The present invention relates to all facets of novel polynucleotides,
the polypeptides they encode, antibodies and specific binding partners
thereto, and their applications to research, diagnosis, drug discovery,
therapy, clinical medicine, forensic science and medicine, etc.
The polynucleotides, Urb-ctf, are expressed in breast cancer and
are therefore useful in variety of ways, including, but not limited
to, as molecular markers, as drug targets, and for detecting, diagnosing,
staging, monitoring, prognosticating, preventing or treating, determining
predisposition to, etc., diseases and conditions especially related
to breast cancer.
What is claimed is:
1. An isolated polynucleotide, comprising a polynucleotide sequence
which codes without interruption for human Urb-ctf comprising amino
acids 1 614 as set forth in SEQ ID NO 2, or the complete complement
2. An isolated polynucleotide of claim 1, comprising the polynucleotide
sequence from nucleotide positions 78 1922 as set forth in SEQ ID
NO 1, or the complete complement thereto.
3. An isolated human polynucleotide, comprising a polynucleotide
sequence which codes without interruption for a full-length human
Urb-crf having 614 amino acids, which has transcriptional regulatory
activity, and which hybridizes to the complete complement of SEQ
ID NO: 1 from nucleotide positions 78 1922 under high stringency
conditions comprising overnight incubation in 5.times.SSC, 0.5%
SDS, 100 .mu.g/ml denatured salmon sperm DNA and 50% formamide,
at 42.degree. C., followed by washing in 0.1% SSC and 0.1% SDS for
30 min at 65.degree. C.
4. An isolated polynucleotide consisting of a polynucleotide sequence
selected from SEQ ID NO 1 wherein said polynucleotide comprises
at least 15 nucleotides and codes for a fragment of SEQ ID NO:2
which comprises amino acid 38 of SEQ ID NO 2, amino acid 68 of SEQ
ID NO 2, amino acids 76 77 of SEQ ID NO 2, amino acid 119 of SEQ
ID NO 2, amino acid 143 144 of SEQ ID NO 2, amino acid 161 of SEQ
ID NO 2, amino acid 583 of SEQ ID NO 2, or amino acid 606 of SEQ
ID NO 2; or the complete complement thereof.
5. An isolated polynucleotide of claim 4, which is a polynucleotide
coding for coding for amino acids 1 263 of SEQ ID NO 2 or 459 614
of SEQ ID NO 2, or a the complete complement thereof.
6. An isolated polynucleotide of claim 4, wherein said polynucleotide
is effective in a polymerase chain reaction.
7. An isolated polynucleotide of claim 4, which codes for a polypeptide
comprising at least eight amino acids in length.
8. An isolated polynucleotide of claim 1, comprising the polynucleotide
sequence from nucleotide positions 1 4372 as set forth in SEQ ID
NO 1, or the complete complement thereto.
9. An isolated polynucleotide of claim 4, which comprises at least
10. An isolated polynucleotide of claim 4, which comprises at least
11. An isolated polynucleotide of claim 4, which comprises at least
12. A method of producing human Urb-ctf polypeptide, comprising
expressing a polynucleotide of claim 1 which codes without interruption
for said polypeptide and which is operably linked to an expression
control sequence under conditions effective to achieve production
of said polypeptide coded for by said polynucleotide.
13. A method of producing human Urb-ctf polypeptide, comprising
expressing a polynucleotide of claim 1 which codes without interruption
for said polypeptide and which is operably linked to an expression
control sequence under conditions effective to achieve production
of said polypeptide coded for by said polynucleotide.
14. A method of producing human Urb-ctf polypeptide, comprising
expressing a polynucleotide of claim 3 which codes without interruption
for said polypeptide and which is operably linked to an expression
control sequence under conditions effective to achieve production
of said polypeptide coded for by said polynucleotide.
15. A method of producing human Urb-ctf polypeptide, comprising:
expressing a polynucleotide of claim 8 which codes without interruption
for said polypeptide and which is operably linked to an expression
control sequence under conditions effective to achieve production
of said polypeptide coded for by said polynucleotide.
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows amino acid alignments between Urb-ctf ("BCU1041,"
SEQ ID NO 2), AK014463 (mouse, SEQ ID NO 4) and XM.sub.--058887
(human, SEQ ID NO 3). Regions of sequence identity are shaded.
DESCRIPTION OF THE INVENTION
The present invention relates to all facets of Urb-ctf, polypeptides
encoded by it, antibodies and specific binding partners thereto,
and their applications to research, diagnosis, drug discovery, therapy,
clinical medicine, forensic science and medicine, etc. Urb-ctf polynucleotides,
polypeptides, antibodies, etc., are useful in variety of ways, including,
but not limited to, as a molecular markers, as drug targets, and
for detecting, diagnosing, staging, monitoring, prognosticating,
preventing or treating, determining predisposition to, etc., diseases
and conditions, such as breast cancer. The identification of specific
genes, and groups of genes, expressed in pathways physiologically
relevant to breast cancer permits the definition of functional and
disease pathways, and the delineation of targets in these pathways
which are useful in diagnostic, therapeutic, and clinical applications.
The present invention also relates to methods of using the polynucleotides
and related products (proteins, antibodies, etc.) in business and
computer-related methods, e.g., advertising, displaying, offering,
selling, etc., such products for sale, commercial use, licensing,
Breast cancer is the second leading cause of cancer death for all
women (after lung cancer), and the leading overall cause of death
in women between the ages of 40 and 55. In 2000, several hundred
thousand new cases of female invasive breast cancer were diagnosed,
and about 40,000 women died from the disease. Nearly 43,000 cases
of female in situ (preinvasive) breast cancer were diagnosed in
There is not one single disease that can be called breast cancer.
Instead, it is highly heterogeneous, exhibiting a wide range of
different phenotypes and genotypes. No single gene or protein has
been identified which is responsible for the etiology of all breast
cancers. A number of different genes have already been identified
which are associated with breast cancer, or a predisposition to
it. It is likely that diagnostic and prognostic markers for breast
cancer disease will involve the identification and use of many different
genes and gene products to reflect its multifactorial origin.
A continuing goal is to characterize the gene expression patterns
of the various breast carcinomas in order to genetically differentiate
them, providing important guidance in preventing, diagnosing, and
treating cancer. For instance, the c-erb-B2 gene codes for a transmembrane
protein which is over-expressed in about 20 30% of all breast cancers.
Based on this information, immunotherapy using an anti-c-erb-B2
antibody has been developed and successfully used to treat breast
cancer. See, e.g., Pegram and Slamon, Semin Oncol., 5, Suppl 9:13,
2000. Molecular pictures of cancer, such as the pattern of up-regulated
genes identified herein, provide an important tool for molecularly
dissecting and classifying cancer, identifying drug targets, providing
prognosis and therapeutic information, etc. For instance, an array
of polynucleotides corresponding to genes differentially regulated
in breast cancer can be used to screen tissue samples for the existence
of cancer, to categorize the cancer (e.g., by the particular pattern
observed), to grade the cancer (e.g., by the number of up-regulated
genes and their levels of expression), to identify the source of
a secondary tumor, to screen for metastatic cells, etc. These arrays
can be used in combination with other markers, e.g., keratin immunophenotyping
(e.g., CK 5/6), c-erb-B2, estrogen receptor (ER) status, etc., or
any grading system desired.
Urb-ctf ("Up-Regulated Breast Cancer Transcription Factor"
or BCU1041FB or FB2847A11) codes for a transcription regulatory
factor having 614 amino acids which is up-regulated in breast cancer.
The nucleotide and amino acid sequences of Urb-ctf are shown in
SEQ ID NOS 1 and 2. It contains a bZIP domain at about amino acid
positions 228 275, conferring DNA-binding activity. It also has
a leucine zipper providing a dimerization activity. There are a
number of UniGene clusters that map close to the gene, including,
e.g., Hs.350229, Hs.272458, Hs.350229, Hs.255286, Hs.184779, and
Hs.276916. Predictions using GenomeScan (e.g., Yeh et al., Genome
Res. 11: 803 816, 2001) revealed at least two different predicted
genes, Hs17.sub.--11001.sub.--27.sub.--4.sub.--1 and Hs17.sub.--11001.sub.--27.sub.--5.sub.--2,
instead of the single gene, Urb-ctf, described herein. A partial
human cDNA (AL049450; XM.sub.--058887; SEQ ID NO 3) for Urb-ctf
was previously identified, but this coded for only 198 amino acids
and contained only a part of the bZIP domain, as well as missing
significant portions of the N- and C-termini. A mouse homolog, AK.sub.--014463
(SEQ ID NO 4), has been cloned.
All or part of Urb-ctf is located in genomic DNA represented by
GenBank ID: AC068669, BAC-ID: RP11-749I16, and Contig ID: NT.sub.--010844.
The present invention relates to any isolated introns and exons
that are present in the gene. Intron and exon boundaries can be
routinely determined, e.g., using the polypeptide and genomic sequences
disclosed herein. Using UniSTS probes, Urb-ctf can be chromosomally
mapped at its 5' end with UniSTS: 155813 to 40.144 Mb, and its 3'
end with UniSTS: 619 to 40.084 Mb. Strikingly, the Urb-ctf overlaps
with the thyroid hormone receptor alpha 2 gene (CAB57886).
As indicated by the presence of a bZIP domain, Urb-ctf has transcriptional
regulatory activity, DNA-binding activity, and dimerization activity.
These activities can be determined routinely. For example, DNA-binding
activity can be determined using gel-shift assays, e.g., as carried
out in, e.g., U.S. Pat. Nos. 6,333,407 and 5,789,538. Transcriptional
activity can be determined using conventional transcriptional assays,
including in vivo and in vitro assays, such as those described in
F. M. Ausubel et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY
(John Wiley & Sons, New York, 1994); de Wet et al., Mol. Cell
Biol. 7:725 (1987); U.S. Pat. No. 6,306,649; U.S. Pat. No. 6,214,588;
Liao, S. M. et al., Genes. Dev. 5:2431 2440 (1991); Nonet, M., et
al., Cell 50:909 915 (1987). The phrase "transcriptional regulatory
activity" indicates that the polypeptide modulates transcription
in analogy to the activity of other bZIP proteins, e.g., by binding
to DNA and interacting with other proteins of the transcription
apparatus. For example, both c-Jun and c-Fos are bZIP proteins that
form a dimer known as the transcriptional activator AP-1, a transcriptional
activator. See, e.g., Genes VII, Lewin, Pages 649 665, 2000. Dimerization
activity, i.e., the ability to form hetero- or homodimers with other
proteins (in analogy to the c-fos and c-jun system), can be measured
routinely, e.g., using the yeast two-hybrid system.
Nucleic acids of the present invention map to chromosomal band
17q21.1. There are a number of different disorders which have been
mapped to, or in close proximity to, this chromosome location. These
include, e.g., Dementia, frontotemporal, with parkinsonism; Neuroblastoma;
Osteoporosis, idiopathic; Ehlers-Danlos syndrome, types I and VIIA;
Osteogenesis imperfecta; Glanzmann thrombasthenia, type B; Renal
cell carcinoma, papillary; Thrombocytopenia, neonatal alloimmune;
Trichodontoosseous syndrome; Hypertension; Epidermolytic hyperkeratosis;
Hemolytic anemia due to band 3 defect; Spherocytosis, hereditary;
Gliosis, familial progressive subcortical; Renal tubular acidosis,
distal; Patella aplasia or hypoplasia; and Pseudohypoaldosteronism
type II. Nucleic acids of the present invention can be used as linkage
markers, diagnostic targets, therapeutic targets, for any of the
mentioned disorders, as well as any disorders or genes mapping in
proximity to it.
In addition to its expression in breast cancer, Urb-ctf can be
detected in most tissues examined, but either none, or at very low
levels, in normal breast tissue. Multiple forms of it can be detected
in the brain, muscle, testes, and thymus. As these results indicate,
Urb-ctf has a normal functional role in most tissues, and can consequently
be involved with diseases associated with them, as well. For instance,
Urb-ctf can be involved in renal cell carcinoma and familial gliosis
disease. As discussed earlier, no single gene is responsible for
all breast cancers. Thus, the fact that Urb-ctf is up-regulated
in the breast cancers examined herein does necessarily mean that
it will be up-regulated in all human breast cancers.
Urb-ctf can be utilized in a number of different ways. Because
it is up-regulated in breast cancers, it can be used as a marker
to determine the presence of breast cancer in normal breast tissue
for diagnostic and therapeutic applications. Methods for detecting
Urb-ctf nucleic acid and polypeptide are described in more detail
below. It can also be used as a therapeutic target, e.g., by down-regulating
or suppressing expression of Urb-ctf, either at the nucleic acid
or protein level. For example, the cancer can be treated by administering
effective amounts of anti-sense to block expression of the gene.
Inhibition of the protein's functional activity can also be achieved.
For example, polyamides, such as those described in Bremer et al.,
Bioorganic Med. Chem., 9:2093 2103, 2001, can be used to inhibit
binding of Urb-ctf to DNA. Specificity to breast cancer cells can
be achieved by conjugating the polyamide, or other therapeutic agent,
to a breast cancer marker, such as c-erb-B2.
Urb-ctf polypeptide and gene can also be used in transcriptional
assays, such as the yeast two-hybrid system. Rather than using the
DNA-binding domain of GAL4, Urb-ctf can be used as the fusion partner
for a protein whose binding partner is to be identified. See, e.g.,
Allen et al., TIBS, December 1995, Pages 511 516. DNA sequences
to which Urb-ctf and other bZIP proteins bind are disclosed, e.g.,
in Kise and Shin, Bioorganic Med. Chem., 9:2485 2491, 2001.
As illustrated in FIG. 1, Urb-ctf is highly conserved between human
and mouse with about 97% amino acid sequence identity between the
two proteins, about 93% nucleotide sequence similarity. The variations
between the polypeptides, e.g., at about amino acid positions 38,
68, and 77, are evidently amino acids which are not stringently
required for biological activity, and therefore can provide guidance
in the kind of mutations/polymorphisms that can be made without
eliminating the activity of the protein.
A mammalian polynucleotide, or fragment thereof of the present
invention is a polynucleotide having a nucleotide sequence obtainable
from a natural source, i.e., the species name indicates that the
polynucleotide or polypeptide is obtainable from a natural source.
It therefore includes naturally-occurring normal, naturally-occurring
mutant, and naturally-occurring polymorphic alleles (e.g., SNPs),
differentially-spliced transcripts, splice-variants, etc. By the
term "naturally-occurring," it is meant that the polynucleotide
is obtainable from a natural source, e.g., animal tissue and cells,
body fluids, tissue culture cells, forensic samples. Natural sources
include, e.g., living cells obtained from tissues and whole organisms,
tumors, cultured cell lines, including primary and immortalized
cell lines. Naturally-occurring mutations can include deletions
(e.g., a truncated amino- or carboxy-terminus), substitutions, inversions,
or additions of nucleotide sequence. These genes can be detected
and isolated by polynucleotide hybridization according to methods
which one skilled in the art would know, e.g., as discussed below.
A polynucleotide according to the present invention can be obtained
from a variety of different sources. It can be obtained from DNA
or RNA, such as polyadenylated mRNA or total RNA, e.g., isolated
from tissues, cells, or whole organism. The polynucleotide can be
obtained directly from DNA or RNA, from a cDNA library, from a genomic
library, etc. The polynucleotide can be obtained from a cell or
tissue (e.g., from an embryonic or adult tissues) at a particular
stage of development, having a desired genotype, phenotype, disease
status, etc. A polynucleotide which "codes without interruption"
refers to a polynucleotide having a continuous open reading frame
("ORF") as compared to an ORF which is interrupted by
introns or other noncoding sequences.
Polynucleotides and polypeptides (including any part of Urb-ctf)
can be excluded as compositions from the present invention if, e.g.,
listed in a publicly available databases on the day this application
was filed and/or disclosed in a patent application having an earlier
filing or priority date than this application and/or conceived and/or
reduced to practice earlier than a polynucleotide in this application.
As described herein, the phrase "an isolated polynucleotide
which is SEQ ID NO," or "an isolated polynucleotide which
is selected from SEQ ID NO," refers to an isolated nucleic
acid molecule from which the recited sequence was derived (e.g.,
a cDNA derived from mRNA; cDNA derived from genomic DNA). Because
of sequencing errors, typographical errors, etc., the actual naturally-occurring
sequence may differ from a SEQ ID listed herein. Thus, the phrase
indicates the specific molecule from which the sequence was derived,
rather than a molecule having that exact recited nucleotide sequence,
analogously to how a culture depository number refers to a specific
cloned fragment in a cryotube.
As explained in more detail below, a polynucleotide sequence of
the invention can contain the complete sequence as shown in SEQ
ID NO 1, degenerate sequences thereof, anti-sense, muteins thereof,
genes comprising said sequences, full-length cDNAs comprising said
sequences, complete genomic sequences, fragments thereof, homologs,
primers, nucleic acid molecules which hybridize thereto, derivatives
The present invention also relates to an isolated polynucleotide
which is specific for human Urb-ctf and which codes for a polypeptide,
said polypeptide comprising, e.g., amino acid 38 of SEQ ID NO 2,
amino acid 68 of SEQ ID NO 2, amino acids 76 77 of SEQ ID NO 2,
amino acid 119 of SEQ ID NO 2, amino acid 143 144 of SEQ ID NO 2,
amino acid 161 of SEQ ID NO 2, amino acid 583 of SEQ ID NO 2, amino
acid 606 of SEQ ID NO 2, or complements thereof. The polynucleotide
can be of any size that is effective to confer specificity to the
sequence, e.g., 15 nucleotides (5 amino acids), 24 nucleotides (8
amino acids), 30 nucleotides (10 amino acids), 45 nucleotides (15
amino acids), etc. It can also comprise much longer sequences, e.g.,
a polynucleotide coding for amino acids 1 263 of SEQ ID NO 2 or
459 614 of SEQ ID NO 2, or a complement thereof.
The present invention also relates genomic DNA from which the polynucleotides
of the present invention can be derived. A genomic DNA coding for
a human, mouse, or other mammalian polynucleotide, can be obtained
routinely, for example, by screening a genomic library (e.g., a
YAC library) with a polynucleotide of the present invention, or
by searching nucleotide databases, such as GenBank and EMBL, for
matches. Promoter and other regulatory regions (including both 5'
and 3' regions) can be identified upstream or downstream of coding
and expressed RNAs, and assayed routinely for activity, e.g., by
joining to a reporter gene (e.g., CAT, GFP, alkaline phosphatase,
luciferase, galatosidase). 3'-untranslated sequences (as well as
introns) can be used, e.g., to stabilize transcripts, to target
A polynucleotide of the present invention can comprise additional
polynucleotide sequences, e.g., sequences to enhance expression,
detection, uptake, cataloging, tagging, etc. A polynucleotide can
include only coding sequence; a coding sequence and additional non-naturally
occurring or heterologous coding sequence (e.g., sequences coding
for leader, signal, secretory, targeting, enzymatic, fluorescent,
antibiotic resistance, and other functional or diagnostic peptides);
coding sequences and non-coding sequences, e.g., untranslated sequences
at either a 5' or 3' end, or dispersed in the coding sequence, e.g.,
A polynucleotide according to the present invention also can comprise
an expression control sequence operably linked to a polynucleotide
as described above. The phrase "expression control sequence"
means a polynucleotide sequence that regulates expression of a polypeptide
coded for by a polynucleotide to which it is functionally ("operably")
linked. Expression can be regulated at the level of the mRNA or
polypeptide. Thus, the expression control sequence includes mRNA-related
elements and protein-related elements. Such elements include promoters,
enhancers (viral or cellular), ribosome binding sequences, transcriptional
terminators, etc. An expression control sequence is operably linked
to a nucleotide coding sequence when the expression control sequence
is positioned in such a manner to effect or achieve expression of
the coding sequence. For example, when a promoter is operably linked
5' to a coding sequence, expression of the coding sequence is driven
by the promoter. Expression control sequences can include an initiation
codon and additional nucleotides to place a partial nucleotide sequence
of the present invention in-frame in order to produce a polypeptide
(e.g., pET vectors from Promega have been designed to permit a molecule
to be inserted into all three reading frames to identify the one
that results in polypeptide expression). Expression control sequences
can be heterologous or endogenous to the normal gene.
A polynucleotide of the present invention can also comprise nucleic
acid vector sequences, e.g., for cloning, expression, amplification,
selection, etc. Any effective vector can be used. A vector is, e.g.,
a polynucleotide molecule which can replicate autonomously in a
host cell, e.g., containing an origin of replication. Vectors can
be useful to perform manipulations, to propagate, and/or obtain
large quantities of the recombinant molecule in a desired host.
A skilled worker can select a vector depending on the purpose desired,
e.g., to propagate the recombinant molecule in bacteria, yeast,
insect, or mammalian cells. The following vectors are provided by
way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,
Phagescript, phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A
(Stratagene); Bluescript KS+II (Stratagene); ptrc99a, pKK223-3,
pKK233-3, pDR54 0, pRIT5 (Pharmacia). Eukaryotic: PWLNEO, pSV2CAT,
pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV, PMSG, pSVL (Pharmacia),
pCR2.1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB, pCMV6-XL4, etc. However,
any other vector, e.g., plasmids, viruses, or parts thereof, may
be used as long as they are replicable and viable in the desired
host. The vector can also comprise sequences which enable it to
replicate in the host whose genome is to be modified.
Polynucleotide hybridization, as discussed in more detail below,
is useful in a variety of applications, including, in gene detection
methods, for identifying mutations, for making mutations, to identify
homologs in the same and different species, to identify related
members of the same gene family, in diagnostic and prognostic assays,
in therapeutic applications (e.g., where an antisense polynucleotide
is used to inhibit expression), etc.
The ability of two single-stranded polynucleotide preparations
to hybridize together is a measure of their nucleotide sequence
complementarity, e.g., base-pairing between nucleotides, such as
A T, G C, etc. The invention thus also relates to polynucleotides,
and their complements, which hybridize to a polynucleotide comprising
a nucleotide sequence as set forth in SEQ ID NO 1 and genomic sequences
thereof. A nucleotide sequence hybridizing to the latter sequence
will have a complementary polynucleotide strand, or act as a template
for one in the presence of a polymerase (i.e., an appropriate polynucleotide
synthesizing enzyme). The present invention includes both strands
of polynucleotide, e.g., a sense strand and an anti-sense strand.
Hybridization conditions can be chosen to select polynucleotides
which have a desired amount of nucleotide complementarity with the
nucleotide sequences set forth in SEQ ID NO 1 and genomic sequences
thereof. A polynucleotide capable of hybridizing to such sequence,
preferably, possesses, e.g., about 70%, 75%, 80%, 85%, 87%, 90%,
92%, 95%, 97%, 99%, or 100% complementarity, between the sequences.
The present invention particularly relates to polynucleotide sequences
which hybridize to the nucleotide sequences set forth in SEQ ID
NO 1 or genomic sequences thereof, under low or high stringency
conditions. These conditions can be used, e.g., to select corresponding
homologs in non-human species.
Polynucleotides which hybridize to polynucleotides of the present
invention can be selected in various ways. Filter-type blots (i.e.,
matrices containing polynucleotide, such as nitrocellulose), glass
chips, and other matrices and substrates comprising polynucleotides
(short or long) of interest, can be incubated in a prehybridization
solution (e.g., 6.times.SSC, 0.5% SDS, 100 .mu.g/ml denatured salmon
sperm DNA, 5.times. Denhardt's solution, and 50% formamide), at
22 68.degree. C., overnight, and then hybridized with a detectable
polynucleotide probe under conditions appropriate to achieve the
desired stringency. In general, when high homology or sequence identity
is desired, a high temperature can be used (e.g., 65.degree. C.).
As the homology drops, lower washing temperatures are used. For
salt concentrations, the lower the salt concentration, the higher
the stringency. The length of the probe is another consideration.
Very short probes (e.g., less than 100 base pairs) are washed at
lower temperatures, even if the homology is high. With short probes,
formamide can be omitted. See, e.g., Current Protocols in Molecular
Biology, Chapter 6, Screening of Recombinant Libraries; Sambrook
et al., Molecular Cloning, 1989, Chapter 9.
For instance, high stringency conditions can be achieved by incubating
the blot overnight (e.g., at least 12 hours) with a long polynucleotide
probe in a hybridization solution containing, e.g., about 5.times.SSC,
0.5% SDS, 100 .mu.g/ml denatured salmon sperm DNA and 50% formamide,
at 42.degree. C. Blots can be washed at high stringency conditions
that allow, e.g., for less than 5% bp mismatch (e.g., wash twice
in 0.1% SSC and 0.1% SDS for 30 min at 65.degree. C.), i.e., selecting
sequences having 95% or greater sequence identity.
Other non-limiting examples of high stringency conditions includes
a final wash at 65.degree. C. in aqueous buffer containing 30 mM
NaCl and 0.5% SDS. Another example of high stringent conditions
is hybridization in 7% SDS, 0.5 M NaPO.sub.4, pH 7, 1 mM EDTA at
50.degree. C., e.g., overnight, followed by one or more washes with
a 1% SDS solution at 42.degree. C. Whereas high stringency washes
can allow for less than 5% mismatch, reduced or low stringency conditions
can permit up to 20% nucleotide mismatch. Hybridization at low stringency
can be accomplished as above, but using lower formamide conditions,
lower temperatures and/or lower salt concentrations, as well as
longer periods of incubation time.
Hybridization can also be based on a calculation of melting temperature
(Tm) of the hybrid formed between the probe and its target, as described
in Sambrook et al. Generally, the temperature Tm at which a short
oligonucleotide (containing 18 nucleotides or fewer) will melt from
its target sequence is given by the following equation: Tm=(number
of A's and T's).times.2.degree. C.+(number of C's and G's).times.4.degree.
C. For longer molecules, Tm=81.5+16.6 log.sub.10[Na.sup.+]+0.41(%
GC)-600/N where [Na.sup.+] is the molar concentration of sodium
ions, % GC is the percentage of GC base pairs in the probe, and
N is the length. Hybridization can be carried out at several degrees
below this temperature to ensure that the probe and target can hybridize.
Mismatches can be allowed for by lowering the temperature even further.
Stringent conditions can be selected to isolate sequences, and
their complements, which have, e.g., at least about 90%, 95%, or
97%, nucleotide complementarity between the probe (e.g., a short
polynucleotide of SEQ ID NO 1 or genomic sequences thereof) and
a target polynucleotide.
Other homologs of polynucleotides of the present invention can
be obtained from mammalian and non-mammalian sources according to
various methods. For example, hybridization with a polynucleotide
can be employed to select homologs, e.g., as described in Sambrook
et al., Molecular Cloning, Chapter 11, 1989. Such homologs can have
varying amounts of nucleotide and amino acid sequence identity and
similarity to such polynucleotides of the present invention. Mammalian
organisms include, e.g., mice, rats, monkeys, pigs, cows, etc. Non-mammalian
organisms include, e.g., vertebrates, invertebrates, zebra fish,
chicken, Drosophila, C. elegans, Xenopus, yeast such as S. pombe,
S. cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, artemia,
Alignments can be accomplished by using any effective algorithm.
For pairwise alignments of DNA sequences, the methods described
by Wilbur-Lipman (e.g., Wilbur and Lipman, Proc. Natl. Acad. Sci.,
80:726 730, 1983) or Martinez/Needleman-Wunsch (e.g., Martinez,
Nucleic Acid Res., 11:4629 4634, 1983) can be used. For instance,
if the Martinez/Needleman-Wunsch DNA alignment is applied, the minimum
match can be set at 9, gap penalty at 1.10, and gap length penalty
at 0.33. The results can be calculated as a similarity index, equal
to the sum of the matching residues divided by the sum of all residues
and gap characters, and then multiplied by 100 to express as a percent.
Similarity index for related genes at the nucleotide level in accordance
with the present invention can be greater than 70%, 80%, 85%, 90%,
95%, 99%, or more. Pairs of protein sequences can be aligned by
the Lipman-Pearson method (e.g., Lipman and Pearson, Science, 227:1435
1441, 1985) with k-tuple set at 2, gap penalty set at 4, and gap
length penalty set at 12. Results can be expressed as percent similarity
index, where related genes at the amino acid level in accordance
with the present invention can be greater than 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99%, or more. Various commercial and free sources
of alignment programs are available, e.g., MegAlign by DNA Star,
BLAST (National Center for Biotechnology Information), BCM (Baylor
College of Medicine) Launcher, etc. BLAST can be used to calculate
amino acid sequence identity, amino acid sequence homology, and
nucleotide sequence identity. These calculations are made along
the entire length of each of the target sequences which are to be
Percent sequence identity can also be determined by other conventional
methods, e.g., as described in Altschul et al., Bull. Math. Bio.
48: 603 616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 10919, 1992.
Specific Polynucleotide Probes
A polynucleotide of the present invention can comprise any continuous
nucleotide sequence of SEQ ID NO 1, sequences which share sequence
identity thereto, or complements thereof. The term "probe"
refers to any substance that can be used to detect, identify, isolate,
etc., another substance. A polynucleotide probe is comprised of
nucleic acid can be used to detect, identify, etc., other nucleic
acids, such as DNA and RNA.
These polynucleotides can be of any desired size that is effective
to achieve the specificity desired. For example, a probe can be
from about 7 or 8 nucleotides to several thousand nucleotides, depending
upon its use and purpose. For instance, a probe used as a primer
PCR can be shorter than a probe used in an ordered array of polynucleotide
probes. Probe sizes vary, and the invention is not limited in any
way by their size, e.g., probes can be from about 7 2000 nucleotides,
7 1000, 8 700, 8 600, 8 500, 8 400, 8 300, 8 150, 8 100, 8 75, 7
50, 10 25, 14 16, at least about 8, at least about 10, at least
about 15, at least about 25, etc. The polynucleotides can have non-naturally-occurring
nucleotides, e.g., inosine, AZT, 3TC, etc. The polynucleotides can
have 100% sequence identity or complementarity to a sequence of
SEQ ID NO 1, or it can have mismatches or nucleotide substitutions,
e.g., 1, 2, 3, 4, or 5 substitutions. The probes can be single-stranded
In accordance with the present invention, a polynucleotide can
be present in a kit, where the kit includes, e.g., one or more polynucleotides,
a desired buffer (e.g., phosphate, tris, etc.), detection compositions,
RNA or cDNA from different tissues to be used as controls, libraries,
etc. The polynucleotide can be labeled or unlabeled, with radioactive
or non-radioactive labels as known in the art. Kits can comprise
one or more pairs of polynucleotides for amplifying nucleic acids
specific for Urb-ctf, e.g., comprising a forward and reverse primer
effective in PCR. These include both sense and anti-sense orientations.
For instance, in PCR-based methods (such as RT-PCR), a pair of primers
are typically used, one having a sense sequence and the other having
an antisense sequence.
Another aspect of the present invention is a nucleotide sequence
that is specific to, or for, a selective polynucleotide. The phrases
"specific for" or "specific to" a polynucleotide
have a functional meaning that the polynucleotide can be used to
identify the presence of one or more target genes in a sample and
distinguish them from non-target genes. It is specific in the sense
that it can be used to detect polynucleotides above background noise
("non-specific binding"). A specific sequence is a defined
order of nucleotides (or amino acids, if it is polypeptide sequence)
which occurs in the polynucleotide, e.g., in the nucleotide sequences
of SEQ ID NO 1, and which is characteristic of that target sequence,
and substantially no non-target sequences. A probe or mixture of
probes can comprise a sequence or sequences that are specific to
a plurality of target sequences, e.g., where the sequence is a consensus
sequence, a functional domain, etc., e.g., capable of recognizing
a family of related genes. Such sequences can be used as probes
in any of the methods described herein or incorporated by reference.
Both sense and antisense nucleotide sequences are included. A specific
polynucleotide according to the present invention can be determined
A polynucleotide comprising a specific sequence can be used as
a hybridization probe to identify the presence of, e.g., human or
mouse polynucleotide, in a sample comprising a mixture of polynucleotides,
e.g., on a Northern blot. Hybridization can be performed under high
stringent conditions (see, above) to select polynucleotides (and
their complements which can contain the coding sequence) having
at least 90%, 95%, 99%, etc., identity (i.e., complementarity) to
the probe, but less stringent conditions can also be used. A specific
polynucleotide sequence can also be fused in-frame, at either its
5' or 3' end, to various nucleotide sequences as mentioned throughout
the patent, including coding sequences for enzymes, detectable markers,
GFP, etc, expression control sequences, etc.
A polynucleotide probe, especially one that is specific to a polynucleotide
of the present invention, can be used in gene detection and hybridization
methods as already described. In one embodiment, a specific polynucleotide
probe can be used to detect whether a particular tissue or cell-type
is present in a target sample. To carry out such a method, a selective
polynucleotide can be chosen which is characteristic of the desired
target tissue. Such polynucleotide is preferably chosen so that
it is expressed or displayed in the target tissue, but not in other
tissues which are present in the sample. For instance, if detection
of is desired, it may not matter whether the selective polynucleotide
is expressed in other tissues, as long as it is not expressed in
cells normally present in blood, e.g., peripheral blood mononuclear
cells. Starting from the selective polynucleotide, a specific polynucleotide
probe can be designed which hybridizes (if hybridization is the
basis of the assay) under the hybridization conditions to the selective
polynucleotide, whereby the presence of the selective polynucleotide
can be determined.
Probes which are specific for polynucleotides of the present invention
can also be prepared using involve transcription-based systems,
e.g., incorporating an RNA polymerase promoter into a selective
polynucleotide of the present invention, and then transcribing anti-sense
RNA using the polynucleotide as a template. See, e.g., U.S. Pat.
A polynucleotide according to the present invention can comprise,
e.g., DNA, RNA, synthetic polynucleotide, peptide polynucleotide,
modified nucleotides, dsDNA, ssDNA, ssRNA, dsRNA, and mixtures thereof.
A polynucleotide can be single- or double-stranded, triplex, DNA:RNA,
duplexes, comprise hairpins, and other secondary structures, etc.
Nucleotides comprising a polynucleotide can be joined via various
known linkages, e.g., ester, sulfamate, sulfamide, phosphorothioate,
phosphoramidate, methylphosphonate, carbamate, etc., depending on
the desired purpose, e.g., resistance to nucleases, such as RNAse
H, improved in vivo stability, etc. See, e.g., U.S. Pat. No. 5,378,825.
Any desired nucleotide or nucleotide analog can be incorporated,
e.g., 6-mercaptoguanine, 8-oxo-guanine, etc.
Various modifications can be made to the polynucleotides, such
as attaching detectable markers (avidin, biotin, radioactive elements,
fluorescent tags and dyes, energy transfer labels, energy-emitting
labels, binding partners, etc.) or moieties which improve hybridization,
detection, and/or stability. The polynucleotides can also be attached
to solid supports, e.g., nitrocellulose, magnetic or paramagnetic
microspheres (e.g., as described in U.S. Pat. No. 5,411,863; U.S.
Pat. No. 5,543,289; for instance, comprising ferromagnetic, supermagnetic,
paramagnetic, superparamagnetic, iron oxide and polysaccharide),
nylon, agarose, diazotized cellulose, latex solid microspheres,
polyacrylamides, etc., according to a desired method. See, e.g.,
U.S. Pat. Nos. 5,470,967, 5,476,925, and 5,478,893.
Polynucleotide according to the present invention can be labeled
according to any desired method. The polynucleotide can be labeled
using radioactive tracers such as .sup.32P, .sup.35S, .sup.3H, or
.sup.14C, to mention some commonly used tracers. The radioactive
labeling can be carried out according to any method, such as, for
example, terminal labeling at the 3' or 5' end using a radiolabeled
nucleotide, polynucleotide kinase (with or without dephosphorylation
with a phosphatase) or a ligase (depending on the end to be labeled).
A non-radioactive labeling can also be used, combining a polynucleotide
of the present invention with residues having immunological properties
(antigens, haptens), a specific affinity for certain reagents (ligands),
properties enabling detectable enzyme reactions to be completed
(enzymes or coenzymes, enzyme substrates, or other substances involved
in an enzymatic reaction), or characteristic physical properties,
such as fluorescence or the emission or absorption of light at a
desired wavelength, etc.
Nucleic Acid Detection Methods
Another aspect of the present invention relates to methods and
processes for detecting Urb-ctf. Detection methods have a variety
of applications, including for diagnostic, prognostic, forensic,
and research applications. To accomplish gene detection, a polynucleotide
in accordance with the present invention can be used as a "probe."
The term "probe" or "polynucleotide probe" has
its customary meaning in the art, e.g., a polynucleotide which is
effective to identify (e.g., by hybridization), when used in an
appropriate process, the presence of a target polynucleotide to
which it is designed. Identification can involve simply determining
presence or absence, or it can be quantitative, e.g., in assessing
amounts of a gene or gene transcript present in a sample. Probes
can be useful in a variety of ways, such as for diagnostic purposes,
to identify homologs, and to detect, quantitate, or isolate a polynucleotide
of the present invention in a test sample.
Assays can be utilized which permit quantification and/or presence/absence
detection of a target nucleic acid in a sample. Assays can be performed
at the single-cell level, or in a sample comprising many cells,
where the assay is "averaging" expression over the entire
collection of cells and tissue present in the sample. Any suitable
assay format can be used, including, but not limited to, e.g., Southern
blot analysis, Northern blot analysis, polymerase chain reaction
("PCR") (e.g., Saiki et al., Science, 241:53, 1988; U.S.
Pat. Nos. 4,683,195, 4,683,202, and 6,040,166; PCR Protocols: A
Guide to Methods and Applications, Innis et al., eds., Academic
Press, New York, 1990), reverse transcriptase polymerase chain reaction
("RT-PCR"), anchored PCR, rapid amplification of cDNA
ends ("RACE") (e.g., Schaefer in Gene Cloning and Analysis:
Current Innovations, Pages 99 115, 1997), ligase chain reaction
("LCR") (EP 320 308), one-sided PCR (Ohara et al., Proc.
Natl. Acad. Sci., 86:5673 5677, 1989), indexing methods (e.g., U.S.
Pat. No. 5,508,169), in situ hybridization, differential display
(e.g., Liang et al., Nucl. Acid. Res., 21:3269 3275, 1993; U.S.
Pat. Nos. 5,262,311, 5,599,672 and 5,965,409; WO97/18454; Prashar
and Weissman, Proc. Natl. Acad. Sci., 93:659 663, and U.S. Pat.
Nos. 6,010,850 and 5,712,126; Welsh et al., Nucleic Acid Res., 20:4965
4970, 1992, and U.S. Pat. No. 5,487,985) and other RNA fingerprinting
techniques, nucleic acid sequence based amplification ("NASBA")
and other transcription based amplification systems (e.g., U.S.
Pat. Nos. 5,409,818 and 5,554,527; WO 88/10315), polynucleotide
arrays (e.g., U.S. Pat. Nos. 5,143,854, 5,424,186; 5,700,637, 5,874,219,
and 6,054,270; PCT WO 92/10092; PCT WO 90/15070), Qbeta Replicase
(PCT/US87/00880), Strand Displacement Amplification ("SDA"),
Repair Chain Reaction ("RCR"), nuclease protection assays,
subtraction-based methods, Rapid-Scan.TM., etc. Additional useful
methods include, but are not limited to, e.g., template-based amplification
methods, competitive PCR (e.g., U.S. Pat. No. 5,747,251), redox-based
assays (e.g., U.S. Pat. No. 5,871,918), Taqman-based assays (e.g.,
Holland et al., Proc. Natl. Acad, Sci., 88:7276 7280, 1991; U.S.
Pat. Nos. 5,210,015 and 5,994,063), real-time fluorescence-based
monitoring (e.g., U.S. Pat. No. 5,928,907), molecular energy transfer
labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129, 5,565,322, 6,030,787,
and 6,117,635; Tyagi and Kramer, Nature Biotech., 14:303 309, 1996).
Any method suitable for single cell analysis of gene or protein
expression can be used, including in situ hybridization, immunocytochemistry,
MACS, FACS, flow cytometry, etc. For single cell assays, expression
products can be measured using antibodies, PCR, or other types of
nucleic acid amplification (e.g., Brady et al., Methods Mol. &
Cell. Biol. 2, 17 25, 1990; Eberwine et al., 1992, Proc. Natl. Acad.
Sci., 89, 3010 3014, 1992; U.S. Pat. No. 5,723,290). These and other
methods can be carried out conventionally, e.g., as described in
the mentioned publications.
Many of such methods may require that the polynucleotide is labeled,
or comprises a particular nucleotide type useful for detection.
The present invention includes such modified polynucleotides that
are necessary to carry out such methods. Thus, polynucleotides can
be DNA, RNA, DNA:RNA hybrids, PNA, etc., and can comprise any modification
or substituent which is effective to achieve detection.
Detection can be desirable for a variety of different purposes,
including research, diagnostic, prognostic, and forensic. For diagnostic
purposes, it may be desirable to identify the presence or quantity
of a polynucleotide sequence in a sample, where the sample is obtained
from tissue, cells, body fluids, etc. In a preferred method as described
in more detail below, the present invention relates to a method
of detecting a polynucleotide comprising, contacting a target polynucleotide
in a test sample with a polynucleotide probe under conditions effective
to achieve hybridization between the target and probe; and detecting
Any test sample in which it is desired to identify a polynucleotide
or polypeptide thereof can be used, including, e.g., blood, urine,
saliva, stool (for extracting nucleic acid, see, e.g., U.S. Pat.
No. 6,177,251), swabs comprising tissue, biopsied tissue, tissue
sections, cultured cells, etc.
Detection can be accomplished in combination with polynucleotide
probes for other genes, e.g., genes which are expressed in other
disease states, tissues, cells, such as brain, heart, kidney, spleen,
thymus, liver, stomach, small intestine, colon, muscle, lung, testis,
placenta, pituitary, thyroid, skin, adrenal gland, pancreas, salivary
gland, uterus, ovary, prostate gland, peripheral blood cells (T-cells,
lymphocytes, etc.), embryo, normal breast fat, adult and embryonic
stem cells, specific cell-types, such as endothelial, epithelial,
myocytes, adipose, luminal epithelial, basoepithelial, myoepithelial,
stromal cells, etc.
Polynucleotides can be used in wide range of methods and compositions,
including for detecting, diagnosing, staging, grading, assessing,
prognosticating, etc. diseases and disorders associated with Urb-ctf,
for monitoring or assessing therapeutic and/or preventative measures,
in ordered arrays, etc. Any method of detecting genes and polynucleotides
of SEQ ID NO 1 can be used; certainly, the present invention is
not to be limited how such methods are implemented.
Along these lines, the present invention relates to methods of
detecting Urb-ctf in a sample comprising nucleic acid. Such methods
can comprise one or more the following steps in any effective order,
e.g., contacting said sample with a polynucleotide probe under conditions
effective for said probe to hybridize specifically to nucleic acid
in said sample, and detecting the presence or absence of probe hybridized
to nucleic acid in said sample, wherein said probe is a polynucleotide
which is SEQ ID NO 1, a polynucleotide having, e.g., about 70%,
80%, 85%, 90%, 95%, 99%, or more sequence identity thereto, effective
or specific fragments thereof, or complements thereto. The detection
method can be applied to any sample, e.g., cultured primary, secondary,
or established cell lines, tissue biopsy, blood, urine, stool, cerebral
spinal fluid, and other bodily fluids, for any purpose.
Contacting the sample with probe can be carried out by any effective
means in any effective environment. It can be accomplished in a
solid, liquid, frozen, gaseous, amorphous, solidified, coagulated,
colloid, etc., mixtures thereof, matrix. For instance, a probe in
an aqueous medium can be contacted with a sample which is also in
an aqueous medium, or which is affixed to a solid matrix, or vice-versa.
Generally, as used throughout the specification, the term "effective
conditions" means, e.g., the particular milieu in which the
desired effect is achieved. Such a milieu, includes, e.g., appropriate
buffers, oxidizing agents, reducing agents, pH, co-factors, temperature,
ion concentrations, suitable age and/or stage of cell (such as,
in particular part of the cell cycle, or at a particular stage where
particular genes are being expressed) where cells are being used,
culture conditions (including substrate, oxygen, carbon dioxide,
etc.). When hybridization is the chosen means of achieving detection,
the probe and sample can be combined such that the resulting conditions
are functional for said probe to hybridize specifically to nucleic
acid in said sample.
The phrase "hybridize specifically" indicates that the
hybridization between single-stranded polynucleotides is based on
nucleotide sequence complementarity. The effective conditions are
selected such that the probe hybridizes to a preselected and/or
definite target nucleic acid in the sample. For instance, if detection
of a polynucleotide set forth in SEQ ID NO 1 is desired, a probe
can be selected which can hybridize to such target gene under high
stringent conditions, without significant hybridization to other
genes in the sample. To detect homologs of a polynucleotide set
forth in SEQ ID NO 1, the effective hybridization conditions can
be less stringent, and/or the probe can comprise codon degeneracy,
such that a homolog is detected in the sample.
As already mentioned, the methods can be carried out by any effective
process, e.g., by Northern blot analysis, polymerase chain reaction
(PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization,
etc., as indicated above. When PCR based techniques are used, two
or more probes are generally used. One probe can be specific for
a defined sequence which is characteristic of a selective polynucleotide,
but the other probe can be specific for the selective polynucleotide,
or specific for a more general sequence, e.g., a sequence such as
polyA which is characteristic of mRNA, a sequence which is specific
for a promoter, ribosome binding site, or other transcriptional
features, a consensus sequence (e.g., representing a functional
domain). For the former aspects, 5' and 3' probes (e.g., polyA,
Kozak, etc.) are preferred which are capable of specifically hybridizing
to the ends of transcripts. When PCR is utilized, the probes can
also be referred to as "primers" in that they can prime
a DNA polymerase reaction.
In addition to testing for the presence or absence of polynucleotides,
the present invention also relates to determining the amounts at
which polynucleotides of the present invention are expressed in
sample and determining the differential expression of such polynucleotides
in samples. Such methods can involve substantially the same steps
as described above for presence/absence detection, e.g., contacting
with probe, hybridizing, and detecting hybridized probe, but using
more quantitative methods and/or comparisons to standards.
The amount of hybridization between the probe and target can be
determined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR,
Northern blot, polynucleotide microarrays, Rapid-Scan, etc., and
includes both quantitative and qualitative measurements. For further
details, see the hybridization methods described above and below.
Determining by such hybridization whether the target is differentially
expressed (e.g., up-regulated or down-regulated) in the sample can
also be accomplished by any effective means. For instance, the target's
expression pattern in the sample can be compared to its pattern
in a known standard, such as in a normal tissue, or it can be compared
to another gene in the same sample. When a second sample is utilized
for the comparison, it can be a sample of normal tissue that is
known not to contain diseased cells. The comparison can be performed
on samples which contain the same amount of RNA (such as polyadenylated
RNA or total RNA), or, on RNA extracted from the same amounts of
starting tissue. Such a second sample can also be referred to as
a control or standard. Hybridization can also be compared to a second
target in the same tissue sample. Experiments can be performed that
determine a ratio between the target nucleic acid and a second nucleic
acid (a standard or control), e.g., in a normal tissue. When the
ratio between the target and control are substantially the same
in a normal and sample, the sample is determined or diagnosed not
to contain cells. However, if the ratio is different between the
normal and sample tissues, the sample is determined to contain cancer
cells. The approaches can be combined, and one or more second samples,
or second targets can be used. Any second target nucleic acid can
be used as a comparison, including "housekeeping" genes,
such as beta-actin, alcohol dehydrogenase, or any other gene whose
expression does not vary depending upon the disease status of the
Methods of Identifying Polymorphisms, Mutations, etc., of Urb-ctf
Polynucleotides of the present invention can also be utilized to
identify mutant alleles, SNPs, gene rearrangements and modifications,
and other polymorphisms of the wild-type gene. Mutant alleles, polymorphisms,
SNPs, etc., can be identified and isolated from cancers that are
known, or suspected to have, a genetic component. Identification
of such genes can be carried out routinely (see, above for more
guidance), e.g., using PCR, hybridization techniques, direct sequencing,
mismatch reactions (see, e.g., above), RFLP analysis, SSCP (e.g.,
Orita et al., Proc. Natl. Acad. Sci., 86:2766, 1992), etc., where
a polynucleotide having a sequence selected from SEQ ID NO 1 is
used as a probe. The selected mutant alleles, SNPs, polymorphisms,
etc., can be used diagnostically to determine whether a subject
has, or is susceptible to a disorder associated with Urb-ctf, as
well as to design therapies and predict the outcome of the disorder.
Methods involve, e.g., diagnosing a disorder associated with Urb-ctf
or determining susceptibility to a disorder, comprising, detecting
the presence of a mutation in a gene represented by a polynucleotide
selected from SEQ ID NO 1. The detecting can be carried out by any
effective method, e.g., obtaining cells from a subject, determining
the gene sequence or structure of a target gene (using, e.g., mRNA,
cDNA, genomic DNA, etc), comparing the sequence or structure of
the target gene to the structure of the normal gene, whereby a difference
in sequence or structure indicates a mutation in the gene in the
subject. Polynucleotides can also be used to test for mutations,
SNPs, polymorphisms, etc., e.g., using mismatch DNA repair technology
as described in U.S. Pat. No. 5,683,877; U.S. Pat. No. 5,656,430;
Wu et al., Proc. Natl. Acad. Sci., 89:8779 8783, 1992.
The present invention also relates to methods of detecting polymorphisms
in Urb-ctf, comprising, e.g., comparing the structure of: genomic
DNA comprising all or part of Urb-ctf, mRNA comprising all or part
of Urb-ctf, cDNA comprising all or part of Urb-ctf, or a polypeptide
comprising all or part of Urb-ctf, with the structure of Urb-ctf
set forth in SEQ ID NO 1. The methods can be carried out on a sample
from any source, e.g., cells, tissues, body fluids, blood, urine,
stool, hair, egg, sperm, cerebral spinal fluid, etc.
These methods can be implemented in many different ways. For example,
"comparing the structure" steps include, but are not limited
to, comparing restriction maps, nucleotide sequences, amino acid
sequences, RFLPs, Dnase sites, DNA methylation fingerprints (e.g.,
U.S. Pat. No. 6,214,556), protein cleavage sites, molecular weights,
electrophoretic mobilities, charges, ion mobility, etc., between
a standard Urb-ctf and a test Urb-ctf. The term "structure"
can refer to any physical characteristics or configurations which
can be used to distinguish between nucleic acids and polypeptides.
The methods and instruments used to accomplish the comparing step
depends upon the physical characteristics which are to be compared.
Thus, various techniques are contemplated, including, e.g., sequencing
machines (both amino acid and polynucleotide), electrophoresis,
mass spectrometer (U.S. Pat. Nos. 6,093,541, 6,002,127), liquid
chromatography, HPLC, etc.
To carry out such methods, "all or part" of the gene
or polypeptide can be compared. For example, if nucleotide sequencing
is utilized, the entire gene can be sequenced, including promoter,
introns, and exons, or only parts of it can be sequenced and compared,
e.g., exon 1, exon 2, etc.
Mutated polynucleotide sequences of the present invention are useful
for various purposes, e.g., to create mutations of the polypeptides
they encode, to identify functional regions of genomic DNA, to produce
probes for screening libraries, etc. Mutagenesis can be carried
out routinely according to any effective method, e.g., oligonucleotide-directed
(Smith, M., Ann. Rev. Genet. 19:423 463, 1985), degenerate oligonucleotide-directed
(Hill et al., Method Enzymology, 155:558 568, 1987), region-specific
(Myers et al., Science, 229:242 246, 1985; Derbyshire et al., Gene,
46:145, 1986; Ner et al., DNA, 7:127, 1988), linker-scanning (McKnight
and Kingsbury, Science, 217:316 324, 1982), directed using PCR,
recursive ensemble mutagenesis (Arkin and Yourvan, Proc. Natl. Acad.
Sci., 89:7811 7815, 1992), random mutagenesis (e.g., U.S. Pat. Nos.
5,096,815; 5,198,346; and 5,223,409), site-directed mutagenesis
(e.g., Walder et al., Gene, 42:133, 1986; Bauer et al., Gene, 37:73,
1985; Craik, Bio Techniques, Jan. 1985, 12 19; Smith et al., Genetic
Engineering: Principles and Methods, Plenum Press, 1981), phage
display (e.g., Lowman et al., Biochem. 30:10832 10837, 1991; Ladner
et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204),
etc. Desired sequences can also be produced by the assembly of target
sequences using mutually priming oligonucleotides (Uhlmann, Gene,
71:29 40, 1988). For directed mutagenesis methods, analysis of the
three-dimensional structure of the Urb-ctf polypeptide can be used
to guide and facilitate making mutants which effect polypeptide
activity. Sites of substrate-enzyme interaction or other biological
activities can also be determined by analysis of crystal structure
as determined by such techniques as nuclear magnetic resonance,
crystallography or photoaffinity labeling. See, for example, de
Vos et al., Science 255:306 312, 1992; Smith et al., J. Mol. Biol.
224:899 904, 1992; Wlodaver et al., FEBS Lett. 309:59 64, 1992.
In addition, libraries of Urb-ctf and fragments thereof can be
used for screening and selection of Urb-ctf variants. For instance,
a library of coding sequences can be generated by treating a double-stranded
DNA with a nuclease under conditions where the nicking occurs, e.g.,
only once per molecule, denaturing the double-stranded DNA, renaturing
it to for double-stranded DNA that can include sense/antisense pairs
from different nicked products, removing single-stranded portions
from reformed duplexes by treatment with S1 nuclease, and ligating
the resulting DNAs into an expression vecore. By this method, xpression
libraries can be made comprising "mutagenized" Urb-ctf.
The entire coding sequence or parts thereof can be used.
Polynucleotide Expression, Polypeptides Produced thereby, and Specific-binding
A polynucleotide according to the present invention can be expressed
in a variety of different systems, in vitro and in vivo, according
to the desired purpose. For example, a polynucleotide can be inserted
into an expression vector, introduced into a desired host, and cultured
under conditions effective to achieve expression of a polypeptide
coded for by the polynucleotide, to search for specific binding
partners. Effective conditions include any culture conditions which
are suitable for achieving production of the polypeptide by the
host cell, including effective temperatures, pH, medium, additives
to the media in which the host cell is cultured (e.g., additives
which amplify or induce expression such as butyrate, or methotrexate
if the coding polynucleotide is adjacent to a dhfr gene), cycloheximide,
cell densities, culture dishes, etc. A polynucleotide can be introduced
into the cell by any effective method including, e.g., naked DNA,
calcium phosphate precipitation, electroporation, injection, DEAE-Dextran
mediated transfection, fusion with liposomes, association with agents
which enhance its uptake into cells, viral transfection. A cell
into which a polynucleotide of the present invention has been introduced
is a transformed host cell. The polynucleotide can be extrachromosomal
or integrated into a chromosome(s) of the host cell. It can be stable
or transient. An expression vector is selected for its compatibility
with the host cell. Host cells include, mammalian cells, e.g., COS,
CV1, BHK, CHO, HeLa, LTK, NIH 3T3, ZR-75-1 (ATCC CRL-1500), ZR-75-30
(ATCC CRL-1504), UACC-812 (ATCC CRL-1897), UACC-893 (ATCC CRL-1902),
HCC38 (ATCC CRL-2314), HCC70 (CRL-2315), and other HCC cell lines
(e.g., as deposited with the ATCC), AU565 (ATCC CRL-2351), Hs 496.T
(ATCC CRL-7303), Hs 748.T (ATCC CRL-7486), SW527 (ATCC CRL-7940),
184A1 (ATCC CRL-8798), MCF cell lines (e.g., 10A and others deposited
with the ATCC), MDA-MB-134-VI (ATCC HTB-23 and other MDA cell lines),
SK-BR-3 (ATCC HTB-30), ME-180 (ATCC HTB-33), Hs 578Bst (ATCC HTB-125),
Hs 578T (ATCC HTB-126), T-47D (ATCC HTB-133), insect cells, such
as Sf9 (S. frugipeda) and Drosophila, bacteria, such as E. coli,
Streptococcus, bacillus, yeast, such as Sacharomyces, S. cerevisiae,
fungal cells, plant cells, embryonic or adult stem cells (e.g.,
mammalian, such as mouse or human).
Expression control sequences are similarly selected for host compatibility
and a desired purpose, e.g., high copy number, high amounts, induction,
amplification, controlled expression. Other sequences which can
be employed include enhancers such as from SV40, CMV, RSV, inducible
promoters, cell-type specific elements, or sequences which allow
selective or specific cell expression. Promoters that can be used
to drive its expression, include, e.g., the endogenous promoter,
MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts;
or alpha factor, alcohol oxidase, or PGH promoters for yeast. RNA
promoters can be used to produced RNA transcripts, such as T7 or
SP6. See, e.g., Melton et al., Polynucleotide Res., 12(18):7035
7056, 1984; Dunn and Studier. J. Mol. Bio., 166:477 435, 1984; U.S.
Pat. No. 5,891,636; Studier et al., Gene Expression Technology,
Methods in Enzymology, 85:60 89, 1987. In addition, as discussed
above, translational signals (including in-frame insertions) can
When a polynucleotide is expressed as a heterologous gene in a
transfected cell line, the gene is introduced into a cell as described
above, under effective conditions in which the gene is expressed.
The term "heterologous" means that the gene has been introduced
into the cell line by the "hand-of-man." Introduction
of a gene into a cell line is discussed above. The transfected (or
transformed) cell expressing the gene can be lysed or the cell line
can be used intact.
For expression and other purposes, a polynucleotide can contain
codons found in a naturally-occurring gene, transcript, or cDNA,
for example, e.g., as set forth in SEQ ID NO 1, or it can contain
degenerate codons coding for the same amino acid sequences. For
instance, it may be desirable to change the codons in the sequence
to optimize the sequence for expression in a desired host. See,
e.g., U.S. Pat. Nos. 5,567,600 and 5,567,862.
A polypeptide according to the present invention can be recovered
from natural sources, transformed host cells (culture medium or
cells) according to the usual methods, including, detergent extraction
(e.g., non-ionic detergent, Triton X-100, CHAPS, octylglucoside,
Igepal CA-630), ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography, hydroxyapatite
chromatography, lectin chromatography, gel electrophoresis. Protein
refolding steps can be used, as necessary, in completing the configuration
of the mature protein. Finally, high performance liquid chromatography
(HPLC) can be employed for purification steps. Another approach
is express the polypeptide recombinantly with an affinity tag (Flag
epitope, HA epitope, myc epitope, 6xHis, maltose binding protein,
chitinase, etc) and then purify by anti-tag antibody-conjugated
The present invention also relates to polypeptides of Urb-ctf,
e.g., an isolated human Urb-ctf polypeptide comprising or having
the amino acid sequence set forth in SEQ ID NO 2, an isolated human
Urb-ctf polypeptide comprising an amino acid sequence having 99%
or more sequence identity to the amino acid sequence set forth in
SEQ ID NO 2, and have having one or more of Urb-ctf activities,
such as transcriptional regulatory activity, DNA-binding activity,
dimerization activity, immunological activity, etc. Fragments specific
to Urb-ctf can also used, e.g., to produce antibodies or other immune
responses, as competitors to DNA-binding, dimerization, or transcriptional
activity, etc. These fragments can be referred to as being "specific
for" Urb-ctf. The latter phrase, as already defined, indicates
that the peptides are characteristic of Urb-ctf, and the defined
sequences are substantially absent from all other protein types.
Such polypeptides can be of any size which is necessary to confer
specificity, e.g., 5, 8, 10, 12, 15, 20, etc. Especially preferred
are polypeptides which comprise the following amino acid residues,
e.g., amino acid 38 of SEQ ID NO 2, amino acid 68 of SEQ ID NO 2,
amino acids 76 77 of SEQ ID NO 2, amino acid 119 of SEQ ID NO 2,
amino acid 143 144 of SEQ ID NO 2, amino acid 161 of SEQ ID NO 2,
amino acid 583 of SEQ ID NO 2, or amino acid 606 of SEQ ID NO 2,
including peptides having amino acids 1 263 of SEQ ID NO 2 or 459
614 of SEQ ID NO 2.
The present invention also relates to antibodies, and other specific-binding
partners, which are specific for polypeptides encoded by polynucleotides
of the present invention, e.g., Urb-ctf. Antibodies, e.g., polyclonal,
monoclonal, recombinant, chimeric, humanized, single-chain, Fab,
and fragments thereof, can be prepared according to any desired
method. See, also, screening recombinant immunoglobulin libraries
(e.g., Orlandi et al., Proc. Natl. Acad. Sci., 86:3833 3837, 1989;
Huse et al., Science, 256:1275 1281, 1989); in vitro stimulation
of lymphocyte populations; Winter and Milstein, Nature, 349: 293
299, 1991. The antibodies can be IgM, IgG, subtypes, IgG2a, IgG1,
etc. Antibodies, and immune responses, can also be generated by
administering naked DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466;
5,580,859. Antibodies can be used from any source, including, goat,
rabbit, mouse, chicken (e.g., IgY; see, Duan, W0/029444 for methods
of making antibodies in avian hosts, and harvesting the antibodies
from the eggs). An antibody specific for a polypeptide means that
the antibody recognizes a defined sequence of amino acids within
or including the polypeptide. Other specific binding partners include,
e.g., aptamers and PNA. Antibodies can be prepared against specific
epitopes or domains of Urb-ctf, e.g., an antibody which is specific
for an epitope comprising, amino acid 38 of SEQ ID NO 2, amino acid
68 of SEQ ID NO 2, amino acids 76 77 of SEQ ID NO 2, amino acid
119 of SEQ ID NO 2, amino acid 143 144 of SEQ ID NO 2, amino acid
161 of SEQ ID NO 2, amino acid 583 of SEQ ID NO 2, amino acid 606
of SEQ ID NO 2, etc. By being specific to an epitope, it means that
the antibody recognizes a defined sequence of amino acids which
includes the particular amino acid residue.
The preparation of polyclonal antibodies is well-known to those
skilled in the art. See, for example, Green et al., Production of
Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.),
pages 1 5 (Humana Press 1992); Coligan et al., Production of Polyclonal
Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS
IN IMMUNOLOGY, section 2.4.1 (1992). The preparation of monoclonal
antibodies likewise is conventional. See, for example, Kohler &
Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1
2.6.7; and Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page
726 (Cold Spring Harbor Pub. 1988).
Antibodies can also be humanized, e.g., where they are to be used
therapeutically. Humanized monoclonal antibodies are produced by
transferring mouse complementarity determining regions from heavy
and light variable chains of the mouse immunoglobulin into a human
variable domain, and then substituting human residues in the framework
regions of the murine counterparts. The use of antibody components
derived from humanized monoclonal antibodies obviates potential
problems associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable domains
are described, for example, by Orlandi et al., Proc. Nat'l Acad.
Sci. USA 86:3833 (1989), which is hereby incorporated in its entirety
by reference. Techniques for producing humanized monoclonal antibodies
are described, for example, in U.S. Pat. No. 6,054,297, Jones et
al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988);
Verhoeyen et al., Science 239: 1534 (1988); Carter et al., Proc.
Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech.
12: 437 (1992); and Singer et al., J. Immunol. 150: 2844 (1993).
Antibodies of the invention also may be derived from human antibody
fragments isolated from a combinatorial immunoglobulin library.
See, for example, Barbas et al., METHODS: A COMPANION TO METHODS
IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann. Rev.
Immunol. 12: 433 (1994). Cloning and expression vectors that are
useful for producing a human immunoglobulin phage library can be
obtained commercially, for example, from STRATAGENE Cloning Systems
(La Jolla, Calif.).
In addition, antibodies of the present invention may be derived
from a human monoclonal antibody. Such antibodies are obtained from
transgenic mice that have been "engineered" to produce
specific human antibodies in response to antigenic challenge. In
this technique, elements of the human heavy and light chain loci
are introduced into strains of mice derived from embryonic stem
cell lines that contain targeted disruptions of the endogenous heavy
and light chain loci. The transgenic mice can synthesize human antibodies
specific for human antigens and can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, e.g., in Green et al., Nature Genet. 7:13 (1994);
Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immunol.
Antibody fragments of the present invention can be prepared by
proteolytic hydrolysis of the antibody or by expression in E. coli
of nucleic acid encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies by conventional
methods. For example, antibody fragments can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage
using pepsin produces two monovalent Fab' fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, and references
contained therein. These patents are hereby incorporated in their
entireties by reference. See also Nisoiihoff et al., Arch. Biochem.
Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman
et al, METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967);
and Coligan et al. at sections 2.8.1 2.8.10 and 2.10.1 2.10.4.
Other methods of cleaving antibodies, such as separation of heavy
chains to form monovalent light-heavy chain fragments, further cleavage
of fragments, or other enzymatic, chemical, or genetic techniques
can also be used. For example, Fv fragments comprise an association
of V.sub.H and V.sub.L chains. This association may be noncovalent,
as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an intermolecular
disulfide bond or cross-linked by chemicals such as glutaraldehyde.
See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise
V.sub.H and V.sub.L chains connected by a peptide linker. These
single-chain antigen binding proteins (sFv) are prepared by constructing
a structural gene comprising nucleic acid sequences encoding the
V.sub.H and V.sub.L domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The recombinant
host cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing sFvs are described,
for example, by Whitlow et al., METHODS: A COMPANION TO METHODS
IN ENZYMOLOGY, VOL. 2, page 97(1991); Bird etal.,Science 242:423
426 (1988); Ladneret al., U.S. Pat. No. 4,946,778; Pack et al.,
Bio/Technology 11: 1271 77 (1993); and Sandhu, supra.
Another form of an antibody fragment is a peptide coding for a
single complementarity-determining region (CDR). CDR peptides ("minimal
recognition units") can be obtained by constructing genes encoding
the CDR of an antibody of interest. Such genes are prepared, for
example, by using the polymerase chain reaction to synthesize the
variable region from RNA of antibody-producing cells. See, for example,
Larrick et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL.
2, page 106 (1991).
The term "antibody" as used herein includes intact molecules
as well as fragments thereof, such as Fab, F(ab')2, and Fv which
are capable of binding to an epitopic determinant present in Bin1
polypeptide. Such antibody fragments retain some ability to selectively
bind with its antigen or receptor. The term "epitope"
refers to an antigenic determinant on an antigen to which the paratope
of an antibody binds. Epitopic determinants usually consist of chemically
active surface groupings of molecules such as amino acids or sugar
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics. Antibodies
can be prepared against specific epitopes or polypeptide domains.
Antibodies which bind to Urb-ctf polypeptides of the present invention
can be prepared using an intact polypeptide or fragments containing
small peptides of interest as the immunizing antigen. For example,
it may be desirable to produce antibodies that specifically bind
to the N- or C-terminal domains of Urb-ctf. The polypeptide or peptide
used to immunize an animal which is derived from translated cDNA
or chemically synthesized which can be conjugated to a carrier protein,
if desired. Such commonly used carriers which are chemically coupled
to the immunizing peptide include keyhole limpet hemocyanin (KLH),
thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
Polyclonal or monoclonal antibodies can be further purified, for
example, by binding to and elution from a matrix to which the polypeptide
or a peptide to which the antibodies were raised is bound. Those
of skill in the art will know of various techniques common in the
immunology arts for purification and/or concentration of polyclonal
antibodies, as well as monoclonal antibodies (See for example, Coligan,
et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,
1994, incorporated by reference).
Anti-idiotype technology can also be used to produce invention
monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic
monoclonal antibody made to a first monoclonal antibody will have
a binding domain in the hypervariable region which is the "image"
of the epitope bound by the first monoclonal antibody.
Methods of Detecting Polypeptides
Polypeptides coded for by Urb-ctf of the present invention can
be detected, visualized, determined, quantitated, etc. according
to any effective method. useful methods include, e.g., but are not
limited to, immunoassays, RIA (radioimmunassay), ELISA, (enzyme-linked-immunosorbent
assay), immunoflourescence, flow cytometry, histology, electron
microscopy, light microscopy, in situ assays, immunoprecipitation,
Western blot, etc.
Immunoassays may be carried in liquid or on biological support.
For instance, a sample (e.g., blood, stool, urine, cells, tissue,
cerebral spinal fluid, body fluids, etc.) can be brought in contact
with and immobilized onto a solid phase support or carrier such
as nitrocellulose, or other solid support that is capable of immobilizing
cells, cell particles or soluble proteins. The support may then
be washed with suitable buffers followed by treatment with the detectably
labeled Urb-ctf specific antibody. The solid phase support can then
be washed with a buffer a second time to remove unbound antibody.
The amount of bound label on solid support may then be detected
by conventional means.
A "solid phase support or carrier" includes any support
capable of binding an antigen, antibody, or other specific binding
partner. Supports or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides, and magnetite. A support material can have any
structural or physical configuration. Thus, the support configuration
may be spherical, as in a bead, or cylindrical, as in the inside
surface of a test tube, or the external surface of a rod. Alternatively,
the surface may be flat such as a sheet, test strip, etc. Preferred
supports include polystyrene beads
One of the many ways in which gene peptide-specific antibody can
be detectably labeled is by linking it to an enzyme and using it
in an enzyme immunoassay (EIA). See, e.g., Voller, A., "The
Enzyme Linked Immunosorbent Assay (ELISA)," 1978, Diagnostic
Horizons 2, 1 7, Microbiological Associates Quarterly Publication,
Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31,
507 520; Butler, J. E., 1981, Meth. Enzymol. 73, 482 523; Maggio,
E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.
The enzyme which is bound to the antibody will react with an appropriate
substrate, preferably a chromogenic substrate, in such a manner
as to produce a chemical moiety that can be detected, for example,
by spectrophotometric, fluorimetric or by visual means. Enzymes
that can be used to detectably label the antibody include, but are
not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast alcohol dehydrogenase, .alpha.-glycerophosphate,
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase, beta.-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase and acetylcholinesterase. The detection can be accomplished
by colorimetric methods that employ a chromogenic substrate for
the enzyme. Detection may also be accomplished by visual comparison
of the extent of enzymatic reaction of a substrate in comparison
with similarly prepared standards.
Detection may also be accomplished using any of a variety of other
immunoassays. For example, by radioactively labeling the antibodies
or antibody fragments, it is possible to detect Urb-ctf peptides
through the use of a radioimmunoassay (RIA). See, e.g., Weintraub,
B., Principles of Radioimmunoassays, Seventh Training Course on
Radioligand Assay Techniques, The Endocrine Society, March, 1986.
The radioactive isotope can be detected by such means as the use
of a gamma counter or a scintillation counter or by autoradiography.
It is also possible to label the antibody with a fluorescent compound.
When the fluorescently labeled antibody is exposed to light of the
proper wave length, its presence can then be detected due to fluorescence.
Among the most commonly used fluorescent labeling compounds are
fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine. The antibody
can also be detectably labeled using fluorescence emitting metals
such as those in the lanthanide series. These metals can be attached
to the antibody using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The antibody also can be detectably labeled by coupling it to a
chemiluminescent compound. The presence of the chemiluminescent-tagged
antibody is then determined by detecting the presence of luminescence
that arises during the course of a chemical reaction. Examples of
useful chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
Likewise, a bioluminescent compound may be used to label the antibody
of the present invention. Bioluminescence is a type of chemiluminescence
found in biological systems in which a catalytic protein increases
the efficiency of the chemiluminescent reaction. The presence of
a bioluminescent protein is determined by detecting the presence
of luminescence. Important bioluminescent compounds for purposes
of labeling are luciferin, luciferase and aequorin.
The present invention also relates to methods and compositions
for diagnosing abreast cancer, or determining susceptibility to
it, using polynucleotides, polypeptides, and specific-binding partners
of the present invention to detect, assess, determine, etc., Urb-ctf.
In such methods, the gene can serve as a marker for the disorder,
e.g., where the gene, when mutant, is a direct cause of the disorder;
where the gene is affected by another gene(s) which is directly
responsible for the disorder, e.g., when the gene is part of the
same signaling pathway as the directly responsible gene; and, where
the gene is chromosomally linked to the gene(s) directly responsible
for the disorder, and segregates with it. Many other situations
are possible. To detect, assess, determine, etc., a probe specific
for the gene can be employed as described above and below. Any method
of detecting and/or assessing the gene can be used, including detecting
expression of the gene using polynucleotides, antibodies, or other
The present invention relates to methods of diagnosing a disorder
associated with Urb-ctf, such as breast cancer, or determining a
subject's susceptibility to such disorder, comprising, e.g., assessing
the expression of Urb-ctf in a tissue sample comprising tissue or
cells suspected of having the disorder (e.g., where the sample comprises
breast cancer). The phrase "diagnosing" indicates that
it is determined whether the sample has the disorder. A "disorder"
means, e.g., any abnormal condition as in a disease or malady. "Determining
a subject's susceptibility to a disease or disorder" indicates
that the subject is assessed for whether s/he is predisposed to
get such a disease or disorder, where the predisposition is indicated
by abnormal expression of the gene (e.g., gene mutation, gene expression
pattern is not normal, etc.). Predisposition or susceptibility to
a disease may result when a such disease is influenced by epigenetic,
environmental, etc., factors. This includes prenatal screening where
samples from the fetus or embryo (e.g., via amniocentesis or CV
sampling) are analyzed for the expression of the gene.
By the phrase "assessing expression of Urb-ctf," it is
meant that the functional status of the gene is evaluated. This
includes, but is not limited to, measuring expression levels of
said gene, determining the genomic structure of said gene, determining
the mRNA structure of transcripts from said gene, or measuring the
expression levels of polypeptide coded for by said gene. Thus, the
term "assessing expression" includes evaluating the all
aspects of the transcriptional and translational machinery of the
gene. For instance, if a promoter defect causes, or is suspected
of causing, the disorder, then a sample can be evaluated (i.e.,
"assessed") by looking (e.g., sequencing or restriction
mapping) at the promoter sequence in the gene, by detecting transcription
products (e.g., RNA), by detecting translation product (e.g., polypeptide).
Any measure of whether the gene is functional can be used, including,
polypeptide, polynucleotide, and functional assays for the gene's
In making the assessment, it can be useful to compare the results
to a normal gene, e.g., a gene which is not associated with the
disorder. The nature of the comparison can be determined routinely,
depending upon how the assessing is accomplished. If, for example,
the mRNA levels of a sample is detected, then the mRNA levels of
a normal can serve as a comparison, or a gene which is known not
to be affected by the disorder. Methods of detecting mRNA are well
known, and discussed above, e.g., but not limited to, Northern blot
analysis, polymerase chain reaction (PCR), reverse transcriptase
PCR, RACE PCR, etc. Similarly, if polypeptide production is used
to evaluate the gene, then the polypeptide in a normal tissue sample
can be used as a comparison, or, polypeptide from a different gene
whose expression is known not to be affected by the disorder. These
are only examples of how such a method could be carried out.
Assessing the effects of therapeutic and preventative interventions
(e.g., administration of a drug, chemotherapy, radiation, etc.)
on breast cancer is a major effort in drug discovery, clinical medicine,
and pharmacogenomics. The evaluation of therapeutic and preventative
measures, whether experimental or already in clinical use, has broad
applicability, e.g., in clinical trials, for monitoring the status
of a patient, for analyzing and assessing animal models, and in
any scenario involving cancer treatment and prevention. Analyzing
the expression profiles of polynucleotides of the present invention
can be utilized as a parameter by which interventions are judged
and measured. Treatment of a disorder can change the expression
profile in some manner which is prognostic or indicative of the
drug's effect on it. Changes in the profile can indicate, e.g.,
drug toxicity, return to a normal level, etc. Accordingly, the present
invention also relates to methods of monitoring or assessing a therapeutic
or preventative measure (e.g., chemotherapy, radiation, anti-neoplastic
drugs, antibodies, etc.) in a subject having breast cancer, or,
susceptible to such disease, comprising, e.g., detecting the expression
levels of Urb-ctf. A subject can be a cell-based assay system, non-human
animal model, human patient, etc. Detecting can be accomplished
as described for the methods above and below. By "therapeutic
or preventative intervention," it is meant, e.g., a drug administered
to a patient, surgery, radiation, chemotherapy, and other measures
taken to prevent, treat, or diagnose a disorder.
Expression can be assessed in any sample comprising any tissue
or cell type, body fluid, etc., as discussed for other methods of
the present invention, including cells from breast cancer can be
used, or cells derived from breast cancer. By the phrase "cells
derived from breast cancer," it is meant that the derived cells
originate from breast cancer, e.g., when metastasis from a primary
tumor site has occurred, when a progenitor-type or pluripotent cell
gives rise to other cells, etc.
Identifying Agent Methods
The present invention also relates to methods of identifying agents,
and the agents themselves, which modulate Urb-ctf. These agents
can be used to modulate the biological activity of the polypeptide
encoded for the gene, or the gene, itself. Agents which regulate
the gene or its product are useful in variety of different environments,
including as medicinal agents to treat or prevent disorders associated
with Urb-ctf and as research reagents to modify the function of
tissues and cell.
Methods of identifying agents generally comprise steps in which
an agent is placed in contact with the gene, transcription product,
translation product, or other target, and then a determination is
performed to assess whether the agent "modulates" the
target. The specific method utilized will depend upon a number of
factors, including, e.g., the target (i.e., is it the gene or polypeptide
encoded by it), the environment (e.g., in vitro or in vivo), the
composition of the agent, etc.
For modulating the expression of Urb-ctf gene, a method can comprise,
in any effective order, one or more of the following steps, e.g.,
contacting a Urb-ctf gene (e.g., in a cell population) with a test
agent under conditions effective for said test agent to modulate
the expression of Urb-ctf, and determining whether said test agent
modulates said Urb-ctf. An agent can modulate expression of Urb-ctf
at any level, including transcription, translation, and/or perdurance
of the nucleic acid (e.g., degradation, stability, etc.) in the
cell. For modulating the biological activity of Urb-ctf polypeptides,
a method can comprise, in any effective order, one or more of the
following steps, e.g., contacting a Urb-ctf polypeptide (e.g., in
a cell, lysate, or isolated) with a test agent under conditions
effective for said test agent to modulate the biological activity
of said polypeptide, and determining whether said test agent modulates
said biological activity.
Contacting Urb-ctf with the test agent can be accomplished by any
suitable method and/or means that places the agent in a position
to functionally control expression or biological activity of Urb-ctf
present in the sample. Functional control indicates that the agent
can exert its physiological effect on Urb-ctf through whatever mechanism
it works. The choice of the method and/or means can depend upon
the nature of the agent and the condition and type of environment
in which the Urb-ctf is presented, e.g., lysate, isolated, or in
a cell population (such as, in vivo, in vitro, organ explants, etc.).
For instance, if the cell population is an in vitro cell culture,
the agent can be contacted with the cells by adding it directly
into the culture medium. If the agent cannot dissolve readily in
an aqueous medium, it can be incorporated into liposomes, or another
lipophilic carrier, and then administered to the cell culture. Contact
can also be facilitated by incorporation of agent with carriers
and delivery molecules and complexes, by injection, by infusion,
After the agent has been administered in such a way that it can
gain access to Urb-ctf, it can be determined whether the test agent
modulates Urb-ctf expression or biological activity. Modulation
can be of any type, quality, or quantity, e.g., increase, facilitate,
enhance, up-regulate, stimulate, activate, amplify, augment, induce,
decrease, down-regulate, diminish, lessen, reduce, etc. The modulatory
quantity can also encompass any value, e.g., 1%, 5%, 10%, 50%, 75%,
1-fold, 2-fold, 5-fold, 10-fold, 100-fold, etc. To modulate Urb-ctf
expression means, e.g., that the test agent has an effect on its
expression, e.g., to effect the amount of transcription, to effect
RNA splicing, to effect translation of the RNA into polypeptide,
to effect RNA or polypeptide stability, to effect polyadenylation
or other processing of the RNA, to effect post-transcriptional or
post-translational processing, etc. To modulate biological activity
means, e.g., that a functional activity of the polypeptide is changed
in comparison to its normal activity in the absence of the agent.
This effect includes, increase, decrease, block, inhibit, enhance,
etc. Biological activities of Urb-ctf include, e.g., transcriptional
regulatory activity (e.g., similar to bZIP proteins c-fos and c-jun).
A test agent can be of any molecular composition, e.g., chemical
compounds, biomolecules, such as polypeptides, lipids, nucleic acids
(e.g., antisense to a polynucleotide sequence selected from SEQ
ID NO 1), carbohydrates, antibodies, ribozymes, double-stranded
RNA, aptamers, etc. For example, polypeptide fragments can be used
to competitively inhibit Urb-ctf from binding to DNA or from forming
dimers. Antibodies can also be used to modulate the biological activity
a polypeptide in a lysate or other cell-free form. Antisense Urb-ctf
can also be used as test agents to modulate gene expression.
The polynucleotides of the present invention can be used with other
markers, especially breast cancer markers, to identity, detect,
stage, diagnosis, determine, prognosticate, treat, etc., tissue,
diseases and conditions, etc, of the breast cancer. Markers can
be polynucleotides, polypeptides, antibodies, ligands, specific
binding partners, etc. The targets for such markers include, but
are not limited genes and polypeptides that are selective for cell
types present in the breast cancer. The targets for such markers
include, but are not limited genes and polypeptides that are selective
for cell types present in the breast. Specific targets include,
BRCA1, BRCA2, ATM, PTEN/MMAC1 (e.g., Ali et al., J. Natl. Cancer
Inst., 91:1922 1932, 1999), MLH2, MSH2, TP53 (e.g., Done et al.,
Cancer Res., 58:785 789, 1998), STK11, myc, cyclin D1 (e.g., Weinstat-Saslow
et al., Nature Med., 1:1257 1260, 1995), c-erb-B2, keratins, such
as 5/6 and 8/18.
Selective polynucleotides, polypeptides, and specific-binding partners
thereto, can be utilized in therapeutic applications, especially
to treat diseases and conditions of breast cancer. Useful methods
include, but are not limited to, immunotherapy (e.g., using specific-binding
partners to polypeptides), vaccination (e.g., using a selective
polypeptide or a naked DNA encoding such polypeptide), protein or
polypeptide replacement therapy, gene therapy (e.g., germ-line correction,
Various immunotherapeutic approaches can be used. For instance,
unlabeled antibody that specifically recognizes a tissue-specific
antigen can be used to stimulate the body to destroy or attack the
cancer, to cause down-regulation, to produce complement-mediated
lysis, to inhibit cell growth, etc., of target cells which display
the antigen, e.g., analogously to how c-erbB-2 antibodies are used
to treat breast cancer. In addition, antibody can be labeled or
conjugated to enhance its deleterious effect, e.g., with radionuclides
and other energy emitting entitities, toxins, such as ricin, exotoxin
A (ETA), and diphtheria, cytotoxic or cytostatic agents, immunomodulators,
chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090.
An antibody or other specific-binding partner can be conjugated
to a second molecule, such as a cytotoxic agent, and used for targeting
the second molecule to a tissue-antigen positive cell (Vitetta,
E. S. et al., 1993, Immunotoxin therapy, in DeVita, Jr., V. T. et
al., eds, Cancer: Principles and Practice of Oncology, 4th ed.,
J. B. Lippincott Co., Philadelphia, 2624 2636). Examples of cytotoxic
agents include, but are not limited to, antimetabolites, alkylating
agents, anthracyclines, antibiotics, anti-mitotic agents, radioisotopes
and chemotherapeutic agents. Further examples of cytotoxic agents
include, but are not limited to ricin, doxorubicin, daunorubicin,
taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, dihydroxy anthracin dione, actinomycin
D, 1-dehydrotestosterone, diptheria toxin, Pseudomonas exotoxin
(PE) A, PE40, abrin, elongation factor-2 and glucocorticoid. Techniques
for conjugating therapeutic agents to antibodies are well.
In addition to immunotherapy, polynucleotides and polypeptides
can be used as targets for non-immunotherapeutic applications, e.g.,
using compounds which interfere with function, expression (e.g.,
antisense as a therapeutic agent), assembly, etc. RNA interference
can be used in vivtro and in vivo to silence Urb-ctf when its expression
contributes to a disease (but also for other purposes, e.g., to
identify the gene's function to change a developmental pathway of
a cell, etc.). See, e.g., Sharp and Zamore, Science, 287:2431 2433,
2001; Grishok et al., Science, 287:2494, 2001.
Delivery of therapeutic agents can be achieved according to any
effective method, including, liposomes, viruses, plasmid vectors,
bacterial delivery systems, orally, systemically, etc. Therapeutic
agents of the present invention can be administered in any form
by any effective route, including, e.g., oral, parenteral, enteral,
intraperitoneal, topical, transdermal (e.g., using any standard
patch), ophthalmic, nasally, local, non-oral, such as aerosal, inhalation,
subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal,
intra-arterial, and intrathecal, etc. They can be administered alone,
or in combination with any ingredient(s), active or inactive.
In addition to therapeutics, per se, the present invention also
relates to methods of treating a disease of breast cancer showing
altered expression of Urb-ctf, comprising, e.g., administering to
a subject in need thereof a therapeutic agent which is effective
for regulating expression of said Urb-ctf and/or which is effective
in treating said disease. The term "treating" is used
conventionally, e.g., the management or care of a subject for the
purpose of combating, alleviating, reducing, relieving, improving
the condition of, etc., of a disease or disorder. Diseases or disorders
which can be treated in accordance with the present invention include,
but are not limited to breast cancer. By the phrase "altered
expression," it is meant that the disease is associated with
a mutation in the gene, or any modification to the gene (or corresponding
product) which affects its normal function. Thus, expression of
Urb-ctf refers to, e.g., transcription, translation, splicing, stability
of the mRNA or protein product, activity of the gene product, differential
Any agent which "treats" the disease can be used. Such
an agent can be one which regulates the expression of the Urb-ctf.
Expression refers to the same acts already mentioned, e.g. transcription,
translation, splicing, stability of the mRNA or protein product,
activity of the gene product, differential expression, etc. For
instance, if the condition was a result of a complete deficiency
of the gene product, administration of gene product to a patient
would be said to treat the disease and regulate the gene's expression.
Many other possible situations are possible, e.g., where the gene
is aberrantly expressed, and the therapeutic agent regulates the
aberrant expression by restoring its normal expression pattern.
For Urb-ctf in cancer, agents can down-regulate the gene, or inhibit
the activity of the protein product in activating gene transcription.
Antisense polynucleotide (e.g., RNA) can also be prepared from
a polynucleotide according to the present invention, preferably
an anti-sense to a sequence of SEQ ID NO 1. Antisense polynucleotide
can be used in various ways, such as to regulate or modulate expression
of the polypeptides they encode, e.g., inhibit their expression,
for in situ hybridization, for therapeutic purposes, for making
targeted mutations (in vivo, triplex, etc.) etc. For guidance on
administering and designing anti-sense, see, e.g., U.S. Pat. Nos.
6,200,960, 6,200,807, 6,197,584, 6,190,869, 6,190,661, 6,187,587,
6,168,950, 6,153,595, 6,150,162, 6,133,246, 6,117,847, 6,096,722,
6,087,343, 6,040,296, 6,005,095, 5,998,383, 5,994,230, 5,891,725,
5,885,970, and 5,840,708. An antisense polynucleotides can be operably
linked to an expression control sequence. A total length of about
35 bp can be used in cell culture with cationic liposomes to facilitate
cellular uptake, but for in vivo use, preferably shorter oligonucleotides
are administered, e.g. 25 nucleotides.
Antisense polynucleotides can comprise modified, nonnaturally-occurring
nucleotides and linkages between the nucleotides (e.g., modification
of the phosphate-sugar backbone; methyl phosphonate, phosphorothioate,
or phosphorodithioate linkages; and 2'-O-methyl ribose sugar units),
e.g., to enhance in vivo or in vitro stability, to confer nuclease
resistance, to modulate uptake, to modulate cellular distribution
and compartmentalization, etc. Any effective nucleotide or modification
can be used, including those already mentioned, as known in the
art, etc., e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533;
6,124,445; 6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites);
4,973,679; Sproat et al., "2'-O-Methyloligoribonucleotides:
synthesis and applications," Oligonucleotides and Analogs A
Practical Approach, Eckstein (ed.), IRL Press, Oxford, 1991, 49
86; Iribarren et al., "2'-O-Alkyl Oligoribonucleotides as Antisense
Probes," Proc. Natl. Acad. Sci. USA, 1990, 87, 7747 7751; Cotton
et al., "2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate
oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent
mRNA processing event," Nucl. Acids Res., 1991, 19, 2629 2635.
The present invention also relates to an ordered array of polynucleotide
probes and specific-binding partners (e.g., antibodies) for detecting
the expression of Urb-ctf in a sample, comprising, one or more polynucleotide
probes or specific binding partners associated with a solid support,
wherein each probe is specific for Urb-ctf, and the probes comprise
a nucleotide sequence of SEQ ID NO 1 which is specific for said
gene, a nucleotide sequence having sequence identity to SEQ ID NO
1 which is specific for said gene or polynucleotide, or complements
thereto, or a specific-binding partner which is specific for Urb-ctf.
The phrase "ordered array" indicates that the probes
are arranged in an identifiable or position-addressable pattern,
e.g., such as the arrays disclosed in U.S. Pat. Nos. 6,156,501,
6,077,673, 6,054,270, 5,723,320, 5,700,637, WO09919711, WO00023803.
The probes are associated with the solid support in any effective
way. For instance, the probes can be bound to the solid support,
either by polymerizing the probes on the substrate, or by attaching
a probe to the substrate. Association can be, covalent, electrostatic,
noncovalent, hydrophobic, hydrophilic, noncovalent, coordination,
adsorbed, absorbed, polar, etc. When fibers or hollow filaments
are utilized for the array, the probes can fill the hollow orifice,
be absorbed into the solid filament, be attached to the surface
of the orifice, etc. Probes can be of any effective size, sequence
identity, composition, etc., as already discussed.
Ordered arrays can further comprise polynucleotide probes or specific-binding
partners which are specific for other genes, including genes specific
for breast cancer or disorders associated with breast cancer.
The present invention also relates to transgenic animals comprising
Urb-ctf genes. Such genes, as discussed in more detail below, include,
but are not limited to, functionally-disrupted genes, mutated genes,
ectopically or selectively-expressed genes, inducible or regulatable
genes, etc. These transgenic animals can be produced according to
any suitable technique or method, including homologous recombination,
mutagenesis (e.g., ENU, Rathkolb et al., Exp. Physiol., 85(6):635
644, 2000), and the tetracycline-regulated gene expression system
(e.g., U.S. Pat. No. 6,242,667). The term "gene" as used
herein includes any part of a gene, i.e., regulatory sequences,
promoters, enhancers, exons, introns, coding sequences, etc. The
Urb-ctf nucleic acid present in the construct or transgene can be
naturally-occurring wild-type, polymorphic, or mutated.
Along these lines, polynucleotides of the present invention can
be used to create transgenic animals, e.g. a non-human animal, comprising
at least one cell whose genome comprises a functional disruption
of Urb-ctf. By the phrases "functional disruption" or
"functionally disrupted," it is meant that the gene does
not express a biologically-active product. It can be substantially
deficient in at least one functional activity coded for by the gene.
Expression of a polypeptide can be substantially absent, i.e., essentially
undetectable amounts are made. However, polypeptide can also be
made, but which is deficient in activity, e.g., where only an amino-terminal
portion of the gene product is produced.
The transgenic animal can comprise one or more cells. When substantially
all its cells contain the engineered gene, it can be referred to
as a transgenic animal "whose genome comprises" the engineered
gene. This indicates that the endogenous gene loci of the animal
has been modified and substantially all cells contain such modification.
Functional disruption of the gene can be accomplished in any effective
way, including, e.g., introduction of a stop codon into any part
of the coding sequence such that the resulting polypeptide is biologically
inactive (e.g., because it lacks a catalytic domain, a ligand binding
domain, etc.), introduction of a mutation into a promoter or other
regulatory sequence that is effective to turn it off, or reduce
transcription of the gene, insertion of an exogenous sequence into
the gene which inactivates it (e.g., which disrupts the production
of a biologically-active polypeptide or which disrupts the promoter
or other transcriptional machinery), deletion of sequences from
the Urb-ctf gene, etc. Examples of transgenic animals having functionally
disrupted genes are well known, e.g., as described in U.S. Pat.
Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849,
6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642,
6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004,
5,817,912, 5,789,654, 5,777,195, and 5,569,824. A transgenic animal
which comprises the functional disruption can also be referred to
as a "knock-out" animal, since the biological activity
of its Urb-ctf genes has been "knocked-out." Knock-outs
can be homozygous or heterozygous.
For creating functional disrupted genes, and other gene mutations,
homologous recombination technology is of special interest since
it allows specific regions of the genome to be targeted. Using homologous
recombination methods, genes can be specifically-inactivated, specific
mutations can be introduced, and exogenous sequences can be introduced
at specific sites. These methods are well known in the art, e.g.,
as described in the patents above. See, also, Robertson, Biol. Reproduc.,
44(2):238 245, 1991. Generally, the genetic engineering is performed
in an embryonic stem (ES) cell, or other pluripotent cell line (e.g.,
adult stem cells, EG cells), and that genetically-modified cell
(or nucleus) is used to create a whole organism. Nuclear transfer
can be used in combination with homologous recombination technologies.
For example, the Urb-ctf locus can be disrupted in mouse ES cells
using a positive-negative selection method (e.g., Mansour et al.,
Nature, 336:348 352, 1988). In this method, a targeting vector can
be constructed which comprises a part of the gene to be targeted.
A selectable marker, such as neomycin resistance genes, can be inserted
into a Urb-ctf exon present in the targeting vector, disrupting
it. When the vector recombines with the ES cell genome, it disrupts
the function of the gene. The presence in the cell of the vector
can be determined by expression of neomycin resistance. See, e.g.,
U.S. Pat. No. 6,239,326. Cells having at least one functionally
disrupted gene can be used to make chimeric and germline animals,
e.g., animals having somatic and/or germ cells comprising the engineered
gene. Homozygous knock-out animals can be obtained from breeding
heterozygous knock-out animals. See, e.g., U.S. Pat. No. 6,225,525.
A transgenic animal, or animal cell, lacking one or more functional
Urb-ctf genes can be useful in a variety of applications, including,
as an animal model for breast cancer diseases, for drug screening
assays, for making a cell deficient in Urb-ctf to study the contribution
of it and other transcription factors, as a source of tissues deficient
in Urb-ctf activity, and any of the utilities mentioned in any issued
U.S. Patent on transgenic animals, including, U.S. Pat. Nos. 6,239,326,
6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610,
6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244,
6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912,
5,789,654, 5,777,195, and 5,569,824. For instance, Urb-ctf deficient
animal cells can be utilized to study transcriptional regulatory
activity. Breast cancer cells display a variety of activities which
are responsive to extracellular and intracellular signals. By knocking-out
transcription factors, e.g., one at a time, the physiological pathways
using transcriptional regulatory activity can be dissected out and
The present invention also relates to non-human, transgenic animal
whose genome comprises recombinant Urb-ctf nucleic acid operatively
linked to an expression control sequence effective to express said
coding sequence, e.g., in breast tissue. such a transgenic animal
can also be referred to as a "knock-in" animal since an
exogenous gene has been introduced, stably, into its genome.
A recombinant Urb-ctf nucleic acid refers to a gene which has been
introduced into a target host cell and optionally modified, such
as cells derived from animals, plants, bacteria, yeast, etc. A recombinant
Urb-ctf includes completely synthetic nucleic acid sequences, semi-synthetic
nucleic acid sequences, sequences derived from natural sources,
and chimeras thereof. "Operable linkage" has the meaning
used through the specification, i.e., placed in a functional relationship
with another nucleic acid. When a gene is operably linked to an
expression control sequence, as explained above, it indicates that
the gene (e.g., coding sequence) is joined to the expression control
sequence (e.g., promoter) in such a way that facilitates transcription
and translation of the coding sequence. As described above, the
phrase "genome" indicates that the genome of the cell
has been modified. In this case, the recombinant Urb-ctf has been
stably integrated into the genome of the animal. The Urb-ctf nucleic
acid in operable linkage with the expression control sequence can
also be referred to as a construct or transgene.
Any expression control sequence can be used depending on the purpose.
For instance, if selective expression is desired, then expression
control sequences which limit its expression can be selected. These
include, e.g., tissue or cell-specific promoters, introns, enhancers,
etc. For various methods of cell and tissue-specific expression,
see, e.g., U.S. Pat. Nos. 6,215,040, 6,210,736, and 6,153,427. These
also include the endogenous promoter, i.e., the coding sequence
can be operably linked to its own promoter. Inducible and regulatable
promoters can also be utilized.
The present invention also relates to a transgenic animal which
contains a functionally disrupted and a transgene stably integrated
into the animals genome. Such an animal can be constructed using
combinations any of the above- and below-mentioned methods. Such
animals have any of the aforementioned uses, including permitting
the knock-out of the normal gene and its replacement with a mutated
gene. Such a transgene can be integrated at the endogenous gene
locus so that the functional disruption and "knock-in"
are carried out in the same step.
In addition to the methods mentioned above, transgenic animals
can be prepared according to known methods, including, e.g., by
pronuclear injection of recombinant genes into pronuclei of 1-cell
embryos, incorporating an artificial yeast chromosome into embryonic
stem cells, gene targeting methods, embryonic stem cell methodology,
cloning methods, nuclear transfer methods. See, also, e.g., U.S.
Pat. Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489;
5,174,986; 5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc.
Natl. Acad. Sci., 77:7380 7384, 1980; Palmiter et al., Cell, 41:343
345, 1985; Palmiter et al., Ann. Rev. Genet., 20:465 499, 1986;
Askew et al., Mol. Cell. Bio., 13:4115 4124, 1993; Games et al.
Nature, 373:523 527, 1995; Valancius and Smithies, Mol. Cell. Bio.,
11:1402 1408, 1991; Stacey et al., Mol. Cell. Bio., 14:1009 1016,
1994; Hasty et al., Nature, 350:243 246, 1995; Rubinstein et al.,
Nucl. Acid Res., 21:2613 2617,1993; Cibelli et al., Science, 280:1256
1258, 1998. For guidance on recombinase excision systems, see, e.g.,
U.S. Pat. Nos. 5,626,159, 5,527,695, and 5,434,066. See also, Orban,
P. C., et al., "Tissue- and Site-Specific DNA Recombination
in Transgenic Mice," Proc. Natl. Acad. Sci. USA, 89:6861 6865
(1992); O'Gorman, S., et al., "Recombinase-Mediated Gene Activation
and Site-Specific Integration in Mammalian Cells," Science,
251:1351 1355 (1991); Sauer, B., et al., "Cre-stimulated recombination
at loxP-Containing DNA sequences placed into the mammalian genome,"
Polynucleotides Research, 17(1):147 161 (1989); Gagneten, S. et
al. (1997) Nucl. Acids Res. 25:3326 3331; Xiao and Weaver (1997)
Nucl. Acids Res. 25:2985 2991; Agah, R. et al. (1997) J. Clin. Invest.
100:169 179; Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543 2545;
Araki, K. et al. (1997) Nucl. Acids Res. 25:868 872; Mortensen,
R. N. et al. (1992) Mol. Cell. Biol. 12:2391 2395 (G418 escalation
method); Lakhlani, P. P. et al. (1997) Proc. Natl. Acad. Sci. USA
94:9950 9955 ("hit and run"); Westphal and Leder (1997)
Curr. Biol. 7:530 533 (transposon-generated "knock-out"
and "knock-in"); Templeton, N. S. et al. (1997) Gene Ther.
4:700 709 (methods for efficient gene targeting, allowing for a
high frequency of homologous recombination events, e.g., without
selectable markers); PCT International Publication WO 93/22443 (functionally-disrupted).
A polynucleotide according to the present invention can be introduced
into any non-human animal, including a non-human mammal, mouse (Hogan
et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986), pig (Hammer
et al., Nature, 315:343 345, 1985), sheep (Hammer et al., Nature,
315:343 345, 1985), cattle, rat, or primate. See also, e.g., Church,
1987, Trends in Biotech. 5:13 19; Clark et al., Trends in Biotech.
5:20 24, 1987); and DePamphilis et al., BioTechniques, 6:662 680,
1988. Transgenic animals can be produced by the methods described
in U.S. Pat. No. 5,994,618, and utilized for any of the utilities
The present invention also relates to electronic forms of polynucleotides,
polypeptides, etc., of the present invention, including computer-readable
medium (e.g., magnetic, optical, etc., stored in any suitable format,
such as flat files or hierarchical files) which comprise such sequences,
or fragments thereof, e-commerce-related means, etc. Along these
lines, the present invention relates to methods of retrieving gene
sequences from a computer-readable medium, comprising, one or more
of the following steps in any effective order, e.g., selecting a
cell or gene expression profile, e.g., a profile that specifies
that said gene is differentially expressed in breast cancer, and
retrieving said differentially expressed gene sequences, where the
gene sequences consist of the genes represented by SEQ ID NO 1.
A "gene expression profile" means the list of tissues,
cells, etc., in which a defined gene is expressed (i.e, transcribed
and/or translated). A "cell expression profile" means
the genes which are expressed in the particular cell type. The profile
can be a list of the tissues in which the gene is expressed, but
can include additional information as well, including level of expression
(e.g., a quantity as compared or normalized to a control gene),
and information on temporal (e.g., at what point in the cell-cycle
or developmental program) and spatial expression. By the phrase
"selecting a gene or cell expression profile," it is meant
that a user decides what type of gene or cell expression pattern
he is interested in retrieving, e.g., he may require that the gene
is differentially expressed in a tissue, or he may require that
the gene is not expressed in blood, but must be expressed in breast
cancer. Any pattern of expression preferences may be selected. The
selecting can be performed by any effective method. In general,
"selecting" refers to the process in which a user forms
a query that is used to search a database of gene expression profiles.
The step of retrieving involves searching for results in a database
that correspond to the query set forth in the selecting step. Any
suitable algorithm can be utilized to perform the search query,
including algorithms that look for matches, or that perform optimization
between query and data. The database is information that has been
stored in an appropriate storage medium, having a suitable computer-readable
format. Once results are retrieved, they can be displayed in any
suitable format, such as HTML.
For instance, the user may be interested in identifying genes that
are differentially expressed in a breast cancer. He may not care
whether small amounts of expression occur in other tissues, as long
as such genes are not expressed in peripheral blood lymphocytes.
A query is formed by the user to retrieve the set of genes from
the database having the desired gene or cell expression profile.
Once the query is inputted into the system, a search algorithm is
used to interrogate the database, and retrieve results.
The present invention also relates to methods of selecting a breast
cancer marker from a database comprising polynucleotide sequences,
comprising displaying, in a computer-readable medium, a polynucleotide
sequence or polypeptide sequence for human Urb-ctf of claim 1, or
complements to the polynucleotides sequence, wherein said displayed
sequences have been retrieved from said database upon selection
by a user. The phrase "upon selection by a user" indicates
that a user of the database has specified or directed a search or
other retrieval feature that results in the retrieval and display
of the target sequences. For example, the user could ask the database
to display polynucleotides or polypeptides expressed in breast cancer
by inputting an appropriate inquiry. The user could also input sequence
information, and request the display of any sequences in the database
that match the inputted sequence information. One or more sequences
can be displayed at a time in response to any user inquiry.
Advertising, Licensing, etc., Methods
The present invention also relates to methods of advertising, licensing,
selling, purchasing, brokering, etc., genes, polynucleotides, specific-binding
partners, antibodies, etc., of the present invention. Methods can
comprises, e.g., displaying a Urb-ctf gene, Urb-ctf polypeptide,
or antibody specific for Urb-ctf in a printed or computer-readable
medium (e.g., on the Web or Internet), accepting an offer to purchase
said gene, polypeptide, or antibody.
A polynucleotide, probe, polypeptide, antibody, specific-binding
partner, etc., according to the present invention can be isolated.
The term "isolated" means that the material is in a form
in which it is not found in its original environment or in nature,
e.g., more concentrated, more purified, separated from component,
etc. An isolated polynucleotide includes, e.g., a polynucleotide
having the sequenced separated from the chromosomal DNA found in
a living animal, e.g., as the complete gene, a transcript, or a
cDNA. This polynucleotide can be part of a vector or inserted into
a chromosome (by specific gene-targeting or by random integration
at a position other than its normal position) and still be isolated
in that it is not in a form that is found in its natural environment.
A polynucleotide, polypeptide, etc., of the present invention can
also be substantially purified. By substantially purified, it is
meant that polynucleotide or polypeptide is separated and is essentially
free from other polynucleotides or polypeptides, i.e., the polynucleotide
or polypeptide is the primary and active constituent. A polynucleotide
can also be a recombinant molecule. By "recombinant,"
it is meant that the polynucleotide is an arrangement or form which
does not occur in nature. For instance, a recombinant molecule comprising
a promoter sequence would not encompass the naturally-occurring
gene, but would include the promoter operably linked to a coding
sequence not associated with it in nature, e.g., a reporter gene,
or a truncation of the normal coding sequence.
The term "marker" is used herein to indicate a means
for detecting or labeling a target. A marker can be a polynucleotide
(usually referred to as a "probe"), polypeptide (e.g.,
an antibody conjugated to a detectable label), PNA, or any effective
The topic headings set forth above are meant as guidance where
certain information can be found in the application, but are not
intended to be the only source in the application where information
on such topic can be found. Reference materials
For other aspects of the polynucleotides, reference is made to
standard textbooks of molecular biology. See, e.g., Hames et al.,
Polynucleotide Hybridization, IL Press, 1985; Davis et al., Basic
Methods in Molecular Biology, Elsevir Sciences Publishing, Inc.,
New York, 1986; Sambrook et al., Molecular Cloning, CSH Press, 1989;
Howe, Gene Cloning and Manipulation, Cambridge University Press,
1995; Ausubel et al., Current Protocols in Molecular Biology, John
Wiley & Sons, Inc., 1994 1998.
The preceding description, utilize the present invention to its
fullest extent. The preceding preferred specific embodiments are,
therefore, to be construed as merely illustrative, and not limiting
the remainder of the disclosure in any way whatsoever. The entire
disclosure of all applications, patents and publications, cited
above and, in the FIGURE are hereby incorporated by reference in