U.S. patent application number 10/028272 was filed with the patent office on 2003-10-02 for tbx3 gene and methods of using it.
Invention is credited to Fan, Wufang, Sun, Zairen.
Application Number | 20030186235 10/028272 |
Document ID | / |
Family ID | 28452157 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186235 |
Kind Code |
A1 |
Sun, Zairen ; et
al. |
October 2, 2003 |
TBX3 GENE AND METHODS OF USING IT
Abstract
The present invention relates to all facets of a novel
polynucleotide, Tbx3-pr408, the polypeptides it encodes, antibodies
and specific binding partners thereto, and their applications to
research, diagnosis, drug discovery, therapy, clinical medicine,
forensic science and medicine, etc. The polynucleotides 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, such as
mammary-ulnar syndrome and other developmental disorders.
Inventors: |
Sun, Zairen; (Rockville,
MD) ; Fan, Wufang; (Germantown, MD) |
Correspondence
Address: |
ORIGENE TECHNOLOGIES, INCORPORATED
6 TAFT COURT
SUITE 100
ROCKVILLE
MD
20850
US
|
Family ID: |
28452157 |
Appl. No.: |
10/028272 |
Filed: |
December 28, 2001 |
Current U.S.
Class: |
435/6.14 ;
435/226; 435/325; 435/6.16; 435/7.23; 536/23.2; 705/2; 705/37 |
Current CPC
Class: |
G16H 20/10 20180101;
Y02A 90/10 20180101; C07K 14/4702 20130101; G06Q 40/04 20130101;
G01N 33/689 20130101; G16H 50/00 20180101 |
Class at
Publication: |
435/6 ; 435/7.23;
435/325; 435/226; 536/23.2; 705/2; 705/37 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; C12N 009/64; G06F 017/60; C12N 005/06 |
Claims
1. An isolated Tbx3-pr408 polynucleotide which codes without
interruption for an amino acid sequence set forth in SEQ ID NO 2,
or a complement thereto.
2. An isolated Tbx3-pr408 polynucleotide comprising, polynucleotide
sequence having 97% or more sequence identity to the polynucleotide
sequence set forth in SEQ ID NO 1, which codes without interruption
for Tbx3-pr408, or a complement thereto.
3. An isolated polynucleotide of claim 2, having 97% or more
sequence identity to the polynucleotide sequence set forth in SEQ
ID NO 1.
4. An isolated polynucleotide of claim 3 having transcriptional
regulatory activity.
5. An isolated Tbx3-pr408 polypeptide comprising, the amino acid
sequence set forth in SEQ ID NO 2.
6. An isolated polypeptide, selected from the amino acid sequence
596-608 of SEQ ID NO 2 which is specific for Tbx3-Pr408.
7. An antibody which is specific-for a polypeptide of claim 5.
8. An isolated polypeptide comprising an amino acid sequence having
99% or more sequence identity to the amino acid sequence set forth
in SEQ ID NO 2.
9. A method of diagnosing a disorder in a subject associated with
Tbx3-pr408, or determining a subject's susceptibility to such
disease, comprising: assessing the expression of Tbx3-pr408 of
claim 1 in a tissue sample from said subject.
10. A method of claim 9, 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
gene.
11. A method of claim 9, wherein the expression of Tbx3-pr408 and
XM.sub.--016321 are assessed.
12. A method of claim 9, 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, 3, 4, or 5, effective specific fragments
thereof, or complements thereto.
13. A method for identifying an agent that modulates the
transcriptional regulatory activity of Tbx3-pr408, comprising,
contacting Tbx3-pr408 of claim 1, or a biologically-active fragment
thereof which comprises Tbx3-pr408 specific sequence, with a test
agent under conditions effective for said test agent to modulate
its transcriptional regulatory activity, and determining whether
said test agent modulates said Tbx3-pr408.
14. A method of claim 13, wherein said agent is an antibody
specific for a polypeptide comprising amino acids 596-608, or a
polypeptide comprising amino acids 215-225.
15. A method of detecting Tbx transcripts in a tissue sample,
comprising assessing the expression of a Tbx3-pr408 transcript of
claim 3 in a tissue sample, and assessing the expression of a Tbx3
transcript comprising a T box insertion.
16. A method of claim 15, 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 gene.
17. A method of claim 15, wherein the expression of Tbx3-pr408 and
XM.sub.--016321 are assessed.
18. A mammalian cell, comprising an exogenous polynucleotide of
claim 1.
19. A method of advertising Tbx3-pr4-8 for sale, commercial use, or
licensing, comprising, displaying in a computer-readable medium a
polynucleotide, or polypeptide sequence thereto, of claim 1,
effective specific fragments thereof, or complements thereto.
Description
DESCRIPTION OF THE DRAWINGS
[0001] SEQ ID NOS 1 and 2 show the nucleotide and amino acid
sequences of Tbx3-pr408. The polynucleotides are human cDNAs.
[0002] FIG. 1 shows the amino acid comparisons between Tbx3-pr408
(SEQ ID NO 2), NM.sub.--005996 (SEQ ID NO 3), XM.sub.--016321 (SEQ
ID NO 4), and NM.sub.--016569 (SEQ ID NO 5).
DESCRIPTION OF THE INVENTION
[0003] The present invention relates to all facets of Tbx3-pr408,
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. Tbx3-pr408 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 mammary-ulnar syndrome and other
developmental disorders associated with it, as well as conditions
associated with the adult tissues in which Tbx3-pr408 is expressed.
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.
[0004] Tbx3-pr408 codes for a polypeptide having 723 amino acids.
It is a transcription factor. The nucleotide and amino acid
sequences of Tbx3-pr408 are shown in SEQ ID NOS 1 and 2. A T box
domain, having DNA-binding activity, is located at about amino acid
positions 102-290.
[0005] Tbx3-pr408 is a transcript of the Tbx3 gene. See, e.g., He
et al., Proc. Natl. Acad. Sci., 96:10212-10217, 1999. It maps to
chromosomal position 12q24.1. Mutations in this gene cause
ulnar-mammary syndrome, a disease associated with anomalies in
limb, apocrine and genital development. Bamshad et al., Nat.
Genet., 16:311-316, 1997. FIG. 1 shows the alignment between
Tbx3-pr408 and previously known transcripts (NM.sub.--005996,
XM.sub.--016321, and NM.sub.--016569) of the Tbx3 gene. As
indicated in FIG. 1, Pr408 is decidedly different. In addition to
other differences, XM.sub.--016321, and NM.sub.--016569 contain a
20 amino acid insertion in the T box domain between amino acids 220
and 221 of Tbx3-pr408. This insertion appears to alter the
DNA-binding activity of the T box. NM.sub.--005996 is at least one
amino acid shorter than Tbx3-pr408 and contains different sequence
at amino acid positions 596-608.
[0006] Tbx3-pr408 has several different biological activities,
including, e.g., transcription modulatory activity, and DNA-binding
activity. Its activity can be determined routinely. For instance,
its transcriptional regulatory activity can be assessed in
transcription reporter assays in which Tbx3-pr408, or fragments
thereof, can be fused DNA-binding elements (e.g., LexA or Gal4)
and/or transactivators, that are used to modulate expression of
reporter genes, e.g., as described in He et al., Proc. Natl. Acad.
Sci., 96:10212-10217, 1999. By the phrase "transcription regulatory
activity," it is meant that the polypeptide modulates transcription
of a gene. This modulatory activity can be activation or repression
(e.g., He et al. describe repression for a Tbx3 domain).
DNA-binding activity can be determined using gel-shift assays.
[0007] Consistent with the phenotype of mammary-ulnar syndrome,
Tbx3-pr408 is expressed in the testes and prostate. Additionally,
it is expressed at high levels in the adrenal gland, heart, lung,
and muscle. Lower levels are observed in other tissues, as well.
Tissues were also assayed for the presence of the Tbx3 transcript
having the insertion between amino acids 220 and 221. Both this
transcript and Tbx3-pr408 were expressed in most tissues, however,
Tbx3-pr408 was more abundant. Heart expression is particularly
striking since mutations in the Tbx5 gene, adjacent to the Tbx3
gene on chromosome 12, is associated with cardiac anomalies,
suggesting that both genes may play a role in heart
development.
[0008] In addition to its association with ulnar-mammary syndrome,
Tbx3-pr408 may also have a role in other developmental disorders,
including, e.g., spinal muscular atrophy (congenital
nonprogressive, of lower limbs), type C brachydactyly, B-cell
non-Hodgkin lymphoma, and scapuloperoneal spinal muscular atrophy
(New England type). 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. Nucleic acids
[0009] A mammalian polynucleotide, or fragment thereof, of the
present invention is a polynucleotide having a nucleotide sequence
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.
[0010] 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. These sequences can be obtained by
any suitable method, e.g., using a partial sequence as a probe to
select a full-length cDNA from a library containing full-length
inserts. 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.
[0011] Polynucleotides and polypeptides (including any part of
Tbx3-pr408) 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.
[0012] 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.
[0013] 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 thereof, etc.
[0014] Constructs
[0015] 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., introns.
[0016] 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.
[0017] A polynucleotide of the present invention can also comprise
nucleic acid vector sequences, e.g., for cloning, expression,
amplification, selection, etc. Any effective vector can be used. A
vector is, e.g., a polynucleotide molecule which can replicate
autonomously in a host cell, e.g., containing an origin of
replication. Vectors can be useful to perform manipulations, to
propagate, and/or obtain large quantities of the recombinant
molecule in a desired host. A skilled worker can select a vector
depending on the purpose desired, e.g., to propagate the
recombinant molecule in bacteria, yeast, insect, or mammalian
cells. The following vectors are provided by way of example.
Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, Phagescript,
phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A (Stratagene);
Bluescript KS+II (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR54
0, pRIT5 (Pharmacia). Eukaryotic: PWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene), pSVK3, PBPV, PMSG, pSVL (Pharmacia), pCR2. 1/TOPO,
pCRII/TOPO, pCR4/TOPO, pTrcHisB, pCMV6-XL4, etc. However, any other
vector, e.g., plasmids, viruses, or parts thereof, may be used as
long as they are replicable and viable in the desired host. The
vector can also comprise sequences which enable it to replicate in
the host whose genome is to be modified.
[0018] Hybridization
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] For instance, high stringency conditions can be achieved by
incubating the blot overnight (e.g., at least 12 hours) with a long
polynucleotide probe in a hybridization solution containing, e.g.,
about 5.times.SSC, 0.5% SDS, 100 .mu.g/ml denatured salmon sperm
DNA and 50% formamide, at 42.degree. C. Blots can be washed at high
stringency conditions that allow, e.g., for less than 5% bp
mismatch (e.g., wash twice in 0.1% SSC and 0.1% SDS for 30 min at
65.degree. C.), i.e., selecting sequences having 95% or greater
sequence identity.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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, viruses,
etc. The degree of nucleotide sequence identity between human and
mouse can be about, e.g. 70% or more, 85% or more for open reading
frames, etc.
[0028] Alignment
[0029] 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.
[0030] 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.
[0031] Specific Polynucleotide Probes
[0032] 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. Useful probes, include,
e.g., probes on either side of amino acid positions 220-221 (i.e.,
where NM.sub.--005996 and XM.sub.--016321 have insertions), e.g., a
probe selected from nucleotide positions 1112-1183 and from
nucleotide positions 1184-1255) to determine whether the insertion
is present or not.
[0033] 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.
[0034] 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 Tbx3-pr408, 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.
[0035] 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. 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 which occurs in the
polynucleotide, e.g., in the nucleotide sequences of SEQ ID NO 1. 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.
[0036] 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.
[0037] 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 prostate 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.
[0038] 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. No. 5,545,522.
[0039] Polynucleotide Composition
[0040] 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.
[0041] 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.
[0042] 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
radio labeled 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.
[0043] Nucleic Acid Detection Methods
[0044] Another aspect of the present invention relates to methods
and processes for detecting Tbx3-pr408. 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.
[0045] 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.
[0046] 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.
[0047] 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
hybridization.
[0048] 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.
[0049] 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.
[0050] 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 Tbx3-pr408, 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.
[0051] Along these lines, the present invention relates to methods
of detecting Tbx3-pr408 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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 cell.
[0058] Methods of Identifying Polymorphisms, Mutations, etc., of
Tbx3-pr408
[0059] 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 Tbx3-pr408, as well as to
design therapies and predict the outcome of the disorder. Methods
involve, e.g., diagnosing a disorder associated with Tbx3-pr408 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.
[0060] The present invention also relates to methods of detecting
polymorphisms in Tbx3-pr408, comprising, e.g., comparing the
structure of: genomic DNA comprising all or part of Tbx3-pr408,
mRNA comprising all or part of Tbx3-pr408, cDNA comprising all or
part of Tbx3-pr408, or a polypeptide comprising all or part of
Tbx3-pr408, with the structure of Tbx3-pr408 gene. 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.
[0061] 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 Tbx3-pr408 and a test Tbx3-pr408. 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.
[0062] 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.
[0063] Mutagenesis
[0064] 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 Tbx3-pr408 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.
[0065] FIG. 1 provides guidance on mutations that can be made in
Tbx3-pr408, e.g., changing a non-conserved amino acid, e., at amino
acid position 295 and/or 609.
[0066] In addition, libraries of Tbx3-pr408 and fragments thereof
can be used for screening and selection of Tbx3-pr408 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" Tbx3-pr408. The entire coding
sequence or parts thereof can be used.
[0067] Polynucleotide Expression, Polypeptides Produced Thereby,
and Specific-binding Partners Thereto.
[0068] 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, 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).
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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, 6.times.His, maltose binding protein,
chitinase, etc) and then purify by anti-tag antibody-conjugated
affinity chromatography.
[0073] 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.,
Tbx3-pr408. Antibodies, e.g., polyclonal, monoclonal, recombinant,
chimeric, humanized, single-chain, Fab, and fragments thereof, can
be prepared according to any desired method. See, also, screening
recombinant immunoglobulin libraries (e.g., Orlandi et al., Proc.
Natl. Acad. Sci., 86:3833-3837, 1989; Huse et al., Science,
256:1275-1281, 1989); in vitro stimulation of lymphocyte
populations; Winter and Milstein, Nature, 349: 293-299, 1991. The
antibodies can be IgM, IgG, subtypes, IgG2a, IgG1, etc. Antibodies,
and immune responses, can also be generated by administering naked
DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466; 5,580,859.
Antibodies can be used from any source, including, goat, rabbit,
mouse, chicken (e.g., IgY; see, Duan, W0/029444 for methods of
making antibodies in avian hosts, and harvesting the antibodies
from the eggs). An antibody specific for a polypeptide means that
the antibody recognizes a defined sequence of amino acids within or
including the polypeptide. Other specific binding partners include,
e.g., aptamers and PNA. antibodies can be prepared against specific
epitopes or domains of Tbx3-pr408, e.g., amino acids 596-608 of SEQ
ID NO 2, or peptides comprising amino acids 220-221 (e.g., where
the Tbx3-pr408 transcript is to be distinguished from the Tbx3
transcripts which contain the 20-amin acid insertion).
[0074] 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).
[0075] 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. lmnmunol. 150: 2844 (1993).
[0076] 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.).
[0077] 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. 6:579
(1994).
[0078] 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 etal,
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.
[0079] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques can also be used. For example, Fv fragments
comprise an association of V.sub.H and V.sub.L chains. This
association may be noncovalent, as described in Inbar et al., Proc.
Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, the variable
chains can be linked by an intermolecular disulfide bond or
cross-linked by chemicals such as glutaraldehyde. See, e.g.,
Sandhu, supra. Preferably, the Fv fragments comprise V.sub.H and
V.sub.L chains connected by a peptide linker. These single-chain
antigen binding proteins (sFv) are prepared by constructing a
structural gene comprising nucleic acid sequences encoding the
V.sub.H and V.sub.L domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow et al., METHODS: A
COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird
etal.,Science 242:423-426 (1988); Ladneret al., U.S. Pat. No.
4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); and
Sandhu, supra.
[0080] 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).
[0081] 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.
[0082] Antibodies which bind to Tbx3-pr408 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
Tbx3-pr408. 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.
[0083] 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).
[0084] 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.
[0085] Methods of Detecting Polypeptides
[0086] Polypeptides coded for by Tbx3-pr408 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 (radioimmunoassay),
ELISA, (enzyme-linked-immunosorbent assay), immunoflourescence,
flow cytometry, histology, electron microscopy, light microscopy,
in situ assays, immunoprecipitation, Western blot, etc.
[0087] 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 Tbx3-pr408 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.
[0088] 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
[0089] 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
calorimetric 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.
[0090] 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
Tbx3-pr408 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.
[0091] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine. The antibody can also be detectably labeled using
fluorescence emitting metals such as those in the lanthanide
series. These metals can be attached to the antibody using such
metal chelating groups as diethylenetriaminepentacetic acid (DTPA)
or ethylenediaminetetraacetic acid (EDTA).
[0092] 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 ester.
[0093] 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.
[0094] Diagnostic
[0095] The present invention also relates to methods and
compositions for diagnosing a developmental disorder, or
determining susceptibility to a disorder, using polynucleotides,
polypeptides, and specific-binding partners of the present
invention to detect, assess, determine, etc., Tbx3-pr408. 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 specific-binding
partners.
[0096] The present invention relates to methods of diagnosing a
disorder associated with Tbx3-pr408 (e.g., of the prostate, heart,
muscle, lung, or adrenal gland), or determining a subject's
susceptibility to such disorder, comprising, e.g., assessing the
expression of Tbx3-pr408 in a tissue sample comprising tissue or
cells suspected of having the disorder.
[0097] 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.
[0098] By the phrase "assessing expression of Tbx3-pr408," 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 biological activity.
[0099] 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.
[0100] Assessing the effects of therapeutic and preventative
interventions (e.g., administration of a drug, chemotherapy,
radiation, etc.) on disorders 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 a
disorder, or, susceptible to such a disorder, comprising, e.g.,
detecting the expression levels of Tbx3-pr408. 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.
[0101] The present invention also relates to methods of detecting
Tbx transcripts in a tissue sample, comprising, e.g., assessing the
expression of a Tbx3-pr408 transcript in a tissue sample, and
assessing the expression of other transcripts of the Tbx3 gene,
such as those represented by NM.sub.--005996 (SEQ ID NO 3),
XM.sub.--016321 (SEQ ID NO 4), and NM.sub.--016569 (SEQ ID NO 5).
Tbx3 transcripts which contain about a 20 amino acid insertion at
about amino acid 220 are referred to "Tbx3 transcripts comprising a
T box insertion." Examples are NM.sub.--005996 (SEQ ID NO 3) and
XM.sub.--016321 (SEQ ID NO 4). Assessing can be accomplished as
described above. This method can be used to determine the relative
levels of Tbx3-pr408 as compared to other Tbx3 transcripts. For
instance, as described above, Tbx3-pr408 is more abundant in most
tissues than NM.sub.--005996 or XM.sub.--016321. Patients having
mammary-ulnar syndrome can be assessed for the relative levels of
both transcript types. If PCR is used to determine mRNA expression,
the following primers can be used which span the insertion, e.g.,
forward primer having the sequence of about 1023-1048 (SEQ ID NO
1), and a reverse primer which is complementary to the sequence of
about 1230-1255. If both transcripts are present in the sample, two
different bands will be detected, the larger corresponding to
NM.sub.--005996 or XM.sub.--016321, and the smaller to
Tbx3-pr408.
[0102] Identifying Agent Methods
[0103] The present invention relates to compositions and methods of
modulating Tbx3-pr48 gene or polypeptide. Assays can be performed
on whole cells, lysates, isolated Tbx3-pr408, etc.
[0104] For example, the present invention also relates to methods
of identifying agents that modulate the expression of Tbx3-pr408 in
a cell population, comprising, in any effective order, one or more
of the following steps, e.g., contacting a cell population with a
test agent under conditions effective for said test agent to
modulate the expression of Tbx3-pr408 in said cell population, and
determining whether said test agent modulates said Tbx3-pr408. An
agent can modulate expression of Tbx3-pr408 at any level, including
transcription, translation, and/or perdurance of the nucleic acid
or polypeptide (e.g., degradation, stability, etc.) product in the
cell.
[0105] In addition, the present invention also relates to methods
of identifying agents that modulate the biological activity of
Tbx3-pr408 polypeptide in a cell population, lysate, etc.,
comprising, in any effective order, one or more of the following
steps, e.g., contacting said polypeptide 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.
[0106] Contacting the cell population 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 Tbx3-pr408 present in cells within the
population, or in the sample mixture. Functional control indicates
that the agent can exert its physiological effect on the cell
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 the 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, etc.
[0107] After the agent has been administered in such a way that it
can gain access to the cells or polypeptide, it can be determined
whether the test agent modulates Tbx3-pr408 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
Tbx3-pr408 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 the 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 Tbx3-pr408
included, e.g., transcription regulatory activity, DNA-binding
activity, etc. Assays for measuring these activities were discussed
previously.
[0108] 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, etc. For example, if a polypeptide to be
modulated is a cell-surface molecule, a test agent can be an
antibody that specifically recognizes it and, e.g., causes the
polypeptide to be internalized, leading to its down regulation on
the surface of the cell. Such an effect does not have to be
permanent, but can require the presence of the antibody to continue
the down-regulatory effect. Antisense Tbx3-pr408 can also be used
as test agents to modulate gene expression.
[0109] The entire Tbx3-pr408 polypeptide can be used, or
biologically-active fragments thereof. If transcriptional activity
is being measured, then a biologically-active fragment would be one
that retains transcriptional regulatory activity, and so on. In
some cases, biologically-active fragments can comprise Tbx3-pr408
specific sequence. By this, it is meant that fragments common to
other Tbx3 transcripts (e.g., NM.sub.--005996, XM.sub.--016321, and
NM.sub.--016569) are excluded. An example of such a fragment would
comprise amino acids from about 596-608, e.g., 1-650, 50-650,
50-723, 300-650 (e.g., fragments deleting the DNA-binding domain),
300-723, etc.
[0110] Therapeutics
[0111] Selective polynucleotides, polypeptides, and
specific-binding partners thereto, can be utilized in therapeutic
applications, especially to treat diseases and conditions of
associated with Tbx-pr408, e.g., mammary-ulnar disease. 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, antisense), etc.
[0112] 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.
[0113] 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.
[0114] 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
Tbx3-pr408 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.
[0115] 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.
[0116] In addition to therapeutics, per se, the present invention
also relates to methods of treating a disease showing altered
expression of Tbx3-pr408, comprising, e.g., administering to a
subject in need thereof a therapeutic agent which is effective for
regulating expression of said Tbx3-pr408 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. 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 Tbx3-pr408 refers to, e.g., transcription,
translation, splicing, stability of the mRNA or protein product,
activity of the gene product, differential expression, etc.
[0117] Any agent which "treats" the disease can be used. Such an
agent can be one which regulates the expression of the Tbx3-pr408.
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.
[0118] Antisense
[0119] 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.
[0120] 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.
[0121] Arrays
[0122] The present invention also relates to an ordered array of
polynucleotide probes and specific-binding partners (e.g.,
antibodies) for detecting the expression of Tbx3-pr408 in a sample,
comprising, one or more polynucleotide probes or specific binding
partners associated with a solid support, wherein each probe is
specific for Tbx3-pr408, and the probes comprise a nucleotide
sequence of SEQ ID NO 1 which is specific for said gene, a
nucleotide sequence having sequence identity to SEQ ID NO 1 which
is specific for said gene or polynucleotide, or complements
thereto, or a specific-binding partner which is specific for
Tbx3-pr408.
[0123] 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, WO0991971 1,
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.
[0124] Transgenic Animals
[0125] The present invention also relates to transgenic animals
comprising Tbx3-pr408 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 Tbx3-pr408 nucleic acid present in the construct or
transgene can be naturally-occurring wild-type, polymorphic, or
mutated.
[0126] Along these lines, polynucleotides of the present invention
can be used to create transgenic animals, e.g. a non-human animal,
comprising at least one cell whose genome comprises a functional
disruption of Tbx3-pr408. By the phrases "functional disruption" or
"functionally disrupted," it is meant that the gene does not
express a biologically-active product. It can be substantially
deficient in at least one functional activity coded for by the
gene. Expression of a polypeptide can be substantially absent,
i.e., essentially undetectable amounts are made. However,
polypeptide can also be made, but which is deficient in activity,
e.g., where only an amino-terminal portion of the gene product is
produced.
[0127] The transgenic animal can comprise one or more cells. When
substantially all its cells contain the engineered gene, it can be
referred to as a transgenic animal "whose genome comprises" the
engineered gene. This indicates that the endogenous gene loci of
the animal has been modified and substantially all cells contain
such modification.
[0128] Functional disruption of the gene can be accomplished in any
effective way, including, e.g., introduction of a stop codon into
any part of the coding sequence such that the resulting polypeptide
is biologically inactive (e.g., because it lacks a catalytic
domain, a ligand binding domain, etc.), introduction of a mutation
into a promoter or other regulatory sequence that is effective to
turn it off, or reduce transcription of the gene, insertion of an
exogenous sequence into the gene which inactivates it (e.g., which
disrupts the production of a biologically-active polypeptide or
which disrupts the promoter or other transcriptional machinery),
deletion of sequences from the Tbx3-pr408 gene, etc. Examples of
transgenic animals having functionally disrupted genes are well
known, e.g., as described in U.S. Pat. Nos. 6,239,326, 6,225,525,
6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445,
6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858,
5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654,
5,777,195, and 5,569,824. A transgenic animal which comprises the
functional disruption can also be referred to as a "knock-out"
animal, since the biological activity of its Tbx3-pr408 genes has
been "knocked-out." Knock-outs can be homozygous or
heterozygous.
[0129] For creating functional disrupted genes, and other gene
mutations, homologous recombination technology is of special
interest since it allows specific regions of the genome to be
targeted. Using homologous recombination methods, genes can be
specifically-inactivated, specific mutations can be introduced, and
exogenous sequences can be introduced at specific sites. These
methods are well known in the art, e.g., as described in the
patents above. See, also, Robertson, Biol. Reproduc.,
44(2):238-245, 1991. Generally, the genetic engineering is
performed in an embryonic stem (ES) cell, or other pluripotent cell
line (e.g., adult stem cells, EG cells), and that
genetically-modified cell (or nucleus) is used to create a whole
organism. Nuclear transfer can be used in combination with
homologous recombination technologies.
[0130] For example, the Tbx3-pr408 locus can be disrupted in mouse
ES cells using a positive-negative selection method (e.g., Mansour
et al., Nature, 336:348-352, 1988). In this method, a targeting
vector can be constructed which comprises a part of the gene to be
targeted. A selectable marker, such as neomycin resistance genes,
can be inserted into a Tbx3-pr408 exon present in the targeting
vector, disrupting it. When the vector recombines with the ES cell
genome, it disrupts the function of the gene. The presence in the
cell of the vector can be determined by expression of neomycin
resistance. See, e.g., U.S. Pat. No. 6,239,326. Cells having at
least one functionally disrupted gene can be used to make chimeric
and germine animals, e.g., animals having somatic and/or germ cells
comprising the engineered gene. Homozygous knock-out animals can be
obtained from breeding heterozygous knock-out animals. See, e.g.,
U.S. Pat. No. 6,225,525.
[0131] A transgenic animal, or animal cell, lacking one or more
functional Tbx3-pr408 genes can be useful in a variety of
applications, including, as an animal model for mammary-ulnar
disease, and any of the utilities mentioned in any issued U.S.
Patent on transgenic animals, including, U.S. Pat. Nos. 6,239,326,
6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610,
6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244,
6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912,
5,789,654, 5,777,195, and 5,569,824
[0132] The present invention also relates to non-human, transgenic
animal whose genome comprises recombinant Tbx3-pr408 nucleic acid
operatively linked to an expression control sequence effective to
express said coding sequence, e.g., in heart, prostate, lung,
adrenal, testes, and/or muscle. Such a transgenic animal can also
be referred to as a "knock-in" animal since an exogenous gene has
been introduced, stably, into its genome.
[0133] A recombinant Tbx3-pr408 nucleic acid refers to a gene which
has been introduced into a target host cell and optionally
modified, such as cells derived from animals, plants, bacteria,
yeast, etc. A recombinant Tbx3-pr408 includes completely synthetic
nucleic acid sequences, semi-synthetic nucleic acid sequences,
sequences derived from natural sources, and chimeras thereof.
"Operable linkage" has the meaning used through the specification,
i.e., placed in a functional relationship with another nucleic
acid. When a gene is operably linked to an expression control
sequence, as explained above, it indicates that the gene (e.g.,
coding sequence) is joined to the expression control sequence
(e.g., promoter) in such a way that facilitates transcription and
translation of the coding sequence. As described above, the phrase
"genome" indicates that the genome of the cell has been modified.
In this case, the recombinant Tbx3-pr408 has been stably integrated
into the genome of the animal. The Tbx3-pr408 nucleic acid in
operable linkage with the expression control sequence can also be
referred to as a construct or transgene.
[0134] Any expression control sequence can be used depending on the
purpose. For instance, if selective expression is desired, then
expression control sequences which limit its expression can be
selected. These include, e.g., tissue or cell-specific promoters,
introns, enhancers, etc. For various methods of cell and
tissue-specific expression, see, e.g., U.S. Pat. Nos. 6,215,040,
6,210,736, and 6,153,427. These also include the endogenous
promoter, i.e., the coding sequence can be operably linked to its
own promoter. Inducible and regulatable promoters can also be
utilized.
[0135] The present invention also relates to a transgenic animal
which contains a functionally disrupted and a transgene stably
integrated into the animals genome. Such an animal can be
constructed using combinations any of the above- and
below-mentioned methods. Such animals have any of the
aforementioned uses, including permitting the knock-out of the
normal gene and its replacement with a mutated gene. Such a
transgene can be integrated at the endogenous gene locus so that
the functional disruption and "knock-in" are carried out in the
same step.
[0136] In addition to the methods mentioned above, transgenic
animals can be prepared according to known methods, including,
e.g., by pronuclear injection of recombinant genes into pronuclei
of 1-cell embryos, incorporating an artificial yeast chromosome
into embryonic stem cells, gene targeting methods, embryonic stem
cell methodology, cloning methods, nuclear transfer methods. See,
also, e.g., U.S. Pat. Nos. 4,736,866; 4,873,191; 4,873,316;
5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; 5,221,778;
Gordon et al., Proc. Natl. Acad. Sci., 77:7380-7384, 1980; Palmiter
et al., Cell, 41:343-345, 1985; Palmiter et al., Ann. Rev. Genet.,
20:465-499, 1986; Askew et al., Mol. Cell. Bio., 13:4115-4124,
1993; Games et al. Nature, 373:523-527, 1995; Valancius and
Smithies, Mol. Cell. Bio., 11: 1402-1408, 1991; Stacey et al., Mol.
Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246,
1995; Rubinstein et al., Nucl. Acid Res., 21:2613-2617,1993;
Cibelli et al., Science, 280:1256-1258, 1998. For guidance on
recombinase excision systems, see, e.g., U.S. Pat. Nos. 5,626,159,
5,527,695, and 5,434,066. See also, Orban, P. C., et al.,
"Tissue-and Site-Specific DNA Recombination in Transgenic Mice,"
Proc. Natl. Acad. Sci. USA, 89:6861-6865 (1992); O'Gorman, S., et
al., "Recombinase-Mediated Gene Activation and Site-Specific
Integration in Mammalian Cells," Science, 251:1351-1355 (1991);
Sauer, B., et al., "Cre-stimulated recombination at loxP-Containing
DNA sequences placed into the mammalian genome," Polynucleotides
Research, 17(1):147-161 (1989); Gagneten, S. et al. (1997) Nucl.
Acids Res. 25:3326-3331; Xiao and Weaver (1997) Nucl. Acids Res.
25:2985-2991; Agah, R. et al. (1997) J. Clin. Invest. 100:169-179;
Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543-2545; Araki, K.
et al. (1997) Nucl. Acids Res. 25:868-872; Mortensen, R. N. et al.
(1992) Mol. Cell. Biol. 12:2391-2395 (G418 escalation method);
Lakhlani, P. P. et al. (1997) Proc. Natl. Acad. Sci. USA
94:9950-9955 ("hit and run"); Westphal and Leder (1997) Curr. Biol.
7:530-533 (transposon-generated "knock-out" and "knock-in");
Templeton, N. S. et al. (1997) Gene Ther. 4:700-709 (methods for
efficient gene targeting, allowing for a high frequency of
homologous recombination events, e.g., without selectable markers);
PCT International Publication WO 93/22443
(functionally-disrupted).
[0137] A polynucleotide according to the present invention can be
introduced into any non-human animal, including a non-human mammal,
mouse (Hogan et al., Manipulating the Mouse Embryo: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1986), pig (Hammer et al., Nature, 315:343-345, 1985), sheep
(Hammer et al., Nature, 315:343-345, 1985), cattle, rat, or
primate. See also, e.g., Church, 1987, Trends in Biotech. 5:13-19;
Clark et al., Trends in Biotech. 5:20-24, 1987); and DePamphilis et
al., BioTechniques, 6:662-680, 1988. Transgenic animals can be
produced by the methods described in U.S. Pat. No. 5,994,618, and
utilized for any of the utilities described therein.
[0138] Database
[0139] The present invention also relates to electronic forms of
polynucleotides, polypeptides, etc., of the present invention,
including computer-readable medium (e.g., magnetic, optical, etc.,
stored in any suitable format, such as flat files or hierarchical
files) which comprise such sequences, or fragments thereof,
e-commerce-related means, etc. Along these lines, the present
invention relates to methods of retrieving gene sequences from a
computer-readable medium, comprising, one or more of the following
steps in any effective order, e.g., selecting a cell or gene
expression profile, e.g., a profile that specifies that said gene
is differentially expressed in, e.g., heart, prostate, and lung,
and retrieving said differentially expressed gene sequences, where
the gene sequences consist of the genes represented by SEQ ID NOS 1
and 2.
[0140] A "gene expression profile" means the list of tissues,
cells, etc., in which a defined gene is expressed (i.e, transcribed
and/or translated). A "cell expression profile" means the genes
which are expressed in the particular cell type. The profile can be
a list of the tissues in which the gene is expressed, but can
include additional information as well, including level of
expression (e.g., a quantity as compared or normalized to a control
gene), and information on temporal (e.g., at what point in the
cell-cycle or developmental program) and spatial expression. By the
phrase "selecting a gene or cell expression profile," it is meant
that a user decides what type of gene or cell expression pattern he
is interested in retrieving, e.g., he may require that the gene is
differentially expressed in a tissue, or he may require that the
gene is not expressed in blood, but must be expressed in prostate.
Any pattern of expression preferences may be selected. The
selecting can be performed by any effective method. In general,
"selecting" refers to the process in which a user forms a query
that is used to search a database of gene expression profiles. The
step of retrieving involves searching for results in a database
that correspond to the query set forth in the selecting step. Any
suitable algorithm can be utilized to perform the search query,
including algorithms that look for matches, or that perform
optimization between query and data. The database is information
that has been stored in an appropriate storage medium, having a
suitable computer-readable format. Once results are retrieved, they
can be displayed in any suitable format, such as HTML.
[0141] For instance, the user may be interested in identifying
genes that are differentially expressed in prostate and heart. He
may not care whether small amounts of expression occur in other
tissues, as long as such genes are not expressed in peripheral
blood lymphocytes. A query is formed by the user to retrieve the
set of genes from the database having the desired gene or cell
expression profile. Once the query is inputted into the system, a
search algorithm is used to interrogate the database, and retrieve
results.
[0142] Advertising, Licensing, etc., Methods
[0143] The present invention also relates to methods of
advertising, licensing, selling, purchasing, brokering, etc.,
genes, polynucleotides, specific-binding partners, antibodies,
etc., of the present invention. Methods can comprises, e.g.,
displaying a Tbx3-pr408 gene, Tbx3-pr408 polypeptide, or antibody
specific for Tbx3-pr408 in a printed or computer-readable medium
(e.g., on the Web or Internet), accepting an offer to purchase said
gene, polypeptide, or antibody.
[0144] Other
[0145] A polynucleotide, probe, polypeptide, antibody,
specific-binding partner, etc., according to the present invention
can be isolated. The term "isolated" means that the material is in
a form in which it is not found in its original environment or in
nature, e.g., more concentrated, more purified, separated from
component, etc. An isolated polynucleotide includes, e.g., a
polynucleotide having the sequenced separated from the chromosomal
DNA found in a living animal, e.g., as the complete gene, a
transcript, or a cDNA. This polynucleotide can be part of a vector
or inserted into a chromosome (by specific gene-targeting or by
random integration at a position other than its normal position)
and still be isolated in that it is not in a form that is found in
its natural environment. A polynucleotide, polypeptide, etc., of
the present invention can also be substantially purified. By
substantially purified, it is meant that polynucleotide or
polypeptide is separated and is essentially free from other
polynucleotides or polypeptides, i.e., the polynucleotide or
polypeptide is the primary and active constituent. A polynucleotide
can also be a recombinant molecule. By "recombinant," it is meant
that the polynucleotide is an arrangement or form which does not
occur in nature. For instance, a recombinant molecule comprising a
promoter sequence would not encompass the naturally-occurring gene,
but would include the promoter operably linked to a coding sequence
not associated with it in nature, e.g., a reporter gene, or a
truncation of the normal coding sequence.
[0146] The term "marker" is used herein to indicate a means for
detecting or labeling a target. A marker can be a polynucleotide
(usually referred to as a "probe"), polypeptide (e.g., an antibody
conjugated to a detectable label), PNA, or any effective
material.
[0147] The topic headings set forth above are meant as guidance
where certain information can be found in the application, but are
not intended to be the only source in the application where
information on such topic can be found. Reference materials
[0148] For other aspects of the polynucleotides, reference is made
to standard textbooks of molecular biology. See, e.g., Hames et
al., Polynucleotide Hybridization, IL Press, 1985; Davis et al.,
Basic Methods in Molecular Biology, Elsevir Sciences Publishing,
Inc., New York, 1986; Sambrook et al., Molecular Cloning, CSH
Press, 1989; Howe, Gene Cloning and Manipulation, Cambridge
University Press, 1995; Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons, Inc., 1994-1998.
[0149] The preceding description, utilize the present invention to
its fullest extent. The preceding preferred specific embodiments
are, therefore, to be construed as merely illustrative, and not
limiting the remainder of the disclosure in any way whatsoever. The
entire disclosure of all applications, patents and publications,
cited above and in the figures are hereby incorporated by reference
in their entirety.
Sequence CWU 1
1
5 1 3113 DNA Homo sapiens CDS (524)..(2695) 1 caccagcgag aaagagggag
aggaagacag atagggggcg ggggaagaag aaaaagaaag 60 gtaaaaagtc
ttctaggaga acctttcaca tttgcaacaa aagacctagg ggctggagag 120
agattcctgg gacgcagggc tggagtgtct atttcgagct cagcggcagg gctcgggcgc
180 gagtcgagac cctgctcgct cctctcgctt ctgaaaccga cgttcaggag
cggcttttta 240 aaaacgcaag gcacaaggac ggtcacccgc gcgactatgt
ttgctgattt ttcgccttgc 300 cctctttaaa agcggcctcc cattccccaa
aagacacttc ccctcctccc tttgaagtgc 360 attagttgtg atttctgcct
ccttttcttt tttctttctt ttttgttttg ctttttcccc 420 ccttttgaat
tatgtgctgc tgttaaacaa caacaaaaaa acaacaaaac acagcagctg 480
cggacttgtc cccggctgga gcccagcgcc ccgcctggag tgg atg agc ctc tcc 535
Met Ser Leu Ser 1 atg aga gat ccg gtc att cct ggg aca agc atg gcc
tac cat ccg ttc 583 Met Arg Asp Pro Val Ile Pro Gly Thr Ser Met Ala
Tyr His Pro Phe 5 10 15 20 cta cct cac cgg gcg ccg gac ttc gcc atg
agc gcg gtg ctg ggt cac 631 Leu Pro His Arg Ala Pro Asp Phe Ala Met
Ser Ala Val Leu Gly His 25 30 35 cag ccg ccg ttc ttc ccc gcg ctg
acg ctg cct ccc aac ggc gcg gcg 679 Gln Pro Pro Phe Phe Pro Ala Leu
Thr Leu Pro Pro Asn Gly Ala Ala 40 45 50 gcg ctc tcg ctg ccg ggc
gcc ctg gcc aag ccg atc atg gat caa ttg 727 Ala Leu Ser Leu Pro Gly
Ala Leu Ala Lys Pro Ile Met Asp Gln Leu 55 60 65 gtg ggg gcg gcc
gag acc ggc atc ccg ttc tcc tcc ctg ggg ccc cag 775 Val Gly Ala Ala
Glu Thr Gly Ile Pro Phe Ser Ser Leu Gly Pro Gln 70 75 80 gcg cat
ctg agg cct ttg aag acc atg gag ccc gaa gaa gag gtg gag 823 Ala His
Leu Arg Pro Leu Lys Thr Met Glu Pro Glu Glu Glu Val Glu 85 90 95
100 gac gac ccc aag gtg cac ctg gag gct aaa gaa ctt tgg gat cag ttt
871 Asp Asp Pro Lys Val His Leu Glu Ala Lys Glu Leu Trp Asp Gln Phe
105 110 115 cac aag cgg ggc acc gag atg gtc att acc aag tcg gga agg
cga atg 919 His Lys Arg Gly Thr Glu Met Val Ile Thr Lys Ser Gly Arg
Arg Met 120 125 130 ttt cct cca ttt aaa gtg aga tgt tct ggg ctg gat
aaa aaa gcc aaa 967 Phe Pro Pro Phe Lys Val Arg Cys Ser Gly Leu Asp
Lys Lys Ala Lys 135 140 145 tac att tta ttg atg gac att ata gct gct
gat gac tgt cgt tat aaa 1015 Tyr Ile Leu Leu Met Asp Ile Ile Ala
Ala Asp Asp Cys Arg Tyr Lys 150 155 160 ttt cac aat tct cgg tgg atg
gtg gct ggt aag gcc gac ccc gaa atg 1063 Phe His Asn Ser Arg Trp
Met Val Ala Gly Lys Ala Asp Pro Glu Met 165 170 175 180 cca aag agg
atg tac att cac ccg gac agc ccc gct act ggg gaa cag 1111 Pro Lys
Arg Met Tyr Ile His Pro Asp Ser Pro Ala Thr Gly Glu Gln 185 190 195
tgg atg tcc aaa gtc gtc act ttc cac aaa ctg aaa ctc acc aac aac
1159 Trp Met Ser Lys Val Val Thr Phe His Lys Leu Lys Leu Thr Asn
Asn 200 205 210 att tca gac aaa cat gga ttt act ata ttg aac tcc atg
cac aaa tac 1207 Ile Ser Asp Lys His Gly Phe Thr Ile Leu Asn Ser
Met His Lys Tyr 215 220 225 cag ccc cgg ttc cac att gta aga gcc aat
gac atc ttg aaa ctc cct 1255 Gln Pro Arg Phe His Ile Val Arg Ala
Asn Asp Ile Leu Lys Leu Pro 230 235 240 tat agt aca ttt cgg aca tac
ttg ttc ccc gaa act gaa ttc atc gct 1303 Tyr Ser Thr Phe Arg Thr
Tyr Leu Phe Pro Glu Thr Glu Phe Ile Ala 245 250 255 260 gtg act gca
tac cag aat gat aag ata acc cag tta aaa ata gac aac 1351 Val Thr
Ala Tyr Gln Asn Asp Lys Ile Thr Gln Leu Lys Ile Asp Asn 265 270 275
aac cct ttt gca aaa ggt ttc cgg gac act gga aat ggc cga aga gaa
1399 Asn Pro Phe Ala Lys Gly Phe Arg Asp Thr Gly Asn Gly Arg Arg
Glu 280 285 290 aaa aga aaa cag ctc acc ctg cag tcc atg agg gtg ttt
gat gaa aga 1447 Lys Arg Lys Gln Leu Thr Leu Gln Ser Met Arg Val
Phe Asp Glu Arg 295 300 305 cac aaa aag gag aat ggg acc tct gat gag
tcc tcc agt gaa caa gca 1495 His Lys Lys Glu Asn Gly Thr Ser Asp
Glu Ser Ser Ser Glu Gln Ala 310 315 320 gct ttc aac tgc ttc gcc cag
gct tct tct cca gcc gcc tcc act gta 1543 Ala Phe Asn Cys Phe Ala
Gln Ala Ser Ser Pro Ala Ala Ser Thr Val 325 330 335 340 ggg aca tcg
aac ctc aaa gat tta tgt ccc agc gag ggt gag agc gac 1591 Gly Thr
Ser Asn Leu Lys Asp Leu Cys Pro Ser Glu Gly Glu Ser Asp 345 350 355
gcc gag gcc gag agc aaa gag gag cat ggc ccc gag gcc tgc gac gcg
1639 Ala Glu Ala Glu Ser Lys Glu Glu His Gly Pro Glu Ala Cys Asp
Ala 360 365 370 gcc aag atc tcc acc acc acg tcg gag gag ccc tgc cgt
gac aag ggc 1687 Ala Lys Ile Ser Thr Thr Thr Ser Glu Glu Pro Cys
Arg Asp Lys Gly 375 380 385 agc ccc gcg gtc aag gct cac ctt ttc gct
gct gag cgg ccc cgg gac 1735 Ser Pro Ala Val Lys Ala His Leu Phe
Ala Ala Glu Arg Pro Arg Asp 390 395 400 agc ggg cgg ctg gac aaa gcg
tcg ccc gac tca cgc cat agc ccc gcc 1783 Ser Gly Arg Leu Asp Lys
Ala Ser Pro Asp Ser Arg His Ser Pro Ala 405 410 415 420 acc atc tcg
tcc agc act cgc ggc ctg ggc gcg gag gag cgc agg agc 1831 Thr Ile
Ser Ser Ser Thr Arg Gly Leu Gly Ala Glu Glu Arg Arg Ser 425 430 435
ccg gtt cgc gag ggc aca gcg ccg gcc aag gtg gaa gag gcg cgc gcg
1879 Pro Val Arg Glu Gly Thr Ala Pro Ala Lys Val Glu Glu Ala Arg
Ala 440 445 450 ctc ccg ggc aag gag gcc ttc gcg ccg ctc acg gtg cag
acg gac gcg 1927 Leu Pro Gly Lys Glu Ala Phe Ala Pro Leu Thr Val
Gln Thr Asp Ala 455 460 465 gcc gcc gcg cac ctg gcc cag ggc ccc ctg
cct ggc ctc ggc ttc gcc 1975 Ala Ala Ala His Leu Ala Gln Gly Pro
Leu Pro Gly Leu Gly Phe Ala 470 475 480 ccg ggc ctg gcg ggc caa cag
ttc ttc aac ggg cac ccg ctc ttc ctg 2023 Pro Gly Leu Ala Gly Gln
Gln Phe Phe Asn Gly His Pro Leu Phe Leu 485 490 495 500 cac ccc agc
cag ttt gcc atg ggg ggc gcc ttc tcc agc atg gcg gcc 2071 His Pro
Ser Gln Phe Ala Met Gly Gly Ala Phe Ser Ser Met Ala Ala 505 510 515
gct ggc atg ggt ccc ctc ctg gcc acg gtt tct ggg gcc tcc acc ggt
2119 Ala Gly Met Gly Pro Leu Leu Ala Thr Val Ser Gly Ala Ser Thr
Gly 520 525 530 gtc tcg ggc ctg gat tcc acg gcc atg gcc tct gcc gct
gcg gcg cag 2167 Val Ser Gly Leu Asp Ser Thr Ala Met Ala Ser Ala
Ala Ala Ala Gln 535 540 545 gga ctg tcc ggg gcg tcc gcg gcc acc ctg
ccc ttc cac ctc cag cag 2215 Gly Leu Ser Gly Ala Ser Ala Ala Thr
Leu Pro Phe His Leu Gln Gln 550 555 560 cac gtc ctg gcc tct cag ggc
ctg gcc atg tcc cct ttc gga agc ctg 2263 His Val Leu Ala Ser Gln
Gly Leu Ala Met Ser Pro Phe Gly Ser Leu 565 570 575 580 ttc cct tac
ccc tac acg tac atg gcc gca gcg gcg gcc gcc tcc tct 2311 Phe Pro
Tyr Pro Tyr Thr Tyr Met Ala Ala Ala Ala Ala Ala Ser Ser 585 590 595
gcg gca gcc tcc agc tcg gtg cac cgc cac ccc ttc ctc aat ctg aac
2359 Ala Ala Ala Ser Ser Ser Val His Arg His Pro Phe Leu Asn Leu
Asn 600 605 610 acc atg cgc ccg cgg ctg cgc tac agc ccc tac tcc atc
ccg gtg ccg 2407 Thr Met Arg Pro Arg Leu Arg Tyr Ser Pro Tyr Ser
Ile Pro Val Pro 615 620 625 gtc ccg gac ggc agc agt ctg ctc acc acc
gcc ctg ccc tcc atg gcg 2455 Val Pro Asp Gly Ser Ser Leu Leu Thr
Thr Ala Leu Pro Ser Met Ala 630 635 640 gcg gcc gcg ggg ccc ctg gac
ggc aaa gtc gcc gcc ctg gcc gcc agc 2503 Ala Ala Ala Gly Pro Leu
Asp Gly Lys Val Ala Ala Leu Ala Ala Ser 645 650 655 660 ccg gcc tcg
gtg gca gtg gac tcg ggc tct gaa ctc aac agc cgc tcc 2551 Pro Ala
Ser Val Ala Val Asp Ser Gly Ser Glu Leu Asn Ser Arg Ser 665 670 675
tcc acg ctc tcc tcc agc tcc atg tcc ttg tcg ccc aaa ctc tgc gcg
2599 Ser Thr Leu Ser Ser Ser Ser Met Ser Leu Ser Pro Lys Leu Cys
Ala 680 685 690 gag aaa gag gcg gcc acc agc gaa ctg cag agc atc cag
cgg ttg gtt 2647 Glu Lys Glu Ala Ala Thr Ser Glu Leu Gln Ser Ile
Gln Arg Leu Val 695 700 705 agc ggc ttg gaa gcc aag ccg gac agg tcc
cgc agc gcg tcc ccg tag 2695 Ser Gly Leu Glu Ala Lys Pro Asp Arg
Ser Arg Ser Ala Ser Pro 710 715 720 acccgtccca gacacgtctt
ttcattccag tccagttcag gctgccgtgc actttgtcgg 2755 atataaaata
aaccacgggc ccgccatggc gttagccctt ccttttgcag ttgcgtctgg 2815
gaaggggccc cggactccct cgagagaatg tgctagagac agcccctgtc ttcttggcgt
2875 ggtttatatg tccgggatct ggatcagatt ctgggggctc agaaacgtcg
gttgcattga 2935 gctactgggg gtaggagttc caacatttat gtccagagca
acttccagca aggctggtct 2995 gggtctctgc ccaccaggcg gggaggtgtt
caaagacatc tccctcagtg cggatttata 3055 tatatatttt tccttcactg
tgtcaagtgg aaacaaaaac aaaaaaaaaa aaaaaaaa 3113 2 723 PRT Homo
sapiens 2 Met Ser Leu Ser Met Arg Asp Pro Val Ile Pro Gly Thr Ser
Met Ala 1 5 10 15 Tyr His Pro Phe Leu Pro His Arg Ala Pro Asp Phe
Ala Met Ser Ala 20 25 30 Val Leu Gly His Gln Pro Pro Phe Phe Pro
Ala Leu Thr Leu Pro Pro 35 40 45 Asn Gly Ala Ala Ala Leu Ser Leu
Pro Gly Ala Leu Ala Lys Pro Ile 50 55 60 Met Asp Gln Leu Val Gly
Ala Ala Glu Thr Gly Ile Pro Phe Ser Ser 65 70 75 80 Leu Gly Pro Gln
Ala His Leu Arg Pro Leu Lys Thr Met Glu Pro Glu 85 90 95 Glu Glu
Val Glu Asp Asp Pro Lys Val His Leu Glu Ala Lys Glu Leu 100 105 110
Trp Asp Gln Phe His Lys Arg Gly Thr Glu Met Val Ile Thr Lys Ser 115
120 125 Gly Arg Arg Met Phe Pro Pro Phe Lys Val Arg Cys Ser Gly Leu
Asp 130 135 140 Lys Lys Ala Lys Tyr Ile Leu Leu Met Asp Ile Ile Ala
Ala Asp Asp 145 150 155 160 Cys Arg Tyr Lys Phe His Asn Ser Arg Trp
Met Val Ala Gly Lys Ala 165 170 175 Asp Pro Glu Met Pro Lys Arg Met
Tyr Ile His Pro Asp Ser Pro Ala 180 185 190 Thr Gly Glu Gln Trp Met
Ser Lys Val Val Thr Phe His Lys Leu Lys 195 200 205 Leu Thr Asn Asn
Ile Ser Asp Lys His Gly Phe Thr Ile Leu Asn Ser 210 215 220 Met His
Lys Tyr Gln Pro Arg Phe His Ile Val Arg Ala Asn Asp Ile 225 230 235
240 Leu Lys Leu Pro Tyr Ser Thr Phe Arg Thr Tyr Leu Phe Pro Glu Thr
245 250 255 Glu Phe Ile Ala Val Thr Ala Tyr Gln Asn Asp Lys Ile Thr
Gln Leu 260 265 270 Lys Ile Asp Asn Asn Pro Phe Ala Lys Gly Phe Arg
Asp Thr Gly Asn 275 280 285 Gly Arg Arg Glu Lys Arg Lys Gln Leu Thr
Leu Gln Ser Met Arg Val 290 295 300 Phe Asp Glu Arg His Lys Lys Glu
Asn Gly Thr Ser Asp Glu Ser Ser 305 310 315 320 Ser Glu Gln Ala Ala
Phe Asn Cys Phe Ala Gln Ala Ser Ser Pro Ala 325 330 335 Ala Ser Thr
Val Gly Thr Ser Asn Leu Lys Asp Leu Cys Pro Ser Glu 340 345 350 Gly
Glu Ser Asp Ala Glu Ala Glu Ser Lys Glu Glu His Gly Pro Glu 355 360
365 Ala Cys Asp Ala Ala Lys Ile Ser Thr Thr Thr Ser Glu Glu Pro Cys
370 375 380 Arg Asp Lys Gly Ser Pro Ala Val Lys Ala His Leu Phe Ala
Ala Glu 385 390 395 400 Arg Pro Arg Asp Ser Gly Arg Leu Asp Lys Ala
Ser Pro Asp Ser Arg 405 410 415 His Ser Pro Ala Thr Ile Ser Ser Ser
Thr Arg Gly Leu Gly Ala Glu 420 425 430 Glu Arg Arg Ser Pro Val Arg
Glu Gly Thr Ala Pro Ala Lys Val Glu 435 440 445 Glu Ala Arg Ala Leu
Pro Gly Lys Glu Ala Phe Ala Pro Leu Thr Val 450 455 460 Gln Thr Asp
Ala Ala Ala Ala His Leu Ala Gln Gly Pro Leu Pro Gly 465 470 475 480
Leu Gly Phe Ala Pro Gly Leu Ala Gly Gln Gln Phe Phe Asn Gly His 485
490 495 Pro Leu Phe Leu His Pro Ser Gln Phe Ala Met Gly Gly Ala Phe
Ser 500 505 510 Ser Met Ala Ala Ala Gly Met Gly Pro Leu Leu Ala Thr
Val Ser Gly 515 520 525 Ala Ser Thr Gly Val Ser Gly Leu Asp Ser Thr
Ala Met Ala Ser Ala 530 535 540 Ala Ala Ala Gln Gly Leu Ser Gly Ala
Ser Ala Ala Thr Leu Pro Phe 545 550 555 560 His Leu Gln Gln His Val
Leu Ala Ser Gln Gly Leu Ala Met Ser Pro 565 570 575 Phe Gly Ser Leu
Phe Pro Tyr Pro Tyr Thr Tyr Met Ala Ala Ala Ala 580 585 590 Ala Ala
Ser Ser Ala Ala Ala Ser Ser Ser Val His Arg His Pro Phe 595 600 605
Leu Asn Leu Asn Thr Met Arg Pro Arg Leu Arg Tyr Ser Pro Tyr Ser 610
615 620 Ile Pro Val Pro Val Pro Asp Gly Ser Ser Leu Leu Thr Thr Ala
Leu 625 630 635 640 Pro Ser Met Ala Ala Ala Ala Gly Pro Leu Asp Gly
Lys Val Ala Ala 645 650 655 Leu Ala Ala Ser Pro Ala Ser Val Ala Val
Asp Ser Gly Ser Glu Leu 660 665 670 Asn Ser Arg Ser Ser Thr Leu Ser
Ser Ser Ser Met Ser Leu Ser Pro 675 680 685 Lys Leu Cys Ala Glu Lys
Glu Ala Ala Thr Ser Glu Leu Gln Ser Ile 690 695 700 Gln Arg Leu Val
Ser Gly Leu Glu Ala Lys Pro Asp Arg Ser Arg Ser 705 710 715 720 Ala
Ser Pro 3 722 PRT Homo sapiens 3 Met Ser Leu Ser Met Arg Asp Pro
Val Ile Pro Gly Thr Ser Met Ala 1 5 10 15 Tyr His Pro Phe Leu Pro
His Arg Ala Pro Asp Phe Ala Met Ser Ala 20 25 30 Val Leu Gly His
Gln Pro Pro Phe Phe Pro Ala Leu Thr Leu Pro Pro 35 40 45 Asn Gly
Ala Ala Ala Leu Ser Leu Pro Gly Ala Leu Ala Lys Pro Ile 50 55 60
Met Asp Gln Leu Val Gly Ala Ala Glu Thr Gly Ile Pro Phe Ser Ser 65
70 75 80 Leu Gly Pro Gln Ala His Leu Arg Pro Leu Lys Thr Met Glu
Pro Glu 85 90 95 Glu Glu Val Glu Asp Asp Pro Lys Val His Leu Glu
Ala Lys Glu Leu 100 105 110 Trp Asp Gln Phe His Lys Arg Gly Thr Glu
Met Val Ile Thr Lys Ser 115 120 125 Gly Arg Arg Met Phe Pro Pro Phe
Lys Val Arg Cys Ser Gly Leu Asp 130 135 140 Lys Lys Ala Lys Tyr Ile
Leu Leu Met Asp Ile Ile Ala Ala Asp Asp 145 150 155 160 Cys Arg Tyr
Lys Phe His Asn Ser Arg Trp Met Val Ala Gly Lys Ala 165 170 175 Asp
Pro Glu Met Pro Lys Arg Met Tyr Ile His Pro Asp Ser Pro Ala 180 185
190 Thr Gly Glu Gln Trp Met Ser Lys Val Val Thr Phe His Lys Leu Lys
195 200 205 Leu Thr Asn Asn Ile Ser Asp Lys His Gly Phe Thr Ile Leu
Asn Ser 210 215 220 Met His Lys Tyr Gln Pro Arg Phe His Ile Val Arg
Ala Asn Asp Ile 225 230 235 240 Leu Lys Leu Pro Tyr Ser Thr Phe Arg
Thr Tyr Leu Phe Pro Glu Thr 245 250 255 Glu Phe Ile Ala Val Thr Ala
Tyr Gln Asn Asp Lys Ile Thr Gln Leu 260 265 270 Lys Ile Asp Asn Asn
Pro Phe Ala Lys Gly Phe Arg Asp Thr Gly Asn 275 280 285 Gly Arg Arg
Glu Lys Arg Lys Gln Leu Thr Leu Gln Ser Met Arg Val 290 295 300 Phe
Asp Glu Arg His Lys Lys Glu Asn Gly Thr Ser Asp Glu Ser Ser 305 310
315 320 Ser Glu Gln Ala Ala Phe Asn Cys Phe Ala Gln Ala Ser Ser Pro
Ala 325 330 335 Ala Ser Thr Val Gly Thr Ser Asn Leu Lys Asp Leu Cys
Pro Ser Glu 340 345 350 Gly Glu Ser Asp Ala Glu Ala Glu Ser Lys Glu
Glu His Gly Pro Glu 355 360 365 Ala Cys Asp Ala Ala Lys Ile Ser Thr
Thr Thr Ser Glu Glu Pro Cys 370 375 380 Arg Asp
Lys Gly Ser Pro Ala Val Lys Ala His Leu Phe Ala Ala Glu 385 390 395
400 Arg Pro Arg Asp Ser Gly Arg Leu Asp Lys Ala Ser Pro Asp Ser Arg
405 410 415 His Ser Pro Ala Thr Ile Ser Ser Ser Thr Arg Gly Leu Gly
Ala Glu 420 425 430 Glu Arg Arg Ser Pro Val Arg Glu Gly Thr Ala Pro
Ala Lys Val Glu 435 440 445 Glu Ala Arg Ala Leu Pro Gly Lys Glu Ala
Phe Ala Pro Leu Thr Val 450 455 460 Gln Thr Asp Ala Ala Ala Ala His
Leu Ala Gln Gly Pro Leu Pro Gly 465 470 475 480 Leu Gly Phe Ala Pro
Gly Leu Ala Gly Gln Gln Phe Phe Asn Gly His 485 490 495 Pro Leu Phe
Leu His Pro Ser Gln Phe Ala Met Gly Gly Ala Phe Ser 500 505 510 Ser
Met Ala Ala Ala Gly Met Gly Pro Leu Leu Ala Thr Val Ser Gly 515 520
525 Ala Ser Thr Gly Val Ser Gly Leu Asp Ser Thr Ala Met Ala Ser Ala
530 535 540 Ala Ala Ala Gln Gly Leu Ser Gly Ala Ser Ala Ala Thr Leu
Pro Phe 545 550 555 560 His Leu Gln Gln His Val Leu Ala Ser Gln Gly
Leu Ala Met Ser Pro 565 570 575 Phe Gly Ser Leu Phe Pro Tyr Pro Tyr
Thr Tyr Met Ala Ala Ala Ala 580 585 590 Ala Ala Ser Leu Arg Gln Pro
Gln Leu Arg Cys Thr Ala Pro Leu Leu 595 600 605 Asn Leu Asn Thr Met
Arg Pro Arg Leu Arg Tyr Ser Pro Tyr Ser Ile 610 615 620 Pro Val Pro
Val Pro Asp Gly Ser Ser Leu Leu Thr Thr Ala Leu Pro 625 630 635 640
Ser Met Ala Ala Ala Ala Gly Pro Leu Asp Gly Lys Ala Ala Ala Leu 645
650 655 Ala Ala Ser Pro Ala Ser Val Ala Val Asp Ser Gly Ser Glu Pro
Asn 660 665 670 Ser Arg Ser Ser Thr Leu Ser Ser Ser Ser Met Ser Leu
Ser Pro Lys 675 680 685 Leu Cys Ala Glu Lys Glu Ala Ala Thr Ser Glu
Leu Gln Ser Ile Gln 690 695 700 Arg Leu Val Ser Gly Leu Glu Ala Lys
Pro Asp Arg Ser Arg Ser Ala 705 710 715 720 Ser Pro 4 743 PRT Homo
sapiens 4 Met Ser Leu Ser Met Arg Asp Pro Val Ile Pro Gly Thr Ser
Met Ala 1 5 10 15 Tyr His Pro Phe Leu Pro His Arg Ala Pro Asp Phe
Ala Met Ser Ala 20 25 30 Val Leu Gly His Gln Pro Pro Phe Phe Pro
Ala Leu Thr Leu Pro Pro 35 40 45 Asn Gly Ala Ala Ala Leu Ser Leu
Pro Gly Ala Leu Ala Lys Pro Ile 50 55 60 Met Asp Gln Leu Val Gly
Ala Ala Glu Thr Gly Ile Pro Phe Ser Ser 65 70 75 80 Leu Gly Pro Gln
Ala His Leu Arg Pro Leu Lys Thr Met Glu Pro Glu 85 90 95 Glu Glu
Val Glu Asp Asp Pro Lys Val His Leu Glu Ala Lys Glu Leu 100 105 110
Trp Asp Gln Phe His Lys Arg Gly Thr Glu Met Val Ile Thr Lys Ser 115
120 125 Gly Arg Arg Met Phe Pro Pro Phe Lys Val Arg Cys Ser Gly Leu
Asp 130 135 140 Lys Lys Ala Lys Tyr Ile Leu Leu Met Asp Ile Ile Ala
Ala Asp Asp 145 150 155 160 Cys Arg Tyr Lys Phe His Asn Ser Arg Trp
Met Val Ala Gly Lys Ala 165 170 175 Asp Pro Glu Met Pro Lys Arg Met
Tyr Ile His Pro Asp Ser Pro Ala 180 185 190 Thr Gly Glu Gln Trp Met
Ser Lys Val Val Thr Phe His Lys Leu Lys 195 200 205 Leu Thr Asn Asn
Ile Ser Asp Lys His Gly Phe Thr Leu Ala Phe Pro 210 215 220 Ser Asp
His Ala Thr Trp Gln Gly Asn Tyr Ser Phe Gly Thr Gln Thr 225 230 235
240 Ile Leu Asn Ser Met His Lys Tyr Gln Pro Arg Phe His Ile Val Arg
245 250 255 Ala Asn Asp Ile Leu Lys Leu Pro Tyr Ser Thr Phe Arg Thr
Tyr Leu 260 265 270 Phe Pro Glu Thr Glu Phe Ile Ala Val Thr Ala Tyr
Gln Asn Asp Lys 275 280 285 Ile Thr Gln Leu Lys Ile Asp Asn Asn Pro
Phe Ala Lys Gly Phe Arg 290 295 300 Asp Thr Gly Asn Gly Arg Arg Glu
Lys Arg Lys Gln Leu Thr Leu Gln 305 310 315 320 Ser Met Arg Val Phe
Asp Glu Arg His Lys Lys Glu Asn Gly Thr Ser 325 330 335 Asp Glu Ser
Ser Ser Glu Gln Ala Ala Phe Asn Cys Phe Ala Gln Ala 340 345 350 Ser
Ser Pro Ala Ala Ser Thr Val Gly Thr Ser Asn Leu Lys Asp Leu 355 360
365 Cys Pro Ser Glu Gly Glu Ser Asp Ala Glu Ala Glu Ser Lys Glu Glu
370 375 380 His Gly Pro Glu Ala Cys Asp Ala Ala Lys Ile Ser Thr Thr
Thr Ser 385 390 395 400 Glu Glu Pro Cys Arg Asp Lys Gly Ser Pro Ala
Val Lys Ala His Leu 405 410 415 Phe Ala Ala Glu Arg Pro Arg Asp Ser
Gly Arg Leu Asp Lys Ala Ser 420 425 430 Pro Asp Ser Arg His Ser Pro
Ala Thr Ile Ser Ser Ser Thr Arg Gly 435 440 445 Leu Gly Ala Glu Glu
Arg Arg Ser Pro Val Arg Glu Gly Thr Ala Pro 450 455 460 Ala Lys Val
Glu Glu Ala Arg Ala Leu Pro Gly Lys Glu Ala Phe Ala 465 470 475 480
Pro Leu Thr Val Gln Thr Asp Ala Ala Ala Ala His Leu Ala Gln Gly 485
490 495 Pro Leu Pro Gly Leu Gly Phe Ala Pro Gly Leu Ala Gly Gln Gln
Phe 500 505 510 Phe Asn Gly His Pro Leu Phe Leu His Pro Ser Gln Phe
Ala Met Gly 515 520 525 Gly Ala Phe Ser Ser Met Ala Ala Ala Gly Met
Gly Pro Leu Leu Ala 530 535 540 Thr Val Ser Gly Ala Ser Thr Gly Val
Ser Gly Leu Asp Ser Thr Ala 545 550 555 560 Met Ala Ser Ala Ala Ala
Ala Gln Gly Leu Ser Gly Ala Ser Ala Ala 565 570 575 Thr Leu Pro Phe
His Leu Gln Gln His Val Leu Ala Ser Gln Gly Leu 580 585 590 Ala Met
Ser Pro Phe Gly Ser Leu Phe Pro Tyr Pro Tyr Thr Tyr Met 595 600 605
Ala Ala Ala Ala Ala Ala Ser Ser Ala Ala Ala Ser Ser Ser Val His 610
615 620 Arg His Pro Phe Leu Asn Leu Asn Thr Met Arg Pro Arg Leu Arg
Tyr 625 630 635 640 Ser Pro Tyr Ser Ile Pro Val Pro Val Pro Asp Gly
Ser Ser Leu Leu 645 650 655 Thr Thr Ala Leu Pro Ser Met Ala Ala Ala
Ala Gly Pro Leu Asp Gly 660 665 670 Lys Val Ala Ala Leu Ala Ala Ser
Pro Ala Ser Val Ala Val Asp Ser 675 680 685 Gly Ser Glu Leu Asn Ser
Arg Ser Ser Thr Leu Ser Ser Ser Ser Met 690 695 700 Ser Leu Ser Pro
Lys Leu Cys Ala Glu Lys Glu Ala Ala Thr Ser Glu 705 710 715 720 Leu
Gln Ser Ile Gln Arg Leu Val Ser Gly Leu Glu Ala Lys Pro Asp 725 730
735 Arg Ser Arg Ser Ala Ser Pro 740 5 596 PRT Homo sapiens 5 Met
Ser Leu Ser Met Arg Asp Pro Val Ile Pro Gly Thr Ser Met Ala 1 5 10
15 Tyr His Pro Phe Leu Pro His Arg Ala Pro Asp Phe Ala Met Ser Ala
20 25 30 Val Leu Gly His Gln Pro Pro Phe Phe Pro Ala Leu Thr Leu
Pro Pro 35 40 45 Asn Gly Ala Ala Ala Leu Ser Leu Pro Gly Ala Leu
Ala Lys Pro Ile 50 55 60 Met Asp Gln Leu Val Gly Ala Ala Glu Thr
Gly Ile Pro Phe Ser Ser 65 70 75 80 Leu Gly Pro Gln Ala His Leu Arg
Pro Leu Lys Thr Met Glu Pro Glu 85 90 95 Glu Glu Val Glu Asp Asp
Pro Lys Val His Leu Glu Ala Lys Glu Leu 100 105 110 Trp Asp Gln Phe
His Lys Arg Gly Thr Glu Met Val Ile Thr Lys Ser 115 120 125 Gly Arg
Arg Met Phe Pro Pro Phe Lys Val Arg Cys Ser Gly Leu Asp 130 135 140
Lys Lys Ala Lys Tyr Ile Leu Leu Met Asp Ile Ile Ala Ala Asp Asp 145
150 155 160 Cys Arg Tyr Lys Phe His Asn Ser Arg Trp Met Val Ala Gly
Lys Ala 165 170 175 Asp Pro Glu Met Pro Lys Arg Met Tyr Ile His Pro
Asp Ser Pro Ala 180 185 190 Thr Gly Glu Gln Trp Met Ser Lys Val Val
Thr Phe His Lys Leu Lys 195 200 205 Leu Thr Asn Asn Ile Ser Asp Lys
His Gly Phe Thr Leu Ala Phe Pro 210 215 220 Ser Asp His Ala Thr Trp
Gln Gly Asn Tyr Ser Phe Gly Thr Gln Thr 225 230 235 240 Ile Leu Asn
Ser Met His Lys Tyr Gln Pro Arg Phe His Ile Val Arg 245 250 255 Ala
Asn Asp Ile Leu Lys Leu Pro Tyr Ser Thr Phe Arg Thr Tyr Leu 260 265
270 Phe Pro Glu Thr Glu Phe Ile Ala Val Thr Ala Tyr Gln Asn Asp Lys
275 280 285 Ile Thr Gln Leu Lys Ile Asp Asn Asn Pro Phe Ala Lys Gly
Phe Arg 290 295 300 Asp Thr Gly Asn Gly Arg Arg Glu Lys Arg Gln Gln
Leu Thr Leu Gln 305 310 315 320 Ser Met Arg Val Phe Asp Glu Arg His
Lys Lys Glu Asn Gly Thr Ser 325 330 335 Asp Glu Ser Ser Ser Glu Gln
Ala Ala Phe Asn Cys Phe Ala Gln Ala 340 345 350 Ser Ser Pro Ala Ala
Ser Thr Val Gly Thr Ser Asn Leu Lys Asp Leu 355 360 365 Cys Pro Ser
Glu Gly Glu Ser Asp Ala Glu Ala Glu Ser Lys Glu Glu 370 375 380 His
Gly Pro Glu Ala Cys Asp Ala Ala Lys Ile Ser Thr Thr Thr Ser 385 390
395 400 Glu Glu Pro Cys Arg Asp Lys Gly Ser Pro Ala Val Lys Ala His
Leu 405 410 415 Phe Ala Ala Glu Arg Pro Arg Asp Ser Gly Arg Leu Asp
Lys Ala Ser 420 425 430 Pro Asp Ser Arg His Ser Pro Ala Thr Ile Ser
Ser Ser Thr Arg Gly 435 440 445 Leu Gly Ala Glu Glu Arg Arg Ser Pro
Val Arg Glu Gly Thr Ala Pro 450 455 460 Ala Lys Val Glu Glu Ala Arg
Ala Leu Pro Gly Lys Glu Ala Phe Ala 465 470 475 480 Pro Leu Thr Val
Gln Thr Asp Ala Ala Arg Ser Ser Val His Arg His 485 490 495 Pro Phe
Arg Asn Leu Asn Thr Met Arg Pro Arg Leu Arg Tyr Ser Pro 500 505 510
Tyr Ser Ile Pro Val Pro Val Pro Asp Gly Ser Ser Leu Leu Thr Thr 515
520 525 Ala Leu Ala Ala Ser Pro Ala Ser Val Ala Val Asp Ser Gly Ser
Glu 530 535 540 Leu Asn Ser Arg Ser Ser Thr Leu Ser Ser Ser Ser Met
Ser Leu Ser 545 550 555 560 Pro Lys Leu Cys Ala Glu Lys Glu Ala Ala
Thr Ser Glu Leu Gln Ser 565 570 575 Ile Gln Arg Leu Val Ser Gly Leu
Glu Ala Lys Pro Asp Arg Ser Arg 580 585 590 Ser Ala Ser Pro 595
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