U.S. patent application number 10/408017 was filed with the patent office on 2003-09-11 for detection of genes regulated by egf in breast cancer.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Faris, Mary, Streeter, David G..
Application Number | 20030170717 10/408017 |
Document ID | / |
Family ID | 26849667 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170717 |
Kind Code |
A1 |
Faris, Mary ; et
al. |
September 11, 2003 |
Detection of genes regulated by EGF in breast cancer
Abstract
The present invention relates to a combination comprising a
plurality of polynucleotide probes that are modulated in response
to EGF and which are associated with breast cancer, and which may
be used in their entirety or in part as to diagnose, to stage, to
treat, or to monitor the treatment of a subject with a breast
cancer.
Inventors: |
Faris, Mary; (Los Angeles,
CA) ; Streeter, David G.; (Boulder Creek,
CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Corporation
3160 Porter Drive
Palo Alto
CA
94304
|
Family ID: |
26849667 |
Appl. No.: |
10/408017 |
Filed: |
April 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10408017 |
Apr 3, 2003 |
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09653119 |
Aug 31, 2000 |
|
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6544742 |
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60152548 |
Sep 3, 1999 |
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Current U.S.
Class: |
435/6.14 ;
435/6.16; 536/23.2 |
Current CPC
Class: |
C12Q 2600/136 20130101;
C12Q 2600/158 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
435/6 ;
536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A combination comprising a plurality of cDNAs, selected from SEQ
ID NOs:1-16 or their complements, whose expression is modulated by
EGF and is associated with breast cancer.
2. The combination of claim 1, wherein the cDNAs are immobilized on
a substrate.
3. A high throughput method for detecting differential expression
of one or more cDNAs in a sample containing nucleic acids, the
method comprising: (a) hybridizing the substrate of claim 2 with
nucleic acids of the sample, thereby forming one or more
hybridization complexes; (b) detecting the hybridization complexes;
and (c) comparing the hybridization complexes with those of a
standard, wherein differences between the standard and sample
hybridization complexes indicate differential expression of cDNAs
in the sample.
4. The method of claim 3, where in the nucleic acids of the sample
are amplified prior to hybridization.
5. The method of claim 3, wherein the sample is from a subject with
a breast carcinoma and comparison with a standard defines an early,
mid, or late stage of that disease.
6. A high throughput method of screening a plurality of molecules
or compounds to identify a ligand which specifically binds a cDNA,
the method comprising: (a) combining the combination of claim 1
with the plurality of molecules or compounds under conditions to
allow specific binding; and (b) detecting specific binding between
each cDNA and at least one molecule or compound, thereby
identifying a ligand that specifically binds to each cDNA.
7. The method of claim 6 wherein the plurality of molecules or
compounds are selected from DNA molecules, RNA molecules, peptide
nucleic acid molecules, mimetics, peptides, transcription factors,
repressors, and regulatory proteins.
8. An isolated cDNA, or the complement thereof, selected from SEQ
ID NOs:12-16.
9. A composition comprising a cDNA of claim 8 in conjunction with a
suitable pharmaceutical carrier.
10. A method of using a cDNA to purify a ligand that specifically
binds the cDNA, the method comprising; a) combining a cDNA of claim
8 with a sample under conditions which allow specific binding; b)
recovering the cDNA bound to the ligand; and c) separating the cDNA
from the ligand thereby obtaining purified ligand.
11. A vector containing a probe selected from the cDNA of claim
8.
12. A host cell containing the vector of claim 11.
13. A method for producing a protein, the method comprising the
steps of: (a) culturing the host cell of claim 12 under conditions
for expression of protein; and (b) recovering the protein from the
host cell culture.
14. A protein or a portion thereof produced by the method of claim
13.
15. A high-throughput method for using a protein to screen a
plurality of molecules or compounds to identify at least one ligand
which specifically binds the protein, the method comprising: (a)
combining the protein of claim 14 with the plurality of molecules
or compounds under conditions to allow specific binding; and (b)
detecting specific binding between the protein and a molecule or
compound, thereby identifying a ligand which specifically binds the
protein.
16. The method of claim 15 wherein the plurality of molecules or
compounds is selected from DNA molecules, RNA molecules, peptide
nucleic acid molecules, mimetics, peptides, proteins, agonists,
antagonists, antibodies or their fragments, immunoglobulins,
inhibitors, drug compounds, and pharmaceutical agents.
17. A method of using a protein to produce an antibody, the method
comprising: a) immunizing an animal with the protein of claim 14
under conditions to elicit an antibody response; b) isolating
animal antibodies; and c) screening the isolated antibodies with
the protein, thereby identifying an antibody which specifically
binds the protein.
18. A method of purifying an antibody, the method comprising: a)
combining the protein of claim 14 with a sample under conditions to
allow specific binding; b) recovering the bound protein; and c)
separating the protein from the antibody, thereby obtaining
purified antibody.
Description
[0001] This application is a continuation application of U.S.
application Ser. No. 09/653,119, filed on Aug. 31, 2000, which
claims the benefit of U.S. Provisional Application No. 60/152,548,
our Docket No. PA-0018 P, filed on Sep. 3, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to a composition comprising a
plurality of polynucleotide probes which may be used in detecting
expression of genes modulated in response to EGF, and which are
associated with breast cancer.
BACKGROUND OF THE INVENTION
[0003] Intercellular communication is essential for the development
and survival of multicellular organisms. Communication is achieved
through the secretion of proteins by signaling cells and the
internalization of these proteins by target cells. Growth factors
are secreted proteins that mediate communication between signaling
and target cells. The secreted growth factors bind to specific
receptors on the surfaces of target cells, and bound receptors
trigger second messenger signal transduction pathways. These signal
transduction pathways elicit specific cellular responses in the
target cells. Such responses can include the modulation of gene
expression and the stimulation or inhibition of cell division, cell
differentiation, and cell motility.
[0004] Epidermal growth factor (EGF) is a member of a broad class
of polypeptide growth factors that generally act as mitogens in
diverse cell types to stimulate wound healing, bone synthesis and
remodeling, extracellular matrix synthesis, and proliferation of
epithelial, epidermal, and connective tissues. In addition, EGF
produces non-mitogenic effects in certain tissues. The EGF receptor
(EGFR), and its stimulation by EGF, has been linked with a number
of cell proliferative disorders or malignancies. These include skin
hyperplasia, erythroblastosis, and fibrosarcoma in animals; and in
humans, benign hyperplasia of the skin, mammary carcinoma,
glioblastoma, and hepatic carcinoma. Other epithelial carcinomas
associated with EGFR activity include prostatic hyperplasia/cancer,
renal carcinoma, bladder cancer, laryngeal cancer, esophageal
tumors, stomach cancer, colon carcinoma, ovarian adenomas, and lung
cancer (Khazaie, K. et al. (1993) Cancer and Metastasis Rev.
12:255-274).
[0005] The relationship of EGFR expression to human mammary
carcinoma has been particularly well studied. (See Khazaie et al.,
supra, and references cited therein for a review of this area.)
Overexpression of EGFR, particularly coupled with down-regulation
of the estrogen receptor (ER), has been a marker of poor prognosis
in breast cancer patients. In addition, EGFR expression in breast
tumor metastases is frequently elevated relative to the primary
tumor, suggesting that EGFR is involved in tumor progression and
metastasis. This is supported by accumulating evidence that EGF has
pleiotropic effects on cell motility, chemotaxis, secretion and
differentiation; cell functions related to metastatic potential.
For example, EGF has been found to influence the expression and
organization of integrins, a family of receptors known to function
in cell attachment to the extracelluar matrix during metastasis
(Nicolson, G. L. (1984) Expl. Cell Res. 150:3-22; Schirrmacher, V
(1985) Adv. Cancer Res. 43:1-73). EGFR may influence cell-cell
adhesion by affecting changes in the phosphorylation of certain
proteins involved in the process, such as B-catenin, fodrin,
spectrin, and tubulin (Khazaie et al., supra). EGF has also been
shown to affect the production and release of various proteinases
involved in cell invasion of the extracelluar matrix, such as
metalloproteinases, aminopeptidases, serine proteases, cysteine
proteases and aspartic proteinases, as well as proteinase
inhibitors such as plasminogen activator inhibitor (PAI-1) and
tissue inhibitors of metalloproteases (TIMP).
[0006] In addition to the various proteins indicated above that are
affected by EGF activity, the EGF signal transduction pathway
itself involves the recruitment and activation of a variety of
molecules including phospholipase C, phosphoinositol-3 kinase, MAP
kinase, raf kinase, and a GTPase-activating protein (GAP). The
expression of these and other molecules effected by EGF activity
may be useful for the prediction or monitoring of cell
proliferative disorders, pre-malignant conditions, or the presence
and progression of malignant diseases in which EGF
participates.
[0007] Array technology can provide a simple way to explore the
expression of a single polymorphic gene or a large number of
related or unrelated genes. When the expression of a single gene is
explored, arrays are employed to detect the expression of specific
gene variants. For example, a p53 tumor suppressor gene array is
used to determine whether individuals are carrying mutations that
predispose them to cancer. The array has over tens of thousands of
DNA probes to analyze more than 400 distinct mutations of p53.
[0008] DNA-based array technology is especially relevant for the
rapid screening of expression of a large number of genes. There is
a growing awareness that gene expression is affected in a global
fashion. A genetic predisposition, disease or therapeutic treatment
may affect, directly or indirectly, the expression of a large
number of genes. In some cases the interactions may be expected,
such as where the genes are part of the same signaling pathway. In
other cases, the interactions may be totally unexpected, such as
when the genes participate in separate signaling pathways.
Therefore, DNA-based arrays can be used to investigate how genetic
predisposition, disease, or therapeutic treatment affects the
expression of a large number of genes.
[0009] The potential application of gene expression profiling to
breast cancer is particularly relevant to improving diagnosis and
prognosis of this disease. The mortality rate for breast cancer
approaches 10% of all deaths in females between the ages of 45-54
(K. Gish (1999) AWIS Magazine, 28:7-10). However the survival rate
based on early diagnosis of localized breast cancer is extremely
high (97%), compared with the advanced stage of the disease in
which the tumor has spread beyond the breast (22%). Current
procedures for clinical breast examination are, however, lacking in
sensitivity and specificity, and efforts are underway in other
laboratories to develop comprehensive gene expression profiles for
breast cancer that may be used in conjunction with conventional
screening methods to improve diagnosis and prognosis of this
disease (Gish, supra).
[0010] It would be advantageous to prepare DNA-based arrays that
can be used for monitoring expression of a large number of genes
associated with cell proliferative disorders and with pre-malignant
and malignant conditions. The present invention provides for a
composition comprising a plurality of polynucleotide probes for use
in detecting changes in expression of a large number of genes
encoding proteins associated with EGFR expression and activity.
Such a microarray can be employed for diagnosis and monitoring of
the treatment of any disease or precondition where EGFR activation
is involved, in particular, breast cancer.
SUMMARY
[0011] The present invention provides a combination comprising a
plurality of cDNAs, wherein each of the cDNAs comprises at least a
fragment of a polynucleotide sequence or a complement thereof whose
expression is modulated by EGF and is associated with breast cancer
and which are selected from SEQ ID NOs:1-16 as presented in the
Sequence Listing. In one aspect, the combination is immobilized on
a substrate.
[0012] The invention also provides a high throughput method to
detect differential expression of one or more of the cDNAs of the
combination. The method comprises hybridizing the substrate
comprising the combination with the nucleic acids of a sample,
thereby forming one or more hybridization complexes, detecting the
hybridization complexes, and comparing the hybridization complexes
with those of a standard, wherein differences in the size and
signal intensity of each hybridization complex indicates
differential expression of nucleic acids in the sample.
[0013] The invention further provides a high throughput method of
screening a library or a plurality of molecules or compounds to
identify a ligand. The method comprises combining the substrate
comprising the combination with a library or a plurality of
molecules or compounds under conditions to allow specific binding
and detecting specific binding, thereby identifying a ligand. The
library or a plurality of molecules or compounds are selected from
DNA molecules, RNA molecules, peptide nucleic acid molecules,
mimetics, peptides, transcription factors, repressors, and other
regulatory proteins.
[0014] The invention still further provides an isolated cDNA
selected from SEQ ID NOs:12-16 as presented in the Sequence
Listing. The invention still further provides a pharmaceutical
composition comprising the cDNA and a suitable pharmaceutical
carrier. The invention also provides a vector comprising the cDNA,
a host cell comprising the vector, and a method for producing a
protein comprising culturing the host cell under conditions for the
expression of a protein and recovering the protein from the host
cell culture. The invention additionally provides a method for
purifying a ligand, the method comprising combining a cDNA of the
invention with a sample under conditions which allow specific
binding, recovering the bound cDNA, and separating the cDNA from
the ligand, thereby obtaining purified ligand.
[0015] The present invention provides a purified protein encoded
and produced by a cDNA of the invention. The invention also
provides a high-throughput method for using a protein to screen a
library or a plurality of molecules or compounds to identify a
ligand. The method comprises combining the protein or a portion
thereof with the library or a plurality of molecules or compounds
under conditions to allow specific binding and detecting specific
binding, thereby identifying a ligand which specifically binds the
protein. A library or a plurality of molecules or compounds are
selected from DNA molecules, RNA molecules, peptide nucleic acid
molecules, mimetics, peptides, proteins, agonists, antagonists,
antibodies or their fragments, immunoglobulins, inhibitors, drug
compounds, and pharmaceutical agents. The invention further
provides for using a protein to purify a ligand. The method
comprises combining the protein or a portion thereof with a sample
under conditions to allow specific binding, recovering the bound
protein, and separating the protein from the ligand, thereby
obtaining purified ligand. The invention yet still further provides
a method for using the protein to produce an antibody. The method
comprises immunizing an animal with the protein or an
antigenically-effective epitope under conditions to elicit an
antibody response, isolating animal antibodies, and screening the
isolated antibodies with the protein to identify an antibody which
specifically binds the protein. The invention yet still further
provides a method for using the protein to purify antibodies which
bind specifically to the protein.
DESCRIPTION OF THE SEQUENCE LISTING AND TABLES
[0016] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure, as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
[0017] The Sequence Listing is a compilation of nucleic acid
sequences obtained by sequencing clone inserts (isolates) of
different cDNAs. Each sequence is identified by a sequence
identification number (SEQ ID NO) and by the clone number from
which it was obtained.
[0018] Table 1 lists genes differentially expressed in human BT20
breast carcinoma cells and in primary breast carcinoma tissue. In
each page, the table contains (by column): 1) SEQ ID NO: as shown
in the Sequence Listing; 2) the Genbank ID and; 3) gene description
to which the Incyte sequence was annotated; 4-10) the differential
expression of the gene in each of seven breast carcinoma tissue
samples, and 5) the maximal differential expression of the gene
measured during the time course of the experiment for BT20
cells.
DESCRIPTION OF THE INVENTION
[0019] Definitions
[0020] "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.
[0021] The "complement" of a nucleic acid molecule of the Sequence
Listing refers to a cDNA which is completely complementary over the
full length of the sequence and which will hybridize to the nucleic
acid molecule under conditions of high stringency.
[0022] A "combination" comprises at least two and up to 16
sequences selected from the group consisting of SEQ ID NOs: 1-16 as
presented in the Sequence Listing.
[0023] "cDNA" refers to a chain of nucleotides, an isolated
polynucleotide, nucleic acid molecule, or any fragment or
complement thereof. It may have originated recombinantly or
synthetically, be double-stranded or single-stranded, coding and/or
noncoding, an exon with or without an intron from a genomic DNA
molecule, and purified or combined with carbohydrate, lipids,
protein or inorganic elements or substances. Preferably, the cDNA
is from about 4000 to about 5000 nucleotides.
[0024] The phrase "cDNA encoding a protein" refers to a nucleic
acid sequence that closely aligns with sequences which encode
conserved regions, motifs or domains that were identified by
employing analyses well known in the art. These analyses include
BLAST (Basic Local Alignment Search Tool; Altschul (1993) J Mol
Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403-410)
which provides identity within the conserved region. Brenner et al.
(1998; Proc Natl Acad Sci 95:6073-6078) who analyzed BLAST for its
ability to identify structural homologs by sequence identity found
30% identity is a reliable threshold for sequence alignments of at
least 150 residues and 40% is a reasonable threshold for alignments
of at least 70 residues (Brenner et al., page 6076, column 2).
[0025] "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.
[0026] "Differential expression" refers to an increased,
upregulated or present, or decreased, downregulated or absent, gene
expression as detected by the absence, presence, or at least
two-fold changes in the amount of transcribed messenger RNA or
translated protein in a sample.
[0027] "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.
[0028] 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.
[0029] "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.
[0030] "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.
[0031] "Portion" refers to any part of a protein used for any
purpose; but especially, to an epitope for the screening of ligands
or for the production of antibodies.
[0032] "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.
[0033] "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.
[0034] "Protein" refers to a polypeptide or any portion thereof. A
"portion" of a protein retains at least one biological or antigenic
characteristic of a native protein. An "oligopeptide" is an amino
acid sequence from about five residues to about 15 residues that is
used as part of a fusion protein to produce an antibody.
[0035] "Purified" refers to any molecule or compound that is
separated from its natural environment and is from about 60% free
to about 90% free from other components with which it is naturally
associated.
[0036] "Sample" is used in its broadest sense as containing nucleic
acids, proteins, antibodies, and the like. A sample may comprise a
bodily fluid; the soluble fraction of a cell preparation, or an
aliquot of media in which cells were grown; a chromosome, an
organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a
tissue; a tissue print; a fingerprint, buccal cells, skin, or hair;
and the like.
[0037] "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.
[0038] "Similarity" as applied to sequences, refers to the
quantification (usually percentage) of nucleotide or residue
matches between at least two sequences aligned using a standardized
algorithm such as Smith-Waterman alignment (Smith and Waterman
(1981) J Mol Biol 147:195-197) or BLAST2 (Altschul et al. (1997)
Nucleic Acids Res 25:3389-3402). BLAST2 may be used in a
standardized and reproducible way to insert gaps in one of the
sequences in order to optimize alignment and to achieve a more
meaningful comparison between them.
[0039] "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.
[0040] "Variant" refers to molecules that are recognized variations
of a cDNA or a protein encoded by the cDNA. Splice variants may be
determined by BLAST score, wherein the score is at least 100, and
most preferably at least 400. Allelic variants have a high percent
identity to the cDNAs and may differ by about three bases per
hundred bases. "Single nucleotide polymorphism" (SNP) refers to a
change in a single base as a result of a substitution, insertion or
deletion. The change may be conservative (purine for purine) or
non-conservative (purine to pyrimidine) and may or may not result
in a change in an encoded amino acid.
[0041] The Invention
[0042] The present invention provides a combination comprising a
plurality of polynucleotide probes, comprising at least a fragment
of a gene whose transcript is modulated in response to EGF.
Preferably, the plurality of probes comprise at least a fragment of
one or more of the sequences, SEQ ID NOs:1-16, presented in the
Sequence Listing; and they are arranged on a substrate, preferably
a microarray.
[0043] The microarray can be used for large scale genetic or gene
expression analysis of a large number of targets. The microarray
can also be used in the diagnosis of diseases and in the monitoring
of treatments where altered expression of genes is associated with
a cell proliferative disorder, in particular, breast cancer.
Further, the microarray can be employed to investigate an
individual's predisposition to a disease, in particular, breast
cancer.
[0044] In a preferred embodiment, the combination provides the
expression of those probes selected from SEQ ID NOs:1-16 which are
associated with breast cancer
[0045] When the composition of the invention is employed as
hybridizable elements in a microarray, the elements are organized
in an ordered fashion so that each element is present at a
specified location on the substrate. Because the elements are at
specified locations on the substrate, the hybridization patterns
and intensities, which together create a unique expression profile,
can be interpreted in terms of expression levels of particular
genes and can be correlated with a particular metabolic process,
condition, disorder, disease, stage of disease, or treatment.
[0046] The combination comprising a plurality of cDNAs can also be
used to identify or purify a molecule or compound which
specifically binds to at least one of the cDNAs. These molecules
may be identified from a sample or in high throughput mode from a
library of mRNAs, cDNAs, genomic fragments, and the like.
Typically, samples or libraries will include targets of diagnostic
or therapeutic interest. If nucleic acids in a particular sample
enhance the hybridization background, it may be advantageous to
remove these nucleic acids. One method for removing additional
nucleic acids is by hybridizing the sample with immobilized probes
and washing away those nucleic acids that do not form hybridization
complexes. At a later point, hybridization complexes can be
dissociated, thereby releasing the purified targets.
[0047] cDNAs and Their Uses
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] When the cDNAs of the invention are employed on a
microarray, the cDNAs are arranged in an ordered fashion so that
each cDNA is present at a specified location. Because the cDNAs are
at specified locations on the substrate, the hybridization patterns
and intensities, which together create a unique expression profile,
can be interpreted in terms of expression levels of particular
genes and can be correlated with a particular metabolic process,
condition, disorder, disease, stage of disease, or treatment.
[0054] Hybridization
[0055] 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.
[0056] 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.
[0057] 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).
[0058] 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.)
[0059] Screening and Purification Assays
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] Protein Production and Uses
[0065] 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.
[0066] The proteins may contain amino acid substitutions, deletions
or insertions made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues involved. Such substitutions may be
conservative in nature when the substituted residue has structural
or chemical properties similar to the original residue (e.g.,
replacement of leucine with isoleucine or valine) or they may be
nonconservative when the replacement residue is radically different
(e.g., a glycine replaced by a tryptophan). Computer programs
included in LASERGENE software (DNASTAR, Madison, Wis.), MACVECTOR
software (Genetics Computer Group, Madison, Wis.) and RasMol
software (www.umass.edu/microbio/rasmol) may be used to help
determine which and how many amino acid residues in a particular
portion of the protein may be substituted, inserted, or deleted
without abolishing biological or immunological activity.
[0067] Expression of Encoded Proteins
[0068] Expression of a particular cDNA may be accomplished by
cloning the cDNA into a vector and transforming this vector into a
host cell. The cloning vector used for the construction of cDNA
libraries in the LIESEQ databases may also be used for expression.
Such vectors usually contain a promoter and a polylinker useful for
cloning, priming, and transcription. An exemplary vector may also
contain the promoter for .beta.-galactosidase, an amino-terminal
methionine and the subsequent seven amino acid residues of
.beta.-galactosidase. The vector may be transformed into competent
E. coli cells. Induction of the isolated bacterial strain with
isopropylthiogalactoside (IPTG) using standard methods will produce
a fusion protein that contains an N terminal methionine, the first
seven residues of .beta.-galactosidase, about 15 residues of
linker, and the protein encoded by the cDNA.
[0069] 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 and expressed.
[0070] 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.
[0071] 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).
[0072] Additionally, the vector may contain promoters or enhancers
that increase gene expression. Many promoters are known and used in
the art. Most promoters are host specific and exemplary promoters
includes SV40 promoters for CHO cells; T7 promoters for bacterial
hosts; viral promoters and enhancers for plant cells; and PGH
promoters for yeast. Adenoviral vectors with the rous sarcoma virus
enhancer or retroviral vectors with long terminal repeat promoters
may be used to drive protein expression in mammalian cell lines.
Once homogeneous cultures of recombinant cells are obtained, large
quantities of secreted soluble protein may be recovered from the
conditioned medium and analyzed using chromatographic methods well
known in the art. An alternative method for the production of large
amounts of secreted protein involves the transformation of
mammalian embryos and the recovery of the recombinant protein from
milk produced by transgenic cows, goats, sheep, and the like.
[0073] 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.
[0074] Screening and Purification Assays
[0075] 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.
[0076] 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.
[0077] 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 purified ligand.
[0078] Production of Antibodies
[0079] 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. No. 4,816,567.
[0080] 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.).
[0081] 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.
[0082] Labeling of Molecules for Assay
[0083] 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.).
[0084] 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.
[0085] Diagnostics
[0086] The cDNAs, or fragments thereof, may be used to detect and
quantify differential gene expression; absence, presence, or excess
expression of mRNAs; or to monitor mRNA levels during therapeutic
intervention associated with breast cancer. These cDNAs can also be
utilized as markers of treatment efficacy against breast cancer
over a period ranging from several days to months. The diagnostic
assay may use hybridization or amplification technology to compare
gene expression in a biological sample from a patient to standard
samples in order to detect altered gene expression. Qualitative or
quantitative methods for this comparison are well known in the
art.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] Gene Expression Profiles
[0091] A gene expression profile comprises a plurality of cDNAs and
a plurality of detectable hybridization complexes, wherein each
complex is formed by hybridization of one or more probes to one or
more complementary sequences in a sample. The cDNAs of the
invention are used as elements on a microarray to analyze gene
expression profiles. In one embodiment, the microarray is used to
monitor the progression of disease. Researchers can assess and
catalog the differences in gene expression between healthy and
diseased tissues or cells. By analyzing changes in patterns of gene
expression, disease can be diagnosed at earlier stages before the
patient is symptomatic. The invention can be used to formulate a
prognosis and to design a treatment regimen. The invention can also
be used to monitor the efficacy of treatment. For treatments with
known side effects, the microarray is employed to improve the
treatment regimen. A dosage is established that causes a change in
genetic expression patterns indicative of successful treatment.
Expression patterns associated with the onset of undesirable side
effects are avoided. This approach may be more sensitive and rapid
than waiting for the patient to show inadequate improvement, or to
manifest side effects, before altering the course of treatment.
[0092] 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.
[0093] Assays Using Antibodies
[0094] 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.
[0095] 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)
[0096] Therapeutics
[0097] The cDNAs and fragments thereof can be used in gene therapy.
cDNAs can be delivered ex vivo to target cells, such as cells of
bone marrow. Once stable integration and transcription and or
translation are confirmed, the bone marrow may be reintroduced into
the subject. Expression of the protein encoded by the cDNA may
correct a disorder associated with mutation of a normal sequence,
reduction or loss of an endogenous target protein, or
overexpression of an endogenous or mutant protein. Alternatively,
cDNAs may be delivered in vivo using vectors such as retrovirus,
adenovirus, adeno-associated virus, herpes simplex virus, and
bacterial plasmids. Non-viral methods of gene delivery include
cationic liposomes, polylysine conjugates, artificial viral
envelopes, and direct injection of DNA (Anderson (1998) Nature
392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al.
(1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) Cell Mol Life
Sci 55(3):334-358; Agruwal (1996) Antisense Therapeutics, Humana
Press, Totowa, N.J.; and August et al. (1997) Gene Therapy
(Advances in Pharmacology, Vol. 40), Academic Press, San Diego,
Calif.).
[0098] 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.
[0099] 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.
[0100] Additionally, any of the proteins, or their ligands, or
complementary nucleic acid sequences may be administered as
pharmaceutical compositions or in combination with other
appropriate therapeutic agents. Selection of the appropriate agents
for use in combination therapy may be made by one of ordinary skill
in the art, according to conventional pharmaceutical principles.
The combination of therapeutic agents may act synergistically to
affect the treatment or prevention of the conditions and disorders
associated with EGF regulation. Using this approach, one may be
able to achieve therapeutic efficacy with lower dosages of each
agent, thus reducing the potential for adverse side effects.
Further, the therapeutic agents may be combined with
pharmaceutically-acceptable carriers including excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Further
details on techniques for formulation and administration used by
doctors and pharmacists may be found in the latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing, Easton,
Pa.).
[0101] Model Systems
[0102] 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.
[0103] Transgenic Animal Models
[0104] Transgenic rodents that overexpress or underexpress a gene
of interest may be inbred and used to model human diseases or to
test therapeutic or toxic agents. (See, e.g., U.S. Pat. No.
5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the
introduced gene may be activated at a specific time in a specific
tissue type during fetal or postnatal development. Expression of
the transgene is monitored by analysis of phenotype, of
tissue-specific mRNA expression, or of serum and tissue protein
levels in transgenic animals before, during, and after challenge
with experimental drug therapies.
[0105] Embryonic Stem Cells
[0106] 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.
[0107] Knockout Analysis
[0108] 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
gene.
[0109] Knockin Analysis
[0110] 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.
[0111] 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.
EXAMPLES
[0112] For purposes of example, the preparation and sequencing of
the breast tissue cDNA library (BRSTNOT07), from which Incyte Clone
1297817 was identified is described. Preparation and sequencing of
cDNAs in libraries in the LIFESEQ database (Incyte Genomics) have
varied over time, and the gradual changes involved use of kits,
plasmids, and machinery available at the particular time the
library was made and analyzed.
[0113] I cDNA Library Preparation
[0114] The BRSTNOT07 cDNA library was constructed from diseased
breast tissue removed from a 43-year-old Caucasian female during
unilateral extended simple mastectomy. Pathology indicated mildly
proliferative fibrocystic changes with epithelial hyperplasia,
papillomatosis, and duct ectasia. Pathology for the matched tumor
tissue indicated invasive grade 4, nuclear grade 3 mammary
adenocarcinoma with extensive comedo necrosis.
[0115] The frozen tissue was homogenized and lysed in a guanidinium
isothiocyanate solution using a POLYTRON homogenizer (PT-3000,
Brinkmann Instruments, Westbury, N.J.). The lysate was centrifuged
over a 5.7 M CsCl cushion using a SW28 rotor in an L8-70M
ultracentrifuge (Beckman Instruments, Fullerton, Calif.) for 18
hours at 25,000 rpm at ambient temperature. The RNA was extracted
with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate
and 2.5 volumes of ethanol, resuspended in RNAse-free water, and
treated with DNase at 37.degree. C. RNA extraction and
precipitation was repeated as before. The mRNA was then isolated
using the OLIGOTEX kit (Qiagen, Inc., Chatsworth, Calif.) and used
to construct the cDNA library.
[0116] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid System (Life Technologies). The cDNA
were fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia
Biotech, Piscataway, N.J.), and those cDNAs exceeding 400 bp were
ligated into pSPORT I. The plasmid was subsequently transformed
into DH5.alpha. competent cells (Life Technologies).
[0117] II. Isolation and Sequencing of cDNA Clones
[0118] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 plasmid kit (Qiagen). This kit enabled the
simultaneous purification of 96 samples in a 96-well block using
multi-channel reagent dispensers. The recommended protocol was
employed except for the following changes: 1) the bacteria were
cultured in 1 ml of sterile Terrific Broth (Life Technologies) with
carbenicillin at 25 mg/l and glycerol at 0.4%; 2) after
inoculation, the cells were cultured for 19 hours and lysed with
0.3 ml of lysis buffer; and 3) following isopropanol precipitation,
the DNA pellet was resuspended in 0.1 ml of distilled water. After
the last step in the protocol, samples were transferred to a
96-well block for storage at 4.degree. C.
[0119] The cDNAs were prepared using a MICROLAB 2200 system
(Hamilton, Reno, Nev.) in combination with DNA ENGINE thermal
cyclers (PTC200; MJ Research, Waltham, Mass.). The cDNAs were
sequenced by the method of Sanger and Coulson (1975; J. Mol. Biol.
94:441f) using ABI PRISM 377 DNA sequencing systems (PE
Biosystems). Most of the sequences were sequenced using standard
ABI protocols and kits (PE Biosystems) at solution volumes of
0.25.times.-1.0.times.. In the alternative, some of the sequences
were sequenced using solutions and dyes from Amersham Pharmacia
Biotech.
[0120] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0121] As used herein, "homology" refers to sequence similarity
between a reference sequence and at least a fragment of a newly
sequenced clone insert, and can refer to either a nucleic acid or
amino acid sequence. The GenBank databases which contain previously
identified and annotated sequences, were searched for regions of
homology using BLAST (Altschul (1993 and 1990) supra).
[0122] BLAST involves first finding similar segments between the
query sequence and a database sequence, then evaluating the
statistical significance of any matches that are found and finally
reporting only those matches that satisfy a user-selectable
threshold of significance. BLAST produces alignments of both
nucleotide and amino acid sequences to determine sequence
similarity. The fundamental unit of the BLAST algorithm output is
the High scoring Segment Pair (HSP). An HSP consists of two
sequence fragments of arbitrary, but equal lengths, whose alignment
is locally maximal and for which the alignment score meets or
exceeds a threshold or cutoff score set by the user.
[0123] The basis of the search is the product score, which is
defined as:
% sequence identity.times.% maximum BLAST score/100
[0124] The product score takes into account both the degree of
identity between two sequences and the length of the sequence match
as reflected in the BLAST score. The BLAST score is calculated by
scoring +5 for every base that matches in an HSP and -4 for every
mismatch. For example, with a product score of 40, the match will
be exact within a 1% to 2% error, and, with a product score of 70,
the match will be exact. Homologous molecules are usually
identified by selecting those which show product scores between 15
and 40, although lower scores may identify related molecules. The
P-value for any given HSP is a function of its expected frequency
of occurrence and the number of HSPs observed against the same
database sequence with scores at least as high. Percent sequence
identity is found in a comparison of two or more amino acid or
nucleic acid sequences. Percent identity can be determined
electronically, e.g., by using the MEGALIGN program (DNASTAR). The
percentage similarity between two amino acid sequences, e.g.,
sequence A and sequence B, is calculated by dividing the length of
sequence A, minus the number of gap residues in sequence A, minus
the number of gap residues in sequence B, into the sum of the
residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no homology between the two amino acid
sequences are not included in determining percentage
similarity.
[0125] Sequences with conserved protein motifs may also be searched
using the BLOCKS search program. This program analyses sequence
information contained in the Swiss-Prot Database and PROSITE and is
useful for determining the classification of uncharacterized
proteins translated from genomic or cDNA sequences (Bairoch, supra;
Attwood, supra). PROSITE is a useful source for identifying
functional or structural domains that are not detected using motifs
due to extreme sequence divergence. Using weight matrices, these
domains are calibrated against the SWISS-PROT database to obtain a
measure of the chance distribution of the matches.
[0126] 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.
[0127] IV Extension of cDNA Clones
[0128] Some of the nucleic acid sequences of SEQ ID NO:1-16 were
produced by extension of an appropriate fragment of the molecule
using oligonucleotide primers designed from this fragment. One
primer was synthesized to initiate 5' extension of the known
fragment, and the other primer, to initiate 3' extension of the
known fragment. The initial primers were designed using OLIGO 4.06
software (National Biosciences), or another appropriate program, to
be about 22 to 30 nucleotides in length, to have a GC content of
about 50% or more, and to anneal to the target sequence at
temperatures of about 68.degree. C. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0129] 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.
[0130] High fidelity amplification was obtained by PCR using
methods well known in the art. PCR was performed in 96-well plates
using the DNA Engine thermal cycler (MJ Research, Inc.). The
reaction mix contained DNA template, 200 nmol of each primer,
reaction buffer containing Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4, and
.beta.-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair PCI A and PCI B: Step 1: 94.degree. C., 3 min; Step 2:
94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68.degree. C., Step 7: storage at 4.degree. C. In the
alternative, the parameters for primer pair T7 and SK+ were as
follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15
sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. C., 2 min;
Step 5: 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C., 5
min; Step 7: storage at 4.degree. C.
[0131] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene, Oreg.) dissolved in 1.times.TE
and 0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Corning Costar, Acton, Mass.), allowing the DNA
to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of
the sample and to quantify the concentration of DNA. A 5 .mu.l to
10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a 1% agarose mini-gel to determine which
reactions were successful in extending the sequence.
[0132] The extended nucleotides were desalted and concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison, Wis.), and
sonicated or sheared prior to religation into pUC 18 vector
(Amersham Pharmacia Biotech). For shotgun sequencing, the digested
nucleotides were separated on low concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar
ACE (Promega). Extended clones were religated using T4 ligase (New
England Biolabs, Beverly, Mass.) into pUC 18 vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction site overhangs, and transfected into competent
E. coli cells. Transformed cells were selected on
antibiotic-containing media, individual colonies were picked and
cultured overnight at 37.degree. C. in 384-well plates in
LB/2.times.carb liquid media.
[0133] The cells were lysed, and DNA was amplified by PCR using Taq
DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated 29
times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the same conditions as described above. Samples were diluted
with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (PE Biosystems).
[0134] V Propagation and Maintenance of Cultured Cells
[0135] The human breast carcinoma cell line (BT-20) was purchased
from ATCC (Manassus, Va.); and primary mammary epithelial cells
(HMEC), from Clonetics (San Diego, Calif.). The cells were
propagated in media according to the supplier's
recommendations.
[0136] VI Experimental Treatment of Cultured Cells
[0137] Cells were plated in culture dishes and grown at 37.degree.
C. in 5% CO.sub.2 till 80% confluent. The cells were then grown for
48 hours in the presence of 1% fetal bovine serum (FBS), following
which the spent media was removed and replaced with either media
alone or media containing 50 ng/ml EGF (R and D system,
Minneapolis, Minn.). Control and EGF-stimulated cells were lysed at
different time points (e.g. 4, 8, 12, 24, 36, and 48 hours for the
BT20 experiment) following treatment.
[0138] VII Preparation of mRNA
[0139] Following the experimental treatments described above, total
RNA was extracted from cell samples using the TRIZOL reagent (Life
Technologies) extraction protocol based on the supplier's
recommendations, and the mRNA purified using the OLIGOTEX kit
(Qiagen) described above.
[0140] The mRNA from four non-diseased breast tissue samples,
prepared from three female patients ages 32-42 and a pooled tissue
sample from two donors ages 43 and 58, were obtained from BioChain
Institute (San Leandro, Calif.). mRNA from seven ductal carcinoma
primary tumors, prepared from six female patients, ages 46-56, and
one pool of 18 donors, ages 40-72, was also obtained from the same
source.
[0141] VIII Labeling of Probes and Hybridization Analyses
[0142] Substrate Preparation
[0143] Target nucleic acids were amplified from bacterial vectors
by thirty cycles of PCR using primers complementary to vector
sequences flanking the insert. Amplified target nucleic acids were
purified using SEPHACRYL-400 beads (Amersham Pharmacia Biotech).
Purified target nucleic acids were robotically arrayed onto a glass
microscope slide (Corning Science Products, Corning, N.Y.). The
slide was previously coated with 0.05% aminopropyl silane
(Sigma-Aldrich, St. Louis, Mo.) and cured at 110.degree. C. The
arrayed glass slide (microarray) was exposed to UV irradiation in a
STRATALINKER UV-crosslinker (Stratagene).
[0144] In an alternative method, a mixture of target nucleic acids,
a restriction digest of genomic DNA, is fractionated by
electrophoresis through an 0.7% agarose gel in 1.times.TAE
[Tris-acetate-ethylenediamine tetraacetic acid (EDTA)] running
buffer and transferred to a nylon membrane by capillary transfer
using 20.times.saline sodium citrate. (SSC). Alternatively, targets
are individually ligated to a vector and inserted into bacterial
host cells to form a library. Target nucleic acids are arranged on
substrate by one of the following methods. In the first method,
bacterial cells containing individual clones are robotically picked
and arranged on a nylon membrane. The membrane is placed on
bacterial growth medium, LB agar containing carbenicillin, and
incubated at 37.degree. C. for 16 hours. Bacterial colonies are
denatured, neutralized, and digested with proteinase K. Nylon
membranes are exposed to UV irradiation in a STRATALINKER
UV-crosslinker (Stratagene) to cross-link DNA to the membrane.
[0145] Probe Preparation
[0146] Each mRNA sample was reverse transcribed using MMLV reverse
transcriptase in the presence of dCTP-Cy3 or dCTP-Cy5 (Amersham
Pharmacia Biotech) according to standard protocol. After incubation
at 37.degree. C., the reaction was stopped with 0.5 M sodium
hydroxide, and RNA was degraded at 85.degree. C. The probes were
then purified using Chroma Spin 30 gel filtration spin columns
(Clontech, Palo Alto, Calif.) and ethanol precipitation.
[0147] Hybridization
[0148] Competitive hybridization was performed using RNA from (1)
Control untreated versus EGF treated cells or (2) Normal breast
versus tumor breast tissue.
[0149] The hybridization mixture, containing 0.2 mg of each of Cy3
and Cy5 labeled cDNA probes as starting material, was heated to
65.degree. C., and added to the microarray surface. The array was
covered with a coverslip and incubated at 60.degree. C. The
microarrays were washed at 45.degree. C. in high stringency buffer
(1.times.SSC and 0.1% SDS) followed by low stringency washes
(0.1.times.SSC) and dried.
[0150] Detection
[0151] A laser microscope was used to detect the
fluorescence-labeled probed. Excitation wavelengths were 488 nm for
Cy3 and 632 nm for Cy5. Each array was scanned twice, one scan per
fluorophore. The emission maxima was 565 nm for Cy3 and 650 nm for
Cy5. The emitted light was split into two photomultiplier tube
detectors based on wavelength. The output of the photomultiplier
tube was digitized and displayed as an image, where the signal
intensity was represented using a linear 20 color transformation,
with red representing a high signal and blue a low signal. The
fluorescence signal for each element was integrated to obtain a
numerical value corresponding to the signal intensity using
GEMTOOLS gene expression analysis software (Incyte Genomics).
[0152] IX Data Analysis and Results
[0153] Data analysis using the GEMTOOLS gene expression analysis
software (Incyte Genomics) was performed to identify those genes
which exhibited a 2-fold or more change in expression in response
to EGF and displayed a signal intensity of over 300. The sequences
found in the Sequence Listing were selected because they showed at
least a 2-fold change in expression in response to EGF treatment of
a human breast tumor cell line, BT-20, and were also differentially
expressed in primary breast carcinoma tissue samples. Comparisons
of expression among these cells and tissues allowed the
identification of genes potentially useful in diagnosing a breast
cancer (differentially expressed in BT20 cells and breast carcinoma
tissue, but not in HMEC cells).
[0154] Table 1 lists genes differentially regulated by EGF
treatment at least 2-fold in BT20 breast carcinoma cells that are
also differentially regulated at least 2-fold in at least four of
seven breast carcinoma tissue samples (BC). Column 1 lists the SEQ
ID NO, column 2 the Genbank ID, and column 3 the description of the
gene by BLAST, where identified. Sequences not identified by BLAST
are indicated as "Incyte unique". Columns 4-10 list the
differential expression of the gene in seven breast carcinoma
tissue samples (BC1-BC7), and column 11 lists the maximal
differential expression of the gene during the time course of the
experiment for BT20 cells (BT20). Positive values indicate
upregulation of the gene, and negative (-) values indicate
downregulation. This comparison shows that genes differentially
regulated in vitro by EGF treatment of BT20 cells are also
differentially regulated in vivo in breast carcinoma.. These genes
may be useful in diagnosing and monitoring the progression of
breast cancer and the response to treatment.
[0155] X Expression of the Encoded Protein
[0156] 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.
[0157] 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.).
[0158] XI Production of Specific Antibodies
[0159] 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.
[0160] 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 IgG.
[0161] 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.
[0162] 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.
[0163] XII Purification of Naturally Occurring Protein Using
Specific Antibodies
[0164] 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.
[0165] XIII Screening Molecules for Specific Binding with the cDNA
or Protein
[0166] 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.
1TABLE 1 SEQ ID Genbank NO: ID Genbank Description BC1 BC2 BC3 BC4
BC5 BC6 BC7 BT20 1 609301 Human keratin 5 (KRT5) gene, intron 8,
5'end. -2.4 2.0 -7.1 -2.8 -2.1 -3.8 -4.2 -3.6 2 531159 Human
dihydrodiol dehydrogenase mRNA, complete cds. -4.3 -4.8 -1.7 -4.1
-3.4 -3.4 -7.8 -9.9 3 4500003 Human bile salt-activated lipase
(BAL) mRNA, complete cds -3.4 -1.4 -1.5 -2.0 -2.4 -2.3 -1.0 -2.2 4
180947 Human carboxylesterase mRNA, complete cds -4.1 -3.2 -3.1
-3.3 -2.5 -2.3 1.1 -4.1 5 219899 Human mRNA for long-chain acyl-CoA
synthetase. -1.7 -3.3 1.2 -2.4 -1.3 -2.0 -2.3 -2.2 6 688296
brain-expressed HHCPA78 -4.4 -1.4 -4.0 -2.1 -1.7 -3.0 -1.5 -2.8 7
4105412 GC4;RTP -6.8 -4.6 -12 -6.3 -5.6 -10 -5.2 -2.4 8 1916005
Human eyes absent homolog (Eab1) mRNA, complete cds -2.4 -1.9 -4.9
-2.0 -2.9 -2.3 -2.6 -2.4 9 3676496 Human mRNA for
6-phosphofructo-2-kinase/fructose- -1.7 -2.2 -1.7 -3.2 -2.9 -3.1
-3.7 -2.1 2,6-bisphosphatase 10 1621606 Human neogenin mRNA,
complete cds. -3.2 -1.1 -2.1 -2.3 -1.9 -2.3 -1.6 -2.5 11 1469919
Human tumor protein D53 (TPD52L1) mRNA, partial cds. -2.8 -2.2 1.2
-2.2 -2.0 -3.2 -1.6 -2.4 12 Incyte Unique -1.6 -1.5 -2.3 -2.3 -2.5
-1.7 -2.1 -3.4 13 Incyte Unique 3.6 1.8 1.1 2.1 -1.1 6.9 5.5 2.2s
14 Incyte Unique -3.5 -1.1 -3.1 -2.0 -2.2 -5.1 -1.8 -3.3 15 Incyte
Unique -2.4 -1.2 -3.4 -1.8 -1.5 -2.0 -2.7 -2.4 16 Incyte Unique
-5.7 -1.3 -4.3 -3.6 -2.0 -5.8 -1.8 -3.5
[0167]
Sequence CWU 1
1
* * * * *