
in humans in the pancreas, colon and pancreas adenocarcinomas, pancreas
epitheliod carcinomas,
lung large cell carcinomas, renal cell carcinomas, placenta choriocarcinomas,
ovary tumor tissue,
brain (including fetal), heart (including fetal), kidney (including fetal),
uterus, and thyroid.
Additionally, splice form 3 is expressed in fetal brain. Specifically, a
virtual northern blot shows
expression in pancreas, colon and pancreas adenocarcinomas, pancreas
epitheliod carcinomas, lung
large cell carcinomas, renal cell carcinomas, placenta choriocarcinomas, and
ovary tumor tissue. In
addition, PCR-based tissue screening panels indicate expression in the brain
(including fetal), heart
(including fetal), kidney (including fetal), uterus, and thyroid. Modulation
includes both up-
regulation (i.e. activation or agonization) or down-regulation (suppression or
antagonization) or
nucleic acid expression.
Alternatively, a modulator for phosphatase nucleic acid expression can be a
small molecule
or drug identified using the screening assays described herein as long as the
drug or small molecule
inhibits the phosphatase nucleic acid expression in the cells and tissues that
express the protein.
Experimental data as provided in Figure 1 indicates that splice form 1 of the
present invention is
expressed in humans in the pancreas, colon and pancreas adenocarcinomas,
pancreas epitheliod
carcinomas, lung large cell carcinomas, renal cell carcinomas, placenta
choriocarcinomas, ovary
tumor tissue, brain (including fetal), heart (including fetal), kidney
(including fetal), uterus, and
thyroid. Additionally, splice form 3 is expressed in fetal brain.
The nucleic acid molecules are also useful for monitoring the effectiveness of
modulating
compounds on the expression or activity of the phosphatase gene in clinical
trials or in a treatment
regimen. Thus, the gene expression pattern can serve as a barometer for the
continuing
effectiveness of treatment with the compound, particularly with compounds to
which a patient can
develop resistance. The gene expression pattern can also serve as a marker
indicative of a
physiological response of the affected cells to the compound. Accordingly,
such monitoring would
allow either increased administration of the compound or the administration of
alternative
compounds to which the patient has not become resistant. Similarly, if the
level of nucleic acid
expression falls below a desirable level, administration of the compound could
be commensurately
decreased.
The nucleic acid molecules are also useful in diagnostic assays for
qualitative changes in
phosphatase nucleic acid expression, and particularly in qualitative changes
that lead to pathology.
The nucleic acid molecules can be used to detect mutations in phosphatase
genes and gene
expression products such as mRNA. The nucleic acid molecules can be used as
hybridization
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
probes to detect naturally occurring genetic mutations in the phosphatase gene
and thereby to
determine whether a subject with the mutation is at risk for a disorder caused
by the mutation.
Mutations include deletion, addition, or substitution of one or more
nucleotides in the gene,
chromosomal rearrangement, such as inversion or transposition, modification of
genomic DNA,
such as aberrant methylation patterns or changes in gene copy number, such as
amplification.
Detection of a mutated form of the phosphatase gene associated with a
dysfimction provides a
diagnostic tool for an active disease or susceptibility to disease when the
disease results from
overexpression, underexpression, or altered expression of a phosphatase
protein.
Individuals carrying mutations in the phosphatase gene can be detected at the
nucleic acid
level by a variety of techniques. Figure 3 provides information on SNPs that
have been found in the
gene encoding the phosphatase proteins of the present invention. The following
SNPs were
identified: G577A, G1451A, and G2641A. 6577A and G1451A are non-synonymous
coding SNPs.
Changes in the amino acid sequence caused by these SNPs is indicated in Figure
3 and can readily
be determined using the universal genetic code and the protein sequence
provided in Figure 2 as a
reference. 62641 A is 3' of the ORF and may affect control/regulatory
elements. The gene
encoding the novel phosphatase proteins of the present invention is located on
public BAC
AP001885, which is known to be mapped to chromosome 11 (as indicated in Figure
3). Genomic
DNA can be analyzed directly or 'can be amplified by using PCR prior to
analysis. RNA or cDNA
can be used in the same way. In some uses, detection of the mutation involves
the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. LJ.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., Science 241:1077-1080 (1988); and Nakazawa et
al., PNAS 91:360-364
(1994)), the latter of which can be particularly useful for detecting point
mutations in the gene (see
Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can
include the steps of
collecting a sample of cells from a patient, isolating nucleic acid (e.g.,
genomic, mRNA or both)
from the cells of the sample, contacting the nucleic acid sample with one or
more primers which
specifically hybridize to a gene under conditions such that hybridization and
amplification of the
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.
Deletions and insertions can be detected by a change in size of the amplified
product compared to
the normal genotype. Point mutations can be identified by hybridizing
amplified DNA to normal
RNA or antisense DNA sequences.
Alternatively, mutations in a phosphatase gene can be directly identified, for
example, by
36
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
alterations in restriction enzyme digestion patterns determined by gel
electrophoresis.
Further, sequence-specific ribozymes (LJ.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. Perfectly
matched sequences can be distinguished from mismatched sequences by nuclease
cleavage
digestion assays or by differences in melting temperature.
Sequence changes at specific locations can also be assessed by nuclease
protection assays
such as RNase and S 1 protection or the chemical cleavage method. Furthermore,
sequence
differences between a mutant phosphatase gene and a wild-type gene can be
determined by direct
DNA sequencing. A variety of automated sequencing procedures can be utilized
when performing
the diagnostic assays (Naeve, C.W., (1995) Biotechniques 19:448), including
sequencing by mass
spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen
et al., Adv.
Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol.
38:147-159 (1993)).
Other methods for detecting mutations in the gene include methods in which
protection
from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA
duplexes
(Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth.
Euzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type
nucleic acid is
compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutest. Res.
285:125-144 (1993); and
Hayashi et al., Gehet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of
mutant or wild-type
fragments in polyacrylamide gels containing a gradient of denaturant is
assayed using denaturing
gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples
of other techniques
for detecting point mutations include selective oligonucleotide hybridization,
selective
amplification, and selective primer extension.
The nucleic acid molecules are also useful for testing an individual for a
genotype that while
not necessarily causing the disease, nevertheless affects the treatment
modality. Thus, the nucleic
acid molecules can be used to study the relationship between an individual's
genotype and the
individual's response to a compound used for treatment (pharmacogenomic
relationship).
Accordingly, the nucleic acid molecules described herein can be used to assess
the mutation content
of the phosphatase gene in an individual in order to select an appropriate
compound or dosage
regimen for treatment. Figure 3 provides information on SNPs that have been
found in the gene
encoding the phosphatase proteins of the present invention. The following SNPs
were identified:
G577A, G1451A, and G2641A. G577A and G1451A are non-synonymous coding SNPs.
Changes
in the amino acid sequence caused by these SNPs is indicated in Figure 3 and
can readily be
determined using the universal genetic code and the protein sequence provided
in Figure 2 as a
37
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
reference. G2641A is 3' of the ORF and may affect control/regulatory elements.
Thus nucleic acid molecules displaying genetic variations that affect
treatment provide a
diagnostic target that can be used to tailor treatment in an individual.
Accordingly, the production
of recombinant cells and animals containing these polymorphisms allow
effective clinical design of
treatment compounds and dosage regimens.
The nucleic acid molecules are thus useful as antisense constructs to control
phosphatase
gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid
molecule is
designed to be complementary to a region of the gene involved in
transcription, preventing
transcription and hence production of phosphatase protein. An antisense RNA or
DNA nucleic acid
molecule would hybridize to the mRNA and thus block translation of mRNA into
phosphatase
protein.
Alternatively, a class of antisense molecules can be used to inactivate mRNA
in order to
decrease expression of phosphatase nucleic acid. Accordingly, these molecules
can treat a disorder
characterized by abnormal or undesired phosphatase nucleic acid expression.
This technique
involves cleavage by means of ribozymes containing nucleotide sequences
complementary to one or
more regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions
include coding regions and particularly coding regions corresponding to the
catalytic and other
functional activities of the phosphatase protein, such as substrate binding.
The nucleic acid molecules also provide vectors for gene therapy in patients
containing cells
that are aberrant in phosphatase gene expression. Thus, recombinant cells,
which include the
patient's cells that have been engineered ex vivo and returned to the patient,
are introduced into an
individual where the cells produce the desired phosphatase protein to treat
the individual.
The invention also encompasses kits for detecting the presence of a
phosphatase nucleic acid
in a biological sample. Experimental data as provided in Figure 1 indicates
that splice form 1 of the
phosphatase protein of the present invention is expressed in humans in the
pancreas, colon and
pancreas adenocarcinomas, pancreas epitheliod carcinomas, lung large cell
carcinomas, renal cell
carcinomas, placenta choriocarcinomas, ovary tumor tissue, brain (including
fetal), heart (including
fetal), kidney (including fetal), uterus, and thyroid. Additionally, splice
form 3 is expressed in fetal
brain. Specifically, a virtual northern blot shows expression in pancreas,
colon and pancreas
adenocarcinomas, pancreas epitheliod carcinomas, lung large cell carcinomas,
renal cell
carcinomas, placenta choriocarcinomas, and ovary tumor tissue. In addition,
PCR-based tissue
screening panels indicate expression in the brain (including fetal), heart
(including fetal), kidney
(including fetal), uterus, and thyroid. For example, the kit can comprise
reagents such as a labeled
38
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
or labelable nucleic acid or agent capable of detecting phosphatase nucleic
acid in a biological
sample; means for determining the amount of phosphatase nucleic acid in the
sample; and means for
comparing the amount of phosphatase nucleic acid 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 phosphatase protein mRNA or DNA.
Nucleic Acid Arrays
The present invention further provides nucleic acid detection kits, such as
arrays or
microarrays of nucleic acid molecules that are based on the sequence
information provided in
Figures l and 3 (SEQ ID NOS:1,3,4,6).
As used herein "Arrays" or "Microarrays" refers to an array of distinct
polynucleotides or
oligonucleotides synthesized on a substrate, such as paper, nylon or other
type of membrane,
filter, chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is
prepared and used according to the methods described in US Patent 5,837,832,
Chee et al., PCT
application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat.
Biotech. 14: 1675-1680)
and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of
which are
incorporated herein in their entirety by reference. In other embodiments, such
arrays axe
produced by the methods described by Brown et al., US Patent No. 5,807,522.
The microarray or detection kit is preferably composed of a large number of
unique,
single-stranded nucleic acid sequences, usually either synthetic antisense
oligonucleotides or
fragments of cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60
nucleotides in length, more preferably 15-30 nucleotides in length, and most
preferably about 20-
nucleotides in length. For a certain type of microarray or detection kit, it
may be preferable to
use oligonucleotides that are only 7-20 nucleotides in length. The microarray
or detection kit
25 may contain oligonucleotides that cover the known 5', or 3', sequence,
sequential
oligonucleotides which cover the full length sequence; or unique
oligonucleotides selected from
particular areas along the length of the sequence. Polynucleotides used in the
microarray or
detection kit may be oligonucleotides that are specific to a gene or genes of
interest.
In order to produce oligonucleotides to a known sequence for a microarray or
detection
kit, the genes) of interest (or an ORF identified from the contigs of the
present invention) is
typically examined using a computer algorithm which starts at the 5' or at the
3' end of the
nucleotide sequence. Typical algorithms will then identify oligomers of
defined length that are
39
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
unique to the gene, have a GC content within a range suitable for
hybridization, and lack
predicted secondary structure that may interfere with hybridization. In
certain situations it may
be appropriate to use pairs of oligonucleotides on a microarray or detection
kit. The "pairs" will
be identical, except for one nucleotide that preferably is located in the
center of the sequence.
The second oligonucleotide in the pair (mismatched by one) serves as a
control. The number of
oligonucleotide pairs may range from two to one million. The oligomers are
synthesized at
designated areas on a substrate using a light-directed chemical process. The
substrate may be
paper, nylon or other type of membrane, filter, chip, glass slide or any other
suitable solid
support.
In another aspect, an oligonucleotide may be synthesized on the surface of the
substrate
by using a chemical coupling procedure and an ink jet application apparatus,
as described in PCT
application W095/251116 (Baldeschweiler et al.) which is incorporated herein
in its entirety by
reference. In another aspect, a "gridded" array analogous to a dot (or slot)
blot may be used to
arrange and link cDNA fragments or oligonucleotides to the surface of a
substrate using a
vacuum system, thermal, LTV, mechanical or chemical bonding procedures. An
array, such as
those described above, may be produced by hand or by using available devices
(slot blot or dot
blot apparatus), materials (any suitable solid support), and maclunes
(including robotic
instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more
oligonucleotides, or any other
number between two and one million which lends itself to the efficient use of
commercially
available instrumentation.
In order to conduct sample analysis using a microarray or detection kit, the
RNA or DNA
from a biological sample is made into hybridization probes. The mRNA is
isolated, and cDNA is
produced and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the
presence of fluorescent nucleotides, and labeled probes are incubated with the
microarray or
detection kit so that the probe sequences hybridize to complementary
oligonucleotides of the
microarray or detection kit. Incubation conditions are adjusted so that
hybridization occurs with
precise complementary matches or with various degrees of less complementarity.
After removal
of nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence.
The scanned images are examined to determine degree of complementarity and the
relative
abundance of each oligonucleotide sequence on the microarray or detection kit.
The biological
samples may be obtained from any bodily fluids (such as blood, urine, saliva,
phlegm, gastric
juices, etc.), cultured cells, biopsies, or other tissue preparations. A
detection system may be
used to measure the absence, presence, and amount of hybridization for all of
the distinct
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
sequences simultaneously. This data may be used for large-scale correlation
studies on the
sequences, expression patterns, mutations, variants, or polymorphisms among
samples.
Using such arrays, the present invention provides methods to identify the
expression of
the phosphatase proteins/peptides of the present invention. In detail, such
methods comprise
incubating a test sample with one or more nucleic acid molecules and assaying
for binding of the
nucleic acid molecule with components within the test sample. Such assays will
typically
involve arrays comprising many genes, at least one of which is a gene of the
present invention
and or alleles of the phosphatase gene of the present invention. Figure 3
provides information on
SNPs that have been found in the gene encoding the phosphatase proteins of the
present
invention. The following SNPs were identified: G577A, G1451A, and G2641A.
G577A and
G1451A are non-synonymous coding SNPs. Changes in the amino acid sequence
caused by
these SNPs is indicated in Figure 3 and can readily be determined using the
universal genetic
code and the protein sequence provided in Figure 2 as a reference. G2641A is
3' of the ORF and
may affect control/regulatory elements.
Conditions for incubating a nucleic acid molecule with a test sample vary.
Incubation
conditions depend on the format employed in the assay, the detection methods
employed, and the
type and nature of the nucleic acid molecule used in the assay. One skilled in
the art will
recognize that any one of the commonly available hybridization, amplification
or array assay
formats can readily be adapted to employ the novel fragments of the Human
genome disclosed
herein. Examples of such assays can be found in Chard, T, Arc Iutroductio~ to
Radioimmu~coassay and Related Techv~iques, Elsevier Science Publishers,
Amsterdam, The
Netherlands (1986); Bullock, G. R. et al., Techniques in Immuhocytochemist~y,
Academic
Press, Orlando, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P.,
Practice ahd
Theory of Enzyme Immunoassays: Laboratory Techniques irc Biochemistry ahd
Molecular
Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
The test samples of the present invention include cells, protein or membrane
extracts of
cells. The test sample used in the above-described method will vary based on
the assay format,
nature of the detection method and the tissues, cells or extracts used as the
sample to be assayed.
Methods for preparing nucleic acid extracts or of cells are well known in the
art and can be
readily be adapted in order to obtain a sample that is compatible with the
system utilized.
In another embodiment of the present invention, kits are provided which
contain the
necessary reagents to carry out the assays of the present invention.
Specifically, the invention provides a compartmentalized kit to receive, in
close
41
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
confinement, one or more containers which comprises: (a) a first container
comprising one of the
nucleic acid molecules that can bind to a fragment of the Human genome
disclosed herein; and
(b) one or more other containers comprising one or more of the following: wash
reagents,
reagents capable of detecting presence of a bound nucleic acid.
In detail, a compartmentalized kit includes any kit in which reagents are
contained in
separate containers. Such containers include small glass containers, plastic
containers, strips of
plastic, glass or paper, or arraying material such as silica. Such containers
allows one to
efficiently transfer reagents from one compartment to another compartment such
that the
samples and reagents are not cross-contaminated, and the agents or solutions
of each container
can be added in a quantitative fashion from one compartment to another. Such
containers will
include a container which will accept the test sample, a container which
contains the nucleic acid
probe, containers which contain wash reagents (such as phosphate buffered
saline, Tris-buffers,
etc.), and containers which contain the reagents used to detect the bound
probe. One skilled in
the art will readily recognize that the previously unidentified phosphatase
gene of the present
invention can be routinely identified using the sequence information disclosed
herein can be
readily incorporated into one of the established kit formats which are well
known in the art,
particularly expression arrays.
Vectors/host cells
The invention also provides vectors containing the nucleic acid molecules
described herein.
The term "vector" refers to a vehicle, preferably a nucleic acid molecule,
which can transport the
nucleic acid molecules. When the vector is a nucleic acid molecule, the
nucleic acid molecules are
covalently linked to the vector nucleic acid. With this aspect of the
invention, the vector includes a
plasmid, single or double stranded phage, a single or double stranded RNA or
DNA viral vector, or
artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
A vector can be maintained in the host cell as an extrachromosomal element
where it
replicates and produces additional copies of the nucleic acid molecules.
Alternatively, the vector
may integrate into the host cell genome and produce additional copies of the
nucleic acid molecules
when the host cell replicates.
The invention provides vectors for the maintenance (cloning vectors) or
vectors for
expression (expression vectors) of the nucleic acid molecules. The vectors can
function in
prokaryotic or eukaryotic cells or in both (shuttle vectors).
42
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WO 02/42436 PCT/USO1/42995
Expression vectors contain cis-acting regulatory regions that are operably
linked in the
vector to the nucleic acid molecules such that transcription of the nucleic
acid molecules is allowed
in a host cell. The nucleic acid molecules can be introduced into the host
cell with a separate
nucleic acid molecule capable of affecting transcription. Thus, the second
nucleic acid molecule
may provide a traps-acting factor interacting with the cis-regulatory control
region to allow
transcription of the nucleic acid molecules from the vector. Alternatively, a
traps-acting factor may
be supplied by the host cell. Finally, a traps-acting factor can be produced
from the vector itself. It
is understood, however, that in some embodiments, transcription and/or
translation of the nucleic
acid molecules can occur in a cell-free system.
The regulatory sequence to which the nucleic acid molecules described herein
can be
operably linked include promoters for directing mRNA transcription. These
include, but are not
limited to, the left promoter from bacteriophage ~,, the lac, TRP, and TAC
promoters from E. coli,
the early and late promoters from SV40, the CMV immediate early promoter, the
adenovirus early
and late promoters, and retrovirus long-terminal repeats.
In addition to control regions that promote transcription, expression vectors
may also
include regions that modulate transcription, such as repressor binding sites
and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate early
enhancer, polyoma
enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
In addition to containing sites for transcription initiation and control,
expression vectors can
also contain sequences necessary for transcription termination and, in the
transcribed region a
ribosome binding site for translation. Other regulatory control elements for
expression include
initiation and termination codons as well as polyadenylation signals. The
person of ordinary skill in
the art would be aware of the numerous regulatory sequences that are useful in
expression vectors.
Such regulatory sequences are described, for example, in Sambrook et al.,
Molecular Cloning: A
Laboratory Manual. 2~d. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY,
(1989).
A variety of expression vectors can be used to express a nucleic acid
molecule. Such
vectors include chromosomal, episomal, and virus-derived vectors, for example
vectors derived
from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast
chromosomal
elements, including yeast artificial chromosomes, from viruses such as
baculoviruses,
papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses,
pseudorabies viruses, and
retroviruses. Vectors may also be derived from combinations of these sources
such as those derived
from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.
Appropriate
43
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
cloning and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et
al., Molecular Clo~i~cg: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold
Spring Harbor, NY, (1989).
The regulatory sequence may provide constitutive expression in one or more
host cells (i.e.
tissue specific) or may provide for inducible expression in one or more cell
types such as by
temperature, nutrient additive, or exogenous factor such as a hormone or other
ligand. A variety of
vectors providing for constitutive and inducible expression in prokaryotic and
eukaryotic hosts are
well known to those of ordinary skill in the art.
The nucleic acid molecules can be inserted into the vector nucleic acid by
well-known
methodology. Generally, the DNA sequence that will ultimately be expressed is
joined to an
expression vector by cleaving the DNA sequence and the expression vector with
one or more
restriction enzymes and then ligating the fragments together. Procedures for
restriction enzyme
digestion and ligation are well known to those of ordinary skill in the art.
The vector containing the appropriate nucleic acid molecule can be introduced
into an
appropriate host cell for propagation or expression using well-known
techniques. Bacterial cells
include, but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells
include, but are not limited to, yeast, insect cells such as Drosophila,
animal cells such as COS and
CHO cells, and plant cells.
As described herein, it may be desirable to express the peptide as a fusion
protein.
Accordingly, the invention provides fusion vectors that allow for the
production of the peptides.
Fusion vectors can increase the expression of a recombinant protein, increase
the solubility of the
recombinant protein, and aid in the purification of the protein by acting for
example as a ligand for
affinity purification. A proteolytic cleavage site may be introduced at the
junction of the fusion
moiety so that the desired peptide can ultimately be separated from the fusion
moiety. Proteolytic
enzymes include, but are not limited to, factor Xa, thrombin, and
enterophosphatase. Typical fusion
expression vectors include pGEX (Smith et al., Geue 67:31-40 (1988)), pMAL
(New England
Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, N~ which fuse
glutathione S-
transferase (GST), maltose E binding protein, or protein A, respectively, to
the target recombinant
protein. Examples of suitable inducible non-fusion E. coli expression vectors
include pTrc (Amann
et al., Gene 69:301-315 (1988)) and pET l 1d (Studier et al., Gene Expression
Technology: Methods
in Ehzymology 185:60-89 (1990)).
Recombinant protein expression can be maximized in host bacteria by providing
a genetic
background wherein the host cell has an impaired capacity to proteolytically
cleave the recombinant
44
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic
Press, San Diego, California (1990) 119-128). Alternatively, the sequence of
the nucleic acid
molecule of interest can be altered to provide preferential codon usage for a
specific host cell, for
example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).
The nucleic acid molecules can also be expressed by expression vectors that
are operative in
yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include
pYepSecl (Baldari, et
al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)),
pJRY88 (Schultz et
al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,
CA).
The nucleic acid molecules can also be expressed in insect cells using, for
example,
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., Mol.
Cell Biol. 3:2156-2165
(1983)) and the pVL series (Lucklow et al., Tli~ology 170:31-39 (1989)).
In certain embodiments of the invention, the nucleic acid molecules described
herein are
expressed in mammalian cells using mammalian expression vectors. Examples of
mammalian
expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufinan et al.,
EMBO J. 6:187-195 (1987)).
The expression vectors listed herein are provided by way of example only of
the well-
known vectors available to those of ordinary skill in the art that would be
useful to express the
nucleic acid molecules. The person of ordinary skill in the art would be aware
of other vectors
suitable for maintenance propagation or expression of the nucleic acid
molecules described herein.
These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T.
Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spying Harbor Labo~ato~y, Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, NY, 1989.
The invention also encompasses vectors in which the nucleic acid sequences
described
herein are cloned into the vector in reverse orientation, but operably linked
to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense transcript can
be produced to all, or
to a portion, of the nucleic acid molecule sequences described herein,
including both coding and
non-coding regions. Expression of this antisense RNA is subject to each of the
parameters
described above in relation to expression of the sense RNA (regulatory
sequences, constitutive or
inducible expression, tissue-specific expression).
The invention also relates to recombinant host cells containing the vectors
described herein.
Host cells therefore include prokaryotic cells, lower eukaryotic cells such as
yeast, other eukaryotic
cells such as insect cells, and higher eukaryotic cells such as mammalian
cells.
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
The recombinant host cells are prepared by introducing the vector constructs
described
herein into the cells by techniques readily available to the person of
ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection, DEAF-dextran-
mediated
transfection, cationic lipid-mediated transfection, electroporation,
transduction, infection,
lipofection, and other techniques such as those 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, 199).
Host cells can contain more than one vector. Thus, different nucleotide
sequences can be
introduced on different vectors of the same cell. Similarly, the nucleic acid
molecules can be
introduced either alone or with other nucleic acid molecules that are not
related to the nucleic acid
molecules such as those providing trans-acting factors for expression vectors.
When more than one
vector is introduced into a cell, the vectors can be introduced independently,
co-introduced or joined
to the nucleic acid molecule vector.
In the case of bacteriophage and viral vectors, these can be introduced into
cells as packaged
or encapsulated virus by standard procedures for infection and transduction.
Viral vectors can be
replication-competent or replication-defective. In the case in which viral
replication is defective,
replication will occur in host cells providing functions that complement the
defects.
Vectors generally include selectable markers that enable the selection of the
subpopulation
of cells that contain the recombinant vector constructs. The marker can be
contained in the same
vector that contains the nucleic acid molecules described herein or may be on
a separate vector.
Markers include tetracycline or ampicillin-resistance genes for prokaryotic
host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host cells.
However, any marker that
provides selection for a phenotypic trait will be effective.
While the mature proteins can be produced in bacteria, yeast, mammalian cells,
and other
cells under the control of the appropriate regulatory sequences, cell- free
transcription and
translation systems can also be used to produce these proteins using RNA
derived from the DNA
constructs described herein.
Where secretion of the peptide is desired, which is difficult to achieve with
multi-
transmembrane domain containing proteins such as phosphatases, appropriate
secretion signals are
incorporated into the vector. The signal sequence can be endogenous to the
peptides or
heterologous to these peptides.
Where the peptide is not secreted into the medium, which is typically the case
with
phosphatases, the protein can be isolated from the host cell by standard
disruption procedures,
46
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
including freeze thaw, sonication, mechanical disruption, use of lysing agents
and the like. The
peptide can then be recovered and purified by well-known purification methods
including
ammonium sulfate precipitation, acid extraction, anion or cationic exchange
chromatography,
phosphbcellulose chromatography, hydrophobic-interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography, lectin chromatography, or high
performance
liquid chromatography.
It is also understood that depending upon the host cell in recombinant
production of the
peptides described herein, the peptides can have various glycosylation
patterns, depending upon the
cell, or maybe non-glycosylated as when produced in bacteria. In addition, the
peptides may
include an initial modified methionine in some cases as a result of a host-
mediated process.
Uses of yectors and host cells
The recombinant host cells expressing the peptides described herein have a
variety of uses.
First, the cells are useful for producing a phosphatase protein or peptide
that can be further purified
to produce desired amounts of phosphatase protein or fragments. Thus, host
cells containing
expression vectors are useful for peptide production.
Host cells are also useful for conducting cell-based assays involving the
phosphatase protein
or phosphatase protein fragments, such as those described above as well as
other formats known in
the art. Thus, a recombinant host cell expressing a native phosphatase protein
is useful for assaying
compounds that stimulate or inhibit phosphatase protein function.
Host cells are also useful for identifying phosphatase protein mutants in
which these
functions are affected. If the mutants naturally occur and give rise to a
pathology, host cells
containing the mutations are useful to assay compounds that have a desired
effect on the mutant
phosphatase protein (for example, stimulating or inhibiting function) which
may not be indicated by
their effect on the native phosphatase protein.
Genetically engineered host cells can be fiu-ther used to produce non-human
transgenic
animals. A transgenic animal is preferably a mammal, for example a rodent,
such as a rat or mouse,
in which one or more of the cells of the animal include a transgene. 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 in one or more cell types or
tissues of the transgenic
animal. These animals are useful for studying the function of a phosphatase
protein and identifying
and evaluating modulators of phosphatase protein activity. Other examples of
transgenic animals
47
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
include non-human primates, sheep, dogs, cows, goats, chickens, and
amphibians.
A transgenic animal can be produced by introducing 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. Any of the phosphatase protein
nucleotide sequences can
be introduced as a transgene into the genome of a non-human animal, such as a
mouse.
Any of the regulatory or other sequences useful in expression vectors can form
part of the
transgenic sequence. This includes intronic sequences and polyadenylation
signals, if not already
included. A tissue-specific regulatory sequences) can be operably linked to
the transgene to direct
expression of the phosphatase protein to particular cells.
Methods for generating transgenic animals via embryo manipulation and
microinjection,
particularly animals such as mice, have become conventional in the art and are
described, for
example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Patent No.
4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo,
(Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are
used for
production of other transgenic animals. A transgenic founder animal can be
identified based upon
the presence of the transgene in its genome and/or expression of transgenic
mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used to breed
additional animals
carrying the transgene. Moreover, transgenic animals carrying a transgene can
further be bred to
other transgenic animals carrying other transgenes. A transgenic animal also
includes animals in
which the entire animal or tissues in the animal have been produced using the
homologously
recombinant host cells described herein.
In another embodiment, transgenic non-human animals can be produced which
contain
selected systems that allow for regulated expression of the transgene. One
example of such a
system is the crelloxP recombinase system of bacteriophage Pl. For a
description of the c~elloxP
recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another
example of a
recombinase system is the FLP recombinase system of S cerevisiae (O'Gorman et
al. Science
251:1351-1355 (1991). 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
is 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 Wilinut, I. et al. Nature 385:810-813
(1997) and PCT
48
CA 02398924 2002-07-19
WO 02/42436 PCT/USO1/42995
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
reconstructed oocyte is then cultured such that it develops to morula or
blastocyst and then
transferred to pseudopregnant female foster animal. The offspring born of this
female foster animal
will be a clone of the animal from which the cell, e.g., the somatic cell, is
isolated.
Transgenic animals containing recombinant cells that express the peptides
described herein
are useful to conduct the assays described herein in an in vivo context.
Accordingly, the various
physiological factors that are present ih vivo and that could effect substrate
binding, kinase protein
activation, and signal transduction, may not be evident from in vitro cell-
free or cell-based assays.
Accordingly, it is useful to provide non-human transgenic animals to assay in
vivo phosphatase
protein function, including substrate interaction, the effect of specific
mutant phosphatase proteins
on phosphatase protein function and substrate interaction, and the effect of
chimeric phosphatase
proteins. It is also possible to assess the efFect of null mutations, that is
mutations that substantially
or completely eliminate one or more phosphatase protein functions.
All publications and patents mentioned in the above specification are herein
incorporated
by reference. Various modifications and variations of the described method and
system of the
invention will be apparent to those skilled in the art without departing from
the scope and spirit
of the invention. Although the invention has been described in connection with
specific
preferred embodiments, it should be understood that the invention as claimed
should not be
unduly limited to such specific embodiments. Indeed, various modifications of
the above-
described modes for carrying out the invention which are obvious to those
skilled in the field of
molecular biology or related fields are intended to be within the scope of the
following claims.
49
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1
SEQUENCE LISTING
<110> PE CORPORATION (NY)
<120> ISOLATED HUMAN PHOSPHATASE PROTEINS,
NUCLEIC ACII? MOLECULES ENCODING HUMAN PHOSPHATASE PROTEINS,
AND USES THEREOF
<130> CL000964PCT
<150> TO BE ASSIGNED
<151> 2001-11-07
<140> 09/761,640
<141> 2001-O1-18
<140> 09/715,177
<141> 2000-11-20
<160> 10
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 2704
<212> DNA
<213> Homo Sapiens
<400> 1
cgtccttcct ggtcctgcgg gtccaggact gtccgcgggg ttgagggaag
gggccgtgcc 60
cggtgccagc ccaggtgctc gcggcctggc tccatggccc tggtcacagt
gagccgttcg 120
cccccgggca gcggcgcctc cacgcccgtg gggccctggg accaggcggt
ccagcgaagg 180
agtcgactcc agcgaaggca gagctttgcg gtgctccgtg gggctgtcct
gggactgcag 240
gatggagggg acaatgatga tgcagcagag gccagttctg agccaacaga
gaaggccccg 300
agtgaggagg agctccacgg ggaccagaca gacttcgggc aaggatccca
gagtccccag 360
aagcaggagg agcagaggca gcacctgcac ctcatggtac agctgctgag
gccgcaggat 420
gacatccgcc tggcagccca gctggaggca ccccggcctc cccggctccg
ctacctgctg 480
gtagtttcta cacgagaagg agaaggtctg agccaggatg agacggtcct
cctgggcgtg 540
gatttccctg acagcagctc ccccagctgc accctgggcc tggtcttgcc
cctctggagt 600
gacacccagg tgtacttaga tggagacggg ggcttcagcg tgacgtctgg
tgggcaaagc 660
cggatcttca agcccatctc catccagacc atgtgggcca cactccaggt
attgcaccaa 720
gcatgtgagg cagctctagg cagcggcctt gtaccgggtg gcagtgccct
cacctgggcc 780
agccactacc aggagagact gaactccgaa cagagctgcc tcaatgagtg
gacggctatg 840
gccgacctgg agtctctgcg gcctcccagc gccgagcctg gcgggtcctc
agaacaggag 900
CA 02398924 2002-07-19
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2
cagatggagc aggcgatccg tgctgagctg tggaaagtgt tggatgtcag
tgacctggag 960
agtgtcactt ccaaagagat ccgccaggct ctggagctgc gcctggggct
ccccctccag 1020
cagtaccgtg acttcatcga caaccagatg ctgctgctgg tggcacagcg
ggaccgagcc 1080
tcccgcatct tcccccacct ctacctgggc tcagagtgga acgcagcaaa
cctggaggag 1140
ctgcagagga acagggtcac ccacatcttg aacatggccc gggagattga
caacttctac 1200
cctgagcgct tcacctacca caatgtgcgc ctctgggatg aggagtcggc
ccagctgctg 1260
ccgcactgga aggagacgca ccgcttcatt gaggctgcaa gagcacaggg
cacccacgtg 1320
ctggtccact gcaagatggg cgtcagccgc tcagcggcca cagtgctggc
ctatgccatg 1380
aagcagtacg aatgcagcct ggagcaggcc ctgcgccacg tgcaggagct
ccggcccatc 1440
gcccgcccca accctggctt cctgcgccag ctgcagatct accagggcat
cctgacggcc 1500
agaacctgag ggtggtgggg aggagaaggt tgtaggcatg gaagagagcc
aggcagcccc 1560
gaaagaagag cctgggccac ggccacgtat aaacctccga ggggtcatga
ggtccatcag 1620
tcttctggag ccctccttgg agctggagag cacctcagag accagtgaca
tgccagaggt 1680
cttctcttcc cacgagtctt cacatgaaga gcctctgca~g cccttcccac
agcttgcaag 1740
gaccaaggga ggccagcagg tggacagggg gcctcagcct gccctgaagt
cccgccagtc 1800
agtggttacc ctccagggca gtgccgtggt ggccaaccgg acccaggcct
tccaggagca 1860
ggagcagggg caggggcagg ggcagggaga gccctgcatt tcctctacgc
ccaggttccg 1920
gaaggtggtg agacaggcca gcgtgcatga cagtggagag gagggcgagg
cctgagccct 1980
cacacatgcc cacgctcccc tgacactgaa gaggatccac aactccttgg
agaaacaccc 2040
tcacgtctgt tgccgcacac attcctctca gctccgcccc atacccgtca
ctacagcctc 2100
acctcccacc cctgtcacta cggcctcacc tcccacccct gtcactacag
cctcacctcc 2160
tacagcctta agtcccaggc ccatgtctgc ctgtccaagg gctcaagact
ttctaactgg 2220
gatgtggtag agggactgaa ggtacctttg ggggcaacag caccctagtt
tcattctcaa 2280
ctctagccct gcacactcac ctgtggcacg gaatgaaaac agagcttccc
gtgcaaaaag 2340
ggtcacgcct cccacccccg ccccctccct gcacctcctg tcctctccca
gttcattcct 2400
ggaaccagcc aggccaggca accagtggcc cccaaaggca ggcaggatcc
tcaggcccca 2460
gccgcgggag gctggaaggg ctggcagatc gcttccctca tccacctcca
ccggtccagg 2520
tctttgctgc tgtccccaga cctcctgtga caccacgcca gatcacaggg
caccaggcca 2580
gagatagtct tctttttgtc ctttctggcc tctggctagt cagtttttca
tagccttaca 2640
gtatctggct ttgtactgag aaataaaaca cattttcata aaaaaaaaaa
aaaaaaaaaa 2700
CA 02398924 2002-07-19
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3
aaaa
2704
<210> 2
<211> 2852
<212> DNA
<213> Homo sapiens
<400> 2
tggttgaggg aaggggccgt gcccggtgcc agcccaggtg ctcgcggcct
ggctccatgg 60
ccctggtcac agtgagccgt tcgcccccgg gcagcggcgc ctccacgccc
gtggggccct 120
gggaccaggc ggtccagcga aggagtcgac tccagcgaag gcagagcttt
gcggtgctcc 180
gtggggctgt cctgggactg caggatggag gggacaatga tgatgcagca
gaggccagtt 240
ctgagccaac agagaaggcc ccgagtgagg aggagctcca cggggaccag
acagacttcg 300
ggcaaggatc ccagagtccc cagaagcagg aggagcagag gcagcacctg
cacctcatgg 360
tacagctgct gaggccgcag gatgacatcc gcctggcagc ccagctggag
gcaccccggc 420
ctccccggct ccgctacctg ctggtagttt ctacacgaga aggagaaggt
ctgagccagg 480
atgagacggt cctcctgggc gtggatttcc ctgacagcag ctcccccagc
tgcaccctgg 540
gcctggtctt gcccctctgg agtgacaccc aggtgtactt agatggagac
gggggcttca 600
gcgtgacgtc tggtgggcaa agccggatct tcaagcccat ctccatccag
accatgtggg 660
ccacactcca ggtattgcac caagcatgtg aggcagctct aggcagcggc
cttgtaccgg 720
gtggcagtgc cctcacctgg gccagccact accaggagag actgaactcc
gaacagagct 780
gcctcaatga gtggacggct atggccgacc tggagtctct gcggcctccc
agcgccgagc 840
ctggcgggtc ctcagaacag gagcagatgg agcaggcgat ccgtgctgag
ctgtggaaag 900
tgttggatgt cagtgacctg gagagtgtca cttccaaaga gatccgccag
gctctggagc 960
tgcgcctggg gctccccctc cagcagtacc gtgacttcat cgacaaccag
atgctgctgc 1020
tggtggcaca gcgggaccga gcctcccgca tcttccccca cctctacctg
ggctcagagt 1080
ggaacgcagc aaacctggag gagctgcaga ggaacagggt cacccacatc
ttgaacatgg 1140
cccgggagat tgacaacttc taccctgagc gcttcaccta ccacaatgtg
cgcctctggg 1200
atgaggagtc ggcccagctg ctgccgcact ggaaggagac gcaccgcttc
attgaggctg 1260
caagagcaca gggcacccac gtgctggtcc actgcaagat gggcgtcagc
cgctcagcgg 1320
ccacagtgct ggcctatgcc atgaagcagt acgaatgcag cctggagcag
gccctgcgcc 1380
acgtgcagga gctccggccc atcgcccgcc ccaaccctgg cttcctgcgc
cagctgcaga 1440
tctaccaggg catcctgacg gccagccgcc agagccatgt ctgggagcag
aaagtgggtg 1500
gggtctcccc agaggagcac ccagcccctg aagtctctac accattccca
cttcttccgc 1560
CA 02398924 2002-07-19
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cagaacctga gggtggtggg gaggagaagg ttgtaggcat ggaagagagc
caggcagccc 1620
cgaaagaaga gcctgggcca cggccacgta taaacctccg aggggtcatg
aggtccatca 1680
gtcttctgga gccctccttg gagctggaga gcacctcaga gaccagtgac
atgccagagg 1740
tcttctcttc ccacgagtct tcacatgaag agcctctgca gcccttccca
cagcttgcaa 1800
ggaccaaggg aggccagcag gtggacaggg ggcctcagcc tgccctgaag
tcccgccagt 1860
cagtggttac cctccagggc agtgccgtgg tggccaaccg gacccaggcc
ttccaggagc 1920
aggagcaggg gcaggggcag gggcagggag agccctgcat ttcctctacg
cccaggttcc 1980
ggaaggtggt gagacaggcc agcgtgcatg acagtggaga ggagggcgag
gcctgagccc 2040
tcacacatgc ccacgctccc ctgacactga agaggatcca caactccttg
gagaaacacc 2100
ctcacgtctg ttgccgcaca cattcctctc agctccgccc catacccgtc
actacagcct 2160
cacctcccac ccctgtcact acggcctcac ctcccacccc tgtcactaca
gcctcacctc 2220
ctacagcctt aagtcccagg cccatgtctg cctgtccaag ggctcaagac
tttctaactg 2280
ggatgtggta gagggactga aggtaccttt gggggcaaca gcaccctagt
ttcattctca 2340
actctagccc tgcacactca cctgtggcac ggaatgaaaa cagagcttcc
cgtgcaaaaa 2400
gggtcacgcc tCCCdCCCCC gCCCCCtCCC tgC3CCrCCt gtCCtCt CCC
agttcattcc 2460
tggaaccagc caggccaggc aaccagtggc ccccaaaggc aggcaggatc
ctcaggcccc 2520
agccgcggga ggctggaagg gctggcagat cgcttccctc atccacctcc
accggtccag 2580
gtctttgctg ctgtccccag acctcctgtg acaccacgcc agatcacagg
gcaccaggcc 2640
agagatagtc ttctttttgt cctttctggc ctctggctag tcagtttttc
atagccttac 2700
agtatctggc tttgtac ga gaaataaaac acattttcat aaaaaaaaaa
aaaaaaaaaa 2760
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2820
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa as
2852
<210> 3
<211> 2540
<212> DNA
<213> Homo sapiens
<400> 3
cctggtcctg cgggtccagg actgtcccgc ggggttgagg gaaggggccg
tgcccggtgc 60
cagcccaggt gctcgcggcc tggctccatg gccctggtca cagtgagccg
ttcgcccccg 120
ggcagcggcg cctccacgcc cgtggggccc tgggaccagg cggtccagcg
aaggagtcga 180
ctccagcgaa ggcagagctt tgcggtgctc cgtggggctg tcctgggact
gcaggatgga 240
ggggacaatg atgatgcagc agaggccagt tctgagccaa cagagaaggc
,...,.,.-,r~n~,.. inn
CA 02398924 2002-07-19
WO 02/42436 5 PCT/USO1/42995
gaggagctcc acggggacca gacagacttc gggcaaggat cccagagtcc
ccagaagcag 360
gaggagcaga ggcagcacct gcacctcatg gtacagctgc tgaggccgca
ggatgacatc 420
cgcctggcag cccagctgga ggcaccccgg cctccccggc tccgctacct
gctggtagtt 480
tctacacgag aaggagaagg tctgagccag gatgagacgg tcctcctggg
cgtggatttc 540
cctgacagca gctcccccag ctgcaccctg ggcctggtct tgcccctctg
gagtgacacc 600
caggtgtact tagatggaga cgggggcttc agcgtgacgt ctggtgggca
aagccggatc 660
ttcaagccca tctccatcca gaccatgtgg tcctcagaac aggagcagat
ggagcaggcg 720
atccgtgctg agctgtggaa agtgttggat gtcagtgacc tggagagtgt
cacttccaaa 780
gagatccgcc aggctctgga gctgcgcctg gggctccccc tccagcagta
ccgtgacttc 840
atcgacaacc agatgctgct gctggtggca cagcgggacc gagcctcccg
catcttcccc 900
cacctctacc tgggctcaga gtggaacgca gcaaacctgg aggagctgca
gaggaacagg 960
gtcacccaca tcttgaacat ggcccgggag attgacaact tctaccctga
gcgcttcacc 1020
taccacaatg tgcgcctctg ggatgaggag tcggcccagc tgctgccgca
ctggaaggag 1080
acgcaccgct tcattgaggc tgcaagagca cagggcaccc acgtgctggt
ccactgcaag 1140
atgggcgtca gccgctcagc ggccacagtg ctggcctatg ccatgaagca
gtacgaatgc 1200
agcctggagc aggccctgcg ccacgtgcag gagctccggc ccatcgcccg
ccccaaccct 1260
ggcttcctgc gccagctgca gatctaccag ggcatcctga cggccagaac
ctgagggtgg 1320
tggggaggag aaggttgtag gcatggaaga gagccaggca gccccgaaag
aagagcctgg 1380
ggccacgggg cacgtataaa cctccgaggg gtcatgaggt ccatcagtct
tctggagccc 1440
tccttgggag ctggagagca cctcagtaga ccagtgacat gccagaggtc
ttCtCttCCC 1500
acgagtcttc acatgaagag cctctgcagc ccttcccaca gcttgcaagg
accaagggag 1560
gccagcaggt ggacaggggg cctcagcctg ccctgaagtc ccgccagtca
gtggttaccc 1620
tccagggcag tgccgtggtg gccaaccgga cccaggcctt ccaggagcag
gagcaggggc 1680
aggggcaggg gcagggagag ccctgcattt cctctacgcc caggttccgg
aaggtggtga 1740
gacaggccag cgtgcatgac agtggagagg agggcgaggc ctgagccctc
acacatgccc 1800
acgctcccct gacactgaag aggatccaca actccttgga gaaacaccct
cacgtctgtt 1860
gccgcacaca ttcctctcag ctccgcccca tacccgtcac tacagcctca
cctcccaccc 1920
ctgtcactac ggcctcacct cccacccctg tcactacagc ctcacctcct
acagccttaa 1980
gtcccaggcc catgtctgcc tgtccaaggg ctcaagactt tctaactggg
atgtggtaga 2040
gggactgaag gtacctttgg gggcaacagc accctagttt cattctcaac
tctagccctg 2100
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cacactcacc tgtggcacgg aatgaaaaca gagcttcccg tgcaaaaagg
gtcacgcctc 2160
ccacccccgc cccctccctg cacctcctgt cctctcccag ttcattcctg
gaaccagcca 2220
ggccaggcaa ccagtggccc ccaaaggcag gcaggatcct caggccccag
ccgcgggagg 2280
ctggaagggc tggcagatcg cttccctcat ccacctccac cggtccaggt
ctttgctgct 2340
gtccccagac ctcctgtgac accacgccag atcacagggc accaggccag
agatagtctt 2400
ctttttgtcc tttctggcct ctggctagtc agtttttcat agccttacag
tatctggctt 2460
tgtactgaga aataaaacac attttcatat ttggttaaaa aaaaaaaaaa
aaaaaaaaaa 2520
aaaaaaaaaa aaaaaaaaaa
2540
<210> 4
<211> 471
<212> PRT
<213> Homo sapiens
<400> 4
Met Ala Leu Val Thr Val Ser Arg Ser Pro Pro Gly Ser Gly Ala Ser
1 5 10 15
Thr Pro Val Gly Pro Trp Asp Gln Ala Val Gln Arg Arg Ser Arg Leu
20 25 30
Gln Arg Arg Gln Ser Phe Ala Val Leu Arg Gly Ala Val Leu Gly Leu
35 40 45
Gln Asp Gly G1y Asp Asn Asp Asp Ala Ala Glu Ala Ser Ser Glu Pro
50 55 60
Thr Glu Lys Ala Pro Ser Glu Glu Glu Leu His Gly Asp Gln Thr Asp
65 70 75 80
Phe Gly Gln Gly Ser Gln Ser Pro Gln Lys Gln Glu Glu Gln Arg G1n
85 90 95
His Leu His Leu Met Val Gln Leu Leu Arg Pro Gln Asp Asp Tle Arg
100 105 110
Leu Ala Ala Gln Leu Glu Ala Pro Arg Pro Pro Arg Leu Arg Tyr Leu
115 120 125
Leu Val Va1 Ser Thr Arg Glu Gly Glu Gly Leu Ser Gln Asp Glu Thr
l30 135 l40
Val Leu Leu Gly Val Asp Phe Pro Asp Ser Ser Ser Pro Ser Cys Thr
145 150 155 160
Leu Gly Leu Val Leu Pro Leu Trp Ser Asp Thr Gln Val Tyr Leu Asp
165 170 175
Gly Asp Gly Gly Phe Ser Val Thr Ser Gly Gly Gln Ser Arg Ile Phe
180 185 l90
Lys Pro Ile Ser Ile Gln Thr Met Trp Ala Thr Leu Gln Val Leu His
l95 200 205
Gln Ala Cys Glu Ala Ala Leu Gly Ser Gly Leu Val Pro G1y Gly Ser
210 215 220
A1a Leu Thr Trp Ala Ser His Tyr Gln Glu Arg Leu Asn Ser Glu Gln
225 230 235 240
Ser Cys Leu Asn Glu Trp Thr Ala Met Ala Asp Leu Glu Ser Leu Arg
245 250 255
Pro Pro Ser Ala Glu Pro Gly Gly Ser Ser Glu Gln Glu Gln Met Glu
260 265 270
Gln Ala Ile Arg Ala Glu Leu Trp Lys Val Leu Asp Val Ser Asp Leu
275 280 285
Glu Ser Va1 Thr Ser Lys Glu Ile Arg Gln Ala Leu Glu Leu Arg Leu
290 295 300
CA 02398924 2002-07-19
WO 02/42436 7 PCT/USO1/42995
Gly Leu Pro Leu Gln Gln Tyr Arg Asp Phe Ile Asp Asn Gln Met Leu
305 310 315 320
Leu Leu Val Ala Gln Arg Asp Arg Ala Ser Arg Tle Phe Pro His Leu
325 330 335
Tyr Leu Gly Ser Glu Trp Asn Ala Ala Asn Leu Glu Glu Leu Gln Arg
340 345 350
Asn Arg Val Thr His Ile Leu Asn Met Ala Arg Glu Ile Asp Asn Phe
355 360 365
Tyr Pro Glu Arg Phe Thr Tyr His Asn Val Arg Leu Trp Asp Glu Glu
370 375 380
Ser A1a Gln Leu Leu Pro His Trp Lys Glu Thr His Arg Phe Ile Glu
385 390 395 400
Ala Ala Arg Ala Gln Gly Thr His Val Leu Val His Cys Lys Met Gly
405 410 415
Val Ser Arg Ser Ala Ala Thr Val Leu Ala Tyr Ala Met Lys Gln Tyr
420 425 430
Glu Cys Ser Leu Glu Gln Ala Leu Arg His Val Gln Glu Leu Arg Pro
435 440 445
Ile Ala Arg Pro Asn Pro Gly Phe Leu Arg Gln Leu Gln Ile Tyr Gln
450 455 460
Gly Ile Leu Thr Ala Arg Thr
465 470
<210> 5
<211> 659
<212> PRT
<213> Homo Sapiens
<400> 5
Met Ala Leu Val Thr Val Ser Arg Ser Pro Pro Gly Ser Gly Ala Ser
1 5 10 15
Thr Pro Val Gly Pro Trp Asp Gln Ala Val Gln Arg Arg Ser Arg Leu
20 25 30
Gln Arg Arg Gln Ser Phe Ala Va1 Leu Arg Gly Ala Val Leu Gly Leu
35 40 45
G1n Asp G1y Gly Asp Asn Asp Asp Ala Ala Glu A1a Ser Ser Glu Pro
50 55 60
Thr Glu Lys Ala Pro Ser Glu Glu Glu Leu His Gly Asp Gln Thr Asp
65 70 75 80
Phe Gly Gln Gly Ser Gln Ser Pro Gln Lys Gln Glu Glu Gln Arg Gln
85 90 95
His Leu His Leu Met Val Gln Leu Leu Arg Pro Gln Asp Asp Ile Arg
100 105 110
Leu Ala Ala G1n Leu Glu Ala Pro Arg Pro Pro Arg Leu Arg Tyr Leu
115 120 125
Leu Val Val Ser Thr Arg Glu Gly Glu Gly Leu Ser Gln Asp Glu Thr
130 135 140
Val Leu Leu Gly Val Asp Phe Pro Asp Ser Ser Ser Pro Ser Cys Thr
145 150 155 160
Leu Gly Leu Val Leu Pro Leu Trp Ser Asp Thr Gln Val Tyr Leu Asp
165 170 175
Gly Asp Gly Gly Phe Ser Val Thr Ser Gly Gly Gln Ser Arg Ile Phe
180 185 190
Lys Pro Ile Ser Ile Gln Thr Met Trp Ala Thr Leu Gln Val Leu His
195 200 205
Gln Ala Cys Glu Ala Ala Leu Gly Ser Gly Leu Val Pro G1y Gly Ser
210 215 220
Ala Leu Thr Trp Ala Ser His Tyr Gln Glu Arg Leu Asn Ser Glu Gln
225 230 235 240
Ser Cys Leu Asn Glu Trp Thr Ala Met Ala Asp Leu Glu Ser Leu Arg
CA 02398924 2002-07-19
WO 02/42436 g PCT/USO1/42995
245 250 255
Pro Pro Ser Ala Glu Pro Gly Gly Ser Ser Glu Gln Glu Gln Met Glu
260 265 270
Gln Ala Ile Arg Ala Glu Leu Trp Lys Val Leu Asp Val Ser Asp Leu
275 280 285
Glu Ser Val Thr Ser Lys Glu Ile Arg Gln Ala Leu Glu Leu Arg Leu
290 295 300
Gly Leu Pro Leu Gln Gln Tyr Arg Asp Phe Ile Asp Asn Gln Met Leu
305 310 315 320
Leu Leu Val Ala Gln Arg Asp Arg Ala Ser Arg Ile Phe Pro His Leu
325 330 335
Tyr Leu Gly Ser Glu Trp Asn A1a Ala Asn Leu Glu Glu Leu Gln Arg
340 345 350
Asn Arg Val Thr His Ile Leu Asn Met Ala Arg Glu Ile Asp Asn Phe
355 360 365
Tyr Pro Glu Arg Phe Thr Tyr His Asn Val Arg Leu Trp Asp Glu Glu
370 375 380
Ser Ala Gln Leu Leu Pro His Trp Lys Glu Thr His Arg Phe Ile Glu
385 390 395 400
Ala A1a Arg Ala Gln Gly Thr His Val Leu Val His Cys Lys Met Gly
405 410 415
Val Ser Arg Ser Ala Ala Thr Val Leu Ala Tyr Ala Met Lys Gln Tyr
420 425 ' 430
Glu Cys Ser Leu Glu Gln A1a Leu Arg His Val Gln Glu Leu Arg Pro
435 440 445
Ile Ala Arg Pro Asn Pro Gly Phe Leu Arg Gln Leu Gln Ile Tyr Gln
450 455 460
Gly Ile Leu Thr Ala Ser Arg Gln Ser His Val Trp Glu Gln Lys Val
465 470 475 480
Gly Gly Val Ser Pro Glu Glu His Pro Ala Pro Glu Val Ser Thr Pro
485 490 495
Phe Pro Leu Leu Pro Pro Glu Pro Glu Gly Gly Gly Glu Glu Lys Val
500 505 510
Val Gly Met G1u Glu Ser Gln Ala Ala Pro Lys Glu Glu Pro Gly Pro
515 520 525
Arg Pro Arg Ile Asn Leu Arg Gly Val Met Arg Ser Ile Ser Leu Leu
530 535 540
Glu Pro Ser Leu Glu Leu Glu Ser Thr Ser Glu Thr Ser Asp Met Pro
545 550 555 560
Glu Val Phe Ser Ser His Glu Ser Ser His Glu Glu Pro Leu Gln Pro
565 570 575
Phe Pro Gln Leu Ala Arg Thr Lys Gly Gly Gln Gln Val Asp Arg Gly
580 585 590
Pro Gln Pro Ala Leu Lys Ser Arg Gln Ser Val Val Thr Leu Gln Gly
595 600 605
Ser Ala Val Val Ala Asn Arg Thr Gln A1a Phe Gln Glu Gln Glu Gln
610 615 620
Gly Gln Gly G1n Gly Gln Gly Glu Pro Cys Ile Ser Ser Thr Pro Arg
625 630 635 640
Phe Arg Lys Val Val Arg Gln Ala Ser Val His Asp Ser G1y Glu Glu
645 650 655
Gly Glu Ala
<210> 6
<211> 408
<212> PRT
<213> Homo Sapiens
<400> 6
CA 02398924 2002-07-19
WO 02/42436 9 PCT/USO1/42995
Met Ala Leu Val Thr Val Ser Arg Ser Pro Pro Gly Ser Gly Ala Ser
1 5 10 15
Thr Pro Val Gly Pro Trp Asp Gln Ala Val Gln Arg Arg Ser Arg Leu
20 25 30
Gln Arg Arg Gln Ser Phe Ala Val Leu Arg Gly Ala Val Leu Gly Leu
35 40 45
Gln Asp Gly Gly Asp Asn Asp Asp Ala Ala Glu Ala Ser Ser Glu Pro
50 55 60
Thr Glu Lys Ala Pro Ser Glu Glu Glu Leu His Gly Asp Gln Thr Asp
65 70 75 80
Phe Gly Gln Gly Ser Gln Ser Pro Gln Lys Gln Glu Glu Gln Arg Gln
85 90 95
His Leu His Leu Met Val Gln Leu Leu Arg Pro Gln Asp Asp Ile Arg
100 105 110
Leu Ala Ala Gln Leu Glu Ala Pro Arg Pro Pro Arg Leu Arg Tyr Leu
115 120 125
Leu Val Val Ser Thr Arg Glu Gly Glu Gly Leu Ser Gln Asp Glu Thr
130 135 140
Val Leu Leu Gly Val Asp Phe Pro Asp Ser Ser Ser Pro Ser Cys Thr
145 150 155 160
Leu Gly Leu Val Leu Pro Leu Trp Ser Asp Thr Gln Val Tyr Leu Asp
165 l70 175
Gly Asp Gly Gly Phe Ser Val Thr Ser Gly Gly Gln Ser Arg Ile Phe
180 185 190
Lys Pro Ile Ser Ile Gln Thr Met Trp Ser Ser Glu Gln Glu Gln Met
195 200 205
Glu Gln Ala Ile Arg Ala Glu Leu Trp Lys Val Leu Asp Val Ser Asp
210 215 220
Leu Glu Ser Val Thr Ser Lys Glu Ile Arg Gln Ala Leu Glu Leu Arg
225 230 235 240
Leu Gly Leu Pro Leu Gln Gln Tyr Arg Asp Phe Ile Asp Asn Gln Met
245 250 255
Leu Leu Leu VaT Ala Gln Arg Asp Arg Ala Ser Arg Ile Phe Pro His
260 265 270
Leu Tyr Leu Gly Ser Glu Trp Asn Ala Ala Asn Leu Glu G1u Leu Gln
275 280 285
Arg Asn Arg Val Thr His I1e Leu Asn Met Ala Arg Glu Ile Asp Asn
290 295 300
Phe Tyr Pro Glu Arg Phe Thr Tyr His Asn Val Arg Leu Trp Asp Glu
305 310 315 320
Glu Ser Ala Gln Leu Leu Pro His Trp Lys Glu Thr His Arg Phe Ile
325 330 335
Glu Ala Ala Arg Ala Gln Gly Thr His Val Leu Val His Cys Lys Met
340 345 350
Gly Val Ser Arg Ser Ala Ala Thr Val Leu Ala Tyr Ala Met Lys Gln
355 360 365
Tyr Glu Cys Ser Leu Glu Gln Ala Leu Arg His Val Gln Glu Leu Arg
370 375 380
Pro Ile Ala Arg Pro Asn Pro Gly Phe Leu Arg Gln Leu Gln Ile Tyr
385 390 395 400
G1n Gly Ile Leu Thr Ala Arg Thr
405
<210> 7
<211> 2704
<212> DNA
<213> Homo Sapiens
<400> 7
CA 02398924 2002-07-19
WO 02/42436 ~ p PCT/USO1/42995
cgtccttcct ggtcctgcgg gtccaggact gtccgcgggg ttgagggaag
gggccgtgcc 60
cggtgccagc ccaggtgctc gcggcctggc tccatggccc tggtcacagt
gagccgttcg 120
cccccgggca gcggcgcctc cacgcccgtg gggccctggg accaggcggt
ccagcgaagg 180
agtcgactcc agcgaaggca gagctttgcg gtgctccgtg gggctgtcct
gggactgcag 240
gatggagggg acaatgatga tgcagcagag gccagttctg agccaacaga
gaaggccccg 300
agtgaggagg agctccacgg ggaccagaca gacttcgggc aaggatccca
gagtccccag 360
aagcaggagg agcagaggca gcacctgcac ctcatggtac agctgctgag
gccgcaggat 420
gacatccgcc tggcagccca gctggaggca ccccggcctc cccggctccg
ctacctgctg 480
gtagtttcta cacgagaagg agaaggtctg agccaggatg agacggtcct
cctgggcgtg 540
gatttccctg acagcagctc ccccagctgc accctgggcc tggtcttgcc
cctctggagt 600
gacacccagg tgtacttaga tggagacggg ggcttcagcg tgacgtctgg
tgggcaaagc 660
cggatcttca agcccatctc catccagacc atgtgggcca cactccaggt
attgcaccaa 720
gcatgtgagg cagctctagg cagcggcctt gtaccgggtg gcagtgccct
cacctgggcc 780
agccactacc aggagagact gaactccgaa cagagctgcc tcaatgagtg
gacggctatg 840
gccgacctgg agtctctgcg gcctcccagc gccgagcctg gcgggtcctc
agaacaggag 900
cagatggagc aggcgatccg tgctgagctg tggaaagtgt tggatgtcag
tgacctggag 960
agtgtcactt ccaaagagat ccgccaggct ctggagctgc gcctggggct
ccccctccag 1020
cagtaccgtg acttcatcga caaccagatg ctgctgctgg tggcacagcg
ggaccgagcc 1080
tcccgcatct tcccccacct ctacctgggc tcagagtgga acgcagcaaa
cctggaggag 1140
ctgcagagga acagggtcac ccacatcttg aacatggccc gggagattga
caacttctac 1200
cctgagcgct tcacctacca caatgtgcgc ctctgggatg aggagtcggc
ccagctgctg 1260
ccgcactgga aggagacgca ccgcttcatt gaggctgcaa gagcacaggg
cacccacgtg 1320
ctggtccact gcaagatggg cgtcagccgc tcagcggcca cagtgctggc
ctatgccatg 1380
aagcagtacg aatgcagcct ggagcaggcc ctgcgccacg tgcaggagct
ccggcccatc 1440
gcccgcccca accctggctt cctgcgccag ctgcagatct accagggcat
cctgacggcc 1500
agaacctgag ggtggtgggg aggagaaggt tgtaggcatg gaagagagcc
aggcagcccc 1560
gaaagaagag cctgggccac ggccacgtat aaacctccga ggggtcatga
ggtccatcag 1620
tcttctggag ccctccttgg agctggagag cacctcagag accagtgaca
tgccagaggt 1680
cttctcttcc cacgagtctt cacatgaaga gcctctgcag cccttcccac
agcttgcaag 1740
gaccaaggga ggccagcagg tggacagggg gcctcagcct gccctgaagt
cccgccagtc 1800
CA 02398924 2002-07-19
WO 02/42436 ~ ~ PCT/USO1/42995
agtggttacc ctccagggca gtgccgtggt ggccaaccgg acccaggcct
tccaggagca 1860
ggagcagggg caggggcagg ggcagggaga gccctgcatt tcctctacgc
ccaggttccg 1920
gaaggtggtg agacaggcca gcgtgcatga cagtggagag gagggcgagg
cctgagccct 1980
cacacatgcc cacgctcccc tgacactgaa gaggatccac aactccttgg
agaaacaccc 2040
tcacgtctgt tgccgcacac attcctctca gctccgoccc atacccgtca
CtaCagCCtC 2100
aCC'tCCCdCC CCtgtCdCta cggcctcacc tCCC3CCCCt gtC3CtaCag
cctcacctcc 2160
tacagcctta agtcccaggc ccatgtctgc ctgtccaagg gctcaagact
ttctaactgg 2220
gatgtggtag agggactgaa ggtacctttg ggggcaacag caccctagtt
tcattctcaa 2280
ctctagccct gcacactcac ctgtggcacg gaatgaaaac agagcttccc
gtgcaaaaag 2340
ggtC3CgCCt CCC3CCCCCg CCCCCt CCCt gC3CC'tCCtg tCCtCtCCCa
gttcattcct 2400
ggaaccagcc aggccaggca accagtggcc cccaaaggca ggcaggatcc
tcaggcccca 2460
gccgcgggag gctggaaggg ctggcagatc gcttccctca tccacctcoa
ccggtccagg 2520
tctttgctgc tgtccccaga cctcctgtga caccacgcca gatcacaggg
caccaggcca 2580
gagatagtct tctttttgtc ctttctggcc tctggctagt cagtttttca
tagccttaca 2640
gtatctggct ttgtactgag aaataaaaca cattttcata aaaaaaaaaa
aaaaaaaaaa 2700
aaaa
2704
<210> 8
<211> 312
<212> PRT
<213> Homo Sapiens
<400> 8
Met Ala Leu Val Thr Val Ser Arg Ser Pro Pro Gly Ser Gly Ala Ser
1 5 10 15
Thr Pro Val Gly Pro Trp Asp Gln Ala Val Gln Arg Arg Ser Arg Leu
20 25 30
Gln Arg Arg Gln Ser Phe Ala Val Leu Arg Gly Ala Val Leu Gly Leu
35 40 45
Gln Asp Gly Gly Asp Asn Asp Asp Ala Ala Glu Ala Ser Ser Glu Pro
50 55 60
Thr Glu Lys Ala Pro Ser Glu Glu Glu Leu His Gly Asp Gln Thr Asp
65 70 75 80
Phe Gly Gln Gly Ser Gln Ser Pro Gln Lys Gln Glu Glu Gln Arg Gln
85 90 95
His Leu His Leu Met Val Gln Leu Leu Arg Pro Gln Asp Asp Ile Arg
100 105 110
Leu Ala A1a Gln Leu Glu Ala Pro Arg Pro Pro Arg Leu Arg Tyr Leu
115 120 125
Leu Val Val Ser Thr Arg Glu Gly Glu Gly Leu Ser Gln Asp Glu Thr
130 135 140
Val Leu Leu Gly Val Asp Phe Pro Asp Ser Ser Ser Pro Ser Cys Thr
145 150 155 160
Leu Gly Leu Val Leu Pro Leu Trp Ser Asp Thr Gln Val Tyr Leu Asp
165 170 175
CA 02398924 2002-07-19
WO 02/42436 12 PCT/USO1/42995
Gly Asp Gly Gly Phe Ser Val Thr Ser Gly Gly Gln Ser Arg Ile Phe
180 185 190
Lys Pro Ile Ser Ile Gln Thr Met Trp Ala Thr Leu Gln Val Leu His
195 200 205
Gln Ala Cys Glu Ala Ala Leu Gly Ser Gly Leu Val Pro G1y Gly Ser
210 215 220
Ala Leu Thr Trp Ala Ser His Tyr Gln Glu Arg Leu Asn Ser Glu Gln
225 230 235 240
Ser Cys Leu Asn Glu Trp Thr Ala Met Ala Asp Leu Glu Ser Leu Arg
245 250 255
Pro Pro Ser Ala G1u Pro Gly Gly Ser Ser Glu Gln Glu Gln Met G1u
260 265 270
Gln Ala Ile Arg Ala Glu Leu Trp Lys Val Leu Glu Leu Glu Ser Thr
275 280 285
Ser Glu Thr Ser Asp Met Pro Glu Val Phe Ser Ser His Glu Ser Ser
290 295 300
His Glu Glu Pro Leu Gln Pro Phe
305 310
<210> 9
<2l1> 524
<212> PRT
<213> Drosophila melanogaster
<400> 9
Met Ala Leu Val Thr Val Gln Arg Ser Pro Ser Val Ala Gly Ser Cys
1 5 10 15
Ser Asn Ser Asp Gly Glu Ser Glu Asp Asp Glu Gly Asn Ser Lys Gly
20 25 30
Asn Asp Arg Ser Glu Cys Phe Phe Ala Gly Lys Gly Thr Ala Leu Val
35 40. 45
Leu Ala Leu Lys Asp Ile Pro Pro Leu Thr Gln Ser Glu Arg Arg Leu
50 55 60
Ser Thr Asp Ser Thr Arg Ser Ser Asn Ser Thr Gln Ser Asn Asn Ser
65 70 75 80
Asp Ile Gln Leu His Leu Gln Ser Met Phe Tyr Leu Leu Gln Arg Glu
85 90 95
Asp Thr Leu Lys Met Ala Val Lys Leu Glu Ser Gln Arg Ser Asn Arg
100 105 110
Thr Arg Tyr Leu Val I1e Ala Ser Arg Ser Cys Cys Arg Ser Gly Thr
115 120 125
Ser Asp Arg Arg Arg His Arg Ile Met Arg His His Ser Val Lys Val
130 135 140
Gly Gly Ser Ala Gly Thr Lys Ser Ser Thr Ser Pro Ala Val Pro Thr
145 150 ~ 155 160
Gln Arg Gln Leu Ser Val Glu Gln Thr A1a Thr Glu Ala Ser Ser Lys
165 170 175
Cys Asp Lys Thr Ala Asp Lys Glu Asn Ala Thr Ala Ala Gly Asp Asn
180 ~ 185 190
Lys Asn Thr Ser G1y Met G1u Glu Ser Cys Leu Leu Gly Ile Asp Cys
195 200 205
Asn Glu Arg Thr Thr Ile Gly Leu Val Val Pro Ile Leu Ala Asp Thr
210 215 220
Thr Ile His Leu Asp Gly Asp Gly Gly Phe Ser Val Lys Val Tyr Glu
225 230 235 240
Lys Thr His Ile Phe Lys Pro Val Ser Val Gln Ala Met Trp Ser Ala
245 250 255
Leu Gln Thr Leu His Lys Val Ser Lys Lys Ala Arg Glu Asn Asn Phe
260 265 270
Tyr Ala Ser Gly Pro Ser His Asp Trp Leu Ser Ser Tyr Glu Arg Arg
CA 02398924 2002-07-19
WO 02/42436 13 PCT/USO1/42995
275 280 285
Ile Glu Ser Asp Gln Ser Cys Leu Asn Glu Trp Asn Ala Met Asp Ala
290 295 300
Leu Glu Ser Arg Arg Pro Pro Ser Pro Asp Ala Ile Arg Asn Lys Pro
305 310 315 320
Pro Glu Lys Glu Glu Thr Glu Ser Val Ile Lys Met Lys Leu Lys Ala
325 330 335
Ile Met Met Ser Va1 Asp Leu Asp Glu Val Thr Ser Lys Tyr Ile Arg
340 - 345 350
Gly Arg Leu Glu Glu Ile Leu Asp Met Asp Leu G1y Glu Tyr Lys Ser
355 360 365
Phe Ile Asp Ala Glu Met Leu Val Ile Leu Gly Gln Met Asp Ala Pro
370 375 380
Thr Lys Ile Phe Glu His Val Tyr Leu Gly Ser Glu Trp Asn Ala Ser
385 390 395 400
Asn Leu Glu Glu Leu Gln Lys Asn Gly Val Arg His Ile Leu Asn Val
405 410 415
Thr Arg Glu Ile Asp Asn Phe Phe Pro Gly Thr Phe Glu Tyr Phe Asn
420 425 430
Val Arg Val Tyr Asp Asp Glu Lys Thr Asn Leu Leu Lys Tyr Trp Asp
435 440 445
Asp Thr Phe Arg Tyr Ile Thr Arg Ala Lys Ala Glu Gly Ser Lys Val
450 455 460
Leu Val His Cys Lys Met Gly Val Ser Arg Ser Ala Ser Val Val Ile
465 470 475 480
Ala Tyr Ala Met Lys Ala Tyr Gln Trp Glu Phe Gln Gln Ala Leu Glu
485 490 495
His Val Lys Lys Arg Arg Ser Cys Ile Lys Pro Asn Lys Asn Phe Leu
500 505 510
Asn Gln Leu Glu Thr Tyr Ser Gly Met Leu Asp Ala
515 520
<210> l0
<211> 111
<212> PRT
<213> Homo Sapiens
<400> l0
Met Ala Arg Glu Ile Asp Asn Phe Tyr Pro Glu Arg Phe Thr Tyr His
1 5 10 15.
Asn Val Arg Leu Trp Asp Glu Glu Ser Ala Gln Leu Leu Pro His Trp
20 25 30
Lys Glu Thr His Arg Phe Ile Glu Ala Ala Arg Ala Gln Gly Thr His
35 40 45
Val Leu Val His Cys Lys Met Gly Va1 Ser Arg Ser Ala A1a Thr Val
50 55 60
Leu Ala Tyr Ala Met Lys Gln Tyr Glu Cys Ser Leu Glu Gln Ala Leu
65 70 75 80
Arg His Val Gln Glu Leu Arg Pro Ile Ala Arg Pro Asn Pro Gly Phe
85 90 95
Leu Arg Gln Leu Gln Ile Tyr Gln Gly Ile Leu Thr Ala Arg Thr
100 105 110
