The present invention relates to a combination comprising a plurality
of cDNAs which are differentially expressed in 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
What is claimed is:
1. A combination comprising a plurality of cDNAs that are differentially
expressed in breast cancer and selected from SEQ ID NOs:1, 3, 5,
7, 8, 10-14, 16, 18, 19, 21-40, 42-48, 50, 51, 53-55, 57-65, 67-70,
72-74, 76-80, 82-85, 87, 88, 90, 92, 94, 96-109, 111, 113, 115,
116, 117, 119, 121, 123-126, 128, 130, 131, 133, 134, 135, 137,
138, 140, 142-144, 146, 148, 150, 152, 153, 155-158, 160, 161, 163-183,
185, 187-192, and 194 or their complements.
2. The combination of claim 1, comprising a plurality of cDNAs
that are differentially expressed in metastatic breast cancer and
selected from SEQ ID NOs:109, 111, 113, 115, 116, 117, 119, 121,
123-126, 128, 130, 131, 133, 134, 135, 137, 138, 140, 142-144, 146,
148, 150, 152, 153, 155-158, 160, 161, 163-183, 185, 187-192, and
194 or their complements.
3. The combination of claim 1, wherein the cDNAs are immobilized
on a substrate.
4. 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 3 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.
5. The method of claim 4, where in the nucleic acids of the sample
are amplified prior to hybridization.
6. The method of claim 4, wherein the sample is from a subject
with breast cancer and comparison with a standard defines a stage
of that disease.
7. 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.
8. The method of claim 7 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.
9. An isolated cDNA selected from SEQ ID NOs:21, 50, 79, 100, 105,
106, 107, 109, 126, 178, 181, 190, and 191.
10. A vector containing the cDNA of claim 9.
11. A host cell containing the vector of claim 10.
12. A method for producing a protein, the method comprising the
steps of: (a) culturing the host cell of claim 11 under conditions
for expression of protein; and (b) recovering the protein from the
host cell culture.
13. A protein or a portion thereof produced by the method of claim
14. 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 13 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.
15. The method of claim 14 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.
16. A method of using a protein to produce a polyclonal antibody,
the method comprising: a) immunizing an animal with the protein
of claim 13 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.
17. A method of using a protein to prepare a monoclonal antibody
comprising: a) immunizing a animal with a protein of claim 1 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 from culture monoclonal antibodies which specifically
bind the protein.
18. A method of purifying an antibody from a sample, the method
comprising: a) combining the protein of claim 13 with a sample under
conditions to allow specific binding; b) recovering the bound protein;
and c) separating the protein from the antibody, thereby obtaining
19. A purified antibody which specifically binds a protein differentially
expressed in breast cancer.
20. A method for using an antibody to detect expression of a protein
in a sample, the method comprising: a) combining the antibody of
claim 19 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
FIELD OF THE INVENTION
 The present invention relates to a composition comprising
a plurality of cDNAs which are differentially expressed in breast
cancer and which may be used entirely or in part to diagnose, to
stage, to treat, or to monitor the progression or treatment of breast
BACKGROUND OF THE INVENTION
 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 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 breast cancer may be compared with
the levels and sequences expressed in normal tissue.
 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 C M 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. 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 effects on cell functions related to metastatic potential, such
as cell motility, chemotaxis, secretion and differentiation. Changes
in expression of other members of the erbB receptor family, of which
EGFR is one, have also been implicated in breast cancer. The abundance
of erbB receptors, such as HER-2/neu, HER-3, and HER-4, and their
ligands in breast cancer points to their functional importance in
the pathogenesis of the disease, and may therefore provide targets
for therapy of the disease (Bacus, S S et al. (1994) Am J Clin Pathol
102:S13-S24). 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).
 Cell lines derived from human mammary epithelial cells at
various stages of breast cancer provide a useful model to study
the process of malignant transformation and tumor progression as
it has been shown that these cell lines retain many of the properties
of their parental tumors for lengthy culture periods (Wistuba II
et.al. (1998) Clin Cancer Res 4:2931-2938). Such a model is particularly
useful for comparing phenotypic and molecular characteristics of
human mammary epithelial cells at various stages of malignant transformation.
 The present invention provides for a combination comprising
a plurality of cDNAs for use in detecting changes in expression
of genes encoding proteins that are associated with breast cancer.
Such a composition can be employed for the diagnosis, prognosis
or treatment of breast cancer correlated with differential gene
expression. The present invention satisfies a need in the art by
providing a set of differentially expressed genes which may be used
entirely or in part to diagnose, to stage, to treat, or to monitor
the progression or treatment of a subject with breast cancer.
 The present invention provides a combination comprising
a plurality of cDNAs and their complements which are differentially
expressed in breast cancer and which are selected from SEQ ID NOs:1,
3, 5, 7, 8, 10-14, 16, 18, 19, 21-40, 42-48, 50, 51, 53-55, 57-65,
67-70, 72-74, 76-80, 82-85, 87, 88, 90, 92, 94, 96-109, 111, 113,
115, 116, 117, 119, 121, 123-126, 128, 130, 131, 133, 134, 135,
137, 138, 140, 142-144, 146, 148, 150, 152, 153, 155-158, 160, 161,
163-183, 185, 187-192, and 194 as presented in the Sequence Listing.
In one aspect, the combination is useful to diagnose a breast cancer.
In another aspect, the combination is immobilized on a substrate.
The invention also provides a combination comprising a subset of
these cDNAs and their complements which are differentially expressed
in metastatic breast cancer and which are selected from SEQ ID NOs:109,
111, 113, 115, 116, 117, 119, 121, 123-126, 128, 130, 131, 133,
134, 135, 137, 138, 140, 142-144, 146, 148, 150, 152, 153, 155-158,
160, 161, 163-183, 185, 187-192, and 194 as presented in the Sequence
Listing. In one aspect, the combination is useful to diagnose and
monitor treatment of an advanced stage of breast cancer. In another
aspect, the combination is immobilized on a substrate.
 The invention provides a high throughput method to detect
expression of a nucleic acid which is complementary at least one
of the cDNAs of the combination in a sample. The method comprises
hybridizing a substrate containing the combination with a sample
containing nucleic acids under conditions to form at least one hybridization
complex and detecting hybridization complex formation, wherein complex
formation indicates expression of at least one complementary nucleic
acid in the sample. In one aspect, the sample is from a subject
with breast cancer and differential expression determines the presence
or the stage of that disorder.
 The invention also 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 plurality of molecules
or compounds under conditions to allow specific binding and detecting
specific binding, thereby identifying a ligand. The library or 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
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 invention further provides an isolated cDNA selected
from SEQ ID NOs:21, 50, 79, 100, 105, 106, 107, 109, 126, 178, 181,
190, and 191 as presented in the Sequence Listing. 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 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 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 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 still further
provides a composition comprising the protein and a pharmaceutical
 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 and purifying
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 from
culture monoclonal antibodies which specifically bind the protein.
 The invention provides a purified antibody that specifically
binds a protein expressed in breast cancer. The invention also provides
a method for using an antibody to detect expression of a protein
in a sample comprising combining the antibody with a sample under
conditions which allow the formation of antibody:protein complexes
and detecting complex formation, wherein complex formation indicates
expression of the protein in the sample.
DESCRIPTION OF THE SEQUENCE LISTING AND TABLES
 A portion of the disclosure of this patent document contains
material that 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 cDNAs and encoded
proteins obtained by sequencing and extension of clone inserts.
Each sequence is identified by a sequence identification number
(SEQ ID NO) and by the Incyte identification number (Incyte ID No)
from which it was obtained.
 Table 1 lists the differential expression values of clones
representing the cDNAs of the present invention that are differentially
expressed in both tumorigenic, nonmetastatic (MCF7, BT20, T47D)
and metastatic (MDA-mb-231) breast carcinoma cells. Columns 1 and
2 show the Incyte Clone ID and differential expression values, respectively.
Columns 3 and 4 show the tumor cell lines in which differential
expression was measured relative to the non-tumorigenic breast cell
lines, MCF10A (column 3), and HMEC (column 4).
 Table 2 lists similar data to that found in Table 1 for
clones representing cDNAs differentially expressed in metastatic
breast adenocarcinoma cells only (MDA-mb-231). Column 1 lists the
Incyte Clone ID, and columns 2 and 3 list the differential expression
value observed in the metastatic cell line, MDA-mb-231, relative
to HMEC cells (column 2) and MCF10A cells (column 3).
 Table 3 links the differentially expressed clones on a microarray
with Incyte cDNA templates. Columns 1 and 2 show the SEQ ID NO and
TEMPLATE ID, respectively. Column 3 shows the CLONE ID and columns
4 and 5 show the first residue (START) and last residue (STOP) encompassed
by the clone on the template.
 Table 4 shows Incyte nucleotide templates presented in the
Sequence Listing and the corresponding protein templates encoded
by these cDNAs, also presented in the Sequence Listing. Columns
1 and 2 show the SEQ ID NO and the Nucleotide Template ID, respectively,
and columns 3 and 4 show the corresponding SEQ ID NO and Protein
Template ID, respectively.
 Table 5 shows the annotation of both nucleotide and protein
Template IDs of the invention to sequences in GenBank. Columns 1
and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns
3, 4, and 5 show the GenBank hit (GI Number), probability score
(E-value), and functional annotation, respectively, as determined
by BLAST analysis (version 1.4 using default parameters; Altschul
(1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol
215:403-410) of the cDNA against GenBank (release 116; National
Center for Biotechnology Information (NCBI), Bethesda Md.).
 Table 6 shows Pfam annotations of the cDNAs and proteins
of the present invention. Columns 1 and 2 show the SEQ ID NO and
TEMPLATE ID, respectively. Columns 3 and 4 show the first residue
(START), last residue (STOP), respectively, for the segment of the
cDNA or protein identified by Pfam analysis. Column 5 shows the
reading frame for cDNA sequences. Columns 6 and 7 show the Pfam
hit and Pfam description, respectively, corresponding to the polypeptide
domain encoded by the cDNA segment or found in the protein sequence,
and column 8 shows the E-value for the annotation.
 Table 7 shows signal peptide and transmembrane regions predicted
within the cDNAs of the present invention and in the proteins of
the invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID,
respectively. Columns 3 and 4 show the first residue (START), last
residue (STOP), respectively, for the segment of the cDNA or the
protein identified as a signal peptide or transmembrane region,
and column 5 shows the reading frame for cDNA sequences. Column
6 identifies the polypeptide region as either a signal peptide (SP)
or transmembrane (TM) domain.
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 nucleotide sequence which is completely
complementary over the full length of the sequence and which will
hybridize to the nucleic acid molecule under conditions of high
 A "combination" comprises at least two and up
to 194 sequences selected from the group consisting of SEQ ID NOs:1-194
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 400 to
about 10,000 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
Evol36: 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
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.
 "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.
 "Identity" 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), CLUSTALW (Thompson et al. (1994) Nucleic
Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997) supra).
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. "Similarity"
as applied to proteins uses the same algorithms but takes into account
conservative substitutions of nucleotides or residues.
 "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 which retains at least one biological or antigenic
characteristic of a native protein; 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. 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 preferably
60% free, and more preferably 90% free from other components with
which it is naturally associated.
 "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 or tissue biopsy; 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.
 "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
maybe 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 for a combination comprising
a plurality of cDNAs or their complements, SEQ ID NOs:1, 3, 5, 7,
8, 10-14, 16, 18, 19, 21-40, 42-48, 50, 51, 53-55, 57-65, 67-70,
72-74, 76-80, 82-85, 87, 88, 90, 92, 94, 96-108, 109, 111, 113,
115, 116, 117, 119, 121, 123-126, 128, 130, 131, 133, 134, 135,
137, 138, 140, 142-144, 146, 148, 150, 152, 153, 155-158, 160, 161,
163-183, 185, 187-192, and 194 which may be used on a substrate
to diagnose, to stage, to treat or to monitor the progression or
treatment of breast cancer. These cDNAs represent known and novel
genes differentially expressed in breast adenocarcinoma cells. SEQ
ID NOs:21, 50, 79, 100, 105, 106, 107, 178, 181, 190, and 191 represent
novel cDNAs associated with breast cancer. Since the novel cDNAs
were identified solely by their differential expression, it is not
essential to know a priori the name, structure, or function of the
gene or it's encoded protein. The usefulness of the novel cDNAs
exists in their immediate value as diagnostics for breast cancer.
 Table 1 shows cDNA clones on an array having at least a
3-fold increase (upregulated) or decrease (downregulated, indicated
by a minus sign) in either tumorigenic, non-metastatic breast adenocarcinoma
cells (MCF7, T47D, or BT20), or in metastatic adenocarcinoma cells
(MDA-mb-231) relative to non-tumorigenic breast cells (MCF10A and
HMEC). Columns 1 and 2 show the Clone ID and differential expression
value, respectively. The value given in column 2 is representative
of the differential expression observed in all of the tumor cell
lines indicated in columns 3 and 4. Columns 3 and 4 show the tumor
cell lines in which differential expression was observed relative
to non-tumorigenic breast cell lines MCF10A (column 3) and HMEC
(column 4), respectively. The numerical designations of the cell
lines given in columns 3 and 4 are as follows: 1=MCF7; 2=T47D; 3=BT20;
4=MDA-mb-231. These genes are useful in the diagnosis of, or monitoring
the treatment of breast cancer, particularly at an early stage,
e.g., prior to metastasis.
 Table 2 shows cDNA clones on an array having at least a
3-fold increase or decrease in only the metastatic breast adenocarcinoma
cell line, MDA-mb-231, relative to non-tumorigenic cells. These
cDNA clones were not observed to be differentially regulated in
either the tumorigenic, non-metastatic cell lines MCF7, T47D, and
BT20, or the non-tumorigenic cells, MCF10A and HMEC. These genes
are particularly useful for the diagnosis or monitoring the treatment
of an advanced stage of breast cancer in which metastasis to other
organs or tissues may have occurred, or has the potential to occur.
 Tables 3 and 4 further link the differentially expressed
cDNA clones to full-length genes and to proteins in the Incyte database,
and Table 5 provides the annotation of these sequences to known
proteins in GenBank. Tables 6 and 7 provide further identification
of encoded protein sequences by Pfam and the presence of signal
peptide or transmembrane regions.
 Of particular interest in Table 5 are several genes annotated
to known breast tumor markers. SEQ ID NO:11 is identified as a human
S100A2 gene; SEQ ID NO:24 is identified as a human maspin mRNA;
SEQ ID NO:28 is identified as a human secreted frizzled related
protein mRNA; SEQ ID NO:48 is identified as a human matrix G1 a
protein mRNA; and SEQ ID NO:80 is identified as a human mRNA for
Drg1 protein. As shown in Tables 1-3, these genes are derived from
Incyte clones that are differentially expressed in tumor cells in
agreement with the previously observed differential expression patterns
of the known genes in human breast tumors or human breast tumor
cell lines. It is also noteworthy that the majority of differentially
expressed genes in the metastatic cell line MDA-mb-231, shown in
Table 2, are upregulated (58 of 62 or approximately 94%). In this
particular cell model, suppressive mechanisms (downregulation) are
operative in the tumorigenic conversion of the cell type, while
inductive mechanisms (upregulation) are involved in the progression
of the cell type to metastasis.
 The cDNAs of the invention define differential expression
patterns against which to compare the expression pattern of biopsied
breast tissue to determine the presence of breast cancer (SEQ ID
NOs:1, 3, 5, 7, 8, 10-14, 16, 18, 19, 21-40, 42-48, 50, 51, 53-55,
57-65, 67-70, 72-74, 76-80, 82-85, 87, 88, 90, 92, 94, 96-109, 111,
113, 115, 116, 117, 119, 121, 123-126, 128, 130, 131, 133, 134,
135, 137, 138, 140, 142-144, 146, 148, 150, 152, 153, 155-158, 160,
161, 163-183, 185, 187-192, and 194), or more particularly, the
presence of an advanced stage of the disease (SEQ ID NOs:109, 111,
113, 115, 116, 117, 119, 121, 123-126, 128, 130, 131, 133, 134,
135, 137, 138, 140, 142-144, 146, 148, 150, 152, 153, 155-158, 160,
161, 163-183, 185, 187-192, and 194). Experimentally, differential
expression of the cDNAs can be evaluated by methods including, but
not limited to, differential display by spatial immobilization or
by gel electrophoresis, genome mismatch scanning, representational
discriminant analysis, clustering, transcript imaging and array
technologies. These methods may be used alone or in combination.
 In one embodiment, an additional set of cDNAs, such as cDNAs
encoding signaling molecules, are arranged on the substrate with
the combination. Such combinations may be useful in the elucidation
of pathways which are affected in a particular disorder or to identify
new, coexpressed, candidate, therapeutic molecules.
 In another embodiment, the combination can be used for large
scale genetic or gene expression analysis of a large number of novel,
nucleic acid molecules. These samples are prepared by methods well
known in the art and are from mammalian cells or tissues which are
in a certain stage of development; have been treated with a known
molecule or compound, such as a cytokine, growth factor, a drug,
and the like; or have been extracted or biopsied from a mammal with
a known or unknown condition, disorder, or disease before or after
treatment. The sample nucleic acid molecules are hybridized to the
combination for the purpose of defining a novel gene profile associated
with that developmental stage, treatment, or disorder.
 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 an array,
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 (Roger Sayle, University
of Massachusetts, Amherst Mass.) 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 LIFESEQ 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
include 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 in breast cancer; absence,
presence, or excess expression of mRNAs; or to monitor mRNA levels
during therapeutic intervention. 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 art.
 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 cDNA composition
of the invention is 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; 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.).
 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 an immune response.
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. Nos. 5,175,383
and 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.
 I Construction of cDNA Libraries
 RNA was purchased from Clontech Laboratories (Palo Alto
Calif.) or isolated from various tissues. Some tissues were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such
as TRIZOL reagent (Life Technologies, Rockville Md.). The resulting
lysates were centrifuged over CsCl cushions or extracted with chloroform.
RNA was precipitated with either isopropanol or ethanol and sodium
acetate, or by other routine methods.
 Phenol extraction and precipitation of RNA were repeated
as necessary to increase RNA purity. In most cases, RNA was treated
with DNase. For most libraries, poly(A) RNA was isolated using oligo
d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles
(Qiagen, Valencia Calif.), or an OLIGOTEX mRNA purification kit
(Qiagen). Alternatively, poly(A) RNA was isolated directly from
tissue lysates using other kits, including the POLY(A)PURE mRNA
purification kit (Ambion, Austin Tex.).
 In some cases, Stratagene (La Jolla Calif.) was provided
with RNA and constructed the corresponding cDNA libraries. Otherwise,
cDNA was synthesized and cDNA libraries were constructed with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system
(Life Technologies) using the recommended procedures or similar
methods known in the art. (See Ausubel, supra, Units 5.1 through
6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double
stranded cDNA, and the cDNA was digested with the appropriate restriction
enzyme or enzymes. For most libraries, the cDNA was size-selected
(300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE
CL4B column chromatography (APB) or preparative agarose gel electrophoresis.
cDNAs were ligated into compatible restriction enzyme sites of the
polylinker of the pBLUESCRIPT phagemid (Stratagene), pSPORT1 plasmid
(Life Technologies), or pINCY plasmid (Incyte Genomics, Inc., Palo
Alto Calif.). Recombinant plasmids were transformed into XL1-BLUE,
XL1-BLUEMRF, or SOLR competent E. coli cells (Stratagene) or DHII5.alpha.,
DH10B, or ELECTROMAX DH10B competent E. coli cells (Life Technologies).
 In some cases, libraries were superinfected with a 5.times.
excess of the helper phage, M13K07, according to the method of Vieira
et al. (1987, Methods Enzymol. 153:3-11) and normalized or subtracted
using a methodology adapted from Soares (1994, Proc Natl Acad Sci
91:9228-9232), Swaroop et al. (1991, Nucl Acids Res 19:1954), and
Bonaldo et al. (1996, Genome Research 6:791-806). The modified Soares
normalization procedure was utilized to reduce the repetitive cloning
of highly expressed high abundance cDNAs while maintaining the overall
sequence complexity of the library. Modification included significantly
longer hybridization times which allowed for increased gene discovery
rates by biasing the normalized libraries toward those infrequently
expressed low-abundance cDNAs which are poorly represented in a
standard transcript image (Soares et al., supra).
 II Isolation and Sequencing of cDNA Clones
 Plasmids were recovered from host cells by in vivo excision
using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids
were purified using one of the following: the Magic or WIZARD MINIPREPS
DNA purification system (Promega); the AGTC MINIPREP purification
kit (Edge BioSystems, Gaithersburg Md.); the QIAWELL 8, QIAWELL
8 Plus, or QIAWELL 8 Ultra plasmid purification systems, or the
REAL PREP 96 plasmid purification kit (QIAGEN). Following precipitation,
plasmids were resuspended in 0.1 ml of distilled water and stored,
with or without lyophilization, at 4.degree. C.
 Alternatively, plasmid DNA was amplified from host cell
lysates using direct link PCR in a high-throughput format (Rao (1994)
Anal Biochem 216:1-14). Host cell lysis and thermal cycling steps
were carried out in a single reaction mixture. Samples were processed
and stored in 384-well plates, and the concentration of amplified
plasmid DNA was quantified fluorometrically using PICOGREEN dye
(Molecular Probes) and a FLUOROSKAN II fluorescence scanner (Labsystems
Oy, Helsinki, Finland).
 cDNA sequencing reactions were processed using standard
methods or high-throughput instrumentation such as the ABI CATALYST
800 thermal cycler (Applied Biosystems) or the DNA ENGINE thermal
cycler (M J Research, Watertown Mass.) in conjunction with the HYDRA
microdispenser (Robbins Scientific, Sunnyvale Calif.) or the MICROLAB
2200 system (Hamilton, Reno Nev.). cDNA sequencing reactions were
prepared using reagents provided by APB or supplied in ABI sequencing
kits such as the ABI PRISM BIGDYE cycle sequencing kit (Applied
Biosystems). Electrophoretic separation of cDNA sequencing reactions
and detection of labeled cDNAs were carried out using the MEGABACE
1000 DNA sequencing system (APB); the ABI PRISM 373 or 377 sequencing
systems (Applied Biosystems) in conjunction with standard ABI protocols
and base calling software; or other sequence analysis systems known
in the art. Reading frames within the cDNA sequences were identified
using standard methods (reviewed in Ausubel, supra, Unit 7.7).
 III Extension of cDNA Sequences
 Nucleic acid sequences were extended using the cDNA clones
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 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 avoided.
 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. Preferred libraries are
ones that have been size-selected to include larger cDNAs. Also,
random primed libraries are preferred because they will contain
more sequences with the 5' and upstream regions of genes. A randomly
primed library is particularly useful if an oligo d(T) library does
not yield a full-length cDNA.
 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). 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: 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., 5 min; Step 7: storage at 4.degree.
C. In the alternative, the parameters for primer pair T7 and SK+
(Stratagene) 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: Steps 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 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 Costar, 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 mini-gel to determine which reactions were successful
in extending the sequence.
 The extended nucleic acids 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 pUC18 vector (APB). For shotgun
sequencing, the digested nucleic acids were separated on low concentration
(0.6 to 0.8%) agarose gels, fragments were excised, and agar digested
with AGARACE enzyme (Promega). Extended clones were religated using
T4 DNA ligase (New England Biolabs, Beverly Mass.) into pUC18 vector
(APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction
site overhangs, and transformed into competent E. coli cells. Transformed
cells were selected on antibiotic-containing media, and individual
colonies were picked and cultured overnight at 37.degree. C. in
384-well plates in LB/2.times. carbenicillin liquid media.
 The cells were lysed, and DNA was amplified by PCR using
Taq DNA polymerase (APB) 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
using PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA recoveries were reamplified using the same 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).
 IV Assembly and Analysis of Sequences
 Component nucleotide sequences from chromatograms were subjected
to PHRED analysis (Phil Green, University of Washington, Seattle
Wash.) and assigned a quality score. The sequences having at least
a required quality score were subject to various pre-processing
algorithms to eliminate low quality 3' ends, vector and linker sequences,
polyA tails, Alu repeats, mitochondrial and ribosomal sequences,
bacterial contamination sequences, and sequences smaller than 50
base pairs. Sequences were screened using the BLOCK 2 program (Incyte
Genomics), a motif analysis program based on sequence information
contained in the SWISS-PROT and PROSITE databases (Bairoch et al.
(1997) Nucleic Acids Res 25:217-221; Attwood et al. (1997) J Chem
Inf Comput Sci 37:417-424).
 Processed sequences were subjected to assembly procedures
in which the sequences were assigned to bins, one sequence per bin.
Sequences in each bin were assembled to produce consensus sequences,
templates. Subsequent new sequences were added to existing bins
using BLAST (Altschul (supra); Altschul et al. (supra); Karlin et
al. (1988) Proc Natl Acad Sci 85:841-845), BLASTn (vers. 1.4, WashU),
and CROSSMATCH software (Phil Green, supra). Candidate pairs were
identified as all BLAST hits having a quality score greater than
or equal to 150. Alignments of at least 82% local identity were
accepted into the bin. The component sequences from each bin were
assembled using PHRAP (Phil Green, supra). Bins with several overlapping
component sequences were assembled using DEEP PHRAP (Phil Green,
 Bins were compared against each other, and those having
local similarity of at least 82% were combined and reassembled.
Reassembled bins having templates of insufficient overlap (less
than 95% local identity) were re-split. Assembled templates were
also subjected to analysis by STITCHER/EXON MAPPER algorithms which
analyzed the probabilities of the presence of splice variants, alternatively
spliced exons, splice junctions, differential expression of alternative
spliced genes across tissue types, disease states, and the like.
These resulting bins were subjected to several rounds of the above
assembly procedures to generate the template sequences found in
the LIFESEQ GOLD database (Incyte Genomics).
 The assembled templates were annotated using the following
procedure. Template sequences were analyzed using BLASTn (vers.
2.0, NCBI) versus GBpri (GenBank vers. 117). "Hits" were
defined as an exact match having from 95% local identity over 200
base pairs through 100% local identity over 100 base pairs, or a
homolog match having an E-value equal to or greater than 1.times.10.sup.-8.
(The "E-value" quantifies the statistical probability
that a match between two sequences occurred by chance). The hits
were subjected to frameshift FASTx versus GENPEPT (GenBank version
117). In this analysis, a homolog match was defined as having an
E-value of 1.times.10.sup.-8. The assembly method used above was
described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999, and
the LIFESEQ GOLD user manual (Incyte Genomics).
 Following assembly, template sequences were subjected to
motif, BLAST, Hidden Markov Model (HMM; Pearson and Lipman (1988)
Proc Natl Acad Sci 85:2444-2448; Smith and Waterman (1981) J Mol
Biol 147:195-197), and functional analyses, and categorized in protein
hierarchies using methods described in U.S. Ser. No. 08/812,290,
filed Mar. 6, 1997; U.S. Ser. No. 08/947,845, filed Oct. 9, 1997;
U.S. Pat. No. 5,953,727; and U.S. Ser. No. 09/034,807, filed Mar.
4, 1998. Template sequences may be further queried against public
databases such as the GenBank rodent, mammalian, vertebrate, eukaryote,
prokaryote, and human EST databases.
 V Selection of Sequences, Microarray Preparation and Use
 Incyte clones represent template sequences derived from
the LIFESEQ GOLD assembled human sequence database (Incyte Genomics).
In cases where more than one clone was available for a particular
template, the 5'-most clone in the template was used on the microarray.
The HUMAN GENOME GEM series 1-3 microarrays (Incyte Genomics) contain
28,626 array elements which represent 10,068 annotated clusters
and 18,558 unannotated clusters. For the UNIGEM series microarrays
(Incyte Genomics), Incyte clones were mapped to non-redundant Unigene
clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol
Med 75:694-698), and the 5' clone with the strongest BLAST alignment
(at least 90% identity and 100 bp overlap) was chosen, verified,
and used in the construction of the microarray. The UNIGEM V microarray
(Incyte Genomics) contains 7075 array elements which represent 4610
annotated genes and 2,184 unannotated clusters. Table 5 shows the
GenBank annotations for SEQ ID NOs:1-194 of this invention as produced
by BLAST analysis.
 To construct microarrays, cDNAs were amplified from bacterial
cells using primers complementary to vector sequences flanking the
cDNA insert. Thirty cycles of PCR increased the initial quantity
of cDNAs from 1-2 ng to a final quantity greater than 5 .mu.g. Amplified
cDNAs were then purified using SEPIIACRYL-400 columns (APB). Purified
cDNAs were immobilized on polymer-coated glass slides. Glass microscope
slides (Corning, Corning N.Y.) were cleaned by ultrasound in 0.1%
SDS and acetone, with extensive distilled water washes between and
after treatments. Glass slides were etched in 4% hydrofluoric acid
(VWR Scientific Products, West Chester Pa.), washed thoroughly in
distilled water, and coated with 0.05% aminopropyl silane (Sigma
Aldrich) in 95% ethanol. Coated slides were cured in a 110.degree.
C. oven. cDNAs were applied to the coated glass substrate using
a procedure described in U.S. Pat. No. 5,807,522. One microliter
of the cDNA at an average concentration of 100 ng/ul was loaded
into the open capillary printing element by a high-speed robotic
apparatus which then deposited about 5 nl of cDNA per slide.
 Microarrays were UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene), and then washed at room temperature once in 0.2% SDS
and three times in distilled water. Non-specific binding sites were
blocked by incubation of microarrays in 0.2% casein in phosphate
buffered saline (Tropix, Bedford Mass.) for 30 minutes at 60.degree.
C. followed by washes in 0.2% SDS and distilled water as before.
 VI Preparation of Samples
 Propagation of Human Epithelial Cell Lines
 HMEC is a human primary mammary epithelial cell strain derived
from normal mammary tissue (Clonetics San Diego, Calif.). The following
cell lines were obtained from ATCC (Manassus, Va.): MCF10A is a
breast mammary gland cell line derived from a 36-year old female
with fibrocystic breast disease; MCF7 is a breast adenocarcinoma
cell line derived from the pleural effusion of a 69-year old female;
T47D is a breast carcinoma cell line derived from a pleural effusion
from a 54-year old female with an infiltrating ductal carcinoma
of the breast; BT20 is a breast carcinoma cell line derived in vitro
from cells emigrating out of thin slices of a tumor mass isolated
from a 74-year old female; MDA-mb-231 is a metastatic breast tumor
cell line derived from the pleural effusion of a 51-year old female
with metastatic breast carcinoma. All cell cultures were propagated
in media according to the supplier's recommendations and grown to
70-80% confluence prior to RNA isolation.
 Isolation and Labeling of Sample cDNAs
 Cells were harvested and lysed in 1 ml of TRIZOL reagent
(5.times.10.sup.6 cells/ml; Life Technologies). The lysates were
vortexed thoroughly and incubated at room temperature for 2-3 minutes
and extracted with 0.5 ml chloroform. The extract was mixed, incubated
at room temperature for 5 minutes, and centrifuged at 15,000 rpm
for 15 minutes at 4.degree. C. The aqueous layer was collected and
an equal volume of isopropanol was added. Samples were mixed, incubated
at room temperature for 10 minutes, and centrifuged at 15,000 rpm
for 20 minutes at 4.degree. C. The supernatant was removed and the
RNA pellet was washed with 1 ml of 70% ethanol, centrifuged at 15,000
rpm at 4.degree. C., and resuspended in RNase-free water. The concentration
of the RNA was determined by measuring the optical density at 260
 Poly(A) RNA was prepared using an OLIGOTEX mRNA kit (QIAGEN)
with the following modifications: OLIGOTEX beads were washed in
tubes instead of on spin columns, resuspended in elution buffer,
and then loaded onto spin columns to recover mRNA. To obtain maximum
yield, the mRNA was eluted twice.
 Each poly(A) RNA sample was reverse transcribed using MMLV
reverse-transcriptase, 0.05 pg/.mu.l oligo-d(T) primer (21 mer),
1.times. first strand buffer, 0.03 units/ul RNase inhibitor, 500
uM dATP, 500 uM dGTP, 500 uM dTTP, 40 uM dCTP, and 40 uM either
dCTP-Cy3 or dCTP-Cy5 (APB). The reverse transcription reaction was
performed in a 25 ml volume containing 200 ng poly(A) RNA using
the GEMBRIGHT kit (Incyte Genomics). Specific control poly(A) RNAs
(YCFR06, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25,
YCFR44, YCFR26) were synthesized by in vitro transcription from
non-coding yeast genomic DNA (W. Lei, unpublished). As quantitative
controls, control mRNAs (YCFR06, YCFR45, YCFR67, and YCFR85) at
0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse transcription
reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100 (w/w) to
sample mRNA, respectively. To sample differential expression patterns,
control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were
diluted into reverse transcription reaction at ratios of 1:3, 3:1,
1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA. Reactions were incubated
at 37.degree. C. for 2 hr, treated with 2.5 ml of 0.5M sodium hydroxide,
and incubated for 20 minutes at 85.degree. C. to the stop the reaction
and degrade the RNA.
 cDNAs were purified using two successive CHROMA SPIN 30
gel filtration spin columns (Clontech). Cy3- and Cy1-labeled reaction
samples were combined as described below and ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300
ml of 100% ethanol. The cDNAs were then dried to completion using
a SpeedVAC system (Savant Instruments, Holbrook N.Y.) and resuspended
in 14 .mu.l 5.times. SSC, 0.2% SDS.
 VII Hybridization and Detection
 Hybridization reactions contained 9 .mu.l of sample mixture
containing 0.2 .mu.g each of Cy3 and Cy5 labeled cDNA synthesis
products in 5.times. SSC, 0.2% SDS hybridization buffer. The mixture
was heated to 65.degree. C. for 5 minutes and was aliquoted onto
the microarray surface and covered with an 1.8 cm.sup.2 coverslip.
The microarrays were transferred to a waterproof chamber having
a cavity just slightly larger than a microscope slide. The chamber
was 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 microarrays was incubated for about 6.5 hours at 60.degree.
C. The microarrays were washed for 10 min at 45.degree. C. in low
stringency wash buffer (1.times. SSC, 0.1% SDS), three times for
10 minutes each at 45.degree. C. in high stringency wash buffer
(0.1.times. SSC), and dried.
 Reporter-labeled hybridization complexes were 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 was focused on the microarray using a 20.times.
microscope objective (Nikon, Melville N.Y.). The slide containing
the microarray was placed on a computer-controlled X-Y stage on
the microscope and raster-scanned past the objective. The 1.8 cm.times.1.8
cm microarray used in the present example was scanned with a resolution
of 20 micrometers.
 In two separate scans, the mixed gas multiline laser excited
the two fluorophores sequentially. Emitted light was split, based
on wavelength, into two photomultiplier tube detectors (PMT R1477;
Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to
the two fluorophores. Appropriate filters positioned between the
microarray and the photomultiplier tubes were used to filter the
signals. The emission maxima of the fluorophores used were 565 nm
for Cy3 and 650 nm for Cy1. Each microarray was typically scanned
twice, one scan per fluorophore using the appropriate filters at
the laser source, although the apparatus was capable of recording
the spectra from both fluorophores simultaneously.
 The sensitivity of the scans was calibrated using the signal
intensity generated by a cDNA control species. Samples of the calibrating
cDNA were separately labeled with the two fluorophores and identical
amounts of each were added to the hybridization mixture. A specific
location on the microarray contained 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 was 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 were displayed as an image where the signal intensity
was mapped using a linear 20-color transformation to a pseudocolor
scale ranging from blue (low signal) to red (high signal). The data
was also analyzed quantitatively. Where two different fluorophores
were excited and measured simultaneously, the data were first corrected
for optical crosstalk (due to overlapping emission spectra) between
the fluorophores using each fluorophore's emission spectrum.
 A grid was superimposed over the fluorescence signal image
such that the signal from each spot was centered in each element
of the grid. The fluorescence signal within each element was then
integrated to obtain a numerical value corresponding to the average
intensity of the signal. The software used for signal analysis was
the GEMTOOLS gene expression analysis program (Incyte Genomics).
Significance was defined as signal to background ratio exceeding
2.times. and area hybridization exceeding 40%.
 VIII Data Analysis and Results
 The expression of cDNAs from the four tumor cell lines representing
various stages of breast tumor progression (MCF7, T47D, BT20, and
MDA-mb-231) were compared with that of two non-malignant mammary
epithelial cell lines, HMEC and MCF10A. Array elements that exhibited
at least a 3-fold change in expression and a signal intensity over
250 units, a signal-to-background ratio of at least 2.5, and an
element spot size of at least 40% were identified as differentially
expressed using the GEMTOOLS program (Incyte Genomics). Moreover,
only array elements were selected that exhibited these changes in
at least two tumor cell lines compared with both non-malignant controls.
The cDNAs that are differentially expressed under these conditions
are shown in Table 1. Table 1 identifies both upregulated and downregulated
cDNAs. Downregulated cDNAs are indicated by a minus (-) sign. The
cDNAs are identified by their Incyte Clone ID. These genes are useful
diagnostic markers or as potential therapeutic targets for breast
cancer, particularly in its early stages prior to metastasis.
 Table 2 identifies upregulated and downregulated cDNAs that
are differentially expressed in the metastatic MDA-mb-231 breast
carcinoma cell line that are not differentially expressed in the
tumorigenic but non-metastatic cell lines MCF7, T47D, and BT20.
These genes are useful diagnostic markers or potential therapeutic
targets for an advanced stage of breast cancer where metastasis
to other organs or tissues may have occurred, or may potentially
 IX Other Hybridization Technologies and Analyses
 Other hybridization technologies utilize a variety of substrates
such as nylon membranes, capillary tubes, etc. Arranging cDNAs on
polymer coated slides is described in Example V; sample cDNA preparation
and hybridization and analysis using polymer coated slides is described
in examples VI and VII, respectively.
 The cDNAs are applied to a membrane 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 37.degree. C. 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
 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.
 Hybridization probes derived from 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 100.degree. C. 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 microliters of [.sup.32P]dCTP is added
to the tube, and the contents are incubated at 37.degree. C. 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 100.degree. C. for five min and then snap cooled for two min
 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 55.degree. C. 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 55.degree. C. for 16 hr. Following hybridization, the membrane
is washed for 15 min at 25.degree. C. in 1 mM Tris (pH 8.0), 1%
Sarkosyl, and four times for 15 min each at 25.degree. C. 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 -70.degree. C., developed, and examined.
 X Further Characterization of Differentially Expressed cDNAs
 Clones were blasted against the LIFESEQ Gold 5.1 database
(Incyte Genomics) and an Incyte template and its sequence variants
were chosen for each clone. The template and variant sequences were
blasted against GenBank database to acquire annotation. The nucleotide
sequences were translated into amino acid sequence which was blasted
against the GenPept and other protein databases to acquire annotation
and characterization, i.e., structural motifs.
 Percent sequence identity can be determined electronically
for two or more amino acid or nucleic acid sequences using the MEGALIGN
program (DNASTAR). The percent identity between two amino acid sequences
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
 Sequences with conserved protein motifs may be searched
using the BLOCKS search program. This program analyses sequence
information contained in the Swiss-Prot and PROSITE databases and
is useful for determining the classification of uncharacterized
proteins translated from genomic or cDNA sequences (Bairoch et al.
(supra); Attwood et al. (supra). PROSITE database 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.
 XI 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.).
 XII 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.
 XIII Purification of Naturally Occurring Protein Using Specific
 Naturally occurring or recombinant protein is substantially
purified by immunoaffinity chromatography using antibodies specific
for 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 protein is collected.
 XIV Screening Molecules for Specific Binding with the cDNA
 The cDNA or fragments thereof and the protein or portions
thereof are labeled with .sup.32P-dCTP, Cy3-dCTP, Cy5-dCTP (APB),
or BIODIPY or FITC (Molecular Probes), respectively. Candidate molecules
or compounds previously arranged on a substrate are incubated in
the presence of labeled nucleic or amino acid. After incubation
under conditions for either a cDNA or a protein, the substrate is
washed, and any position on the substrate retaining label, which
indicates specific binding or complex formation, is assayed. The
binding molecule is identified by its arrayed position on the substrate.
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. High throughput screening
using very small assay volumes and very small amounts of test compound
is fully described in Burbaum et al. U.S. Pat. No. 5,876,946.
 All patents and publications mentioned in the specification
are incorporated herein by reference. Various modifications and
variations of the described method and system of the invention will
be apparent to those skilled in the art without departing from the
scope and spirit of the invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various modifications
of the 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.