The present invention relates to a combination comprising a plurality
of polynucleotide probes that are modulated in response to EGF and
which are associated with breast cancer, and which may be used in
their entirety or in part as to diagnose, to stage, to treat, or
to monitor the treatment of a subject with a breast cancer.
What is claimed is:
1. A combination comprising a plurality of cDNAs, selected from
SEQ ID NOs:1-16 or their complements, whose expression is modulated
by EGF and is associated with breast cancer.
2. The combination of claim 1, wherein the cDNAs are immobilized
on a substrate.
3. A high throughput method for detecting differential expression
of one or more cDNAs in a sample containing nucleic acids, the method
comprising: (a) hybridizing the substrate of claim 2 with nucleic
acids of the sample, thereby forming one or more hybridization complexes;
(b) detecting the hybridization complexes; and (c) comparing the
hybridization complexes with those of a standard, wherein differences
between the standard and sample hybridization complexes indicate
differential expression of cDNAs in the sample.
4. The method of claim 3, where in the nucleic acids of the sample
are amplified prior to hybridization.
5. The method of claim 3, wherein the sample is from a subject
with a breast carcinoma and comparison with a standard defines an
early, mid, or late stage of that disease.
6. A high throughput method of screening a plurality of molecules
or compounds to identify a ligand which specifically binds a cDNA,
the method comprising: (a) combining the combination of claim 1
with the plurality of molecules or compounds under conditions to
allow specific binding; and (b) detecting specific binding between
each cDNA and at least one molecule or compound, thereby identifying
a ligand that specifically binds to each cDNA.
7. The method of claim 6 wherein the plurality of molecules or
compounds are selected from DNA molecules, RNA molecules, peptide
nucleic acid molecules, mimetics, peptides, transcription factors,
repressors, and regulatory proteins.
8. An isolated cDNA, or the complement thereof, selected from SEQ
9. A composition comprising a cDNA of claim 8 in conjunction with
a suitable pharmaceutical carrier.
10. A method of using a cDNA to purify a ligand that specifically
binds the cDNA, the method comprising; a) combining a cDNA of claim
8 with a sample under conditions which allow specific binding; b)
recovering the cDNA bound to the ligand; and c) separating the cDNA
from the ligand thereby obtaining purified ligand.
11. A vector containing a probe selected from the cDNA of claim
12. A host cell containing the vector of claim 11.
13. A method for producing a protein, the method comprising the
steps of: (a) culturing the host cell of claim 12 under conditions
for expression of protein; and (b) recovering the protein from the
host cell culture.
14. A protein or a portion thereof produced by the method of claim
15. A high-throughput method for using a protein to screen a plurality
of molecules or compounds to identify at least one ligand which
specifically binds the protein, the method comprising: (a) combining
the protein of claim 14 with the plurality of molecules or compounds
under conditions to allow specific binding; and (b) detecting specific
binding between the protein and a molecule or compound, thereby
identifying a ligand which specifically binds the protein.
16. The method of claim 15 wherein the plurality of molecules or
compounds is selected from DNA molecules, RNA molecules, peptide
nucleic acid molecules, mimetics, peptides, proteins, agonists,
antagonists, antibodies or their fragments, immunoglobulins, inhibitors,
drug compounds, and pharmaceutical agents.
17. A method of using a protein to produce an antibody, the method
comprising: a) immunizing an animal with the protein of claim 14
under conditions to elicit an antibody response; b) isolating animal
antibodies; and c) screening the isolated antibodies with the protein,
thereby identifying an antibody which specifically binds the protein.
18. A method of purifying an antibody, the method comprising: a)
combining the protein of claim 14 with a sample under conditions
to allow specific binding; b) recovering the bound protein; and
c) separating the protein from the antibody, thereby obtaining purified
 This application is a continuation application of U.S. application
Ser. No. 09/653,119, filed on Aug. 31, 2000, which claims the benefit
of U.S. Provisional Application No. 60/152,548, our Docket No. PA-0018
P, filed on Sep. 3, 1999.
FIELD OF THE INVENTION
 The present invention relates to a composition comprising
a plurality of polynucleotide probes which may be used in detecting
expression of genes modulated in response to EGF, and which are
associated with breast cancer.
BACKGROUND OF THE INVENTION
 Intercellular communication is essential for the development
and survival of multicellular organisms. Communication is achieved
through the secretion of proteins by signaling cells and the internalization
of these proteins by target cells. Growth factors are secreted proteins
that mediate communication between signaling and target cells. The
secreted growth factors bind to specific receptors on the surfaces
of target cells, and bound receptors trigger second messenger signal
transduction pathways. These signal transduction pathways elicit
specific cellular responses in the target cells. Such responses
can include the modulation of gene expression and the stimulation
or inhibition of cell division, cell differentiation, and cell motility.
 Epidermal growth factor (EGF) is a member of a broad class
of polypeptide growth factors that generally act as mitogens in
diverse cell types to stimulate wound healing, bone synthesis and
remodeling, extracellular matrix synthesis, and proliferation of
epithelial, epidermal, and connective tissues. In addition, EGF
produces non-mitogenic effects in certain tissues. The EGF receptor
(EGFR), and its stimulation by EGF, has been linked with a number
of cell proliferative disorders or malignancies. These include skin
hyperplasia, erythroblastosis, and fibrosarcoma in animals; and
in humans, benign hyperplasia of the skin, mammary carcinoma, glioblastoma,
and hepatic carcinoma. Other epithelial carcinomas associated with
EGFR activity include prostatic hyperplasia/cancer, renal carcinoma,
bladder cancer, laryngeal cancer, esophageal tumors, stomach cancer,
colon carcinoma, ovarian adenomas, and lung cancer (Khazaie, K.
et al. (1993) Cancer and Metastasis Rev. 12:255-274).
 The relationship of EGFR expression 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 (ER), has been a marker of poor prognosis in breast cancer
patients. In addition, EGFR expression in breast tumor metastases
is frequently elevated relative to the primary tumor, suggesting
that EGFR is involved in tumor progression and metastasis. This
is supported by accumulating evidence that EGF has pleiotropic effects
on cell motility, chemotaxis, secretion and differentiation; cell
functions related to metastatic potential. For example, EGF has
been found to influence the expression and organization of integrins,
a family of receptors known to function in cell attachment to the
extracelluar matrix during metastasis (Nicolson, G. L. (1984) Expl.
Cell Res. 150:3-22; Schirrmacher, V (1985) Adv. Cancer Res. 43:1-73).
EGFR may influence cell-cell adhesion by affecting changes in the
phosphorylation of certain proteins involved in the process, such
as B-catenin, fodrin, spectrin, and tubulin (Khazaie et al., supra).
EGF has also been shown to affect the production and release of
various proteinases involved in cell invasion of the extracelluar
matrix, such as metalloproteinases, aminopeptidases, serine proteases,
cysteine proteases and aspartic proteinases, as well as proteinase
inhibitors such as plasminogen activator inhibitor (PAI-1) and tissue
inhibitors of metalloproteases (TIMP).
 In addition to the various proteins indicated above that
are affected by EGF activity, the EGF signal transduction pathway
itself involves the recruitment and activation of a variety of molecules
including phospholipase C, phosphoinositol-3 kinase, MAP kinase,
raf kinase, and a GTPase-activating protein (GAP). The expression
of these and other molecules effected by EGF activity may be useful
for the prediction or monitoring of cell proliferative disorders,
pre-malignant conditions, or the presence and progression of malignant
diseases in which EGF participates.
 Array technology can provide a simple way to explore the
expression of a single polymorphic gene or a large number of related
or unrelated genes. When the expression of a single gene is explored,
arrays are employed to detect the expression of specific gene variants.
For example, a p53 tumor suppressor gene array is used to determine
whether individuals are carrying mutations that predispose them
to cancer. The array has over tens of thousands of DNA probes to
analyze more than 400 distinct mutations of p53.
 DNA-based array technology is especially relevant for the
rapid screening of expression of a large number of genes. There
is a growing awareness that gene expression is affected in a global
fashion. A genetic predisposition, disease or therapeutic treatment
may affect, directly or indirectly, the expression of a large number
of genes. In some cases the interactions may be expected, such as
where the genes are part of the same signaling pathway. In other
cases, the interactions may be totally unexpected, such as when
the genes participate in separate signaling pathways. Therefore,
DNA-based arrays can be used to investigate how genetic predisposition,
disease, or therapeutic treatment affects the expression of a large
number of genes.
 The potential application of gene expression profiling to
breast cancer is particularly relevant to improving diagnosis and
prognosis of this disease. 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, however, lacking in sensitivity
and specificity, and efforts are underway in other laboratories
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 (Gish, supra).
 It would be advantageous to prepare DNA-based arrays that
can be used for monitoring expression of a large number of genes
associated with cell proliferative disorders and with pre-malignant
and malignant conditions. The present invention provides for a composition
comprising a plurality of polynucleotide probes for use in detecting
changes in expression of a large number of genes encoding proteins
associated with EGFR expression and activity. Such a microarray
can be employed for diagnosis and monitoring of the treatment of
any disease or precondition where EGFR activation is involved, in
particular, breast cancer.
 The present invention provides a combination comprising
a plurality of cDNAs, wherein each of the cDNAs comprises at least
a fragment of a polynucleotide sequence or a complement thereof
whose expression is modulated by EGF and is associated with breast
cancer and which are selected from SEQ ID NOs:1-16 as presented
in the Sequence Listing. In one aspect, the combination is immobilized
on a substrate.
 The invention also provides a high throughput method to
detect differential expression of one or more of the cDNAs of the
combination. The method comprises hybridizing the substrate comprising
the combination with the nucleic acids of a sample, thereby forming
one or more hybridization complexes, detecting the hybridization
complexes, and comparing the hybridization complexes with those
of a standard, wherein differences in the size and signal intensity
of each hybridization complex indicates differential expression
of nucleic acids in the sample.
 The invention further provides a high throughput method
of screening a library or a plurality of molecules or compounds
to identify a ligand. The method comprises combining the substrate
comprising the combination with a library or a plurality of molecules
or compounds under conditions to allow specific binding and detecting
specific binding, thereby identifying a ligand. The library or a
plurality of molecules or compounds are selected from DNA molecules,
RNA molecules, peptide nucleic acid molecules, mimetics, peptides,
transcription factors, repressors, and other regulatory proteins.
 The invention still further provides an isolated cDNA selected
from SEQ ID NOs:12-16 as presented in the Sequence Listing. The
invention still further provides a pharmaceutical composition comprising
the cDNA and a suitable pharmaceutical carrier. The invention also
provides a vector comprising the cDNA, a host cell comprising the
vector, and a method for producing a protein comprising culturing
the host cell under conditions for the expression of a protein and
recovering the protein from the host cell culture. The invention
additionally provides a method for purifying a ligand, the method
comprising combining a cDNA of the invention with a sample under
conditions which allow specific binding, recovering the bound cDNA,
and separating the cDNA from the ligand, thereby obtaining purified
 The present invention provides a purified protein encoded
and produced by a cDNA of the invention. The invention also provides
a high-throughput method for using a protein to screen a library
or a plurality of molecules or compounds to identify a ligand. The
method comprises combining the protein or a portion thereof with
the library or a plurality of molecules or compounds under conditions
to allow specific binding and detecting specific binding, thereby
identifying a ligand which specifically binds the protein. A library
or a plurality of molecules or compounds are selected from DNA molecules,
RNA molecules, peptide nucleic acid molecules, mimetics, peptides,
proteins, agonists, antagonists, antibodies or their fragments,
immunoglobulins, inhibitors, drug compounds, and pharmaceutical
agents. The invention further provides for using a protein to purify
a ligand. The method comprises combining the protein or a portion
thereof with a sample under conditions to allow specific binding,
recovering the bound protein, and separating the protein from the
ligand, thereby obtaining purified ligand. The invention yet still
further provides a method for using the protein to produce an antibody.
The method comprises immunizing an animal with the protein or an
antigenically-effective epitope under conditions to elicit an antibody
response, isolating animal antibodies, and screening the isolated
antibodies with the protein to identify an antibody which specifically
binds the protein. The invention yet still further provides a method
for using the protein to purify antibodies which bind specifically
to the protein.
DESCRIPTION OF THE SEQUENCE LISTING AND TABLES
 A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure, as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
 The Sequence Listing is a compilation of nucleic acid sequences
obtained by sequencing clone inserts (isolates) of different cDNAs.
Each sequence is identified by a sequence identification number
(SEQ ID NO) and by the clone number from which it was obtained.
 Table 1 lists genes differentially expressed in human BT20
breast carcinoma cells and in primary breast carcinoma tissue. In
each page, the table contains (by column): 1) SEQ ID NO: as shown
in the Sequence Listing; 2) the Genbank ID and; 3) gene description
to which the Incyte sequence was annotated; 4-10) the differential
expression of the gene in each of seven breast carcinoma tissue
samples, and 5) the maximal differential expression of the gene
measured during the time course of the experiment for BT20 cells.
DESCRIPTION OF THE INVENTION
 "Array" refers to an ordered arrangement of at
least two cDNAs on a substrate. At least one of the cDNAs represents
a control or standard sequence, and the other, a cDNA of diagnostic
interest. The arrangement of from about two to about 40,000 cDNAs
on the substrate assures that the size and signal intensity of each
labeled hybridization complex formed between a cDNA and a sample
nucleic acid is individually distinguishable.
 The "complement" of a nucleic acid molecule of
the Sequence Listing refers to a cDNA which is completely complementary
over the full length of the sequence and which will hybridize to
the nucleic acid molecule under conditions of high stringency.
 A "combination" comprises at least two and up
to 16 sequences selected from the group consisting of SEQ ID NOs:
1-16 as presented in the Sequence Listing.
 "cDNA" refers to a chain of nucleotides, an isolated
polynucleotide, nucleic acid molecule, or any fragment or complement
thereof. It may have originated recombinantly or synthetically,
be double-stranded or single-stranded, coding and/or noncoding,
an exon with or without an intron from a genomic DNA molecule, and
purified or combined with carbohydrate, lipids, protein or inorganic
elements or substances. Preferably, the cDNA is from about 4000
to about 5000 nucleotides.
 The phrase "cDNA encoding a protein" refers to
a nucleic acid 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; Altschul (1993) J Mol
Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403-410)
which provides identity within the conserved region. Brenner et
al. (1998; Proc Natl Acad Sci 95:6073-6078) who analyzed BLAST for
its ability to identify structural homologs by sequence identity
found 30% identity is a reliable threshold for sequence alignments
of at least 150 residues and 40% is a reasonable threshold for alignments
of at least 70 residues (Brenner et al., page 6076, column 2).
 "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,
upregulated or present, or decreased, downregulated or absent, gene
expression as detected by the absence, presence, or at least two-fold
changes in the amount of transcribed messenger RNA or translated
protein in a sample.
 "Fragment" refers to a chain of consecutive nucleotides
from about 200 to about 700 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.
Nucleic acids and their ligands identified in this manner 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'. The degree of
complementarity and the use of nucleotide analogs affect the efficiency
and stringency of hybridization reactions.
 "Ligand" refers to any agent, molecule, or compound
which will bind specifically to a complementary site on a cDNA molecule
or polynucleotide, or to an epitope or a protein. Such ligands stabilize
or modulate the activity of polynucleotides or proteins and may
be composed of inorganic or organic substances including 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. Substantially equivalent
terms are amplimer, primer, and oligomer.
 "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 molecule 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 retains at least one
biological or antigenic characteristic of a native protein. 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.
 "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.
 "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, the hydrogen bonding along the backbone between
two single stranded nucleic acids, or the binding between an epitope
of a protein and an agonist, antagonist, or antibody.
 "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
 "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.
 "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 amino acid.
 The Invention
 The present invention provides a combination comprising
a plurality of polynucleotide probes, comprising at least a fragment
of a gene whose transcript is modulated in response to EGF. Preferably,
the plurality of probes comprise at least a fragment of one or more
of the sequences, SEQ ID NOs:1-16, presented in the Sequence Listing;
and they are arranged on a substrate, preferably a microarray.
 The microarray can be used for large scale genetic or gene
expression analysis of a large number of targets. The microarray
can also be used in the diagnosis of diseases and in the monitoring
of treatments where altered expression of genes is associated with
a cell proliferative disorder, in particular, breast cancer. Further,
the microarray can be employed to investigate an individual's predisposition
to a disease, in particular, breast cancer.
 In a preferred embodiment, the combination provides the
expression of those probes selected from SEQ ID NOs:1-16 which are
associated with breast cancer
 When the composition of the invention is employed as hybridizable
elements in a microarray, the elements are organized in an ordered
fashion so that each element is present at a specified location
on the substrate. Because the elements are at specified locations
on the substrate, the hybridization patterns and intensities, which
together create a unique expression profile, can be interpreted
in terms of expression levels of particular genes and can be correlated
with a particular metabolic process, condition, disorder, disease,
stage of disease, or treatment.
 The combination comprising a plurality of cDNAs can also
be used to identify or purify a molecule or compound which specifically
binds to at least one of the cDNAs. These molecules may be identified
from a sample or in high throughput mode from a library of mRNAs,
cDNAs, genomic fragments, and the like. Typically, samples or libraries
will include targets of diagnostic or therapeutic interest. If nucleic
acids in a particular sample enhance the hybridization background,
it may be advantageous to remove these nucleic acids. One method
for removing additional nucleic acids is by hybridizing the sample
with immobilized probes and washing away those nucleic acids that
do not form hybridization complexes. At a later point, hybridization
complexes can be dissociated, thereby releasing the purified targets.
 cDNAs and Their Uses
 cDNAs can be prepared by a variety of synthetic or enzymatic
methods well known in the art. cDNAs can be synthesized, in whole
or in part, using chemical methods well known in the art (Caruthers
et al. (1980) Nucleic Acids Symp. Ser. (7)215-233). Alternatively,
cDNAs can be produced enzymatically or recombinantly, by in vitro
or in vivo transcription.
 Nucleotide analogs can be incorporated into cDNAs by methods
well known in the art. The only requirement is that the incorporated
analog must base pair with native purines or pyrimidines. For example,
2, 6-diaminopurine can substitute for adenine and form stronger
bonds with thymidine than those between adenine and thymidine. A
weaker pair is formed when hypoxanthine is substituted for guanine
and base pairs with cytosine. Additionally, cDNAs can include nucleotides
that have been derivatized chemically or enzymatically.
 cDNAs can be synthesized on a substrate. Synthesis on the
surface of a substrate may be accomplished using a chemical coupling
procedure and a piezoelectric printing apparatus as described by
Baldeschweiler et al. (PCT publication WO95/251116). Alternatively,
the cDNAs can be synthesized on a substrate surface using a self-addressable
electronic device that controls when reagents are added as described
by Heller et al. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized
directly on a substrate by sequentially dispensing reagents for
their synthesis on the substrate surface or by dispensing preformed
DNA fragments to the substrate surface. Typical dispensers include
a micropipette delivering solution to the substrate with a robotic
system to control the position of the micropipette with respect
to the substrate. There can be a multiplicity of dispensers so that
reagents can be delivered to the reaction regions efficiently.
 cDNAs can be immobilized on a substrate by covalent means
such as by chemical bonding procedures or UV irradiation. In one
method, a cDNA is bound to a glass surface which has been modified
to contain epoxide or aldehyde groups. In another method, a cDNA
is placed on a polylysine coated surface and UV cross-linked to
it as described by Shalon et al. (WO95/35505). In yet another method,
a cDNA is actively transported from a solution to a given position
on a substrate by electrical means (Heller, supra). cDNAs do not
have to be directly bound to the substrate, but rather can be bound
to the substrate through a linker group. The linker groups are typically
about 6 to 50 atoms long to provide exposure of the attached cDNA.
Preferred linker groups include ethylene glycol oligomers, diamines,
diacids and the like. Reactive groups on the substrate surface react
with a terminal group of the linker to bind the linker to the substrate.
The other terminus of the linker is then bound to the cDNA. Alternatively,
polynucleotides, plasmids or cells can be arranged on a filter.
In the latter case, cells are lysed, proteins and cellular components
degraded, and the DNA is coupled to the filter by UV cross-linking.
 The cDNAs may be used for a variety of purposes. For example,
the combination of the invention may be used on an array. The array,
in turn, can be used in high-throughput methods for detecting a
related polynucleotide in a sample, screening a plurality of molecules
or compounds to identify a ligand, diagnosing a breast cancer, or
inhibiting or inactivating a therapeutically relevant gene related
to the cDNA.
 When the cDNAs of the invention are employed on a microarray,
the cDNAs are arranged in an ordered fashion so that each cDNA is
present at a specified location. Because the cDNAs are at specified
locations on the substrate, the hybridization patterns and intensities,
which together create a unique expression profile, can be interpreted
in terms of expression levels of particular genes and can be correlated
with a particular metabolic process, condition, disorder, disease,
stage of disease, or treatment.
 The cDNAs or fragments or complements thereof may be used
in various hybridization technologies. The cDNAs may be labeled
using a variety of reporter molecules by either PCR, recombinant,
or enzymatic techniques. For example, a commercially available vector
containing the cDNA is transcribed in the presence of an appropriate
polymerase, such as T7 or SP6 polymerase, and at least one labeled
nucleotide. Commercial kits are available for labeling and cleanup
of such cDNAs. Radioactive (Amersham Pharmacia Biotech (APB), Piscataway,
N.J.), fluorescent (Operon Technologies, Alameda, Calif.), and chemiluminescent
labeling (Promega, Madison, Wis.) are well known in the art.
 A cDNA may represent the complete coding region of an mRNA
or be designed or derived from unique regions of the mRNA or genomic
molecule, an intron, a 3' untranslated region, or from a conserved
motif. The cDNA is at least 18 contiguous nucleotides in length
and is usually single stranded. Such a cDNA may be used under hybridization
conditions that allow binding only to an identical sequence, a naturally
occurring molecule encoding the same protein, or an allelic variant.
Discovery of related human and mammalian sequences may also be accomplished
using a pool of degenerate cDNAs and appropriate hybridization conditions.
Generally, a cDNA for use in Southern or northern hybridizations
may be from about 400 to about 6000 nucleotides long. Such cDNAs
have high binding specificity in solution-based or substrate-based
hybridizations. An oligonucleotide, a fragment of the cDNA, may
be used to detect a polynucleotide in a sample using PCR.
 The stringency of hybridization is determined by G+C content
of the cDNA, salt concentration, and temperature. In particular,
stringency is increased by reducing the concentration of salt or
raising the hybridization temperature. In solutions used for some
membrane based hybridizations, addition of an organic solvent such
as formamide allows the reaction to occur at a lower temperature.
Hybridization may be performed with buffers, such as 5.times.saline
sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60.degree.
C., that permit the formation of a hybridization complex between
nucleic acid sequences that contain some mismatches. Subsequent
washes are performed with buffers such as 0.2.times.SSC with 0.1%
SDS at either 45.degree. C. (medium stringency) or 65.degree.-68.degree.
C. (high stringency). At high stringency, hybridization complexes
will remain stable only where the nucleic acid molecules are completely
complementary. In some membrane-based hybridizations, preferably
35% or most preferably 50%, formamide may be added to the hybridization
solution to reduce the temperature at which hybridization is performed.
Background signals may 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 et al. (1997, Short
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y., Units 2.8-2.11, 3.18-3.19 and 4-6-4.9).
 Dot-blot, slot-blot, low density and high density arrays
are prepared and analyzed using methods known in the art. cDNAs
from about 18 consecutive nucleotides to about 5000 consecutive
nucleotides in length are contemplated by the invention and used
in array technologies. The preferred number of cDNAs on an array
is at least about 100,000, a more preferred number is at least about
40,000, an even more preferred number is at least about 10,000,
and a most preferred number is at least about 600 to about 800.
The array may be used to monitor the expression level of large numbers
of genes simultaneously and to identify genetic variants, mutations,
and SNPs. Such information may be used to determine gene function;
to understand the genetic basis of a disorder; to diagnose a disorder;
and to develop and monitor the activities of therapeutic agents
being used to control or cure a disorder. (See, e.g., U.S. Pat.
No. 5,474,796; WO95/11995; WO95/35505; U.S. Pat. No. 5,605,662;
and U.S. Pat. No. 5,958,342.)
 Screening and Purification Assays
 A cDNA may be used to screen a library or a plurality of
molecules or compounds for a ligand which specifically binds the
cDNA. Ligands may be DNA molecules, RNA molecules, peptide nucleic
acid molecules, peptides, proteins such as transcription factors,
promoters, enhancers, repressors, and other proteins that regulate
replication, transcription, or translation of the polynucleotide
in the biological system. The assay involves combining the cDNA
or a fragment thereof with the molecules or compounds under conditions
that allow specific binding and detecting the bound cDNA to identify
at least one ligand that specifically binds the cDNA.
 In one embodiment, the cDNA may be incubated with a library
of isolated and purified molecules or compounds and binding activity
determined by methods such as 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. Protein binding may
be confirmed by raising antibodies against the protein and adding
the antibodies to the gel-retardation assay where specific binding
will cause a supershift in the 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.
 The cDNA may be used to purify a ligand from a sample. A
method for using a cDNA to purify a ligand would involve combining
the cDNA or a fragment thereof with a sample under conditions to
allow specific binding, recovering the bound cDNA, and using an
appropriate agent to separate the cDNA from the purified ligand.
 Protein Production and Uses
 The full length cDNAs or fragment thereof may be used to
produce purified proteins using recombinant DNA technologies described
herein and taught in Ausubel et al. (supra; Units 16.1-16.62). One
of the advantages of producing proteins by these procedures is the
ability to obtain highly-enriched sources of the proteins thereby
simplifying purification procedures.
 The proteins may contain amino acid substitutions, deletions
or insertions made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues involved. Such substitutions may be conservative
in nature when the substituted residue has structural or chemical
properties similar to the original residue (e.g., replacement of
leucine with isoleucine or valine) or they may be nonconservative
when the replacement residue is radically different (e.g., a glycine
replaced by a tryptophan). Computer programs included in LASERGENE
software (DNASTAR, Madison, Wis.), MACVECTOR software (Genetics
Computer Group, Madison, Wis.) and RasMol software (www.umass.edu/microbio/rasmol)
may be used to help determine which and how many amino acid residues
in a particular portion of the protein may be substituted, inserted,
or deleted without abolishing biological or immunological activity.
 Expression of Encoded Proteins
 Expression of a particular cDNA may be accomplished by cloning
the cDNA into a vector and transforming this vector into a host
cell. The cloning vector used for the construction of cDNA libraries
in the LIESEQ databases may also be used for expression. Such vectors
usually contain a promoter and a polylinker useful for cloning,
priming, and transcription. An exemplary vector may also contain
the promoter for .beta.-galactosidase, an amino-terminal methionine
and the subsequent seven amino acid residues of .beta.-galactosidase.
The vector may be transformed into competent E. coli cells. Induction
of the isolated bacterial strain with isopropylthiogalactoside (IPTG)
using standard methods will produce a fusion protein that contains
an N terminal methionine, the first seven residues of .beta.-galactosidase,
about 15 residues of linker, and the protein encoded by the cDNA.
 The cDNA may be shuttled into other vectors known to be
useful for expression of protein in specific hosts. Oligonucleotides
containing cloning sites and fragments of DNA sufficient to hybridize
to stretches at both ends of the cDNA may be chemically synthesized
by standard methods. These primers may then be used to amplify the
desired fragments by PCR. The fragments may be digested with appropriate
restriction enzymes under standard conditions and isolated using
gel electrophoresis. Alternatively, similar fragments are produced
by digestion of the cDNA with appropriate restriction enzymes and
filled in with chemically synthesized oligonucleotides. Fragments
of the coding sequence from more than one gene may be ligated together
 Signal sequences that dictate secretion of soluble proteins
are particularly desirable as component parts of a recombinant sequence.
For example, a chimeric protein may be expressed that includes one
or more additional purification-facilitating domains. Such domains
include, but are not limited to, metal-chelating domains that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex, Seattle,
Wash.). The inclusion of a cleavable-linker sequence such as ENTEROKINASEMAX
(Invitrogen, San Diego, Calif.) between the protein and the purification
domain may also be used to recover the protein.
 Suitable host cells may include, but are not limited to,
mammalian cells such as Chinese Hamster Ovary (CHO) and human 293
cells, insect cells such as Sf9 cells, plant cells such as Nicotiana
tabacum, yeast cells such as Saccharomyces cerevisiae, and bacteria
such as E. coli. For each of these cell systems, a useful vector
may also include an origin of replication and one or two selectable
markers to allow selection in bacteria as well as in a transformed
eukaryotic host. Vectors for use in eukaryotic host cells may require
the addition of 3' poly(A) tail if the cDNA lacks poly(A).
 Additionally, the vector may contain promoters or enhancers
that increase gene expression. Many promoters are known and used
in the art. Most promoters are host specific and exemplary promoters
includes SV40 promoters for CHO cells; T7 promoters for bacterial
hosts; viral promoters and enhancers for plant cells; and PGH promoters
for yeast. Adenoviral vectors with the rous sarcoma virus enhancer
or retroviral vectors with long terminal repeat promoters may be
used to drive protein expression in mammalian cell lines. Once homogeneous
cultures of recombinant cells are obtained, large quantities of
secreted soluble protein may be recovered from the conditioned medium
and analyzed using chromatographic methods well known in the art.
An alternative method for the production of large amounts of secreted
protein involves the transformation of mammalian embryos and the
recovery of the recombinant protein from milk produced by transgenic
cows, goats, sheep, and the like.
 In addition to recombinant production, proteins or portions
thereof may be produced manually, using solid-phase techniques (Stewart
et al. (1969) Solid-Phase Peptide Synthesis, W H Freeman, San Francisco,
Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machines
such as the ABI 431A peptide synthesizer (Applied Biosystems, Foster
City, Calif.). Proteins produced by any of the above methods may
be used as pharmaceutical compositions to treat disorders associated
with null or inadequate expression of the genomic sequence.
 Screening and Purification Assays
 A protein or a portion thereof encoded by the cDNA may be
used to screen a library or a plurality of molecules or compounds
for a ligand with specific binding affinity or to purify a molecule
or compound from a sample. The protein or portion thereof employed
in such screening may be free in solution, affixed to an abiotic
or biotic substrate, or located intracellularly. For example, viable
or fixed prokaryotic host cells that are stably transformed with
recombinant nucleic acids that have expressed and positioned a protein
on their cell surface can be used in screening assays. The cells
are screened against a library or a plurality of ligands and the
specificity of binding or formation of complexes between the expressed
protein and the ligand may be measured. The ligands may be DNA,
RNA, or PNA molecules, agonists, antagonists, antibodies, immunoglobulins,
inhibitors, peptides, pharmaceutical agents, proteins, drugs, or
any other test molecule or compound that specifically binds the
protein. An exemplary assay involves combining the mammalian protein
or a portion thereof with the molecules or compounds under conditions
that allow specific binding and detecting the bound protein to identify
at least one ligand that specifically binds the protein.
 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 or oligopeptide or fragment thereof. One
method for high throughput screening using very small assay volumes
and very small amounts of test compound is described in U.S. Pat.
No. 5,876,946. Molecules or compounds identified by screening may
be used in a model system to evaluate their toxicity, diagnostic,
or therapeutic potential.
 The protein may be used to purify a ligand from a sample.
A method for using a protein to purify a ligand would involve combining
the protein or a portion thereof with a sample under conditions
to allow specific binding, recovering the bound protein, and using
an appropriate chaotropic agent to separate the protein from the
 Production of Antibodies
 A protein encoded by a cDNA of the invention may be used
to produce specific antibodies. Antibodies may be produced using
an oligopeptide or a portion of the protein with inherent immunological
activity. Methods for producing antibodies include: 1) injecting
an animal, usually goats, rabbits, or mice, with the protein, or
an antigenically-effective portion or an oligopeptide thereof, to
induce an immune response; 2) engineering hybridomas to produce
monoclonal antibodies; 3) inducing in vivo production in the lymphocyte
population; or 4) screening libraries of recombinant immunoglobulins.
Recombinant immunoglobulins may be produced as taught in U.S. Pat.
 Antibodies produced using the proteins of the invention
are useful for the diagnosis of prepathologic disorders as well
as the diagnosis of chronic or acute diseases characterized by abnormalities
in the expression, amount, or distribution of the protein. A variety
of protocols for competitive binding or immunoradiometric assays
using either polyclonal or monoclonal antibodies specific for proteins
are well known in the art. Immunoassays typically involve the formation
of complexes between a protein and its specific binding molecule
or compound and the measurement of complex formation. Immunoassays
may employ a two-site, monoclonal-based assay that utilizes monoclonal
antibodies reactive to two noninterfering epitopes on a specific
protein or a competitive binding assay (Pound (1998) Immunochemical
Protocols, Humana Press, Totowa, N.J.).
 Immunoassay procedures may be used to quantify expression
of the protein in cell cultures, in subjects with a particular disorder
or in model animal systems under various conditions. Increased or
decreased production of proteins as monitored by immunoassay may
contribute to knowledge of the cellular activities associated with
developmental pathways, engineered conditions or diseases, or treatment
efficacy. The quantity of a given protein in a given tissue may
be determined by performing immunoassays on freeze-thawed detergent
extracts of biological samples and comparing the slope of the binding
curves to binding curves generated by purified protein.
 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
cDNA, polynucleotide, protein, peptide or antibody assays. Synthesis
of labeled molecules may be achieved using commercial kits for incorporation
of a labeled nucleotide such as .sup.32P-dCTP, Cy3-dCTP or Cy5-dCTP
or amino acid such as .sup.35S-methionine. Polynucleotides, cDNAs,
proteins, or antibodies may be directly labeled with a reporter
molecule by chemical conjugation to amines, thiols and other groups
present in the molecules using reagents such as BIODIPY or FITC
(Molecular Probes, Eugene, Oreg.).
 The proteins and antibodies may be labeled for purposes
of assay by joining them, either covalently or noncovalently, with
a reporter molecule that provides for a detectable signal. A wide
variety of labels and conjugation techniques are known and have
been reported in the scientific and patent literature including,
but not limited to U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241.
 The cDNAs, or fragments thereof, may be used to detect and
quantify differential gene expression; absence, presence, or excess
expression of mRNAs; or to monitor mRNA levels during therapeutic
intervention associated with breast cancer. These cDNAs can also
be utilized as markers of treatment efficacy against breast cancer
over a period ranging from several days to months. 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 altered gene expression. Qualitative
or quantitative methods for this comparison are well known in the
 For example, the cDNA may be labeled by standard methods
and added to a biological sample from a patient under conditions
for hybridization complex formation. 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 the amount of label in the patient sample is significantly
altered in comparison to the standard value, then the presence of
the associated condition, disease or disorder is indicated.
 In order to provide a basis for the diagnosis of a condition,
disease or disorder associated with gene expression, a normal or
standard expression profile is established. This may be accomplished
by combining a biological sample taken from normal subjects, either
animal or human, with a probe under conditions for hybridization
or amplification. Standard hybridization may be quantified by comparing
the values obtained using normal subjects with values from an experiment
in which a known amount of a substantially purified target sequence
is used. Standard values obtained in this manner may be compared
with values obtained from samples from patients who are symptomatic
for a particular condition, disease, or disorder. Deviation from
standard values toward those associated with a particular condition
is used to diagnose that condition.
 Such assays may also be used to evaluate the efficacy of
a particular therapeutic treatment regimen in animal studies and
in clinical trial 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 months.
 Gene Expression Profiles
 A gene expression profile comprises a plurality of cDNAs
and a plurality of detectable hybridization complexes, wherein each
complex is formed by hybridization of one or more probes to one
or more complementary sequences in a sample. The cDNAs of the invention
are used as elements on a microarray to analyze gene expression
profiles. In one embodiment, the microarray is used to monitor the
progression of disease. Researchers can assess and catalog the differences
in gene expression between healthy and diseased tissues or cells.
By analyzing changes in patterns of gene expression, disease can
be diagnosed at earlier stages before the patient is symptomatic.
The invention can be used to formulate a prognosis and to design
a treatment regimen. The invention can also be used to monitor the
efficacy of treatment. For treatments with known side effects, the
microarray is employed to improve the treatment regimen. A dosage
is established that causes a change in genetic expression patterns
indicative of successful treatment. Expression patterns associated
with the onset of undesirable side effects are avoided. This approach
may be more sensitive and rapid than waiting for the patient to
show inadequate improvement, or to manifest side effects, before
altering the course of treatment.
 In another embodiment, animal models which mimic a human
disease can be used to characterize expression profiles associated
with a particular condition, disorder or disease; or treatment of
the condition, disorder or disease. Novel treatment regimens may
be tested in these animal models using microarrays to establish
and then follow expression profiles over time. In addition, microarrays
may be used with cell cultures or tissues removed from animal models
to rapidly screen large numbers of candidate drug molecules, looking
for ones that produce an expression profile similar to those of
known therapeutic drugs, with the expectation that molecules with
the same expression profile will likely have similar therapeutic
effects. Thus, the invention provides the means to rapidly determine
the molecular mode of action of a drug.
 Assays Using Antibodies
 Antibodies directed against epitopes on a protein encoded
by a cDNA of the invention may be used in assays to quantify the
amount of protein found in a particular human cell. Such assays
include methods utilizing the antibody and a label to detect expression
level under normal or disease conditions. The antibodies may be
used with or without modification, and labeled by joining them,
either covalently or noncovalently, with a labeling moiety.
 Protocols for detecting and measuring protein expression
using either polyclonal or monoclonal antibodies are well known
in the art. Examples include ELISA, RIA, and fluorescent activated
cell sorting (FACS). Such immunoassays typically involve the formation
of complexes between the protein and its specific antibody and the
measurement of such complexes. These and other assays are described
in Pound (supra). The method may employ a two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two non-interfering
epitopes, or a competitive binding assay. (See, e.g., Coligan et
al. (1997) Current Protocols in Immunology, Wiley-Interscience,
New York, N.Y.; Pound, supra)
 The cDNAs and fragments thereof 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 overexpression 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; Agruwal (1996) Antisense Therapeutics,
Humana Press, Totowa, N.J.; and August et al. (1997) Gene Therapy
(Advances in Pharmacology, Vol. 40), Academic Press, San Diego,
 In addition, expression of a particular protein can be regulated
through the specific binding of a fragment of a cDNA to a genomic
sequence or an mRNA which encodes the protein or directs its transcription
or translation. The cDNA can be modified or derivatized to any RNA-like
or DNA-like material including peptide nucleic acids, branched nucleic
acids, and the like. These sequences can be produced biologically
by transforming an appropriate host cell with a vector containing
the sequence of interest.
 Molecules which regulate the activity of the cDNA or encoded
protein are useful as therapeutics for breast cancer. Such molecules
include agonists which increase the expression or activity of the
polynucleotide or encoded protein, respectively; or antagonists
which decrease expression or activity of the polynucleotide or encoded
protein, respectively. In one aspect, an antibody which specifically
binds the protein may be used directly as an antagonist or indirectly
as a delivery mechanism for bringing a pharmaceutical agent to cells
or tissues which express the protein.
 Additionally, any of the proteins, or their ligands, or
complementary nucleic acid sequences may be administered as pharmaceutical
compositions or in combination with other appropriate therapeutic
agents. Selection of the appropriate agents for use in combination
therapy may be made by one of ordinary skill in the art, according
to conventional pharmaceutical principles. The combination of therapeutic
agents may act synergistically to affect the treatment or prevention
of the conditions and disorders associated with EGF regulation.
Using this approach, one may be able to achieve therapeutic efficacy
with lower dosages of each agent, thus reducing the potential for
adverse side effects. Further, the therapeutic agents may be combined
with pharmaceutically-acceptable carriers including excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Further details
on techniques for formulation and administration used by doctors
and pharmacists may be found in the latest edition of Remington's
Pharmaceutical Sciences (Maack 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 underexpression or overexpression of genes of interest and for
the development of methods for diagnosis and treatment of diseases.
A mammal inbred to overexpress a particular gene (for example, secreted
in milk) may also serve as a convenient source of the protein expressed
by that gene.
 Transgenic Animal Models
 Transgenic rodents that overexpress or underexpress 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 such as the
mouse 129/SvJ cell line are placed in a blastocyst from the C57BL/6
mouse strain, they resume normal development and contribute to tissues
of the live-born animal. ES cells are preferred for use in the creation
of experimental knockout and knockin animals. The method for this
process is well known in the art and the steps are: the cDNA is
introduced into a vector, the vector is transformed into ES cells,
transformed cells are identified and microinjected into mouse cell
blastocysts, blastocysts are surgically transferred to pseudopregnant
dams. The resulting chimeric progeny are genotyped and bred to produce
heterozygous or homozygous strains.
 Knockout Analysis
 In gene knockout analysis, a region of a gene is enzymatically
modified to include a non-natural intervening sequence 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
 Knockin Analysis
 ES cells can be used to create knockin humanized animals
or transgenic animal models 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. Transgenic
progeny or inbred lines are studied and treated with potential pharmaceutical
agents to obtain information on the progression and treatment of
the analogous human condition.
 As described herein, the uses of the cDNAs, provided in
the Sequence Listing of this application, and their encoded proteins
are exemplary of known techniques and are not intended to reflect
any limitation on their use in any technique that would be known
to the person of average skill in the art. Furthermore, the cDNAs
provided in this application may be used in molecular biology techniques
that have not yet been developed, provided the new techniques rely
on properties of nucleotide sequences that are currently known to
the person of ordinary skill in the art, e.g., the triplet genetic
code, specific base pair interactions, and the like. Likewise, reference
to a method may include combining more than one method for obtaining
or assembling full length cDNA sequences that will be known to those
skilled in the art. It is also to be understood that this invention
is not limited to the particular methodology, protocols, and reagents
described, as these may vary. It is also understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present invention
which will be limited only by the appended claims. The examples
below are provided to illustrate the subject invention and are not
included for the purpose of limiting the invention.
 For purposes of example, the preparation and sequencing
of the breast tissue cDNA library (BRSTNOT07), from which Incyte
Clone 1297817 was identified is described. Preparation and sequencing
of cDNAs in libraries in the LIFESEQ database (Incyte Genomics)
have varied over time, and the gradual changes involved use of kits,
plasmids, and machinery available at the particular time the library
was made and analyzed.
 I cDNA Library Preparation
 The BRSTNOT07 cDNA library was constructed from diseased
breast tissue removed from a 43-year-old Caucasian female during
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
 The frozen tissue was homogenized and lysed in a guanidinium
isothiocyanate solution using a POLYTRON homogenizer (PT-3000, Brinkmann
Instruments, Westbury, N.J.). The lysate was centrifuged over a
5.7 M CsCl cushion using a SW28 rotor in an L8-70M ultracentrifuge
(Beckman Instruments, 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 treated with DNase
at 37.degree. C. RNA extraction and precipitation was repeated as
before. The mRNA was then isolated using the OLIGOTEX kit (Qiagen,
Inc., Chatsworth, Calif.) and used to construct the cDNA library.
 The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid System (Life Technologies). The cDNA
were fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia
Biotech, Piscataway, N.J.), and those cDNAs exceeding 400 bp were
ligated into pSPORT I. The plasmid was subsequently transformed
into DH5.alpha. competent cells (Life Technologies).
 II. Isolation and Sequencing of cDNA Clones
 Plasmid DNA was released from the cells and purified using
the REAL Prep 96 plasmid kit (Qiagen). This kit enabled the simultaneous
purification of 96 samples in a 96-well block using multi-channel
reagent dispensers. The recommended protocol was employed except
for the following changes: 1) the bacteria were cultured in 1 ml
of sterile Terrific Broth (Life Technologies) with carbenicillin
at 25 mg/l and glycerol at 0.4%; 2) after inoculation, the cells
were cultured for 19 hours and lysed with 0.3 ml of lysis buffer;
and 3) following isopropanol precipitation, the 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 4.degree.
 The cDNAs were prepared using a MICROLAB 2200 system (Hamilton,
Reno, Nev.) in combination with DNA ENGINE thermal cyclers (PTC200;
MJ Research, Waltham, Mass.). The cDNAs were sequenced by the method
of Sanger and Coulson (1975; J. Mol. Biol. 94:441f) using ABI PRISM
377 DNA sequencing systems (PE Biosystems). Most of the sequences
were sequenced using standard ABI protocols and kits (PE Biosystems)
at solution volumes of 0.25.times.-1.0.times.. In the alternative,
some of the sequences were sequenced using solutions and dyes from
Amersham Pharmacia Biotech.
 III Homology Searching of cDNA Clones and Their Deduced
 As used herein, "homology" refers to sequence
similarity between a reference sequence and at least a fragment
of a newly sequenced clone insert, and can refer to either a nucleic
acid or amino acid sequence. The GenBank databases which contain
previously identified and annotated sequences, were searched for
regions of homology using BLAST (Altschul (1993 and 1990) supra).
 BLAST involves first finding similar segments between the
query sequence and a database sequence, then evaluating the statistical
significance of any matches that are found and finally reporting
only those matches that satisfy a user-selectable threshold of significance.
BLAST produces alignments of both nucleotide and amino acid sequences
to determine sequence similarity. The fundamental unit of the BLAST
algorithm output is the High scoring Segment Pair (HSP). An HSP
consists of two sequence fragments of arbitrary, but equal lengths,
whose alignment is locally maximal and for which the alignment score
meets or exceeds a threshold or cutoff score set by the user.
 The basis of the search is the product score, which is defined
% sequence identity.times.% maximum BLAST score/100
 The product score takes into account both the degree of
identity between two sequences and the length of the sequence match
as reflected in the BLAST score. The BLAST score is calculated by
scoring +5 for every base that matches in an HSP and -4 for every
mismatch. For example, with a product score of 40, the match will
be exact within a 1% to 2% error, and, with a product score of 70,
the match will be exact. Homologous molecules are usually identified
by selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules. The P-value
for any given HSP is a function of its expected frequency of occurrence
and the number of HSPs observed against the same database sequence
with scores at least as high. Percent sequence identity is found
in a comparison of two or more amino acid or nucleic acid sequences.
Percent identity can be determined electronically, e.g., by using
the MEGALIGN program (DNASTAR). The percentage similarity between
two amino acid sequences, e.g., sequence A and sequence B, is calculated
by dividing the length of sequence A, minus the number of gap residues
in sequence A, minus the number of gap residues in sequence B, into
the sum of the residue matches between sequence A and sequence B,
times one hundred. Gaps of low or of no homology between the two
amino acid sequences are not included in determining percentage
 Sequences with conserved protein motifs may also be searched
using the BLOCKS search program. This program analyses sequence
information contained in the Swiss-Prot Database and PROSITE and
is useful for determining the classification of uncharacterized
proteins translated from genomic or cDNA sequences (Bairoch, supra;
Attwood, supra). PROSITE is a useful source for identifying functional
or structural domains that are not detected using motifs due to
extreme sequence divergence. Using weight matrices, these domains
are calibrated against the SWISS-PROT database to obtain a measure
of the chance distribution of the matches.
 The PRINTS database can be searched using the BLIMPS search
program to obtain protein family "fingerprints". The PRINTS
database complements the PROSITE database by exploiting groups of
conserved motifs within sequence alignments to build characteristic
signatures of different protein families. For both BLOCKS and PRINTS
analyses, the cutoff scores for local similarity were: >1300=strong,
1000-1300=suggestive; for global similarity were: p<exp-3; and
for strength (degree of correlation) were: >1300=strong, 1000-1300=weak.
 IV Extension of cDNA Clones
 Some of the nucleic acid sequences of SEQ ID NO:1-16 were
produced by extension of an appropriate fragment of the molecule
using oligonucleotide primers designed from this fragment. One primer
was synthesized to initiate 5' extension of the known fragment,
and the other primer, to initiate 3' extension of the known fragment.
The initial primers were designed using OLIGO 4.06 software (National
Biosciences), or another appropriate program, 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 68.degree.
C. to about 72.degree. C. Any stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was
 Selected human cDNA libraries were used to extend the sequence.
If more than one extension was necessary or desired, additional
or nested sets of primers were designed.
 High fidelity amplification was obtained by PCR using methods
well known in the art. PCR was performed in 96-well plates using
the DNA Engine thermal cycler (MJ Research, Inc.). 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 (Amersham Pharmacia Biotech), ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters for primer pair PCI A and PCI B: Step 1: 94.degree.
C., 3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C.,
1 min; Step 4: 68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated
20 times; Step 6: 68.degree. C., Step 7: storage at 4.degree. C.
In the alternative, the parameters for primer pair T7 and SK+ were
as follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C.,
15 sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. C., 2 min;
Step 5: 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C., 5
min; Step 7: storage at 4.degree. C.
 The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene, Oreg.) dissolved in 1.times.TE
and 0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Corning Costar, Acton, Mass.), allowing the DNA
to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) 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 mini-gel to determine which reactions were successful
in extending the sequence.
 The extended nucleotides were desalted and 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 pUC 18 vector (Amersham Pharmacia
Biotech). For shotgun sequencing, the digested nucleotides were
separated on low concentration (0.6 to 0.8%) agarose gels, fragments
were excised, and agar digested with Agar ACE (Promega). Extended
clones were religated using T4 ligase (New England Biolabs, Beverly,
Mass.) into pUC 18 vector (Amersham Pharmacia Biotech), treated
with Pfu DNA polymerase (Stratagene) to fill-in restriction site
overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on antibiotic-containing media, individual colonies
were picked and cultured overnight at 37.degree. C. in 384-well
plates in LB/2.times.carb liquid media.
 The cells were lysed, and DNA was amplified by PCR using
Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated
29 times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the same conditions as described above. Samples were diluted
with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham
Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing
ready reaction kit (PE Biosystems).
 V Propagation and Maintenance of Cultured Cells
 The human breast carcinoma cell line (BT-20) was purchased
from ATCC (Manassus, Va.); and primary mammary epithelial cells
(HMEC), from Clonetics (San Diego, Calif.). The cells were propagated
in media according to the supplier's recommendations.
 VI Experimental Treatment of Cultured Cells
 Cells were plated in culture dishes and grown at 37.degree.
C. in 5% CO.sub.2 till 80% confluent. The cells were then grown
for 48 hours in the presence of 1% fetal bovine serum (FBS), following
which the spent media was removed and replaced with either media
alone or media containing 50 ng/ml EGF (R and D system, Minneapolis,
Minn.). Control and EGF-stimulated cells were lysed at different
time points (e.g. 4, 8, 12, 24, 36, and 48 hours for the BT20 experiment)
 VII Preparation of mRNA
 Following the experimental treatments described above, total
RNA was extracted from cell samples using the TRIZOL reagent (Life
Technologies) extraction protocol based on the supplier's recommendations,
and the mRNA purified using the OLIGOTEX kit (Qiagen) described
 The mRNA from four non-diseased breast tissue samples, prepared
from three female patients ages 32-42 and a pooled tissue sample
from two donors ages 43 and 58, were obtained from BioChain Institute
(San Leandro, Calif.). mRNA from seven ductal carcinoma primary
tumors, prepared from six female patients, ages 46-56, and one pool
of 18 donors, ages 40-72, was also obtained from the same source.
 VIII Labeling of Probes and Hybridization Analyses
 Substrate Preparation
 Target nucleic acids were amplified from bacterial vectors
by thirty cycles of PCR using primers complementary to vector sequences
flanking the insert. Amplified target nucleic acids were purified
using SEPHACRYL-400 beads (Amersham Pharmacia Biotech). Purified
target nucleic acids were robotically arrayed onto a glass microscope
slide (Corning Science Products, Corning, N.Y.). The slide was previously
coated with 0.05% aminopropyl silane (Sigma-Aldrich, St. Louis,
Mo.) and cured at 110.degree. C. The arrayed glass slide (microarray)
was exposed to UV irradiation in a STRATALINKER UV-crosslinker (Stratagene).
 In an alternative method, a mixture of target nucleic acids,
a restriction digest of genomic DNA, is fractionated by electrophoresis
through an 0.7% agarose gel in 1.times.TAE [Tris-acetate-ethylenediamine
tetraacetic acid (EDTA)] running buffer and transferred to a nylon
membrane by capillary transfer using 20.times.saline sodium citrate.
(SSC). Alternatively, targets are individually ligated to a vector
and inserted into bacterial host cells to form a library. Target
nucleic acids are arranged on 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 bacterial growth medium, LB agar containing
carbenicillin, and incubated at 37.degree. C. for 16 hours. Bacterial
colonies are denatured, neutralized, and digested with proteinase
K. Nylon membranes are exposed to UV irradiation in a STRATALINKER
UV-crosslinker (Stratagene) to cross-link DNA to the membrane.
 Probe Preparation
 Each mRNA sample was reverse transcribed using MMLV reverse
transcriptase in the presence of dCTP-Cy3 or dCTP-Cy5 (Amersham
Pharmacia Biotech) according to standard protocol. After incubation
at 37.degree. C., the reaction was stopped with 0.5 M sodium hydroxide,
and RNA was degraded at 85.degree. C. The probes were then purified
using Chroma Spin 30 gel filtration spin columns (Clontech, Palo
Alto, Calif.) and ethanol precipitation.
 Competitive hybridization was performed using RNA from (1)
Control untreated versus EGF treated cells or (2) Normal breast
versus tumor breast tissue.
 The hybridization mixture, containing 0.2 mg of each of
Cy3 and Cy5 labeled cDNA probes as starting material, was heated
to 65.degree. C., and added to the microarray surface. The array
was covered with a coverslip and incubated at 60.degree. C. The
microarrays were washed at 45.degree. C. in high stringency buffer
(1.times.SSC and 0.1% SDS) followed by low stringency washes (0.1.times.SSC)
 A laser microscope was used to detect the fluorescence-labeled
probed. Excitation wavelengths were 488 nm for Cy3 and 632 nm for
Cy5. Each array was scanned twice, one scan per fluorophore. The
emission maxima was 565 nm for Cy3 and 650 nm for Cy5. The emitted
light was split into two photomultiplier tube detectors based on
wavelength. The output of the photomultiplier tube was digitized
and displayed as an image, where the signal intensity was represented
using a linear 20 color transformation, with red representing a
high signal and blue a low signal. The fluorescence signal for each
element was integrated to obtain a numerical value corresponding
to the signal intensity using GEMTOOLS gene expression analysis
software (Incyte Genomics).
 IX Data Analysis and Results
 Data analysis using the GEMTOOLS gene expression analysis
software (Incyte Genomics) was performed to identify those genes
which exhibited a 2-fold or more change in expression in response
to EGF and displayed a signal intensity of over 300. The sequences
found in the Sequence Listing were selected because they showed
at least a 2-fold change in expression in response to EGF treatment
of a human breast tumor cell line, BT-20, and were also differentially
expressed in primary breast carcinoma tissue samples. Comparisons
of expression among these cells and tissues allowed the identification
of genes potentially useful in diagnosing a breast cancer (differentially
expressed in BT20 cells and breast carcinoma tissue, but not in
 Table 1 lists genes differentially regulated by EGF treatment
at least 2-fold in BT20 breast carcinoma cells that are also differentially
regulated at least 2-fold in at least four of seven breast carcinoma
tissue samples (BC). Column 1 lists the SEQ ID NO, column 2 the
Genbank ID, and column 3 the description of the gene by BLAST, where
identified. Sequences not identified by BLAST are indicated as "Incyte
unique". Columns 4-10 list the differential expression of the
gene in seven breast carcinoma tissue samples (BC1-BC7), and column
11 lists the maximal differential expression of the gene during
the time course of the experiment for BT20 cells (BT20). Positive
values indicate upregulation of the gene, and negative (-) values
indicate downregulation. This comparison shows that genes differentially
regulated in vitro by EGF treatment of BT20 cells are also differentially
regulated in vivo in breast carcinoma.. These genes may be useful
in diagnosing and monitoring the progression of breast cancer and
the response to treatment.
 X Expression of the Encoded Protein
 Expression and purification of a protein encoded by a cDNA
of the invention is achieved using bacterial or virus-based expression
systems. For expression in bacteria, cDNA is subcloned into a vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into bacterial hosts, such as BL21(DE3). Antibiotic
resistant bacteria express the protein upon induction with IPTG.
Expression in eukaryotic cells is achieved by infecting Spodoptera
frugiperda (Sf9) insect cells with recombinant baculovirus, Autographica
californica nuclear polyhedrosis virus. The polyhedrin gene of baculovirus
is replaced with the cDNA by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is maintained and the strong polyhedrin promoter
drives high levels of transcription.
 For ease of purification, the protein is synthesized as
a fusion protein with glutathione-S-transferase (GST; APB) or a
similar alternative such as FLAG. The fusion protein is purified
on immobilized glutathione under conditions that maintain protein
activity and antigenicity. After purification, the GST moiety is
proteolytically cleaved from the protein with thrombin. A fusion
protein with FLAG, an 8-amino acid peptide, is purified using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak, Rochester, N.Y.).
 XI Production of Specific Antibodies
 A denatured protein from a reverse phase HPLC separation
is obtained in quantities up to 75 mg. This denatured protein is
used to immunize mice or rabbits following standard protocols. About
100 .mu.g is used to immunize a mouse, while up to 1 mg is used
to immunize a rabbit. The denatured protein is radioiodinated and
incubated with murine B-cell hybridomas to screen for monoclonal
antibodies. About 20 mg of protein is sufficient for labeling and
screening several thousand clones.
 In another approach, the amino acid sequence translated
from a cDNA of the invention is analyzed using PROTEAN software
(DNASTAR) to determine regions of high antigenicity, essentially
antigenically-effective epitopes of the protein. The optimal sequences
for immunization are usually at the C-terminus, the N-terminus,
and those intervening, hydrophilic regions of the protein that are
likely to be exposed to the external environment when the protein
is in its natural conformation. Typically, oligopeptides about 15
residues in length are synthesized using an ABI 431 peptide synthesizer
(Applied Biosystems) using Fmoc-chemistry and then coupled to keyhole
limpet hemocyanin (KLH; Sigma Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide
ester. If necessary, a cysteine may be introduced at the N-terminus
of the peptide to permit coupling to KLH. Rabbits are immunized
with the oligopeptide-KLH complex in complete Freund's adjuvant.
The resulting antisera are tested for antipeptide activity by binding
the peptide to plastic, blocking with 1% BSA, reacting with rabbit
antisera, washing, and reacting with radioiodinated goat anti-rabbit
 Hybridomas are prepared and screened using standard techniques.
Hybridomas of interest are detected by screening with radioiodinated
protein to identify those fusions producing a monoclonal antibody
specific for the protein. In a typical protocol, wells of 96 well
plates (FAST, Becton-Dickinson, Palo Alto, Calif.) are coated with
affinity-purified, specific rabbit-anti-mouse (or suitable anti-species
Ig) antibodies at 10 mg/ml. The coated wells are blocked with 1%
BSA and washed and exposed to supernatants from hybridomas. After
incubation, the wells are exposed to radiolabeled protein at 1 mg/ml.
Clones producing antibodies bind a quantity of labeled protein that
is detectable above background.
 Such clones are expanded and subjected to 2 cycles of cloning
at 1 cell/3 wells. Cloned hybridomas are injected into pristane-treated
mice to produce ascites, and monoclonal antibody is purified from
the ascitic fluid by affinity chromatography on protein A (APB).
Monoclonal antibodies with affinities of at least 10.sup.8 M.sup.-1,
preferably 10.sup.9 to 10.sup.10 M.sup.-1 or stronger, are made
by procedures well known in the art.