The invention provides a cDNA which encodes a protein differentially
expressed in breast cancer. It also provides for the use of the
cDNA, fragments, complements, and variants thereof and of the encoded
protein, portions thereof and antibodies thereto for diagnosis and
treatment of cancer, particularly a breast cancer. The invention
additionally provides expression vectors and host cells for the
production of the protein and a transgenic model system.
What is claimed is:
1. An isolated cDNA, or the complement thereof, comprising a nucleic
acid sequence encoding a protein selected from: a) amino acid sequence
of SEQ ID NO: 1, b) an immunogenic fragment of SEQ ID NO: 1, and
c) a variant of SEQ ID NO: 1 having at least 90% sequence identity
to SEQ ID NO: 1
2. An isolated cDNA comprising a nucleic acid sequence selected
from: a) SEQ ID NO: 2 or the complement thereof; and b) a variant
of SEQ ID NO: 2 having at least 85% identity to SEQ ID NO: 2.
3. A composition comprising the cDNA of claim 1 and a labeling
4. A vector comprising the cDNA of claim 1.
5. A host cell comprising the vector of claim 4.
6. A method for using a cDNA to produce a protein, the method comprising:
a) culturing the host cell of claim 5 under conditions for protein
expression; and b) recovering the protein from the host cell culture.
7. A method for using a cDNA to detect expression of a nucleic
acid in a sample comprising: a) hybridizing the composition of claim
3 to nucleic acids of the sample under conditions to form at least
one hybridization complex; and b) detecting hybridization complex
formation, wherein complex formation indicates expression of the
cDNA in the sample.
8. The method of claim 7 further comprising amplifying the nucleic
acids of the sample prior to hybridization.
9. The method of claim 7 wherein the composition is attached to
10. The method of claim 7 wherein complex formation is compared
with at least one standard to determine differential expression.
11. A method of using a cDNA to screen a plurality of molecules
or compounds, the method comprising: a) combining the cDNA of claim
1 with a plurality of molecules or compounds under conditions to
allow specific binding; and b) detecting specific binding, thereby
identifying a molecule or compound which specifically binds the
12. The method of claim 11 wherein the molecules or compounds are
selected from DNA molecules, RNA molecules, peptide nucleic acids,
artificial chromosome constructions, peptides, transcription factors,
repressors, and regulatory molecules.
13. A purified protein or a portion thereof produced by the method
of claim 6 and selected from: a) an amino acid sequence of SEQ ID
NO: 1; b) an antigenic epitope of SEQ ID NO: 1 from about amino
acid S31 to about amino acid Q50 of SEQ ID NO: 1; and c) a variant
of SEQ ID NO: 1 having at least 90% amino acid identity to SEQ ID
14. A purified protein comprising an amino acid sequence of SEQ
ID NO: 1
15. A composition comprising the protein of claim 13 and a pharmaceutical
16. A method for using a protein to screen a plurality of molecules
or compounds to identify at least one ligand, the method comprising:
a) combining the protein of claim 13 with the molecules or compounds
under conditions to allow specific binding; and b) detecting specific
binding, thereby identifying a ligand which specifically binds the
17. The method of claim 16 wherein the molecules or compounds are
selected from DNA molecules, RNA molecules, peptide nucleic acids,
peptides, proteins, mimetics, agonists, antagonists, antibodies,
immunoglobulins, inhibitors, and drugs.
18. A method of using a protein to prepare and purify a polyclonal
antibody comprising: a) immunizing a animal with a protein of claim
13 under conditions to elicit an antibody response; b) isolating
animal antibodies; c) attaching the protein to a substrate; d) contacting
the substrate with isolated antibodies under conditions to allow
specific binding to the protein; e) dissociating the antibodies
from the protein, thereby obtaining purified polyclonal antibodies.
19. A method of using a protein to prepare and purify a monoclonal
antibody comprising: a) immunizing a animal with a protein of claim
13 under conditions to elicit an antibody response; b) isolating
antibody-producing cells from the animal; c) fusing the antibody-producing
cells with immortalized cells in culture to form monoclonal antibody
producing hybridoma cells; d) culturing the hybridoma cells; and
e) isolating monoclonal antibodies from culture.
20. An isolated antibody which specifically binds to a protein
of claim 13.
21. The antibody of claim 20, wherein the antibody is selected
from an intact immunoglobulin molecule, a polyclonal antibody, a
monoclonal antibody, a chimeric antibody, a recombinant antibody,
a humanized antibody, a single chain antibody, a Fab fragment, an
F(ab').sub.2 fragment, an Fv fragment; and an antibody-peptide fusion
22. A polyclonal antibody produced by the method of claim 18.
23. A monoclonal antibody produced by the method of claim 19.
24. A method for using an antibody to detect expression of a protein
in a sample, the method comprising: a) combining the antibody of
claim 20 with a sample under conditions which allow the formation
of antibody:protein complexes; and b) detecting complex formation,
wherein complex formation indicates expression of the protein in
25. A method for using an antibody to detect expression of a protein
in a sample, the method comprising: a) combining the antibody of
claim 20 with a sample under conditions which allow the formation
of antibody:protein complexes; and b) detecting complex formation,
wherein complex formation indicates expression of the protein in
26. The method of claim 25 wherein complex formation is compared
with standards and is diagnostic of a breast cancer.
27. A composition comprising an antibody of claim 20 and a labeling
28. A composition comprising an antibody of claim 20 and a pharmaceutical
 This application is continuation-in-part of U.S. Ser. No.
09/232,160, Incyte Docket No. PA-0003 US, filed Jan. 15, 1999, all
of which application is hereby incorporated by reference.
FIELD OF THE INVENTION
 This invention relates to a cDNA which encodes a breast
cancer marker and to the use of the cDNA and the encoded protein
in the diagnosis and treatment of breast cancer, in particular,
an invasive, potentially metastatic stage of the disease.
BACKGROUND OF THE INVENTION
 Phylogenetic relationships among organisms have been demonstrated
many times, and studies from a diversity of prokaryotic and eukaryotic
organisms suggest a more or less gradual evolution of molecules,
biochemical and physiological mechanisms, and metabolic pathways.
Despite different evolutionary pressures, the proteins of nematode,
fly, rat, and man have common chemical and structural features and
generally perform the same cellular function. Comparisons of the
nucleic acid and protein sequences from organisms where structure
and/or function are known accelerate the investigation of human
sequences and allow the development of model systems for testing
diagnostic and therapeutic agents for human conditions, diseases,
 Array technology can provide a simple way to explore the
expression of a single polymorphic gene or the expression profile
of a large number of related or unrelated genes. When the expression
of a single gene is examined, arrays are employed to detect the
expression of a specific gene or its variants. When an expression
profile is examined, arrays provide a platform for examining which
genes are tissue specific, carrying out housekeeping functions,
parts of a signaling cascade, or specifically related to a particular
genetic predisposition, condition, disease, or disorder.
 The potential application of gene and protein expression
profiling is particularly relevant to improving diagnosis, prognosis,
and treatment of disease. For example, both the levels and sequences
expressed in tissues from subjects with cancer may be compared with
the levels and sequences expressed in normal tissue.
 Cancers and malignant tumors are characterized by continuous
cell proliferation and cell death and are causally related to both
genetics and the environment. Cancer markers are of great importance
in determining familial predisposition to cancers and in the early
diagnosis and prognosis of various cancers.
 There are more than 180,000 new cases of breast cancer diagnosed
each year, and the mortality rate for breast cancer approaches 10%
of all deaths in females between the ages of 45-54 (K. Gish (1999)
AWIS Magazine 28:7-10). However the survival rate based on early
diagnosis of localized breast cancer is extremely high (97%), compared
with the advanced stage of the disease in which the tumor has spread
beyond the breast (22%). Current procedures for clinical breast
examination are lacking in sensitivity and specificity, and efforts
are underway to develop comprehensive gene expression profiles for
breast cancer that may be used in conjunction with conventional
screening methods to improve diagnosis and prognosis of this disease
(Perou CM et al. (2000) Nature 406:747-752).
 Breast cancer is a genetic disease commonly caused by mutations
in cellular disease. Mutations in two genes, BRCA1 and BRCA2, are
known to greatly predispose a woman to breast cancer and may be
passed on from parents to children (Gish, supra). However, this
type of hereditary breast cancer accounts for only about 5% to 9%
of breast cancers, while the vast majority of breast cancer is due
to noninherited mutations that occur in breast epithelial cells.
 A good deal is already known about the expression of specific
genes associated with breast cancer. For example, the relationship
between expression of epidermal growth factor (EGF) and its receptor,
EGFR, to human mammary carcinoma has been particularly well studied.
(See Khazaie et al., supra, and references cited therein for a review
of this area.) Overexpression of EGFR, particularly coupled with
down-regulation of the estrogen receptor, is a marker of poor prognosis
in breast cancer patients. Other known markers of breast cancer
include a human secreted frizzled protein mRNA that is downregulated
in breast tumors; the matrix G1 a protein which is overexpressed
is human breast carcinoma cells; Drg1 or RTP, a gene whose expression
is diminished in colon, breast, and prostate tumors; maspin, a tumor
suppressor gene downregulated in invasive breast carcinomas; and
CaN19, a member of the S100 protein family, all of which are down
regulated in mammary carcinoma cells relative to normal mammary
epithelial cells (Zhou Z et al. (1998) Int J Cancer 78:95-99; Chen,
L et al. (1990) Oncogene 5:1391-1395; Ulrix W et al (1999) FEBS
Lett 455:23-26; Sager, R et al. (1996) Curr Top Microbiol Immunol
213:51-64; and Lee, S W et al. (1992) Proc Natl Acad Sci USA 89:2504-2508).
 The discovery of a cDNA encoding a protein differentially
expressed in breast cancer satisfies a need in the art by providing
a differentially expressed gene and its encoded protein which may
be used to diagnose, to stage, to treat, or to monitor the progression
or treatment of a subject with breast cancer, in particular, an
invasive, potentially metastatic stage of the disease.
SUMMARY OF THE INVENTION
 The invention is based on the discovery of a cDNA encoding
a protein differentially expressed in breast cancer, PDEBC, which
is useful in the diagnosis and treatment of a breast cancer, particularly
an invasive, potentially metastatic stage of the disease.
 The invention provides an isolated a cDNA comprising a nucleic
acid sequence encoding a protein having the amino acid sequence
of SEQ ID NO: 1. The invention also provides an isolated cDNA or
the complement thereof selected from the group consisting of a nucleic
acid sequence of SEQ ID NO: 2, and a variant of SEQ ID NO: 2 selected
from SEQ ID NOs: 3-5. The invention additionally provides a composition,
a substrate, and a probe comprising the cDNA, or the complement
of the cDNA, encoding PDEBC. The invention further provides a vector
containing the cDNA, a host cell containing the vector and a method
for using the cDNA to make PDEBC. The invention still further provides
a transgenic cell line or organism comprising the vector containing
the cDNA encoding PDEBC. In one aspect, the invention provides a
substrate containing at least one of the cDNAs of SEQ ID NOs: 2-5
or the complements thereof. In a second aspect, the invention provides
a probe comprising a cDNA or the complement thereof which can be
used in methods of detection, screening, and purification. In a
further aspect, the probe is a single-stranded complementary RNA
or DNA molecule.
 The invention provides a method for using a cDNA to detect
the differential expression of a nucleic acid in a sample comprising
hybridizing a probe to the nucleic acids, thereby forming hybridization
complexes and comparing hybridization complex formation with a standard,
wherein the comparison indicates the differential expression of
the cDNA in the sample. In one aspect, the method of detection further
comprises amplifying the nucleic acids of the sample prior to hybridization.
In another aspect, the method showing differential expression of
the cDNA is used to diagnose breast cancer. In another aspect, the
cDNA or a fragment or a variant or the complements thereof may comprise
an element on an array.
 The invention additionally provides a method for using a
cDNA or a fragment or a variant or the complements thereof to screen
a library or plurality of molecules or compounds to identify at
least one ligand which specifically binds the cDNA, the method comprising
combining the cDNA with the molecules or compounds under conditions
allowing specific binding, and detecting specific binding to the
cDNA, thereby identifying a ligand which specifically binds the
cDNA. In one aspect, the molecules or compounds are selected from
DNA molecules, RNA molecules, peptide nucleic acids, artificial
chromosome constructions, peptides, transcription factors, repressors,
and regulatory molecules.
 The invention provides a purified protein or a portion thereof
selected from the group consisting of an amino acid sequence of
SEQ ID NO: 1, a variant having at least 90% identity to the amino
acid sequence of SEQ ID NO: 1, and an antigenic epitope of SEQ ID
NO: 1. The invention also provides a composition comprising the
purified protein and a pharmaceutical carrier. The invention still
further provides a method for using a protein to screen a library
or a plurality of molecules or compounds to identify at least one
ligand, the method comprising combining the protein with the molecules
or compounds under conditions to allow specific binding and detecting
specific binding, thereby identifying a ligand which specifically
binds the protein. In one aspect, the molecules or compounds are
selected from DNA molecules, RNA molecules, peptide nucleic acids,
peptides, proteins, mimetics, agonists, antagonists, antibodies,
immunoglobulins, inhibitors, and drugs. In another aspect, the ligand
is used to treat a subject with breast cancer.
 The invention provides a method for using a protein to screen
a plurality of antibodies to identify an antibody which specifically
binds the protein comprising contacting a plurality of antibodies
with the protein under conditions to form an antibody:protein complex,
and dissociating the antibody from the antibody:protein complex,
thereby obtaining antibody which specifically binds the protein.
 The invention also provides methods for using a protein
to prepare and purify polyclonal and monoclonal antibodies which
specifically bind the protein. The method for preparing a polyclonal
antibody comprises immunizing a animal with protein under conditions
to elicit an antibody response, isolating animal antibodies, attaching
the protein to a substrate, contacting the substrate with isolated
antibodies under conditions to allow specific binding to the protein,
dissociating the antibodies from the protein, thereby obtaining
purified polyclonal antibodies. The method for preparing a monoclonal
antibodies comprises immunizing a animal with a protein under conditions
to elicit an antibody response, isolating antibody producing cells
from the animal, fusing the antibody producing cells with immortalized
cells in culture to form monoclonal antibody producing hybridoma
cells, culturing the hybridoma cells, and isolating monoclonal antibodies
 The invention further provides purified antibodies which
bind specifically to a protein. The invention also provides a method
for using an antibody to detect expression of a protein in a sample,
the method comprising combining the antibody with a sample under
conditions for formation of antibody:protein complexes; and detecting
complex formation, wherein complex formation indicates expression
of the protein in the sample. In one aspect, the amount of complex
formation when compared to standards is diagnostic of a breast cancer.
 The invention still further provides a method for immunopurification
of a protein comprising attaching an antibody to a substrate, exposing
the antibody to a sample containing protein under conditions to
allow antibody:protein complexes to form, dissociating the protein
from the complex, and collecting purified protein. The invention
yet still further provides an array containing an antibody which
specifically binds the protein.
 The invention provides a method for inserting a heterologous
marker gene into the genomic DNA of a mammal to disrupt the expression
of the endogenous polynucleotide. The invention also provides a
method for using a cDNA to produce a mammalian model system, the
method comprising constructing a vector containing the cDNA selected
from SEQ ID NOs: 2-5, transforming the vector into an embryonic
stem cell, selecting a transformed embryonic stem cell, microinjecting
the transformed embryonic stem cell into a mammalian blastocyst,
thereby forming a chimeric blastocyst, transferring the chimeric
blastocyst into a pseudopregnant dam, wherein the dam gives birth
to a chimeric offspring containing the cDNA in its germ line, and
breeding the chimeric mammal to produce a homozygous, mammalian
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
 FIGS. 1A, 1B, 1C, 1D, and 1E show the PDEBC (SEQ ID NO:
1) encoded by the cDNA (SEQ ID NO: 2). The alignment was produced
using MACDNASIS PRO software (Hitachi Software Engineering, South
San Francisco Calif.).
 Table 1 shows the northern analysis for PDEBC in breast
tissues produced using the LIFESEQ Gold database (Incyte Genomics,
Palo Alto Calif.). The first column lists the library name, the
second column, the number of cDNAs sequenced for that library; the
third column, a brief description of the tissue; the fourth column,
the absolute abundance of the transcript; and the fifth column,
the percent abundance of the transcript.
 Table 2 presents a detailed description of the breast tissue
libraries cited in Table 1.
DESCRIPTION OF THE INVENTION
 It is understood that this invention is not limited to the
particular machines, materials and methods described. It is also
to be understood that the terminology used herein is for the purpose
of describing particular embodiments and is not intended to limit
the scope of the present invention which will be limited only by
the appended claims. As used herein, the singular forms "a",
"an", and "the" include plural reference unless
the context clearly dictates otherwise. For example, a reference
to "a host cell" includes a plurality of such host cells
known to those skilled in the art.
 Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one
of ordinary skill in the art to which this invention belongs. All
publications mentioned herein are cited for the purpose of describing
and disclosing the cell lines, protocols, reagents and vectors which
are reported in the publications and which might be used in connection
with the invention. Nothing herein is to be construed as an admission
that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
 "PDEBC" refers to a purified protein obtained
from any mammalian species, including bovine, canine, murine, ovine,
porcine, rodent, simian, and preferably the human species, and from
any source, whether natural, synthetic, semi-synthetic, or recombinant.
 "Antibody" refers to intact immunoglobulin molecule,
a polyclonal antibody, a monoclonal antibody, a chimeric antibody,
a recombinant antibody, a humanized antibody, single chain antibodies,
a Fab fragment, an F(ab').sub.2 fragment, an Fv fragment; and an
antibody-peptide fusion protein.
 "Antigenic determinant" refers to an immunogenic
epitope, structural feature, or region of an oligopeptide, peptide,
or protein which is capable of inducing formation of an antibody
which specifically binds the protein. Biological activity is not
a prerequisite for immunogenicity.
 "Array" refers to an ordered arrangement of at
least two cDNAs, proteins, or antibodies on a substrate. At least
one of the cDNAs, proteins, or antibodies represents a control or
standard, and the other cDNA, protein, or antibody of diagnostic
or therapeutic interest. The arrangement of two to about 40,000
cDNAs, proteins, or antibodies on the substrate assures that the
size and signal intensity of each labeled complex, formed between
each cDNA and at least one nucleic acid, each protein and at least
one ligand or antibody, or each antibody and at least one protein
to which the antibody specifically binds, is individually distinguishable.
 The "complement" of a cDNA of the Sequence Listing
refers to a nucleic acid molecule which is completely complementary
over its full length and which will hybridize to the cDNA or an
mRNA under conditions of high stringency.
 "cDNA" refers to an isolated polynucleotide, nucleic
acid molecule, or any fragment or complement thereof. It may have
originated recombinantly or synthetically, may be double-stranded
or single-stranded, represents coding and noncoding 3' or 5' sequence,
and lacks introns.
 The phrase "cDNA encoding a protein" refers to
a nucleotide sequence that closely aligns with sequences which encode
conserved regions, motifs or domains that were identified by employing
analyses well known in the art. These analyses include BLAST (Basic
Local Alignment Search Tool) which provides identity within the
conserved region (Altschul (1993) J Mol Evol 36: 290-300; Altschul
et al. (1990) J Mol Biol 215:403-410).
 A "composition" refers to the polynucleotide and
a labeling moiety; a purified protein and a pharmaceutical carrier
or a heterologous, labeling or purification moiety; an antibody
and a labeling moiety or pharmaceutical agent; and the like.
 "Derivative" refers to a cDNA or a protein that
has been subjected to a chemical modification. Derivatization of
a cDNA can involve substitution of a nontraditional base such as
queosine or of an analog such as hypoxanthine. These substitutions
are well known in the art. Derivatization of a protein involves
the replacement of a hydrogen by an acetyl, acyl, alkyl, amino,
formyl, or morpholino group. Derivative molecules retain the biological
activities of the naturally occurring molecules but may confer advantages
such as longer lifespan or enhanced activity.
 "Differential expression" refers to an increased
or upregulated or a decreased or downregulated expression as detected
by absence, presence, or at least two-fold change in the amount
of transcribed messenger RNA or translated protein in a sample.
 An "expression profile" is a representation of
gene expression in a sample. A nucleic acid expression profile is
produced using sequencing, hybridization, or amplification technologies
and mRNAs or cDNAs from a sample. A protein expression profile mirrors
the nucleic acid expression profile and uses labeling moieties or
antibodies to quantify the protein expression in a sample. The nucleic
acids, proteins, or antibodies may be used in solution or attached
to a substrate, and their detection is based on methods and labeling
moieties well known in the art.
 "Disorder" refers to conditions, diseases or syndromes
in which the cDNAs and PDEBC are differentially expressed. Such
a disorder includes a breast cancer, in particular, an invasive
and potentially metastatic stage of the disease.
 "Fragment" refers to a chain of consecutive nucleotides
from about 50 to about 4000 base pairs in length. Fragments may
be used in PCR or hybridization technologies to identify related
nucleic acid molecules and in binding assays to screen for a ligand.
Such ligands are useful as therapeutics to regulate replication,
transcription or translation.
 A "hybridization complex" is formed between a
cDNA and a nucleic acid of a sample when the purines of one molecule
hydrogen bond with the pyrimidines of the complementary molecule,
e.g., 5'-A-G-T-C-3' base pairs with 3'-T-C-A-G-5'. Hybridization
conditions, degree of complementarity and the use of nucleotide
analogs affect the efficiency and stringency of hybridization reactions.
 "Labeling moiety" refers to any visible or radioactive
label than can be attached to or incorporated into a cDNA or protein.
Visible labels include but are not limited to anthocyanins, green
fluorescent protein (GFP), .beta. glucuronidase, luciferase, Cy3
and Cy5, and the like. Radioactive markers include radioactive forms
of hydrogen, iodine, phosphorous, sulfur, and the like.
 "Ligand" refers to any agent, molecule, or compound
which will bind specifically to a polynucleotide or to an epitope
of a protein. Such ligands stabilize or modulate the activity of
polynucleotides or proteins and may be composed of inorganic and/or
organic substances including minerals, cofactors, nucleic acids,
proteins, carbohydrates, fats, and lipids. "Oligonucleotide"
refers a single-stranded molecule from about 18 to about 60 nucleotides
in length which may be used in hybridization or amplification technologies
or in regulation of replication, transcription or translation. Equivalent
terms are amplimer, primer, and oligomer.
 An "oligopeptide" is an amino acid sequence from
about five residues to about 15 residues that is used as part of
a fusion protein to produce an antibody.
 "Portion" refers to any part of a protein used
for any purpose; but especially, to an epitope for the screening
of ligands or for the production of antibodies.
 "Post-translational modification" of a protein
can involve lipidation, glycosylation, phosphorylation, acetylation,
racemization, proteolytic cleavage, and the like. These processes
may occur synthetically or biochemically. Biochemical modifications
will vary by cellular location, cell type, pH, enzymatic milieu,
and the like.
 "Probe" refers to a cDNA that hybridizes to at
least one nucleic acid in a sample. Where targets are single-stranded,
probes are complementary single strands. Probes can be labeled with
reporter molecules for use in hybridization reactions including
Southern, northern, in situ, dot blot, array, and like technologies
or in screening assays.
 "Protein" refers to a polypeptide or any portion
thereof. A "portion" of a protein refers to that length
of amino acid sequence which would retain at least one biological
activity, a domain identified by PFAM or PRINTS analysis or an antigenic
epitope of the protein identified using Kyte-Doolittle algorithms
of the PROTEAN program (DNASTAR, Madison Wis.).
 "Purified" refers to any molecule or compound
that is separated from its natural environment and is from about
60% free to about 90% free from other components with which it is
 "Sample" is used in its broadest sense as containing
nucleic acids, proteins, antibodies, and the like. A sample may
comprise a bodily fluid; the soluble fraction of a cell preparation,
or an aliquot of media in which cells were grown; a chromosome,
an organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a
tissue; a tissue print; a fingerprint, buccal cells, skin, or hair;
and the like.
 "Similarity" as applied to sequences, refers to
the quantification (usually percentage) of nucleotide or residue
matches between at least two sequences aligned using a standardized
algorithm such as Smith-Waterman alignment (Smith and Waterman (1981)
J Mol Biol 147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic
Acids Res 25:3389-3402). BLAST2 may be used in a standardized and
reproducible way to insert gaps in one of the sequences in order
to optimize alignment and to achieve a more meaningful comparison
between them Particularly in proteins, similarity is greater than
identity in that conservative substitutions, for example, valine
for leucine or isoleucine, are counted in calculating the reported
percentage. Substitutions which are considered to be conservative
are well known in the art.
 "Specific binding" refers to a special and precise
interaction between two molecules which is dependent upon their
structure, particularly their molecular side groups. For example,
the intercalation of a regulatory protein into the major groove
of a DNA molecule or the binding between an epitope of a protein
and an agonist, antagonist, or antibody.
 "Substrate" refers to any rigid or semi-rigid
support to which cDNAs or proteins are bound and includes membranes,
filters, chips, slides, wafers, fibers, magnetic or nonmagnetic
beads, gels, capillaries or other tubing, plates, polymers, and
microparticles with a variety of surface forms including wells,
trenches, pins, channels and pores.
 A "transcript image" (TI) is a profile of gene
transcription activity in a particular tissue at a particular time.
TI provides assessment of the relative abundance of expressed polynucleotides
in the cDNA libraries of an EST database as described in U.S. Pat.
No. 5,840,484, incorporated herein by reference.
 "Variant" refers to molecules that are recognized
variations of a cDNA or a protein encoded by the cDNA. Splice variants
may be determined by BLAST score, wherein the score is at least
100, and most preferably at least 400. Allelic variants have a high
percent identity to the cDNAs and may differ by about three bases
per hundred bases. "Single nucleotide polymorphism" (SNP)
refers to a change in a single base as a result of a substitution,
insertion or deletion. The change may be conservative (purine for
purine) or non-conservative (purine to pyrimidine) and may or may
not result in a change in an encoded to amino acid or its secondary,
tertiary, or quaternary structure.
 The Invention
 The invention is based on the discovery of a cDNA which
encodes PDEBC and on the use of the cDNA, or fragments thereof,
and protein, or portions thereof, directly or as compositions in
the characterization, diagnosis, and treatment of a breast cancer,
in particular, an invasive and potentially metastatic stage of breast
cancer. cDNAs encoding PDEBC of the present invention were first
discovered as differentially expressed in breast tisssue samples
from patients with ductal carcinomas of the breast relative to non-diseased
breast tissue. The identification and characterization of these
cDNAs and and their encoded proteins was described in U.S. Ser.
 In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO: 1 encoded by the
polynucleotide of SEQ ID NO: 2 as shown in FIGS. 1A, 1B, 1C, 1D
and 1E. Table 1 shows the expression of polynucleotides encoding
PDEBC in breast tissue libraries from the LIFESEQ Gold database
(Incyte Genomics). The analysis was performed as described in Example
VI. The results show the expression of the polynucleotides encoding
PDEBC exclusively in breast tissue associated with breast cancer
or with proliferative breast disease (PF). In particular, the expression
of PDEBC was at least four-fold more abundant in a breast tumor
sample associated with a poorly differentiated, invasive form of
the disease (BRSTTUP03) then in any other type of diseased breast
tissue. See Table 2. PDEBC was not found in various normal breast
tissue samples unassociated with disease, e.g., BRSENOP01, BRSTNOM01,
BRSTNOM02, BRSTNON02, BRSTNOP01, BRSTNOT01, BRSTNOT25, and BRSTNOT35.
An antibody which specifically binds PDEBC is therefore useful in
a diagnostic assay to identify a breast cancer, in particular, to
identify an invasive, potentially metastatic stage of the disease.
A fragment of SEQ ID NO: 1 about amino acid S31 to about amino acid
Q50 of SEQ ID NO: 1 is a useful antigenic epitope for the production
of an antibody specific for SEQ ID NO: 1.
 Mammalian variants of the cDNA encoding PDEBC were identified
using BLAST2 with default parameters and the ZOOSEQ databases (Incyte
Genomics). These preferred variants have from about 84% to about
86% identity as shown in the table below. The first column shows
the SEQ IDvar for variant cDNAs; the second column, the clone number
for the variant cDNAs; the third column, the percent identity to
the human cDNA; the fourth column, the species of the variant cDNA;
and the fifth column, the alignment of the variant cDNA to the human
1 SEQ ID.sub.Var cDNA.sub.Var Identity species Nt.sub.H Alignment
3 702127782H1 84% Rat 773-1151 4 701647942H1 86% Rat 882-1151 5
704113673J1 86% Dog 961-1317
 It will be appreciated by those skilled in the art that
as a result of the degeneracy of the genetic code, a multitude of
cDNAs encoding PDEBC, some bearing minimal similarity to the cDNAs
of any known and naturally occurring gene, may be produced. Thus,
the invention contemplates each and every possible variation of
cDNA that could be made by selecting combinations based on possible
codon choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide encoding
naturally occurring PDEBC, and all such variations are to be considered
as being specifically disclosed.
 The cDNAs of SEQ ID NOs: 2-5 may be used in hybridization,
amplification, and screening technologies to identify and distinguish
among SEQ ID NO: 2 and related molecules in a sample. The mammalian
cDNAs, SEQ ID NOS: 2-5, may be used to produce transgenic cell lines
or organisms which are model systems for human breast cancer and
upon which the toxicity and efficacy of potential therapeutic treatments
may be tested. Toxicology studies, clinical trials, and subject/patient
treatment profiles may be performed and monitored using the cDNAs,
proteins, antibodies and molecules and compounds identified using
the cDNAs and proteins of the present invention.
 The identification and characterization of the cDNAs (SEQ
ID NO: 2) and protein (SEQ ID NO: 1) of the invention were described
in U.S. Ser. No. 09/232,260, incorporated by reference herein in
 Characterization and Use of the Invention
 cDNA Libraries
 In a particular embodiment disclosed herein, mRNA is isolated
from mammalian cells and tissues using methods which are well known
to those skilled in the art and used to prepare the cDNA libraries.
The Incyte cDNAs were isolated from mammalian cDNA libraries aprepared
as described in the EXAMPLES. The consensus sequences are chemically
and/or electronically assembled from fragments including Incyte
cDNAs and extension and/or shotgun sequences using computer programs
such as PHRAP (P Green, University of Washington, Seattle Wash.),
and AUTOASSEMBLER application (Applied Biosystems, Foster City Calif.).
After verification of the 5' and 3' sequence, at least one representative
cDNA which encodes PDEBC is designated a reagent.
 Methods for sequencing nucleic acids are well known in the
art and may be used to practice any of the embodiments of the invention.
These methods employ enzymes such as the Klenow fragment of DNA
polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7
DNA polymerase (Amersham Pharmacia Biotech (APB), Piscataway N.J.),
or combinations of polymerases and proofreading exonucleases such
as those found in the ELONGASE amplification system (Life Technologies,
Gaithersburg Md.). Preferably, sequence preparation is automated
with machines such as the MICROLAB 2200 system (Hamilton, Reno Nev.)
and the DNA ENGINE thermal cycler (M J Research, Watertown Mass.).
Machines commonly used for sequencing include the ABI PRISM 3700,
377 or 373 DNA sequencing systems (Applied Biosystems), the MEGABACE
1000 DNA sequencing system (APB), and the like. The sequences may
be analyzed using a variety of algorithms well known in the art
and described in Ausubel et al. (1997; Short Protocols in Molecular
Biology, John Wiley & Sons, New York N.Y., unit 7.7) and in
Meyers (1995; Molecular Biology and Biotechnology, Wiley V C H,
New York N.Y., pp. 856-853).
 Shotgun sequencing may also be used to complete the sequence
of a particular cloned insert of interest. Shotgun strategy involves
randomly breaking the original insert into segments of various sizes
and cloning these fragments into vectors. The fragments are sequenced
and reassembled using overlapping ends until the entire sequence
of the original insert is known. Shotgun sequencing methods are
well known in the art and use thermostable DNA polymerases, heat-labile
DNA polymerases, and primers chosen from representative regions
flanking the cDNAs of interest. Incomplete assembled sequences are
inspected for identity using various algorithms or programs such
as CONSED (Gordon (1998) Genome Res 8:195-202) which are well known
in the art. Contaminating sequences, including vector or chimeric
sequences, or deleted sequences can be removed or restored, respectively,
organizing the incomplete assembled sequences into finished sequences.
 Extension of a Nucleic Acid Sequence
 The sequences of the invention may be extended using various
PCR-based methods known in the art. For example, the XL-PCR kit
(Applied Biosystems), nested primers, and commercially available
cDNA or genomic DNA libraries may be used to extend the nucleic
acid sequence. For all PCR-based methods, primers may be designed
using commercially available primer analysis software to be about
22 to 30 nucleotides in length, to have a GC content of about 50%
or more, and to anneal to a target molecule at temperatures from
about 55C. to about 68C. When extending a sequence to recover regulatory
elements, it is preferable to use genomic, rather than cDNA libraries.
 The cDNA and fragments thereof can be used in hybridization
technologies for various purposes. A probe may be designed or derived
from unique regions such as the 5' regulatory region or from a nonconserved
region (i.e., 5' or 3' of the nucleotides encoding the conserved
catalytic domain of the protein) and used in protocols to identify
naturally occurring molecules encoding the PDEBC, allelic variants,
or related molecules. The probe may be DNA or RNA, may be single-stranded,
and should have at least 50% sequence identity to any of the nucleic
acid sequences, SEQ ID NOs: 2-5. Hybridization probes may be produced
using oligolabeling, nick translation, end-labeling, or PCR amplification
in the presence of a reporter molecule. A vector containing the
cDNA or a fragment thereof may be used to produce an mRNA probe
in vitro by addition of an RNA polymerase and labeled nucleotides.
These procedures may be conducted using commercially available kits
such as those provided by APB.
 The stringency of hybridization is determined by G+C content
of the probe, salt concentration, and temperature. In particular,
stringency can be increased by reducing the concentration of salt
or raising the hybridization temperature. Hybridization can be performed
at low stringency with buffers, such as 5.times.SSC with 1% sodium
dodecyl sulfate (SDS) at 60C., which permits the formation of a
hybridization complex between nucleic acid sequences that contain
some mismatches. Subsequent washes are performed at higher stringency
with buffers such as 0.2.times.SSC with 0.1% SDS at either 45C.
(medium stringency) or 68C. (high stringency). At high stringency,
hybridization complexes will remain stable only where the nucleic
acids are completely complementary. In some membrane-based hybridizations,
preferably 35% or most preferably 50%, formamide can be added to
the hybridization solution to reduce the temperature at which hybridization
is performed, and background signals can be reduced by the use of
detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St.
Louis Mo.) and a blocking agent such as denatured salmon sperm DNA.
Selection of components and conditions for hybridization are well
known to those skilled in the art and are reviewed in Ausubel (sura)
and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Press, Plainview N.Y.
 Arrays may be prepared and analyzed using methods well known
in the art. Oligonucleotides or cDNAs may be used as hybridization
probes or targets to monitor the expression level of large numbers
of genes simultaneously or to identify genetic variants, mutations,
and single nucleotide polymorphisms. Arrays may be used to determine
gene function; to understand the genetic basis of a condition, disease,
or disorder; to diagnose a condition, disease, or disorder; and
to develop and monitor the activities of therapeutic agents. (See,
e.g., Brennan et al. (1995) U.S. Pat. No. 5,474,796; Schena et al.
(1996) Proc Natl Acad Sci 93:10614-10619; Heller et al. (1997) Proc
Natl Acad Sci 94:2150-2155; and Heller et al. (1997) U.S. Pat. No.
 Hybridization probes are also useful in mapping the naturally
occurring genomic sequence. The probes may be hybridized to a particular
chromosome, a specific region of a chromosome, or an artificial
chromosome construction. Such constructions include human artificial
chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial
artificial chromosomes (BAC), bacterial P1 constructions, or the
cDNAs of libraries made from single chromosomes.
 Quantitative PCR (TAQMAN, ABI)
 Quantitative real-time PCR (QPCR) is a method for quantifying
a nucleic acid molecule based on detection of a fluorescent signal
produced during PCR amplification (Gibson et al. (1996) Genome Res
6:995-1001; Heid et al. (1996) Genome Res 6:986-994). Amplification
is carried out on machines such as the ABI PRISM 7700 detection
system which consists of a 96-well thermal cycler connected to a
laser and charge-coupled device (CCD) optics system. To perform
QPCR, a PCR reaction is carried out in the presence of a doubly
labeled "TAQMAN" probe. The probe, which is designed to
anneal between the standard forward and reverse PCR primers, is
labeled at the 5' end by a flourogenic reporter dye such as 6-carboxyfluorescein
(6-FAM) and at the 3' end by a quencher molecule such as 6-carboxy-tetramethyl-rhodamine
(TAMRA). As long as the probe is intact, the 3' quencher extinguishes
fluorescence by the 5' reporter. However, during each primer extension
cycle, the annealed probe is degraded as a result of the intrinsic
5' to 3' nuclease activity of Taq polymerase (Holland et al. (1991)
Proc Natl Acad Sci 88:7276-7280). This degradation separates the
reporter from the quencher, and fluorescence is detected every few
seconds by the CCD. The higher the starting copy number of the nucleic
acid, the sooner a significant increase in fluorescence is observed.
A cycle threshold (C.sub.T) value, representing the cycle number
at which the PCR product crosses a fixed threshold of detection
is determined by the instrument software. The C.sub.T is inversely
proportional to the copy number of the template and can therefore
be used to calculate either the relative or absolute initial concentration
of the nucleic acid molecule in the sample. The relative concentration
of two different molecules can be calculated by determining their
respective C.sub.T values (comparative C.sub.T method). Alternatively,
the absolute concentration of the nucleic acid molecule can be calculated
by constructing a standard curve using a housekeeping molecule of
known concentration. The process of calculating C.sub.Ts, preparing
a standard curve, and determining starting copy number is performed
by the SEQUENCE DETECTOR 1.7 software (ABI).
 Any one of a multitude of cDNAs encoding PDEBC may be cloned
into a vector and used to express the protein, or portions thereof,
in host cells. The nucleic acid sequence can be engineered by such
methods as DNA shuffling (U.S. Pat. No. 5,830,721) and site-directed
mutagenesis to create new restriction sites, alter glycosylation
patterns, change codon preference to increase expression in a particular
host, produce splice variants, extend half-life, and the like. The
expression vector may contain transcriptional and translational
control elements (promoters, enhancers, specific initiation signals,
and polyadenylated 3' sequence) from various sources which have
been selected for their efficiency in a particular host. The vector,
cDNA, and regulatory elements are combined using in vitro recombinant
DNA techniques, synthetic techniques, and/or in vivo genetic recombination
techniques well known in the art and described in Sambrook (supra,
ch. 4, 8, 16 and 17).
 A variety of host systems may be transformed with an expression
vector. These include, but are not limited to, bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems transformed with baculovirus expression vectors; plant
cell systems transformed with expression vectors containing viral
and/or bacterial elements, or animal cell systems (Ausubel supra,
unit 16). For example, an adenovirus transcription/translation complex
may be utilized in mammalian cells. After sequences are ligated
into the E1 or E3 region of the viral genome, the infective virus
is used to transform and express the protein in host cells. The
Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also
be used for high-level protein expression.
 Routine cloning, subcloning, and propagation of nucleic
acid sequences can be achieved using the multifunctional PBLUESCRIPT
vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies).
Introduction of a nucleic acid sequence into the multiple cloning
site of these vectors disrupts the lacZ gene and allows calorimetric
screening for transformed bacteria. In addition, these vectors may
be useful for in vitro transcription, dideoxy sequencing, single
strand rescue with helper phage, and creation of nested deletions
in the cloned sequence.
 For long term production of recombinant proteins, the vector
can be stably transformed into cell lines along with a selectable
or visible marker gene on the same or on a separate vector. After
transformation, cells are allowed to grow for about 1 to 2 days
in enriched media and then are transferred to selective media. Selectable
markers, antimetabolite, antibiotic, or herbicide resistance genes,
confer resistance to the relevant selective agent and allow growth
and recovery of cells which successfully express the introduced
sequences. Resistant clones identified either by survival on selective
media or by the expression of visible markers may be propagated
using culture techniques. Visible markers are also used to estimate
the amount of protein expressed by the introduced genes. Verification
that the host cell contains the desired cDNA is based on DNA-DNA
or DNA-RNA hybridizations or PCR amplification techniques.
 The host cell may be chosen for its ability to modify a
recombinant protein in a desired fashion. Such modifications include
acetylation, carboxylation, glycosylation, phosphorylation, lipidation,
acylation and the like. Post-translational processing which cleaves
a "prepro" form may also be used to specify protein targeting,
folding, and/or activity. Different host cells available from the
ATCC (Manassas Va.) which have specific cellular machinery and characteristic
mechanisms for post-translational activities may be chosen to ensure
the correct modification and processing of the recombinant protein.
 Recovery of Proteins from Cell Culture
 Heterologous moieties engineered into a vector for ease
of purification include glutathione S-transferase (GST), 6.times.His,
FLAG, MYC, and the like. GST and 6-His are purified using commercially
available affinity matrices such as immobilized glutathione and
metal-chelate resins, respectively. FLAG and MYC are purified using
commercially available monoclonal and polyclonal antibodies. For
ease of separation following purification, a sequence encoding a
proteolytic cleavage site may be part of the vector located between
the protein and the heterologous moiety. Methods for recombinant
protein expression and purification are discussed in Ausubel (supra,
unit 16) and are commercially available.
 Protein Identification
 Several techniques have been developed which permit rapid
identification of proteins using high performance liquid chromatography
and mass spectrometry. Beginning with a sample containing proteins,
the major steps involved are: 1) proteins are separated using two-dimensional
gel electrophoresis (2-DE), 2) selected proteins are excised from
the gel and digested with a protease to produce a set of peptides;
and 3) the peptides are subjected to mass spectral (MS) analysis
to derive peptide ion mass and spectral pattern information. The
MS information is used to identify the protein by comparing it with
information in a protein database (Shevenko et al.(1996) Proc Natl
Acad Sci 93:14440-14445). A more detailed description follows.
 Proteins are separated by 2DE employing isoelectric focusing
(IEF) in the first dimension followed by SDS-PAGE in the second
dimension. For IEF, an immobilzed pH gradient strip is useful to
increase reproducibility and resolution of the separation. Alternative
techniques may be used to improve resolution of very basic, hydrophobic,
or high molecular weight proteins. The separated proteins are detected
using a stain or dye such as silver stain, Coomassie blue, or spyro
red (Molecular Bioprobes, Eugene Oreg.) that is compatible with
mass spectrometry Gels may be blotted onto a PVDF membrane for western
analysis and optically scanned using a STORM scanner (APB) to produce
a computer-readable output which is analyzed by pattern recognition
software such as MELANIE (GeneBio, Geneva, Switzerland). The software
annotates individual spots by assigning a unique identifier and
calculating their respective x,y coordinates, molecular masses,
isoelectric points, and signal intensity. Individual spots of interest,
such as those representing differentially expressed proteins, are
excised and proteolytically digested with a site-specific protease
such as trypsin or chymotrypsin, singly or in combination, to generate
a set of small peptides, preferably in the range of 1-2 kDa. Prior
to digestion, samples may be treated with reducing and alkylating
agents, and following digestion, the peptides are then separated
by liquid chromatography or capillary electrophoresis and analyzed
 MS converts components of a sample into gaseous ions, separates
the ions based on their mass-to-charge ratio, and determines relative
abundance. For peptide mass fingerprinting analysis, a mass spectrometer
of the MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time
of Flight), ESI (Electrospray Ionization), and TOF-TOF (Time of
Flight/Time of Flight) machines are used to determine a set of highly
accurate peptide masses. Using analytical programs, such as TURBOSEQUEST
software (Finnigan, San Jose Calif.), the MS data is compared against
a database of theoretical MS data derived from known or predicted
proteins. A minimum match of three peptide masses is usually required
for reliable protein identification. If additional information is
needed for identification, Tandem-MS may be used to derive information
about individual peptides. In tandem-MS a first stage of MS is performed
to determine individual peptide masses. Then selected peptide ions
are subjected to fragmentation using a technique such as collision
induced dissociation (CID) to produce an ion series. The resulting
fragmentation ions are analyzed in a second round of MS, and their
spectral pattern may be used to determine a short stretch of amino
acid sequence (Dancik et al. (1999) J Comput Biol 6:327-342).
 Assuming the protein is represented in the database, a combination
of peptide mass and fragmentation data, together with the calculated
MW and pI of the protein, will usually yield an unambiguous identification.
If no match is found, protein sequence can be obtained using direct
chemical sequencing procedures well known in the art (cf Creighton
(1984) Proteins, Structures and Molecular Properties, W H Freeman,
New York N.Y.).
 Chemical Synthesis of Peptides
 Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in
a batchwise or continuous flow process which sequentially adds .alpha.-amino-
and side chain-protected amino acid residues to an insoluble polymeric
support via a linker group. A linker group such as methylamine-derivatized
polyethylene glycol is attached to poly(styrene-co-divinylbenzene)
to form the support resin. The amino acid residues are N-.alpha.-protected
by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl).
The carboxyl group of the protected amino acid is coupled to the
amine of the linker group to anchor the residue to the solid phase
support resin. Trifluoroacetic acid or piperidine are used to remove
the protecting group in the case of Boc or Fmoc, respectively. Each
additional amino acid is added to the anchored residue using a coupling
agent or pre-activated amino acid derivative, and the resin is washed.
The full length peptide is synthesized by sequential deprotection,
coupling of derivitized amino acids, and washing with dichloromethane
and/or N, N-dimethylformamide. The peptide is cleaved between the
peptide carboxy terminus and the linker group to yield a peptide
acid or amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis
Handbook, San Diego Calif. pp. S1-S20). Automated synthesis may
also be carried out on machines such as the ABI 431A peptide synthesizer
(Applied Biosystems). A protein or portion thereof may be purified
by preparative high performance liquid chromatography and its composition
confirmed by amino acid analysis or by sequencing (Creighton (1984)
Proteins, Structures and Molecular Properties, W H Freeman, New
 Antibodies, or immunoglobulins (Ig), are components of immune
response expressed on the surface of or secreted into the circulation
by B cells. The prototypical antibody is a tetramer composed of
two identical heavy polypeptide chains (H-chains) and two identical
light polypeptide chains (L-chains) interlinked by disulfide bonds
which binds and neutralizes foreign antigens. Based on their H-chain,
antibodies are classified as IgA, IgD, IgE, IgG or IgM. The most
common class, IgG, is tetrameric while other classes are variants
or multimers of the basic structure.
 Antibodies are described in terms of their two main functional
domains. Antigen recognition is mediated by the Fab (antigen binding
fragment) region of the antibody, while effector functions are mediated
by the Fc (crystallizable fragment) region. The binding of antibody
to antigen triggers destruction of the antigen by phagocytic white
blood cells such as macrophages and neutrophils. These cells express
surface Fc receptors that specifically bind to the Fc region of
the antibody and allow the phagocytic cells to destroy antibody-bound
antigen. Fc receptors are single-pass transmembrane glycoproteins
containing about 350 amino acids whose extracellular portion typically
contains two or three Ig domains (Sears et al. (1990) J Immunol
 Preparation and Screening of Antibodies
 Various hosts including mice, rats, rabbits, goats, llamas,
camels, and human cell lines may be immunized by injection with
an antigenic determinant. Adjuvants such as Freund's, mineral gels,
and surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH;
Sigma-Aldrich, St. Louis Mo.), and dinitrophenol may be used to
increase immunological response. In humans, BCG (bacilli Calmette-Guerin)
and Corynebacterium parvum are preferable. The antigenic determinant
may be an oligopeptide, peptide, or protein. When the amount of
antigenic determinant allows immunization to be repeated, specific
polyclonal antibody with high affinity can be obtained (Klinman
and Press (1975) Transplant Rev 24:41-83). Oligopepetides which
may contain between about five and about fifteen amino acids identical
to a portion of the endogenous protein may be fused with proteins
such as KLH in order to produce antibodies to the chimeric molecule.
 Monoclonal antibodies may be prepared using any technique
which provides for the production of antibodies by continuous cell
lines in culture. These include the hybridoma technique, the human
B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler
et al (1975) Nature 256:495-497; Kozbor et al (1985) J Immunol Methods
81:31-42; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030; and
Cole et al (1984) Mol Cell Biol 62:109-120).
 "Chimeric antibodies" may be produced by techniques
such as splicing of mouse antibody genes to human antibody genes
to obtain a molecule with appropriate antigen specificity and biological
activity (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855;
Neuberger et al. (1984) Nature 312:604-608; and Takeda et al. (1985)
Nature 314:452-454). Alternatively, techniques described for antibody
production may be adapted, using methods known in the art, to produce
specific, single chain antibodies. Antibodies with related specificity,
but of distinct idiotypic composition, may be generated by chain
shuffling from random combinatorial immunoglobulin libraries (Burton
(1991) Proc Natl Acad Sci 88:10134-10137). Antibody fragments which
contain specific binding sites for an antigenic determinant may
also be produced. For example, such fragments include, but are not
limited to, F(ab')2 fragments produced by pepsin digestion of the
antibody molecule and Fab fragments generated by reducing the disulfide
bridges of the F(ab')2 fragments. Alternatively, Fab expression
libraries may be constructed to allow rapid and easy identification
of monoclonal Fab fragments with the desired specificity (Huse et
al (1989) Science 246:1275-1281).
 Antibodies may also be produced by inducing production in
the lymphocyte population or by screening immunoglobulin libraries
or panels of highly specific binding reagents as disclosed in Orlandi
et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al.
(1991; Nature 349:293-299). A protein may be used in screening assays
of phagemid or B-lymphocyte immunoglobulin libraries to identify
antibodies having a desired specificity. Numerous protocols for
competitive binding or immunoassays using either polyclonal or monoclonal
antibodies with established specificities are well known in the
 Antibody Specificity
 Various methods such as Scatchard analysis combined with
radioimmunoassay techniques may be used to assess the affinity of
particular antibodies for a protein. Affinity is expressed as an
association constant, K.sub.a, which is defined as the molar concentration
of protein-antibody complex divided by the molar concentrations
of free antigen and free antibody under equilibrium conditions.
The K.sub.a determined for a preparation of polyclonal antibodies,
which are heterogeneous in their affinities for multiple antigenic
determinants, represents the average affinity, or avidity, of the
antibodies. The K.sub.a determined for a preparation of monoclonal
antibodies, which are specific for a particular antigenic determinant,
represents a true measure of affinity. High-affinity antibody preparations
with K.sub.a ranging from about 10.sup.9 to 10.sup.12 L/mole are
preferred for use in immunoassays in which the protein-antibody
complex must withstand rigorous manipulations. Low-affinity antibody
preparations with K.sub.a ranging from about 10.sup.6 to 10.sup.7
L/mole are preferred for use in immunopurification and similar procedures
which ultimately require dissociation of the protein, preferably
in active form, from the antibody (Catty (1988) Antibodies Volume
I: A Practical Approach, IRL Press, Washington D.C.; Liddell and
Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley
& Sons, New York N.Y.).
 The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For example,
a polyclonal antibody preparation containing about 5-10 mg specific
antibody/ml, is generally employed in procedures requiring precipitation
of protein-antibody complexes. Procedures for making antibodies,
evaluating antibody specificity, titer, and avidity, and guidelines
for antibody quality and usage in various applications, are widely
available (Catty (supra); Ausubel (sura) pp. 11.1-11.31).
 Immunological Assays
 Immunological methods for detecting and measuring complex
formation as a measure of protein expression using either specific
polyclonal or monoclonal antibodies are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), fluorescence-activated cell sorting (FACS)
and antibody arrays. Such immunoassays typically involve the measurement
of complex formation between the protein and its specific antibody.
A two-site, monoclonal-based immunoassay utilizing antibodies reactive
to two non-interfering epitopes is preferred, but a competitive
binding assay may be employed (Pound (1998) Immunochemical Protocols,
Humana Press, Totowa N.J.).
 These methods are also useful for diagnosing diseases that
show differential protein expression. Normal or standard values
for protein expression are established by combining body fluids
or cell extracts taken from a normal mammalian or human subject
with specific antibodies to a protein under conditions for complex
formation. Standard values for complex formation in normal and diseased
tissues are established by various methods, often photometric means.
Then complex formation as it is expressed in a subject sample is
compared with the standard values. Deviation from the normal standard
and toward the diseased standard provides parameters for disease
diagnosis or prognosis while deviation away from the diseased and
toward the normal standard may be used to evaluate treatment efficacy.
 Labeling of Molecules for Assay
 A wide variety of reporter molecules and conjugation techniques
are known by those skilled in the art and may be used in various
nucleic acid, amino acid, and antibody assays. Synthesis of labeled
molecules may be achieved using commercially available kits (Promega,
Madison Wis.) for incorporation of a labeled nucleotide such as
.sup.32P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Operon Technologies,
Alameda Calif.), or amino acid such as .sup.35S-methionine (APB).
Nucleotides and amino acids may be directly labeled with a variety
of substances including fluorescent, chemiluminescent, or chromogenic
agents, and the like, by chemical conjugation to araines, thiols
and other groups present in the molecules using reagents such as
BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
 Nucleic Acid Assays
 The cDNAs, fragments, oligonucleotides, complementary RNA
and DNA molecules, and PNAs and may be used to detect and quantify
differential gene expression for diagnosis of a disorder. Similarly
antibodies which specifically bind PDEBC may be used to quantitate
the protein. Disorders associated with differential expression include
breast cancer and, in particular, an invasive and potentially metastatic
stage of the disease. The diagnostic assay may use hybridization
or amplification technology to compare gene expression in a biological
sample from a patient to standard samples in order to detect differential
gene expression. Qualitative or quantitative methods for this comparison
are well known in the art.
 For example, the cDNA or probe may be labeled by standard
methods and added to a biological sample from a patient under conditions
for the formation of hybridization complexes. After an incubation
period, the sample is washed and the amount of label (or signal)
associated with hybridization complexes, is quantified and compared
with a standard value. If complex formation in the patient sample
is significantly altered (higher or lower) in comparison to either
a normal or disease standard, then differential expression indicates
the presence of a disorder.
 In order to provide standards for establishing differential
expression, normal and disease expression profiles are established.
This is accomplished by combining a sample taken from normal subjects,
either animal or human, with a cDNA under conditions for hybridization
to occur. Standard hybridization complexes may be quantified by
comparing the values obtained using normal subjects with values
from an experiment in which a known amount of a purified sequence
is used. Standard values obtained in this manner may be compared
with values obtained from samples from patients who were diagnosed
with a particular condition, disease, or disorder. Deviation from
standard values toward those associated with a particular disorder
is used to diagnose that disorder.
 Such assays may also be used to evaluate the efficacy of
a particular therapeutic treatment regimen in animal studies or
in clinical trials or to monitor the treatment of an individual
patient. Once the presence of a condition is established and a treatment
protocol is initiated, diagnostic assays may be repeated on a regular
basis to determine if the level of expression in the patient begins
to approximate that which is observed in a normal subject. The results
obtained from successive assays may be used to show the efficacy
of treatment over a period ranging from several days to years.
 Protein Assays
 Detection and quantification of a protein using either labeled
amino acids or specific polyclonal or monoclonal antibodies are
known in the art. Examples of such techniques include two-dimensional
polyacrylamide gel electrophoresis, enzyme-linked immunosorbent
assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated
cell sorting (FACS). These assays and their quantitation against
purifed, labeled standards are well known in the art (Ausubel, supra,
unit 10.1-10.6). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes is
preferred, but a competitive binding assay may be employed. (See,
e.g., Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience,
New York N.Y.; and Pound, supra.)
 Recently, antibody arrays have allowed the development of
techniques for high-throughput screening of recombinant antibodies.
Such methods use robots to pick and grid bacteria containing antibody
genes, and a filter-based ELISA to screen and identify clones that
express antibody fragments. Because liquid handling is eliminated
and the clones are arrayed from master stocks, the same antibodies
can be spotted multiple times and screened against multiple antigens
simultaneously. Antibody arrays are highly useful in the identification
of differentially expressed proteins. (See de Wildt et al. (2000)
Nat Biotechnol 18:989-94.)
 Differential expression of PDEBC as detected using any of
the above assays is diagnostic of a breast cancer.
 Differential expression of PDEBC (SEQ ID NO: 1) is highly
associated with breast cancer, as shown in Table 1, and in particular
with an invasive, potentially metastatic stage of the disease. PDEBC
clearly plays a role in breast cancer.
 In one embodiment, when decreased expression of activity
of the protein is desired, an inhibitor, antagonist, antibody and
the like or a pharmaceutical agent containing one or more of these
molecules may be delivered. Such delivery may be effected by methods
well known in the art and may include delivery by an antibody specifically
targeted to the protein. Neutralizing antibodies which inhibit dimer
formation are generally preferred for therapeutic use.
 In another embodiment, when increased expression or activity
of the protein is desired, the protein, an agonist, an enhancer
and the like or a pharmaceutical agent containing one or more of
these molecules may be delivered. Such delivery may be effected
by methods well known in the art and may include delivery of a pharmaceutical
agent by an antibody specifically targeted to the protein.
 Any of the cDNAs, complementary molecules, or fragments
thereof, proteins or portions thereof, vectors delivering these
nucleic acid molecules or expressing the proteins, and their ligands
may be administered in combination with other therapeutic agents.
Selection of the agents for use in combination therapy may be made
by one of ordinary skill in the art according to conventional pharmaceutical
principles. A combination of therapeutic agents may act synergistically
to affect treatment of a particular disorder at a lower dosage of
 Modification of Gene Expression Using Nucleic Acids
 Gene expression may be modified by designing complementary
or antisense molecules (DNA, 0RNA, or PNA) to the control, 5', 3',
or other regulatory regions of the gene encoding PDEBC. Oligonucleotides
designed to inhibit transcription initiation are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing which
inhibits the binding of polymerases, transcription factors, or regulatory
molecules (Gee et al. In: Huber and Carr (1994) Molecular and Immunologic
Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177). A complementary
molecule may also be designed to block translation by preventing
binding between ribosomes and mRNA. In one alternative, a library
or plurality of cDNAs may be screened to identify those which specifically
bind a regulatory, nontranslated sequence.
 Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA followed by endonucleolytic
cleavage at sites such as GUA, GUU, and GUC. Once such sites are
identified, an oligonucleotide with the same sequence may be evaluated
for secondary structural features which would render the oligonucleotide
inoperable. The suitability of candidate targets may also be evaluated
by testing their hybridization with complementary oligonucleotides
using ribonuclease protection assays.
 Complementary nucleic acids and ribozymes of the invention
may be prepared via recombinant expression, in vitro or in vivo,
or using solid phase phosphoramidite chemical synthesis. In addition,
RNA molecules may be modified to increase intracellular stability
and half-life by addition of flanking sequences at the 5' and/or
3' ends of the molecule or by the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. Modification is inherent in the production of PNAs
and can be extended to other nucleic acid molecules. Either the
inclusion of nontraditional bases such as inosine, queosine, and
wybutosine, or the modification of adenine, cytidine, guanine, thymine,
and uridine with acetyl-, methyl-, thio-groups renders the molecule
less available to endogenous endonucleases.
 cDNA Therapeutics
 The cDNAs of the invention can be used in gene therapy.
cDNAs can be delivered ex vivo to target cells, such as cells of
bone marrow. Once stable integration and transcription and or translation
are confirmed, the bone marrow may be reintroduced into the subject.
Expression of the protein encoded by the cDNA may correct a disorder
associated with mutation of a normal sequence, reduction or loss
of an endogenous target protein, or overepression of an endogenous
or mutant protein. Alternatively, cDNAs may be delivered in vivo
using vectors such as retrovirus, adenovirus, adeno-associated virus,
herpes simplex virus, and bacterial plasmids. Non-viral methods
of gene delivery include cationic liposomes, polylysine conjugates,
artificial viral envelopes, and direct injection of DNA (Anderson
(1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325;
Chu et al. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999)
Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics,
Humana Press, Totowa N.J.; and August et al (1997) Gene Therapy
(Advances in Pharmacology, Vol. 40), Academic Press, San Diego Calif.).
 Screening and Purification Assays
 The cDNA encoding PDEBC may be used to screen a library
or a plurality of molecules or compounds for specific binding affinity.
The libraries may be DNA molecules, RNA molecules, PNAs, peptides,
proteins such as transcription factors, enhancers, or repressors,
and other ligands which regulate the activity, replication, transcription,
or translation of the endogenous gene. The assay involves combining
a polynucleotide with a library or plurality of molecules or compounds
under conditions allowing specific binding, and detecting specific
binding to identify at least one molecule which specifically binds
the single-stranded or double-stranded molecule.
 In one embodiment, the cDNA of the invention may be incubated
with a plurality of purified molecules or compounds and binding
activity determined by methods well known in the art, e.g., a gel-retardation
assay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional
assay. In another embodiment, the cDNA may be incubated with nuclear
extracts from biopsied and/or cultured cells and tissues. Specific
binding between the cDNA and a molecule or compound in the nuclear
extract is initially determined by gel shift assay and may be later
confirmed by recovering and raising antibodies against that molecule
or compound. When these antibodies are added into the assay, they
cause a supershift in the gel-retardation assay.
 In another embodiment, the cDNA may be used to purify a
molecule or compound using affinity chromatography methods well
known in the art. In one embodiment, the cDNA is chemically reacted
with cyanogen bromide groups on a polymeric resin or gel. Then a
sample is passed over and reacts with or binds to the cDNA. The
molecule or compound which is bound to the cDNA may be released
from the cDNA by increasing the salt concentration of the flow-through
medium and collected.
 In a further embodiment, the protein or a portion thereof
may be used to purify a ligand from a sample. A method for using
a protein or a portion thereof to purify a ligand would involve
combining the protein or a portion thereof with a sample under conditions
to allow specific binding, detecting specific binding between the
protein and ligand, recovering the bound protein, and using a chaotropic
agent to separate the protein from the purified ligand.
 In a preferred embodiment, PDEBC may be used to screen a
plurality of molecules or compounds in any of a variety of screening
assays. The portion of the protein employed in such screening may
be free in solution, affixed to an abiotic or biotic substrate (e.g.
borne on a cell surface), or located intracellularly. For example,
in one method, viable or fixed prokaryotic host cells that are stably
transformed with recombinant nucleic acids that have expressed and
positioned a peptide on their cell surface can be used in screening
assays. The cells are screened against a plurality or libraries
of ligands, and the specificity of binding or formation of complexes
between the expressed protein and the ligand can be measured. Depending
on the particular kind of molecules or compounds being screened,
the assay may be used to identify DNA molecules, RNA molecules,
peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists,
antibodies, immunoglobulins, inhibitors, and drugs or any other
ligand, which specifically binds the protein.
 In one aspect, this invention comtemplates a method for
high throughput screening using very small assay volumes and very
small amounts of test compound as described in U.S. Pat. No. 5,876,946,
incorporated herein by reference. This method is used to screen
large numbers of molecules and compounds via specific binding. In
another aspect, this invention also contemplates the use of competitive
drug screening assays in which neutralizing antibodies capable of
binding the protein specifically compete with a test compound capable
of binding to the protein. Molecules or compounds identified by
screening may be used in a mammalian model system to evaluate their
toxicity, diagnostic, or therapeutic potential.
 Pharmaceutical Compositions
 Pharmaceutical compositions may be formulated and administered,
to a subject in need of such treatment, to attain a therapeutic
effect. Such compositions contain the instant protein, agonists,
antibodies specifically binding the protein, antagonists, inhibitors,
or mimetics of the protein. Compositions may be manufactured by
conventional means such as mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, or lyophilizing.
The composition may be provided as a salt, formed with acids such
as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic, or as a lyophilized powder which may be combined with
a sterile buffer such as saline, dextrose, or water. These compositions
may include auxiliaries or excipients which facilitate processing
of the active compounds.
 Auxiliaries and excipients may include coatings, fillers
or binders including sugars such as lactose, sucrose, mannitol,
glycerol, or sorbitol; starches from corn, wheat, rice, or potato;
proteins such as albumin, gelatin and collagen; cellulose in the
form of hydroxypropylmethyl-cellulose, methyl cellulose, or sodium
carboxymethylcellulose; gums including arabic and tragacanth; lubricants
such as magnesium stearate or talc; disintegrating or solubilizing
agents such as the, agar, alginic acid, sodium alginate or cross-linked
polyvinyl pyrrolidone; stabilizers such as carbopol gel, polyethylene
glycol, or titanium dioxide; and dyestuffs or pigments added for
identify the product or to characterize the quantity of active compound
 These compositions may be administered by any number of
routes including oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.
 The route of administration and dosage will determine formulation;
for example, oral administration may be accomplished using tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries, or suspensions;
parenteral administration may be formulated in aqueous, physiologically
compatible buffers such as Hanks' solution, Ringer's solution, or
physiologically buffered saline. Suspensions for injection may be
aqueous, containing viscous additives such as sodium carboxymethyl
cellulose or dextran to increase the viscosity, or oily, containing
lipophilic solvents such as sesame oil or synthetic fatty acid esters
such as ethyl oleate or triglycerides, or liposomes. Penetrants
well known in the art are used for topical or nasal administration.
 Toxicity and Therapeutic Efficacy
 A therapeutically effective dose refers to the amount of
active ingredient which ameliorates symptoms or condition. For any
compound, a therapeutically effective dose can be estimated from
cell culture assays using normal and neoplastic cells or in animal
models. Therapeutic efficacy, toxicity, concentration range, and
route of administration may be determined by standard pharmaceutical
procedures using experimental animals.
 The therapeutic index is the dose ratio between therapeutic
and toxic effects--LD50 (the dose lethal to 50% of the population)/ED50
(the dose therapeutically effective in 50% of the population)--and
large therapeutic indices are preferred. Dosage is within a range
of circulating concentrations, includes an ED50 with little or no
toxicity, and varies depending upon the composition, method of delivery,
sensitivity of the patient, and route of administration. Exact dosage
will be determined by the practitioner in light of factors related
to the subject in need of the treatment.
 Dosage and administration are adjusted to provide active
moiety that maintains therapeutic effect. Factors for adjustment
include the severity of the disease state, general health of the
subject, age, weight, and gender of the subject, diet, time and
frequency of administration, drug combination(s), reaction sensitivities,
and tolerance/response to therapy. Long-acting pharmaceutical compositions
may be administered every 3 to 4 days, every week, or once every
two weeks depending on half-life and clearance rate of the particular
 Normal dosage amounts may vary from 0.1 .mu.g, up to a total
dose of about 1 g, depending upon the route of administration. The
dosage of a particular composition may be lower when administered
to a patient in combination with other agents, drugs, or hormones.
Guidance as to particular dosages and methods of delivery is provided
in the pharmaceutical literature and generally available to practitioners.
Further details on techniques for formulation and administration
may be found in the latest edition of Remington's Pharmaceutical
Sciences (Mack Publishing, Easton Pa.).
 Model Systems
 Animal models may be used as bioassays where they exhibit
a phenotypic response similar to that of humans and where exposure
conditions are relevant to human exposures. Mammals are the most
common models, and most infectious agent, cancer, drug, and toxicity
studies are performed on rodents such as rats or mice because of
low cost, availability, lifespan, reproductive potential, and abundant
reference literature. Inbred and outbred rodent strains provide
a convenient model for investigation of the physiological consequences
of under- or over-expression of genes of interest and for the development
of methods for diagnosis and treatment of diseases. A mammal inbred
to over-express a particular gene (for example, secreted in milk)
may also serve as a convenient source of the protein expressed by
 Toxicology is the study of the effects of agents on living
systems. The majority of toxicity studies are performed on rats
or mice. Observation of qualitative and quantitative changes in
physiology, behavior, homeostatic processes, and lethality in the
rats or mice are used to generate a toxicity profile and to assess
potential consequences on human health following exposure to the
 Genetic toxicology identifies and analyzes the effect of
an agent on the rate of endogenous, spontaneous, and induced genetic
mutations. Genotoxic agents usually have common chemical or physical
properties that facilitate interaction with nucleic acids and are
most harmful when chromosomal aberrations are transmitted to progeny.
Toxicological studies may identify agents that increase the frequency
of structural or functional abnormalities in the tissues of the
progeny if administered to either parent before conception, to the
mother during pregnancy, or to the developing organism. Mice and
rats are most frequently used in these tests because their short
reproductive cycle allows the production of the numbers of organisms
needed to satisfy statistical requirements.
 Acute toxicity tests are based on a single administration
of an agent to the subject to determine the symptomology or lethality
of the agent. Three experiments are conducted: 1) an initial dose-range-finding
experiment, 2) an experiment to narrow the range of effective doses,
and 3) a final experiment for establishing the dose-response curve.
 Subchronic toxicity tests are based on the repeated administration
of an agent. Rat and dog are commonly used in these studies to provide
data from species in different families. With the exception of carcinogenesis,
there is considerable evidence that daily administration of an agent
at high-dose concentrations for periods of three to four months
will reveal most forms of toxicity in adult animals.
 Chronic toxicity tests, with a duration of a year or more,
are used to demonstrate either the absence of toxicity or the carcinogenic
potential of an agent. When studies are conducted on rats, a minimum
of three test groups plus one control group are used, and animals
are examined and monitored at the outset and at intervals throughout
 Transgenic Animal Models
 Transgenic rodents that over-express or under-express a
gene of interest may be inbred and used to model human diseases
or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. No.
5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the introduced
gene may be activated at a specific time in a specific tissue type
during fetal or postnatal development. Expression of the transgene
is monitored by analysis of phenotype, of tissue-specific mRNA expression,
or of serum and tissue protein levels in transgenic animals before,
during, and after challenge with experimental drug therapies.
 Embryonic Stem Cells
 Embryonic (ES) stem cells isolated from rodent embryos retain
the potential to form embryonic tissues. When ES cells are placed
inside a carrier embryo, they resume normal development and contribute
to tissues of the live-born animal. ES cells are the preferred cells
used in the creation of experimental knockout and knockin rodent
strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are
derived from the early mouse embryo and are grown under culture
conditions well known in the art. Vectors used to produce a transgenic
strain contain a disease gene candidate and a marker gen, the latter
serves to identify the presence of the introduced disease gene.
The vector is transformed into ES cells by methods well known in
the art, and transformed ES cells are identified and microinjected
into mouse cell blastocysts such as those from the C57BL/6 mouse
strain. The blastocysts are surgically transferred to pseudopregnant
dams, and the resulting chimeric progeny are genotyped and bred
to produce heterozygous or homozygous strains.
 ES cells derived from human blastocysts may be manipulated
in vitro to differentiate into at least eight separate cell lineages.
These lineages are used to study the differentiation of various
cell types and tissues in vitro, and they include endoderm, mesoderm,
and ectodermal cell types which differentiate into, for example,
neural cells, hematopoietic lineages, and cardiomyocytes.
 Knockout Analysis
 In gene knockout analysis, a region of a mammalian gene
is enzymatically modified to include a non-mammalian gene such as
the neomycin phosphotransferase gene (neo; Capecchi (1989) Science
244:1288-1292). The modified gene is transformed into cultured ES
cells and integrates into the endogenous genome by homologous recombination.
The inserted sequence disrupts transcription and translation of
the endogenous gene. Transformed cells are injected into rodent
blastulae, and the blastulae are implanted into pseudopregnant dams.
Transgenic progeny are crossbred to obtain homozygous inbred lines
which lack a functional copy of the mammalian gene. In one example,
the mammalian gene is a human gene.
 Knockin Analysis
 ES cells can be used to create knockin humanized animals
(pigs) or transgenic animal models (mice or rats) of human diseases.
With knockin technology, a region of a human gene is injected into
animal ES cells, and the human sequence integrates into the animal
cell genome. Transformed cells are injected into blastulae and the
blastulae are implanted as described above. Transgenic progeny or
inbred lines are studied and treated with potential pharmaceutical
agents to obtain information on treatment of the analogous human
condition. These methods have been used to model several human diseases.
 Non-Human Primate Model
 The field of animal testing deals with data and methodology
from basic sciences such as physiology, genetics, chemistry, pharmacology
and statistics. These data are paramount in evaluating the effects
of therapeutic agents on non-human primates as they can be related
to human health. Monkeys are used as human surrogates in vaccine
and drug evaluations, and their responses are relevant to human
exposures under similar conditions. Cynomolgus and Rhesus monkeys
(Macaca fascicularis and Macaca mulatta, respectively) and Common
Marmosets (Callithrix jacchus) are the most common non-human primates
(NHPs) used in these investigations. Since great cost is associated
with developing and maintaining a colony of NHPs, early research
and toxicological studies are usually carried out in rodent models.
In studies using behavioral measures such as drug addiction, NHPs
are the first choice test animal. In addition, NHPs and individual
humans exhibit differential sensitivities to many drugs and toxins
and can be classified as a range of phenotypes from "extensive
metabolizers" to "poor metabolizers" of these agents.
 In additional embodiments, the cDNAs which encode the protein
may be used in any molecular biology techniques that have yet to
be developed, provided the new techniques rely on properties of
cDNAs that are currently known, including, but not limited to, such
properties as the triplet genetic code and specific base pair interactions.
 The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the invention.
The preparation of the human breast tumor-associated library, BRSTDIT01,
will be described.
 I cDNA Library Construction
 The BRSTDIT01 library was constructed from diseased breast
tissue removed from a 48-year-old Caucasian female during a local
excision of breast lesion. Pathology indicated proliferative fibrocystic
changes without atypia characterized by epithelial ductal hyperplasia,
and microcalcifications. Pathology for the matched tumor tissue
indicated intraductal cancer.
 The frozen tissue was homogenized and lysed in guanidinium
isothiocyanate solution using a POLYTRON homogenizer (Brinkmann
Instruments, Westbury N.J.). The lysate was centrifuged over a 5.7
M CsCl cushion using an SW28 rotor in an L8-70M ultracentrifuge
(Beckman Coulter, Fullerton Calif.) for 18 hours at 25,000 rpm at
ambient temperature. The RNA was extracted with acid phenol, pH
4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of
ethanol, resuspended in RNAse-free water, and DNAse treated at 37.degree.
C. Extraction with acid phenol, pH 4.7, and precipitation with sodium
acetate and ethanol was repeated. The mRNA was isolated with the
OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used to construct the
 The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system (Life Technologies) which contains
a NotI primer-adaptor designed to prime the first strand cDNA synthesis
at the poly(A) tail of mRNAs. Double stranded cDNA was blunted,
ligated to EcoRI adaptors and digested with NotI (New England Biolabs,
Beverly Mass.). The cDNAs were fractionated on a SEPHAROSE CL4B
column (APB), and those cDNAs exceeding 400 bp were ligated into
pINCY plasmid (Incyte Genomics). The plasmid pINCY was subsequently
transformed into DH5.alpha. competent cells (Life Technologies).
 II Construction of pINCY Plasmid
 The plasmid was constructed by digesting the pSPORT1 plasmid
(Life Technologies) with EcoRI restriction enzyme (New England Biolabs,
Beverly Mass.) and filling the overhanging ends using Klenow enzyme
(New England Biolabs) and 2'-deoxynucleotide 5'-triphosphates (dNTPs).
The plasmid was self-ligated and transformed into the bacterial
host, E. coli strain JM109.
 An intermediate plasmid, pSPORT 1-.DELTA.RI, which showed
no digestion with EcoRI, was digested with Hind III (New England
Biolabs); and the overhanging ends were filled in with Klenow and
dNTPs. A linker sequence was phosphorylated, ligated onto the 5'
blunt end, digested with EcoRI, and self-ligated. Following transformation
into JM109 host cells, plasmids were isolated and tested for preferential
digestibility with EcoRI, but not with Hind III. A single colony
that met this criteria was designated pINCY plasmid.
 After testing the plasmid for its ability to incorporate
cDNAs from a library prepared using NotI and EcoRI restriction enzymes,
several clones were sequenced; and a single clone containing an
insert of approximately 0.8 kb was selected from which to prepare
a large quantity of the plasmid. After digestion with NotI and EcoRI,
the plasmid was isolated on an agarose gel and purified using a
QIAQUICK column (Qiagen) for use in library construction.
 III Isolation and Sequencing of cDNA Clones
 Plasmid DNA was released from the cells and purified using
either the MINIPREP kit (Edge Biosystems, Gaithersburg Md.) or the
REAL PREP 96 plasmid kit (Qiagen). A kit consists of a 96-well block
with reagents for 960 purifications. The recommended protocol was
employed except for the following changes: 1) the bacteria were
cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, Sparks
Md.) with carbenicillin at 25 mg/l and glycerol at 0.4%; 2) after
inoculation, the cells were cultured for 19 hours and then lysed
with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation,
the plasmid DNA pellet was resuspended in 0.1 ml of distilled water.
After the last step in the protocol, samples were transferred to
a 96-well block for storage at 4C.
 The cDNAs were prepared for sequencing using the MICROLAB
2200 system (Hamilton) in combination with the DNA ENGINE thermal
cyclers (M J Research). The cDNAs were sequenced by the method of
Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM
377 sequencing system (Applied Biosystems) or the MEGABACE 1000
DNA sequencing system (APB). Most of the isolates were sequenced
according to standard ABI protocols and kits (Applied Biosystems)
with solution volumes of 0.25.times.-1.0.times. concentrations.
In the alternative, cDNAs were sequenced using solutions and dyes
 IV Extension of cDNA Sequences
 The cDNAs were extended using the cDNA clone and oligonucleotide
primers. One primer was synthesized to initiate 5' extension of
the known fragment, and the other, to initiate 3' extension of the
known fragment. The initial primers were designed using commercially
available primer analysis software to be about 22 to 30 nucleotides
in length, to have a GC content of about 50% or more, and to anneal
to the target sequence at temperatures of about 68C. to about 72C.
Any stretch of nucleotides that would result in hairpin structures
and primer-primer dimerizations was avoided.
 Selected cDNA libraries were used as templates to extend
the sequence. If more than one extension was necessary, additional
or nested sets of primers were designed. Preferred libraries have
been size-selected to include larger cDNAs and random primed to
contain more sequences with 5' or upstream regions of genes. Genomic
libraries are used to obtain regulatory elements, especially extension
into the 5' promoter binding region.
 High fidelity amplification was obtained by PCR using methods
such as that taught in U.S. Pat. No. 5,932,451. PCR was performed
in 96-well plates using the DNA ENGINE thermal cycler (M J Research).
The reaction mix contained DNA template, 200 nmol of each primer,
reaction buffer containing Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4,
and .beta.-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters for primer pair PCI A and PCI B (Incyte Genomics):
Step 1: 94C., three min; Step 2: 94C., 15 sec; Step 3: 60C., one
min; Step 4: 68C., two min; Step 5: Steps 2, 3, and 4 repeated 20
times; Step 6: 68C., five min; Step 7: storage at 4C. In the alternative,
the parameters for primer pair T7 and SK+(Stratagene) were as follows:
Step 1: 94C., three min; Step 2: 94C., 15 sec; Step 3: 57C., one
min; Step 4: 68C., two min; Step 5: Steps 2, 3, and 4 repeated 20
times; Step 6: 68C., five min; Step 7: storage at 4C.
 The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% reagent
in 1.times. TE, v/v; Molecular Probes) and 0.5 .mu.l of undiluted
PCR product into each well of an opaque fluorimeter plate (Corning,
Acton Mass.) and allowing the DNA to bind to the reagent. The plate
was scanned in a Fluoroskan II (Labsystems Oy) to measure the fluorescence
of the sample and to quantify the concentration of DNA. A 5 .mu.l
to 10 .mu.l aliquot of the reaction mixture was analyzed by electrophoresis
on a 1% agarose minigel to determine which reactions were successful
in extending the sequence.
 The extended clones were desalted, concentrated, transferred
to 384-well plates, digested with CviJI cholera virus endonuclease
(Molecular Biology Research, Madison Wis.), and sonicated or sheared
prior to religation into pUC18 vector (APB). For shotgun sequences,
the digested nucleotide sequences were separated on low concentration
(0.6 to 0.8%) agarose gels, fragments were excised, and the agar
was digested with AGARACE enzyme (Promega). Extended clones were
religated using T4 DNA ligase (New England Biolabs) into pUC18 vector
(APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction
site overhangs, and transfected into E. coli competent cells. Transformed
cells were selected on antibiotic-containing media, and individual
colonies were picked and cultured overnight at 37C. in 384-well
plates in LB/2.times. carbenicillin liquid media.
 The cells were lysed, and DNA was amplified using primers,
Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with
the following parameters: Step 1: 94C., three min; Step 2: 94C.,
15 sec; Step 3: 60C., one min; Step 4: 72C., two min; Step 5: steps
2, 3, and 4 repeated 29 times; Step 6: 72C., five min; Step 7: storage
at 4C. DNA was quantified using PICOGREEN quantitation reagent (Molecular
Probes) as described above. Samples with low DNA recoveries were
reamplified using the conditions described above. Samples were diluted
with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using
DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT
cycle sequencing kit (APB) or the ABI PRISM BIGDYE terminator cycle
sequencing kit (Applied Biosystems).
 V Homology Searching of cDNA Clones and Their Deduced Proteins
 The cDNAs of the Sequence Listing or their deduced amino
acid sequences were used to query databases such as GenBank, SwissProt,
BLOCKS, and the like. These databases that contain previously identified
and annotated sequences or domains were searched using BLAST or
BLAST2 to produce alignments and to determine which sequences were
exact matches or homologs. The alignments were to sequences of prokaryotic
(bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively,
algorithms such as the one described in Smith and Smith (1992, Protein
Engineering 5:35-51) could have been used to deal with primary sequence
patterns and secondary structure gap penalties. All of the sequences
disclosed in this application have lengths of at least 49 nucleotides,
and no more than 12% uncalled bases (where N is recorded rather
than A, C, G, or T).
 As detailed in Karlin and Altschul (1993; Proc Natl Acad
Sci 90:5873-5877), BLAST matches between a query sequence and a
database sequence were evaluated statistically and only reported
when they satisfied the threshold of 10.sup.-25 for nucleotides
and 10.sup.-14 for peptides. Homology was also evaluated by product
score calculated as follows: the % nucleotide or amino acid identity
[between the query and reference sequences] in BLAST is multiplied
by the % maximum possible BLAST score [based on the lengths of query
and reference sequences] and then divided by 100. In comparison
with hybridization procedures used in the laboratory, the stringency
for an exact match was set from a lower limit of about 40 (with
1-2% error due to uncalled bases) to a 100% match of about 70.
 The BLAST software suite (NCBI, Bethesda Md.; http://www.ncbi.nlm.nih.gov/gorf/bl2.html),
includes various sequence analysis programs including "blastn"
that is used to align nucleotide sequences and BLAST2 that is used
for direct pairwise comparison of either nucleotide or amino acid
sequences. BLAST programs are commonly used with gap and other parameters
set to default settings, e.g.: Matrix: BLOSUM62; Reward for match:
1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties;
Gap.times.drop-off: 50; Expect: 10; Word Size: 11; and Filter: on.
Identity is measured over the entire length of a sequence. Brenner
et al. (1998; Proc Natl Acad Sci 95:6073-6078, incorporated herein
by reference) analyzed BLAST for its ability to identify structural
homologs by sequence identity and found 30% identity is a reliable
threshold for sequence alignments of at least 150 residues and 40%,
for alignments of at least 70 residues.
 The cDNAs of this application were compared with assembled
consensus sequences or templates found in the LIFESEQ GOLD database
(Incyte Genomics). Component sequences from cDNA, extension, full
length, and shotgun sequencing projects were subjected to PHRED
analysis and assigned a quality score. All sequences with an acceptable
quality score were subjected to various pre-processing and editing
pathways to remove low quality 3' ends, vector and linker sequences,
polyA tails, Alu repeats, mitochondrial and ribosomal sequences,
and bacterial contamination sequences. Edited sequences had to be
at least 50 bp in length, and low-information sequences and repetitive
elements such as dinucleotide repeats, Alu repeats, and the like,
were replaced by "Ns" or masked.
 Edited sequences were subjected to assembly procedures in
which the sequences were assigned to gene bins. Each sequence could
only belong to one bin, and sequences in each bin were assembled
to produce a template. Newly sequenced components were added to
existing bins using BLAST and CROSSMATCH. To be added to a bin,
the component sequences had to have a BLAST quality score greater
than or equal to 150 and an alignment of at least 82% local identity.
The sequences in each bin were assembled using PHRAP. Bins with
several overlapping component sequences were assembled using DEEP
PHRAP. The orientation of each template was determined based on
the number and orientation of its component sequences.
 Bins were compared to one another, and those having local
similarity of at least 82% were combined and reassembled. Bins having
templates with less than 95% local identity were split. Templates
were subjected to analysis by STITCHER/EXON MAPPER algorithms that
determine the probabilities of the presence of splice variants,
alternatively spliced exons, splice junctions, differential expression
of alternative spliced genes across tissue types or disease states,
and the like. Assembly procedures were repeated periodically, and
templates were annotated using BLAST against GenBank databases such
as GBpri. An exact match was defined as having from 95% local identity
over 200 base pairs through 100% local identity over 100 base pairs
and a homolog match as having an E-value (or probability score)
of .ltoreq.1.times.10.sup.-8. The templates were also subjected
to frameshift FAST.times. against GENPEPT, and homolog match was
defined as having an E-value of .ltoreq.1.times.10.sup.-8. Template
analysis and assembly was described in U.S. Ser. No. 09/276,534,
filed Mar. 25, 1999.
 Following assembly, templates were subjected to BLAST, motif,
and other functional analyses and categorized in protein hierarchies
using methods described in U.S. Ser. No. 08/812,290 and U.S. Ser.
No. 08/811,758, both filed Mar. 6, 1997; in U.S. Ser. No. 08/947,845,
filed October 9, 1997; and in U.S. Ser. No. 09/034,807, filed Mar.
4, 1998. Then templates were analyzed by translating each template
in all three forward reading frames and searching each translation
against the PFAM database of hidden Markov model-based protein families
and domains using the HMMER software package (Washington University
School of Medicine, St. Louis Mo.; http://pfam.wustl.edu/). The
cDNA was further analyzed using MACDNASIS PRO software (Hitachi
Software Engineering), and LASERGENE software (DNASTAR) and queried
against public databases such as the GenBank rodent, mammalian,
vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS,
PRINTS, PFAM, and Prosite.
 VI Transcript Imaging
 A transcript image was performed using the LIFESEQ GOLD
database (January 2002 release, Incyte Genomics). This process allowed
assessment of the relative abundance of the expressed polynucleotides
in all of the cDNA libraries and was described in U.S. Pat. No.
5,840,484 incorporated herein by reference. All sequences and cDNA
libraries in the LIFESEQ database were categorized by system, organ/tissue
and cell type. The categories included cardiovascular system, connective
tissue, digestive system, embryonic structures, endocrine system,
exocrine glands, female and male genitalia, germ cells, hemic/immune
system, liver, musculoskeletal system, nervous system, pancreas,
respiratory system, sense organs, skin, stomatognathic system, unclassified/mixed,
and the urinary tract. Criteria for transcript imaging can be selected
from category, number of cDNAs per library, library description,
disease indication, clinical relevance of sample, and the like.
 All sequences and cDNA libraries in the LIFESEQ database
have been categorized by system, organ/tissue and cell type. For
each category, the number of libraries in which the sequence was
expressed were counted and shown over the total number of libraries
in that category. For each library, the number of cDNAs were counted
and shown over the total number of cDNAs in that library. In some
transcript images, all normalized or subtracted libraries, which
have high copy number sequences removed prior to processing, and
all mixed or pooled tissues, which are considered non-specific in
that they contain more than one tissue type or more than one subject's
tissue, can be excluded from the analysis. Treated and untreated
cell lines and/or fetal tissue data can also be excluded where clinical
relevance is emphasized. Conversely, fetal tissue can be emphasized
wherever elucidation of inherited disorders or differentiation of
particular adult or embryonic stem cells into tissues or organs
such as heart, kidney, nerves or pancreas would be aided by removing
clinical samples from the analysis. Transcript imaging can also
be used to support data from other methodologies such as guilt-by-association
and hybridization analyses. The results of this analysis are presented
in Table 1.
 VII Chromosome Mapping
 Radiation hybrid and genetic mapping data available from
public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for Genome Research (WIGR), and Gnthon are used
to determine if any of the cDNAs presented in the Sequence Listing
have been mapped. Any of the fragments of the cDNA encoding PDEBC
that have been mapped result in the assignment of all related regulatory
and coding sequences to the same location. The genetic map locations
are described as ranges, or intervals, of human chromosomes. The
map position of an interval, in cM (which is roughly equivalent
to 1 megabase of human DNA), is measured relative to the terminus
of the chromosomal p-arm.
 VIII Hybridization Technologies and Analyses
 Immobilization of cDNAs on a Substrate
 The cDNAs are applied to a substrate by one of the following
methods. A mixture of cDNAs is fractionated by gel electrophoresis
and transferred to a nylon membrane by capillary transfer. Alternatively,
the cDNAs are individually ligated to a vector and inserted into
bacterial host cells to form a library. The cDNAs are then arranged
on a substrate by one of the following methods. In the first method,
bacterial cells containing individual clones are robotically picked
and arranged on a nylon membrane. The membrane is placed on LB agar
containing selective agent (carbenicillin, kanamycin, ampicillin,
or chloramphenicol depending on the vector used) and incubated at
37C. for 16 hr. The membrane is removed from the agar and consecutively
placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl,
0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0),
and twice in 2.times.SSC for 10 min each. The membrane is then UV
irradiated in a STRATALINKER UV-crosslinker (Stratagene).
 In the second method, cDNAs are amplified from bacterial
vectors by thirty cycles of PCR using primers complementary to vector
sequences flanking the insert. PCR amplification increases a starting
concentration of 1-2 ng nucleic acid to a final quantity greater
than 5 .mu.g. Amplified nucleic acids from about 400 bp to about
5000 bp in length are purified using SEPHACRYL-400 beads (APB).
Purified nucleic acids are arranged on a nylon membrane manually
or using a dot/slot blotting manifold and suction device and are
immobilized by denaturation, neutralization, and UV irradiation
as described above. Purified nucleic acids are robotically arranged
and immobilized on polymer-coated glass slides using the procedure
described in U.S. Pat. No. 5,807,522. Polymer-coated slides are
prepared by cleaning glass microscope slides (Corning, Acton Mass.)
by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric
acid (VWR Scientific Products, West Chester Pa.), coating with 0.05%
aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in
a 110C. oven. The slides are washed extensively with distilled water
between and after treatments. The nucleic acids are arranged on
the slide and then immobilized by exposing the array to UV irradiation
using a STRATALINKER UV-crosslinker (Stratagene). Arrays are then
washed at room temperature in 0.2% SDS and rinsed three times in
distilled water. Non-specific binding sites are blocked by incubation
of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix,
Bedford Mass.) for 30 min at 60C.; then the arrays are washed in
0.2% SDS and rinsed in distilled water as before.
 Probe Preparation for Membrane Hybridization
 Hybridization probes derived from the cDNAs of the Sequence
Listing are employed for screening cDNAs, mRNAs, or genomic DNA
in membrane-based hybridizations. Probes are prepared by diluting
the cDNAs to a concentration of 40-50 ng in 45 .mu.l TE buffer,
denaturing by heating to 100C. for five min, and briefly centrifuging.
The denatured cDNA is then added to a REDIPRIME tube (APB), gently
mixed until blue color is evenly distributed, and briefly centrifuged.
Five .mu.l of [.sup.32P]dCTP is added to the tube, and the contents
are incubated at 37C. for 10 min. The labeling reaction is stopped
by adding 5 .mu.l of 0.2M EDTA, and probe is purified from unincorporated
nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified
probe is heated to 100C. for five min, snap cooled for two min on
ice, and used in membrane-based hybridizations as described below.
 Probe Preparation for Polymer Coated Slide Hybridization
 So Hybridization probes derived from mRNA isolated from
samples are employed for screening cDNAs of the Sequence Listing
in array-based hybridizations. Probe is prepared using the GEMbright
kit (Incyte Genomics) by diluting mRNA to a concentration of 200
ng in 9 .mu.l TE buffer and adding 5 .mu.l 5.times. buffer, 1 .mu.l
0.1 M DTT, 3 .mu.l Cy3 or Cy5 labeling mix, 1 .mu.l RNase inhibitor,
1 .mu.l reverse transcriptase, and 5 .mu.l 1.times. yeast control
mRNAs. Yeast control mRNAs are synthesized by in vitro transcription
from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative
controls, one set of control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng,
and 2 ng are diluted into reverse transcription reaction mixture
at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample
mRNA respectively. To examine mRNA differential expression patterns,
a second set of control mRNAs are diluted into reverse transcription
reaction mixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1
(w/w). The reaction mixture is mixed and incubated at 37C. for two
hr. The reaction mixture is then incubated for 20 min at 85C., and
probes are purified using two successive CHROMA SPIN+TE 30 columns
(Clontech, Palo Alto Calif.). Purified probe is ethanol precipitated
by diluting probe to 90 .mu.l in DEPC-treated water, adding 2 .mu.l
mg/ml glycogen, 60 .mu.l 5 M sodium acetate, and 300 .mu.l 100%
ethanol. The probe is centrifuged for 20 min at 20,800.times.g,
and the pellet is resuspended in 12 .mu.l resuspension buffer, heated
to 65C. for five min, and mixed thoroughly. The probe is heated
and mixed as before and then stored on ice. Probe is used in high
density array-based hybridizations as described below.
 Membrane-based Hybridization
 Membranes are pre-hybridized in hybridization solution containing
1% Sarkosyl and 1.times. high phosphate buffer (0.5 M NaCl, 0.1
M Na.sub.2HPO.sub.4, 5 mM EDTA, pH 7) at 55C. for two hr. The probe,
diluted in 15 ml fresh hybridization solution, is then added to
the membrane. The membrane is hybridized with the probe at 55C.
for 16 hr. Following hybridization, the membrane is washed for 15
min at 25C. in 1 mM Tris (pH 8.0), 1% Sarkosyl, and four times for
15 min each at 25C. in 1 mM Tris (pH 8.0). To detect hybridization
complexes, XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed
to the membrane overnight at -70C., developed, and examined visually.
 Polymer Coated Slide-based Hybridization
 Probe is heated to 65C. for five min, centrifuged five min
at 9400 rpm in a 5415C. microcentrifuge (Eppendorf Scientific, Westbury
N.Y.), and then 18 .mu.l is aliquoted onto the array surface and
covered with a coverslip. The arrays are transferred to a waterproof
chamber having a cavity just slightly larger than a microscope slide.
The chamber is kept at 100% humidity internally by the addition
of 140 .mu.l of 5.times.SSC in a corner of the chamber. The chamber
containing the arrays is incubated for about 6.5 hr at 60C. The
arrays are washed for 10 min at 45C. in 1.times.SSC, 0.1% SDS, and
three times for 10 min each at 45C. in 0.1.times.SSC, and dried.
 Hybridization reactions are performed in absolute or differential
hybridization formats. In the absolute hybridization format, probe
from one sample is hybridized to array elements, and signals are
detected after hybridization complexes form. Signal strength correlates
with probe mRNA levels in the sample. In the differential hybridization
format, differential expression of a set of genes in two biological
samples is analyzed. Probes from the two samples are prepared and
labeled with different labeling moieties. A mixture of the two labeled
probes is hybridized to the array elements, and signals are examined
under conditions in which the emissions from the two different labels
are individually detectable. Elements on the array that are hybridized
to equal numbers of probes derived from both biological samples
give a distinct combined fluorescence (Shalon WO95/35505).
 Hybridization complexes are detected with a microscope equipped
with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.)
capable of generating spectral lines at 488 nm for excitation of
Cy3 and at 632 nm for excitation of Cy5. The excitation laser light
is focused on the array using a 20.times. microscope objective (Nikon,
Melville N.Y.). The slide containing the array is placed on a computer-controled
X-Y stage on the microscope and raster-scanned past the objective
with a resolution of 20 micrometers. In the differential hybridization
format, the two fluorophores are sequentially excited by the laser.
Emitted light is split, based on wavelength, into two photomultiplier
tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater
N.J.) corresponding to the two fluorophores. Filters positioned
between the array and the photomultiplier tubes are used to separate
the signals. The emission maxima of the fluorophores used are 565
nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated
using the signal intensity generated by the yeast control mRNAs
added to the probe mix. A specific location on the array contains
a complementary DNA sequence, allowing the intensity of the signal
at that location to be correlated with a weight ratio of hybridizing
species of 1:100,000.
 The output of the photomultiplier tube is digitized using
a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog
Devices, Norwood Mass.) installed in an IBM-compatible PC computer.
The digitized data are displayed as an image where the signal intensity
is mapped using a linear 20-color transformation to a pseudocolor
scale ranging from blue (low signal) to red (high signal). The data
is also analyzed quantitatively. Where two different fluorophores
are excited and measured simultaneously, the data are first corrected
for optical crosstalk (due to overlapping emission spectra) between
the fluorophores using the emission spectrum for each fluorophore.
A grid is superimposed over the fluorescence signal image such that
the signal from each spot is centered in each element of the grid.
The fluorescence signal within each element is then integrated to
obtain a numerical value corresponding to the average intensity
of the signal. The software used for signal analysis is the GEMTOOLS
program (Incyte Genomics).
 IX Complementary Molecules
 Molecules complementary to the cDNA, from about 5 (PNA)
to about 5000 bp (complement of a cDNA insert), are used to detect
or inhibit gene expression. Detection is described in Example VII.
To inhibit transcription by preventing promoter binding, the complementary
molecule is designed to bind to the most unique 5' sequence and
includes nucleotides of the 5' UTR upstream of the initiation codon
of the open reading frame. Complementary molecules include genomic
sequences (such as enhancers or introns) and are used in "triple
helix" base pairing to compromise the ability of the double
helix to open sufficiently for the binding of polymerases, transcription
factors, or regulatory molecules. To inhibit translation, a complementary
molecule is designed to prevent ribosomal binding to the mRNA encoding
 Complementary molecules are placed in expression vectors
and used to transform a cell line to test efficacy; into an organ,
tumor, synovial cavity, or the vascular system for transient or
short term therapy; or into a stem cell, zygote, or other reproducing
lineage for long term or stable gene therapy. Transient expression
lasts for a month or more with a non-replicating vector and for
three months or more if elements for inducing vector replication
are used in the transformation/expression system.
 Stable transformation of dividing cells with a vector encoding
the complementary molecule produces a transgenic cell line, tissue,
or organism (U.S. Pat. No. 4,736,866). Those cells that assimilate
and replicate sufficient quantities of the vector to allow stable
integration also produce enough complementary molecules to compromise
or entirely eliminate activity of the cDNA encoding the protein.
 X Expression of PDEBC
 Expression and purification of the protein are achieved
using either a mammalian cell expression system or an insect cell
expression system. The pUB6/V5-His vector system (Invitrogen, Carlsbad
Calif.) is used to express PDEBC in CHO cells. The vector contains
the selectable bsd gene, multiple cloning sites, the promoter/enhancer
sequence from the human ubiquitin C gene, a C-terminal V5 epitope
for antibody detection with anti-V5 antibodies, and a C-terminal
polyhistidine (6.times.His) sequence for rapid purification on PROBOND
resin (Invitrogen). Transformed cells are selected on media containing
 Spodoptera frugiperda (Sf9) insect cells are infected with
recombinant Autographica californica nuclear polyhedrosis virus
(baculovirus). The polyhedrin gene is replaced with the cDNA by
homologous recombination and the polyhedrin promoter drives cDNA
transcription. The protein is synthesized as a fusion protein with
6.times.his which enables purification as described above. Purified
protein is used in the following activity and to make antibodies
 XI Production of Specific Antibodies
 Purification using polyacrylamide gel electrophoresis or
similar techniques is used to isolate protein for immunization of
hosts or host cells to produce antibodies using standard protocols.
 Alternatively, the amino acid sequence of the protein is
analyzed using readily available commercial software to determine
regions of high immunogenicity. A peptide with high immunogenicity
is cleaved, recombinantly-produced, or synthesized and used to raise
antibodies by means known to those of skill in the art. Methods
for selection of appropriate antigenic determinants such as those
near the C-terminus or in hydrophilic regions are well described
in the art (Ausubel, supra, Chap. 11).
 Oligopeptides of about 15 residues in length are synthesized
using an ABI 431A peptide synthesizer (ABI) using FMOC chemistry
and coupled to carriers such as BSA, thyroglobulin, or KLH (Sigma-Aldrich)
by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to
increase immunogenicity. The coupled peptide is then used to immunize
the host. Rabbits are immunized with the oligopeptide-KLH complex
in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity by binding the peptide to a substrate, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG.
 XII Immunopurification Using Antibodies
 Naturally occurring or recombinantly produced protein is
purified by immunoaffinity chromatography using antibodies which
specifically bind the protein. An immunoaffinity column is constructed
by covalently coupling the antibody to CNBr-activated SEPHAROSE
resin (APB). Media containing the protein is passed over the immunoaffinity
column, and the column is washed using high ionic strength buffers
in the presence of detergent to allow preferential absorbance of
the protein. After coupling, the protein is eluted from the column
using a buffer of pH 2-3 or a high concentration of urea or thiocyanate
ion to disrupt antibody/protein binding, and the purified protein
 XIII Antibody Arrays
 Protein:protein Interactions
 In an alternative to yeast two hybrid system analysis of
proteins, an antibody array can be used to study protein-protein
interactions and phosphorylation. A variety of protein ligands are
immobilized on a membrane using methods well known in the art. The
array is incubated in the presence of cell lysate until protein:antibody
complexes are formed. Proteins of interest are identified by exposing
the membrane to an antibody specific to the protein of interest.
In the alternative, a protein of interest is labeled with digoxigenin
(DIG) and exposed to the membrane; then the membrane is exposed
to anti-DIG antibody which reveals where the protein of interest
forms a complex. The identity of the proteins with which the protein
of interest interacts is determined by the position of the protein
of interest on the membrane.
 Proteomic Profiles
 Antibody arrays can also be used for high-throughput screening
of recombinant antibodies. Bacteria containing antibody genes are
robotically-picked and gridded at high density (up to 18,342 different
double-spotted clones) on a filter. Up to 15 antigens at a time
are used to screen for clones to identify those that express binding
antibody fragments. These antibody arrays can also be used to identify
proteins which are differentially expressed in samples (de Wildt,
 XIV Screening Molecules for Specific Binding with the cDNA
 The cDNA, or fragments thereof, or the protein, or portions
thereof, are labeled with .sup.32P-dCTP, Cy3-dCTP, or Cy5-dCTP (APB),
or with BIODIPY or FITC (Molecular Probes, Eugene Oreg.), respectively.
Libraries of candidate molecules or compounds previously arranged
on a substrate are incubated in the presence of labeled cDNA or
protein. After incubation under conditions for either a nucleic
acid or amino acid sequence, the substrate is washed, and any position
on the substrate retaining label, which indicates specific binding
or complex formation, is assayed, and the ligand is identified.
Data obtained using different concentrations of the nucleic acid
or protein are used to calculate affinity between the labeled nucleic
acid or protein and the bound molecule.
 XV Two-Hybrid Screen
 A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system
(Clontech Laboratories, Palo Alto Calif.), is used to screen for
peptides that bind the protein of the invention. A cDNA encoding
the protein is inserted into the multiple cloning site of a pLexA
vector, ligated, and transformed into E. coli. cDNA, prepared from
mRNA, is inserted into the multiple cloning site of a pB42AD vector,
ligated, and transformed into E. coli to construct a cDNA library.
The pLexA plasmid and pB42AD-cDNA library constructs are isolated
from E. coli and used in a 2:1 ratio to co-transform competent yeast
EGY48[p8op-lacZ] cells using a polyethylene glycol/lithium acetate
protocol. Transformed yeast cells are plated on synthetic dropout
(SD) media lacking histidine (-His), tryptophan (-Trp), and uracil
(-Ura), and incubated at 30C. until the colonies have grown up and
are counted. The colonies are pooled in a minimal volume of 1.times.TE
(pH 7.5), replated on SD/-His/-Leu/-Trp/-Ura media supplemented
with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl
.beta.-d-galactopyranoside (X-Gal), and subsequently examined for
growth of blue colonies. Interaction between expressed protein and
cDNA fusion proteins activates expression of a LEU2 reporter gene
in EGY48 and produces colony growth on media lacking leucine (-Leu).
Interaction also activates expression of .beta.-galactosidase from
the p8op-lacZ reporter construct that produces blue color in colonies
grown on X-Gal.
 Positive interactions between expressed protein and cDNA
fusion proteins are verified by isolating individual positive colonies
and growing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at
30C. A sample of the culture is plated on SD/-Trp/-Ura media and
incubated at 30C. until colonies appear. The sample is replica-plated
on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow
on SD containing histidine but not on media lacking histidine have
lost the pLexA plasmid. Histidine-requiring colonies are grown on
SD/Gal/Raf/X-Gal/-Trp/-Ura, and white colonies are isolated and
propagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding
a protein that physically interacts with the protein, is isolated
from the yeast cells and characterized.
 XVI Demonstration of Protein Activity
 Cell Proliferation
 PDEBC can be expressed in a mammalian cell line such as
DLD-1 or HCT116 (ATCC; Manassas Va.) by transforming the cells with
a eukaryotic expression vector encoding PDEBC. Other eukaryotic
expression vectors, such as those mentioned in EXAMPLE X above,
are commercially available, and the techniques to introduce them
into cells are well known to those skilled in the art. The effect
of PDEBC on cell morphology can be visualized by microscopy; the
effect on cell growth can be determined by measuring cell doubling-time;
and the effect on tumorigenicity can be assessed by the ability
of transformed cells to grow in a soft agar growth assay (Groden
et al. (1995) Cancer Res. 55:1531-1539).
 All patents and publications mentioned in the specification
are incorporated by reference herein. 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 described modes for carrying out the invention that 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.
2TABLE 1 Library cDNAs Description of Breast Tissue Abundance %
Abundance BRSTTUP03 828 breast tumor, ductal, poorly differentiated,
1 0.1208 F, 3' CGAP BRSTDIT01 3394 breast, PF changes, mw/intraductal
1 0.0295 cancer, 48F BRSTNOT13 3859 breast, mw/neoplasm, 36F 1 0.0259
BRSTNOT27 3936 breast, mw/ductal adenoCA, intraductal CA, 1 0.0254
aw/node mets, 57F BRSTNOT18 3999 breast, PF breast disease, 57F
1 0.0250 BRSTNOT05 13198 breast, mw/lobular CA, 58F, m/BRSTTUT03
3 0.0227 BRSTNOT02 9074 breast, PF changes, mw/adenoCA, 1 0.0110
55F, m/BRSTTUT01 BRSTNOT07 10052 breast, PF changes, mw/adenoCA,
1 0.0099 intraductal CA, 43F BRSTNOT04 10308 breast, mw/ductal CA,
CA in situ, 1 0.0097 aw/node mets, 62F
3 TABLE 2 The BRSTTUP03 library was obtained from the Cancer Genome
Anatomy Project (CGAP) (PD Name: NCI_CGAP_Br3). Starting RNA was
made from poorly differentiated invasive ductal breast tumor tissue
removed from an adult female. The BRSTDIT01 library was constructed
from diseased breast tissue removed from a 48-year-old Caucasian
female during a local excision of breast lesion. Pathology indicated
proliferative fibrocystic changes without atypia characterized by
epithelial ductal hyperplasia, and microcalcifications. Pathology
for the matched tumor tissue indicated intraductal cancer. The patient
presented with a malignant neoplasm of the breast and unspecified
breast symptoms. The BRSTNOT13 library was constructed from breast
tissue removed from the left medial lateral breast of a 36-year-old
Caucasian female during bilateral simple mastectomy and total breast
reconstruction. Pathology indicated benign breast tissue. Patient
history included a breast neoplasm. The BRSTNOT27 library was constructed
from right breast tissue removed from a 57-year-old Caucasian female
during a unilateral extended simple mastectomy. Pathology indicated
benign fat replaced breast parenchyma. Pathology for the matched
tumor tissue indicated residual microscopic infiltrating grade 3
ductal adenocarcinoma and extensive grade 2 intraductal carcinoma.
Multiple (9 of 19) axillary lymph nodes were positive for metastatic
adenocarcinoma with minimal extranodal extension. The largest nodal
metastasis measured less than 1 cm in greatest dimension. Immunoperoxidase
stains for estrogen and progesterone receptors were positive. Patient
history included benign hypertension, hyperlipidemia, cardiac dysrhythmia,
a benign colon neoplasm, a solitary breast cyst, and a breast neoplasm
of uncertain behavior. The BRSTNOT18 library was constructed from
diseased breast tissue removed from a 57-year-old Caucasian female
during a unilateral simple extended mastectomy. Pathology indicated
a biopsy cavity in the upper outer quadrant of the right breast.
No residual tumor was seen. The non-neoplastic breast showed mildly
proliferative breast disease. In addition, there were multiple inflammatory
axillary lymph nodes identified. Patient history included breast
cancer. The BRSTNOT05 library was constructed from breast tissue
removed from a 58-year-old Caucasian female during a unilateral
extended simple mastectomy. Pathology indicated all surgical margins,
including the skin, nipple, and fascia were negative for tumor.
Pathology for the matched tumor tissue indicated multicentric invasive
grade 4 lobular carcinoma. The mass was identified in the upper
outer quadrant of the left breast. Three separate nodules were also
found in the lower outer quadrant of the left breast. No evidence
of vascular invasion was found. All axiliary lymph nodes were negative
for tumor. Patient history included skin cancer, rheumatic heart
disease, osteoarthritis, and tuberculosis. The BRSTNOT02 library
was constructed from diseased breast tissue removed from a 55-year-old
Caucasian female during a unilateral extended simple mastectomy.
Pathology indicated proliferative fibrocystic changes characterized
by apocrine metaplasia, sclerosing adenosis, cyst formation, and
ductal hyperplasia without atypia. Pathology for the matched tumor
tissue indicated an invasive grade 4 mammary adenocarcinoma of mixed
lobular and ductal type, extensively involving all four quadrants
of the left breast. The tumor was identified in the deep dermis
near the lactiferous ducts with extracapsular extension. Surgical
margins were negative. Seven mid and low and five high axillary
lymph nodes were positive fo tumor. Patient history included atrial
tachycardia, blood in the stool, and a benign breast neoplasm. The
BRSTNOT07 library was constructed from diseased breast tissue removed
from a 43-year-old Caucasian female during a unilateral extended
simple mastectomy. Pathology indicated mildly proliferative fibrocystic
changes with epithelial hyperplasia, papillomatosis, and duct ectasia.
Pathology for the matched tumor tissue indicated invasive grade
4, nuclear grade 3 mammary adenocarcinoma with extensive comedo
necrosis. Approximately 50 percent of the tumor was intraductal
(comedo carcinoma). A microscopic focus of residual intraductal
carcinoma was identified at the biopsy site in the lower inner quadrant
of the right breast. The overlying skin, nipple, deep fascia, and
axillary lymph nodes were negative for tumor. The BRSTNOT04 library
was constructed from breast tissue removed from a 62-year-old East
Indian female during a unilateral extended simple mastectomy. Pathology
indicated the surgical margins were negative for tumor. Pathology
for the matched tumor tissue indicated an invasive grade 3 ductal
carcinoma. A 0.4 cm focus of carcinoma in situ was identified in
the lower outer quadrant of the breast. Multiple mid and low axillary
lymph nodes contained micrometastasis, and estrogen/progesterone
receptors were positive. Patient history included benign hypertension,
hyperlipidemia, and hematuria.