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.
1. An isolated human Urb-ctf polynucleotide which codes without
interruption for an amino acid sequence set forth in SEQ ID NO 2,
or a complement thereto.
2. An isolated human Urb-ctf polynucleotide of claim 1, having
the polynucleotide sequence set forth in SEQ ID NO 1, or a complement
3. An isolated human Urb-ctf polynucleotide comprising, polynucleotide
sequence having 97% or more nucleotide sequence identity to the
polynucleotide sequence set forth in SEQ ID NO 1, which codes without
interruption for Urb-ctf, and which has transcriptional regulatory
4. An isolated polynucleotide of claim 3 having 99% or more sequence
identity to the polynucleotide sequence set forth in SEQ ID NO 1.
5. An isolated polynucleotide which is specific for human Urb-ctf
and which codes for a polypeptide, said polypeptide 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, or complements
6. An isolated polynucleotide of claim 5, comprising 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.
7. An isolated polynucleotide of claim 5, wherein said polynucleotide
is effective in a polymerase chain reaction.
8. An isolated polynucleotide of claim 5, which codes for a polypeptide
comprising at least eight amino acids in length.
9. An isolated human Urb-ctf polypeptide of claim 1 comprising,
the amino acid sequence set forth in SEQ ID NO 2.
10. An isolated human Urb-ctf polypeptide of claim 3 comprising,
an amino acid sequence having 99% or more sequence identity to the
amino acid sequence set forth in SEQ ID NO 2.
11. An isolated human Urb-ctf polypeptide of claim 10, which has
transcriptional regulatory activity.
12. An isolated human polypeptide which is specific for Urb-ctf
of claim 8, said polypeptide 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, or amino acid 606 of SEQ ID NO 2.
13. An isolated polypeptide of claim 12, comprising, a polypeptide
coding for amino acids 1-263 of SEQ ID NO 2 or 459-614 of SEQ ID
14. A method of treating breast cancer showing altered expression
of human Urb-ctf of claim 1, comprising: administering to a subject
in need thereof a therapeutic agent which is effective for regulating
expression of said Urb-ctf gene or polypeptide.
15. A method of claim 14, wherein said agent is an antisense which
is effective to inhibit translation of the gene coding for human
16. A method of diagnosing human breast cancer disease associated
with abnormal Urb-ctf expression, or determining a subject's susceptibility
to such disease, comprising: assessing the expression of human Urb-ctf
of claim 1 in a tissue sample comprising breast cancer cells.
17. A method of claim 16, wherein assessing is: 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
18. A method of claim 16, wherein said assessing detecting is performed
by: Northern blot analysis, polymerase chain reaction (PCR), reverse
transcriptase PCR, RACE PCR, or in situ hybridization, and using
a polynucleotide probe having a sequence selected from SEQ ID NO
1, a polynucleotide having 99% sequence identity or more to a sequence
set forth in SEQ ID NO 1, or complements thereto.
19. A method of assessing a therapeutic or preventative intervention
in a human subject having breast cancer, comprising, determining
the expression levels of human Urb-ctf of claim 1 in a tissue sample
comprising breast cancer cells, or cells derived from breast cancer.
20. A method for identifying an agent that modulates the expression
of human Urb-ctf of claim 1 in cells, comprising, contacting a cell
population with a test agent under conditions effective for said
test agent to modulate the expression of the gene coding for human
Urb-ctf in cells, and determining whether said test agent modulates
21. A method of claim 20, wherein said agent is an antisense polynucleotide
to a target polynucleotide sequence selected from SEQ ID NO 1 and
which is effective to inhibit translation of said gene.
22. A method for identifying an agent that modulates the biological
activity of human Urb-ctf of claim 8, comprising, contacting human
Urb-ctf polypeptide of claim 8 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
23. A non-human, transgenic mammal whose genome comprises a recombinant
polynucleotide coding for a human Urb-ctf of claim 1 operatively
linked to an expression control sequence effective to express said
gene in breast tissue.
24. A non-human transgenic mammal of claim 22, wherein said expression
control sequence is an inducible promoter.
25. An antibody which is specific for human Urb-ctf of claim 1,
which antibody 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, or amino acid 606 of SEQ ID NO 2.
26. A method of advertising human Urb-ctf of claim 1 for sale,
commercial use, or licensing, comprising, displaying in a computer-readable
medium a polynucleotide sequence set forth in SEQ ID NO 1, or complements
thereto, or a polypeptide sequence set forth in sequence in SEQ
ID NO 2.
27. A method 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.
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, etc.
 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 2000.
 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: RP 1-749116, 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.
 Nucleic Acids
 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, be effective for
 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, pDR540, 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
 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 or double-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 routinely.
 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.
 Polynucleotide Composition
 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
 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, January 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 Partners Thereto.
 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 be included.
 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, WO/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 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 et al., Science 242:423-426 (1988); Ladneret al., U.S. Pat.
No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); and
 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
 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
 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.
 Transgenic Animals
 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.