U.S. patent application number 11/800457 was filed with the patent office on 2007-09-13 for 32263, a novel human biotin enzyme and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Rosana Kapeller-Libermann, Rachel E. Meyers, Laura A. Rudolph-Owen.
Application Number | 20070212728 11/800457 |
Document ID | / |
Family ID | 27586316 |
Filed Date | 2007-09-13 |
United States Patent
Application |
20070212728 |
Kind Code |
A1 |
Meyers; Rachel E. ; et
al. |
September 13, 2007 |
32263, a novel human biotin enzyme and uses thereof
Abstract
The invention provides isolated nucleic acids molecules,
designated BRE nucleic acid molecules, which encode novel biotin
enzyme-related molecules. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
BRE nucleic acid molecules, host cells into which the expression
vectors have been introduced, and nonhuman transgenic animals in
which a BRE gene has been introduced or disrupted. The invention
still further provides isolated BRE proteins, fusion proteins,
antigenic peptides and anti-BRE antibodies. Diagnostic methods
utilizing compositions of the invention are also provided.
Inventors: |
Meyers; Rachel E.; (Newton,
MA) ; Rudolph-Owen; Laura A.; (Jamaica Plain, MA)
; Kapeller-Libermann; Rosana; (Chestnut Hill,
MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
27586316 |
Appl. No.: |
11/800457 |
Filed: |
May 4, 2007 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10160501 |
May 30, 2002 |
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11800457 |
May 4, 2007 |
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09838573 |
Apr 18, 2001 |
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10160501 |
May 30, 2002 |
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09870133 |
May 29, 2001 |
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10160501 |
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09870130 |
May 29, 2001 |
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10160501 |
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09862535 |
May 21, 2001 |
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10160501 |
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09870383 |
May 29, 2001 |
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10160501 |
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09860821 |
May 18, 2001 |
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10160501 |
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09870110 |
May 29, 2001 |
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10160501 |
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09907509 |
Jul 16, 2001 |
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10160501 |
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09945327 |
Aug 31, 2001 |
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10160501 |
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60197747 |
Apr 18, 2000 |
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60207649 |
May 26, 2000 |
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60207640 |
May 26, 2000 |
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60205961 |
May 19, 2000 |
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60207506 |
May 26, 2000 |
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60205449 |
May 19, 2000 |
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60207650 |
May 26, 2000 |
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60218385 |
Jul 14, 2000 |
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60229425 |
Aug 31, 2000 |
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60318581 |
Sep 10, 2001 |
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Current U.S.
Class: |
435/6.11 ;
435/320.1; 435/325; 435/69.1; 435/7.1; 530/350; 530/387.1;
536/23.2 |
Current CPC
Class: |
C12N 9/16 20130101; A61K
38/00 20130101; C12N 9/6421 20130101; C12Q 1/527 20130101; C12N
9/0022 20130101; C12N 9/00 20130101; C12Q 1/25 20130101; C12N 9/88
20130101; C12N 9/0006 20130101; C12N 15/52 20130101; C07K 2319/00
20130101; A01K 2217/075 20130101; A01K 2217/05 20130101; C12Q 1/008
20130101; C12N 9/20 20130101 |
Class at
Publication: |
435/006 ;
435/320.1; 435/325; 435/069.1; 435/007.1; 530/350; 530/387.1;
536/023.2 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07H 21/04 20060101 C07H021/04; C07K 14/00 20060101
C07K014/00; C07K 16/00 20060101 C07K016/00; G01N 33/53 20060101
G01N033/53; C12Q 1/68 20060101 C12Q001/68; C12N 15/00 20060101
C12N015/00; C12N 5/06 20060101 C12N005/06 |
Claims
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence which is at least 80% identical to the nucleotide sequence
of SEQ ID NO:1 or 3; b) a nucleic acid molecule comprising a
fragment of at least 50 nucleotides of a nucleic acid comprising
the nucleotide sequence of SEQ ID NO:1 or 3; c) a nucleic acid
molecule which which hybridizes to a nucleic acid molecule
consisting of SEQ ID NO:1 or 3 under stringent conditions; d) a
nucleic acid molecule which encodes a fragment of a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, wherein the
fragment comprises at least 15 contiguous amino acid residues of
the amino acid sequence of SEQ ID NO: 2; and e) the complement of
the nucleic acid molecule of any of a), b), c) or d).
2. An isolated nucleic acid molecule comprising the nucleic acid
molecule of claim 1 and a nucleotide sequence encoding a
heterologous polypeptide.
3. A vector comprising the nucleic acid molecule of claim 1.
4. A host cell which expresses the nucleic acid molecule of claim
1.
5. A method of producing a polypeptide comprising culturing the
host cell of claim 4 in an appropriate culture medium to, thereby,
produce the polypeptide.
6. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising a fragment of the amino acid sequence
of SEQ ID NO: 2, wherein the fragment comprises at least 15
contiguous amino acids of SEQ ID NO: 2; b) a polypeptide comprising
a variant of the amino acid sequence of SEQ ID NO:2, wherein the
variant is encoded by a nucleic acid molecule which hybridizes to a
nucleic acid molecule consisting of SEQ ID NO:1 or 3 under
stringent conditions; c) a polypeptide which is encoded by a
nucleic acid molecule comprising a nucleotide sequence which is at
least 80% identical to the nucleotide sequence of SEQ ID NO:1 or 3;
d) a polypeptide comprising an amino acid sequence which is at
least 80% identical to the amino acid sequence of SEQ ID NO: 2.
7. The polypeptide of claim 6, further comprising heterologous
amino acid sequences.
8. An antibody which selectively binds to the polypeptide of claim
6.
9. A method for detecting the presence of the polypeptide of claim
6 in a sample comprising: a) contacting the sample with a compound
which selectively binds to the polypeptide; and b) determining
whether the compound binds to the polypeptide in the sample to
thereby detect the presence of the polypeptide of claim 6 in the
sample.
10. The method of claim 9, wherein the compound which binds to the
polypeptide is an antibody.
11. A kit comprising a compound which selectively binds to the
polypeptide of claim 6 and instructions for use.
12. A method for detecting the presence of the nucleic acid
molecule of claim 1 in a sample comprising: a) contacting the
sample with a nucleic acid probe or primer which selectively
hybridizes to the nucleic acid molecule; and b) determining whether
the nucleic acid probe or primer binds to the nucleic acid molecule
in the sample to thereby detect the presence of a nucleic acid
molecule of claim 1 in the sample.
13. The method of claim 12, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
14. A kit comprising a compound which selectively hybridizes to the
nucleic acid molecule of claim 1 and instructions for use.
15. A method for identifying a compound which binds to the
polypeptide of claim 6 comprising: a) contacting the polypeptide,
or a cell expressing the polypeptide with a test compound; and b)
determining whether the polypeptide binds to the test compound.
16. The method of claim 15, wherein the binding of the test
compound to the polypeptide is detected by a method selected from
the group consisting of: a) detection of binding by direct
detection of test compound/polypeptide binding; b) detection of
binding using a competition binding assay; and c) detection of
binding using an assay for BRE activity.
17. A method for modulating the activity of the polypeptide of
claim 6 comprising contacting the polypeptide or a cell expressing
the polypeptide with a compound which binds to the polypeptide in a
sufficient concentration to modulate the activity of the
polypeptide.
18. A method for identifying a compound which modulates the
activity of the polypeptide of claim 6 comprising: a) contacting
the polypeptide of claim 6 with a test compound; and b) determining
the effect of the test compound on the activity of the polypeptide
to thereby identify a compound which modulates the activity of the
polypeptide.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/160,501, filed May 30, 2002 (allowed), which: [0002] is
a continuation-in-part of U.S. patent application Ser. No.
09/838,573, filed Apr. 18, 2001 (abandoned), which claims the
benefit of U.S. Provisional Application Ser. No. 60/197,747, filed
Apr. 18, 2000; [0003] and a continuation-in-part of U.S. patent
application Ser. No. 09/870,133, filed May 29, 2001 (abandoned),
which claims the benefit of U.S. Provisional Application Ser. No.
60/207,649, filed May 26, 2000; [0004] and a continuation-in-part
of U.S. patent application Ser. No. 09/870,130, filed May 29, 2001
(abandoned), which claims the benefit of U.S. Provisional
Application Ser. No. 60/207,640, filed May 26, 2000; [0005] and a
continuation-in-part of U.S. patent application Ser. No.
09/862,535, filed May 21, 2001 (abandoned), which claims the
benefit of U.S. Provisional Application Ser. No. 60/205,961, filed
May 19, 2000; [0006] and a continuation-in-part of U.S. patent
application Ser. No. 09/870,383, filed May 29, 2001 (abandoned),
which claims the benefit of U.S. Provisional Application Ser. No.
60/207,506, filed May 26, 2000; [0007] and a continuation-in-part
of U.S. patent application Ser. No. 09/860,821, filed May 18, 2001
(abandoned), which claims the benefit of U.S. Provisional
Application Ser. No. No. 60/205,449, filed May 19, 2000; [0008] and
a continuation-in-part of U.S. patent application Ser. No.
09/870,110, filed May 29, 2001 (abandoned), which claims the
benefit of U.S. Provisional Application Ser. No. 60/207,650, filed
May 26, 2000; [0009] and a continuation-in-part of U.S. patent
application Ser. No. 09/907,509, filed Jul. 16, 2001 (abandoned),
which claims the benefit of U.S. Provisional Application Ser. No.
60/218,385, filed Jul. 14, 2000; [0010] and a continuation-in-part
of U.S. patent application Ser. No. 09/945,327, filed Aug. 31, 2001
(abandoned), which claims the benefit of U.S. Provisional
Application Ser. No. 60/229,425, filed Aug. 31, 2000; [0011] and
also claims the benefit of U.S. Provisional Application Ser. No.
60/318,581, filed Sep. 10, 2001.
[0012] The entire contents of each of the above-referenced patent
applications are incorporated herein by this reference.
BACKGROUND OF THE INVENTION
[0013] Biotin is an essential water-soluble vitamin of the
B-complex group which is synthesized by plants, most prokaryotes
and virtually all eukaryotes. Also known as vitamin H, biotin is
well characterized in its role as a coenzyme or prosthetic group of
a number of enzymes. The biotin group can serve as a carrier of
activated CO.sub.2 and is often covalently attached to enzymes at a
biotin-attachment domain through the .epsilon.-amino group of a
lysine residue. The addition of a carboxyl group to an acceptor
molecule (carboxylase reaction), a reaction which is catalyzed by
such biotin enzymes, generally occurs in two steps: ##STR1## Biotin
enzymes are also involved in the reverse (decarboxylase)
reaction.
[0014] The manipulation of biomolecules by addition and removal of
carboxyl bonds is of critical importance in most metabolic (e.g.,
catabolic and anabolic) pathways in cells. A large family of
enzymes which catalyze such reactions has been described, generally
called biotin carboxylases and biotin decarboxylases in humans
(see, e.g., Knowles (1989) Ann. Rev. Biochem. 58:195-221; Samols et
al (1988) J. Biol. Chem. 263:6461-6464). The biotin carboxylases
are key enzymes in such pathways as gluconeogenesis, lipogenesis,
amino acid metabolism, the urea cycle, and energy transduction. In
addition, other biotin enzymes have been identified which are not
carboxylases, for example the Biotin Protein Ligases (BPL), which
are responsible for specific covalent attachment of biotin to its
cognate proteins (Chapman-Smith and Cronan (1999) J. of Nutrition
129:477S-484S).
[0015] Biotin enzymes play important roles in the synthesis and
breakdown of a great number of metabolic intermediates, which may
implicate them in a number of pathologies. Several inherited and
acquired disorders involving errant biotin metabolism have been
described (Baumgartner and Suormala (1997) Int. J. Vitam Nutr Res
67:377-384; Baumgartner and Suormala (1999) Biofactors 10:287-290).
These disorders can manifest themselves in a number of symptoms
including severe nutritional difficulties, organic aciduria,
neurologic abnormalities, and cutaneous distress (rash, alopecia,
etc). Accordingly, proteins which are involved with biotin-related
metabolism may hold significant therapeutic value.
[0016] Given the importance of biotin enzymes in a wide range of
cellular processes, there exists a need to identify novel biotin
enzymes as well as modulators of such enzymes for use in a variety
of processes.
SUMMARY OF THE INVENTION
[0017] The present invention is based, at least in part, on the
discovery of novel members of the family of biotin proteins,
referred to herein as Biotin Enzyme-1 (or BRE) nucleic acid and
protein molecules. The BRE nucleic acid and protein molecules of
the present invention are useful as modulating agents in regulating
a variety of cellular processes, e.g., cellular proliferation,
growth, differentiation, protein synthesis, or energy transduction.
Accordingly, in one aspect, this invention provides isolated
nucleic acid molecules encoding BRE proteins or biologically active
portions thereof, as well as nucleic acid fragments suitable as
primers or hybridization probes for the detection of BRE-encoding
nucleic acids.
[0018] In one embodiment, a BRE nucleic acid molecule of the
invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or more identical to the nucleotide
sequence (e.g., to the entire length of the nucleotide sequence)
shown in SEQ ID NO:1 or 3, or a complement thereof.
[0019] In a preferred embodiment, the isolated nucleic acid
molecule includes the nucleotide sequence shown in SEQ ID NO:1 or
3, or a complement thereof. In another embodiment, the nucleic acid
molecule includes SEQ ID NO:3 and nucleotides 1-166 of SEQ ID NO:1.
In yet a further embodiment, the nucleic acid molecule includes SEQ
ID NO:3 and nucleotides 2179-2577 of SEQ ID NO:1. In another
preferred embodiment, the nucleic acid molecule consists of the
nucleotide sequence shown in SEQ ID NO:1 or 3.
[0020] In another embodiment, a BRE nucleic acid molecule includes
a nucleotide sequence encoding a protein having an amino acid
sequence sufficiently identical to the amino acid sequence of SEQ
ID NO:2. In a preferred embodiment, a BRE nucleic acid molecule
includes a nucleotide sequence encoding a protein having an amino
acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or more identical to the entire length of
the amino acid sequence of SEQ ID NO:2.
[0021] In another preferred embodiment, an isolated nucleic acid
molecule encodes the amino acid sequence of human BRE. In yet
another preferred embodiment, the nucleic acid molecule includes a
nucleotide sequence encoding a protein having the amino acid
sequence of SEQ ID NO. In yet another preferred embodiment, the
nucleic acid molecule is at least 50-100, 100-250, 250-500,
500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000,
2000-2250, 2250-2500 or more nucleotides in length. In a further
preferred embodiment, the nucleic acid molecule is at least 50-100,
100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500,
1500-1750, 1750-2000, 2000-2250, 2250-2500, or more nucleotides in
length and encodes a protein having a BRE activity (as described
herein).
[0022] Another embodiment of the invention features nucleic acid
molecules, preferably BRE nucleic acid molecules, which
specifically detect BRE nucleic acid molecules relative to nucleic
acid molecules encoding non-BRE proteins. For example, in one
embodiment, such a nucleic acid molecule is at least 50-100,
100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500,
1500-1750, 1750-2000, 2000-2250, 2250-2500 or more nucleotides in
length and hybridizes under stringent conditions to a complement of
a nucleic acid molecule comprising the nucleotide sequence shown in
SEQ ID NO:1.
[0023] In preferred embodiments, the nucleic acid molecules are at
least 15 (e.g., 15 contiguous) nucleotides in length and hybridize
under stringent conditions to a complement of the nucleotide
molecules set forth in SEQ ID NO:1.
[0024] In other preferred embodiments, the nucleic acid molecule
encodes a naturally occurring allelic variant of a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, wherein the
nucleic acid molecule hybridizes to a complement of a nucleic acid
molecule comprising SEQ ID NO:1 or 3, respectively, under stringent
conditions.
[0025] Another embodiment of the invention provides an isolated
nucleic acid molecule which is antisense to a BRE nucleic acid
molecule, e.g., the coding strand of a BRE nucleic acid
molecule.
[0026] Another aspect of the invention provides a vector comprising
a BRE nucleic acid molecule. In certain embodiments, the vector is
a recombinant expression vector. In another embodiment, the
invention provides a host cell containing a vector of the
invention. In yet another embodiment, the invention provides a host
cell containing a nucleic acid molecule of the invention. The
invention also provides a method for producing a protein,
preferably a BRE protein, by culturing in a suitable medium, a host
cell, e.g., a mammalian host cell such as a non-human mammalian
cell, of the invention containing a recombinant expression vector,
such that the protein is produced.
[0027] Another aspect of this invention features isolated or
recombinant BRE proteins and polypeptides. In one embodiment, an
isolated BRE protein includes at least one or more of the following
domains: a carbamoyl-phosphate synthase L chain, ATP binding domain
(or CPSase domain), and/or a biotin-requiring enzyme domain.
[0028] In a preferred embodiment, a BRE protein includes at least
one or more of the following domains: a CPSase domain, a
biotin-requiring enzyme domain, and has an amino acid sequence at
least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence of SEQ ID NO:2. In another preferred embodiment, a BRE
protein includes at least one or more of the following domains: a
CPSase domain, a biotin-requiring enzyme domain and has a BRE
activity (as described herein).
[0029] In yet another preferred embodiment, a BRE protein includes
at least one or more of the following domains: a CPSase domain, a
biotin-requiring enzyme domain, and is encoded by a nucleic acid
molecule having a nucleotide sequence which hybridizes under
stringent hybridization conditions to a complement of a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or
3.
[0030] In another embodiment, the invention features fragments of
the protein having the amino acid sequence of SEQ ID NO:2, wherein
the fragment comprises at least 15 amino acids (e.g., contiguous
amino acids) of the amino acid sequence of SEQ ID NO:2. In another
embodiment, a BRE protein has the amino acid sequence of SEQ ID
NO:2.
[0031] In another embodiment, the invention features a BRE protein
which is encoded by a nucleic acid molecule consisting of a
nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a
nucleotide sequence of SEQ ID NO: 1 or 3, or a complement thereof.
This invention further features a BRE protein which is encoded by a
nucleic acid molecule consisting of a nucleotide sequence which
hybridizes under stringent hybridization conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or
3, or a complement thereof.
[0032] The proteins of the present invention or portions thereof,
e.g., biologically active portions thereof, can be operatively
linked to a non-BRE polypeptide (e.g., heterologous amino acid
sequences) to form fusion proteins. The invention further features
antibodies, such as monoclonal or polyclonal antibodies, that
specifically bind proteins of the invention, preferably BRE
proteins. In addition, the BRE proteins or biologically active
portions thereof can be incorporated into pharmaceutical
compositions, which optionally include pharmaceutically acceptable
carriers.
[0033] In another aspect, the present invention provides a method
for detecting the presence of a BRE nucleic acid molecule, protein,
or polypeptide in a biological sample by contacting the biological
sample with an agent capable of detecting a BRE nucleic acid
molecule, protein, or polypeptide such that the presence of a BRE
nucleic acid molecule, protein or polypeptide is detected in the
biological sample.
[0034] In another aspect, the present invention provides a method
for detecting the presence of BRE activity in a biological sample
by contacting the biological sample with an agent capable of
detecting an indicator of BRE activity such that the presence of
BRE activity is detected in the biological sample.
[0035] In another aspect, the invention provides a method for
modulating BRE activity comprising contacting a cell capable of
expressing BRE with an agent that modulates BRE activity such that
BRE activity in the cell is modulated. In one embodiment, the agent
inhibits BRE activity. In another embodiment, the agent stimulates
BRE activity. In one embodiment, the agent is an antibody that
specifically binds to a BRE protein. In another embodiment, the
agent modulates expression of BRE by modulating transcription of a
BRE gene or translation of a BRE mRNA. In yet another embodiment,
the agent is a nucleic acid molecule having a nucleotide sequence
that is antisense to the coding strand of a BRE mRNA or a BRE
gene.
[0036] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
or unwanted BRE protein or nucleic acid expression or activity by
administering an agent which is a BRE modulator to the subject. In
one embodiment, the BRE modulator is a BRE protein. In another
embodiment the BRE modulator is a BRE nucleic acid molecule. In yet
another embodiment, the BRE modulator is a peptide, peptidomimetic,
or other small molecule. In a preferred embodiment, the disorder
characterized by aberrant or unwanted BRE protein or nucleic acid
expression is a BRE-associated disorder (e.g., a carboxylase
associated disorder, a decarboxylase-associated disorder).
[0037] The present invention also provides diagnostic assays for
identifying the presence or absence of a genetic alteration
characterized by at least one of (i) aberrant modification or
mutation of a gene encoding a BRE protein; (ii) mis-regulation of
the gene; and (iii) aberrant post-translational modification of a
BRE protein, wherein a wild-type form of the gene encodes a protein
with a BRE activity.
[0038] In another aspect the invention provides methods for
identifying a compound that binds to or modulates the activity of a
BRE protein, by providing an indicator composition comprising a BRE
protein having BRE activity, contacting the indicator composition
with a test compound, and determining the effect of the test
compound on BRE activity in the indicator composition to identify a
compound that modulates the activity of a BRE protein.
[0039] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1A-1E depict the cDNA sequence and predicted amino
acid sequence of human BRE (clone Fbh32263). The nucleotide
sequence corresponds to nucleic acids 1 to 2577 of SEQ ID NO:1. The
amino acid sequence corresponds to amino acids 1 to 725 of SEQ ID
NO: 2. The coding region without the 3' untranslated region of the
human BRE gene is shown in SEQ ID NO: 3.
[0041] FIGS. 2A-2B depict a structural, hydrophobicity, and
antigenicity analysis of the human Fbh32263 protein.
[0042] FIGS. 3A-3F depict the results of a search which was
performed against the HMM database and which resulted in the
identification of a "CPSase domain" and a "biotin requiring enzyme
domain".
[0043] FIGS. 4A-4D depict an alignment of human BRE (SEQ ID NO:2,
depicted as "32263.pro") with known transcarboxylases. These are
3-methylcrotonyl-CoA carboxylase precursor from Arabidopsis (SEQ ID
NO:4, GenBank No. AAA67356; depicted as "thal.pro"); a protein
similar to propionyl-CoA carboxylase alpha chain from C. elegans
(SEQ ID NO:5, GenBank No. AAA93384; depicted as "celegans.pro");
and proprionyl-CoA carboxylase alpha chain precursor from H.
sapiens (SEQ ID NO:6, GenBank No. P05165; depicted as "human.pro").
The CPSase domain of the human BRE is indicated in italics. The
biotin-requiring enzyme domain of the human BRE is underlined. The
alignment was performed using the Clustal algorithm which is part
of the MEGALIGN program (e.g., version 3.1.7) which is part of the
DNASTAR sequence analysis software package. The pairwise alignment
parameters are as follows: K-tuple=1; Gap Penalty=3; Window=5;
Diagonals saved=5. The multiple alignment parameters are as
follows: Gap Penalty=10; and Gap length penalty=10.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as "Biotin
Requiring Enzyme" or "BRE" nucleic acid and protein molecules,
which are novel members of a family of enzymes which possess the
ability to associate with biotin molecules (e.g. to associate
covalently with a biotin coenzyme, to associate non-covalently with
a biotin cofactor) in order to function in their biological
capacity (e.g., to convert BRE substrates and metabolites into
their corresponding BRE-mediated products). These novel molecules
are capable of participating in metabolic pathways (e.g., as a
carboxylase, as a decarboxylase, as a transcarboxylase) and, thus,
play a role in or function in a variety of cellular processes,
e.g., gluconeogenesis, lipogenesis, amino acid metabolism, nucleic
acid metabolism, the urea cycle, and energy transduction.
[0045] As used herein, the term "biotin requiring enzyme", also
called "biotin enzyme", (referred to herein interchangeably as
"BRE") includes a protein, peptide, or enzyme which is able to
interact with one or more molecules of biotin in order to carry out
its function(s), e.g., specific reactions in catabolic or anabolic
pathways. BRE molecules are involved in the anabolism and
catabolism of metabolically important biomolecules, including the
metabolism of biochemical molecules necessary for energy production
or storage (e.g., carbohydrate metabolism, lipid metabolism),
important cellular metabolites (e.g. amino acids, nucleic acids,
urea cycle intermediates), as well as the detoxification (e.g.,
catabolism) of potentially harmful compounds (e.g., toxins,
carcinogens). Examples of BRE molecules include prokaryotic, plant,
and mammalian carboxylases, decarboxylases, transcarboxylases, and
biotin protein ligases. As biotin enzymes, the BRE molecules of the
present invention provide novel diagnostic targets and therapeutic
agents to control BRE-associated disorders.
[0046] Preferably such BRE proteins comprise a family of BRE
molecules. The term "family" when referring to the protein and
nucleic acid molecules of the invention is intended to mean two or
more proteins or nucleic acid molecules having a common structural
domain or motif and having sufficient amino acid or nucleotide
sequence homology as defined herein. Such family members can be
naturally or non-naturally occurring and can be from either the
same or different species. For example, a family can contain a
first protein of human origin, as well as other, distinct proteins
of human origin or alternatively, can contain homologues of
non-human origin, e.g., monkey proteins. Members of a family may
also have common functional characteristics.
[0047] In another embodiment, a BRE molecule of the present
invention is identified based on the presence of at least one
"CPSase" domain in the protein or corresponding nucleic acid
molecule. As used herein, the term "CPSase" or "CPSase domain"
includes a protein domain having an amino acid sequence of about
64-669 amino acid residues and a bit score of at least 286 when
compared against a CPSase domain Markov Model (HMM), e.g., PFAM
accession number PF00289. In a preferred embodiment, a CPSase
domain includes a protein domain having an amino acid sequence of
about 169-569 amino acid residues and a bit score of at least 386.
Preferably, a CPSase domain includes a protein domain having an
amino acid sequence of about 269-469 amino acid residues and a bit
score of at least about 486 (e.g., 500, 525, 550, 575, 586.6, 600
or more). A CPSase domain preferably includes a sufficient number
of amino acid residues for the enzymatic function of the
polypeptide sequence
[0048] Alternatively, in another embodiment, a BRE molecule of the
present invention is identified based on the presence of at least
one carbamoyl phosphate synthase L chain, N-terminal ("CPSase
N-terminal") domain in the protein or corresponding nucleic acid
molecule. As used herein, the term "CPSase N-terminal domain"
includes a protein domain having an amino acid sequence of about
100-125 amino acid residues and a bit score of at least 144 when
compared against a CPSase domain Markov Model (HMM), e.g., PFAM
accession number PF00289. In a preferred embodiment, a CPSase
N-terminal domain includes a protein domain having an amino acid
sequence of about 105-120 amino acid residues and a bit score of at
least 164. Preferably, a CPSase domain includes a protein domain
having an amino acid sequence of about 100-125 amino acid residues
(e.g., 113) and a bit score of at least about 184.
[0049] In another embodiment, a BRE molecule of the present
invention is identified based on the presence of at least one
carbamoyl phosphate synthase ATP-binding ("CPSase ATP-binding")
domain in the protein or corresponding nucleic acid molecule. As
used herein, the term "CPSase ATP_binding domain" includes a
protein domain having an amino acid sequence of about 190-240 amino
acid residues and a bit score of at least 190 when compared against
a CPSase domain Markov Model (HMM), e.g., PFAM accession number
PF02786. In a preferred embodiment, a CPSase domain includes a
protein domain having an amino acid sequence of about 200-230 amino
acid residues and a bit score of at least 330. Preferably, a CPSase
domain includes a protein domain having an amino acid sequence of
about 210-220 amino acid residues (e.g.,214 amino acid residues)
and a bit score of at least about 550 (e.g., 353 or more).
Preferably, a "carbamoyl-phosphate synthase L chain, ATP binding
domain" ("CPSase ATP-binding domain") contains a "carbamoyl
phosphate synthase subdomain signature". This domain is implicated
in ATP binding and/or catalytic activity.
[0050] A CPSase domain can include, for example, amino acid
residues essential for the enzymatic function of the BRE proteins
of the present invention. CPSase domains have been found, for
example, in the carbamoylase CPSase (e.g., in duplicate) as well as
in a variety of biotin-dependent enzymes (e.g., in single copy) for
example acetyl-CoA carboxylase, propionyl-CoA carboxylase, pyruvate
carboxylase and urea carboxylase. To identify the presence of a
CPSase domain in a BRE protein, the amino acid sequence of the
protein is used to search a database of known Hidden Markov Models
(HMMs e.g., the PFAM HMM database). The CPSase HMM has been
assigned the PFAM Accession PF00289 (http://pfam.wustl.edu),
InterPro accession number IPR000901
(http://www.ebi.ac.uk/interpro), and Prosite accession numbers
PS00866 and PS00867 (http://www.expasy.ch/prosite). For example, a
search was performed against the HMM database using the amino acid
sequence (SEQ ID NO:2) of human BRE resulting in the identification
of a CPSase domain in the amino acid sequence of human BRE (SEQ ID
NO: 2) at about residues 51-419 having a score of 586.6. The
results of the search are set forth in FIGS. 3A-3F.
[0051] In another embodiment, a BRE molecule of the present
invention is identified based on the presence of at least one
"biotin-requiring enzyme domain" in the protein or corresponding
nucleic acid molecule. As used herein, the term "biotin-requiring
enzyme domain" includes a protein domain having an amino acid
sequence of about 35-95 amino acid residues and a bit score of at
least 38 when compared against a biotin-requiring enzyme domain
Markov Model (HMM), e.g., PFAM accession number PF00364. In a
preferred embodiment, a biotin-requiring enzyme domain includes a
protein domain having an amino acid sequence of about 45-85 amino
acid residues and a bit score of at least 48. In another preferred
embodiment, a biotin-requiring enzyme domain includes a protein
domain having an amino acid sequence of about 55-75 amino acid
residues and a bit score of at least 58. Preferably, a
biotin-requiring enzyme domain includes a protein domain having an
amino acid sequence of about 60-70 amino acid residues and a bit
score of at least about 65 (e.g., 66, 67.8, 69, 70, 75, 100 or
more). Preferably, the biotin requiring enzyme domain binds biotin
and contains, or can be characterized by, the presence of a "biotin
requiring enzyme attachment site", which itself is characterized by
the inclusion of a conserved lysine residue. To identify the
presence of a biotin-requiring enzyme domain in a BRE protein, the
amino acid sequence of the protein is used to search a database of
known Hidden Markov Models (HMMs e.g., the PFAM HMM database). The
biotin-requiring enzyme domain HMM has been assigned the PFAM
Accession PF00364 (http://pfam.wustl.edu), InterPro accession
number IPR000089 (http://www.ebi.ac.uk/interpro), and Prosite
accession number PS00188 (http://www.expasy.ch/prosite). For
example, a search was performed against the HMM database using the
amino acid sequence (SEQ ID NO:2) of human BRE resulting in the
identification of a biotin-requiring enzyme domain in the amino
acid sequence of human BRE (SEQ ID NO: 2) at about residues 650-714
having a score of 67.8. The results of the search are set forth in
FIG. 3A.
[0052] In a preferred embodiment, a biotin-requiring enzyme domain
as described herein is characterized by the presence of a
"biotin-requiring enzyme attachment site." As used herein, the term
"biotin-requiring enzyme attachment site" includes a motif having
the consensus sequence
[GN]-[DEQTR]-X-[LIVMFY]-X(2)-[LIVM]-X-[AIV]-M-K-[LMAT]-X
(3)-[LIVM]-X-[SAV] and is described under Prosite entry number
PS00188 (http://www.expasy.ch/prosite). A biotin-requiring enzyme
attachment site can be found, for example, within the
biotin-requiring enzyme domain of the BRE protein of SEQ ID NO:2 at
about residues 671-688. The consensus sequences described herein
are described according to standard Prosite Signature designation
(e.g., all amino acids are indicated according to their universal
single letter designation; X designates any amino acid; X(n)
designates any n amino acids, e.g., X (2) designates any 2 amino
acids; [LIVM] indicates any one of the amino acids appearing within
the brackets, e.g., any one of L, I, V, or M, in the alternative,
any one of Leu, Ile, Val, or Met.); and {LIVM} indicates any amino
acid except the amino acids appearing within the brackets, e.g.,
not L, not I, not V, and not M.
[0053] Isolated proteins of the present invention, for example BRE
proteins, preferably have an amino acid sequence sufficiently
identical to the amino acid sequence of SEQ ID NO:2, or are encoded
by a nucleotide sequence sufficiently identical to SEQ ID NO:1 or
3. As used herein, the term "sufficiently identical" refers to a
first amino acid or nucleotide sequence which contains a sufficient
or minimum number of identical or equivalent (e.g., an amino acid
residue which has a similar side chain) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences share
common structural domains or motifs and/or a common functional
activity. For example, amino acid or nucleotide sequences which
share common structural domains have at least 30%, 40%, or 50%
homology, preferably 60% homology, more preferably 70%-80%, and
even more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more homology across the amino acid sequences of the domains
and contain at least one and preferably two structural domains or
motifs, are defined herein as sufficiently identical. Furthermore,
amino acid or nucleotide sequences which share at least 30%, 40%,
or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology
(and ranges intermediate therein) and share a common functional
activity are defined herein as sufficiently identical.
[0054] As used interchangeably herein, a "BRE activity",
"biological activity of BRE" or "functional activity of BRE",
refers to an activity exhibited by a BRE protein, polypeptide or
nucleic acid molecule (e.g., in a BRE expressing cell or tissue),
on a BRE substrate, as determined in vivo, or in vitro, according
to standard techniques. In one embodiment, a BRE activity is a
direct activity, such as processing of a BRE-substrate (e.g.,
carboxylation, decarboxylation). As used herein, a "BRE substrate"
is a molecule or a metabolite which is processed by a BRE molecule.
Exemplary substrates include, but are not limited to, energy
metabolites, lipid metabolism intermediates, activated CO.sub.2,
carbonyl groups, urea cycle intermediates, amino acid precursors,
and nucleic acid precursors. Examples of BRE substrates also
include molecules that are essential for BRE function, e.g.,
biotin, ATP, acetyl CoA. Alternatively, a BRE activity is an
indirect activity, such as a cellular signaling or feedback
activity mediated by the processing of a BRE substrate by BRE. In a
preferred embodiment, the BRE proteins of the present invention
have one or more of the following activities: 1) modulate the
bioenergetic activities of a cell (e.g., storage or yielding of
chemical energy, 2) modulate intra- or intercellular signaling or
feedback mechanisms, 3) removal of potentially harmful compounds
(e.g., cytotoxic substances) from the cell, or facilitate the
neutralization of these molecules through enzymatic alteration
(e.g., carboxylation, decarboxylation), 4) modulate the production
or breakdown of amino acids or nucleic acids, or modulate the
homeostatic balance of available amino acid or nucleic acid pools,
5) specific attachment of biotin to its cognate enzyme, e.g.,
biotin protein ligase activity.
[0055] Accordingly, another embodiment of the invention features
isolated BRE proteins and polypeptides having a BRE activity. Other
preferred proteins are BRE proteins having one or more of the
following domains: a CPSase domain, a biotin-requiring enzyme
domain and, preferably, a BRE activity.
[0056] Additional preferred proteins have at least one CPSase
domain, one biotin-requiring enzyme domain, and are, preferably,
encoded by a nucleic acid molecule having a nucleotide sequence
which hybridizes under stringent hybridization conditions to a
complement of a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:1 or 3.
[0057] The nucleotide sequence of the isolated human BRE cDNA and
the predicted amino acid sequence of the human BRE polypeptide are
shown in FIGS. 1A-1E and in SEQ ID NOs:1 and 2, respectively.
[0058] The human BRE gene, which is approximately 2577 nucleotides
in length, encodes a protein having a molecular weight of
approximately 79.8 kD and which is approximately 725 amino acid
residues in length.
[0059] Various aspects of the invention are described in further
detail in the following subsections:
I. Isolated Nucleic Acid Molecules
[0060] One aspect of the invention pertains to isolated nucleic
acid molecules that encode BRE proteins or biologically active
portions thereof, as well as nucleic acid fragments sufficient for
use as hybridization probes to identify BRE-encoding nucleic acid
molecules (e.g., BRE mRNA) and fragments for use as PCR primers for
the amplification or mutation of BRE nucleic acid molecules. As
used herein, the term "nucleic acid molecule" is intended to
include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules
(e.g., mRNA) and analogs of the DNA or RNA generated using
nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0061] The term "isolated nucleic acid molecule" includes nucleic
acid molecules which are separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. For example, with regards to genomic DNA, the term "isolated"
includes nucleic acid molecules which are separated from the
chromosome with which the genomic DNA is naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genornic DNA of the
organism from which the nucleic acid is derived. For example, in
various embodiments, the isolated BRE nucleic acid molecule can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1
kb of nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover, an "isolated" nucleic acid molecule, such as a
cDNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized.
[0062] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1
or 3, or a portion thereof, can be isolated using standard
molecular biology techniques and the sequence information provided
herein. Using all or portion of the nucleic acid sequence of SEQ ID
NO:1 or 3 as a hybridization probe, BRE nucleic acid molecules can
be isolated using standard hybridization and cloning techniques
(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis,
T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989).
[0063] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO:1 or 3 can be isolated by the polymerase chain
reaction (PCR) using synthetic oligonucleotide primers designed
based upon the sequence of SEQ ID NO:1 or 3.
[0064] A nucleic acid of the invention can be amplified using cDNA,
mRNA or, alternatively, genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to BRE nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0065] In a preferred embodiment, an isolated nucleic acid molecule
of the invention comprises the nucleotide sequence shown in SEQ ID
NO:1 or 3. This cDNA may comprise sequences encoding the human BRE
protein (i.e., "the coding region", from nucleotides 165-6599), as
well as 5' untranslated sequences (nucleotides 1-166) and 3'
untranslated sequences (nucleotides 2179-2577) of SEQ ID NO:1.
Alternatively, the nucleic acid molecule can comprise only the
coding region of SEQ ID NO:1 (e.g., nucleotides 167-2178,
corresponding to SEQ ID NO:3).
[0066] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
is a complement of the nucleotide sequence shown in SEQ ID NO:1 or
3, or a portion of any of these nucleotide sequences. A nucleic
acid molecule which is complementary to the nucleotide sequence
shown in SEQ ID NO:1 or 3, is one which is sufficiently
complementary to the nucleotide sequence shown in SEQ ID NO:1 or 3,
such that it can hybridize to the nucleotide sequence shown in SEQ
ID NO:1 or 3, respectively, thereby forming a stable duplex.
[0067] In still another preferred embodiment, an isolated nucleic
acid molecule of the present invention comprises a nucleotide
sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire
length of the nucleotide sequence shown in SEQ ID NO:1 or 3, or a
portion of any of these nucleotide sequences.
[0068] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the nucleic acid sequence of SEQ ID NO:1
or 3, for example, a fragment which can be used as a probe or
primer or a fragment encoding a portion of a BRE protein, e.g., a
biologically active portion of a BRE protein. The nucleotide
sequence determined from the cloning of the BRE gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning other BRE family members, as well as BRE homologues
from other species. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12 or 15, preferably
about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60,
65, or 75 consecutive nucleotides of a sense sequence of SEQ ID
NO:1 or 3, of an anti-sense sequence of SEQ ID NO:1 or 3, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:1 or 3.
In one embodiment, a nucleic acid molecule of the present invention
comprises a nucleotide sequence which is greater than 50-100,
100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500,
1500-1750, 1750-2000, 2000-2250, 2250-2500, or more nucleotides in
length and hybridizes under stringent hybridization conditions to a
nucleic acid molecule of SEQ ID NO:1 or 3.
[0069] Probes based on the BRE nucleotide sequences can be used to
detect transcripts or genomic sequences encoding the same or
homologous proteins. In preferred embodiments, the probe further
comprises a label group attached thereto, e.g., the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a BRE protein,
such as by measuring a level of a BRE-encoding nucleic acid in a
sample of cells from a subject e.g., detecting BRE mRNA levels or
determining whether a genomic BRE gene has been mutated or
deleted.
[0070] A nucleic acid fragment encoding a "biologically active
portion of a BRE protein" can be prepared by isolating a portion of
the nucleotide sequence of SEQ ID NO:1 or 3, which encodes a
polypeptide having a BRE biological activity (the biological
activities of the BRE proteins are described herein), expressing
the encoded portion of the BRE protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the BRE protein.
[0071] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1 or 3,
due to degeneracy of the genetic code and thus encode the same BRE
proteins as those encoded by the nucleotide sequence shown in SEQ
ID NO:1 or 3. In another embodiment, an isolated nucleic acid
molecule of the invention has a nucleotide sequence encoding a
protein having an amino acid sequence shown in SEQ ID NO:2.
[0072] In addition to the BRE nucleotide sequences shown in SEQ ID
NO:1 or 3, it will be appreciated by those skilled in the art that
DNA sequence polymorphisms that lead to changes in the amino acid
sequences of the BRE proteins may exist within a population (e.g.,
the human population). Such genetic polymorphism in the BRE genes
may exist among individuals within a population due to natural
allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules which include an
open reading frame encoding a BRE protein, preferably a mammalian
BRE protein, and can further include non-coding regulatory
sequences, and introns.
[0073] Allelic variants of human BRE include both functional and
non-functional BRE proteins. Functional allelic variants are
naturally occurring amino acid sequence variants of the human BRE
protein that maintain the ability to process a BRE substrate (e.g.,
carboxylation, decarboxylation). Functional allelic variants will
typically contain only conservative substitution of one or more
amino acids of SEQ ID NO:2, or substitution, deletion or insertion
of non-critical residues in non-critical regions of the
protein.
[0074] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the human BRE protein that do not
have the ability to bind or process a BRE substrate substrate
(e.g., carboxylation, decarboxylation), and/or carry out any of the
BRE activities described herein. Non-functional allelic variants
will typically contain a non-conservative substitution, a deletion,
or insertion or premature truncation of the amino acid sequence of
SEQ ID NO:2, or a substitution, insertion or deletion in critical
residues or critical regions of the protein.
[0075] The present invention further provides non-human orthologues
of the human BRE protein. Orthologues of the human BRE protein are
proteins that are isolated from non-human organisms and possess the
same BRE substrate binding and/or modulation of membrane
excitability activities of the human BRE protein. Orthologues of
the human BRE protein can readily be identified as comprising an
amino acid sequence that is substantially identical to SEQ ID
NO:2.
[0076] Moreover, nucleic acid molecules encoding other BRE family
members and, thus, which have a nucleotide sequence which differs
from the BRE sequences of SEQ ID NO:1 or 3 are intended to be
within the scope of the invention. For example, another BRE cDNA
can be identified based on the nucleotide sequence of human BRE.
Moreover, nucleic acid molecules encoding BRE proteins from
different species, and which, thus, have a nucleotide sequence
which differs from the BRE sequences of SEQ ID NO:1 or 3 are
intended to be within the scope of the invention. For example, a
mouse BRE cDNA can be identified based on the nucleotide sequence
of a human BRE.
[0077] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the BRE cDNAs of the invention can be
isolated based on their homology to the BRE nucleic acids disclosed
herein using the cDNAs disclosed herein, or a portion thereof, as a
hybridization probe according to standard hybridization techniques
under stringent hybridization conditions. Nucleic acid molecules
corresponding to natural allelic variants and homologues of the BRE
cDNAs of the invention can further be isolated by mapping to the
same chromosome or locus as the BRE gene.
[0078] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 15, 20, 25, 30 or more
nucleotides in length and hybridizes under stringent conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO:1 or 3. In other embodiment, the nucleic acid is at least
50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500,
1500-1750, 1750-2000, 2000-2250, 2250-2500, or more nucleotides in
length. As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences that are significantly
identical or homologous to each other remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% identical to each other remain hybridized
to each other. Such stringent conditions are known to those skilled
in the art and can be found in Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4 and 6. Additional stringent conditions can be found
in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9
and 11. A preferred, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
hybridization in 4.times. SSC plus 50% formamide at about
42-50.degree. C.) followed by one or more washes in 1.times. SSC,
at about 65-70.degree. C. A preferred, non-limiting example of
highly stringent hybridization conditions includes hybridization in
1.times. SSC, at about 65-70.degree. C. (or hybridization in
1.times. SSC plus 50% formamide at about 42-50.degree. C.) followed
by one or more washes in 0.3.times. SSC, at about 65-70.degree. C.
A preferred, non-limiting example of reduced stringency
hybridization conditions includes hybridization in 4.times. SSC, at
about 50-60.degree. C. (or alternatively hybridization in 6.times.
SSC plus 50% formamide at about 40-45.degree. C.) followed by one
or more washes in 2.times. SSC, at about 50-60.degree. C. Ranges
intermediate to the above-recited values, e.g., at 65-70.degree. C.
or at 42-50.degree. C. are also intended to be encompassed by the
present invention. SSPE (1.times. SSPE is 0.15 M NaCl, 10 mM
NaH.sub.2PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for
SSC (1.times. SSC is 0.15 M NaCl and 15 mM sodium citrate) in the
hybridization and wash buffers; washes are performed for 15 minutes
each after hybridization is complete. The hybridization temperature
for hybrids anticipated to be less than 50 base pairs in length
should be 5-10.degree. C. less than the melting temperature
(T.sub.m) of the hybrid, where T.sub.m is determined according to
the following equations. For hybrids less than 18 base pairs in
length, T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases).
For hybrids between 18 and 49 base pairs in length,
T.sub.m(.degree. C.)=81.5+16.6(log.sub.10[Na+])+0.41(%
G+C)-(600/N), where N is the number of bases in the hybrid, and
[Na.sup.+] is the concentration of sodium ions in the hybridization
buffer ([Na.sup.+] for 1.times. SSC=0.165 M). It will also be
recognized by the skilled practitioner that additional reagents may
be added to hybridization and/or wash buffers to decrease
non-specific hybridization of nucleic acid molecules to membranes,
for example, nitrocellulose or nylon membranes, including but not
limited to blocking agents (e.g., BSA or salmon or herring sperm
carrier DNA), detergents (e.g., SDS), chelating agents (e.g.,
EDTA), Ficoll, PVP and the like. When using nylon membranes, in
particular, an additional preferred, non-limiting example of
stringent hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.,
see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995, (or alternatively 0.2.times. SSC, 1% SDS).
[0079] Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:1 or 3 and corresponds to a
naturally-occurring nucleic acid molecule. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein).
[0080] In addition to naturally-occurring allelic variants of the
BRE sequences that may exist in the population, the skilled artisan
will further appreciate that changes can be introduced by mutation
into the nucleotide sequences of SEQ ID NO:1 or 3, thereby leading
to changes in the amino acid sequence of the encoded BRE proteins,
without altering the functional ability of the BRE proteins. For
example, nucleotide substitutions leading to amino acid
substitutions at "non-essential" amino acid residues can be made in
the sequence of SEQ ID NO:1 or 3. A "non-essential" amino acid
residue is a residue that can be altered from the wild-type
sequence of BRE (e.g., the sequence of SEQ ID NO:2) without
altering the biological activity, whereas an "essential" amino acid
residue is required for biological activity. For example, amino
acid residues that are conserved among the BRE proteins of the
present invention (for example, those present in a biotin-requiring
enzyme domain or in a carbomoyl-phosphate synthase domain), are
predicted to be particularly unamenable to alteration. Furthermore,
additional amino acid residues that are conserved between the BRE
proteins of the present invention and other members of the BRE
family are not likely to be amenable to alteration.
[0081] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding BRE proteins that contain changes
in amino acid residues that are not essential for activity. Such
BRE proteins differ in amino acid sequence from SEQ ID NO:2, yet
retain biological activity. In one embodiment, the isolated nucleic
acid molecule comprises a nucleotide sequence encoding a protein,
wherein the protein comprises an amino acid sequence at least about
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or more identical to SEQ ID NO:2.
[0082] An isolated nucleic acid molecule encoding a BRE protein
identical to the protein of SEQ ID NO:2 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of SEQ ID NO:1 or 3, such
that one or more amino acid substitutions, additions or deletions
are introduced into the encoded protein. Mutations can be
introduced into SEQ ID NO:1 or 3 by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
predicted non-essential amino acid residues. A "conservative amino
acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in a BRE protein is preferably
replaced with another amino acid residue from the same side chain
family. Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of a BRE coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for BRE biological activity to identify mutants that
retain activity. Following mutagenesis of SEQ ID NO:1 or 3, the
encoded protein can be expressed recombinantly and the activity of
the protein can be determined.
[0083] In a preferred embodiment, a mutant BRE protein can be
assayed for the ability to metabolize or catabolize biochemical
molecules necessary for energy production or storage, permit intra-
or intercellular signaling, metabolize or catabolize metabolically
important biomolecules (e.g. amino acids, nucleic acids), and to
detoxify potentially harmful compounds, or to facilitate the
neutralization of these molecules.
[0084] In addition to the nucleic acid molecules encoding BRE
proteins described above, another aspect of the invention pertains
to isolated nucleic acid molecules which are antisense thereto. An
"antisense" nucleic acid comprises a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. Accordingly, an
antisense nucleic acid can hydrogen bond to a sense nucleic acid.
The antisense nucleic acid can be complementary to an entire BRE
coding strand, or to only a portion thereof. In one embodiment, an
antisense nucleic acid molecule is antisense to a "coding region"
of the coding strand of a nucleotide sequence encoding a BRE. The
term "coding region" refers to the region of the nucleotide
sequence comprising codons which are translated into amino acid
residues (e.g., the coding region of human BRE corresponds to SEQ
ID NO:3). In another embodiment, the antisense nucleic acid
molecule is antisense to a "noncoding region" of the coding strand
of a nucleotide sequence encoding BRE. The term "noncoding region"
refers to 5' and 3' sequences which flank the coding region that
are not translated into amino acids (i.e., also referred to as 5'
and 3' untranslated regions).
[0085] Given the coding strand sequences encoding BRE disclosed
herein (e.g., SEQ ID NO:3), antisense nucleic acids of the
invention can be designed according to the rules of Watson and
Crick base pairing. The antisense nucleic acid molecule can be
complementary to the entire coding region of BRE mRNA, but more
preferably is an oligonucleotide which is antisense to only a
portion of the coding or noncoding region of BRE mRNA. For example,
the antisense oligonucleotide can be complementary to the region
surrounding the translation start site of BRE mRNA. An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid
of the invention can be constructed using chemical synthesis and
enzymatic ligation reactions using procedures known in the art. For
example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleic
acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0086] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a BRE protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention include direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[0087] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0088] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave BRE mRNA transcripts to thereby
inhibit translation of BRE mRNA. A ribozyme having specificity for
a BRE-encoding nucleic acid can be designed based upon the
nucleotide sequence of a BRE cDNA disclosed herein (i.e., SEQ ID
NO:1 or 3). For example, a derivative of a Tetrahymena L-19 IVS RNA
can be constructed in which the nucleotide sequence of the active
site is complementary to the nucleotide sequence to be cleaved in a
BRE-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;
and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, BRE mRNA
can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.
[0089] Alternatively, BRE gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the BRE (e.g., the BRE promoter and/or enhancers; e.g.,
nucleotides 1-107 of SEQ ID NO:1) to form triple helical structures
that prevent transcription of the BRE gene in target cells. See
generally, Helene, C. (1991) Anticancer Drug Des. 6(6): 569-84;
Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,
L. J. (1992) Bioassays 14(12):807-15.
[0090] In yet another embodiment, the BRE nucleic acid molecules of
the present invention can be modified at the base moiety, sugar
moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility of the molecule. For example, the
deoxyribose phosphate backbone of the nucleic acid molecules can be
modified to generate peptide nucleic acids (see Hyrup B. et al.
(1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used
herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural nucleobases are retained. The neutral backbone of
PNAs has been shown to allow for specific hybridization to DNA and
RNA under conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using standard solid phase peptide
synthesis protocols as described in Hyrup B. et al. (1996) supra;
Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
[0091] PNAs of BRE nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of BRE nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene, (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases (Hyrup B.
(1996) supra)); or as probes or primers for DNA sequencing or
hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe
supra).
[0092] In another embodiment, PNAs of BRE can be modified, (e.g.,
to enhance their stability or cellular uptake), by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
BRE nucleic acid molecules can be generated which may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, (e.g., RNAse H and DNA polymerases), to
interact with the DNA portion while the PNA portion would provide
high binding affinity and specificity. PNA-DNA chimeras can be
linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA
chimeras can be performed as described in Hyrup B. (1996) supra and
Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For
example, a DNA chain can be synthesized on a solid support using
standard phosphoramidite coupling chemistry and modified nucleoside
analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can be used as a between the PNA and the 5' end of
DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
P. J. et al. (1996) supra). Alternatively, chimeric molecules can
be synthesized with a 5' DNA segment and a 3' PNA segment
(Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:
1119-11124).
[0093] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.
(1988) Bio-Techniques 6:958-976) or intercalating agents. (See,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the
oligonucleotide may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0094] Alternatively, the expression characteristics of an
endogenous BRE gene within a cell line or microorganism may be
modified by inserting a heterologous DNA regulatory element into
the genome of a stable cell line or cloned microorganism such that
the inserted regulatory element is operatively linked with the
endogenous BRE gene. For example, an endogenous BRE gene which is
normally "transcriptionally silent", i.e., a BRE gene which is
normally not expressed, or is expressed only at very low levels in
a cell line or microorganism, may be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed gene product in that cell line or
microorganism. Alternatively, a transcriptionally silent,
endogenous BRE gene may be activated by insertion of a promiscuous
regulatory element that works across cell types.
[0095] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with an endogenous BRE gene, using techniques,
such as targeted homologous recombination, which are well known to
those of skill in the art, and described, e.g., in Chappel, U.S.
Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May
16, 1991.
II. Isolated BRE Proteins and Anti-BRE Antibodies
[0096] One aspect of the invention pertains to isolated BRE
proteins, and biologically active portions thereof, as well as
polypeptide fragments suitable for use as immunogens to raise
anti-BRE antibodies. In one embodiment, native BRE proteins can be
isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, BRE proteins are produced by recombinant DNA
techniques. Alternative to recombinant expression, a BRE protein or
polypeptide can be synthesized chemically using standard peptide
synthesis techniques.
[0097] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the BRE protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of BRE protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
BRE protein having less than about 30% (by dry weight) of non-BRE
protein (also referred to herein as a "contaminating protein"),
more preferably less than about 20% of non-BRE protein, still more
preferably less than about 10% of non-BRE protein, and most
preferably less than about 5% non-BRE protein. When the BRE protein
or biologically active portion thereof is recombinantly produced,
it is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation.
[0098] The language "substantially free of chemical precursors or
other chemicals" includes preparations of BRE protein in which the
protein is separated from chemical precursors or other chemicals
which are involved in the synthesis of the protein. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of BRE protein having
less than about 30% (by dry weight) of chemical precursors or
non-BRE chemicals, more preferably less than about 20% chemical
precursors or non-BRE chemicals, still more preferably less than
about 10% chemical precursors or non-BRE chemicals, and most
preferably less than about 5% chemical precursors or non-BRE
chemicals.
[0099] As used herein, a "biologically active portion" of a BRE
protein includes a fragment of a BRE protein which participates in
an interaction between a BRE molecule and a non-BRE molecule.
Biologically active portions of a BRE protein include peptides
comprising amino acid sequences sufficiently identical to or
derived from the amino acid sequence of the BRE protein, e.g., the
amino acid sequence shown in SEQ ID NO:2, which include less amino
acids than the full length BRE protein, and exhibit at least one
activity of a BRE protein. Typically, biologically active portions
comprise a domain or motif with at least one activity of the BRE
protein, e.g., carboxylase activity, decarboxylase activity,
transcarboxylase activity. A biologically active portion of a BRE
protein can be a polypeptide which is, for example, 25, 50, 75,
100, 125, 150, 175, 200, 250, 300, 325, 350, 375, 400, 425, 450,
475, 500, 525, 550, 575, 600, 625, 650, 675, 700 or more amino
acids in length. Biologically active portions of a BRE protein can
be used as targets for developing agents which modulate a BRE
mediated activity, e.g., intercellular signaling.
[0100] It is to be understood that a preferred biologically active
portion of a BRE protein of the present invention may contain one
or more of the following domains: a CPSase domain, and/or a
biotin-requiring enzyme domain. Moreover, other biologically active
portions, in which other regions of the protein are deleted, can be
prepared by recombinant techniques and evaluated for one or more of
the functional activities of a native BRE protein.
[0101] In a preferred embodiment, the BRE protein has an amino acid
sequence shown in SEQ ID NO:2. In other embodiments, the BRE
protein is substantially identical to SEQ ID NO:2, and retains the
functional activity of the protein of SEQ ID NO:2, yet differs in
amino acid sequence due to natural allelic variation or
mutagenesis, as described in detail in subsection I above.
Accordingly, in another embodiment, the BRE protein is a protein
which comprises an amino acid sequence at least about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
identical to SEQ ID NO:2.
[0102] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the BRE amino acid sequence of SEQ ID NO:2 having 725 amino acid
residues, at least 218, preferably at least 290, more preferably at
least 363, even more preferably at least 435, and even more
preferably at least 508, 580, 653 or more amino acid residues are
aligned). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are identical at that position
(as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0103] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the
percent identity between two amino acid or nucleotide sequences is
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0104] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to BRE nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=100, wordlength=3 to obtain amino
acid sequences homologous to BRE protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
[0105] The invention also provides BRE chimeric or fusion proteins.
As used herein, a BRE "chimeric protein" or "fusion protein"
comprises a BRE polypeptide operatively linked to a non-BRE
polypeptide. An "BRE polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to a BRE molecule, whereas a
"non-BRE polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous to the BRE protein, e.g., a protein which is different
from the BRE protein and which is derived from the same or a
different organism. Within a BRE fusion protein the BRE polypeptide
can correspond to all or a portion of a BRE protein. In a preferred
embodiment, a BRE fusion protein comprises at least one
biologically active portion of a BRE protein. In another preferred
embodiment, a BRE fusion protein comprises at least two
biologically active portions of a BRE protein. Within the fusion
protein, the term "operatively linked" is intended to indicate that
the BRE polypeptide and the non-BRE polypeptide are fused in-frame
to each other. The non-BRE polypeptide can be fused to the
N-terminus or C-terminus of the BRE polypeptide.
[0106] For example, in one embodiment, the fusion protein is a
GST-BRE fusion protein in which the BRE sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant BRE.
[0107] In another embodiment, the fusion protein is a BRE protein
containing a heterologous signal sequence at its N-terminus. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of BRE can be increased through use of a heterologous
signal sequence.
[0108] The BRE fusion proteins of the invention can be incorporated
into pharmaceutical compositions and administered to a subject in
vivo. The BRE fusion proteins can be used to affect the
bioavailability of a BRE substrate. Use of BRE fusion proteins may
be useful therapeutically for the treatment of disorders caused by,
for example, (i) aberrant modification or mutation of a gene
encoding a BRE protein; (ii) mis-regulation of the BRE gene; and
(iii) aberrant post-translational modification of a BRE
protein.
[0109] Moreover, the BRE-fusion proteins of the invention can be
used as immunogens to produce anti-BRE antibodies in a subject for
use in screening assays to identify molecules which inhibit the
interaction of BRE with a BRE substrate.
[0110] Preferably, a BRE chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A BRE-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the BRE protein.
[0111] The present invention also pertains to variants of the BRE
proteins which function as either BRE agonists (mimetics) or as BRE
antagonists. Variants of the BRE proteins can be generated by
mutagenesis, e.g., discrete point mutation or truncation of a BRE
protein. An agonist of the BRE proteins can retain substantially
the same, or a subset, of the biological activities of the
naturally occurring form of a BRE protein. An antagonist of a BRE
protein can inhibit one or more of the activities of the naturally
occurring form of the BRE protein by, for example, competitively
modulating a BRE-mediated activity of a BRE protein. Thus, specific
biological effects can be elicited by treatment with a variant of
limited function. In one embodiment, treatment of a subject with a
variant having a subset of the biological activities of the
naturally occurring form of the protein has fewer side effects in a
subject relative to treatment with the naturally occurring form of
the BRE protein.
[0112] In one embodiment, variants of a BRE protein which function
as either BRE agonists (mimetics) or as BRE antagonists can be
identified by screening combinatorial libraries of mutants, e.g.,
truncation mutants, of a BRE protein for BRE protein agonist or
antagonist activity. In one embodiment, a variegated library of BRE
variants is generated by combinatorial mutagenesis at the nucleic
acid level and is encoded by a variegated gene library. A
variegated library of BRE variants can be produced by, for example,
enzymatically ligating a mixture of synthetic oligonucleotides into
gene sequences such that a degenerate set of potential BRE
sequences is expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage
display) containing the set of BRE sequences therein. There are a
variety of methods which can be used to produce libraries of
potential BRE variants from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be performed
in an automatic DNA synthesizer, and the synthetic gene then
ligated into an appropriate expression vector. Use of a degenerate
set of genes allows for the provision, in one mixture, of all of
the sequences encoding the desired set of potential BRE sequences.
Methods for synthesizing degenerate oligonucleotides are known in
the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura
et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)
Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
[0113] In addition, libraries of fragments of a BRE protein coding
sequence can be used to generate a variegated population of BRE
fragments for screening and subsequent selection of variants of a
BRE protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a BRE coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA
which 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 fragment
library into an expression vector. By this method, an expression
library can be derived which encodes N-terminal, C-terminal and
internal fragments of various sizes of the BRE protein.
[0114] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of BRE proteins. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a new technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify BRE variants (Arkin and Yourvan (1992) Proc. Natl. Acad.
Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering
6(3): 327-331).
[0115] In one embodiment, cell based assays can be exploited to
analyze a variegated BRE library. For example, a library of
expression vectors can be transfected into a cell line, e.g., a
neuronal cell line, which ordinarily responds to a BRE ligand in a
particular BRE ligand-dependent manner. The transfected cells are
then contacted with a BRE ligand and the effect of expression of
the mutant on, e.g., membrane excitability of BRE can be detected.
Plasmid DNA can then be recovered from the cells which score for
inhibition, or alternatively, potentiation of signaling by the BRE
ligand, and the individual clones further characterized.
[0116] An isolated BRE protein, or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind BRE
using standard techniques for polyclonal and monoclonal antibody
preparation. A full-length BRE protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of BRE for use as immunogens. The antigenic peptide of BRE
comprises at least 8 amino acid residues of the amino acid sequence
shown in SEQ ID NO:2 and encompasses an epitope of BRE such that an
antibody raised against the peptide forms a specific immune complex
with the BRE protein. Preferably, the antigenic peptide comprises
at least 10 amino acid residues, more preferably at least 15 amino
acid residues, even more preferably at least 20 amino acid
residues, and most preferably at least 30 amino acid residues.
[0117] Preferred epitopes encompassed by the antigenic peptide are
regions of BRE that are located on the surface of the protein,
e.g., hydrophilic regions, as well as regions with high
antigenicity.
[0118] An BRE immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other
mammal) with the immunogen. An appropriate immunogenic preparation
can contain, for example, recombinantly expressed BRE protein or a
chemically synthesized BRE polypeptide. The preparation can further
include an adjuvant, such as Freund's complete or incomplete
adjuvant, or similar immunostimulatory agent. Immunization of a
suitable subject with an immunogenic BRE preparation induces a
polyclonal anti-BRE antibody response.
[0119] Accordingly, another aspect of the invention pertains to
anti-BRE antibodies. The term "antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site which specifically binds (immunoreacts with) an
antigen, such as a BRE. Examples of immunologically active portions
of immunoglobulin molecules include F(ab) and F(ab').sub.2
fragments which can be generated by treating the antibody with an
enzyme such as pepsin. The invention provides polyclonal and
monoclonal antibodies that bind BRE molecules. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of BRE. A monoclonal antibody composition thus
typically displays a single binding affinity for a particular BRE
protein with which it immunoreacts.
[0120] Polyclonal anti-BRE antibodies can be prepared as described
above by immunizing a suitable subject with a BRE immunogen. The
anti-BRE antibody titer in the immunized subject can be monitored
over time by standard techniques, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized BRE. If desired, the
antibody molecules directed against BRE can be isolated from the
mammal (e.g., from the blood) and further purified by well known
techniques, such as protein A chromatography to obtain the IgG
fraction. At an appropriate time after immunization, e.g., when the
anti-BRE antibody titers are highest, antibody-producing cells can
be obtained from the subject and used to prepare monoclonal
antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature
256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46;
Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976)
Proc. Natl. Acad. Sci. USA 76:2927-31;and Yeh et al. (1982) Int. J.
Cancer 29:269-75), the more recent human B cell hybridoma technique
(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma
technique (Cole et al. (1985), Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The
technology for producing monoclonal antibody hybridomas is well
known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New
Dimension In Biological Analyses, Plenum Publishing Corp., New
York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med.,
54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet.
3:231-36). Briefly, an immortal cell line (typically a myeloma) is
fused to lymphocytes (typically splenocytes) from a mammal
immunized with a BRE immunogen as described above, and the culture
supernatants of the resulting hybridoma cells are screened to
identify a hybridoma producing a monoclonal antibody that binds
BRE.
[0121] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-BRE monoclonal antibody (see, e.g.,
G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic
Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra;
Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind BRE, e.g., using a standard
ELISA assay.
[0122] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-BRE antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with BRE to
thereby isolate immunoglobulin library members that bind BRE. Kits
for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening antibody display library can be found in,
for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al. PCT
International Publication No. WO 91/17271; Winter et al. PCT
International Publication WO 92/20791; Markland et al. PCT
International Publication No. WO 92/15679; Breitling et al. PCT
International Publication WO 93/01288; McCafferty et al. PCT
International Publication No. WO 92/01047; Garrard et al. PCT
International Publication No. WO 92/09690; Ladner et al. PCT
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J.
Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad
et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991)
Nuc. Acid Res. 19:4133-4137;Barbas et al. (1991) Proc. Natl. Acad.
Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990)
348:552-554.
[0123] Additionally, recombinant anti-BRE antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in Robinson et al. International Application No.
PCT/US86/02269; Akira, et al. European Patent Application 184,187;
Taniguchi, M., European Patent Application 171,496; Morrison et al.
European Patent Application 173,494; Neuberger et al. PCT
International Publication No. WO 86/01533; Cabilly et al. U.S. Pat.
No. 4,816,567; Cabilly et al. European Patent Application 125,023;
Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc.
Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.
139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA
84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et
al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl.
Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science
229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S.
Pat. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan
et al. (1988) Science 239:1534; and Beidler et al. (1988) J.
Immunol. 141:4053-4060.
[0124] An anti-BRE antibody (e.g., monoclonal antibody) can be used
to isolate BRE by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-BRE antibody can
facilitate the purification of natural BRE from cells and of
recombinantly produced BRE expressed in host cells. Moreover, an
anti-BRE antibody can be used to detect BRE protein (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the BRE protein. Anti-BRE
antibodies can be used diagnostically to monitor protein levels in
tissue as part of a clinical testing procedure, e.g., to, for
example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by coupling (i.e., physically linking)
the antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidintbiotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
III. Recombinant Expression Vectors and Host Cells
[0125] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
BRE protein (or a portion thereof). As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0126] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cells and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, and the
like. The expression vectors of the invention can be introduced
into host cells to thereby produce proteins or peptides, including
fusion proteins or peptides, encoded by nucleic acids as described
herein (e.g., BRE proteins, mutant forms of BRE proteins, fusion
proteins, and the like).
[0127] The recombinant expression vectors of the invention can be
designed for expression of BRE proteins in prokaryotic or
eukaryotic cells. For example, BRE proteins can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0128] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[0129] Purified fusion proteins can be utilized in BRE activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for BRE proteins,
for example. In a preferred embodiment, a BRE fusion protein
expressed in a retroviral expression vector of the present
invention can be utilized to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six (6) weeks).
[0130] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gn1 gene under the transcriptional control
of the lacUV 5 promoter.
[0131] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially utilized in E.
coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0132] In another embodiment, the BRE expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari, et al., (1987) Embo J.
6:229-234), pMNa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San
Diego, Calif.).
[0133] Alternatively, BRE proteins can be expressed in insect cells
using baculovirus expression vectors. Baculovirus vectors available
for expression of proteins in cultured insect cells (e.g., Sf 9
cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.
3:2156-2165) and the pVL series (Lucklow and Summers (1989)
Virology 170:31-39).
[0134] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0135] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0136] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to BRE mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0137] Another aspect of the invention pertains to host cells into
which a BRE nucleic acid molecule of the invention is introduced,
e.g., a BRE nucleic acid molecule within a recombinant expression
vector or a BRE nucleic acid molecule containing sequences which
allow it to homologously recombine into a specific site of the host
cell's genome. The terms "host cell" and "recombinant host cell"
are used interchangeably herein. It is understood that such terms
refer not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0138] A host cell can be any prokaryotic or eukaryotic cell. For
example, a BRE protein can be expressed in bacterial cells such as
E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0139] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0140] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding a BRE protein or can be introduced on a separate
vector. Cells stably transfected with the introduced nucleic acid
can be identified by drug selection (e.g., cells that have
incorporated the selectable marker gene will survive, while the
other cells die).
[0141] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) a BRE protein. Accordingly, the invention further provides
methods for producing a BRE protein using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of the invention (into which a recombinant expression
vector encoding a BRE protein has been introduced) in a suitable
medium such that a BRE protein is produced. In another embodiment,
the method further comprises isolating a BRE protein from the
medium or the host cell.
[0142] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which BRE-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic
animals in which exogenous BRE sequences have been introduced into
their genome or homologous recombinant animals in which endogenous
BRE sequences have been altered. Such animals are useful for
studying the function and/or activity of a BRE and for identifying
and/or evaluating modulators of BRE activity. As used herein, a
"transgenic animal" is a non-human animal, preferably a mammal,
more preferably a rodent such as a rat or mouse, in which one or
more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, and the like. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous BRE gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0143] A transgenic animal of the invention can be created by
introducing a BRE-encoding nucleic acid into the male pronuclei of
a fertilized oocyte, e.g., by microinjection, retroviral infection,
and allowing the oocyte to develop in a pseudopregnant female
foster animal. The BRE cDNA sequence of SEQ ID NO:1 can be
introduced as a transgene into the genome of a non-human animal.
Alternatively, a nonhuman homologue of a human BRE gene, such as a
mouse or rat BRE gene, can be used as a transgene. Alternatively, a
BRE gene homologue, such as another BRE family member, can be
isolated based on hybridization to the BRE cDNA sequences of SEQ ID
NO:1 or 3, and used as a transgene. Intronic sequences and
polyadenylation signals can also be included in the transgene to
increase the efficiency of expression of the transgene. A
tissue-specific regulatory sequence(s) can be operably linked to a
BRE transgene to direct expression of a BRE protein to particular
cells. Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of a BRE transgene
in its genome and/or expression of BRE mRNA in tissues or cells of
the animals. A transgenic founder animal can then be used to breed
additional animals carrying the transgene. Moreover, transgenic
animals carrying a transgene encoding a BRE protein can further be
bred to other transgenic animals carrying other transgenes.
[0144] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a BRE gene into which
a deletion, addition or substitution has been introduced to thereby
alter, e.g., functionally disrupt, the BRE gene. The BRE gene can
be a human gene (e.g., the cDNA of SEQ ID NO:3), but more
preferably, is a non-human homologue of a human BRE gene (e.g., a
cDNA isolated by stringent hybridization with the nucleotide
sequence of SEQ ID NO:1). For example, a mouse BRE gene can be used
to construct a homologous recombination nucleic acid molecule,
e.g., a vector, suitable for altering an endogenous BRE gene in the
mouse genome. In a preferred embodiment, the homologous
recombination nucleic acid molecule is designed such that, upon
homologous recombination, the endogenous BRE gene is functionally
disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock out" vector). Alternatively, the homologous
recombination nucleic acid molecule can be designed such that, upon
homologous recombination, the endogenous BRE gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous BRE protein). In the homologous
recombination nucleic acid molecule, the altered portion of the BRE
gene is flanked at its 5' and 3' ends by additional nucleic acid
sequence of the BRE gene to allow for homologous recombination to
occur between the exogenous BRE gene carried by the homologous
recombination nucleic acid molecule and an endogenous BRE gene in a
cell, e.g., an embryonic stem cell. The additional flanking BRE
nucleic acid sequence is of sufficient length for successful
homologous recombination with the endogenous gene. Typically,
several kilobases of flanking DNA (both at the 5' and 3' ends) are
included in the homologous recombination nucleic acid molecule
(see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503
for a description of homologous recombination vectors). The
homologous recombination nucleic acid molecule is introduced into a
cell, e.g., an embryonic stem cell line (e.g., by electroporation)
and cells in which the introduced BRE gene has homologously
recombined with the endogenous BRE gene are selected (see e.g., Li,
E. et al. (1992) Cell 69:915). The selected cells can then injected
into a blastocyst of an animal (e.g., a mouse) to form aggregation
chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,
Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
into a suitable pseudopregnant female foster animal and the embryo
brought to term. Progeny harboring the homologously recombined DNA
in their germ cells can be used to breed animals in which all cells
of the animal contain the homologously recombined DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination nucleic acid molecules, e.g., vectors, or homologous
recombinant animals are described further in Bradley, A. (1991)
Current Opinion in Biotechnology 2:823-829 and in PCT International
Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by
Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by
Berns et al.
[0145] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a
cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[0146] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter Go phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyte and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
V. Pharmaceutical Compositions
[0147] The BRE nucleic acid molecules, fragments of BRE proteins,
and anti-BRE antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the nucleic acid molecule, protein,
or antibody and a pharmaceutically acceptable carrier. As used
herein the language "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0148] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0149] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0150] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a fragment of a BRE
protein or an anti-BRE antibody) in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0151] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0152] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0153] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0154] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0155] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0156] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0157] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0158] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0159] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[0160] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[0161] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e,. including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecule agents depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention.
[0162] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0163] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0164] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
alpha-interferon, beta-interferon, nerve growth factor, platelet
derived growth factor, tissue plasminogen activator; or, biological
response modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophase colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[0165] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[0166] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0167] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
V. Uses and Methods of the Invention
[0168] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic). As described herein, a BRE protein
of the invention has one or more of the following activities: 1)
modulation the bioenergetic activities of a cell (e.g., storage or
yielding of chemical energy) 2) modulation of intra- or
intercellular signaling or feedback mechanisms, 3) removal of
potentially harmful compounds (e.g., cytotoxic substances) from the
cell, or facilitate the neutralization of these molecules through
enzymatic alteration (e.g., carboxylation, decarboxylation), 4)
modulation the production or breakdown of amino acids or nucleic
acids, or modulate the homeostatic balance of available amino acid
or nucleic acid pools, 5) the specific attachment of biotin to its
cognate enzyme, e.g., biotin protein ligase activity.
[0169] In a preferred embodiment, the BRE molecules of the
invention are useful for catalyzing carboxylase, decarboxylase, and
transcarboxylase reactions. As such, these molecules may be
employed in small or large-scale synthesis of either carboxylated
moieties or decarboxylated substrate, or in chemical processes that
require the production or interconversion of these compounds. Such
processes are known in the art (see, e.g., Ullmann et al. (1999)
Ullmann's Encyclopedia of Industrial Chemistry, 6th ed. VCH.
Weinheim; Gutcho (1983) Chemicals by Fermentation. Park ridge,
N.J.: Noyes Data Corporation (ISBN 0818805086); Rehm et al. (eds.)
(1993) Biotechnology, 2nd ed. VCH: Weinheim; and Michal, G. (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology. New York: John Wiley & Sons, and references contained
therein.)
[0170] The isolated nucleic acid molecules of the invention can be
used, for example, to express BRE protein (e.g., via a recombinant
expression vector in a host cell in gene therapy applications), to
detect BRE mRNA (e.g., in a biological sample) or a genetic
alteration in a BRE gene, and to modulate BRE activity, as
described further below. The BRE proteins can be used to treat
disorders characterized by insufficient or excessive production of
a BRE substrate or production of BRE inhibitors. In addition, the
BRE proteins can be used to screen for naturally occurring BRE
substrates, to screen for drugs or compounds which modulate BRE
activity, as well as to treat disorders characterized by
insufficient or excessive production of BRE protein or production
of BRE protein forms which have decreased, aberrant or unwanted
activity compared to BRE wild type protein, preferably a
BE-associated disorder. As used herein, a "BE-associated disorder"
includes a disorder, disease or condition which is caused or
characterized by a misregulation (e.g., downregulation or
upregulation) of a biotin enzyme-mediated activity (e.g.,
BE-mediated activity), for example, carboxylase activity or a
decarboxylase activity. Biotin-associated disorders can
detrimentally affect cellular functions such as cellular
proliferation, growth, differentiation, or migration, cellular
regulation of homeostasis, inter- or intra-cellular communication;
tissue function, such as cardiac function or musculoskeletal
function; systemic responses in an organism, such as nervous system
responses, hormonal responses (e.g., insulin response), or immune
responses; and protection of cells from toxic compounds (e.g.,
carcinogens, toxins, mutagens, and toxic byproducts of metabolic
activity (e.g., reactive oxygen species)). Examples of
biotin-associated disorders include CNS disorders such as cognitive
and neurodegenerative disorders, examples of which include, but are
not limited to, Alzheimer's disease, dementias related to
Alzheimer's disease (such as Pick's disease), Parkinson's and other
Lewy diffuse body diseases, senile dementia, Huntington's disease,
Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic
lateral sclerosis, progressive supranuclear palsy, epilepsy, and
Jakob-Creutzfieldt disease; autonomic function disorders such as
hypertension and sleep disorders, and neuropsychiatric disorders,
such as depression, schizophrenia, schizoaffective disorder,
korsakoff's psychosis, mania, anxiety disorders, or phobic
disorders; learning or memory disorders, e.g., amnesia or
age-related memory loss, attention deficit disorder, dysthymic
disorder, major depressive disorder, mania, obsessive-compulsive
disorder, psychoactive substance use disorders, anxiety, phobias,
panic disorder, as well as bipolar affective disorder, e.g., severe
bipolar affective (mood) disorder (BP-1), and bipolar affective
neurological disorders, e.g., migraine and obesity. Further
CNS-related disorders include, for example, those listed in the
American Psychiatric Association's Diagnostic and Statistical
manual of Mental Disorders (DSM), the most current version of which
is incorporated herein by reference in its entirety.
[0171] Further examples of biotin-associated disorders include
cardiac-related disorders. Cardiovascular system disorders in which
the BRE molecules of the invention may be directly or indirectly
involved include arteriosclerosis, ischemia reperfusion injury,
restenosis, arterial inflammation, vascular wall remodeling,
ventricular remodeling, rapid ventricular pacing, coronary
microembolism, tachycardia, bradycardia, pressure overload, aortic
bending, coronary artery ligation, vascular heart disease, atrial
fibrilation, Jervell syndrome, Lange-Nielsen syndrome, long-QT
syndrome, congestive heart failure, sinus node dysfunction, angina,
heart failure, hypertension, atrial fibrillation, atrial flutter,
dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial
infarction, coronary artery disease, coronary artery spasm, and
arrhythmia. BRE-mediated or related disorders also include
disorders of the musculoskeletal system such as paralysis and
muscle weakness, e.g., ataxia, myotonia, and myokymia.
[0172] BRE-associated disorders also include cellular
proliferation, growth, differentiation, or migration disorders.
Cellular proliferation, growth, differentiation, or migration
disorders include those disorders that affect cell proliferation,
growth, differentiation, or migration processes. As used herein, a
"cellular proliferation, growth, differentiation, or migration
process" is a process by which a cell increases in number, size or
content, by which a cell develops a specialized set of
characteristics which differ from that of other cells, or by which
a cell moves closer to or further from a particular location or
stimulus. The BRE molecules of the present invention are involved
in signal transduction mechanisms, which are known to be involved
in cellular growth, differentiation, and migration processes. Thus,
the BRE molecules may modulate cellular growth, differentiation, or
migration, and may play a role in disorders characterized by
aberrantly regulated growth, differentiation, or migration. Such
disorders include cancer, e.g., carcinoma, sarcoma, or leukemia;
tumor angiogenesis and metastasis; skeletal dysplasia; hepatic
disorders; and hematopoietic and/or myeloproliferative
disorders.
[0173] BRE-associated or related disorders also include hormonal
disorders, such as conditions or diseases in which the production
and/or regulation of hormones in an organism is aberrant. Examples
of such disorders and diseases include type I and type II diabetes
mellitus, pituitary disorders (e.g., growth disorders), thyroid
disorders (e.g., hypothyroidism or hyperthyroidism), and
reproductive or fertility disorders (e.g., disorders which affect
the organs of the reproductive system, e.g., the prostate gland,
the uterus, or the vagina; disorders which involve an imbalance in
the levels of a reproductive hormone in a subject; disorders
affecting the ability of a subject to reproduce; and disorders
affecting secondary sex characteristic development, e.g., adrenal
hyperplasia).
[0174] BRE-associated or related disorders also include immune
disorders, such as autoimmune disorders or immune deficiency
disorders, e.g., congenital X-linked infantile
hypogammaglobulinemia, transient hypogammaglobulinemia, common
variable immunodeficiency, selective IgA deficiency, chronic
mucocutaneous candidiasis, or severe combined immunodeficiency.
BRE-associated or related disorders also include disorders
affecting tissues in which BRE protein is expressed.
[0175] A. Screening Assays:
[0176] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) which bind to BRE proteins, have a
stimulatory or inhibitory effect on, for example, BRE expression or
BRE activity, or have a stimulatory or inhibitory effect on, for
example, the expression or activity of BRE substrate.
[0177] These assays are designed to identify compounds that bind to
a BRE protein, bind to other inter- or extra-cellular proteins that
interact with a BRE protein, and/or interfere with the interaction
of the BRE protein with other inter- or extra-cellular proteins.
For example, in the case of the BRE protein, such techniques can be
used to identify ligands for such a protein. A BRE protein
modulator can, for example, be used to ameliorate cellular growth
or proliferation diseases or disorders, e.g., cancer, or
nutritional difficulties, organic aciduria, neurologic
abnormalities, and cutaneous distress. Such compounds may include,
but are not limited to BRE peptides, anti-BRE antibodies, or small
organic or inorganic compounds. Such compounds may also include
other cellular proteins or peptides.
[0178] Compounds identified via assays such as those described
herein may be useful, for example, for ameliorating cellular growth
and proliferation diseases or disorders. In instances whereby a
cellular growth or proliferation disease condition results from an
overall lower level of BRE gene expression and/or BRE protein in a
cell or tissue, compounds that interact with the BRE protein may
include compounds which accentuate or amplify the activity of the
bound BRE protein. Such compounds would bring about an effective
increase in the level of BRE protein activity, thus ameliorating
symptoms. In other instances, mutations within the BRE gene may
cause aberrant types or excessive amounts of BRE proteins to be
made which have a deleterious effect that leads to a cellular
growth or proliferation disease or disorder. Similarly,
physiological conditions may cause an excessive increase in BRE
gene expression leading to a cellular growth or proliferation
disease or disorder. In such cases, compounds that bind to a BRE
protein may be identified that inhibit the activity of the BRE
protein. Assays for testing the effectiveness of compounds
identified by techniques such as those described in this section
are discussed herein.
[0179] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a BRE
protein or polypeptide or biologically active portion thereof
(e.g., energy transduction metabolites, urea cycle metabolites,
lipid metabolism metabolites, amino acid precursors, nucleic acid
precursors). In another embodiment, the invention provides assays
for screening candidate or test compounds which bind to or modulate
the activity of a BRE protein or polypeptide or biologically active
portion thereof. The test compounds of the present invention can be
obtained using any of the numerous approaches in combinatorial
library methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K.S. (1997) Anticancer
Drug Des. 12:145).
[0180] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al.
(1994)Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J.
Med. Chem. 37:1233.
[0181] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.).
[0182] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a BRE protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to modulate BRE activity is determined. Determining
the ability of the test compound to modulate BRE activity can be
accomplished by monitoring, for example, the production of one or
more specific metabolites in a cell which expresses BRE (see, e.g.,
Saada et al. (2000) Biochem Biophys. Res. Commun. 269: 382-386).
The cell, for example, can be of mammalian origin, e.g., an
epithelial or neuronal cell. The ability of the test compound to
modulate BRE binding to a substrate (e.g., an energy transduction
metabolite, a urea cycle metabolite, a lipid metabolism metabolite,
an amino acid precursor, a nucleic acid precursor) or to bind to
BRE can also be determined. Determining the ability of the test
compound to modulate BRE binding to a substrate can be
accomplished, for example, by coupling the BRE substrate with a
radioisotope or enzymatic label such that binding of the BRE
substrate to BRE can be determined by detecting the labeled BRE
substrate in a complex. Alternatively, BRE could be coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate BRE binding to a BRE substrate in a complex.
Determining the ability of the test compound to bind BRE can be
accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to BRE can be determined by detecting the labeled compound in a
complex. For example, compounds (e.g., BRE substrates) can be
labeled with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0183] It is also within the scope of this invention to determine
the ability of a compound (e.g., a BRE substrate) to interact with
BRE without the labeling of any of the interactants. For example, a
microphysiometer can be used to detect the interaction of a
compound with BRE without the labeling of either the compound or
the BRE. McConnell, H. M. et al. (1992) Science 257:1906-1912. As
used herein, a "microphysiometer" (e.g., Cytosensor) is an
analytical instrument that measures the rate at which a cell
acidifies its environment using a light-addressable potentiometric
sensor (LAPS). Changes in this acidification rate can be used as an
indicator of the interaction between a compound and BRE.
[0184] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a BRE target molecule
(e.g., a BRE substrate) with a test compound and determining the
ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the BRE target molecule. Determining the
ability of the test compound to modulate the activity of a BRE
target molecule can be accomplished, for example, by determining
the ability of the BRE protein to bind to or interact with the BRE
target molecule.
[0185] Determining the ability of the BRE protein, or a
biologically active fragment thereof, to bind to or interact with a
BRE target molecule can be accomplished by one of the methods
described above for determining direct binding. In a preferred
embodiment, determining the ability of the BRE protein to bind to
or interact with a BRE target molecule can be accomplished by
determining the activity of the target molecule. For example, the
activity of the target molecule can be determined by detecting
induction of a cellular response (i.e., cell proliferation,
migration and/or metabolic activity), detecting catalytic/enzymatic
activity of the target on an appropriate substrate, detecting the
induction of a reporter gene (comprising a target-responsive
regulatory element operatively linked to a nucleic acid encoding a
detectable marker, e.g., luciferase), or detecting a
target-regulated cellular response.
[0186] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a BRE protein or biologically active
portion thereof is contacted with a test compound and the ability
of the test compound to bind to the BRE protein or biologically
active portion thereof is determined. Preferred biologically active
portions of the BRE proteins to be used in assays of the present
invention include fragments which participate in interactions with
non-BRE molecules, e.g., fragments with high surface probability
scores (see, for example, FIGS. 2A-2B). Binding of the test
compound to the BRE protein can be determined either directly or
indirectly as described above. In a preferred embodiment, the assay
includes contacting the BRE protein or biologically active portion
thereof with a known compound which binds BRE to form an assay
mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to interact with a BRE
protein, wherein determining the ability of the test compound to
interact with a BRE protein comprises determining the ability of
the test compound to preferentially bind to BRE or biologically
active portion thereof as compared to the known compound.
[0187] In another embodiment, the assay is a cell-free assay in
which a BRE protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to modulate (e.g., stimulate or inhibit) the activity of the BRE
protein or biologically active portion thereof is determined.
Determining the ability of the test compound to modulate the
activity of a BRE protein can be accomplished, for example, by
determining the ability of the BRE protein to bind to a BRE target
molecule by one of the methods described above for determining
direct binding. Determining the ability of the BRE protein to bind
to a BRE target molecule can also be accomplished using a
technology such as real-time Biomolecular Interaction Analysis
(BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem.
63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705. As used herein, "BIA" is a technology for studying
biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the optical phenomenon of
surface plasmon resonance (SPR) can be used as an indication of
real-time reactions between biological molecules.
[0188] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of a BRE protein can be
accomplished by determining the ability of the BRE protein to
interact with and/or convert a BRE substrate (e.g., to produce a
specific metabolite).
[0189] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of a BRE protein can be
accomplished by determining the ability of the BRE protein to
further modulate the activity of a downstream effector of a BRE
target molecule. For example, the activity of the effector molecule
on an appropriate target can be determined or the binding of the
effector to an appropriate target can be determined as previously
described.
[0190] In yet another embodiment, the cell-free assay involves
contacting a BRE protein or biologically active portion thereof
with a known compound (e.g., a BRE substrate) which binds the BRE
protein to form an assay mixture, contacting the assay mixture with
a test compound, and determining the ability of the test compound
to interact with the BRE protein, wherein determining the ability
of the test compound to interact with the BRE protein comprises
determining the ability of the BRE protein to preferentially bind
to or modulate the activity of a BRE target protein, e.g., catalyze
the cleavage, e.g., the hydrolytic cleavage, of a chemical bond
within the target protein.
[0191] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either BRE
or its target molecule to facilitate separation of complexed from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of a test compound to
a BRE protein, or interaction of a BRE protein with a target
molecule in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/BRE fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or BRE protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of BRE binding or activity
determined using standard techniques.
[0192] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a BRE protein or a BRE target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated BRE
protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with BRE
protein or target molecules but which do not interfere with binding
of the BRE protein to its target molecule can be derivatized to the
wells of the plate, and unbound target or BRE protein trapped in
the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the BRE protein or target molecule,
as well as enzyme-linked assays which rely on detecting an
enzymatic activity associated with the BRE protein or target
molecule.
[0193] In another embodiment, modulators of BRE expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of BRE mRNA or protein in the cell is
determined. The level of expression of BRE mRNA or protein in the
presence of the candidate compound is compared to the level of
expression of BRE mRNA or protein in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of BRE expression based on this comparison. For example,
when expression of BRE mRNA or protein is greater (statistically
significantly greater) in the presence of the candidate compound
than in its absence, the candidate compound is identified as a
stimulator of BRE mRNA or protein expression. Alternatively, when
expression of BRE mRNA or protein is less (statistically
significantly less) in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of BRE mRNA or protein expression. The level of BRE mRNA
or protein expression in the cells can be determined by methods
described herein for detecting BRE mRNA or protein.
[0194] In yet another aspect of the invention, the BRE proteins can
be used as "bait proteins" in a two-hybrid assay or three-hybrid
assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993)
Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with BRE
("BRE binding proteins" or "HYDL-1-bp") and are involved in BRE
activity. Such BRE binding proteins are also likely to be involved
in the propagation of signals by the BRE proteins or BRE targets
as, for example, downstream elements of a BRE-mediated signaling
pathway. Alternatively, such BRE binding proteins are likely to be
BRE inhibitors.
[0195] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a BRE protein
is fused to a gene encoding the DNA binding domain of a known
transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a BRE-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the BRE protein.
[0196] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a BRE protein can be confirmed in vivo, e.g., in an animal such
as an animal model for cellular transformation and/or
tumorigenesis, or an animal model for a metabolic disorder.
[0197] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a BRE modulating agent,
an antisense BRE nucleic acid molecule, a BRE-specific antibody, or
a BRE binding partner) can be used in an animal model to determine
the efficacy, toxicity, or side effects of treatment with such an
agent. Alternatively, an agent identified as described herein can
be used in an animal model to determine the mechanism of action of
such an agent. Furthermore, this invention pertains to uses of
novel agents identified by the above-described screening assays for
treatments as described herein. In one embodiment, the invention
features a method of treating a subject having a cellular growth or
proliferation disease or disorder that involves administering to
the subject a BRE modulator such that treatment occurs. In another
embodiment, the invention features a method of treating a subject
having cancer, e.g., colon cancer or lung cancer, that involves
treating a subject with a BRE modulator, such that treatment
occurs. Preferred BRE modulators include, but are not limited to,
BRE proteins or biologically active fragments, BRE nucleic acid
molecules, BRE antibodies, ribozymes, and BRE antisense
oligonucleotides designed based on the BRE nucleotide sequences
disclosed herein, as well as peptides, organic and non-organic
small molecules identified as being capable of modulating BRE
expression and/or activity, for example, according to at least one
of the screening assays described herein.
[0198] Any of the compounds, including but not limited to compounds
such as those identified in the foregoing assay systems, may be
tested for the ability to ameliorate cellular growth or
proliferation disease or disorder symptoms. Cell-based and animal
model-based assays for the identification of compounds exhibiting
such an ability to ameliorate cellular growth or proliferation
disease or disorder systems are described herein.
[0199] In one aspect, cell-based systems, as described herein, may
be used to identify compounds which may act to ameliorate cellular
growth or proliferation disease or disorder symptoms. For example,
such cell systems may be exposed to a compound, suspected of
exhibiting an ability to ameliorate cellular growth or
proliferation disease or disorder symptoms, at a sufficient
concentration and for a time sufficient to elicit such an
amelioration of cellular growth or proliferation disease or
disorder symptoms in the exposed cells. After exposure, the cells
are examined to determine whether one or more of the cellular
growth or proliferation disease or disorder cellular phenotypes has
been altered to resemble a more normal or more wild type, non-
cellular growth or proliferation disease or disorder phenotype.
Cellular phenotypes that are associated with cellular growth and/or
proliferation disease states include aberrant proliferation,
growth, and migration, anchorage independent growth, and loss of
contact inhibition.
[0200] In addition, animal-based cellular growth or proliferation
disease or disorder systems, such as those described herein, may be
used to identify compounds capable of ameliorating cellular growth
or proliferation disease or disordel symptoms. Such animal models
may be used as test substrates for the identification of drugs,
pharmaceuticals, therapies, and interventions which may be
effective in treating cellular growth or proliferation disorders or
diseases. For example, animal models may be exposed to a compound,
suspected of exhibiting an ability to cellular growth or
proliferation disease or disorder symptoms, at a sufficient
concentration and for a time sufficient to elicit such an
amelioration of cellular growth or proliferation disease or
disorder symptoms in the exposed animals. The response of the
animals to the exposure may be monitored by assessing the reversal
of disorders or symptoms associated with cellular growth or
proliferation disease, for example, reduction in tumor burden,
tumor size, and invasive and/or metastatic potential before and
after treatment.
[0201] With regard to intervention, any treatments which reverse
any aspect of cellular growth or proliferation disease or disorder
symptoms should be considered as candidates for human cellular
growth or proliferation disease or disorder therapeutic
intervention. Dosages of test agents may be determined by deriving
dose-response curves.
[0202] Additionally, gene expression patterns may be utilized to
assess the ability of a compound to ameliorate cellular growth
and/or proliferation disease symptoms. For example, the expression
pattern of one or more genes may form part of a "gene expression
profile" or "transcriptional profile" which may be then be used in
such an assessment. "Gene expression profile" or "transcriptional
profile", as used herein, includes the pattern of mRNA expression
obtained for a given tissue or cell type under a given set of
conditions. Such conditions may include, but are not limited to,
cell growth, proliferation, differentiation, transformation,
tumorigenesis, metastasis, and carcinogen exposure. Gene expression
profiles may be generated, for example, by utilizing a differential
display procedure, Northern analysis and/or RT-PCR. In one
embodiment, BRE gene sequences may be used as probes and/or PCR
primers for the generation and corroboration of such gene
expression profiles.
[0203] Gene expression profiles may be characterized for known
states within the cell- and/or animal-based model systems.
Subsequently, these known gene expression profiles may be compared
to ascertain the effect a test compound has to modify such gene
expression profiles, and to cause the profile to more closely
resemble that of a more desirable profile.
[0204] For example, administration of a compound may cause the gene
expression profile of a cellular growth or proliferation disease or
disorder model system to more closely resemble the control system.
Administration of a compound may, alternatively, cause the gene
expression profile of a control system to begin to mimic a cellular
growth and/or proliferation disease state. Such a compound may, for
example, be used in further characterizing the compound of
interest, or may be used in the generation of additional animal
models.
[0205] B. Detection Assays
[0206] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of
a biological sample. These applications are described in the
subsections below.
[0207] 1. Chromosome Mapping
[0208] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the BRE nucleotide
sequences, described herein, can be used to map the location of the
BRE genes on a chromosome. The mapping of the BRE sequences to
chromosomes is an important first step in correlating these
sequences with genes associated with disease.
[0209] Briefly, BRE genes can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp in length) from the BRE nucleotide
sequences. Computer analysis of the BRE sequences can be used to
predict primers that do not span more than one exon in the genomic
DNA, thus complicating the amplification process. These primers can
then be used for PCR screening of somatic cell hybrids containing
individual human chromosomes. Only those hybrids containing the
human gene corresponding to the BRE sequences will yield an
amplified fragment.
[0210] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio P. et al. (1983)
Science 220:919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[0211] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the BRE nucleotide sequences to design
oligonucleotide primers, sublocalization can be achieved with
panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a BRE sequence to its
chromosome include in situ hybridization (described in Fan, Y. et
al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening
with labeled flow-sorted chromosomes, and pre-selection by
hybridization to chromosome specific cDNA libraries.
[0212] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical such as colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual of Basic
Techniques (Pergamon Press, New York 1988).
[0213] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0214] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[0215] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the BRE gene can be determined. If a mutation is observed in some
or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0216] 2. Tissue Typing
[0217] The BRE sequences of the present invention can also be used
to identify individuals from minute biological samples. The United
States military, for example, is considering the use of restriction
fragment length polymorphism (RFLP) for identification of its
personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0218] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the BRE nucleotide sequences described
herein can be used to prepare two PCR primers from the 5' and 3'
ends of the sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0219] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The BRE nucleotide
sequences of the invention uniquely represent portions of the human
genome. Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. It is estimated that allelic variation between
individual humans occurs with a frequency of about once per each
500 bases. Each of the sequences described herein can, to some
degree, be used as a standard against which DNA from an individual
can be compared for identification purposes. Because greater
numbers of polymorphisms occur in the noncoding regions, fewer
sequences are necessary to differentiate individuals. The noncoding
sequences of SEQ ID NO:1 can comfortably provide positive
individual identification with a panel of perhaps 10 to 1,000
primers which each yield a noncoding amplified sequence of 100
bases. If predicted coding sequences, such as those in SEQ ID NO:3
or 6 are used, a more appropriate number of primers for positive
individual identification would be 500-2,000.
[0220] If a panel of reagents from BRE nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0221] 3. Use of BRE Sequences in Forensic Biology
[0222] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0223] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of SEQ ID NO:1 are particularly appropriate for
this use as greater numbers of polymorphisms occur in the noncoding
regions, making it easier to differentiate individuals using this
technique. Examples of polynucleotide reagents include the BRE
nucleotide sequences or portions thereof, e.g., fragments derived
from the noncoding regions of SEQ ID NO:1 having a length of at
least 20 bases, preferably at least 30 bases.
[0224] The BRE nucleotide sequences described herein can further be
used to provide polynucleotide reagents, e.g., labeled or labelable
probes which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue, e.g., thymus or brain
tissue. This can be very useful in cases where a forensic
pathologist is presented with a tissue of unknown origin. Panels of
such BRE probes can be used to identify tissue by species and/or by
organ type.
[0225] In a similar fashion, these reagents, e.g., BRE primers or
probes can be used to screen tissue culture for contamination (i.e.
screen for the presence of a mixture of different types of cells in
a culture).
[0226] C. Predictive Medicine:
[0227] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining BRE protein and/or nucleic acid
expression as well as BRE activity, in the context of a biological
sample (e.g., blood, serum, cells, tissue) to thereby determine
whether an individual is afflicted with a disease or disorder, or
is at risk of developing a disorder, associated with aberrant or
unwanted BRE expression or activity. The invention also provides
for prognostic (or predictive) assays for determining whether an
individual is at risk of developing a disorder associated with BRE
protein, nucleic acid expression or activity. For example,
mutations in a BRE gene can be assayed in a biological sample. Such
assays can be used for prognostic or predictive purpose to thereby
phophylactically treat an individual prior to the onset of a
disorder characterized by or associated with BRE protein, nucleic
acid expression or activity.
[0228] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on the expression or
activity of BRE in clinical trials.
[0229] These and other agents are described in further detail in
the following sections.
[0230] 1. Diagnostic Assays
[0231] The present invention encompasses methods for diagnostic and
prognostic evaluation of cellular growth or proliferation disorders
or diseases, e.g., cancer, including, but not limited to colon
cancer and lung cancer, and for the identification of subjects
exhibiting a predisposition to such conditions.
[0232] An exemplary method for detecting the presence or absence of
BRE protein or nucleic acid in a biological sample involves
obtaining a biological sample from a test subject and contacting
the biological sample with a compound or an agent capable of
detecting BRE protein or nucleic acid (e.g., mRNA, or genomic DNA)
that encodes BRE protein such that the presence of BRE protein or
nucleic acid is detected in the biological sample. A preferred
agent for detecting BRE mRNA or genomic DNA is a labeled nucleic
acid probe capable of hybridizing to BRE niRNA or genomic DNA. The
nucleic acid probe can be, for example, the BRE nucleic acid set
forth in SEQ ID NO:1 or 3, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and sufficient to specifically hybridize under stringent
conditions to BRE mRNA or genomic DNA. Other suitable probes for
use in the diagnostic assays of the invention are described
herein.
[0233] A preferred agent for detecting BRE protein is an antibody
capable of binding to BRE protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab')2) can be used. The term "labeled", with regard to the probe
or antibody, is intended to encompass direct labeling of the probe
or antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect BRE mRNA, protein, or
genomic DNA in a biological sample in vitro as well as in vivo. For
example, in vitro techniques for detection of BRE mRNA include
Northern hybridizations and in situ hybridizations. In vitro
techniques for detection of BRE protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. In vitro techniques for detection of BRE
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of BRE protein include introducing into a
subject a labeled anti-BRE antibody. For example, the antibody can
be labeled with a radioactive marker whose presence and location in
a subject can be detected by standard imaging techniques.
[0234] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a serum sample isolated by conventional means from a
subject.
[0235] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting BRE
protein, mRNA, or genomic DNA, such that the presence of BRE
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of BRE protein, mRNA or genomic DNA in
the control sample with the presence of BRE protein, mRNA or
genomic DNA in the test sample.
[0236] The invention also encompasses kits for detecting the
presence of BRE in a biological sample. For example, the kit can
comprise a labeled compound or agent capable of detecting BRE
protein or mRNA in a biological sample; means for determining the
amount of BRE in the sample; and means for comparing the amount of
BRE in the sample with a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect BRE protein or nucleic
acid.
[0237] In one embodiment, increased levels of BRE protein, mRNA or
DNA (e.g., cDNA or genomic DNA) in the test sample as compared to
the control sample is determinative or predictive of a BRE-related
aberrancy (e.g., a cellular growth or proliferation disease or
disorder, for example, cancer). For example, 2-fold levels of
expression of BRE in the test sample as compared to the control
sample may be determinative or predictive of a BRE-related
aberrancy. Preferably, 5-fold, 10-fold, 100-fold, 500-fold or
1000-fold levels of expression of BRE in the test sample as
compared to the control sample may be determinative or predictive
of a BRE-related aberrancy.
[0238] 2. Prognostic Assays
[0239] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
disease or disorder associated with aberrant or unwanted BRE
expression or activity. As used herein, the term "aberrant"
includes a BRE expression or activity which deviates from the wild
type BRE expression or activity. Aberrant expression or activity
includes increased or decreased expression or activity, as well as
expression or activity which does not follow the wild type
developmental pattern of expression or the subcellular pattern of
expression. For example, aberrant BRE expression or activity is
intended to include the cases in which a mutation in the BRE gene
causes the BRE gene to be under-expressed or over-expressed and
situations in which such mutations result in a non-functional BRE
protein or a protein which does not function in a wild-type
fashion, e.g., a protein which does not interact with a BRE
substrate, or one which interacts with a non-BRE substrate. As used
herein, the term "unwanted" includes an unwanted phenomenon
involved in a biological response such as cellular proliferation.
For example, the term unwanted includes a BRE expression or
activity which is undesirable in a subject.
[0240] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a disorder
associated with a misregulation in BRE protein activity or nucleic
acid expression, such as a CNS disorder (e.g., a cognitive or
neurodegenerative disorder), a cellular proliferation, growth,
differentiation, or migration disorder, a cardiovascular disorder,
musculoskeletal disorder, an immune disorder, or a hormonal
disorder. Alternatively, the prognostic assays can be utilized to
identify a subject having or at risk for developing a disorder
associated with a misregulation in BRE protein activity or nucleic
acid expression, such as a CNS disorder, a cellular proliferation,
growth, differentiation, or migration disorder, a musculoskeletal
disorder, a cardiovascular disorder, an immune disorder, or a
hormonal disorder. Thus, the present invention provides a method
for identifying a disease or disorder associated with aberrant or
unwanted BRE expression or activity in which a test sample is
obtained from a subject and BRE protein or nucleic acid (e.g., mRNA
or genomic DNA) is detected, wherein the presence of BRE protein or
nucleic acid is diagnostic for a subject having or at risk of
developing a disease or disorder associated with aberrant or
unwanted BRE expression or activity. As used herein, a "test
sample" refers to a biological sample obtained from a subject of
interest. For example, a test sample can be a biological fluid
(e.g., cerebrospinal fluid or serum), cell sample, or tissue.
[0241] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant or unwanted BRE
expression or activity. For example, such methods can be used to
determine whether a subject can be effectively treated with an
agent for a CNS disorder, a muscular disorder, a cellular
proliferation, growth, differentiation, or migration disorder, an
immune disorder, or a hormonal disorder. Thus, the present
invention provides methods for determining whether a subject can be
effectively treated with an agent for a disorder associated with
aberrant or unwanted BRE expression or activity in which a test
sample is obtained and BRE protein or nucleic acid expression or
activity is detected (e.g., wherein the abundance of BRE protein or
nucleic acid expression or activity is diagnostic for a subject
that can be administered the agent to treat a disorder associated
with aberrant or unwanted BRE expression or activity).
[0242] The methods of the invention can also be used to detect
genetic alterations in a BRE gene, thereby determining if a subject
with the altered gene is at risk for a disorder characterized by
misregulation in BRE protein activity or nucleic acid expression,
such as a CNS disorder, a musculoskeletal disorder, a cellular
proliferation, growth, differentiation, or migration disorder, a
cardiovascular disorder, an immune disorder, or a hormonal
disorder. In preferred embodiments, the methods include detecting,
in a sample of cells from the subject, the presence or absence of a
genetic alteration characterized by at least one of an alteration
affecting the integrity of a gene encoding a BRE-protein, or the
mis-expression of the BRE gene. For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of 1) a deletion of one or more nucleotides from a BRE
gene; 2) an addition of one or more nucleotides to a BRE gene; 3) a
substitution of one or more nucleotides of a BRE gene, 4) a
chromosomal rearrangement of a BRE gene; 5) an alteration in the
level of a messenger RNA transcript of a BRE gene, 6) aberrant
modification of a BRE gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of a BRE gene, 8) a non-wild
type level of a BRE-protein, 9) allelic loss of a BRE gene, and 10)
inappropriate post-translational modification of a BRE-protein. As
described herein, there are a large number of assays known in the
art which can be used for detecting alterations in a BRE gene. A
preferred biological sample is a tissue or serum sample isolated by
conventional means from a subject.
[0243] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364),
the latter of which can be particularly useful for detecting point
mutations in a BRE gene (see Abravaya et al. (1995) Nucleic Acids
Res 23:675-682). This method can include the steps of collecting a
sample of cells from a subject, isolating nucleic acid (e.g.,
genomic, mRNA or both) from the cells of the sample, contacting the
nucleic acid sample with one or more primers which specifically
hybridize to a BRE gene under conditions such that hybridization
and amplification of the BRE gene (if present) occurs, and
detecting the presence or absence of an amplification product, or
detecting the size of the amplification product and comparing the
length to a control sample. It is anticipated that PCR and/or LCR
may be desirable to use as a preliminary amplification step in
conjunction with any of the techniques used for detecting mutations
described herein.
[0244] Alternative amplification methods include: self sustained
sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh, D. Y. etal., (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988)
Bio-Technology 6:1197), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers.
[0245] In an alternative embodiment, mutations in a BRE gene from a
sample cell can be identified by alterations in restriction enzyme
cleavage patterns. For example, sample and control DNA is isolated,
amplified (optionally), digested with one or more restriction
endonucleases, and fragment length sizes are determined by gel
electrophoresis and compared. Differences in fragment length sizes
between sample and control DNA indicates mutations in the sample
DNA. Moreover, the use of sequence specific ribozymes (see, for
example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0246] In other embodiments, genetic mutations in BRE can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high density arrays containing hundreds or thousands
of oligonucleotides probes (Cronin, M. T. et al. (1996) Human
Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:
753-759). For example, genetic mutations in BRE can be identified
in two dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0247] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the BRE
gene and detect mutations by comparing the sequence of the sample
BRE with the corresponding wild-type (control) sequence. Examples
of sequencing reactions include those based on techniques developed
by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or
Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also
contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Biotechniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT International Publication No. WO
94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and
Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
[0248] Other methods for detecting mutations in the BRE gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes of formed by
hybridizing (labeled) RNA or DNA containing the wild-type BRE
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digesting the mismatched regions. In other embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or
osmium tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992)
Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[0249] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in BRE
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a BRE sequence, e.g., a wild-type BRE
sequence, is hybridized to a cDNA or other DNA product from a test
cell(s). The duplex is treated with a DNA mismatch repair enzyme,
and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[0250] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in BRE genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144;
and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control BRE nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[0251] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[0252] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[0253] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0254] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a BRE gene.
[0255] Furthermore, any cell type or tissue in which BRE is
expressed may be utilized in the prognostic assays described
herein.
[0256] 3. Monitoring of Effects During Clinical Trials
[0257] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a BRE protein (e.g., the maintainence of
cellular homeostasis) can be applied not only in basic drug
screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as
described herein to increase BRE gene expression, protein levels,
or upregulate BRE activity, can be monitored in clinical trials of
subjects exhibiting decreased BRE gene expression, protein levels,
or downregulated BRE activity. Alternatively, the effectiveness of
an agent determined by a screening assay to decrease BRE gene
expression, protein levels, or downregulate BRE activity, can be
monitored in clinical trials of subjects exhibiting increased BRE
gene expression, protein levels, or upregulated BRE activity. In
such clinical trials, the expression or activity of a BRE gene, and
preferably, other genes that have been implicated in, for example,
a BRE-associated disorder can be used as a "read out" or markers of
the phenotype of a particular cell.
[0258] For example, and not by way of limitation, genes, including
BRE, that are modulated in cells by treatment with an agent (e.g.,
compound, drug or small molecule) which modulates BRE activity
(e.g., identified in a screening assay as described herein) can be
identified. Thus, to study the effect of agents on BRE-associated
disorders (e.g., disorders characterized by deregulated cell
proliferation and/or migration), for example, in a clinical trial,
cells can be isolated and RNA prepared and analyzed for the levels
of expression of BRE and other genes implicated in the
BRE-associated disorder, respectively. The levels of gene
expression (e.g., a gene expression pattern) can be quantified by
northern blot analysis or RT-PCR, as described herein, or
alternatively by measuring the amount of protein produced, by one
of the methods as described herein, or by measuring the levels of
activity of BRE or other genes. In this way, the gene expression
pattern can serve as a marker, indicative of the physiological
response of the cells to the agent. Accordingly, this response
state may be determined before, and at various points during
treatment of the individual with the agent.
[0259] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
including the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a BRE protein, mRNA, or genomic DNA in
the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the BRE protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the BRE protein, mRNA, or
genomic DNA in the pre-administration sample with the BRE protein,
mRNA, or genomic DNA in the post administration sample or samples;
and (vi) altering the administration of the agent to the subject
accordingly. For example, increased administration of the agent may
be desirable to increase the expression or activity of BRE to
higher levels than detected, i.e., to increase the effectiveness of
the agent. Alternatively, decreased administration of the agent may
be desirable to decrease expression or activity of BRE to lower
levels than detected, i.e. to decrease the effectiveness of the
agent. According to such an embodiment, BRE expression or activity
may be used as an indicator of the effectiveness of an agent, even
in the absence of an observable phenotypic response.
[0260] D. Methods of Treatment:
[0261] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted BRE expression or activity, e.g., a
biotin-associated disorder such as a CNS disorder; a cellular
proliferation, growth, differentiation, or migration disorder; a,
musculoskeletal disorder; a cardiovascular disorder; an immune
disorder; or a hormonal disorder. With regard to both prophylactic
and therapeutic methods of treatment, such treatments may be
specifically tailored or modified, based on knowledge obtained from
the field of pharmacogenomics. "Pharmacogenomics", as used herein,
refers to the application of genomics technologies such as gene
sequencing, statistical genetics, and gene expression analysis to
drugs in clinical development and on the market. More specifically,
the term refers the study of how a patient's genes determine his or
her response to a drug (e.g., a patient's "drug response
phenotype", or "drug response genotype"). Thus, another aspect of
the invention provides methods for tailoring an individual's
prophylactic or therapeutic treatment with either the BRE molecules
of the present invention or BRE modulators according to that
individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
[0262] 1. Prophylactic Methods
[0263] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted BRE expression or activity, by administering
to the subject a BRE or an agent which modulates BRE expression or
at least one BRE activity. Subjects at risk for a disease which is
caused or contributed to by aberrant or unwanted BRE expression or
activity can be identified by, for example, any or a combination of
diagnostic or prognostic assays as described herein. Administration
of a prophylactic agent can occur prior to the manifestation of
symptoms characteristic of the BRE aberrancy, such that a disease
or disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of BRE aberrancy, for example, a
BRE, BRE agonist or BRE antagonist agent can be used for treating
the subject. The appropriate agent can be determined based on
screening assays described herein.
[0264] 2. Therapeutic Methods
[0265] Another aspect of the invention pertains to methods of
modulating BRE expression or activity for therapeutic purposes.
Accordingly, in an exemplary embodiment, the modulatory method of
the invention involves contacting a cell with a BRE or agent that
modulates one or more of the activities of BRE protein activity
associated with the cell. An agent that modulates BRE protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring substrate molecule of a
BRE protein (e.g., energy transduction metabolites, urea cycle
metabolites, lipid metabolism metabolites, amino acid precursors,
nucleic acid precursors), a BRE antibody, a BRE agonist or
antagonist, a peptidomimetic of a BRE agonist or antagonist, or
other small molecule. In one embodiment, the agent stimulates one
or more BRE activities. Examples of such stimulatory agents include
active BRE protein and a nucleic acid molecule encoding BRE that
has been introduced into the cell. In another embodiment, the agent
inhibits one or more BRE activities. Examples of such inhibitory
agents include antisense BRE nucleic acid molecules, anti-BRE
antibodies, and BRE inhibitors. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant or unwanted expression or activity of a
BRE protein or nucleic acid molecule. In one embodiment, the method
involves administering an agent (e.g., an agent identified by a
screening assay described herein), or combination of agents that
modulates (e.g., upregulates or downregulates) BRE expression or
activity. In another embodiment, the method involves administering
a BRE protein or nucleic acid molecule as therapy to compensate for
reduced, aberrant, or unwanted BRE expression or activity.
[0266] Stimulation of BRE activity is desirable in situations in
which BRE is abnormally downregulated and/or in which increased BRE
activity is likely to have a beneficial effect. Likewise,
inhibition of BRE activity is desirable in situations in which BRE
is abnormally upregulated and/or in which decreased BRE activity is
likely to have a beneficial effect.
[0267] (i) Methods for Inhibiting Target Gene Expression,
Synthesis, or Activity
[0268] As discussed above, genes involved in cellular growth or
proliferation diseases or disorders may cause such disorders via an
increased level of gene activity. In some cases, such up-regulation
may have a causative or exacerbating effect on the disease state. A
variety of techniques may be used to inhibit the expression,
synthesis, or activity of such genes and/or proteins.
[0269] For example, compounds such as those identified through
assays described above, which exhibit inhibitory activity, may be
used in accordance with the invention to ameliorate cellular growth
or proliferation disease or disorder symptoms. Such molecules may
include, but are not limited to, small organic molecules, peptides,
antibodies, and the like.
[0270] For example, compounds can be administered that compete with
endogenous ligand for the BRE protein. The resulting reduction in
the amount of ligand-bound BRE protein will modulate endothelial
cell physiology. Compounds that can be particularly useful for this
purpose include, for example, soluble proteins or peptides, such as
peptides comprising one or more of the extracellular domains, or
portions and/or analogs thereof, of the BRE protein, including, for
example, soluble fusion proteins such as Ig-tailed fusion proteins.
(For a discussion of the production of Ig-tailed fusion proteins,
see, for example, U.S. Pat. No. 5,116,964). Alternatively,
compounds, such as ligand analogs or antibodies, that bind to the
BRE receptor site, but do not activate the protein, (e.g.,
receptor-ligand antagonists) can be effective in inhibiting BRE
protein activity.
[0271] Further, antisense and ribozyme molecules, as described
herein, which inhibit expression of the BRE gene may also be used
in accordance with the invention to inhibit aberrant BRE gene
activity. Still further, triple helix molecules may be utilized in
inhibiting aberrant BRE gene activity.
[0272] Antibodies that are both specific for the BRE protein and
interfere with its activity may also be used to modulate or inhibit
BRE protein function. Such antibodies may be generated using
standard techniques described herein, against the BRE protein
itself or against peptides corresponding to portions of the
protein. Such antibodies include but are not limited to polyclonal,
monoclonal, Fab fragments, single chain antibodies, or chimeric
antibodies.
[0273] In instances where the target gene protein is intracellular
and whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin liposomes may be used to deliver the antibody
or a fragment of the Fab region which binds to the target epitope
into cells. Where fragments of the antibody are used, the smallest
inhibitory fragment which binds to the target protein's binding
domain is preferred. For example, peptides having an amino acid
sequence corresponding to the domain of the variable region of the
antibody that binds to the target gene protein may be used. Such
peptides may be synthesized chemically or produced via recombinant
DNA technology using methods well known in the art (described in,
for example, Creighton (1983), supra; and Sambrook et al. (1989)
supra). Single chain neutralizing antibodies which bind to
intracellular target gene epitopes may also be administered. Such
single chain antibodies may be administered, for example, by
expressing nucleotide sequences encoding single-chain antibodies
within the target cell population by utilizing, for example,
techniques such as those described in Marasco et al. (1993) Proc.
Natl. Acad. Sci. USA 90:7889-7893).
[0274] Any of the administration techniques described below which
are appropriate for peptide administration may be utilized to
effectively administer inhibitory target gene antibodies to their
site of action.
[0275] (ii) Methods for Restoring or Enhancing Target Gene
Activity
[0276] Genes that cause cellular growth or proliferation diseases
or disorders may be underexpressed within cellular growth or
proliferative situations. Alternatively, the activity of the
protein products of such genes may be decreased, leading to the
development of cellular growth or proliferation disease or disorder
symptoms. Such down-regulation of gene expression or decrease of
protein activity might have a causative or exacerbating effect on
the disease state.
[0277] In some cases, genes that are up-regulated in the disease
state might be exerting a protective effect. A variety of
techniques may be used to increase the expression, synthesis, or
activity of genes and/or proteins that exert a protective effect in
response to cellular growth or proliferation disease or disorder
conditions.
[0278] Described in this section are methods whereby the level BRE
activity may be increased to levels wherein cellular growth or
proliferation disease or disorder symptoms are ameliorated. The
level of BRE activity may be increased, for example, by either
increasing the level of BRE gene expression or by increasing the
level of active BRE protein which is present.
[0279] For example, a BRE protein, at a level sufficient to
ameliorate cellular growth or proliferation disease or disorder
symptoms may be administered to a patient exhibiting such symptoms.
Any of the techniques discussed below may be used for such
administration. One of skill in the art will readily be able to
ascertain the concentration of effective, non-toxic doses of the
BRE protein, utilizing techniques such as those described
above.
[0280] Additionally, RNA sequences encoding a BRE protein may be
directly administered to a patient exhibiting cellular growth or
proliferation disease or disorder symptoms, at a concentration
sufficient to produce a level of BRE protein such that cellular
growth or proliferation disease or disorder symptoms are
ameliorated. Any of the techniques discussed below, which achieve
intracellular administration of compounds, such as, for example,
liposome administration, may be used for the administration of such
RNA molecules. The RNA molecules may be produced, for example, by
recombinant techniques such as those described herein.
[0281] Further, subjects may be treated by gene replacement
therapy. One or more copies of a BRE gene, or a portion thereof,
that directs the production of a normal BRE protein with BRE
function, may be inserted into cells using vectors which include,
but are not limited to adenovirus, adeno-associated virus, and
retrovirus vectors, in addition to other particles that introduce
DNA into cells, such as liposomes. Additionally, techniques such as
those described above may be used for the introduction of BRE gene
sequences into human cells.
[0282] Cells, preferably, autologous cells, containing BRE
expressing gene sequences may then be introduced or reintroduced
into the subject at positions which allow for the amelioration of
cellular growth or proliferation disease or disorder symptoms. Such
cell replacement techniques may be preferred, for example, when the
gene product is a secreted, extracellular gene product.
[0283] 3. Pharmacogenomics
[0284] The BRE molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on BRE activity (e.g., BRE gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) BRE-associated
disorders (e.g., proliferative disorders, CNS disorders, cardiac
disorders, metabolic disorders, or muscular disorders) associated
with aberrant or unwanted BRE activity. In conjunction with such
treatment, pharmacogenomics (i.e., the study of the relationship
between an individual's genotype and that individual's response to
a foreign compound or drug) may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a
physician or clinician may consider applying knowledge obtained in
relevant pharmacogenomics studies in determining whether to
administer a BRE molecule or BRE modulator as well as tailoring the
dosage and/or therapeutic regimen of treatment with a BRE molecule
or BRE modulator.
[0285] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp.Pharmacol. Physiol.
23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem.
43(2):254-266. In general, two types of pharmacogenetic conditions
can be differentiated. Genetic conditions transmitted as a single
factor altering the way drugs act on the body (altered drug action)
or genetic conditions transmitted as single factors altering the
way the body acts on drugs (altered drug metabolism). These
pharmacogenetic conditions can occur either as rare genetic defects
or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0286] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0287] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug
target is known (e.g., a BRE protein of the present invention), all
common variants of that gene can be fairly easily identified in the
population and it can be determined if having one version of the
gene versus another is associated with a particular drug
response.
[0288] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0289] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a BRE molecule or BRE modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0290] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a BRE molecule or BRE modulator, such
as a modulator identified by one of the exemplary screening assays
described herein.
[0291] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Figures, are
incorporated herein by reference.
EXAMPLES
Example 1
Identification and Characterization of Human BRE cDNA
[0292] In this example, the identification and characterization of
the gene encoding human BRE (clone Fbh32263) is described.
Isolation of the BRE cDNA
[0293] The invention is based, at least in part, on the discovery
of a human genes encoding a novel protein, referred to herein as
BRE. The entire sequence of human clones Fbh32263, was determined
and found to contain an open reading frame termed human "BRE", set
forth in FIGS. 1A-1E. The amino acid sequence of the human BRE
expression product is set forth in FIGS. 1A-1E. The BRE protein
sequence set forth in SEQ ID NO:2 comprises about 2577 amino acids
and is shown in FIGS. 1A-1E. The coding region (open reading frame)
of SEQ ID NO:1, is set forth as SEQ ID NO:3.
Analysis of the Human BRE Molecule
[0294] An analysis of the possible cellular localization of the BRE
protein based on its amino acid sequence was performed using the
methods and algorithms described in Nakai and Kanehisa (1992)
Genomics 14:897-911, and at http://psort.nibb.ac jp. The results of
the analysis predict that human BRE (SEQ ID NO:2) is localized
intracellularly (probablilities are shown for localization to e.g.,
73.9% in the mitochondria, 13.0% in the cytoplasm, 4.3% in the
Golgi apparatus, 4.3% in extracellular spaces (e.g., cell wall),
and 4.3% in the endoplasmic reticulum).
[0295] A search of the amino acid sequence of BRE was also
performed against the HMM database (FIGS. 3A-3F). This search
resulted in the identification of a "carbamoyl-phosphate synthase L
chain, N-terminal domain" ("CPSase N-terminal domain") in the amino
acid sequence of BRE (SEQ ID NO:2) at about residues 48-160 (score:
184.0). This search also resulted in the identification of a
"carbamoyl-phosphate synthase L chain, ATP binding domain" ("CPSase
ATP-binding domain") in the amino acid sequence of BRE (SEQ ID
NO:2) at about residues 163-376, which is characterized by a
"carbamoyl phosphate synthase subdomain signature". This domain is
implicated in ATP binding and/or catalytic activity. This search
also resulted in the identification of a "biotin/lipoyl attachment
domain" ("Biotin requiring enzyme domain") in the amino acid
sequence of BRE (SEQ ID NO:2) at about residues 650-714 (score:
67.8). This domain binds biotin and contains, or can be
characterized by, the presence of a "biotin requiring enzyme
attachment site", which itself is characterized by the inclusion of
a conserved lysine residue. This search also resulted in the
identification of a "Biotin carboxylase C-terminal domain" in the
amino acid sequence of BRE (SEQ ID NO:2) at about residues 383-490,
which is implicated in enzymatic activity. This search also
resulted in the identification of a "D-ala D-ala ligase" in the
amino acid sequence of BRE (SEQ ID NO:2) at about residues 163-233
(score: 11.1).
[0296] Further domain motifs were identified by using the amino
acid sequence of BRE (SEQ ID NO:2) to search through the ProDom
database (http://protein.toulouse.inra.fr/ prodom.html). Numerous
matches against protein domains described as "Biotin synthetase,
Acetyl-CoA biotin ligase, Biotin dihydrolipoaide pyruvate
dehydrogenase carboxylase, and the like were identified. A search
was also performed against the Prosite database, and resulted in
the identification of a "biotin requiring enzyme attachment site"
at residues 671- 688, (Prosite accession number PS00188).
[0297] A structural, hydrophobicity, and antigenicity analysis of
the human Fbh32263 protein was undertaken. The results of this
analysis are set forth in FIGS. 2A-2B.
[0298] A global comparison of human BRE (SEQ ID NO:2, depicted as
"32263.pro") with known transcarboxylases was completed. The
results of this alignment are set forth in FIGS. 4A-4D. The known
transcarboxylases used in the comparison are 3-methylcrotonyl-CoA
carboxylase precursor from Arabidopsis (SEQ ID NO:4, GenBank No.
AAA67356; depicted as "thal.pro"); a protein similar to
propionyl-CoA carboxylase alpha chain from C. elegans (SEQ ID NO:5,
GenBank No. AAA93384; depicted as "celegans.pro"); and
proprionyl-CoA carboxylase alpha chain precursor from H. sapiens
(SEQ ID NO:6, GenBank No. P05165; depicted as "human.pro"). The
CPSase domain of the human BRE is indicated in italics. The
biotin-requiring enzyme domain of the human BRE is underlined.
Example 2
Expression of Recombinant BRE Protein in Bacterial Cells
[0299] In this example, BRE is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
BRE is fused to GST and this fusion polypeptide is expressed in E.
coli, e.g., strain PEB199. Expression of the GST-BRE fusion protein
in PEB199 is induced with IPTG. The recombinant fusion polypeptide
is purified from crude bacterial lysates of the induced PEB199
strain by affinity chromatography on glutathione beads. Using
polyacrylamide gel electrophoretic analysis of the polypeptide
purified from the bacterial lysates, the molecular weight of the
resultant fusion polypeptide is determined.
Example 3
Expression of Recombinant BRE Protein in COS Cells
[0300] To express the BRE gene in COS cells, the pcDNA/Amp vector
by Invitrogen Corporation (San Diego, Calif.) is used. This vector
contains an SV40 origin of replication, an ampicillin resistance
gene, an E. coli replication origin, a CMV promoter followed by a
polylinker region, and an SV40 intron and polyadenylation site. A
DNA fragment encoding the entire BRE protein and an HA tag (Wilson
et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3'
end of the fragment is cloned into the polylinker region of the
vector, thereby placing the expression of the recombinant protein
under the control of the CMV promoter.
[0301] To construct the plasmid, the BRE DNA sequence is amplified
by PCR using two primers. The 5' primer contains the restriction
site of interest followed by approximately twenty nucleotides of
the BRE coding sequence starting from the initiation codon; the 3'
end sequence contains complementary sequences to the other
restriction site of interest, a translation stop codon, the HA tag
or FLAG tag and the last 20 nucleotides of the BRE coding sequence.
The PCR amplified fragment and the pCDNA/Amp vector are digested
with the appropriate restriction enzymes and the vector is
dephosphorylated using the CIAP enzyme (New England Biolabs,
Beverly, Mass.). Preferably the two restriction sites chosen are
different so that the BRE gene is inserted in the correct
orientation. The ligation mixture is transformed into E. coli cells
(strains HB101, DH5.alpha., SURE, available from Stratagene Cloning
Systems, La Jolla, Calif., can be used), the transformed culture is
plated on ampicillin media plates, and resistant colonies are
selected. Plasmid DNA is isolated from transformants and examined
by restriction analysis for the presence of the correct
fragment.
[0302] COS cells are subsequently transfected with the
BRE-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium
chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of
the BRE polypeptide is detected by radiolabelling
(.sup.35S-methionine or .sup.35S-cysteine available from NEN,
Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and
Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA
specific monoclonal antibody. Briefly, the cells are labeled for 8
hours with .sup.35S-methionine (or .sup.35S-cysteine). The culture
media are then collected and the cells are lysed using detergents
(RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM
Tris, pH 7.5). Both the cell lysate and the culture media are
precipitated with an HA-specific monoclonal antibody. Precipitated
polypeptides are then analyzed by SDS-PAGE.
[0303] Alternatively, DNA containing the BRE coding sequence is
cloned directly into the polylinker of the pCDNA/Amp vector using
the appropriate restriction sites. The resulting plasmid is
transfected into COS cells in the manner described above, and the
expression of the BRE polypeptide is detected by radiolabelling and
immunoprecipitation using a BRE specific monoclonal antibody.
Example 3
Tissue Distribution of BRE by Taqman Expression Analysis
Tissue Expression Analysis of BRE mRNA Using Tagman Analysis
[0304] This example describes the tissue distribution of human BRE
mRNA (huBRE) in a variety of cells and tissues, as determined using
the TaqMan.TM. procedure. The Taqman.TM. procedure is a
quantitative, reverse transcription PCR-based approach for
detecting mRNA. The RT-PCR reaction exploits the 5' nuclease
activity of AmpliTaq Gold.TM. DNA Polymerase to cleave a TaqMan.TM.
probe during PCR. Briefly, cDNA was generated from the samples of
interest, e.g., tumor samples and normal samples, cell lines and
the like,and used as the starting material for PCR amplification.
In addition to the 5' and 3' gene-specific primers, a gene-specific
oligonucleotide probe (complementary to the region being amplified)
was included in the reaction (i.e., the Taqman.TM. probe). The
TaqMan.TM. probe includes the oligonucleotide with a fluorescent
reporter dye covalently linked to the 5' end of the probe (such as
FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2',7'-
tetrachlorofluorescein), JOE
(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a
quencher dye (TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at
the 3' end of the probe.
[0305] When the fluorescently tagged oligonucleotide is intact, the
fluorescent signal from the 5' dye is quenched. As PCR proceeds,
the 5' to 3' nucleolytic activity of taq polymerase digests the
labeled primer, producing a free nucleotide labeled with 6-FAM,
which is now detected as a fluorescent signal. The PCR cycle where
fluorescence is first released and detected is directly
proportional to the starting amount of the gene of interest in the
test sample, thus providing a way of quantitating the initial
template concentration. Accumulation of PCR products is detected
directly by monitoring the increase in fluorescence of the reporter
dye. When the probe is intact, the proximity of the reporter dye to
the quencher dye results in suppression of the reporter
fluorescence. During PCR, if the target of interest is present, the
probe specifically anneals between the forward and reverse primer
sites. The 5'-3' nucleolytic activity of the AmpliTaq.TM. Gold DNA
Polymerase cleaves the probe between the reporter and the quencher
only if the probe hybridizes to the target. The probe fragments are
then displaced from the target, and polymerization of the strand
continues. The 3' end of the probe is blocked to prevent extension
of the probe during PCR. This process occurs in every cycle and
does not interfere with the exponential accumulation of product.
Samples can be internally controlled by the addition of a second
set of primers/probe specific for a housekeeping gene such as 2
microglobulin which has been labeled with a different fluor on the
5' end (typically JOE).
[0306] To determine the level of BRE in various tissues a
primer/probe set was designed using Primer Express software and
primary cDNA sequence information. Total RNA was prepared from a
series of tissues using an RNeasy kit from Qiagen First strand cDNA
was prepared from one .mu.g total RNA using an oligo dT primer and
Superscript II reverse transcriptase (GIBCO-BRL). cDNA obtained
from approximately 50 ng total RNA was used per TaqMan reaction.
Mock cDNA synthesis in the absence of reverse transcriptase
resulted in samples with no detectable PCR amplification of the
control gene confirms efficient removal of genomic DNA
contamination.
[0307] An array of human tissues were tested. The results of one
such analysis are depicted in Table I. Expression was greatest in
brain, kidney, pancreas, ovary and thymus, and also high in nerve
tissues, including dorsal root ganglion and glial cells.
TABLE-US-00001 TABLE I Expression on BRE in various types of human
tissues. Tissue Type Mean .beta. 2 Mean
.differential..differential. Ct Expression Artery normal 34.07 23.3
10.77 0.57 Vein normal 35.05 21.2 13.85 0.07 Aortic SMC EARLY 32.38
24.59 7.78 4.55 Aortic SMC LATE 31.38 24.06 7.32 6.26 Static HUVEC
27.61 21.66 5.94 16.29 Shear HUVEC 28.46 21.45 7.01 7.76 Heart
normal 26.72 20.05 6.67 9.85 Heart CHF 25.55 19.98 5.57 21.12
Kidney 25.71 21.43 4.28 51.47 Skeletal Muscle 30.73 21.9 8.84 2.18
Adipose normal 27.94 20.43 7.51 5.49 Pancreas 26.97 22.17 4.79
36.02 primary osteoblasts 29.45 20.08 9.37 1.52 Osteoclasts (diff)
34.77 18.34 16.43 0.01 Skin normal 29.38 22.16 7.22 6.71 Spinal
cord normal 27.71 20.54 7.17 6.92 Brain Cortex normal 25.59 22.18
3.42 93.75 Brain Hypothalamus normal 27.25 22.13 5.12 28.76 Nerve
30.9 24.7 6.2 13.65 DRG (Dorsal Root Ganglion) 28.98 22.91 6.07
14.88 Glial Cells (Astrocytes) 26.77 21.03 5.74 18.71 Glioblastoma
27.18 19.04 8.14 3.54 Breast normal 30.06 21.5 8.55 2.66 Breast
tumor 26.09 19.32 6.77 9.16 Ovary normal 26.45 20.91 5.53 21.64
Ovary Tumor 30.02 21.16 8.86 2.16 Prostate Normal 27.38 20.36 7.02
7.7 Prostate Tumor 26.43 18.73 7.71 4.79 Epithelial Cells
(Prostate) 28.87 22.16 6.71 9.59 Colon normal 29.62 18.91 10.71 0.6
Colon Tumor 25.76 19.77 5.99 15.79 Lung normal 30.06 19.52 10.54
0.67 Lung tumor 25.48 19.57 5.91 16.63 Lung COPD 27.38 19.61 7.76
4.6 Colon IBD 33.4 19.18 14.22 0.05 Liver normal 27.32 20.76 6.56
10.6 Liver fibrosis 29.02 22.59 6.42 11.64 Dermal Cells-fibroblasts
28.39 20.13 8.27 3.25 Spleen normal 29.41 19.36 10.05 0.94 Tonsil
normal 27.07 18.26 8.81 2.23 Lymph node 27.15 19.72 7.43 5.8 Thymus
normal 27.95 22.1 5.84 17.4 Skin-Decubitus 30.24 21.88 8.36 3.04
Synovium 35.66 21.22 14.45 0.04 BM-MNC (Bone marrow 28.1 17.94
10.16 0.87 mononuclear cells) Activated PBMC 30.24 16.93 13.32
0.1
[0308] Moreover, increased expression of BRE was observed in tumors
of colon, breast and lung as compared to normal colon, breast and
lung respectively. Also, BRE was observed to be decreased in ovary
tumors versus non-cancerous ovarian tissue. Therefore, arrays
including additional samples of cancerous and non-cancerous human
tissues were tested for BRE expression according to the
above-described Taqman procedure. The results of such analyses are
depicted in Tables II and III TABLE-US-00002 TABLE II Expression on
BRE in various types of cancerous and non-cancerous tissues. Tissue
Type Mean .beta. 2 Mean .differential..differential. Ct Expression
NDR 13 Breast N 31.05 21.69 9.36 1.53 PIT 400 Breast N 30.96 19.59
11.37 0.38 PIT 56 Breast N 31.8 23.56 8.23 3.32 MDA 106 Breast T
30.16 22.09 8.07 3.71 MDA 234 Breast T 30.01 18.56 11.45 0.36 NDR
57 Breast T 31.93 20.14 11.8 0.28 MDA 304 Breast T 31.41 20.27
11.13 0.45 NDR 58 Breast T 25.96 18.64 7.33 6.24 NDR 132 Breast T
29.52 21.87 7.64 5 NDR 07 Breast T 32.48 20.31 12.18 0.22 PIT 208
Ovary N 27.41 20.26 7.15 7.04 CHT 620 Ovary N 29.09 20.39 8.71 2.4
CHT 619 Ovary N 27.24 20.6 6.64 10.03 MDA 293 Ovary N 31.38 24.74
6.64 10.03 CLN 03 Ovary T 30.73 20.27 10.46 0.71 CLN 05 Ovary T
30.66 20.01 10.65 0.62 CLN 17 Ovary T 29 21.06 7.95 4.04 CLN 07
Ovary T 31.05 19.98 11.07 0.47 CLN 08 Ovary T 29.88 19.49 10.39
0.75 MDA 216 Ovary T 32.1 21.74 10.36 0.76 CLN 012 Ovary T 29.7
22.23 7.47 5.62 MDA 25 Ovary T 30.7 22.97 7.73 4.71 MDA 183 Lung N
36.07 18.79 17.29 0 CLN 930 Lung N 32.81 21.36 11.46 0.36 MDA 185
Lung N 32.95 20.38 12.57 0.16 CHT 816 Lung N 33.88 18.11 15.77 0.02
CHT 814 Lung T 25.25 17.56 7.7 4.83 MDA 262 Lung T 29.52 23.85 5.67
19.64 CHT 911 Lung T 25 19.52 5.48 22.41 CHT 726 Lung T 30.16 18.52
11.64 0.31 MDA 259 Lung T 26.82 20.36 6.46 11.32 CHT 845 Lung T
28.09 21.43 6.66 9.92 CHT 832 Lung T 29.7 19.93 9.77 1.15 MDA 253
Lung T 25 19.16 5.84 17.46
[0309] TABLE-US-00003 TABLE III Expression on BRE in various types
of cancerous and non-cancerous tissues. Tissue Type Mean .beta. 2
Mean .differential..differential. Ct Expression CHT 396 Colon N
36.17 19.27 16.9 0 CHT 519 Colon N 40 20.75 19.25 0 CHT 416 Colon N
34.14 19.88 14.27 0.05 CHT 452 Colon N 38.46 18.33 20.13 0 CHT 398
Colon T 27.14 20.11 7.02 7.7 CHT 807 Colon T 33.47 17.08 16.39 0.01
CHT 805 Colon T 32.24 18.96 13.29 0.1 CHT 528 Colon T 34.58 18.27
16.31 0.01 CHT 368 Colon T 34.48 18.17 16.32 0.01 CHT 372 Colon T
32.49 20.16 12.33 0.19 CHT 01 Liver Met 29.61 18.99 10.62 0.64 CHT
3 Liver Met 28.56 21.02 7.54 5.39 CHT 896 Liver Met 29.43 19.66
9.77 1.15 CHT 340 Liver Met 29.51 21.38 8.13 3.57 PIT 260 Liver N
28.27 18.18 10.09 0.92 PIT 229 Liver N 32.4 25.54 6.86 8.61 MGH 16
Brain N 30.47 24.46 6 15.57 MCL 53 Brain N 28.64 24.29 4.36 48.87
MCL 377 Brain N 30.52 25.02 5.51 21.94 MCL 390 Brain N 28.32 23.72
4.6 41.23 Astrocytes 27.73 20.48 7.24 6.62 CHT 201 Brain T 35.27
21.15 14.12 0 CHT 216 Brain T 27.36 17.74 9.63 1.27 CHT 501 Brain T
29.84 20.98 8.86 2.15 CHT 1273 Brain T 27.02 22.11 4.92 33.03 CHT
828 Brain T 34.14 22.36 11.79 0.28 A24 HMVEC-Arr 29.25 18.73 10.53
0.68 C48 HMVEC-Prol 29.18 20.4 8.79 2.27 BWH 54 Fetal Liver 28.85
22.45 6.41 11.8 BWH 75 Fetal Liver 27.56 20.15 7.41 5.88
[0310] Notably, expression was upregulated in 3 of 7 breast tumor
samples as compared to normal, in 6 of 8 lung tumor samples versus
normal, in one colon tumor sample versus normal, and downregulated
in brain, and ovary tumor samples versus normal brain and ovary
respectively. Differential expression was also noted in liver
metastasis as compared to normal liver samples.
[0311] To further investigate the underying cause of the change in
expression in cancerous tissue, e.g. angiogenesis, BRE expression
levels were measured in various cancerous samples by quantitative
PCR using the TaqmanTM procedure as described above. The relative
levels of BRE expression in various samples is depicted in Table IV
below. TABLE-US-00004 TABLE IV Expression of BRE in various
cancerous samples. Relative Average Expres- Average32263 Beta 2 D
Ct sion ONC 101 Hemangioma 31.25 18.88 12.37 0.19 ONC 102
Hemangioma 28.17 18.32 9.85 1.08 ONC 103 Hemangioma 29.05 19.13
9.92 1.04 NDR 203 Normal Kidney 26.77 20.60 6.17 13.94 PIT 213
Renal Cell 29.75 20.08 9.67 1.23 Carcinoma CHT 732 Wilms Tumor
26.25 19.51 6.75 9.32 CHT 765 Wilms Tumor 27.62 21.83 5.79 18.14
NDR 295 Skin 29.84 21.28 8.56 2.66 CHT Uterine 26.25 18.98 7.27
6.48 1424 Adenocarcinoma CHT Neuroblastoma 26.96 19.10 7.87 4.29
1238 BWH 78 Fetal Adrenal 26.38 18.87 7.51 5.49 BWH 74 Fetal Kidney
26.02 20.30 5.72 19.04 BWH 4 Fetal Heart 25.26 18.00 7.26 6.55 MPI
849 Normal Heart 27.42 19.13 8.30 3.18 NDR 764 Cartilage 31.57
24.28 7.29 6.41 CLN 746 Spinal cord 28.50 21.05 7.45 5.72 CHT
lymphangiona 31.71 23.66 8.05 3.77 1753 CLN 944 Endometrial 33.29
25.26 8.03 3.84 polyps NEB 3 Synovium (RA) 31.79 22.38 9.41 1.47
CLN Hyperkeratotic 30.59 22.62 7.97 3.99 1221 skin
[0312] Expression was greatest in fetal and normal kidney and
Wilm's tumor cells, and also high in tissues such as uterine
adenocarcinoma, heart, cartilage and spinal cord.
[0313] To further investigate the expression of BRE in tumorigenic
cells, BRE expression levels were measured in various cell types
sutiable for animal transplantation by quantitative PCR using the
Taqman.TM. procedure as described above. The relative levels of BRE
expression in various samples is depicted in Table V below.
TABLE-US-00005 TABLE V Expression of BRE in a xenograft panel.
Tissue Type 32263Mean .beta. 2 Mean .differential..differential. Ct
Expression MCF-7 Breast T 24.11 20.97 3.15 113.05 ZR75 Breast T
26.66 22.97 3.69 77.21 T47D Breast T 23.36 20.74 2.63 162.10 MDA
231 Breast T 25.32 19.5 5.83 17.58 MDA 435 Breast T 26.08 19.32
6.75 9.26 SKBr3 Breast 25.52 21.66 3.87 68.63 DLD 1 ColonT (stageC)
26.91 22.7 4.2 54.41 SW480 Colon T 27.5 23.43 4.08 59.33 (stage B)
SW620 ColonT (stageC) 25.39 20.7 4.69 38.74 HCT116 (colon) 29.17
23.45 5.71 19.04 HT29 (colon) 26.16 19.05 7.12 7.21 Colo 205
(colon) 24.72 18.06 6.66 9.89 NCIH125 (lung) 27.45 22.07 5.38 23.93
NCIH67 (lung) 27.13 22.61 4.52 43.59 NCIH322 (lung) 25.93 22.41
3.52 87.47 NCIH460 (lung) 29.22 21.32 7.9 4.19 A549 (lung) 28.35
23.34 5.01 31.03 NHBE (lung) 28.66 22.51 6.15 14.08 SKOV-3 ovary
29.67 20.16 9.51 1.37 OVCAR-3 ovary 26.78 22.81 3.98 63.59 293 Baby
Kidney 27.5 22.63 4.87 34.32 293T Baby Kidney 28.33 23.92 4.41
47.04
[0314] Notably, BRE expression was highest in cells such as breast
tumors and breast cancer cell lines and NCIH322. Expression was
also noted in colon tumors, ovary and kidney cells.
Example 4
Tissue Distribution of BRE by In situ Analysis
[0315] For in situ analysis, various tissues, e.g. tissues obtained
from normal lung and colon and lung and colon tumors, were first
frozen on dry ice. Ten-micrometer-thick sections of the tissues
were post-fixed with 4% formaldehyde in DEPC treated 1.times.
phosphate- buffered saline at room temperature for 10 minutes
before being rinsed twice in DEPC 1.times. phosphate-buffered
saline and once in 0.1 M triethanolamine-HCl (pH 8.0). Following
incubation in 0.25% acetic anhydride-0.1 M triethanolamine-HCl for
10 minutes, sections were rinsed in DEPC 2.times. SSC (1.times. SSC
is 0.15M NaCl plus 0.015M sodium citrate). Tissue was then
dehydrated through a series of ethanol washes, incubated in 100%
chloroform for 5 minutes, and then rinsed in 100% ethanol for 1
minute and 95% ethanol for 1 minute and allowed to air dry.
[0316] Hybridizations were performed with .sup.35S-radiolabeled
(5.times.10.sup.7 cpm/ml) cRNA probes. Probes were incubated in the
presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5),
1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05%
yeast total RNA type X1, 1.times. Denhardt's solution, 50%
formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium
dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at
55.degree. C.
[0317] After hybridization, slides were washed with 2.times. SSC.
Sections are then sequentially incubated at 37.degree. C. in TNE (a
solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM
EDTA), for 10 minutes, in TNE with 10 .mu.g of RNase A per ml for
30 minutes, and finally in TNE for 10 minutes. Slides were then
rinsed with 2.times. SSC at room temperature, washed with 2.times.
SSC at 50.degree. C. for 1 hour, washed with 0.2.times. SSC at
55.degree. C. for 1 hour, and 0.2.times. SSC at 60.degree. C. for 1
hour. Sections were then dehydrated rapidly through serial
ethanol-0.3 M sodium acetate concentrations before being air dried
and exposed to Kodak Biomax MR scientific imaging film for 24 hours
and subsequently dipped in NB-2 photoemulsion and exposed at
4.degree. C. for 7 days before being developed and counter
stained.
[0318] As depicted in Table VI below, the in situ hybridization
results essentially agreed with the results of the Taqman analysis.
In situ hybridization results a moderate signal in 3 of 4 primary
colon tumors, and a strong signal in 3 of 4 colon tumors which had
metastasized, e.g. to the liver. Similary, BRE expression was
undetectable in 1 normal lung sample tested, and moderate in 5 of 6
lung tumors tested. Ovary and breast tumor samples showed no
expression. Expression was also strongly detectable in kidney
tumors, e.g. Wilm's tumor and renal cell carcinoma. TABLE-US-00006
Spectrum Tissue Diagnosis Expression BREAST: 0/1 normals; 0/2
tumors NDR 17 Breast Tumor: WD-IDC, foci of DCIS (-/-) NDR 7 Breast
IDC (-/-) CLN 100 Breast Normal (-/-) COLON: 0/1 normals; 3/4
primary tumors; 2/3 metastases CHT 910 Colon Tumor: MD-Invasive AC
(++/+) CHT 890 Colon Tumor: M-PD Invasive AC (+/-) NDR 99 Colon
Tumor: MID Invasive AC (+/-) CLN 609 Colon Tumor (-/-) NDR 77 Colon
Met: Liver mets (+/+) NDR 100 Colon Met: AC in liver (-) CHT 1
Colon Met (++/+) CHT 72 Colon Met: MD-AC (+/-) CHT 521 Colon Normal
(-/-) LUNG: 0/1 normal; 5/6 tumors CHT 446 Lung Tumor: Adeno M-WD
invasive (-/-) CHT 799 Lung Tumor: PD-NSCC, squamous (+/-) features
CHT 800 Lung Tumor: PD-NSCC, squamous (++/+) features MPI 323 Lung
Tumor: SCLC (+/-) MPI 215 Lung Tumor: PD-SCLC (++/+) CHT 813 Lung
Tumore: MD-squamous cell LC (+/+) MPI 216 Lung Normal (-/-) OVARY:
0/1 normal; 0/2 tumors MDA 300 Ovary Tumor: MD-AC (-/-) MDA 28
Ovary Tumor: low-grade serous (-/-) MDA 201 Ovary Normal (-/-)
OTHER TISSUES: 2/4 CHT 734 Kidney Wilm's: blue cell tumor (+++/+)
BWH 36 Adrenal Fetal: normal developing gland (-/-) PIT 213 Kidney
RCC: F grade 3-4 (++/+) NEB 3 Synovium Inflamm: plasma cell
infiltrates (-/-)
Equivalents
[0319] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
3 1 2577 DNA Homo sapiens CDS (167)..(2341) 1 gtagcgaggc ttgggtggcg
aactcggcac gaggcccaaa ggtaggctca ggctccgacg 60 gtggccggcg
ggggtcacga ggcttcgtag tggaggaacg ggtttggcgt gtgggacgca 120
gctgcctctg tactggggag tcacggagtg gccgggctcc agggac atg gcg gcg 175
Met Ala Ala 1 gcc tct gcg gtg tcg gtg ctg ctg gtg gcg gcg gag agg
aac cgg tgg 223 Ala Ser Ala Val Ser Val Leu Leu Val Ala Ala Glu Arg
Asn Arg Trp 5 10 15 cat cgt ctc ccg agc ctg ctc ctg ccg ccg agg aca
tgg gtg tgg agg 271 His Arg Leu Pro Ser Leu Leu Leu Pro Pro Arg Thr
Trp Val Trp Arg 20 25 30 35 caa aga acc atg aag tac aca aca gcc aca
gga aga aac att acc aag 319 Gln Arg Thr Met Lys Tyr Thr Thr Ala Thr
Gly Arg Asn Ile Thr Lys 40 45 50 gtc ctc att gca aac aga gga gaa
att gcc tgc agg gtg atg cgc aca 367 Val Leu Ile Ala Asn Arg Gly Glu
Ile Ala Cys Arg Val Met Arg Thr 55 60 65 gcc aaa aaa ctg ggt gta
cag act gtg gcg gtt tat agt gag gct gac 415 Ala Lys Lys Leu Gly Val
Gln Thr Val Ala Val Tyr Ser Glu Ala Asp 70 75 80 aga aat tcc atg
cat gta gat atg gca gat gaa gca tat tcc atc ggc 463 Arg Asn Ser Met
His Val Asp Met Ala Asp Glu Ala Tyr Ser Ile Gly 85 90 95 ccc gct
ccc tcc cag cag agc tac cta tct atg gag aaa atc att caa 511 Pro Ala
Pro Ser Gln Gln Ser Tyr Leu Ser Met Glu Lys Ile Ile Gln 100 105 110
115 gtg gcc aag acc tct gct gca cag gct atc cat cca gga tgc ggt ttt
559 Val Ala Lys Thr Ser Ala Ala Gln Ala Ile His Pro Gly Cys Gly Phe
120 125 130 ctt tca gaa aac atg gaa ttt gct gaa ctt tgt aag caa gaa
gga att 607 Leu Ser Glu Asn Met Glu Phe Ala Glu Leu Cys Lys Gln Glu
Gly Ile 135 140 145 att ttt ata ggc cct cct cca tct gca att aga gac
atg ggt ata aag 655 Ile Phe Ile Gly Pro Pro Pro Ser Ala Ile Arg Asp
Met Gly Ile Lys 150 155 160 agc aca tcc aaa tcc ata atg gct gct gct
gga gta cct gtt gtg gag 703 Ser Thr Ser Lys Ser Ile Met Ala Ala Ala
Gly Val Pro Val Val Glu 165 170 175 ggt tat cat ggt gag gac caa tca
gac cag tgc ctg aag gaa cac gcc 751 Gly Tyr His Gly Glu Asp Gln Ser
Asp Gln Cys Leu Lys Glu His Ala 180 185 190 195 agg aga att ggc tat
cct gtc atg att aaa gcc gtc cgg ggt gga gga 799 Arg Arg Ile Gly Tyr
Pro Val Met Ile Lys Ala Val Arg Gly Gly Gly 200 205 210 gga aaa gga
atg agg att gtt aga tca gaa caa gaa ttt caa gaa cag 847 Gly Lys Gly
Met Arg Ile Val Arg Ser Glu Gln Glu Phe Gln Glu Gln 215 220 225 tta
gag tca gca cgg aga gaa gct aag aag tct ttc aat gat gat gct 895 Leu
Glu Ser Ala Arg Arg Glu Ala Lys Lys Ser Phe Asn Asp Asp Ala 230 235
240 atg ctg atc gag aag ttt gta gac aca ccg agg cat gta gaa gtc cag
943 Met Leu Ile Glu Lys Phe Val Asp Thr Pro Arg His Val Glu Val Gln
245 250 255 gtg ttt ggt gat cac cat ggc aat gct gtg tac ttg ttt gaa
aga gac 991 Val Phe Gly Asp His His Gly Asn Ala Val Tyr Leu Phe Glu
Arg Asp 260 265 270 275 tgt agt gtg cag agg cga cat cag aag atc att
gag gag gcc cca gcg 1039 Cys Ser Val Gln Arg Arg His Gln Lys Ile
Ile Glu Glu Ala Pro Ala 280 285 290 cct ggt att aaa tct gaa gta aga
aaa aag ctg gga gaa gct gca gtc 1087 Pro Gly Ile Lys Ser Glu Val
Arg Lys Lys Leu Gly Glu Ala Ala Val 295 300 305 aga gct gct aaa gct
gta aat tat gtt gga gca ggg act gtg gag ttt 1135 Arg Ala Ala Lys
Ala Val Asn Tyr Val Gly Ala Gly Thr Val Glu Phe 310 315 320 att atg
gac tca aaa cat aat ttc tgt ttc atg gag atg aat aca agg 1183 Ile
Met Asp Ser Lys His Asn Phe Cys Phe Met Glu Met Asn Thr Arg 325 330
335 ctg caa gtg gaa cat cct gtt act gag atg atc aca gga act gac ttg
1231 Leu Gln Val Glu His Pro Val Thr Glu Met Ile Thr Gly Thr Asp
Leu 340 345 350 355 gtg gag tgg cag ctt aga att gca gca gga gag aag
att cct ttg agc 1279 Val Glu Trp Gln Leu Arg Ile Ala Ala Gly Glu
Lys Ile Pro Leu Ser 360 365 370 cag gaa gaa ata act ctg cag ggc cat
gcc ttc gaa gct aga ata tat 1327 Gln Glu Glu Ile Thr Leu Gln Gly
His Ala Phe Glu Ala Arg Ile Tyr 375 380 385 gca gaa gat cct agc aat
aac ttc atg cct gtg gca ggc cca tta gtg 1375 Ala Glu Asp Pro Ser
Asn Asn Phe Met Pro Val Ala Gly Pro Leu Val 390 395 400 cac ctc tct
act cct cga gca gac cct tcc acc agg att gaa act gga 1423 His Leu
Ser Thr Pro Arg Ala Asp Pro Ser Thr Arg Ile Glu Thr Gly 405 410 415
gta cgg caa gga gac gaa gtt tcc gtg cat tat gac ccc atg att gcg
1471 Val Arg Gln Gly Asp Glu Val Ser Val His Tyr Asp Pro Met Ile
Ala 420 425 430 435 aag ctg gtc gtg tgg gca gca gat cgc cag gcg gca
ttg aca aaa ctg 1519 Lys Leu Val Val Trp Ala Ala Asp Arg Gln Ala
Ala Leu Thr Lys Leu 440 445 450 agg tac agc ctt cgt cag tac aat att
gtt gga ctg ccc acc aac att 1567 Arg Tyr Ser Leu Arg Gln Tyr Asn
Ile Val Gly Leu Pro Thr Asn Ile 455 460 465 gac ttc tta ctc aac ctg
tct ggc cac cca gag ttt gaa gct ggg aac 1615 Asp Phe Leu Leu Asn
Leu Ser Gly His Pro Glu Phe Glu Ala Gly Asn 470 475 480 gtg cac act
gat ttc atc cct caa cac cac aaa cag ttg ttg ctc agt 1663 Val His
Thr Asp Phe Ile Pro Gln His His Lys Gln Leu Leu Leu Ser 485 490 495
cgg aag gct gca gcc aaa gag tct tta tgc cag gca gcc ctg ggt ctc
1711 Arg Lys Ala Ala Ala Lys Glu Ser Leu Cys Gln Ala Ala Leu Gly
Leu 500 505 510 515 atc ctc aag gag aaa gcc atg acc gac act ttc act
ctt cag gca cat 1759 Ile Leu Lys Glu Lys Ala Met Thr Asp Thr Phe
Thr Leu Gln Ala His 520 525 530 gat caa ttc tct cca ttt tcg tct agc
agt gga aga aga ctg aat atc 1807 Asp Gln Phe Ser Pro Phe Ser Ser
Ser Ser Gly Arg Arg Leu Asn Ile 535 540 545 tcg tat acc aga aac atg
act ctt aaa gat ggt aaa aac aat gta gcc 1855 Ser Tyr Thr Arg Asn
Met Thr Leu Lys Asp Gly Lys Asn Asn Val Ala 550 555 560 ata gct gta
acg tat aac cat gat ggg tct tat agc atg cag att gaa 1903 Ile Ala
Val Thr Tyr Asn His Asp Gly Ser Tyr Ser Met Gln Ile Glu 565 570 575
gat aaa act ttc caa gtc ctt ggt aat ctt tac agc gag gga gac tgc
1951 Asp Lys Thr Phe Gln Val Leu Gly Asn Leu Tyr Ser Glu Gly Asp
Cys 580 585 590 595 act tac ctg aaa tgt tct gtt aat gga gtt gct agt
aaa gcg aag ctg 1999 Thr Tyr Leu Lys Cys Ser Val Asn Gly Val Ala
Ser Lys Ala Lys Leu 600 605 610 att atc ctg gaa aac act att tac cta
ttt tcc aag gaa gga agt att 2047 Ile Ile Leu Glu Asn Thr Ile Tyr
Leu Phe Ser Lys Glu Gly Ser Ile 615 620 625 gag att gac att cca gtc
ccc aaa tac tta tct tct gtg agc tca caa 2095 Glu Ile Asp Ile Pro
Val Pro Lys Tyr Leu Ser Ser Val Ser Ser Gln 630 635 640 gaa act cag
ggc ggc ccc tta gct cct atg act gga acc att gaa aag 2143 Glu Thr
Gln Gly Gly Pro Leu Ala Pro Met Thr Gly Thr Ile Glu Lys 645 650 655
gtg ttt gtc aaa gct gga gac aaa gtg aaa gcg gga gat tcc ctc atg
2191 Val Phe Val Lys Ala Gly Asp Lys Val Lys Ala Gly Asp Ser Leu
Met 660 665 670 675 gtt atg atc gcc atg aag atg gag cat acc ata aag
tct cca aag gat 2239 Val Met Ile Ala Met Lys Met Glu His Thr Ile
Lys Ser Pro Lys Asp 680 685 690 ggc aca gta aag aaa gtg ttc tac aga
gaa ggt gct cag gcc aac aga 2287 Gly Thr Val Lys Lys Val Phe Tyr
Arg Glu Gly Ala Gln Ala Asn Arg 695 700 705 cac act cct tta gtc gag
ttt gag gag gaa gaa tca gac aaa agg gaa 2335 His Thr Pro Leu Val
Glu Phe Glu Glu Glu Glu Ser Asp Lys Arg Glu 710 715 720 tcg gaa
taaactccag caaggaaatg gccagttaag tagtgtcttc tctctccacc 2391 Ser Glu
725 aaaaagagga agtgcctcca gcttttctgg gggtctcata aagagcagtt
ttactaaatg 2451 attgtatgct tatgctgaac acctttcata ttggagaatc
atgcatttgg gtcactaatt 2511 atctcaaaat atttcatact aataaagttg
aattattttt tattggaagc caaaaaaaaa 2571 aaaagg 2577 2 725 PRT Homo
sapiens 2 Met Ala Ala Ala Ser Ala Val Ser Val Leu Leu Val Ala Ala
Glu Arg 1 5 10 15 Asn Arg Trp His Arg Leu Pro Ser Leu Leu Leu Pro
Pro Arg Thr Trp 20 25 30 Val Trp Arg Gln Arg Thr Met Lys Tyr Thr
Thr Ala Thr Gly Arg Asn 35 40 45 Ile Thr Lys Val Leu Ile Ala Asn
Arg Gly Glu Ile Ala Cys Arg Val 50 55 60 Met Arg Thr Ala Lys Lys
Leu Gly Val Gln Thr Val Ala Val Tyr Ser 65 70 75 80 Glu Ala Asp Arg
Asn Ser Met His Val Asp Met Ala Asp Glu Ala Tyr 85 90 95 Ser Ile
Gly Pro Ala Pro Ser Gln Gln Ser Tyr Leu Ser Met Glu Lys 100 105 110
Ile Ile Gln Val Ala Lys Thr Ser Ala Ala Gln Ala Ile His Pro Gly 115
120 125 Cys Gly Phe Leu Ser Glu Asn Met Glu Phe Ala Glu Leu Cys Lys
Gln 130 135 140 Glu Gly Ile Ile Phe Ile Gly Pro Pro Pro Ser Ala Ile
Arg Asp Met 145 150 155 160 Gly Ile Lys Ser Thr Ser Lys Ser Ile Met
Ala Ala Ala Gly Val Pro 165 170 175 Val Val Glu Gly Tyr His Gly Glu
Asp Gln Ser Asp Gln Cys Leu Lys 180 185 190 Glu His Ala Arg Arg Ile
Gly Tyr Pro Val Met Ile Lys Ala Val Arg 195 200 205 Gly Gly Gly Gly
Lys Gly Met Arg Ile Val Arg Ser Glu Gln Glu Phe 210 215 220 Gln Glu
Gln Leu Glu Ser Ala Arg Arg Glu Ala Lys Lys Ser Phe Asn 225 230 235
240 Asp Asp Ala Met Leu Ile Glu Lys Phe Val Asp Thr Pro Arg His Val
245 250 255 Glu Val Gln Val Phe Gly Asp His His Gly Asn Ala Val Tyr
Leu Phe 260 265 270 Glu Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys
Ile Ile Glu Glu 275 280 285 Ala Pro Ala Pro Gly Ile Lys Ser Glu Val
Arg Lys Lys Leu Gly Glu 290 295 300 Ala Ala Val Arg Ala Ala Lys Ala
Val Asn Tyr Val Gly Ala Gly Thr 305 310 315 320 Val Glu Phe Ile Met
Asp Ser Lys His Asn Phe Cys Phe Met Glu Met 325 330 335 Asn Thr Arg
Leu Gln Val Glu His Pro Val Thr Glu Met Ile Thr Gly 340 345 350 Thr
Asp Leu Val Glu Trp Gln Leu Arg Ile Ala Ala Gly Glu Lys Ile 355 360
365 Pro Leu Ser Gln Glu Glu Ile Thr Leu Gln Gly His Ala Phe Glu Ala
370 375 380 Arg Ile Tyr Ala Glu Asp Pro Ser Asn Asn Phe Met Pro Val
Ala Gly 385 390 395 400 Pro Leu Val His Leu Ser Thr Pro Arg Ala Asp
Pro Ser Thr Arg Ile 405 410 415 Glu Thr Gly Val Arg Gln Gly Asp Glu
Val Ser Val His Tyr Asp Pro 420 425 430 Met Ile Ala Lys Leu Val Val
Trp Ala Ala Asp Arg Gln Ala Ala Leu 435 440 445 Thr Lys Leu Arg Tyr
Ser Leu Arg Gln Tyr Asn Ile Val Gly Leu Pro 450 455 460 Thr Asn Ile
Asp Phe Leu Leu Asn Leu Ser Gly His Pro Glu Phe Glu 465 470 475 480
Ala Gly Asn Val His Thr Asp Phe Ile Pro Gln His His Lys Gln Leu 485
490 495 Leu Leu Ser Arg Lys Ala Ala Ala Lys Glu Ser Leu Cys Gln Ala
Ala 500 505 510 Leu Gly Leu Ile Leu Lys Glu Lys Ala Met Thr Asp Thr
Phe Thr Leu 515 520 525 Gln Ala His Asp Gln Phe Ser Pro Phe Ser Ser
Ser Ser Gly Arg Arg 530 535 540 Leu Asn Ile Ser Tyr Thr Arg Asn Met
Thr Leu Lys Asp Gly Lys Asn 545 550 555 560 Asn Val Ala Ile Ala Val
Thr Tyr Asn His Asp Gly Ser Tyr Ser Met 565 570 575 Gln Ile Glu Asp
Lys Thr Phe Gln Val Leu Gly Asn Leu Tyr Ser Glu 580 585 590 Gly Asp
Cys Thr Tyr Leu Lys Cys Ser Val Asn Gly Val Ala Ser Lys 595 600 605
Ala Lys Leu Ile Ile Leu Glu Asn Thr Ile Tyr Leu Phe Ser Lys Glu 610
615 620 Gly Ser Ile Glu Ile Asp Ile Pro Val Pro Lys Tyr Leu Ser Ser
Val 625 630 635 640 Ser Ser Gln Glu Thr Gln Gly Gly Pro Leu Ala Pro
Met Thr Gly Thr 645 650 655 Ile Glu Lys Val Phe Val Lys Ala Gly Asp
Lys Val Lys Ala Gly Asp 660 665 670 Ser Leu Met Val Met Ile Ala Met
Lys Met Glu His Thr Ile Lys Ser 675 680 685 Pro Lys Asp Gly Thr Val
Lys Lys Val Phe Tyr Arg Glu Gly Ala Gln 690 695 700 Ala Asn Arg His
Thr Pro Leu Val Glu Phe Glu Glu Glu Glu Ser Asp 705 710 715 720 Lys
Arg Glu Ser Glu 725 3 2175 DNA Homo sapiens CDS (1)..(2175) 3 atg
gcg gcg gcc tct gcg gtg tcg gtg ctg ctg gtg gcg gcg gag agg 48 Met
Ala Ala Ala Ser Ala Val Ser Val Leu Leu Val Ala Ala Glu Arg 1 5 10
15 aac cgg tgg cat cgt ctc ccg agc ctg ctc ctg ccg ccg agg aca tgg
96 Asn Arg Trp His Arg Leu Pro Ser Leu Leu Leu Pro Pro Arg Thr Trp
20 25 30 gtg tgg agg caa aga acc atg aag tac aca aca gcc aca gga
aga aac 144 Val Trp Arg Gln Arg Thr Met Lys Tyr Thr Thr Ala Thr Gly
Arg Asn 35 40 45 att acc aag gtc ctc att gca aac aga gga gaa att
gcc tgc agg gtg 192 Ile Thr Lys Val Leu Ile Ala Asn Arg Gly Glu Ile
Ala Cys Arg Val 50 55 60 atg cgc aca gcc aaa aaa ctg ggt gta cag
act gtg gcg gtt tat agt 240 Met Arg Thr Ala Lys Lys Leu Gly Val Gln
Thr Val Ala Val Tyr Ser 65 70 75 80 gag gct gac aga aat tcc atg cat
gta gat atg gca gat gaa gca tat 288 Glu Ala Asp Arg Asn Ser Met His
Val Asp Met Ala Asp Glu Ala Tyr 85 90 95 tcc atc ggc ccc gct ccc
tcc cag cag agc tac cta tct atg gag aaa 336 Ser Ile Gly Pro Ala Pro
Ser Gln Gln Ser Tyr Leu Ser Met Glu Lys 100 105 110 atc att caa gtg
gcc aag acc tct gct gca cag gct atc cat cca gga 384 Ile Ile Gln Val
Ala Lys Thr Ser Ala Ala Gln Ala Ile His Pro Gly 115 120 125 tgc ggt
ttt ctt tca gaa aac atg gaa ttt gct gaa ctt tgt aag caa 432 Cys Gly
Phe Leu Ser Glu Asn Met Glu Phe Ala Glu Leu Cys Lys Gln 130 135 140
gaa gga att att ttt ata ggc cct cct cca tct gca att aga gac atg 480
Glu Gly Ile Ile Phe Ile Gly Pro Pro Pro Ser Ala Ile Arg Asp Met 145
150 155 160 ggt ata aag agc aca tcc aaa tcc ata atg gct gct gct gga
gta cct 528 Gly Ile Lys Ser Thr Ser Lys Ser Ile Met Ala Ala Ala Gly
Val Pro 165 170 175 gtt gtg gag ggt tat cat ggt gag gac caa tca gac
cag tgc ctg aag 576 Val Val Glu Gly Tyr His Gly Glu Asp Gln Ser Asp
Gln Cys Leu Lys 180 185 190 gaa cac gcc agg aga att ggc tat cct gtc
atg att aaa gcc gtc cgg 624 Glu His Ala Arg Arg Ile Gly Tyr Pro Val
Met Ile Lys Ala Val Arg 195 200 205 ggt gga gga gga aaa gga atg agg
att gtt aga tca gaa caa gaa ttt 672 Gly Gly Gly Gly Lys Gly Met Arg
Ile Val Arg Ser Glu Gln Glu Phe 210 215 220 caa gaa cag tta gag tca
gca cgg aga gaa gct aag aag tct ttc aat 720 Gln Glu Gln Leu Glu Ser
Ala Arg Arg Glu Ala Lys Lys Ser Phe Asn 225 230 235 240 gat gat gct
atg ctg atc gag aag ttt gta gac aca ccg agg cat gta 768 Asp Asp Ala
Met Leu Ile Glu Lys Phe Val Asp Thr Pro Arg His Val 245 250 255 gaa
gtc cag gtg ttt ggt gat cac cat ggc aat gct gtg tac ttg ttt 816 Glu
Val Gln Val Phe Gly Asp His His Gly Asn Ala Val Tyr Leu Phe 260 265
270 gaa aga gac tgt agt gtg cag agg cga cat cag aag atc att gag gag
864 Glu Arg Asp Cys Ser Val Gln
Arg Arg His Gln Lys Ile Ile Glu Glu 275 280 285 gcc cca gcg cct ggt
att aaa tct gaa gta aga aaa aag ctg gga gaa 912 Ala Pro Ala Pro Gly
Ile Lys Ser Glu Val Arg Lys Lys Leu Gly Glu 290 295 300 gct gca gtc
aga gct gct aaa gct gta aat tat gtt gga gca ggg act 960 Ala Ala Val
Arg Ala Ala Lys Ala Val Asn Tyr Val Gly Ala Gly Thr 305 310 315 320
gtg gag ttt att atg gac tca aaa cat aat ttc tgt ttc atg gag atg
1008 Val Glu Phe Ile Met Asp Ser Lys His Asn Phe Cys Phe Met Glu
Met 325 330 335 aat aca agg ctg caa gtg gaa cat cct gtt act gag atg
atc aca gga 1056 Asn Thr Arg Leu Gln Val Glu His Pro Val Thr Glu
Met Ile Thr Gly 340 345 350 act gac ttg gtg gag tgg cag ctt aga att
gca gca gga gag aag att 1104 Thr Asp Leu Val Glu Trp Gln Leu Arg
Ile Ala Ala Gly Glu Lys Ile 355 360 365 cct ttg agc cag gaa gaa ata
act ctg cag ggc cat gcc ttc gaa gct 1152 Pro Leu Ser Gln Glu Glu
Ile Thr Leu Gln Gly His Ala Phe Glu Ala 370 375 380 aga ata tat gca
gaa gat cct agc aat aac ttc atg cct gtg gca ggc 1200 Arg Ile Tyr
Ala Glu Asp Pro Ser Asn Asn Phe Met Pro Val Ala Gly 385 390 395 400
cca tta gtg cac ctc tct act cct cga gca gac cct tcc acc agg att
1248 Pro Leu Val His Leu Ser Thr Pro Arg Ala Asp Pro Ser Thr Arg
Ile 405 410 415 gaa act gga gta cgg caa gga gac gaa gtt tcc gtg cat
tat gac ccc 1296 Glu Thr Gly Val Arg Gln Gly Asp Glu Val Ser Val
His Tyr Asp Pro 420 425 430 atg att gcg aag ctg gtc gtg tgg gca gca
gat cgc cag gcg gca ttg 1344 Met Ile Ala Lys Leu Val Val Trp Ala
Ala Asp Arg Gln Ala Ala Leu 435 440 445 aca aaa ctg agg tac agc ctt
cgt cag tac aat att gtt gga ctg ccc 1392 Thr Lys Leu Arg Tyr Ser
Leu Arg Gln Tyr Asn Ile Val Gly Leu Pro 450 455 460 acc aac att gac
ttc tta ctc aac ctg tct ggc cac cca gag ttt gaa 1440 Thr Asn Ile
Asp Phe Leu Leu Asn Leu Ser Gly His Pro Glu Phe Glu 465 470 475 480
gct ggg aac gtg cac act gat ttc atc cct caa cac cac aaa cag ttg
1488 Ala Gly Asn Val His Thr Asp Phe Ile Pro Gln His His Lys Gln
Leu 485 490 495 ttg ctc agt cgg aag gct gca gcc aaa gag tct tta tgc
cag gca gcc 1536 Leu Leu Ser Arg Lys Ala Ala Ala Lys Glu Ser Leu
Cys Gln Ala Ala 500 505 510 ctg ggt ctc atc ctc aag gag aaa gcc atg
acc gac act ttc act ctt 1584 Leu Gly Leu Ile Leu Lys Glu Lys Ala
Met Thr Asp Thr Phe Thr Leu 515 520 525 cag gca cat gat caa ttc tct
cca ttt tcg tct agc agt gga aga aga 1632 Gln Ala His Asp Gln Phe
Ser Pro Phe Ser Ser Ser Ser Gly Arg Arg 530 535 540 ctg aat atc tcg
tat acc aga aac atg act ctt aaa gat ggt aaa aac 1680 Leu Asn Ile
Ser Tyr Thr Arg Asn Met Thr Leu Lys Asp Gly Lys Asn 545 550 555 560
aat gta gcc ata gct gta acg tat aac cat gat ggg tct tat agc atg
1728 Asn Val Ala Ile Ala Val Thr Tyr Asn His Asp Gly Ser Tyr Ser
Met 565 570 575 cag att gaa gat aaa act ttc caa gtc ctt ggt aat ctt
tac agc gag 1776 Gln Ile Glu Asp Lys Thr Phe Gln Val Leu Gly Asn
Leu Tyr Ser Glu 580 585 590 gga gac tgc act tac ctg aaa tgt tct gtt
aat gga gtt gct agt aaa 1824 Gly Asp Cys Thr Tyr Leu Lys Cys Ser
Val Asn Gly Val Ala Ser Lys 595 600 605 gcg aag ctg att atc ctg gaa
aac act att tac cta ttt tcc aag gaa 1872 Ala Lys Leu Ile Ile Leu
Glu Asn Thr Ile Tyr Leu Phe Ser Lys Glu 610 615 620 gga agt att gag
att gac att cca gtc ccc aaa tac tta tct tct gtg 1920 Gly Ser Ile
Glu Ile Asp Ile Pro Val Pro Lys Tyr Leu Ser Ser Val 625 630 635 640
agc tca caa gaa act cag ggc ggc ccc tta gct cct atg act gga acc
1968 Ser Ser Gln Glu Thr Gln Gly Gly Pro Leu Ala Pro Met Thr Gly
Thr 645 650 655 att gaa aag gtg ttt gtc aaa gct gga gac aaa gtg aaa
gcg gga gat 2016 Ile Glu Lys Val Phe Val Lys Ala Gly Asp Lys Val
Lys Ala Gly Asp 660 665 670 tcc ctc atg gtt atg atc gcc atg aag atg
gag cat acc ata aag tct 2064 Ser Leu Met Val Met Ile Ala Met Lys
Met Glu His Thr Ile Lys Ser 675 680 685 cca aag gat ggc aca gta aag
aaa gtg ttc tac aga gaa ggt gct cag 2112 Pro Lys Asp Gly Thr Val
Lys Lys Val Phe Tyr Arg Glu Gly Ala Gln 690 695 700 gcc aac aga cac
act cct tta gtc gag ttt gag gag gaa gaa tca gac 2160 Ala Asn Arg
His Thr Pro Leu Val Glu Phe Glu Glu Glu Glu Ser Asp 705 710 715 720
aaa agg gaa tcg gaa 2175 Lys Arg Glu Ser Glu 725
* * * * *
References