U.S. patent application number 10/172585 was filed with the patent office on 2003-09-04 for 32142, 21481, 25964, 21686, novel human dehydrogenase molecules and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Cook, William James, Meyers, Rachel.
Application Number | 20030166200 10/172585 |
Document ID | / |
Family ID | 26887626 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166200 |
Kind Code |
A1 |
Meyers, Rachel ; et
al. |
September 4, 2003 |
32142, 21481, 25964, 21686, novel human dehydrogenase molecules and
uses thereof
Abstract
The invention provides isolated nucleic acids molecules,
designated DHDR nucleic acid molecules, which encode novel
DHDR-related dehydrogenase molecules. The invention also provides
antisense nucleic acid molecules, recombinant expression vectors
containing DHDR nucleic acid molecules, host cells into which the
expression vectors have been introduced, and nonhuman transgenic
animals in which a DHDR gene has been introduced or disrupted. The
invention still further provides isolated DHDR proteins, fusion
proteins, antigenic peptides and anti-DHDR antibodies. Diagnostic
methods utilizing compositions of the invention are also
provided.
Inventors: |
Meyers, Rachel; (Newton,
MA) ; Cook, William James; (Natick, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS INC
75 SIDNEY STREET
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
26887626 |
Appl. No.: |
10/172585 |
Filed: |
June 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10172585 |
Jun 14, 2002 |
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09634955 |
Aug 8, 2000 |
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6511834 |
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60192002 |
Mar 24, 2000 |
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Current U.S.
Class: |
435/190 ;
435/320.1; 435/325; 435/6.14; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/0004 20130101;
C07K 2319/00 20130101 |
Class at
Publication: |
435/190 ;
435/320.1; 435/325; 435/69.1; 536/23.2; 435/6 |
International
Class: |
C12N 009/04; C12Q
001/68; C07H 021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed:
1. An isolated nucleic acid molecule selected from the group
consisting of: (a) a nucleic acid molecule comprising the
nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID
NO:7, or SEQ ID NO:10; and (b) a nucleic acid molecule comprising
the nucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ
ID NO:9 or SEQ ID NO:12.
2. An isolated nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ
ID NO:5, SEQ ID NO:8, or SEQ ID NO:11.
3. An isolated nucleic acid molecule comprising the nucleotide
sequence contained in the plasmid deposited with ATCC.RTM. as
Accession Number ______.
4. An isolated nucleic acid molecule which encodes a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence set forth in SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO:8,
or SEQ ID NO:11.
5. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence which is at least 60% identical to the nucleotide sequence
of SEQ ID NO:1 or 3, SEQ ID NO: 4 or 6, SEQ ID NO:7 or 9, or SEQ ID
NO: 10 or 12, or a complement thereof; 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, SEQ ID NO:
4 or 6, SEQ ID NO:7 or 9, or SEQ ID NO:10 or 12, or a complement
thereof; c) a nucleic acid molecule which encodes a polypeptide
comprising an amino acid sequence at least about 60% identical to
the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,
or SEQ ID NO:12; and d) a nucleic acid molecule which encodes a
fragment of a polypeptide comprising the amino acid sequence of SEQ
ID NO: 2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11, wherein the
fragment comprises at least 16 contiguous amino acid residues of
the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO:8,
or SEQ ID NO:11.
6. An isolated nucleic acid molecule which hybridizes to the
nucleic acid molecule of any one of claims 1, 2, 3, 4, or 5 under
stringent conditions.
7. An isolated nucleic acid molecule comprising a nucleotide
sequence which is complementary to the nucleotide sequence of the
nucleic acid molecule of any one of claims 1, 2, 3, 4, or 5.
8. An isolated nucleic acid molecule comprising the nucleic acid
molecule of any one of claims 1, 2, 3, 4, or 5, and a nucleotide
sequence encoding a heterologous polypeptide.
9. A vector comprising the nucleic acid molecule of any one of
claims 1, 2, 3, 4, or 5.
10. The vector of claim 9, which is an expression vector.
11. A host cell transfected with the expression vector of claim
10.
12. A method of producing a polypeptide comprising culturing the
host cell of claim 11 in an appropriate culture medium to, thereby,
produce the polypeptide.
13. An isolated polypeptide selected from the group consisting of:
a) a fragment of a polypeptide comprising the amino acid sequence
of SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11, wherein
the fragment comprises at least 16 contiguous amino acids of SEQ ID
NO: 2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11; b) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID
NO:11, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule consisting of
SEQ ID NO:1 or 3, SEQ ID NO: 4 or 6, SEQ ID NO:7 or 9, or SEQ ID
NO:10 or 12 under stringent conditions; c) a polypeptide which is
encoded by a nucleic acid molecule comprising a nucleotide sequence
which is at least 60% identical to a nucleic acid comprising the
nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12; d) a
polypeptide comprising an amino acid sequence which is at least 60%
identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:5,
SEQ ID NO:8, or SEQ ID NO:11.
14. The isolated polypeptide of claim 13 comprising the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID
NO:11.
15. The polypeptide of claim 13, further comprising heterologous
amino acid sequences.
16. An antibody which selectively binds to a polypeptide of claim
13.
17. A method for detecting the presence of a polypeptide of claim
13 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 a polypeptide of claim 13 in the
sample.
18. The method of claim 17, wherein the compound which binds to the
polypeptide is an antibody.
19. A kit comprising a compound which selectively binds to a
polypeptide of claim 13 and instructions for use.
20. A method for detecting the presence of a nucleic acid molecule
of any one of claims 1, 2, 3, 4, or 5 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 a
nucleic acid molecule in the sample to thereby detect the presence
of a nucleic acid molecule of any one of claims 1, 2, 3, 4, or 5 in
the sample.
21. The method of claim 20, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
22. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of any one of claims 1, 2, 3, 4, or 5 and
instructions for use.
23. A method for identifying a compound which binds to a
polypeptide of claim 13 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.
24. The method of claim 23, 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 DHDR activity.
25. A method for modulating the activity of a polypeptide of claim
13 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.
26. A method for identifying a compound which modulates the
activity of a polypeptide of claim 13 comprising: a) contacting a
polypeptide of claim 13 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.
27. A method of identifying a subject having a viral disorder, or
at risk for developing a viral disorder comprising: a) contacting a
sample obtained from said subject comprising nucleic acid molecules
with a hybridization probe comprising at least 25 contiguous
nucleotides of SEQ ID NO:10; and b) detecting the presence of a
nucleic acid molecule in said sample that hybridizes to said probe,
thereby identifying a subject having a viral disorder, or at risk
for developing a viral disorder.
28. The method of claim 27, wherein said hybridization probe is
detectably labeled.
29. The method of claim 27, wherein said sample comprising nucleic
acid molecules is subjected to agarose gel electrophoresis and
southern blotting prior to contacting with said hybridization
probe.
30. The method of claim 29, wherein said method is used to detect
genomic DNA in said sample.
31. The method of claim 27, wherein said sample comprising nucleic
acid molecules is subjected to agarose gel electrophoresis and
northern blotting prior to contacting with said hybridization
probe.
32. The method of claim 31, wherein said method is used to detect
mRNA in the sample.
33. The method of claim 27, wherein said detecting is by in situ
hybridization.
34. A method of identifying a subject having a viral disorder, or
at risk for developing a viral disorder comprising: a) contacting a
sample obtained from said subject comprising nucleic acid molecules
with a first and a second amplification primer, said first primer
comprising at least 25 contiguous nucleotides of SEQ ID NO:10 and
said second primer comprising at least 25 contiguous nucleotides
from the complement of SEQ ID NO:10; b) incubating said sample
under conditions that allow nucleic acid amplification; and c)
detecting the presence of a nucleic acid molecule in said sample
that is amplified, thereby identifying a subject having a viral
disorder, or at risk for developing a viral disorder.
35. The method of claim 34, wherein said sample comprising nucleic
acid molecules is subjected to agarose gel electrophoresis after
said incubation step.
36. The method of any one of claims 34, wherein said method is used
to detect mRNA in said sample.
37. The method of any one of claims 34, wherein said method is used
to detect genomic DNA in said sample.
38. A method of identifying a subject having a viral disorder, or
at risk for developing a viral disorder comprising: a) contacting a
sample obtained from said subject comprising polypeptides with a
DHDR binding substance; and b) detecting the presence of a
polypeptide in said sample that binds to said DHDR binding
substance, thereby identifying a subject having a viral disorder or
at risk for developing a viral disorder.
39. The method of claim 38, wherein said binding substance is an
antibody.
40. The method of claim 38, wherein said binding substance is
detectably labeled.
41. A method for identifying a compound capable of treating a viral
disorder characterized by aberrant DHDR nucleic acid expression or
DHDR polypeptide activity comprising assaying the ability of the
compound to modulate DHDR nucleic acid expression or DHDR
polypeptide activity, thereby identifying a compound capable of
treating a viral disorder characterized by aberrant DHDR nucleic
acid expression or DHDR polypeptide activity.
42. The method of claim 41, wherein the disorder is associated with
hepatitis B virus infection.
43. A method for treating a subject having a viral disorder
characterized by aberrant DHDR polypeptide activity or aberrant
DHDR nucleic acid expression comprising administering to the
subject a DHDR modulator, thereby treating said subject having a
viral disorder.
44. The method of claim 43, wherein the DHDR modulator is a small
molecule.
45. The method of claim 43, wherein the DHDR modulator is an
antisense oligonucleotide.
46. The method of claim 43, wherein the DHDR modulator is a
ribozyme.
47. The method of claim 43, wherein the DHDR modulator is a
polypeptide.
48. The method of claim 43, wherein the DHDR modulator is an
antibody.
49. The method of claim 43, wherein the disorder is associated with
hepatitis B virus infection.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
Application No. 60/192,002, filed on Mar. 24, 2000, incorporated
herein in its entirety by this reference.
BACKGROUND OF THE INVENTION
[0002] The oxidation and reduction of molecules is of critical
importance in most metabolic and catabolic pathways in cells. A
large family of enzymes which facilitate these molecular
alterations, termed dehydrogenases, have been identified. In the
forward reaction, these enzymes catalyze the transfer of a hydride
ion from the target substrate to the enzyme or a cofactor of the
enzyme (e.g., NAD.sup.+ or NADP.sup.+), thereby forming a carbonyl
group on the substrate. These enzymes are also able to participate
in the reverse reaction, wherein a carbonyl group on the target
molecule is reduced by the transfer of a hydride group from the
enzyme. Members of the dehydrogenase family are found in nearly all
organisms, from microbes to Drosophila to humans. Both between
species and within the same species, dehydrogenases vary widely,
and structural similarities between distant dehydrogenase family
members are most frequently found in the cofactor binding site of
the enzyme. Even within a particular subclass of dehydrogenase
molecules, e.g., the short-chain dehydrogenase molecules, members
typically display only 15-30% amino acid sequence identity, and
this is limited to the cofactor binding site and the catalytic site
(Jornvall et al. (1995) Biochemistry 34:6003-6013).
[0003] Different classes of dehydrogenases are specific for an
array of biological and chemical substrates. For example, there
exist dehydrogenases specific for alcohols, for aldehydes, for
steroids, and for lipids, with particularly important classes of
dehydrogenases including the short-chain dehydrogenase/reductases,
the medium-chain dehydrogenases, the aldehyde dehydrogenases, the
alcohol dehydrogenases, and the steroid dehydrogenases. Within each
of these classes, each enzyme is specific for a particular
substrate (e.g., ethanol or isopropanol, but not both with
equivalent affinity). This exquisite specificity not only permits
tight regulation of the metabolic and catabolic pathways in which
these enzymes participate, without affecting similar but separate
biochemical pathways in the same cell or tissue. The short-chain
dehydrogenases, part of the alcohol oxidoreductase superfamily
(Reid et al. (1994) Crit. Rev. Microbiol. 20:13-56), are
Zn.sup.++-independent enzymes with an N-terminal cofactor binding
site and a C-terminal catalytic domain (Persson et al. (1995) Adv.
Exp. Med. Biol. 372:383-395; Jornvall et al.(1995) supra), whereas
the medium chain dehydrogenases are Zn.sup.++-dependent enzymes
with an N-terminal catalytic domain and a C-terminal coenzyme
binding domain (Jornvall et al.(1995) supra; Jornvall et al. (1999)
FEBS Lett. 445:261-264). The steroid dehydrogenases are a subclass
of the short-chain dehydrogenases, and are known to be involved in
a variety of biochemical pathways, affecting mammalian
reproduction, hypertension, neoplasia, and digestion (Duax et al.
(2000) Vitamins and Hormones 58:121-148). Aldehyde dehydrogenases
show heterogeneity in the placement of these domains, and also
heterogeneity in their substrates, which include toxic substances,
retinoic acid, betaine, biogenic amine, and neurotransmitters (Hsu
et al. (1997) Gene 189:89-94). It is common in higher organisms for
different dehydrogenase molecules to be expressed in different
tissues, according to the localization of the substrate for which
the enzyme is specific. For example, different mammalian aldehyde
dehydrogenases are localized to different tissues, e.g., salivary
gland, stomach, and kidney (Hsu et al. (1997) supra).
[0004] Dehydrogenases play important roles in the production and
breakdown of nearly all major metabolic intermediates, including
amino acids, vitamins, energy molecules (e.g., glucose, sucrose,
and their breakdown products), signal molecules (e.g.,
transcription factors and neurotransmitters), and nucleic acids. As
such, their activity contributes to the ability of the cell to grow
and differentiate, to proliferate, and to communicate and interact
with other cells. Dehydrogenases also are important in the
detoxification of compounds to which the organism is exposed, such
as alcohols, toxins, carcinogens, and mutagens.
[0005] A dehydrogenase of the short-chain family,
11-beta-hydroxysteroid dehydrogenase, activates glucocorticoids in
the liver. Glucocorticoids are known to induce transcription of
hepatitis B virus (HBV) genes, probably by direct binding of the
ligand-glucorcorticoid receptor complex to an enhancer element in
the HBV genome. There is also evidence that short chain
dehydrogenases are transcriptional cofactors for retrovirus gene
activation.
SUMMARY OF THE INVENTION
[0006] The present invention is based, at least in part, on the
discovery of novel members of the family of dehydrogenase
molecules, referred to herein as DHDR nucleic acid and protein
molecules (e.g., DHDR-1, DHDR-2, DHDR-3, and DHDR-4). The DHDR
nucleic acid and protein molecules of the present invention are
useful as modulating agents in regulating a variety of cellular
processes, e.g., viral infection, cellular proliferation, growth,
differentiation, or migration. Accordingly, in one aspect, this
invention provides isolated nucleic acid molecules encoding DHDR
proteins or biologically active portions thereof, as well as
nucleic acid fragments suitable as primers or hybridization probes
for the detection of DHDR-encoding nucleic acids.
[0007] In one embodiment, a DHDR 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, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______, or a complement thereof.
[0008] In a preferred embodiment, the isolated nucleic acid
molecule includes the nucleotide sequence shown in SEQ ID NO:1, 3,
4, 6, 7, 9, 10, or 12, or a complement thereof. In another
embodiment, the nucleic acid molecule includes SEQ ID NO:3 and
nucleotides 1-62 of SEQ ID NO:1. In another embodiment, the nucleic
acid molecule includes SEQ ID NO:6 and nucleotides 1-330 of SEQ ID
NO:4. In yet another embodiment, the nucleic acid molecule includes
SEQ ID NO:9 and nucleotides 1-280 of SEQ ID NO:7. In another
embodiment, the nucleic acid molecule includes SEQ ID NO:12 and
nucleotides 1-60 of SEQ ID NO:10. In yet a further embodiment, the
nucleic acid molecule includes SEQ ID NO:3 and nucleotides
2472-2660 of SEQ ID NO:1. In another embodiment, the nucleic acid
molecule includes SEQ ID NO:6 and nucleotides 1267-1379 of SEQ ID
NO:4. In another embodiment, the nucleic acid molecule includes SEQ
ID NO:9 and nucleotides 1391-1725 of SEQ ID NO:7. In another
embodiment, the nucleic acid molecule includes SEQ ID NO:12 and
nucleotides 1030-1209 of SEQ ID NO:10. In another preferred
embodiment, the nucleic acid molecule consists of the nucleotide
sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12.
[0009] In another embodiment, a DHDR 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, 5, 8, or 11, or an amino acid sequence encoded by the DNA
insert of the plasmid deposited with ATCC as Accession Number
______. In a preferred embodiment, a DHDR 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, 5, 8, or 11, or the amino
acid sequence encoded by the DNA insert of the plasmid deposited
with ATCC as Accession Number ______.
[0010] In another preferred embodiment, an isolated nucleic acid
molecule encodes the amino acid sequence of human DHDR-1, DHDR-2,
DHDR-3, or DHDR-4. 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:2, 5, 8, or 11, or the
amino acid sequence encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number ______. In yet another
preferred embodiment, the nucleic acid molecule is at least 50,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1000 or more nucleotides in length. In a
further preferred embodiment, the nucleic acid molecule is at least
50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000 or more nucleotides in length
and encodes a protein having a DHDR activity (as described
herein).
[0011] Another embodiment of the invention features nucleic acid
molecules, preferably DHDR nucleic acid molecules, which
specifically detect DHDR nucleic acid molecules relative to nucleic
acid molecules encoding non-DHDR proteins. For example, in one
embodiment, such a nucleic acid molecule is at least 20, 30, 40,
50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000 or more nucleotides in length
and hybridizes under stringent conditions to a nucleic acid
molecule comprising the nucleotide sequence shown in SEQ ID NO:1,
4, 7, or 10, the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______, or a
complement thereof.
[0012] In preferred embodiments, the nucleic acid molecules are at
least 15 (e.g., 15 contiguous) nucleotides in length and hybridize
under stringent conditions to the nucleotide molecules set forth in
SEQ ID NO:1, 4, 7, or 10.
[0013] 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, 5, 8, or 11, or
an amino acid sequence encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, wherein the nucleic
acid molecule hybridizes to a nucleic acid molecule comprising SEQ
ID NO:1, 3, 4, 6, 7, 9, 10, or 12, respectively, under stringent
conditions.
[0014] Another embodiment of the invention provides an isolated
nucleic acid molecule which is antisense to a DHDR nucleic acid
molecule, e.g., the coding strand of a DHDR nucleic acid
molecule.
[0015] Another aspect of the invention provides a vector comprising
a DHDR 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 DHDR 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.
[0016] Another aspect of this invention features isolated or
recombinant DHDR proteins and polypeptides. In one embodiment, an
isolated DHDR protein includes at least one or more of the
following domains: a transmembrane domain, a signal peptide domain,
an aldehyde dehydrogenase oxidoreductase domain, an aldehyde
dehydrogenase family domain, a short chain dehydrogenase domain, an
oxidoreductase protein dehydrogenase domain, a 3-beta
hydroxysteroid dehydrogenase domain, a NAD-dependent
epimerase/dehydratase domain, a short chain dehydrogenase/reductase
domain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,
and/or a glucose-1-dehydrogenase domain.
[0017] In a preferred embodiment, a DHDR protein includes at least
one or more of the following domains: a transmembrane domain, a
signal peptide domain, an aldehyde dehydrogenase oxidoreductase
domain, an aldehyde dehydrogenase family domain, a short chain
dehydrogenase domain, an oxidoreductase protein dehydrogenase
domain, a 3-beta hydroxysteroid dehydrogenase domain, a
NAD-dependent epimerase/dehydratase domain, a short chain
dehydrogenase/reductase domain, a shikimate 5-dehydrogenase domain,
a dehydrogenase domain, and/or a glucose-1-dehydrogenase 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, 5, 8, or 11,
or the amino acid sequence encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number ______. In another
preferred embodiment, a DHDR protein includes at least one or more
of the following domains: a transmembrane domain, a signal peptide
domain, an aldehyde dehydrogenase oxidoreductase domain, an
aldehyde dehydrogenase family domain, a short chain dehydrogenase
domain, an oxidoreductase protein dehydrogenase domain, a 3-beta
hydroxysteroid dehydrogenase domain, a NAD-dependent
epimerase/dehydratase domain, a short chain dehydrogenase/reductase
domain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,
and/or a glucose-1-dehydrogenase domain, and has a DHDR activity
(as described herein).
[0018] In yet another preferred embodiment, a DHDR protein includes
at least one or more of the following domains: a transmembrane
domain, a signal peptide domain, an aldehyde dehydrogenase
oxidoreductase domain, an aldehyde dehydrogenase family domain, a
short chain dehydrogenase domain, an oxidoreductase protein
dehydrogenase domain, a 3-beta hydroxysteroid dehydrogenase domain,
a NAD-dependent epimerase/dehydratase domain, a short chain
dehydrogenase/reductase domain, a shikimate 5-dehydrogenase domain,
a dehydrogenase domain, and/or a glucose-1-dehydrogenase domain,
and is encoded by a nucleic acid molecule having a nucleotide
sequence which hybridizes under stringent hybridization conditions
to a nucleic acid molecule comprising the nucleotide sequence of
SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12.
[0019] In another embodiment, the invention features fragments of
the protein having the amino acid sequence of SEQ ID NO:2, 5, 8, or
11, wherein the fragment comprises at least 16 amino acids (e.g.,
contiguous amino acids) of the amino acid sequence of SEQ ID NO:2,
5, 8, or 11, or an amino acid sequence encoded by the DNA insert of
the plasmid deposited with the ATCC as Accession Number ______. In
another embodiment, a DHDR protein has the amino acid sequence of
SEQ ID NO:2, 5, 8, or 11.
[0020] In another embodiment, the invention features a DHDR 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, 3, 4, 6, 7, 9, 10, or 12, or a
complement thereof. This invention further features a DHDR 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, 3, 4, 6, 7, 9, 10, or 12, or a complement
thereof.
[0021] The proteins of the present invention or portions thereof,
e.g., biologically active portions thereof, can be operatively
linked to a non-DHDR 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 DHDR
proteins. In addition, the DHDR proteins or biologically active
portions thereof can be incorporated into pharmaceutical
compositions, which optionally include pharmaceutically acceptable
carriers.
[0022] In another aspect, the present invention provides a method
for detecting the presence of a DHDR nucleic acid molecule,
protein, or polypeptide in a biological sample by contacting the
biological sample with an agent capable of detecting a DHDR nucleic
acid molecule, protein, or polypeptide such that the presence of a
DHDR nucleic acid molecule, protein or polypeptide is detected in
the biological sample.
[0023] In another aspect, the present invention provides a method
for detecting the presence of DHDR activity in a biological sample
by contacting the biological sample with an agent capable of
detecting an indicator of DHDR activity such that the presence of
DHDR activity is detected in the biological sample.
[0024] In another aspect, the invention provides a method for
modulating DHDR activity comprising contacting a cell capable of
expressing DHDR with an agent that modulates DHDR activity such
that DHDR activity in the cell is modulated. In one embodiment, the
agent inhibits DHDR activity. In another embodiment, the agent
stimulates DHDR activity. In one embodiment, the agent is an
antibody that specifically binds to a DHDR protein. In another
embodiment, the agent modulates expression of DHDR by modulating
transcription of a DHDR gene or translation of a DHDR 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
DHDR mRNA or a DHDR gene.
[0025] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
or unwanted DHDR protein or nucleic acid expression or activity by
administering an agent which is a DHDR modulator to the subject. In
one embodiment, the DHDR modulator is a DHDR protein. In another
embodiment the DHDR modulator is a DHDR nucleic acid molecule. In
yet another embodiment, the DHDR modulator is a peptide,
peptidomimetic, or other small molecule. In a preferred embodiment,
the disorder characterized by aberrant or unwanted DHDR protein or
nucleic acid expression is a dehydrogenase-associated disorder,
e.g., a viral disorder, a CNS disorder, a cardiovascular disorder,
a muscular disorder, or a cell proliferation, growth,
differentiation, or migration disorder.
[0026] 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 DHDR protein; (ii) mis-regulation of
the gene; and (iii) aberrant post-translational modification of a
DHDR protein, wherein a wild-type form of the gene encodes a
protein with a DHDR activity.
[0027] In another aspect the invention provides methods for
identifying a compound that binds to or modulates the activity of a
DHDR protein, by providing an indicator composition comprising a
DHDR protein having DHDR activity, contacting the indicator
composition with a test compound, and determining the effect of the
test compound on DHDR activity in the indicator composition to
identify a compound that modulates the activity of a DHDR
protein.
[0028] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 depicts the cDNA sequence and predicted amino acid
sequence of human DHDR-1 (clone FBH32142). The nucleotide sequence
corresponds to nucleic acids 1 to 2660 of SEQ ID NO:1. The amino
acid sequence corresponds to amino acids 1 to 802 of SEQ ID NO: 2.
The coding region without the 3' untranslated region of the human
DHDR-1 gene is shown in SEQ ID NO: 3.
[0030] FIG. 2 depicts a structural, hydrophobicity, and
antigenicity analysis of the human DHDR-1 protein.
[0031] FIG. 3 depicts the results of a search which was performed
against the MEMSAT database and which resulted in the
identification of one "transmembrane domains" in the human DHDR-1
protein (SEQ ID NO:2).
[0032] FIG. 4 depicts the results of a search which was performed
against the HMM database and which resulted in the identification
of an "aldehyde dehydrogenase family domain" in the human DHDR-1
protein.
[0033] FIG. 5 depicts the results of a search which was performed
against the ProDom database and which resulted in the
identification of a "aldehyde dehydrogenase oxidoreductase domain"
in the human DHDR-1 protein (SEQ ID NO:2).
[0034] FIG. 6 depicts the cDNA sequence and predicted amino acid
sequence of human DHDR-2 (clone Fbh21481). The nucleotide sequence
corresponds to nucleic acids 1379 of SEQ ID NO:4. The amino acid
sequence corresponds to amino acids 1 to 311 of SEQ ID NO: 5. The
coding region without the 3' untranslated region of the human
DHDR-2 gene is shown in SEQ ID NO: 6.
[0035] FIG. 7 depicts a structural, hydrophobicity, and
antigenicity analysis of the human DHDR-2 protein.
[0036] FIG. 8 depicts the results of a signal peptide prediction
and a search which was performed against the MEMSAT database and
which resulted in the identification of a signal peptide and one
"transmembrane domain" in the human DHDR-2 protein (SEQ ID
NO:5).
[0037] FIG. 9 depicts the results of a search which was performed
against the HMM database and which resulted in the identification
of a "short-chain dehydrogenase domain" in the human DHDR-2
protein.
[0038] FIG. 10 depicts the results of a search which was performed
against the ProDom database and which resulted in the
identification of a "oxidoreductase protein dehydrogenase domain"
in the human DHDR-2 protein (SEQ ID NO:5).
[0039] FIG. 11 depicts the cDNA sequence and predicted amino acid
sequence of human DHDR-3 (clone Fbh25964). The nucleotide sequence
corresponds to nucleic acids 1 to 1725 of SEQ ID NO:7. The amino
acid sequence corresponds to amino acids 1 to 369 of SEQ ID NO: 8.
The coding region without the 3' untranslated region of the human
DHDR-3 gene is shown in SEQ ID NO: 9.
[0040] FIG. 12 depicts a structural, hydrophobicity, and
antigenicity analysis of the human DHDR-3 protein.
[0041] FIG. 13 depicts the results of a search which was performed
against the MEMSAT database and which resulted in the
identification of four "transmembrane domains" in the human DHDR-3
protein (SEQ ID NO:8).
[0042] FIG. 14 depicts the results of a search which was performed
against the HMM database and which resulted in the identification
of a "3-beta hydroxysteroid dehydrogenase domain", a "short chain
dehydrogenase domain", and a "NAD-dependent epimerase/dehydratase
domain" in the human DHDR-3 protein.
[0043] FIG. 15 depicts the results of a search which was performed
against the ProDom database and which resulted in the
identification of a "3-beta hydroxysteroid dehydrogenase domain" in
the human DHDR-3 protein (SEQ ID NO:8).
[0044] FIG. 16 depicts the cDNA sequence and predicted amino acid
sequence of human DHDR-4 (clone Fbh21686). The nucleotide sequence
corresponds to nucleic acids 1 to 1209 of SEQ ID NO:10. The amino
acid sequence corresponds to amino acids 1 to 322 of SEQ ID NO: 11.
The coding region without the 3' untranslated region of the human
DHDR-4 gene is shown in SEQ ID NO: 12.
[0045] FIG. 17 depicts an alignment of the human DHDR-4 amino acid
sequence with the amino acid sequences of Rattus norvegicus
putative short-chain dehydrogenase/reductase (Accession Number
AF099742) using the CLUSTAL W (1.74) multiple sequence alignment
program.
[0046] FIG. 18 depicts a structural, hydrophobicity, and
antigenicity analysis of the human DHDR-4 protein.
[0047] FIG. 19 depicts the results of a signal peptide prediction
and a search which was performed against the MEMSAT database and
which resulted in the identification of a "signal peptide" and four
"transmembrane domains" in the human DHDR-4 protein (SEQ ID
NO:11).
[0048] FIG. 20 depicts the results of a search which was performed
against the HMM database and which resulted in the identification
of a "short chain dehydrogenase domain" and a "short chain
dehydrogenase/reductase domain" in the human DHDR-4 protein.
[0049] FIG. 21 depicts the results of a search which was performed
against the ProDom database and which resulted in the
identification of a "oxidoreductase protein dehydrogenase domain",
a "shikimate 5-dehydrogenase domain", a "dehydrogenase domain" and
a "glucose-1-dehydrogenase domain" in the human DHDR-4 protein (SEQ
ID NO:11).
DETAILED DESCRIPTION OF THE INVENTION
[0050] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as "dehydrogenase"
or "DHDR" nucleic acid and protein molecules, which are novel
members of a family of enzymes possessing dehydrogenase activity.
These novel molecules are capable of oxidizing or reducing
biological molecules by catalyzing the transfer of a hydride moiety
and, thus, play a role in or function in a variety of cellular
processes, e.g., proliferation, growth, differentiation, migration,
immune responses, hormonal responses, inter- or intra-cellular
communication, and viral infection.
[0051] As used herein, the term "dehydrogenase" includes a molecule
which is involved in the oxidation or reduction of a biochemical
molecule (e.g., an amino acid, a vitamin, a steroid such as a
glucocorticoid, or a nucleic acid), by catalyzing the transfer of a
hydride ion to or from the biochemical molecule. Dehydrogenase
molecules are involved in the metabolism and catabolism of
biochemical molecules necessary for energy production or storage,
for intra- or inter-cellular signaling, for metabolism or
catabolism of metabolically important biomolecules, and for
detoxification of potentially harmful compounds. Examples of
dehydrogenases include alcohol dehydrogenases, aldehyde
dehydrogenases, steroid dehydrogenases, and lipid dehydrogenases.
Thus, the DHDR molecules of the present invention provide novel
diagnostic targets and therapeutic agents to control
dehydrogenase-associated disorders.
[0052] As used herein, a "dehydrogenase-associated disorder"
includes a disorder, disease or condition which is caused or
characterized by a misregulation (e.g., downregulation or
upregulation) of dehydrogenase activity. Dehydrogenase-associated
disorders can detrimentally affect cellular functions such as
cellular proliferation, growth, differentiation, or migration,
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), susceptibility to pathogenic infections
(e.g., viral infections), or immune responses; and protection of
cells from toxic compounds (e.g., carcinogens, toxins, or
mutagens). Examples of dehydrogenase-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.
[0053] Further examples of dehydrogenase-associated disorders
include cardiac-related disorders. Cardiovascular system disorders
in which the DHDR 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. DHDR-mediated or related
disorders also include disorders of the musculoskeletal system such
as paralysis and muscle weakness, e.g., ataxia, myotonia, and
myokymia.
[0054] Dehydrogenase-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 DHDR 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 DHDR 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., carcinomas, sarcomas, leukemias,
and lymphomas; tumor angiogenesis and metastasis; skeletal
dysplasia; hepatic disorders; and hematopoietic and/or
myeloproliferative disorders.
[0055] DHDR-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).
[0056] DHDR-associated or related disorders also include immune
disorders, such as autoimmune disorders or immune deficiency
disorders, e.g., allergies, transplant rejection, responses to
pathogenic infection (e.g., bacterial, viral, or parasitic
infection), lupus, multiple sclerosis, congenital X-linked
infantile hypogammaglobulinemia, transient hypogammaglobulinemia,
common variable immunodeficiency, selective IgA deficiency, chronic
mucocutaneous candidiasis, or severe combined immunodeficiency.
[0057] DHDR-associated or related disorders also include viral
disorders, i.e., disorders affected or caused by infection by
viruses (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis
delta, and other hepadnaviruses; Coxsackie B viruses; Epstein-Barr
virus; adenovirus; rhinoviruses; human immunodeficiency virus;
vaccinia virus; human T cell leukemia virus; RD114 virus; herpes
simplex, herpes zoster, and other herpesviruses; Marek's disease
virus; Yamaguchi sarcoma virus; human papillomaviruses; poliovirus;
poxviruses; influenza virus; cytomegalovirus; encephalitis viruses;
measles viruses; and ebola and other hemorrhagic viruses). Such
disorders include, but are not limited to, hepatocellularcarcinoma,
cirrhosis of the liver, cervical carcinoma, Burkitt's lymphoma,
lymphoproliferative disease, Kaposi's sarcoma, T cell leukemia, B
cell lymphoma, plasmablastic lymphoma, Rasmussen's syndrome,
Marek's disease, warts (including common, genital, and plantar
warts), genital herpes, common colds, acquired immune deficiency
syndrome (AIDS), polymyositis, immunorestitution disease, chicken
pox, shingles, ebola and other hemorrhagic fever diseases, cold
sores, transient or acute hepatitis, chronic hepatitis, influenza,
Reye syndrome, measles, Paget's disease, viral encephalitis, viral
pneumonia, and viral meningitis.
[0058] DHDR-associated or related disorders also include disorders
affecting tissues in which DHDR protein is expressed, e.g., liver,
hepatocytes, hepatitis B-infected hepatocytes, HepG2 cells,
hepatitis B-infected HepG2.2.15 cells, kidney, brain, primary
osteoblasts, pituitary, CaCO cells, keratinocytes, aortic
endothelial cells, fetal kidney, fetal lung, mammary epithelium,
fetal spleen, fetal liver, umbilical smooth muscle, RAII Burkitt
Lymphoma cells, lung, prostate, K53 red blood cells, fetal dorsal
spinal cord, insulinoma cells, normal breast and ovarian epithelia,
retina, HMC-1 mast cells, ovarian ascites, d8 dendritic cells,
megakaryocytes, human mobilized bone morrow, mammary carcinoma,
melanoma cells, lymph, vein, U937/A70p B cells, A549con cells, WT
LN Cap testosterone cells, and esophagus.
[0059] As used herein, a "dehydrogenase-mediated activity" includes
an activity which involves the oxidation or reduction of one or
more biochemical molecules, e.g., biochemical molecules (e.g.,
glucocorticoids) in a neuronal cell, a muscle cell, or a liver cell
associated with the regulation of one or more cellular processes.
Dehydrogenase-mediated activities include the oxidation or
reduction of biochemical molecules necessary for energy production
or storage, for intra- or inter-cellular signaling, for metabolism
or catabolism of metabolically important biomolecules, for viral
infection, and for detoxification of potentially harmful
compounds.
[0060] 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.
[0061] For example, the family of DHDR proteins comprises at least
one "transmembrane domain". As used herein, the term "transmembrane
domain" includes an amino acid sequence of about 15 amino acid
residues in length which spans the plasma membrane. More
preferably, a transmembrane domain includes about at least 20, 25,
30, 35, 40, or 45 amino acid residues and spans the plasma
membrane. Transmembrane domains are rich in hydrophobic residues,
and typically have an alpha-helical structure. In a preferred
embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the
amino acids of a transmembrane domain are hydrophobic, e.g.,
leucines, isoleucines, tyrosines, or tryptophans. Transmembrane
domains are described in, for example, Zagotta W. N. et al, (1996)
Annual Rev. Neurosci. 19: 235-263, the contents of which are
incorporated herein by reference. Amino acid residues 159-175 of
the native DHDR-1 protein are predicted to comprise a transmembrane
domain (see FIG. 3). Amino acid residues 7-23 of the native DHDR-2
protein and residues 265-283 of the mature DHDR-2 protein are
predicted to comprise a transmembrane domain (see FIG. 8). Amino
acid residues 10-26, 73-90, 289-305, and 312-333 of the native
DHDR-3 protein are predicted to comprise transmembrane domains (see
FIG. 13). Amino acid residues 29-50, 170-188, 108-224, and 258-275
of the native DHDR-4 protein and residues 10-31, 151-169, 189-205,
and 239-256 of the mature DHDR-4 protein are predicted to comprise
transmembrane domains (see FIG. 19). Accordingly, DHDR proteins
having at least 50-60% homology, preferably about 60-70%, more
preferably about 70-80%, or about 80-90% homology with a
transmembrane domain of human DHDR are within the scope of the
invention.
[0062] In another embodiment of the invention, a DHDR protein of
the present invention is identified based on the presence of a
signal peptide. The prediction of such a signal peptide can be
made, for example, utilizing the computer algorithm SignalP
(Henrik, et al. (1997) Protein Engineering 10:1-6). As used herein,
a "signal sequence" or "signal peptide" includes a peptide
containing about 15 or more amino acids which occurs at the
N-terminus of secretory and membrane bound proteins and which
contains a large number of hydrophobic amino acid residues. For
example, a signal sequence contains at least about 10-30 amino acid
residues, preferably about 15-25 amino acid residues, more
preferably about 18-20 amino acid residues, and more preferably
about 19 amino acid residues, and has at least about 35-65%,
preferably about 38-50%, and more preferably about 40-45%
hydrophobic amino acid residues (e.g., Valine, Leucine, Isoleucine
or Phenylalanine). Such a "signal sequence", also referred to in
the art as a "signal peptide", serves to direct a protein
containing such a sequence to a lipid bilayer, and is cleaved in
secreted and membrane bound proteins. A signal sequence was
identified in the amino acid sequence of human DHDR-2 at about
amino acids 1-18 of SEQ ID NO:5. A signal sequence was also
identified in the amino acid sequence of human DHDR-4 at about
amino acids 1-19 of SEQ ID NO:11.
[0063] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of an "aldehyde
dehydrogenase family domain" in the protein or corresponding
nucleic acid molecule. As used herein, the term "aldehyde
dehydrogenase family domain" includes a protein domain having an
amino acid sequence of about 350-550 amino acid residues and a bit
score of at least 149.8. Preferably, an aldehyde dehydrogenase
family domain includes at least about 400-500, or more preferably
about 448 amino acid residues, and a bit score of about 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,
220, 230, 240, or 250 or more. To identify the presence of an
aldehyde dehydrogenase family domain in a DHDR protein, and make
the determination that a protein of interest has a particular
profile, the amino acid sequence of the protein is searched against
a database of known protein domains (e.g., the HMM database). The
aldehyde dehydrogenase family domain (HMM) has been assigned the
PFAM Accession PF00171 (http://genome.wustl.edu/Pfam/.html). A
search was performed against the HMM database resulting in the
identification of an aldehyde dehydrogenase family domain in the
amino acid sequence of human DHDR-1 (SEQ ID NO: 2) at about
residues 47-494 of SEQ ID NO: 2. The results of the search are set
forth in FIG. 4.
[0064] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of an "aldehyde
dehydrogenase oxidoreductase domain" in the protein or
corresponding nucleic acid molecule. As used herein, the term
"aldehyde dehydrogenase oxidoreductase domain" includes a protein
domain having an amino acid sequence of about 550-750 amino acid
residues and having a bit score for the alignment of the sequence
to the aldehyde dehydrogenase oxidoreductase domain of at least
280. Preferably, an aldehyde dehydrogenase oxidoreductase domain
includes at least about 600-700, or more preferably about 670 amino
acid residues, and has a bit score for the alignment of the
sequence to the aldehyde dehydrogenase oxidoreductase domain of at
least 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 or
higher. The aldehyde dehydrogenase oxidoreductase domain has been
assigned ProDom entry 135. To identify the presence of an aldehyde
dehydrogenase oxidoreductase domain in a DHDR protein, and to make
the determination that a protein of interest has a particular
profile, the amino acid sequence of the protein is searched against
a database of known protein domains (e.g., the ProDom database)
using the default parameters (available at
http://www.toulouse.inra.fr/prodom.html). A search was performed
against the ProDom database resulting in the identification of an
aldehyde dehydrogenase oxidoreductase domain in the amino acid
sequence of human DHDR-1 (SEQ ID NO: 2) at about residues 101-770
of SEQ ID NO: 2. The results of the search are set forth in FIG.
5.
[0065] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of a "short chain
dehydrogenase domain" in the protein or corresponding nucleic acid
molecule. As used herein, the term "short chain dehydrogenase
domain" includes a protein domain having an amino acid sequence of
about 100-300 amino acid residues, and a bit score of at least
120.0-162.5. Preferably, a short chain dehydrogenase domain
includes at least about 150-250, or more preferably about 187-195
amino acid residues, and has a bit score of at least 40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
210, 220, or more. To identify the presence of a short chain
dehydrogenase domain in a DHDR protein, and make the determination
that a protein of interest has a particular profile, the amino acid
sequence of the protein is searched against a database of known
protein domains (e.g., the HMM database). The short chain
dehydrogenase domain (HMM) has been assigned the PFAM Accession
PF00106 (http://genome.wustl.edu/Pfam/ht- ml). A search was
performed against the HMM database resulting in the identification
of a short chain dehydrogenase domain in the amino acid sequence of
human DHDR-2 (SEQ ID NO: 5) at about residues 38-227 of SEQ ID NO:
5. The results of the search are set forth in FIG. 9. A search was
also performed against the HMM database resulting in the
identification of a short chain dehydrogenase domain in the amino
acid sequence of human DHDR-3 (SEQ ID NO:8) at about residues
10-197 of SEQ ID NO:8. The results of this search are set forth in
FIG. 14. Another search performed against the HMM database resulted
in the identification of a short chain dehydrogenase domain in the
amino acid sequence of human DHDR-4 (SEQ ID NO:11) at about
residues 38-226 of SEQ ID NO:11. The results of this search are set
forth in FIG. 20.
[0066] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of an "oxidoreductase
protein dehydrogenase domain" in the protein or corresponding
nucleic acid molecule. As used herein, the term "oxidoreductase
protein dehydrogenase domain" includes a protein domain having an
amino acid sequence of about 50-300 amino acid residues and having
a bit score for the alignment of the sequence to the oxidoreductase
protein dehydrogenase domain of at least 113. Preferably, an
oxidoreductase protein dehydrogenase domain includes at least about
100-250, or more preferably about 120-200 amino acid residues, and
has a bit score for the alignment of the sequence to the
oxidoreductase protein dehydrogenase domain of at least 40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or higher.
The oxidoreductase protein dehydrogenase domain has been assigned
ProDom entry 11. To identify the presence of an oxidoreductase
protein dehydrogenase domain in a DHDR protein, and to make the
determination that a protein of interest has a particular profile,
the amino acid sequence of the protein is searched against a
database of known protein domains (e.g., the ProDom database) using
the default parameters (available at
http://www.toulouse.inra.fr/prodom.html). A search was performed
against the ProDom database resulting in the identification of an
oxidoreductase protein dehydrogenase domain in the amino acid
sequence of human DHDR-2 (SEQ ID NO: 5) at about residues 99-219 of
SEQ ID NO: 5. The results of the search are set forth in FIG. 10.
Another search was performed against the ProDom database, resulting
in the identification of an oxidoreductase protein dehydrogenase
domain in the amino acid sequence of human DHDR-4 (SEQ ID NO:11) at
about residues 37-231 of SEQ ID NO:11. The results of this search
are set forth in FIG. 21.
[0067] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of an "NAD-dependent
epimerase/dehydratase domain" in the protein or corresponding
nucleic acid molecule. As used herein, the term "NAD-dependent
epimerase/dehydratase domain" includes a protein domain having an
amino acid sequence of about 250-450 amino acid residues.
Preferably, an NAD-dependent epimerase/dehydratase domain includes
at least about 300-400, or more preferably about 354 amino acid
residues. To identify the presence of an NAD-dependent
epimerase/dehydratase domain in a DHDR protein, and to make the
determination that a protein of interest has a particular profile,
the amino acid sequence of the protein is searched against a
database of known protein domains (e.g., the HMM database). The
NAD-dependent epimerase/dehydratase domain (HMM) has been assigned
the PFAM Accession PF01370 (http://genome.wustl.edu/Pfam/html). A
search was performed against the HMM database resulting in the
identification of an NAD-dependent epimerase/dehydratase domain in
the amino acid sequence of human DHDR-3 (SEQ ID NO: 8) at about
residues 12-365 of SEQ ID NO: 8. The results of the search are set
forth in FIG. 14.
[0068] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of a "3-beta
hydroxysteroid dehydrogenase domain" in the protein or
corresponding nucleic acid molecule. As used herein, the term
"3-beta hydroxysteroid dehydrogenase domain" includes a protein
domain having an amino acid sequence of about 250-450 amino acid
residues and having a bit score for the alignment of the sequence
to the 3-beta hydroxysteroid dehydrogenase domain of at least
395-676.9. Preferably, a 3-beta hydroxysteroid dehydrogenase domain
includes at least about 300-400, or more preferably about 352-365
amino acid residues, and has a bit score for the alignment of the
sequence to the 3-beta hydroxysteroid dehydrogenase domain of at
least 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,
600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or higher. The
3-beta hydroxysteroid dehydrogenase domain has been assigned ProDom
entry 1280. To identify the presence of a 3-beta hydroxysteroid
dehydrogenase domain in a DHDR protein, and to make the
determination that a protein of interest has a particular profile,
the amino acid sequence of the protein is searched against a
database of known protein domains (e.g., the ProDom database) using
the default parameters (available at
http://www.toulouse.inra.fr/pr- odom.html). A search was performed
against the ProDom database resulting in the identification of a
3-beta hydroxysteroid dehydrogenase domain in the amino acid
sequence of human DHDR-3 (SEQ ID NO: 8) at about residues 11-362 of
SEQ ID NO: 8. The results of the search are set forth in FIG. 15. A
search was also performed against the HMM database resulting in the
identification of a 3-beta hydroxysteroid dehydrogenase domain
(PFAM accession PF01073, see http://genome.wustl.edu/Pfam/html) in
the amino acid sequence of human DHDR-3 (SEQ ID NO: 8) at about
residues 1-365 of SEQ ID NO:8. The results of the search are set
forth in FIG. 14.
[0069] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of a "short-chain
dehydrogenase/reductase domain" in the protein or corresponding
nucleic acid molecule. As used herein, the term "short-chain
dehydrogenase/reductase domain" includes a protein domain having an
amino acid sequence of about 10-100 amino acid residues, and a bit
score of at least 47.2. Preferably, a short-chain
dehydrogenase/reductase domain includes at least about 20-75, or
more preferably about 31 amino acid residues, and has a bit score
of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95 or more. To identify the presence of a
short-chain dehydrogenase/reductase domain in a DHDR protein, and
to make the determination that a protein of interest has a
particular profile, the amino acid sequence of the protein is
searched against a database of known protein domains (e.g., the HMM
database). The short-chain dehydrogenase/reductase domain (HMM) has
been assigned the PFAM Accession PF00678
(http://genome.wustl.edu/Pfam/html). A search was performed against
the HMM database resulting in the identification of a short-chain
dehydrogenase/reductase domain in the amino acid sequence of human
DHDR-4 (SEQ ID NO: 11) at about residues 250-280 of SEQ ID NO: 11.
The results of the search are set forth in FIG. 20.
[0070] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of a "shikimate
5-dehydrogenase domain" in the protein or corresponding nucleic
acid molecule. As used herein, the term "shikimate 5-dehydrogenase
domain" includes a protein domain having an amino acid sequence of
about 10-100 amino acid residues and having a bit score for the
alignment of the sequence to the shikimate 5-dehydrogenase domain
of at least 86. Preferably, a shikimate 5-dehydrogenase domain
includes at least about 25-75, or more preferably about 48 amino
acid residues, and has a bit score for the alignment of the
sequence to the shikimate 5-dehydrogenase domain of at least 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or higher. The
shikimate 5-dehydrogenase domain has been assigned ProDom entry
95301. To identify the presence of a shikimate 5-dehydrogenase
domain in a DHDR protein, and to make the determination that a
protein of interest has a particular profile, the amino acid
sequence of the protein is searched against a database of known
protein domains (e.g., the ProDom database) using the default
parameters (available at http://www.toulouse.inra.fr/prodom.html)-
. A search was performed against the ProDom database resulting in
the identification of a shikimate 5-dehydrogenase domain in the
amino acid sequence of human DHDR-4 (SEQ ID NO: 11) at about
residues 35-82 of SEQ ID NO: 11. The results of the search are set
forth in FIG. 21.
[0071] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of a "dehydrogenase
domain" in the protein or corresponding nucleic acid molecule. As
used herein, the term "dehydrogenase domain" includes a protein
domain having an amino acid sequence of about 10-100 amino acid
residues and having a bit score for the alignment of the sequence
to the dehydrogenase domain of at least 84. Preferably, a
dehydrogenase domain includes at least about 25-75, or more
preferably about 50 amino acid residues, and has a bit score for
the alignment of the sequence to the dehydrogenase domain of at
least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or higher.
The dehydrogenase domain has been assigned ProDom entry 73753. To
identify the presence of a dehydrogenase domain in a DHDR protein,
and to make the determination that a protein of interest has a
particular profile, the amino acid sequence of the protein is
searched against a database of known protein domains (e.g., the
ProDom database) using the default parameters (available at
http://www.toulouse.inra.fr/prodom.html). A search was performed
against the ProDom database resulting in the identification of a
dehydrogenase domain in the amino acid sequence of human DHDR-4
(SEQ ID NO: 11) at about residues 237-286 of SEQ ID NO: 11. The
results of the search are set forth in FIG. 21.
[0072] In another embodiment, a DHDR molecule of the present
invention is identified based on the presence of a
"glucose-1-dehydrogenase domain" in the protein or corresponding
nucleic acid molecule. As used herein, the term
"glucose-1-dehydrogenase domain" includes a protein domain having
an amino acid sequence of about 10-100 amino acid residues and
having a bit score for the alignment of the sequence to the
glucose-1-dehydrogenase domain of at least 92. Preferably, a
dehydrogenase domain includes at least about 25-75, or more
preferably about 45 amino acid residues, and has a bit score for
the alignment of the sequence to the dehydrogenase domain of at
least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or
higher. The glucose-1-dehydrogenase domain has been assigned ProDom
entry 77223. To identify the presence of a glucose-1-dehydrogenase
domain in a DHDR protein, and to make the determination that a
protein of interest has a particular profile, the amino acid
sequence of the protein is searched against a database of known
protein domains (e.g., the ProDom database) using the default
parameters (available at http://www.toulouse.inra.fr/prodom.html).
A search was performed against the ProDom database resulting in the
identification of a dehydrogenase domain in the amino acid sequence
of human DHDR-4 (SEQ ID NO: 11) at about residues 243-287 of SEQ ID
NO: 11. The results of the search are set forth in FIG. 21.
[0073] In a preferred embodiment, the DHDR molecules of the
invention include at least one or more of the following domains: a
transmembrane domain, a signal peptide domain, an aldehyde
dehydrogenase oxidoreductase domain, an aldehyde dehydrogenase
family domain, a short chain dehydrogenase domain, an
oxidoreductase protein dehydrogenase domain, a 3-beta
hydroxysteroid dehydrogenase domain, a NAD-dependent
epimerase/dehydratase domain, a short chain dehydrogenase/reductase
domain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,
and a glucose-1-dehydrogenase domain.
[0074] Isolated proteins of the present invention, preferably DHDR
proteins, have an amino acid sequence sufficiently identical to the
amino acid sequence of SEQ ID NO:2, 5, 8, or 11, or are encoded by
a nucleotide sequence sufficiently identical to SEQ ID NO:1, 3, 4,
6, 7, 9, 10, or 12. 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 90-95% 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 share a common
functional activity are defined herein as sufficiently
identical.
[0075] As used interchangeably herein, an "DHDR activity",
"biological activity of DHDR" or "functional activity of DHDR",
refers to an activity exerted by a DHDR protein, polypeptide or
nucleic acid molecule on a DHDR responsive cell or tissue, or on a
DHDR protein substrate, as determined in vivo, or in vitro,
according to standard techniques. In one embodiment, a DHDR
activity is a direct activity, such as an association with a
DHDR-target molecule. As used herein, a "target molecule" or
"binding partner" is a molecule with which a DHDR protein binds or
interacts in nature, such that DHDR-mediated function is achieved.
A DHDR target molecule can be a non-DHDR molecule or a DHDR protein
or polypeptide of the present invention (e.g., NAD+, NADP+, or
other cofactor). In an exemplary embodiment, a DHDR target molecule
is a DHDR ligand (e.g., an alcohol, an aldehyde, a lipid, or a
steroid (e.g., a glucocorticoid)). Alternatively, a DHDR activity
is an indirect activity, such as a cellular signaling activity
mediated by interaction of the DHDR protein with a DHDR ligand. The
biological activities of DHDR are described herein. For example,
the DHDR proteins of the present invention can have one or more of
the following activities: 1) modulate metabolism and catabolism of
biochemical molecules necessary for energy production or storage,
2) modulate intra- or inter-cellular signaling, 3) modulate
metabolism or catabolism of metabolically important biomolecules
(e.g., glucocorticoids), 4) modulate detoxification of potentially
harmful compounds, 5) modulate viral infection (e.g., by modulating
viral gene expression), and 6) act as a transcriptional cofactor
for viral gene activation.
[0076] Accordingly, another embodiment of the invention features
isolated DHDR proteins and polypeptides having a DHDR activity.
Other preferred proteins are DHDR proteins having one or more of
the following domains: a transmembrane domain, a signal peptide
domain, an aldehyde dehydrogenase oxidoreductase domain, an
aldehyde dehydrogenase family domain, a short chain dehydrogenase
domain, an oxidoreductase protein dehydrogenase domain, a 3-beta
hydroxysteroid dehydrogenase domain, a NAD-dependent
epimerase/dehydratase domain, a short chain dehydrogenase/reductase
domain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,
or a glucose-1-dehydrogenase domain and, preferably, a DHDR
activity.
[0077] Additional preferred proteins have at least one
transmembrane domain, and one or more of a signal peptide domain,
an aldehyde dehydrogenase oxidoreductase domain, an aldehyde
dehydrogenase family domain, a short chain dehydrogenase domain, an
oxidoreductase protein dehydrogenase domain, a 3-beta
hydroxysteroid dehydrogenase domain, a NAD-dependent
epimerase/dehydratase domain, a short chain dehydrogenase/reductase
domain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,
or a glucose-1-dehydrogenase domain., and are, preferably, encoded
by a nucleic acid molecule having a nucleotide sequence which
hybridizes under stringent hybridization conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3,
4, 6, 7, 9, 10, or 12.
[0078] The nucleotide sequence of the isolated human DHDR-1 cDNA
and the predicted amino acid sequence of the human DHDR-1
polypeptide are shown in FIG. 1 and in SEQ ID NOs:1 and 2,
respectively. The nucleotide sequence of the isolated human DHDR-2
cDNA and the predicted amino acid sequence of the human DHDR-2
polypeptide are shown in FIG. 6 and in SEQ ID NOs: 4 and 5,
respectively. The nucleotide sequence of the isolated human DHDR-3
cDNA and the predicted amino acid sequence of the human DHDR-3
polypeptide are shown in FIG. 11 and in SEQ ID NOs:7 and 8,
respectively. The nucleotide sequence of the isolated human DHDR-4
cDNA and the predicted amino acid sequence of the human DHDR-4
polypeptide are shown in FIG. 16 and in SEQ ID NOs:10 and 11,
respectively. Plasmids containing the nucleotide sequence encoding
human DHDR-1, DHDR-2, DHDR-3, and DHDR-4 were deposited with the
American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, on ______ and assigned
Accession Numbers ______. These deposits will be maintained under
the terms of the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for the Purposes of Patent
Procedure. These deposits were made merely as a convenience for
those of skill in the art and are not an admission that deposits
are required under 35 U.S.C. .sctn.112.
[0079] The human DHDR-1 gene, which is approximately 2660
nucleotides in length, encodes a protein having a molecular weight
of approximately 88.0 kD and which is approximately 802 amino acid
residues in length. The human DHDR-2 gene, which is approximately
1379 nucleotides in length, encodes a protein having a molecular
weight of approximately 34.2 kD and which is approximately 311
amino acid residues in length. The human DHDR-3 gene, which is
approximately 1725 nucleotides in length, encodes a protein having
a molecular weight of approximately 40.5 kD and which is
approximately 369 amino acid residues in length. The human DHDR-4
gene, which is approximately 1209 nucleotides in length, encodes a
protein having a molecular weight of approximately 35.4 kD and
which is approximately 322 amino acid residues in length.
[0080] Various aspects of the invention are described in further
detail in the following subsections:
[0081] I. Isolated Nucleic Acid Molecules
[0082] One aspect of the invention pertains to isolated nucleic
acid molecules that encode DHDR proteins or biologically active
portions thereof, as well as nucleic acid fragments sufficient for
use as hybridization probes to identify DHDR-encoding nucleic acid
molecules (e.g., DHDR mRNA) and fragments for use as PCR primers
for the amplification or mutation of DHDR 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.
[0083] 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 genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated DHDR 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.
[0084] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, 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, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______ as a hybridization probe, DHDR 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).
[0085] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______ can be isolated by the polymerase chain
reaction (PCR) using synthetic oligonucleotide primers designed
based upon the sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12,
or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______.
[0086] 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 DHDR nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0087] In a preferred embodiment, an isolated nucleic acid molecule
of the invention comprises the nucleotide sequence shown in SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, or 12. This cDNA may comprise sequences
encoding the human DHDR-1 protein (i.e., "the coding region", from
nucleotides 63-2471), as well as 5' untranslated sequences
(nucleotides 1-62) and 3' untranslated sequences (nucleotides
2472-2660) of SEQ ID NO:1. This cDNA may comprise sequences
encoding the human DHDR-2 protein (i.e., "the coding region", from
nucleotides 331-1266), as well as 5' untranslated sequences
(nucleotides 1-330) and 3' untranslated sequences (nucleotides
1267-1379) of SEQ ID NO:4. This cDNA may comprise sequences
encoding the human DHDR-3 protein (i.e., "the coding region", from
nucleotides 281-1390), as well as 5' untranslated sequences
(nucleotides 1-280) and 3' untranslated sequences (nucleotides
1391-1725) of SEQ ID NO:7. This cDNA may comprise sequences
encoding the human DHDR-4 protein (i.e., "the coding region", from
nucleotides 61-1029), as well as 5' untranslated sequences
(nucleotides 1-60) and 3' untranslated sequences (nucleotides
1030-1209) of SEQ ID NO:10. Alternatively, the nucleic acid
molecule can comprise only the coding region of SEQ ID NO:1 (e.g.
nucleotides 63-2471, corresponding to SEQ ID NO:3), only the coding
region of SEQ ID NO:4 (e.g., nucleotides 331-1266, corresponding to
SEQ ID NO:6), only the coding region of SEQ ID NO:7 (e.g.,
nucleotides 281-1390, corresponding to SEQ ID NO:9), or only the
coding region of SEQ ID NO:10 (e.g., nucleotides 61-1029,
corresponding to SEQ ID NO:12).
[0088] 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, 3,
4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number ______, 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, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence
of the DNA insert of the plasmid deposited with ATCC as Accession
Number ______, is one which is sufficiently complementary to the
nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12,
or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______ such that it can
hybridize to the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6,
7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of
the plasmid deposited with ATCC as Accession Number ______,
respectively, thereby forming a stable duplex.
[0089] 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, 3, 4, 6, 7,
9, 10, or 12, or the entire length of the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC as Accession
Number ______, or a portion of any of these nucleotide
sequences.
[0090] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the nucleic acid sequence of SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, for example, a fragment which can be used as a probe or
primer or a fragment encoding a portion of a DHDR protein, e.g, a
biologically active portion of a DHDR protein. The nucleotide
sequences determined from the cloning of the DHDR-1, DHDR-2,
DHDR-3, and DHDR-4 genes allow for the generation of probes and
primers designed for use in identifying and/or cloning other DHDR
family members, as well as DHDR 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, 3, 4, 6, 7, 9, 10,
or 12, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______ of an antisense
sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12, or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number ______ or of a naturally occurring allelic
variant or mutant of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12, or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number ______. In one embodiment, a nucleic acid
molecule of the present invention comprises a nucleotide sequence
which is greater than 50-100, 100-150, 150-200, 200-250, 250-300,
300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650,
650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000 or
more nucleotides in length and hybridizes under stringent
hybridization conditions to a nucleic acid molecule of SEQ ID NO:1,
3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
______.
[0091] Probes based on the DHDR 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 DHDR
protein, such as by measuring a level of a DHDR-encoding nucleic
acid in a sample of cells from a subject e.g., detecting DHDR mRNA
levels or determining whether a genomic DHDR gene has been mutated
or deleted.
[0092] A nucleic acid fragment encoding a "biologically active
portion of a DHDR protein" can be prepared by isolating a portion
of the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or
12, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______ which encodes a
polypeptide having a DHDR biological activity (the biological
activities of the DHDR proteins are described herein), expressing
the encoded portion of the DHDR protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the DHDR protein.
[0093] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1, 3,
4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number ______ due
to degeneracy of the genetic code and thus encode the same DHDR
proteins as those encoded by the nucleotide sequence shown in SEQ
ID NO:1, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC as Accession
Number ______. 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, 5, 8,
or 11.
[0094] In addition to the DHDR nucleotide sequences shown in SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, 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 DHDR proteins may exist within a population (e.g.,
the human population). Such genetic polymorphism in the DHDR 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 DHDR protein, preferably a mammalian
DHDR protein, and can further include non-coding regulatory
sequences, and introns.
[0095] Allelic variants of human DHDR include both functional and
non-functional DHDR proteins. Functional allelic variants are
naturally occurring amino acid sequence variants of the human DHDR
protein that maintain the ability to bind a DHDR ligand or
substrate and/or modulate cell proliferation and/or migration
mechanisms. Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:2, 5, 8, or 11, or substitution, deletion or insertion of
non-critical residues in non-critical regions of the protein.
[0096] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the human DHDR protein that do not
have the ability to either bind a DHDR ligand and/or modulate any
of the DHDR 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, 5, 8, or 11, or a substitution, insertion
or deletion in critical residues or critical regions of the
protein.
[0097] The present invention further provides non-human orthologues
of the human DHDR protein. Orthologues of the human DHDR protein
are proteins that are isolated from non-human organisms and possess
the same DHDR ligand binding and/or modulation of membrane
excitability activities of the human DHDR protein. Orthologues of
the human DHDR protein can readily be identified as comprising an
amino acid sequence that is substantially identical to SEQ ID NO:2,
5, 8, or 11.
[0098] Moreover, nucleic acid molecules encoding other DHDR family
members and, thus, which have a nucleotide sequence which differs
from the DHDR sequences of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12,
or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______ are intended to be
within the scope of the invention. For example, another DHDR cDNA
can be identified based on the nucleotide sequence of human DHDR.
Moreover, nucleic acid molecules encoding DHDR proteins from
different species, and which, thus, have a nucleotide sequence
which differs from the DHDR sequences of SEQ ID NO:1, 3, 4, 6, 7,
9, 10, or 12, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______ are intended
to be within the scope of the invention. For example, a mouse DHDR
cDNA can be identified based on the nucleotide sequence of a human
DHDR.
[0099] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the DHDR cDNAs of the invention can be
isolated based on their homology to the DHDR 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 DHDR cDNAs of the invention can further be
isolated by mapping to the same chromosome or locus as the DHDR
gene.
[0100] 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, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC as Accession
Number ______. In other embodiment, the nucleic acid is at least
50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400,
400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750,
750-800, 800-850, 850-900, 900-950, 950-1000 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 at least 60% identical
to each other typically 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 typically 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, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
A preferred, non-limiting example of stringent hybridization
conditions are hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times. SSC, 0.1% SDS at 50.degree. C., preferably at
55.degree. C., more preferably at 60.degree. C., and even more
preferably at 65.degree. C. Ranges intermediate to the
above-recited values, e.g., at 60-65.degree. C. or at 55-60.degree.
C. are also intended to be encompassed by the present invention.
Preferably, an isolated nucleic acid molecule of the invention that
hybridizes under stringent conditions to the sequence of SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, or 12, 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).
[0101] In addition to naturally-occurring allelic variants of the
DHDR 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, 3, 4, 6, 7,
9, 10, or 12, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______, thereby
leading to changes in the amino acid sequence of the encoded DHDR
proteins, without altering the functional ability of the DHDR
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, 3, 4, 6, 7, 9, 10, or 12, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______. A "non-essential" amino acid
residue is a residue that can be altered from the wild-type
sequence of DHDR (e.g., the sequence of SEQ ID NO:2, 5, 8, or 11)
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 DHDR
proteins of the present invention, e.g., those present in a
transmembrane domain, are predicted to be particularly unamenable
to alteration. Furthermore, additional amino acid residues that are
conserved between the DHDR proteins of the present invention and
other members of the DHDR family are not likely to be amenable to
alteration.
[0102] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding DHDR proteins that contain changes
in amino acid residues that are not essential for activity. Such
DHDR proteins differ in amino acid sequence from SEQ ID NO:2, 5, 8,
or 11, 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, 5,
8, or 11.
[0103] An isolated nucleic acid molecule encoding a DHDR protein
identical to the protein of SEQ ID NO:2, 5, 8, or 11 can be created
by introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7,
9, 10, or 12, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______ 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, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______ 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 DHDR 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 DHDR coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for DHDR biological activity to identify mutants that
retain activity. Following mutagenesis of SEQ ID NO:1, 3, 4, 6, 7,
9, 10, or 12, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______, the encoded
protein can be expressed recombinantly and the activity of the
protein can be determined.
[0104] In a preferred embodiment, a mutant DHDR protein can be
assayed for the ability to metabolize or catabolize biochemical
molecules necessary for energy production or storage, permit intra-
or inter-cellular signaling, metabolize or catabolize metabolically
important biomolecules, and to detoxify potentially harmful
compounds.
[0105] In addition to the nucleic acid molecules encoding DHDR
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 DHDR
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 DHDR. 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 DHDR corresponds to SEQ
ID NO:3, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO:12). In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding DHDR. 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).
[0106] Given the coding strand sequences encoding DHDR disclosed
herein (e.g., SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID
NO:12), 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 DHDR mRNA, but more preferably is an
oligonucleotide which is antisense to only a portion of the coding
or noncoding region of DHDR mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of DHDR 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-carboxymethylaminomet- hyluracil, 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-isopenten- yladenine,
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).
[0107] 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 DHDR 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.
[0108] 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).
[0109] 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 DHDR mRNA transcripts to thereby
inhibit translation of DHDR mRNA. A ribozyme having specificity for
a DHDR-encoding nucleic acid can be designed based upon the
nucleotide sequence of a DHDR cDNA disclosed herein (i.e., SEQ ID
NO:1, 3, 4, 6, 7, 9, 10, or 12, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______). 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
DHDR-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, DHDR 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.
[0110] Alternatively, DHDR gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the DHDR (e.g., the DHDR promoter and/or enhancers; e.g.,
nucleotides 1-62 of SEQ ID NO:1, nucleotides 1-330 of SEQ ID NO: 4,
nucleotides 1-280 of SEQ ID NO:7, or nucleotides 1-60 of SEQ ID
NO:10) to form triple helical structures that prevent transcription
of the DHDR 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.
[0111] In yet another embodiment, the DHDR 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.
[0112] PNAs of DHDR 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 DHDR 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).
[0113] In another embodiment, PNAs of DHDR 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
DHDR 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-thy- midine
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).
[0114] 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. W088/09810) or the
blood-brain barrier (see, e.g, PCT Publication No. W089/10134). 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).
[0115] Alternatively, the expression characteristics of an
endogenous DHDR 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 DHDR gene. For example, an endogenous DHDR gene which is
normally "transcriptionally silent", i.e., a DHDR 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 DHDR gene may be activated by insertion of a promiscuous
regulatory element that works across cell types.
[0116] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with an endogenous DHDR 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.
[0117] II. Isolated DHDR Proteins and Anti-DHDR Antibodies
[0118] One aspect of the invention pertains to isolated DHDR
proteins, and biologically active portions thereof, as well as
polypeptide fragments suitable for use as immunogens to raise
anti-DHDR antibodies. In one embodiment, native DHDR proteins can
be isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, DHDR proteins are produced by recombinant
DNA techniques. Alternative to recombinant expression, a DHDR
protein or polypeptide can be synthesized chemically using standard
peptide synthesis techniques.
[0119] 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 DHDR 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 DHDR 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
DHDR protein having less than about 30% (by dry weight) of non-DHDR
protein (also referred to herein as a "contaminating protein"),
more preferably less than about 20% of non-DHDR protein, still more
preferably less than about 10% of non-DHDR protein, and most
preferably less than about 5% non-DHDR protein. When the DHDR
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.
[0120] The language "substantially free of chemical precursors or
other chemicals" includes preparations of DHDR 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 DHDR protein having
less than about 30% (by dry weight) of chemical precursors or
non-DHDR chemicals, more preferably less than about 20% chemical
precursors or non-DHDR chemicals, still more preferably less than
about 10% chemical precursors or non-DHDR chemicals, and most
preferably less than about 5% chemical precursors or non-DHDR
chemicals.
[0121] As used herein, a "biologically active portion" of a DHDR
protein includes a fragment of a DHDR protein which participates in
an interaction between a DHDR molecule and a non-DHDR molecule.
Biologically active portions of a DHDR protein include peptides
comprising amino acid sequences sufficiently identical to or
derived from the amino acid sequence of the DHDR protein, e.g., the
amino acid sequence shown in SEQ ID NO:2, 5, 8, or 11, which
include less amino acids than the full length DHDR proteins, and
exhibit at least one activity of a DHDR protein. Typically,
biologically active portions comprise a domain or motif with at
least one activity of the DHDR protein, e.g., modulating membrane
excitability. A biologically active portion of a DHDR protein can
be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150,
175, 200, 250, 300 or more amino acids in length. Biologically
active portions of a DHDR protein can be used as targets for
developing agents which modulate a DHDR mediated activity, e.g., a
proliferative response.
[0122] In one embodiment, a biologically active portion of a DHDR
protein comprises at least one transmembrane domain. It is to be
understood that a preferred biologically active portion of a DHDR
protein of the present invention may contain at least one
transmembrane domain and one or more of the following domains: a
signal peptide domain, an aldehyde dehydrogenase oxidoreductase
domain, an aldehyde dehydrogenase family domain, a short chain
dehydrogenase domain, an oxidoreductase protein dehydrogenase
domain, a 3-beta hydroxysteroid dehydrogenase domain, a
NAD-dependent epimerase/dehydratase domain, a short chain
dehydrogenase/reductase domain, a shikimate 5-dehydrogenase domain,
a dehydrogenase domain, or a glucose-1-dehydrogenase 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 DHDR protein.
[0123] In a preferred embodiment, the DHDR protein has an amino
acid sequence shown in SEQ ID NO:2, 5, 8, or 11. In other
embodiments, the DHDR protein is substantially identical to SEQ ID
NO:2, 5, 8, or 11, and retains the functional activity of the
protein of SEQ ID NO:2, 5, 8, or 11, 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 DHDR 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,
5, 8, or 11.
[0124] 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 DHDR amino acid sequence of SEQ ID NO:2, 5, 8, or 11 having 400
amino acid residues, at least 50, preferably at least 100, more
preferably at least 150, even more preferably at least 200, and
even more preferably at least 300 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.
[0125] 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 or 2.0U), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0126] 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 DHDR 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 DHDR 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.
[0127] The invention also provides DHDR chimeric or fusion
proteins. As used herein, a DHDR "chimeric protein" or "fusion
protein" comprises a DHDR polypeptide operatively linked to a
non-DHDR polypeptide. An "DHDR polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a DHDR molecule,
whereas a "non-DHDR polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially homologous to the DHDR protein, e.g., a protein which
is different from the DHDR protein and which is derived from the
same or a different organism. Within a DHDR fusion protein the DHDR
polypeptide can correspond to all or a portion of a DHDR protein.
In a preferred embodiment, a DHDR fusion protein comprises at least
one biologically active portion of a DHDR protein. In another
preferred embodiment, a DHDR fusion protein comprises at least two
biologically active portions of a DHDR protein. Within the fusion
protein, the term "operatively linked" is intended to indicate that
the DHDR polypeptide and the non-DHDR polypeptide are fused
in-frame to each other. The non-DHDR polypeptide can be fused to
the N-terminus or C-terminus of the DHDR polypeptide.
[0128] For example, in one embodiment, the fusion protein is a
GST-DHDR fusion protein in which the DHDR sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant DHDR.
[0129] In another embodiment, the fusion protein is a DHDR protein
containing a heterologous signal sequence at its N-terminus. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of DHDR can be increased through use of a heterologous
signal sequence.
[0130] The DHDR fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The DHDR fusion proteins can be used to affect the
bioavailability of a DHDR substrate. Use of DHDR 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 DHDR protein; (ii) mis-regulation of the DHDR gene; and
(iii) aberrant post-translational modification of a DHDR
protein.
[0131] Moreover, the DHDR-fusion proteins of the invention can be
used as immunogens to produce anti-DHDR antibodies in a subject, to
purify DHDR ligands and in screening assays to identify molecules
which inhibit the interaction of DHDR with a DHDR substrate.
[0132] Preferably, a DHDR 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 DHDR-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the DHDR protein.
[0133] The present invention also pertains to variants of the DHDR
proteins which function as either DHDR agonists (mimetics) or as
DHDR antagonists. Variants of the DHDR proteins can be generated by
mutagenesis, e.g., discrete point mutation or truncation of a DHDR
protein. An agonist of the DHDR proteins can retain substantially
the same, or a subset, of the biological activities of the
naturally occurring form of a DHDR protein. An antagonist of a DHDR
protein can inhibit one or more of the activities of the naturally
occurring form of the DHDR protein by, for example, competitively
modulating a DHDR-mediated activity of a DHDR 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 DHDR protein.
[0134] In one embodiment, variants of a DHDR protein which function
as either DHDR agonists (mimetics) or as DHDR antagonists can be
identified by screening combinatorial libraries of mutants, e.g.,
truncation mutants, of a DHDR protein for DHDR protein agonist or
antagonist activity. In one embodiment, a variegated library of
DHDR variants is generated by combinatorial mutagenesis at the
nucleic acid level and is encoded by a variegated gene library. A
variegated library of DHDR variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential DHDR sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of DHDR sequences therein. There
are a variety of methods which can be used to produce libraries of
potential DHDR 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 DHDR 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.
[0135] In addition, libraries of fragments of a DHDR protein coding
sequence can be used to generate a variegated population of DHDR
fragments for screening and subsequent selection of variants of a
DHDR protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a DHDR 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 DHDR protein.
[0136] 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 DHDR 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 DHDR variants (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3): 327-331).
[0137] In one embodiment, cell based assays can be exploited to
analyze a variegated DHDR 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 DHDR ligand in a
particular DHDR ligand-dependent manner. The transfected cells are
then contacted with a DHDR ligand and the effect of expression of
the mutant on, e.g., membrane excitability of DHDR can be detected.
Plasmid DNA can then be recovered from the cells which score for
inhibition, or alternatively, potentiation of signaling by the DHDR
ligand, and the individual clones further characterized.
[0138] An isolated DHDR protein, or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind DHDR
using standard techniques for polyclonal and monoclonal antibody
preparation. A full-length DHDR protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of DHDR for use as immunogens. The antigenic peptide of DHDR
comprises at least 8 amino acid residues of the amino acid sequence
shown in SEQ ID NO:2, 5, 8, or 11 and encompasses an epitope of
DHDR such that an antibody raised against the peptide forms a
specific immune complex with the DHDR 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.
[0139] Preferred epitopes encompassed by the antigenic peptide are
regions of DHDR that are located on the surface of the protein,
e.g., hydrophilic regions, as well as regions with high
antigenicity (see, for example, FIGS. 2, 7, 12, and 18).
[0140] A DHDR 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 DHDR protein or a
chemically synthesized DHDR 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 DHDR
preparation induces a polyclonal anti-DHDR antibody response.
[0141] Accordingly, another aspect of the invention pertains to
anti-DHDR 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 DHDR. 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 DHDR 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 DHDR. A monoclonal
antibody composition thus typically displays a single binding
affinity for a particular DHDR protein with which it
immunoreacts.
[0142] Polyclonal anti-DHDR antibodies can be prepared as described
above by immunizing a suitable subject with a DHDR immunogen. The
anti-DHDR 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 DHDR. If desired, the
antibody molecules directed against DHDR 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-DHDR 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. Lemer (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 DHDR 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
DHDR.
[0143] 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-DHDR 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 DHDR, e.g., using a standard
ELISA assay.
[0144] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-DHDR antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with DHDR to
thereby isolate immunoglobulin library members that bind DHDR. 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.
[0145] Additionally, recombinant anti-DHDR 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. No. 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.
[0146] An anti-DHDR antibody (e.g., monoclonal antibody) can be
used to isolate DHDR by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-DHDR antibody can
facilitate the purification of natural DHDR from cells and of
recombinantly produced DHDR expressed in host cells. Moreover, an
anti-DHDR antibody can be used to detect DHDR protein (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the DHDR protein. Anti-DHDR
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 avidin/biotin; 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.
[0147] II. Recombinant Expression Vectors and Host Cells
[0148] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
DHDR 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.
[0149] 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., DHDR proteins, mutant forms of DHDR proteins, fusion
proteins, and the like).
[0150] The recombinant expression vectors of the invention can be
designed for expression of DHDR proteins in prokaryotic or
eukaryotic cells. For example, DHDR 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.
[0151] 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.
[0152] Purified fusion proteins can be utilized in DHDR activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for DHDR
proteins, for example. In a preferred embodiment, a DHDR 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).
[0153] 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.
[0154] 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.
[0155] In another embodiment, the DHDR 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), pMFa (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.).
[0156] Alternatively, DHDR 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).
[0157] 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.
[0158] 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).
[0159] 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 DHDR 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.
[0160] Another aspect of the invention pertains to host cells into
which a DHDR nucleic acid molecule of the invention is introduced,
e.g., a DHDR nucleic acid molecule within a recombinant expression
vector or a DHDR 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.
[0161] A host cell can be any prokaryotic or eukaryotic cell. For
example, a DHDR 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.
[0162] 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.
[0163] 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 DHDR 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).
[0164] 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 DHDR protein. Accordingly, the invention further
provides methods for producing a DHDR 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 DHDR protein has been introduced) in a suitable
medium such that a DHDR protein is produced. In another embodiment,
the method further comprises isolating a DHDR protein from the
medium or the host cell.
[0165] 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 DHDR-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic
animals in which exogenous DHDR sequences have been introduced into
their genome or homologous recombinant animals in which endogenous
DHDR sequences have been altered. Such animals are useful for
studying the function and/or activity of a DHDR and for identifying
and/or evaluating modulators of DHDR 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 DHDR 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.
[0166] A transgenic animal of the invention can be created by
introducing a DHDR-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 DHDR cDNA sequence of SEQ ID NO:1, SEQ ID NO:4,
SEQ ID NO:7, or SEQ ID NO:9 can be introduced as a transgene into
the genome of a non-human animal. Alternatively, a nonhuman
homologue of a human DHDR gene, such as a mouse or rat DHDR gene,
can be used as a transgene. Alternatively, a DHDR gene homologue,
such as another DHDR family member, can be isolated based on
hybridization to the DHDR cDNA sequences of SEQ ID NO:1 or 3, SEQ
ID NO:4 or 6, SEQ ID NO:7 or 9, or SEQ ID NO: 10 or 12, or the DNA
insert of the plasmid deposited with ATCC as Accession Number______
(described further in subsection I above) 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 DHDR transgene to direct expression of a DHDR 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 DHDR
transgene in its genome and/or expression of DHDR 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 DHDR protein can
further be bred to other transgenic animals carrying other
transgenes.
[0167] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a DHDR gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the DHDR gene. The DHDR
gene can be a human gene (e.g., the cDNA of SEQ ID NO:3, SEQ ID NO:
6, SEQ ID NO:9 or SEQ ID NO:12), but more preferably, is a
non-human homologue of a human DHDR gene (e.g., a cDNA isolated by
stringent hybridization with the nucleotide sequence of SEQ ID
NO:1, SEQ ID NO: 4, SEQ ID NO:7 or SEQ ID NO:10). For example, a
mouse DHDR gene can be used to construct a homologous recombination
nucleic acid molecule, e.g., a vector, suitable for altering an
endogenous DHDR gene in the mouse genome. In a preferred
embodiment, the homologous recombination nucleic acid molecule is
designed such that, upon homologous recombination, the endogenous
DHDR 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 DHDR 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
DHDR protein). In the homologous recombination nucleic acid
molecule, the altered portion of the DHDR gene is flanked at its 5'
and 3' ends by additional nucleic acid sequence of the DHDR gene to
allow for homologous recombination to occur between the exogenous
DHDR gene carried by the homologous recombination nucleic acid
molecule and an endogenous DHDR gene in a cell, e.g., an embryonic
stem cell. The additional flanking DHDR 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 DHDR gene has
homologously recombined with the endogenous DHDR 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.
[0168] 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.
[0169] 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 G.sub.0 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.
[0170] IV. Pharmaceutical Compositions
[0171] The DHDR nucleic acid molecules, fragments of DHDR proteins,
and anti-DHDR 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.
[0172] 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.
[0173] 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.
[0174] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a fragment of a DHDR
protein or an anti-DHDR 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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).
[0188] 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.
[0189] 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.
[0190] 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.
[0191] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0192] V. Uses and Methods of the Invention
[0193] 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 DHDR protein
of the invention has one or more of the following activities: 1) it
modulates metabolism or catabolism of biochemical molecules
necessary for energy production or storage, 2) it modulates intra-
or inter-cellular signaling, 3) it modulates metabolism or
catabolism of metabolically important biomolecules (e.g.,
glucocorticoids), 4) it modulates detoxification of potentially
harmful compounds, 5) it modulates viral infection (e.g., by
modulating viral gene expression), and 6) it acts as a
transcriptional cofactor for viral gene activation.
[0194] The isolated nucleic acid molecules of the invention can be
used, for example, to express DHDR protein (e.g., via a recombinant
expression vector in a host cell in gene therapy applications), to
detect DHDR mRNA (e.g., in a biological sample) or a genetic
alteration in a DHDR gene, and to modulate DHDR activity, as
described further below. The DHDR proteins can be used to treat
disorders characterized by insufficient or excessive production of
a DHDR substrate or production of DHDR inhibitors. In addition, the
DHDR proteins can be used to screen for naturally occurring DHDR
substrates, to screen for drugs or compounds which modulate DHDR
activity, as well as to treat disorders characterized by
insufficient or excessive production of DHDR protein or production
of DHDR protein forms which have decreased, aberrant or unwanted
activity compared to DHDR wild type protein (e.g.,
dehydrogenase-associated disorders, such as CNS disorders (e.g.,
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, and bipolar affective disorder (e.g., severe
bipolar affective (mood) disorder (BP-1) and bipolar affective
neurological disorders (e.g., migraine and obesity)); cardiac
disorders (e.g., 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); muscular disorders (e.g., paralysis, muscle weakness
(e.g., ataxia, myotonia, and myokymia), muscular dystrophy (e.g.,
Duchenne muscular dystrophy or myotonic dystrophy), spinal muscular
atrophy, congenital myopathies, central core disease, rod myopathy,
central nuclear myopathy, Lambert-Eaton syndrome, denervation, and
infantile spinal muscular atrophy (Werdnig-Hoffman disease);
cellular growth, differentiation, or migration disorders (e.g.,
cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis
and metastasis; skeletal dysplasia; neuronal deficiencies resulting
from impaired neural induction and patterning); hepatic disorders;
hematopoietic and/or myeloproliferative disorders; neurological
disorders (e.g., Sjogren-Larsson syndrome, disorders in GABA
processing or reception), immune disorders (e.g., immune responses
to pathogens, autoimmune disorders or immune deficit disorders);
hormonal disorders (e.g., pituitary, insulin-dependent, thyroid, or
fertility or reproductive disorders); and viral disorders (e.g.,
disorders caused or affected by infection by a virus, such as
hepatocellularcarcinoma, cirrhosis of the liver, cervical
carcinoma, Burkitt's lymphoma, lymphoproliferative disease,
Kaposi's sarcoma, T cell leukemia, B cell lymphoma, plasmablastic
lymphoma, Rasmussen's syndrome, Marek's disease, warts (including
common, genital, and plantar warts), genital herpes, common colds,
acquired immune deficiency syndrome (AIDS), polymyositis,
immunorestitution disease, chicken pox, shingles, ebola and other
hemorrhagic fever diseases, cold sores, transient or acute
hepatitis, chronic hepatitis, influenza, Reye syndrome, measles,
Paget's disease, viral encephalitis, viral pneumonia, and viral
meningitis. Moreover, the anti-DHDR antibodies of the invention can
be used to detect and isolate DHDR proteins, regulate the
bioavailability of DHDR proteins, and modulate DHDR activity.
[0195] A. Screening Assays:
[0196] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, e.g., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) which bind to DHDR proteins, have a
stimulatory or inhibitory effect on, for example, DHDR expression
or DHDR activity, or have a stimulatory or inhibitory effect on,
for example, the expression or activity of DHDR substrate.
[0197] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
DHDR protein or polypeptide or biologically active portion thereof
(e.g., aldehydes, alcohols, or steroids (e.g., glucocorticoids)).
In another embodiment, the invention provides assays for screening
candidate or test compounds which bind to or modulate the activity
of a DHDR protein or polypeptide or biologically active portion
thereof (e.g., cofactor or coenzyme analogs, or inhibitory
molecules). 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).
[0198] 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.
[0199] 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.
No. '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.).
[0200] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a DHDR protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to modulate DHDR activity is determined. Determining
the ability of the test compound to modulate DHDR activity can be
accomplished by monitoring, for example, the production of one or
more specific metabolites in a cell which expresses DHDR (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.,
a liver cell, a neuronal cell, or a thymus cell. The ability of the
test compound to modulate DHDR binding to a substrate (e.g., an
alcohol, an aldehyde, or a steroid (e.g., a glucocorticoid)) or to
bind to DHDR can also be determined. Determining the ability of the
test compound to modulate DHDR binding to a substrate can be
accomplished, for example, by coupling the DHDR substrate with a
radioisotope or enzymatic label such that binding of the DHDR
substrate to DHDR can be determined by detecting the labeled DHDR
substrate in a complex. Alternatively, DHDR could be coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate DHDR binding to a DHDR substrate in a complex.
Determining the ability of the test compound to bind DHDR can be
accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to DHDR can be determined by detecting the labeled DHDR compound in
a complex. For example, compounds (e.g., DHDR 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.
[0201] It is also within the scope of this invention to determine
the ability of a compound (e.g., a DHDR substrate) to interact with
DHDR without the labeling of any of the interactants. For example,
a microphysiometer can be used to detect the interaction of a
compound with DHDR without the labeling of either the compound or
the DHDR. 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 DHDR.
[0202] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a DHDR target molecule
(e.g., a DHDR substrate) with a test compound and determining the
ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the DHDR target molecule. Determining the
ability of the test compound to modulate the activity of a DHDR
target molecule can be accomplished, for example, by determining
the ability of the DHDR protein to bind to or interact with the
DHDR target molecule.
[0203] Determining the ability of the DHDR protein, or a
biologically active fragment thereof, to bind to or interact with a
DHDR 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 DHDR protein to bind to
or interact with a DHDR 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., changes in intracellular
K.sup.+ levels or induction of viral gene expression), 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.
[0204] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a DHDR protein or biologically active
portion thereof is contacted with a test compound and the ability
of the test compound to bind to the DHDR protein or biologically
active portion thereof is determined. Preferred biologically active
portions of the DHDR proteins to be used in assays of the present
invention include fragments which participate in interactions with
non-DHDR molecules, e.g., fragments with high surface probability
scores (see, for example, FIGS. 2, 7, 12, and 18). Binding of the
test compound to the DHDR protein can be determined either directly
or indirectly as described above. In a preferred embodiment, the
assay includes contacting the DHDR protein or biologically active
portion thereof with a known compound which binds DHDR 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
DHDR protein, wherein determining the ability of the test compound
to interact with a DHDR protein comprises determining the ability
of the test compound to preferentially bind to DHDR or biologically
active portion thereof as compared to the known compound.
[0205] In another embodiment, the assay is a cell-free assay in
which a DHDR 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 DHDR
protein or biologically active portion thereof is determined.
Determining the ability of the test compound to modulate the
activity of a DHDR protein can be accomplished, for example, by
determining the ability of the DHDR protein to bind to a DHDR
target molecule by one of the methods described above for
determining direct binding. Determining the ability of the DHDR
protein to bind to a DHDR 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.
[0206] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of a DHDR protein can be
accomplished by determining the ability of the DHDR protein to
further modulate the activity of a downstream effector of a DHDR
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.
[0207] In yet another embodiment, the cell-free assay involves
contacting a DHDR protein or biologically active portion thereof
with a known compound which binds the DHDR 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
DHDR protein, wherein determining the ability of the test compound
to interact with the DHDR protein comprises determining the ability
of the DHDR protein to preferentially bind to or catalyze the
transfer of a hydride moiety to or from the target substrate.
[0208] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
DHDR 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 DHDR protein, or interaction of a DHDR 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/DHDR fusion proteins or
glutathione-S-transfera- se/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 DHDR 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 DHDR binding or activity
determined using standard techniques.
[0209] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a DHDR protein or a DHDR target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated DHDR
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 DHDR
protein or target molecules but which do not interfere with binding
of the DHDR protein to its target molecule can be derivatized to
the wells of the plate, and unbound target or DHDR 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 DHDR protein or target molecule,
as well as enzyme-linked assays which rely on detecting an
enzymatic activity associated with the DHDR protein or target
molecule.
[0210] In another embodiment, modulators of DHDR expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of DHDR mRNA or protein in the cell is
determined. The level of expression of DHDR mRNA or protein in the
presence of the candidate compound is compared to the level of
expression of DHDR mRNA or protein in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of DHDR expression based on this comparison. For example,
when expression of DHDR 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 DHDR mRNA or protein expression. Alternatively, when
expression of DHDR 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 DHDR mRNA or protein expression. The level of DHDR
mRNA or protein expression in the cells can be determined by
methods described herein for detecting DHDR mRNA or protein.
[0211] In yet another aspect of the invention, the DHDR 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 DHDR
("DHDR-binding proteins" or "DHDR-6-bp" ) and are involved in DHDR
activity.
[0212] Such DHDR-binding proteins are also likely to be involved in
the propagation of signals by the DHDR proteins or DHDR targets as,
for example, downstream elements of a DHDR-mediated signaling
pathway. Alternatively, such DHDR-binding proteins are likely to be
DHDR inhibitors.
[0213] 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 DHDR
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 DHDR-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 DHDR protein.
[0214] 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 DHDR 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 viral infection.
[0215] There are many animal models for viral infection known in
the art. For example, a transgenic mouse model for hepatitis B
virus infection (HBV) (Guidotti, L. G. et al. (1995) J. Viorology
69:6158-6169) may be used. High-level viral gene expression is
present in the liver and kidney tissues of these mice, and the
hepatocytes of the mice replicate the virus at levels comparable to
those in the infected livers of patients with chronic
hepatitis.
[0216] Another mouse model for HBV infection that may be used
includes the mouse model made by transplanting primary human
hepatocytes into mice in a matrix under the kidney capsule along
with administration of an agonistic antibody against c-Met (Ohashi,
K. et al. (2000) Nat. Med 6:327-331). These mice are susceptible to
HBV infection. Additionally, they are susceptible to
super-infection with hepatitis delta virus (HDV).
[0217] Other mouse models for HBV infection that may be used
include the mice described in Babinet, C. et al. (1985) Science
230:1160-3; Lee, T.-H. et al. (1990) J. Virol. 64:5939-5947;
Madden, C. R. et al. (2000) J. Virol. 74:5266-5272; Brown, J. J. et
al. (2000) Hepatology 31:173-181; Larkin, J. (1999) Nat. Med.
5:907-912; and Araki, K. et al. (1989) Proc. Natl. Acad Sci. USA
86:207-11.
[0218] Chronic HBV infection is a major risk factor for
hepatocellular carcinoma (Beasley, R. P. (1988) Cancer
61:1942-1956; Slagle, B. et al. (1994) In Viruses and cancer,
Minson, A. et al., eds., University of Cambridge, Cambridge,
England 51:149-171), and mice transgenic for the HBV X gene have
increased sensitivity to hepatocarcinogens (Slagle, B. L. et al.
(1996) Mol. Carcinog. 15:261-269). The double transgenic mouse
strain described in Madden et al. (supra) can be used to study the
effects of test compounds identified by the screening methods of
the invention in modulating HBV X-mediated hepatocarcinogen
sensitivity. For example, the mice can be treated with a
hepatocarcinogen and a test compound, and the effect of the test
compound on the hepatocarcinogen-mediated mutation rate of the host
DNA can be assayed by functional analysis of a bacteriophage lambda
transgene. Briefly, DNA isolated from the livers of such treated
mice can be packaged into lambda phage particles and used to infect
E. coli bacteria. Mutation rates of the lambda particles (methods
for determination of which are known in the art) are directly
related to the HBV X-mediated host DNA mutation rates in response
to the hepatorcarcinogen in the treated mice.
[0219] Other mouse models for HBV infection and HBV immunity that
may be used include those made by trasplanting human peripheral
blood mononuclear cells (PBMC) from chronic HBV carriers and
HBV-immunized donors, respectively, into lethally-irradiated Balb/c
mice (Bocher, W. O. et al. (2000) Hepatology 31:480-487; Ilan, E.
et al. (1999) Hepatology 29:553-562). Such human/mouse radiation
chimeras, called Trimera mice, may be used to study the effects of
test compounds identified by the screening methods of the invention
on human antibody and T cell responses to HBV infection in vivo
(Marcus, H. et al. (1995) Blood 86:398-406; Reisner, Y. et al.
(1998) Trends Biotechnol 16:242-246; Segall, H. et al. (1996) Blood
88:721-730; Bocher, W. O. et al. (1999) Immunology 96:634-641).
[0220] The effects of a modulating agent on HBV infection can also
be studied in other hepadnavirus animal models: the woodchuck
hepatitis virus (WHV) model (Korba, B. E. et al. (2000) Hepatology
31:1165-1175; Cote, P. J. et al. (2000) Hepatology 31:190-200), the
duck hepatitis B virus (DHBV) model (Le Guerhier, F. et al. (2000)
Antimicrob. Agents. Chemother. 44:111-122; Vickery, K. et al.
(1999) J. Med. Virol. 58:19-25), and the chimpanzee and ground and
tree squirrel models (Caselmann, W. H. (1994) Antiviral Res.
24:121-129).
[0221] While an animal model for hepatitis C virus (HCV) infection
that adequately reproduces the characteristics of HCV infection in
humans does not yet exist, there is an HCV Trimera mouse model
(Dekel, B. et al. (1995) J. Infect. Dis. 172:25-30), and there are
some mouse strains that are transgenic for certain HCV proteins,
and thus, may be useful for testing compounds that can modulate
DHDR activity in vivo (Pasquinelli, C. et al. (1997) Hepatology
25:719-727).
[0222] Other animal models for viral infection are also known in
the art and may be used in the screening assays of the present
invention. For example, there are many animal models for
Epstein-Barr virus (EBV) associated lymphoproliferative disease.
Such models have been made in rabbits, common marmosets (Callithrix
jacchus), cottontop tamarins (Saguinus oedipus oedipus), rhesus
monkeys, and the severe combined immunodeficient (SCID) mouse
(Johannessen, I. and Crawford, D. H. (1999) Rev. Med. Virol.
9:263-77; H layashi, K. and Akagi, T. (2000) Path. International
50:85-97). The mouse .gamma.-herpesvirus 68 infection model (Speck,
S. H. and Virgin, H. W. (1999) Curr. Opin. Microbiol. 2:403-9;
Virgin, H. W. and Speck, S. H. (1999) Curr. Opin. Immunol.
11:371-379) and the cotton rat model for measles virus infection
(Niewiesk, S. (1999) Immunol. Lett. 65:47-50) present other
examples of animal models that may be used in the methods of the
invention. Macaques infected with live attenuated simian
immunodeficiency virus (SIV) (Geretti, A. M. (1999) Rev. Med.
Virol. 9:57-67; Almond, N. and Stott, J. (1999) Immunol. Lett.
66:167-170) as well as the chimpanzee HIV model (Murthy, K. K. et
al. (1998) AIDS Res. Hum. Retroviruses 14 Suppl 3:S271-6) can be
used as models for human immunodeficiency virus (HIV)
infection.
[0223] Other examples of animal models that may be used in the
methods of the invention include the transgenic mouse model for an
AIDS-like disease (Renkema, H. G. and Saksela, K. (2000) Front.
Biosci. 5:D268-83); the chicken model for lymphoma-inducing
herpesviruses (Schat, K. A. and Xing, Z. (2000) Dev. Comp. Immunol.
24:201-21); and the mouse model of cytomegalovirus infection
(Sweet, C. (1999) FEMS Microbiol. Rev. 23:457-82).
[0224] 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, as described
above. For example, an agent identified as described herein (e.g.,
a DHDR modulating agent, an antisense DHDR nucleic acid molecule, a
DHDR-specific antibody, or a DHDR-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.
[0225] B. Detection Assays
[0226] 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.
[0227] 1. Chromosome Mapping
[0228] 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 DHDR nucleotide
sequences, described herein, can be used to map the location of the
DHDR genes on a chromosome. The mapping of the DHDR sequences to
chromosomes is an important first step in correlating these
sequences with genes associated with disease.
[0229] Briefly, DHDR genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the DHDR
nucleotide sequences. Computer analysis of the DHDR 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 DHDR sequences will
yield an amplified fragment.
[0230] 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.
[0231] 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 DHDR 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 DHDR 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.
[0232] 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).
[0233] 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.
[0234] 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.
[0235] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the DHDR 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.
[0236] 2. Tissue Typing
[0237] The DHDR 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).
[0238] 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 DHDR 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.
[0239] 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 DHDR 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, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:10
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.
[0240] If a panel of reagents from DHDR 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.
[0241] 3. Use of DHDR Sequences in Forensic Biology
[0242] 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.
[0243] 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, SEQ ID NO:4, SEQ ID NO:7 or SEQ
ID NO:10 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 DHDR nucleotide sequences or
portions thereof, e.g., fragments derived from the noncoding
regions of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:10
having a length of at least 20 bases, preferably at least 30
bases.
[0244] The DHDR 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 DHDR probes can be used to identify tissue by
species and/or by organ type.
[0245] In a similar fashion, these reagents, e.g., DHDR 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).
[0246] C. Predictive Medicine:
[0247] 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 DHDR protein and/or nucleic acid
expression as well as DHDR 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 DHDR 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 DHDR
protein, nucleic acid expression or activity. For example,
mutations in a DHDR 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 DHDR protein,
nucleic acid expression or activity.
[0248] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on the expression or
activity of DHDR in clinical trials.
[0249] These and other agents are described in further detail in
the following sections.
[0250] 1. Diagnostic Assays
[0251] An exemplary method for detecting the presence or absence of
DHDR 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 DHDR protein or nucleic acid (e.g., mRNA, or genomic DNA)
that encodes DHDR protein such that the presence of DHDR protein or
nucleic acid is detected in the biological sample. A preferred
agent for detecting DHDR mRNA or genomic DNA is a labeled nucleic
acid probe capable of hybridizing to DHDR mRNA or genomic DNA. The
nucleic acid probe can be, for example, the DHDR nucleic acid set
forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, or 12, or the DNA insert
of the plasmid deposited with ATCC as Accession Number ______, 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 DHDR mRNA or
genomic DNA. Other suitable probes for use in the diagnostic assays
of the invention are described herein.
[0252] A preferred agent for detecting DHDR protein is an antibody
capable of binding to DHDR 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 DHDR mRNA, protein,
or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of DHDR mRNA include
Northern hybridizations and in situ hybridizations. In vitro
techniques for detection of DHDR protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. In vitro techniques for detection of DHDR
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of DHDR protein include introducing into a
subject a labeled anti-DHDR 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.
[0253] 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.
[0254] 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 DHDR
protein, mRNA, or genomic DNA, such that the presence of DHDR
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of DHDR protein, mRNA or genomic DNA in
the control sample with the presence of DHDR protein, mRNA or
genomic DNA in the test sample.
[0255] The invention also encompasses kits for detecting the
presence of DHDR in a biological sample. For example, the kit can
comprise a labeled compound or agent capable of detecting DHDR
protein or mRNA in a biological sample; means for determining the
amount of DHDR in the sample; and means for comparing the amount of
DHDR 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 DHDR protein or nucleic
acid.
[0256] 2. Prognostic Assays
[0257] 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 DHDR
expression or activity. As used herein, the term "aberrant"
includes a DHDR expression or activity which deviates from the wild
type DHDR 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 DHDR expression or activity is
intended to include the cases in which a mutation in the DHDR gene
causes the DHDR gene to be under-expressed or over-expressed and
situations in which such mutations result in a non-functional DHDR
protein or a protein which does not function in a wild-type
fashion, e.g., a protein which does not interact with a DHDR
substrate, or one which interacts with a non-DHDR 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 DHDR expression or
activity which is undesirable in a subject.
[0258] 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 DHDR 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, a viral 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 DHDR 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, a viral disorder, or a hormonal disorder. Thus, the
present invention provides a method for identifying a disease or
disorder associated with aberrant or unwanted DHDR expression or
activity in which a test sample is obtained from a subject and DHDR
protein or nucleic acid (e.g., mRNA or genomic DNA) is detected,
wherein the presence of DHDR protein or nucleic acid is diagnostic
for a subject having or at risk of developing a disease or disorder
associated with aberrant or unwanted DHDR 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 sample (e.g., a liver sample).
[0259] 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 DHDR
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, a viral 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 DHDR expression or activity in
which a test sample is obtained and DHDR protein or nucleic acid
expression or activity is detected (e.g., wherein the abundance of
DHDR 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 DHDR expression or
activity).
[0260] The methods of the invention can also be used to detect
genetic alterations in a DHDR gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in DHDR 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,
a viral 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 DHDR-protein, or the mis-expression
of the DHDR 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 DHDR gene; 2) an
addition of one or more nucleotides to a DHDR gene; 3) a
substitution of one or more nucleotides of a DHDR gene, 4) a
chromosomal rearrangement of a DHDR gene; 5) an alteration in the
level of a messenger RNA transcript of a DHDR gene, 6) aberrant
modification of a DHDR 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 DHDR gene, 8) a non-wild
type level of a DHDR-protein, 9) allelic loss of a DHDR gene, and
10) inappropriate post-translational modification of a
DHDR-protein. As described herein, there are a large number of
assays known in the art which can be used for detecting alterations
in a DHDR gene. A preferred biological sample is a tissue (e.g., a
liver sample) or serum sample isolated by conventional means from a
subject.
[0261] 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 DHDR 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 DHDR gene under conditions such that hybridization
and amplification of the DHDR 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.
[0262] 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. et al., (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.
[0263] In an alternative embodiment, mutations in a DHDR 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.
[0264] In other embodiments, genetic mutations in DHDR 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 DHDR 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.
[0265] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
DHDR gene and detect mutations by comparing the sequence of the
sample DHDR 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).
[0266] Other methods for detecting mutations in the DHDR 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 DHDR
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.
[0267] 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 DHDR
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 DHDR sequence, e.g., a wild-type
DHDR 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.
[0268] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in DHDR 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 DHDR 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).
[0269] 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).
[0270] 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.
[0271] 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.
[0272] 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 DHDR gene.
[0273] Furthermore, any cell type or tissue in which DHDR is
expressed may be utilized in the prognostic assays described
herein.
[0274] 3. Monitoring of Effects During Clinical Trials
[0275] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a DHDR protein (e.g., the modulation of a
virus infection and/or the modulation of cell proliferation and/or
migration) 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
DHDR gene expression, protein levels, or upregulate DHDR activity,
can be monitored in clinical trials of subjects exhibiting
decreased DHDR gene expression, protein levels, or downregulated
DHDR activity. Alternatively, the effectiveness of an agent
determined by a screening assay to decrease DHDR gene expression,
protein levels, or downregulate DHDR activity, can be monitored in
clinical trials of subjects exhibiting increased DHDR gene
expression, protein levels, or upregulated DHDR activity. In such
clinical trials, the expression or activity of a DHDR gene, and
preferably, other genes that have been implicated in, for example,
a DHDR-associated disorder can be used as a "read out" or markers
of the phenotype of a particular cell.
[0276] For example, and not by way of limitation, genes, including
DHDR, that are modulated in cells by treatment with an agent (e.g.,
compound, drug or small molecule) which modulates DHDR activity
(e.g., identified in a screening assay as described herein) can be
identified. Thus, to study the effect of agents on DHDR-associated
disorders (e.g., disorders characterized by viral infection and/or
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 DHDR and other genes implicated in
the DHDR-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 DHDR 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.
[0277] 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 DHDR 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 DHDR protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the DHDR protein, mRNA, or
genomic DNA in the pre-administration sample with the DHDR 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 DHDR 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 DHDR to lower
levels than detected, i.e. to decrease the effectiveness of the
agent. According to such an embodiment, DHDR expression or activity
may be used as an indicator of the effectiveness of an agent, even
in the absence of an observable phenotypic response.
[0278] D. Methods of Treatment:
[0279] 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 DHDR expression or activity, e.g., a
dehydrogenase-associated disorder such as a CNS disorder; a
cellular proliferation, growth, differentiation, or migration
disorder; a, musculoskeletal disorder; a cardiovascular disorder;
an immune disorder; a viral 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 DHDR molecules of the present invention
or DHDR 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.
[0280] 1. Prophylactic Methods
[0281] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted DHDR expression or activity, by administering
to the subject a DHDR or an agent which modulates DHDR expression
or at least one DHDR activity. Subjects at risk for a disease which
is caused or contributed to by aberrant or unwanted DHDR 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 DHDR aberrancy,
such that a disease or disorder is prevented or, alternatively,
delayed in its progression. Depending on the type of DHDR
aberrancy, for example, a DHDR, DHDR agonist or DHDR antagonist
agent can be used for treating the subject. The appropriate agent
can be determined based on screening assays described herein.
[0282] 2. Therapeutic Methods
[0283] Another aspect of the invention pertains to methods of
modulating DHDR expression or activity for therapeutic purposes.
Accordingly, in an exemplary embodiment, the modulatory method of
the invention involves contacting a cell with a DHDR or agent that
modulates one or more of the activities of DHDR protein activity
associated with the cell. An agent that modulates DHDR protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a DHDR
protein (e.g., a DHDR substrate), a DHDR antibody, a DHDR agonist
or antagonist, a peptidomimetic of a DHDR agonist or antagonist, or
other small molecule. In one embodiment, the agent stimulates one
or more DHDR activities. Examples of such stimulatory agents
include active DHDR protein and a nucleic acid molecule encoding
DHDR that has been introduced into the cell. In another embodiment,
the agent inhibits one or more DHDR activities. Examples of such
inhibitory agents include antisense DHDR nucleic acid molecules,
anti-DHDR antibodies, and DHDR 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
DHDR 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) DHDR expression
or activity. In another embodiment, the method involves
administering a DHDR protein or nucleic acid molecule as therapy to
compensate for reduced, aberrant, or unwanted DHDR expression or
activity.
[0284] Stimulation of DHDR activity is desirable in situations in
which DHDR is abnormally downregulated and/or in which increased
DHDR activity is likely to have a beneficial effect. Likewise,
inhibition of DHDR activity is desirable in situations in which
DHDR is abnormally upregulated and/or in which decreased DHDR
activity is likely to have a beneficial effect.
[0285] 3. Pharmacogenomics
[0286] The DHDR molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on DHDR activity (e.g., DHDR gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) DHDR-associated
disorders (e.g., proliferative disorders, CNS disorders, cardiac
disorders, metabolic disorders, or muscular disorders) associated
with aberrant or unwanted DHDR 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 DHDR molecule or DHDR modulator as well as tailoring
the dosage and/or therapeutic regimen of treatment with a DHDR
molecule or DHDR modulator.
[0287] 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.
[0288] 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.
[0289] 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 drugs
target is known (e.g., a DHDR 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.
[0290] 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.
[0291] 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 DHDR molecule or DHDR modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0292] 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 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 DHDR molecule or DHDR modulator, such as
a modulator identified by one of the exemplary screening assays
described herein.
[0293] 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 and the
sequence listing, are incorporated herein by reference.
EXAMPLES
Example 1
Identification and Characterization of Human DHDR cDNA
[0294] In this example, the identification and characterization of
the gene encoding human DHDR-1 (clone Fbh32142), DHDR-2 (clone
Fbh21481), DHDR-3 (clone Fbh25964) and DHDR-4 (clone Fbh21686) is
described.
[0295] Isolation of the DHDR cDNA
[0296] The invention is based, at least in part, on the discovery
of several human genes encoding novel proteins, referred to herein
as DHDR-1, DHDR-2, DHDR-3 and DHDR-4. The entire sequences of human
clones Fbh32142, Fbh21481, Fbh25964, and Fbh21686 were determined
and found to contain open reading frames termed human "DHDR-1 ",
"DHDR-2", "DHDR-3", and DHDR-4", respectively, set forth in FIGS.
1, 6, 11, and 16, respectively. The amino acid sequences of these
human DHDR expression products are set forth in FIGS. 1, 6, 11, and
16, respectively. The DHDR-1 protein sequence set forth in SEQ ID
NO:2 comprises about 802 amino acids and is shown in FIG. 1. The
DHDR-2 protein sequence set forth in SEQ ID NO: 5 comprises about
311 amino acids and is shown in FIG. 6. The DHDR-3 protein sequence
set forth in SEQ ID NO:8 comprises about 369 amino acids and is
shown in FIG. 11. The DHDR-4 protein sequence set forth in SEQ ID
NO:11 comprises about 322 amino acids and is shown in FIG. 16. The
coding regions (open reading frames) of SEQ ID NOs:1, 4, 7 and 10
are set forth as SEQ ID NOs:3, 6, 9 and 12. Clones Fbh32142,
Fbh21481, Fbh25964 and Fbh21686, comprising the coding region of
human DHDR-1, DHDR-2, DHDR-3, and DHDR-4, respectively, were
deposited with the American Type Culture Collection (ATCC.RTM.),
10801 University Boulevard, Manassas, Va. 20110-2209, on ______,
and assigned Accession Nos. ______.
[0297] Analysis of the Human DHDR Molecules
[0298] The amino acid sequences of human DHDR-1, DHDR-2, DHDR-3,
and DHDR-4 were analyzed using the program PSORT
(http://www.psort.nibb.ac.jp- ) to predict the localization of the
proteins within the cell. This program assesses the presence of
different targeting and localization amino acid sequences within
the query sequence. The results of the analyses show that human
DHDR-1 (SEQ ID NO:2) may be localized to the mitochondrion, to the
endoplasmic reticulum, to the nucleus, or to secretory vesicles.
The results of the analyses further show that human DHDR-2 (SEQ ID
NO:5) may be localized to the mitochondrion, to the cytoplasm, to
extracellular spaces or the cell wall, to vacuoles, to the nucleus,
or to the endoplasmic reticulum. The results of the analyses
further show that human DHDR-3 (SEQ ID NO:8) may be localized to
the cytoplasm, to the mitochondrion, to the Golgi, to the
endoplasmic reticulum, to the extracellular space or cell wall, to
vacuoles, to the nucleus, or to secretory vesicles. The results of
the analyses further show that human DHDR-4 (SEQ ID NO:11) may be
localized to the nucleus, the cytoplasm, to the Golgi, to the
mitochondrion, to peroxisomes, to the endoplasmic reticulum, or to
secretory vesicles.
[0299] An alignment of the human DHDR-4 amino acid sequence with
the amino acid sequence of Rattus norvegicus putative short-chain
dehydrogenase/reductase (Accession Number AF099742) using the
CLUSTAL W (1.74) multiple sequence alignment program is set forth
in FIG. 17.
[0300] Each of the amino acid sequences of DHDR-1, DHDR-2, DHDR-3,
and DHDR-4 were analyzed by the SignalP program (Henrik, et al.
(1997) Protein Engineering 10:1-6) for the presence of a signal
peptide. These analyses revealed the presence of a signal peptide
in the amino acid sequence of DHDR-2 from residues 1-18 (FIG. 8).
These analyses further revealed the possible presence of a signal
peptide in the amino acid sequence of DHDR-4, from residues 1-19
(FIG. 19).
[0301] Searches of each of the amino acid sequences of DHDR-1,
DHDR-2, DHDR-3, and DHDR-4 were performed against the Memsat
database (FIGS. 3, 8, 13, and 19). These searches resulted in the
identification of one transmembrane domain in the amino acid
sequence of human DHDR-1 (SEQ ID NO:2) at about residues 159-175,
and one transmembrane domain in the amino acid sequence of human
DHDR-2 (SEQ ID NO:5) at about residues 7-23 in the native molecule,
or about residues 265-283 of the predicted mature protein. These
searches further identified four transmembrane domains in the amino
acid sequence of human DHDR-3 (SEQ ID NO:8) at about residues
10-26, 73-90, 289-305, and 312-333, and four transmembrane domains
in the amino acid sequence of human DHDR-4 (SEQ ID NO:11) at about
residues 29-50, 170-188, 208-224, and 258-275 of the native
molecule, and at about residues 10-31, 151-169, 189-205, and
239-256 of the predicted mature protein.
[0302] Searches of each of the amino acid sequences of DHDR-1,
DHDR-2, DHDR-3, and DHDR-4 were also performed against the HMM
database (FIGS. 4, 9, 14, and 20). These searches resulted in the
identification of an "aldehyde dehydrogenase family domain" in the
amino acid sequence of DHDR-1 (SEQ ID NO:2) at about residues
47-494 (score=149.8) (FIG. 4); the identification of a "short-chain
dehydrogenase domain" in the amino acid sequence of DHDR-2 (SEQ ID
NO:5) at about residues 38-227 (score=120.0) (FIG. 9), and the
identification of a "3-beta hydroxysteroid dehydrogenase domain" at
about residues 1-365 (score=676.9), a "short chain dehydrogenase
domain" at about residues 10-197, and a "NAD-dependent
epimerase/dehydratase domain" at about residues 12-365 of the amino
acid sequence of DHDR-3 (SEQ ID NO:8) (FIG. 14). These searches
further resulted in the identification of a "short chain
dehydrogenase domain" at about residues 38-226 (score=162.5), and a
"short chain dehydrogenase/reductase domain" at about residues
250-280 (score=47.2) of the amino acid sequence of DHDR-4 (SEQ ID
NO:11) (FIG. 20).
[0303] Searches of each of the amino acid sequences of DHDR-1,
DHDR-2, DHDR-3, and DHDR-4 were also performed against the ProDom
database (FIGS. 5, 10, 15, and 21). These searches resulted in the
identification of an "aldehyde dehydrogenase oxidoreductase domain"
in the amino acid sequence of human DHDR-1 (SEQ ID NO:2) at about
residues 101-770 (score=280) (FIG. 5), and the identification of an
"oxidoreductase protein dehydrogenase domain" in the amino acid
sequence of human DHDR-2 (SEQ ID NO:5) at about residues 99-219
(score=113) (FIG. 10). These searches further resulted in the
identification of a "3-beta hydroxysteroid dehydrogenase domain" in
the amino acid sequence of human DHDR-3 (SEQ ID NO:8) at about
residues 11-362 (score=395) (FIG. 15). These searches further
resulted in the identification of an "oxidoreductase protein
dehydrogenase domain" at about residues 37-231 (score=157), a
"shikimate 5-dehydrogenase domain" at about residues 35-82
(score=86), a "dehydrogenase domain" at about residues 237-286
(score=84), and a "glucose-1-dehydrogenase domain" at about
residues 243-287 (score=92) of the amino acid sequence of DHDR-4
(SEQ ID NO:11) (FIG. 21).
[0304] Tissue Distribution of DHDR-4 mRNA
[0305] This example describes the tissue distribution of human
DHDR-4 cDNA, as determined using the TaqMan.TM. procedure. The
Taqman.TM. procedure is a quantitative, real-time PCR-based
approach to 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 is generated from the
samples of interest and serves 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) is 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. During the PCR reaction, cleavage of the
probe separates the reporter dye and the quencher dye, resulting in
increased fluorescence of the reporter. 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. RNA was prepared using the trizol method
and treated with DNAse to remove contaminating genomic DNA. cDNA
was synthesized using standard techniques. Mock cDNA synthesis in
the absence of reverse transcriptase resulted in samples with no
detectable PCR amplification of the control GAPDH gene confirming
efficient removal of genomic DNA contamination.
[0306] The human DHDR-4 gene is highly expressed in liver, kidney,
brain, primary osteoblasts, in pituitary, in CaCO cells, in
keratinocytes, in aortic endothelial cells, in fetal kidney, in
fetal lung, in mammary epithelium, in fetal spleen, in fetal liver,
in umbilical smooth muscle, in RAII Burkitt Lymphoma cells, in
lung, in prostate, in K53 red blood cells, in fetal dorsal spinal
cord, in insulinoma cells, in normal breast and ovarian epithelia,
in retina, in HMC-1 mast cells, in ovarian ascites, in d8 dendritic
cells, in megakaryocytes, in human mobilized bone morrow, in
mammary carcinoma, in melanoma cells, in lymph, in vein, in
U937/A70p B cells, in A549con cells, in WT LN Cap testosterone
cells, and in esophagus. Significant expression of DHDR-4 was also
observed in aorta, in breast, in liver, in lung, in small
intestine, and in thymus. Some expression of DHDR-4 was observed in
brain, in cervix, in colon, in heart, in kidney, in muscle, in
ovary, in placenta, in testes, and in thyroid.
[0307] Human DHDR-4 is also greatly induced in situations of
hepatitis B virus (HBV) infection. Human DHDR-4 is expressed at
4-18 fold higher levels in HBV-infected liver than in normal liver.
DHDR-4 expression levels are 12-25 fold higher in HBV-expressing
HepG2.2.15 cells than in HepG2 control cells. Additionally,
transfection of the HBV X transcription factor alone can induce a
5-fold increase in DHDR-4 expression.
[0308] For in situ analysis, various tissues, e.g. tissues obtained
from liver, are first frozen on dry ice. Ten-micrometer-thick
sections of the tissues are postfixed 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 are rinsed in DEPC
2.times.SSC (1.times.SSC is 0.15M NaCl plus 0.015M sodium citrate).
Tissue is 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.
[0309] Hybridizations are performed with .sup.35S-radiolabeled
(5.times.10.sup.7 cpm/ml) cRNA probes. Probes are 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.
[0310] After hybridization, slides are 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 are 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 are 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.
[0311] In situ hybridization analysis revealed that DHDR-4 is
expressed at a much higher level in HBV positive liver than in
normal liver.
Example 2
Expression of Recombinant DHDR Protein in Bacterial Cells
[0312] In this example, DHDR is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
DHDR is fused to GST and this fusion polypeptide is expressed in E.
coli, e.g., strain PEB199. Expression of the GST-DHDR 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 DHDR Protein in COS Cells
[0313] To express the DHDR 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 DHDR 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.
[0314] To construct the plasmid, the DHDR 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 DHDR 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 DHDR 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 DHDR 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.
[0315] COS cells are subsequently transfected with the
DHDR-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 DHDR 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.
[0316] Alternatively, DNA containing the DHDR 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 DHDR polypeptide is detected by radiolabelling
and immunoprecipitation using a DHDR specific monoclonal
antibody.
[0317] Equivalents
[0318] 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 0
0
* * * * *
References