U.S. patent application number 12/001737 was filed with the patent office on 2009-11-12 for novel nucleic acid sequences encoding adenylate kinases, alcohol dehydrogenases, ubiquitin proteases, lipases, adenylate cyclases, and gtpase activators.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Miyoung Chun, Maria Alexandra Glucksmann, Rosana Kapeller-Libermann, Rachel E. Meyers.
Application Number | 20090280482 12/001737 |
Document ID | / |
Family ID | 46324955 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090280482 |
Kind Code |
A1 |
Glucksmann; Maria Alexandra ;
et al. |
November 12, 2009 |
Novel nucleic acid sequences encoding adenylate kinases, alcohol
dehydrogenases, ubiquitin proteases, lipases, adenylate cyclases,
and GTPase activators
Abstract
The invention provides isolated nucleic acids molecules that
encode novel polypeptides. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
the nucleic acid molecules of the invention, host cells into which
the expression vectors have been introduced, and nonhuman
transgenic animals in which a sequence of the invention has been
introduced or disrupted. The invention still further provides
isolated proteins, fusion proteins, antigenic peptides and
antibodies. Diagnostic methods utilizing compositions of the
invention are also provided.
Inventors: |
Glucksmann; Maria Alexandra;
(Lexington, MA) ; Kapeller-Libermann; Rosana;
(Chestnut Hill, MA) ; Meyers; Rachel E.; (Newton,
MA) ; Chun; Miyoung; (Belmont, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
46324955 |
Appl. No.: |
12/001737 |
Filed: |
December 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10165231 |
Jun 6, 2002 |
7329529 |
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12001737 |
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09390038 |
Sep 3, 1999 |
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10165231 |
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09796089 |
Feb 28, 2001 |
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09390038 |
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09464039 |
Dec 15, 1999 |
7094565 |
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09796089 |
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09972525 |
Oct 5, 2001 |
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10165231 |
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09408865 |
Sep 30, 1999 |
6329171 |
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09972525 |
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09963908 |
Sep 26, 2001 |
6797502 |
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10165231 |
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09434613 |
Nov 5, 1999 |
6337187 |
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09963908 |
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09461076 |
Dec 14, 1999 |
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10165231 |
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09802127 |
Feb 26, 2001 |
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09461076 |
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60185611 |
Feb 29, 2000 |
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Current U.S.
Class: |
435/6.13 ;
435/320.1; 435/325; 435/69.1; 435/7.1; 530/350; 530/387.9;
536/23.1 |
Current CPC
Class: |
C12N 9/6472
20130101 |
Class at
Publication: |
435/6 ; 536/23.1;
435/320.1; 435/325; 530/350; 530/387.9; 435/69.1; 435/7.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C12N 5/00 20060101 C12N005/00; C07K 14/00 20060101
C07K014/00; C07K 16/00 20060101 C07K016/00; C12P 21/06 20060101
C12P021/06; G01N 33/53 20060101 G01N033/53 |
Claims
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence that is at least 60% identical to the nucleotide sequence
set forth in SEQ ID NO:1, 6, 8, 10, 12, 14, 16, 18, 20, 21, 23, 24,
or 26, or the nucleotide sequence of the cDNA insert of the plasmid
deposited with ATCC as Patent Deposit Number PTA-1850, PTA-2170,
PTA-2812, PTA-2171, PTA-2813, PTA-2011, PTA-2012, PTA-2341,
PTA-1849, PTA-1915, PTA-1871, or PTA-1918, wherein said nucleotide
sequence encodes a polypeptide having biological activity; b) a
nucleic acid molecule comprising a fragment of at least 20
contiguous nucleotides of the nucleotide sequence set forth in SEQ
ID NO:1, 6, 8, 10, 12, 14, 16, 18, 20, 21, 23, 24, or 26, or the
nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Patent Deposit Number PTA-1850, PTA-2170, PTA-2812,
PTA-2171, PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849,
PTA-1915, PTA-1871, or PTA-1918; c) a nucleic acid molecule
encoding a polypeptide comprising the amino acid sequence set forth
in SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25 or the amino
acid sequence encoded by the nucleotide sequence of the cDNA insert
of the plasmid deposited with ATCC as Patent Deposit Number
PTA-1850, PTA-2170, PTA-2812, PTA-2171, PTA-2813, PTA-2011,
PTA-2012, PTA-2341, PTA-1849, PTA-1915, PTA-1871, or PTA-1918; d) a
nucleic acid molecule which encodes a fragment of a polypeptide
comprising the amino acid sequence set forth in SEQ ID NO:2, 5, 7,
9, 11, 13, 15, 17, 19, 22, or 25, or the amino acid sequence
encoded by the nucleotide sequence of the cDNA insert of the
plasmid deposited with ATCC as Patent Deposit Number PTA-1850,
PTA-2170, PTA-2812, PTA-2171, PTA-2813, PTA-2011, PTA-2012,
PTA-2341, PTA-1849, PTA-1915, PTA-1871, or PTA-1918, wherein the
fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25 or the amino acid
sequence encoded by the nucleotide sequence of the cDNA insert of
the plasmid deposited with ATCC as Patent Deposit Number PTA-1850,
PTA-2170, PTA-2812, PTA-2171, PTA-2813, PTA-2011, PTA-2012,
PTA-2341, PTA-1849, PTA-1915, PTA-1871, or PTA-1918; e) a nucleic
acid molecule encoding a biologically active variant of the amino
acid sequence set forth in SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17,
19, 22, or 25 or the amino acid sequence encoded by the nucleotide
sequence of the cDNA insert of the plasmid deposited with ATCC as
Patent Deposit Number PTA-1850, PTA-2170, PTA-2812, PTA-2171,
PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849, PTA-1915, or
PTA-1871, or PTA-1918, wherein the nucleic acid molecule hybridizes
the complement of the nucleotide sequence set forth in SEQ ID NO:1,
6, 8, 10, 12, 14, 16, 18, 20, 21, 23, 24, or 26, or the nucleotide
sequence of the cDNA insert of the plasmid deposited with ATCC as
Patent Deposit Number PTA-1850, PTA-2170, PTA-2812, PTA-2171,
PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849, PTA-1915,
PTA-1871, or PTA-1918 under stringent conditions; and f) a nucleic
acid molecule comprising the complement of the nucleic acid
molecule of a), b), c), d), or e).
2. The isolated nucleic acid molecule of claim 1, wherein said
nucleic acid molecule is selected from the group consisting of: a)
a nucleic acid molecule comprising the nucleotide sequence set
forth in SEQ ID NO:1, 6, 8, 10, 12, 14, 16, 18, 20, 21, 23, 24, or
26, or a complement thereof; b) a nucleic acid molecule comprising
the nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Patent Deposit Number PTA-1850, PTA-2170, PTA-2812,
PTA-2171, PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849,
PTA-1915, or PTA-1871; c) a nucleic acid molecule encoding a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, 5,
7, 9, 11, 13, 15, 17, 19, 22, or 25, or a complement thereof; and
d) a nucleic acid molecule encoding the polypeptide encoded by the
nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Patent Deposit Number PTA-1850, PTA-2170, PTA-2812,
PTA-2171, PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849,
PTA-1915, or PTA-1871.
3. The nucleic acid molecule of claim 1, further comprising vector
nucleic acid sequences.
4. The nucleic acid molecule of claim 1 further comprising nucleic
acid sequences encoding a heterologous polypeptide.
5. A host cell that contains the nucleic acid molecule of claim
3.
6. An isolated polypeptide selected from the group consisting of:
a) a biologically active 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
set forth in SEQ ID NO:1, 6, 8, 10, 12, 14, 16, 18, 20, 21, 23, 24,
or 26, or the nucleotide sequence of the cDNA insert of the plasmid
deposited with ATCC as Patent Deposit Number PTA-1850, PTA-2170,
PTA-2812, PTA-2171, PTA-2813, PTA-2011, PTA-2012, PTA-2341,
PTA-1849, PTA-1915, PTA-1871, or PTA-1918; b) a naturally occurring
allelic variant of a polypeptide comprising the amino acid sequence
set forth in SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25,
or the amino acid sequence encoded by the nucleotide sequence of
the cDNA insert of the plasmid deposited with ATCC as Patent
Deposit Number PTA-1850, PTA-2170, PTA-2812, PTA-2171, PTA-2813,
PTA-2011, PTA-2012, PTA-2341, PTA-1849, PTA-1915, PTA-1871, or
PTA-1918, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising the
complement of SEQ ID NO:1, 6, 8, 10, 12, 14, 16, 18, 20, 21, 23,
24, or 26, or the nucleotide sequence of the cDNA insert of the
plasmid deposited with ATCC as Patent Deposit Number PTA-1850,
PTA-2170, PTA-2812, PTA-2171, PTA-2813, PTA-2011, PTA-2012,
PTA-2341, PTA-1849, PTA-1915, PTA-1871, or PTA-1918 under stringent
conditions; and, c) a fragment of a polypeptide comprising the
amino acid sequence set forth in SEQ ID NO:2, 5, 7, 9, 11, 13, 15,
17, 19, 22, or 25, or the amino acid sequence encoded by the
nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Patent Deposit Number PTA-1850, PTA-2170, PTA-2812,
PTA-2171, PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849,
PTA-1915, PTA-1871, or PTA-1918, wherein the fragment comprises at
least 15 contiguous amino acids of the amino acid sequence set
forth in SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25, or
the amino acid sequence encoded by the nucleotide sequence of the
cDNA insert of the plasmid deposited with ATCC as Patent Deposit
Number PTA-1850, PTA-2170, PTA-2812, PTA-2171, PTA-2813, PTA-2011,
PTA-2012, PTA-2341, PTA-1849, PTA-1915, PTA-1871, or PTA-1918; and
d) a polypeptide having at least 60% sequence identity to the amino
acid sequence SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25,
wherein the polypeptide has biological activity.
7. The isolated polypeptide of claim 6 comprising the amino acid
sequence of SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or
25.
8. The polypeptide of claim 6 further comprising heterologous amino
acid sequences.
9. An antibody which selectively binds to a polypeptide of claim
6.
10. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
set forth in SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25,
or the amino acid sequence encoded by the nucleotide sequence of
the cDNA insert of the plasmid deposited with ATCC as Patent
Deposit Number PTA-1850, PTA-2170, PTA-2812, PTA-2171, PTA-2813,
PTA-2011, PTA-2012, PTA-2341, PTA-1849, PTA-1915, PTA-1871, or
PTA-1918; b) a polypeptide comprising a fragment of the amino acid
sequence set forth in SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22,
or 25, or the amino acid sequence encoded by the nucleotide
sequence of the cDNA insert of the plasmid deposited with ATCC as
Patent Deposit Number PTA-1850, PTA-2170, PTA-2812, PTA-2171,
PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849, PTA-1915,
PTA-1871, or PTA-1918, wherein the fragment comprises at least 15
contiguous amino acids of SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19,
22, or 25, or the amino acid sequence encoded by the nucleotide
sequence of the cDNA insert of the plasmid deposited with ATCC as
Patent Deposit Number PTA-1850, PTA-2170, PTA-2812, PTA-2171,
PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849, PTA-1915,
PTA-1871, or PTA-1918; c) a biologically active variant of a
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25, or the amino acid
sequence encoded by the nucleotide sequence of the cDNA insert of
the plasmid deposited with ATCC as Patent Deposit Number PTA-1850,
PTA-2170, PTA-2812, PTA-2171, PTA-2813, PTA-2011, PTA-2012,
PTA-2341, PTA-1849, PTA-1915, PTA-1871, or PTA-1918, wherein the
polypeptide is encoded by a nucleic acid molecule which hybridizes
to a nucleic acid molecule comprising the complement of SEQ ID
NO:1, 6, 8, 10, 12, 14, 16, 18, 20, 21, 23, 24, or 26, or the
nucleotide sequence of the cDNA insert of the plasmid deposited
with ATCC as Patent Deposit Number PTA-1850, PTA-2170, PTA-2812,
PTA-2171, PTA-2813, PTA-2011, PTA-2012, PTA-2341, PTA-1849,
PTA-1915, PTA-1871, or PTA-1918; and d) a polypeptide having at
least 60% sequence identity to the amino acid sequence of SEQ ID
NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 22, or 25, wherein said
polypeptide has biological activity; comprising culturing the host
cell of claim 5 under conditions in which the nucleic acid molecule
is expressed.
11. A method for detecting the presence of a polypeptide of claim 6
in a sample, comprising: a) contacting the sample with a compound
which selectively binds to a polypeptide of claim 6; and b)
determining whether the compound binds to the polypeptide in the
sample.
12. The method of claim 11, wherein the compound which binds to the
polypeptide is an antibody.
13. A kit comprising a compound which selectively binds to a
polypeptide of claim 6 and instructions for use.
14. A method for detecting the presence of a nucleic acid molecule
of claim 1 in a sample, comprising the steps of: 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.
15. The method of claim 14, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
16. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 and instructions for use.
17. A method for identifying a compound that binds to a polypeptide
of claim 6 comprising the steps of: a) contacting a polypeptide, or
a cell expressing a polypeptide of claim 6 with a test compound;
and b) determining whether the polypeptide binds to the test
compound.
18. The method of claim 17, 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
detecting of test compound/polypeptide binding; b) detection of
binding using a competition binding assay; c) detection of binding
using an assay for GAP mediated nucleotide exchange.
19. A method for modulating the activity of a polypeptide of claim
6 comprising contacting a polypeptide or a cell expressing a
polypeptide of claim 6 with a compound which binds to the
polypeptide in a sufficient concentration to modulate the activity
of the polypeptide.
20. A method for identifying a compound which modulates the
activity of a polypeptide of claim 6, comprising: a) contacting a
polypeptide of claim 6 with a test compound; and b) determining the
effect of the test compound on the activity of the polypeptide to
thereby identify a compound that modulates the activity of the
polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 10/165,231, filed Jun. 6, 2002 (pending),
which is a continuation-in-part of U.S. patent application Ser. No.
09/390,038, filed Sep. 3, 1999 (abandoned). U.S. patent application
Ser. No. 10/165,231 is also a continuation-in-part of U.S. patent
application Ser. No. 09/796,089, filed Feb. 28, 2001 (abandoned),
which is a continuation-in-part of U.S. patent application Ser. No.
09/464,039, filed Dec. 15, 1999, now U.S. Pat. No. 7,094,565, and
claims the benefit of International Application No. 371
PCT/US00/33873, filed Dec. 15, 2000. U.S. patent application Ser.
No. 10/165,231 is also a continuation-in-part of U.S. patent
application Ser. No. 09/972,525, filed Oct. 5, 2001 (abandoned),
which is a divisional of U.S. patent application Ser. No.
09/408,865, filed Sep. 30, 1999, now U.S. Pat. No. 6,329,171. U.S.
patent application Ser. No. 10/165,231 is also a
continuation-in-part of Ser. No. 09/963,908, filed Sep. 26, 2001,
now, U.S. Pat. No. 6,797,502, which is a divisional of U.S. patent
application Ser. No. 09/434,613, filed Nov. 5, 1999, now U.S. Pat.
No. 6,337,187. U.S. patent application Ser. No. 10/165,231 is also
a continuation-in-part of U.S. patent application Ser. No.
09/461,076, filed Dec. 14, 1999 (abandoned). U.S. patent
application Ser. No. 10/165,231 is also a continuation-in-part of
U.S. patent application Ser. No. 09/802,127, filed Feb. 26, 2001
(abandoned), which claims the benefit of U.S. Provisional
Application Ser. No. 60/185,611, filed Feb. 29, 2000 (abandoned).
The entire contents of each of the above-referenced patent
applications are incorporated herein by this reference.
FIELD OF THE INVENTION
[0002] The invention relates to novel nucleic acid sequences and
polypeptides. Also provided are vectors, host cells, and
recombinant methods for making and using the novel molecules.
TABLE OF CONTENTS
[0003] Chapter 1 23552, A Novel Adenylate Kinase [0004] i) SEQ ID
NOS: 1-4 [0005] ii) FIGS. 1-6 [0006] iii) Continuation-In-Part of
Ser. No. 09/390,038, filed Sep. 3, 1999 [0007] Chapter 2 21612,
21615, 21620, 21676, 33756, Novel Human Alcohol Dehydrogenases
[0008] i) SEQ ID NOS: 5-14 [0009] ii) FIGS. 7A-32 [0010] iii)
Continuation-In-Part of Ser. No. 09/796,089, filed Feb. 28, 2001,
which is a continuation-in-part of Ser. No. 09/464,039, filed Dec.
15, 1999, and claims the benefit of 371 PCT/US00/33873, filed Dec.
15, 2000 [0011] Chapter 3 23484, A Novel Human Ubiquitin Protease
[0012] i) SEQ ID NOS: 15-16 [0013] ii) FIGS. 33A-38 [0014] iii)
Continuation-In-Part of Ser. No. 09/972,525, filed Oct. 5, 2001,
which is a divisional of Ser. No. 09/408,865, filed Sep. 30, 1999,
now U.S. Pat. No. 6,329,171 [0015] Chapter 4 18891, A Novel Human
Lipase [0016] i) SEQ ID NOS: 17-18 [0017] ii) FIGS. 39A-45 [0018]
iii) Continuation-In-Part of Ser. No. 09/963,908, filed Sep. 26,
2001, which is a divisional of Ser. No. 09/434,613, filed Nov. 5,
1999, now U.S. Pat. No. 6,337,187 [0019] Chapter 5 25678, A Novel
Human Adenylate Cyclase [0020] i) SEQ ID NOS: 19-20 [0021] ii)
FIGS. 46A-51 [0022] iii) Continuation-In-Part of Ser. No.
09/461,076, filed Dec. 14, 1999 [0023] Chapter 6 Novel Human GTPase
Activators [0024] i) SEQ ID NOS: 21-30 [0025] ii) FIGS. 52A-64B
[0026] iii) Continuation-In-Part of Ser. No. 09/802,127, filed Feb.
26, 2001, which claims the benefit of U.S. Provisional 60/185,611,
filed Feb. 29, 2000
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows the amino acid sequence alignment for the
protein (h23552; SEQ ID NO:2) encoded by human 23552 (SEQ ID NO:1)
with the porcine UMP-CMP kinase (SP Accession Number Q29561; SEQ ID
NO:3). The sequence alignment was generated using the Clustal
method. The two sequences share approximately 97.4% identity over a
196 amino acid overlap as determined by pairwise alignment.
Asterisks indicate identical residues.
[0028] FIG. 2 shows the 23552 nucleotide sequence (SEQ ID NO:1) and
amino acids 1 to 609 of the amino acid sequence set forth in SEQ ID
NO:2.
[0029] FIG. 3 shows an analysis of the 23552 amino acid sequence:
.alpha..beta. turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index; and surface probability
plot.
[0030] FIG. 4 shows a 23552 receptor hydrophobicity plot and the
23552 amino acid sequence (SEQ ID NO:2).
[0031] FIG. 5 shows an analysis of the 23552 open reading frame for
amino acids corresponding to specific functional sites. These sites
are relevant with regard to providing fragments of the 23552
nucleic acid or peptide as disclosed herein. The 23552 amino acid
sequence contains an N-glycosylation site from amino acids 137 to
140 of SEQ ID NO:2; protein kinase C phosphorylation sites at amino
acids 21-23, 29-31, 170-172, 190-192 of SEQ ID NO:2; casein kinase
II phosphorylation sites at amino acids 65-68 and 212-215 of SEQ ID
NO:2; tyrosine kinase phosphorylation sites at amino acids 54-61
and 74-81 of SEQ ID NO:2; N-myristoylation sites at amino acids
42-47 and 49-54 of SEQ ID NO:2; and an adenylate kinase signature
at amino acids 121-132 of SEQ ID NO:2.
[0032] FIG. 6 shows a comparison of the 23552 protein against the
prosite database of protein patterns, specifically showing a high
score against an adenylate kinase consensus sequence set forth in
SEQ ID NO:4.
[0033] FIGS. 7A-7B show the nucleotide sequence (SEQ ID NO:6) and
the deduced amino acid sequence (SEQ ID NO:5) of the novel 21620
ADH.
[0034] FIG. 8 shows an analysis of the 21620 ADH amino acid
sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0035] FIG. 9 shows a hydrophobicity plot of the 21620 ADH amino
acid sequence (SEQ ID NO:5). Also shown is the predicted
transmembrane segment from about amino acid 13 to about amino acid
32. In addition, a graphical representation of the functional
domain of ADH short chain is also shown.
[0036] FIG. 10 shows an analysis of the 21620 ADH open reading
frame for amino acids (SEQ ID NO:5) corresponding to specific
functional sites. A putative protein kinase C phosphorylation site
is found from about amino acid 135 to about amino acid 137.
Putative casein kinase II phosphorylation sites are found from
about amino acid 72 to about amino acid 75, from about amino acid
89 to about amino acid 92, and from about amino acid 135 to about
amino acid 138. Putative N-myristoylation sites are found from
about amino acid 18 to about amino acid 23, from about amino acid
24 to about amino acid 29, from about amino acid 40 to about amino
acid 45, from about amino acid 90 to about amino acid 95, from
about amino acid 109 to about amino acid 114, and from about amino
acid 199 to about amino acid 204. In addition, amino acids
corresponding to the short-chain alcohol dehydrogenase family
signature are found in the sequence at about amino acids 166 to
176.
[0037] FIG. 11 shows expression of the 21620 ADH mRNA in various
tissues. 21620 expression levels were determined by quantitative
PCR (Taqman.RTM. brand quantitative PCR kit, Applied Biosystems).
The quantitative PCR reactions were performed according to the kit
manufacturer's instructions.
[0038] FIG. 12 shows expression of the 21620 ADH mRNA in normal and
malignant breast, lung, liver and colon tissues. The liver
metastases are derived from malignant colonic tissue. 21620
expression levels were determined by quantitative PCR (Taqman.RTM.
brand quantitative PCR kit, Applied Biosystems).
[0039] FIG. 13 shows the nucleotide sequence (SEQ ID NO:8) and the
deduced amino acid sequence (SEQ ID NO:7) of the novel 33756
ADH.
[0040] FIG. 14 shows an analysis of the 33756 ADH amino acid
sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0041] FIG. 15 shows a hydrophobicity plot of the 33756 ADH amino
acid sequence (SEQ ID NO:7). Also shown is a graphical
representation of the functional domain of ADH short chain.
[0042] FIG. 16 shows an analysis of the 33756 ADH open reading
frame (SEQ ID NO:7) for amino acids corresponding to specific
functional sites. A putative N-glycosylation site is found from
about amino acid 100 to about amino acid 103. Putative protein
kinase C phosphorylation sites are found from about amino acid 29
to about amino acid 31, from about amino acid 32 to about amino
acid 34, from about amino acid 120 to about amino acid 122, from
about amino acid 144 to about amino acid 146, from about amino acid
213 to about amino acid 215, from about amino acid 242 to about
amino acid 244, and from about amino acid 252 to about amino acid
254. Putative casein kinase II phosphorylation sites are found from
about amino acid 32 to about amino acid 35, from about amino 63 to
about amino acid 66, and from about amino acid 252 to about amino
acid 255. Putative N-myristoylation sites are found from about
amino acid 149 to about amino acid 154, from about amino acid 160
to about amino acid 165, and from about amino acid 171 to about
amino acid 176.
[0043] FIGS. 17A-17B show the nucleotide sequence (SEQ ID NO:9) and
the deduced amino acid sequence (SEQ ID NO:10) of the novel 21676
ADH.
[0044] FIG. 18 shows an analysis of the 21676 ADH amino acid
sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0045] FIG. 19 shows a hydrophobicity plot of the 21676 ADH amino
acid sequence (SEQ ID NO:9). Also shown is the predicted amino
terminus signal peptide sequence. In addition, two transmembrane
segments are predicted for the full-length polypeptide from about
amino acid 8 to about amino acid 25 and from about amino acid 242
to about amino acid 261. In the mature form of the polypeptide the
transmembrane domain is predicted from about amino acid 226 to
about amino acid 245. Also shown is a graphical representation of
the functional domain of ADH short chain.
[0046] FIG. 20 shows an analysis of the 21676 ADH open reading
frame (SEQ ID NO:9) for amino acids corresponding to specific
functional sites. A putative N-glycosylation site is found from
about amino acid 171 to about amino acid 174. A putative protein
kinase C phosphorylation sites are found from about amino acid 100
to about amino acid 102, from about amino acid 103 to about amino
acid 105, from about amino acid 191 to about amino acid 193, from
about amino acid 215 to about amino acid 217, from about amino acid
284 to about amino acid 286, from about amino acid 313 to about
amino acid 315, and from about amino acid 323 to about amino acid
325. A putative casein kinase II phosphorylation sites are found
from about amino acid 54 to about amino acid 57, from about amino
103 to about amino acid 106, from about amino acid 134 to about
amino acid 137, and from about amino acid 323 to about amino acid
326. Putative N-myristoylation sites are found from about amino
acid 12 to about amino acid 17, from about amino acid 28 to about
amino acid 33, from about amino acid 45 to about amino acid 50,
from about amino acid 220 to about amino acid 225, from about amino
acid 231 to about amino acid 236, and from about amino acid 242 to
about amino acid 247.
[0047] FIGS. 21A-21B show the nucleotide sequence (SEQ ID NO:12)
and the deduced amino acid sequence (SEQ ID NO:11) of the novel
21612 ADH.
[0048] FIG. 22 shows an analysis of the 21612 ADH amino acid
sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0049] FIG. 23 shows a hydrophobicity plot of the 21612 ADH amino
acid sequence (SEQ ID NO:11). Also shown is a graphical
representation of the functional domain of ADH short chain.
[0050] FIG. 24 shows an analysis of the 21612 ADH open reading
frame (SEQ ID NO:11) for amino acids corresponding to specific
functional sites. A putative N-glycosylation site is found from
about amino acid 101 to about amino acid 104. A putative protein
kinase C phosphorylation sites are found from about amino acid 5 to
about amino acid 7, from about amino acid 115 to about amino acid
117, from about amino acid 282 to about amino acid 284, from about
amino acid 313 to about amino acid 315, from about amino acid 381
to about amino acid 383, and from about amino acid 392 to about
amino acid 394. A putative casein kinase II phosphorylation sites
are found from about amino acid 56 to about amino acid 59, from
about amino 320 to about amino acid 323, from about amino acid 338
to about amino acid 341, and from about amino acid 372 to about
amino acid 375. A putative N-myristoylation sites are found from
about amino acid 17 to about amino acid 22, from about amino acid
52 to about amino acid 57, from about amino acid 128 to about amino
acid 133, and from about amino acid 353 to about amino acid 358. In
addition, a microbodies C-terminal targeting signal is found from
about amino acid 416 to about amino acid 418.
[0051] FIGS. 25A-25B show the nucleotide sequence (SEQ ID NO:14)
and the deduced amino acid sequence (SEQ ID NO:13) of the novel
21615 ADH.
[0052] FIG. 26 shows an analysis of the 21615 ADH amino acid
sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0053] FIG. 27 shows a hydrophobicity plot of the 21615 ADH amino
acid sequence (SEQ ID NO:13). Also shown is the predicted
transmembrane segment from about amino acid 8 to about amino acid
27. In addition, a graphical representation of the functional
domain of ADH short chain is also shown.
[0054] FIG. 28 shows an analysis of the 21615 ADH open reading
frame (SEQ ID NO:13) for amino acids corresponding to specific
functional sites. Putative N-glycosylation sites are found from
about amino acid 39 to about amino acid 42 and from about amino
acid 130 to about 133. Putative protein kinase C phosphorylation
site are found from about amino acid 60 to about amino acid 62,
from about amino acid 137 to about amino acid 139, from about amino
acid 149 to about amino acid 151, and from about amino acid 208 to
about amino acid 210. Putative casein kinase II phosphorylation
sites are found from about amino acid 89 to about amino acid 92,
from about amino 184 to about amino acid 187, from about amino acid
213 to about amino acid 216. A putative tyrosine kinase site is
found from about amino acid 42 to about amino acid 49. Putative
N-myristoylation sites are found from about amino acid 17 to about
amino acid 22, from about amino acid 126 to about amino acid 131,
from about amino acid 156 to about amino acid 161, and from about
amino acid 169 to about amino acid 174. In addition, a short-chain
alcohol dehydrogenase family signature is found from about amino
acid 147 to about amino acid 157.
[0055] FIG. 29 shows a time course of levels of 21612 mRNA
expression in the human colon cancer cell line HCT-116 following
synchronization with nocodazole. 21612 mRNA levels rose steadily as
the HCT-116 cell re-entered the cell cycle. 21612 expression levels
were determined by quantitative PCR (Taqman.RTM. brand quantitative
PCR kit, Applied Biosystems). The quantitative PCR reactions were
performed according to the kit manufacturer's instructions.
[0056] FIG. 30 shows 21612 mRNA expression in a panel of normal and
oncological tissues. 21612 expression is shown for 3 normal breast
tissue samples (columns 1-3); 6 breast tumor samples (columns 4-9),
including invasive ductal carcinomas (columns 4, 6, 7, and 8) and
moderately differentiated invasive ductal carcinomas (column 5); 2
normal ovary tissue samples (columns 10 and 11); 5 ovarian cancer
samples (columns 12-16); 3 normal lung samples (columns 17-19); 7
lung cancer samples (columns 20-26), including small cell carcinoma
(column 20), poorly differentiated non small cell carcinoma of the
lung (columns 21-23), adenocarcinoma (column 25 and 26), 3 normal
colon samples (columns 27-29); 4 colon cancer samples (columns
30-33), including moderately differentiated (columns 30 and 31) and
moderately to partially differentiated tumor (column 33); two colon
cancer liver metastases samples (columns 34 and 35); one normal
liver sample (column 36); one hemanginoma sample (column 37), and
two human pulmonary microvascular endothelial cell samples; one
arresting (column 38) and one proliferating (column 39). 21612
expression levels were determined by quantitative PCR (Taqman.RTM.
brand quantitative PCR kit, Applied Biosystems).
[0057] FIG. 31 shows the levels of 21612 mRNA in samples from
normal colon (columns 1-3), colon cancer tissue (columns 4-7),
colon cancer liver metastases (8 and 9), and normal liver (column
10). 21612 expression levels were determined by quantitative PCR as
described above. Note that 21612 mRNA levels were significantly
higher in 5 of 6 colon cancer and colon cancer liver metastases
samples, in comparison with the expression levels in normal colon
samples.
[0058] FIG. 32 shows the levels of 21612 mRNA in the human colon
cancer cell line HCT116 following cell cycle synchronization with
nocodazole. The first panel of this figure shows a time course the
level of 21612 mRNA following synchronization. Note the 21612
levels increase for approximately 21 hours following removal of
nocodazole. The second panel of this figure shows the time course
of 21612 expression in a population of HCT116 cells in the G.sub.0,
G.sub.1, or S phase of the cell cycle. Cell populations containing
cells in these phase of the cell cycle were isolated by
fluorescence-activated cell sorting. 21612 expression intensity at
each time point was determined by microarray hybridization. Note
that the levels of 21612 mRNA in G.sub.0/G.sub.1/S phase HCT116
cells increase significantly in the first three hours following
withdrawal of nocodazole.
[0059] FIGS. 33A-33D show the nucleotide sequence (SEQ ID NO:16)
and the deduced amino acid sequence (SEQ ID NO:15) of the novel
ubiquitin protease. The underlined amino acids designate the
conserved cysteine region and conserved histidine region. These
regions are conserved among thiol protease members of the UBP and
UCH protein families.
[0060] FIG. 34 shows an analysis of the ubiquitin protease amino
acid sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0061] FIG. 35 shows a hydrophobicity plot of the ubiquitin
protease (SEQ ID NO:15).
[0062] FIGS. 36A-36D show an analysis of the ubiquitin protease
open reading frame for amino acids corresponding to specific
functional sites of SEQ ID NO:15. Glycosylation sites are found
from about amino acid 134 to 137, with the modified amino acid at
position 134; about amino acid 333 to 336, with the modified amino
acid at position 333; from about amino acid 398 to 401 with the
modified amino acid at position 398, from about 492 to 495 with the
modified amino acid at position 492, from about 560 to 563 with the
modified amino acid at position 560, from about 644 to 647 with the
modified amino acid at position 644, and from about 672 to 675 with
the modified amino acid at position 672. Cyclic AMP and cyclic
GMP-dependent protein kinase phosphorylation sites are found from
about amino acid 15 to 18 with the modified amino acid at position
18, from about amino acid 313 to 316 with the modified amino acid
at position 316, from about 607 to 610 with the modified amino acid
at position 610; from about amino acid 694 to 697 with the modified
amino acid at position 697; from about amino acid 812 to 815 with
the modified amino acid at position 815. Protein kinase C
phosphorylation sites are found from about amino acid 31 to 33,
with the modified amino acid at position 31; from about amino acid
107 to 109, with the modified amino acid at position 107; from
about amino acid 111 to 113, with the modified amino acid at
position 111; from about amino acid 312 to 314, with the modified
amino acid at position 312; from about amino acid 327 to 329, with
the modified amino acid at position 327; from about amino acid 426
to 428, with the modified amino acid at position 426; from about
amino acid 453 to 455, with the modified amino acid at position
453; from about amino acid 467 to 469, with the modified amino acid
at position 467; from about amino acid 475 to 477, with the
modified amino acid at position 475; from about amino acid 515 to
517, with the modified amino acid at position 515; from about amino
acid 546 to 548, with the modified amino acid at position 546; from
about amino acid 561 to 563, with the modified amino acid at
position 561; from about amino acid 556 to 568, with the modified
amino acid at position 566; from about amino acid 582 to 584, with
the modified amino acid at position 582; from about amino acid 623
to 625, with the modified amino acid at position 623; from about
amino acid 629 to 631, with the modified amino acid at position
629; from about amino acid 662 to 664, with the modified amino acid
at position 662; from about 692 to 694, with the modified amino
acid at position 692; from about amino acid 748 to 750, with the
modified amino acid at position 748; from about amino acid 765 to
767, with the modified amino acid at position 765; from about amino
acid 809 to 811, with the modified amino acid at position 809; from
about amino acid 865 to 867, with the modified amino acid at
position 865; from about amino acid 911 to 913, with the modified
amino acid at position 911; from about amino acid 952 to 954, with
the modified amino acid at position 952; from about amino acid 965
to 967, with the modified amino acid at position 965; from about
amino acid 980 to 982, with the modified amino acid at position
980; from about amino acid 1034 to 1036, with the modified amino
acid at position 1034; from about amino acid 1103 to 1105, with the
modified amino acid at position 1103; and from about amino acid
1120 to 1122, with the modified amino acid at 1120. Casein kinase
II phosphorylation sites are found from about amino acid 18 to 21;
from amino acid 75 to 78; from amino acid 92 to 95; from amino acid
260 to 263; from amino acid 481 to 484; from amino acid 527 to 530;
from amino acid 613 to 616; from amino acid 656 to 659; from amino
acid 673 to 676; from amino acid 703 to 706; from amino acid 807 to
810; and from amino acid 1067 to 1070. Tyrosine kinase
phosphorylation sites are found from about amino acid 83 to 90,
with the modified amino acid at position 90; from about amino acid
338 to 345, with the modified amino acid at position 345; and from
about amino acid 1031 to 1038, with the modified amino acid at
position 1038. N-myristoylation sites are found from about amino
acid from about 85 to 90, with the modified amino acid at position
85; from about amino acid 336 to 341, with the modified amino acid
at position 336; from about amino acid 486 to 491, with the
modified amino acid at position 486, from about amino acid 493 to
498, with the modified amino acid at position 493, from about amino
acid 552 to 557, with the modified amino acid at position 552; from
amino acid 570 to 575, with the modified amino acid at position
570; from amino acid 595 to 600, with the modified amino acid at
position 595; from amino acid 609 to 614, with the modified amino
acid at position 609; and from amino acid 898 to 903, with the
modified amino acid at position 898. Amidation sites are found from
about amino acid 13 to 16, from about amino acid 467 to 470, from
about amino acid 532 to 535; from about amino acid 841 to 844; and
from about amino acids 1038 to 1041. In addition, an amino acid
signature corresponding to the MHC immunoglobulins and major
histocompatibility complex proteins is found from amino acids 376
to 382. The amino acids corresponding to the UCH family 2 signature
are found at amino acids 365-383.
[0063] FIG. 37 shows expression of the protease in normal and
malignant breast, lung, liver and colon tissues. The liver
metastases are derived from malignant colonic tissue.
[0064] FIG. 38 shows expression of the protease in various tissues
and cell types in culture. The expression data was derived from
RT-PCR of various cDNA libraries.
[0065] FIGS. 39A-39B show the nucleotide sequence (SEQ ID NO:18)
and the deduced amino acid sequence (SEQ ID NO:17) of the novel
lipase.
[0066] FIG. 40 shows an analysis of the lipase amino acid sequence:
.alpha..beta.turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index; and surface probability
plot.
[0067] FIG. 41 shows a hydrophobicity plot of the lipase (SEQ ID
NO:17). The analysis predicted a 35 amino acid signal peptide
sequence at the amino terminus of the protein. Transmembrane
segments of both the full length lipase and the mature lipase are
also shown.
[0068] FIG. 42 shows an analysis of the lipase open reading frame
for amino acids corresponding to specific functional sites of SEQ
ID NO:17. Protein kinase C phosphorylation sites are found from
about amino acid 63 to 65; from about amino acid 111 to 113; from
about amino acid 252 to 254; from about amino acid 316 to 318.
Casein kinase II phosphorylation sites are found from about amino
acid 114 to 117; from amino acid 205 to 208; from amino acid 284 to
287. N-myristoylation sites are found from about amino acid from
about 13 to 18, with the modified amino acid at position 13; from
about amino acid 110 to 115, with the modified amino acid at
position 110; from about amino acid 146 to 151, with the modified
amino acid at position 146, from about amino acid 155 to 160, with
the modified amino acid at position 155, from about amino acid 175
to 180, with the modified amino acid at position 175.
[0069] FIG. 43 shows expression of the lipase mRNA in various
tissues and cell types in culture. The expression data was derived
from RT-PCR of various cDNA libraries. The primers used were
designed to amplify coding sequences.
[0070] FIG. 44 shows expression of the lipase mRNA in normal and
malignant breast, lung, liver and colon tissues. The liver
metastases are derived from malignant colonic tissue. The
expression data was derived from RT-PCR designed to amplify coding
sequences.
[0071] FIG. 45 shows expression of the lipase mRNA in normal and
malignant breast, lung, liver and colon tissues. The liver
metastases are derived from malignant colonic tissue. The
expression data was derived from RT-PCR designed to amplify the
untranslated region of the lipase.
[0072] FIGS. 46A-46D show the adenylate cyclase nucleotide sequence
(SEQ ID NO:20) and the deduced amino acid sequence (SEQ ID
NO:19).
[0073] FIG. 47 shows an analysis of the adenylate cyclase amino
acid sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0074] FIG. 48 shows a hydrophobicity plot of the adenylate
cyclase.
[0075] FIGS. 49A-49C show an analysis of the adenylate cyclase open
reading frame for amino acids corresponding to specific functional
sites of SEQ ID NO:19. Glycosylation sites are shown in the figure
with the actual modified residue being the first amino acid.
Protein kinase C phosphorylation sites are shown in the figure with
the actual modified residue being the first amino acid. Casein
kinase II phosphorylation sites are shown in the figure with the
actual modified residue being the first amino acid. Tyrosine kinase
phosphorylation sites are shown in the figure with the actual
modified residue being the last amino acid. N-myristoylation sites
are shown in the figure, with the actual modified residue being the
first amino acid. In addition, amino acids corresponding to the
guanylate cyclase signature are found at amino acids 394-417 and
1009-1032.
[0076] FIG. 50 shows expression of the 25678 adenylate cyclase in
various normal human tissues.
[0077] FIG. 51 shows expression of the 25678 adenylate cyclase in
various cardiovascular tissues. Int. Mamm: internal mammary artery;
CHF: congestive heart failure; ISCH: ischemic heart; myop:
myopathic heart.
[0078] FIGS. 52A-52B show the 26651 nucleotide sequence (SEQ ID
NO:21) and the deduced amino acid sequence (SEQ ID NO:22). The
coding sequence for 26651 is set forth in SEQ ID NO:23.
[0079] FIG. 53 shows a 26651 protein hydrophobicity plot. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:22) of human 26651 are indicated. Polypeptides of the
invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or as N-glycosylation
site.
[0080] FIG. 54 shows an analysis of the 26651 amino acid sequence:
.alpha..beta.turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index; and surface probability
plot.
[0081] FIG. 55 shows an analysis of the 26651 open reading frame
for amino acids corresponding to predicted functional sites. For
the cAMP- and cGMP-dependent protein kinase phosphorylation site,
the actual modified residue is the last amino acid. For the protein
kinase C phosphorylation sites, the actual modified residue is the
first amino acid. For the casein kinase II phosphorylation sites,
the actual modified residue is the first amino acid. For the
tyrosine kinase phosphorylation site, the actual modified residue
is the last amino acid.
[0082] FIGS. 56A-56B depict an alignment of the rho-GAP domain of
human 26651 with a consensus amino acid sequence derived from a
hidden Markov model. The upper sequence is the consensus amino acid
sequence (SEQ ID NO:27), while the lower amino acid sequence
corresponds to amino acids 236 to 397 of SEQ ID NO:22. The top half
of the figure was obtained by searching for complete domains using
PFAM. In the lower portion of the figure, a portion of human 26651
(amino acids 233 to 423 of SEQ ID NO:22) is aligned with a
consensus rho-GAP.sub.--3 domain (SEQ ID NO:28). The lower half of
the figure was obtained by searching for complete domains in
SMART.
[0083] FIGS. 57A-57C show the 26138 nucleotide sequence (SEQ ID
NO:24) and the deduced 26138 amino acid sequence (SEQ ID NO:25).
The coding sequence for 26138 is set forth in SEQ ID NO:26.
[0084] FIG. 58 shows a 26138 protein hydrophobicity plot. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:25) of human 26138 are indicated. Polypeptides of the
invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or as N-glycosylation
site.
[0085] FIG. 59 shows an analysis of the 26138 amino acid sequence:
.alpha..beta.turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index; and surface probability
plot.
[0086] FIGS. 60A-60B show an analysis of the 26138 open reading
frame for amino acids corresponding to predicted functional sites.
For the N-glycosylation site, the actual modified residue is the
first amino acid. For the N-myristoylation, the actual modified
residue is the first amino acid. For the cAMP- and cGMP-dependent
protein kinase phosphorylation site, the actual modified residue is
the first amino acid. For the protein kinase C phosphorylation
sites, the actual modified residue is the first amino acid. For the
casein kinase II phosphorylation sites, the actual modified residue
is the first amino acid. In addition there is a Ras GTPase
activating protein signature.
[0087] FIGS. 61A-61B depict an alignment of the ras-GAP domain of
human 26138 with a consensus amino acid sequence derived from a
hidden Markov model. The upper sequence is the consensus amino acid
sequence (SEQ ID NO:29), while the lower amino acid sequence
corresponds to amino acids 473 to 645 of SEQ ID NO:25. The top half
of the figure shows the results of a search for complete domains
using PFAM for the 26138 protein. In the lower portion of the
figure, a portion of human 26138 (amino acids 401 to 723 of SEQ ID
NO:25) is aligned with a consensus ras-GAP.sub.--2 domain (SEQ ID
NO:30). The lower half of the figure was obtained by searching for
complete domains in SMART.
[0088] FIGS. 62A-62B show a PSORT prediction of protein
localization for the 26138 GAP protein.
[0089] FIG. 63 shows chromosome mapping information for the 26138
GAP gene.
[0090] FIGS. 64A-64B depict expression of 26138 in various human
tissues and cell types: lung (column 1); kidney (column 2); brain
(column 3); heart (column 4); colon (column 5); tonsil (column 6);
spleen (column 7); fetal liver (column 8); pooled liver samples
(column 9); stellate cells treated with 1,25-dihydroxyvitamin D3
(column 10); serum reactivated stellate cells (column 11); NHLF-CTN
(column 12); NHLF-TGF, normal human lung fibroblasts treated with
TGF-.beta. (column 13); hepG2 CTN (column 14); hepG2 TGF, hepG2
cells treated with TGF-.beta. (column 15); LF NDR 190, fibrotic
liver (column 16); LF NDR 191, fibrotic liver (column 17); LF NDR
194, fibrotic liver (column 18); LF NDR 113 (column 19); Th1 48 hr
M4 (column 20); Th1 48 hr M5 (column 21); Th2 48 hr M5 (column 22);
granulocytes (column 23); CD19+ cells (column 24); CD14+ cells
(column 25); PBMC mock, peripheral blood mononuclear cells (column
26); PBMC PHA, PBMC treated with phytohaemagglutinin (column 27);
PBMC IL10, PBMC producing IL10 (column 28); PBMC 1113 (column 29);
NHBE mock, normal human bronchial epithelial cells (column 30);
NHBE IL13-1 (column 31); BM-MNC, bone marrow mononuclear cells
(column 32); mPB CD34, CD34+ cells from mobilized peripheral blood
(column 33); ABM CD34+, CD34+ cells from adult bone marrow (column
34); erythroid cells (column 35); megakaryocytes (column 36);
neutrophils (column 37); mBM CD11b+, CD11b+ cells from human
mobilized bone marrow (column 38); mBM CD15+, CD15+ mobilized human
bone marrow (column 39); mBM CD11b-, CD11b-cells from human
mobilized bone marrow (column 40); BM/GPA, GPA+ cells from human
bone marrow (column 41); BM CD71, CD71 positive bone marrow cells
(column 42); HepG2A (column 43); HepG2 2.21-a (column 44); and no
template control (column 45). Expression levels were determined by
quantitative RT-PCR (Taqman.RTM. brand quantitative PCR kit,
Applied Biosystems). The quantitative RT-PCR reactions were
performed according to the kit manufacturer's instructions.
CHAPTER 1
23552, A Novel Adenylate Kinase
BACKGROUND OF THE INVENTION
[0091] Adenylate kinases play a key role in the regulation of
energy balance within cells, particularly maintenance of the ratio
of ATP with its diphosphate (ADP) and monophosphate forms (AMP).
ATP serves as the primary source of energy for biochemical
reactions in cells and is also a key precursor in DNA and RNA
synthesis during cellular growth and replication. The energy
associated with the terminal phosphate bonds of ATP may be
transferred to other nucleotides using a nucleoside monophosphate
kinase such as adenlyate kinase. In this manner, the terminal
energy-rich phosphate bonds of ATP may be transferred to the
appropriate nucleotides for use in a variety of biosynthetic and
energy-requiring processes, such as biosynthesis of macromolecules,
active ion transport, muscle contraction, thermogenesis, etc. A
number of these energy-requiring biosynthetic reactions hydrolyze
ATP into AMP plus pyrophosphate. Reutilization of the resulting AMP
requires conversion back into the triphosphate form following
conversion to ADP. Various nucleotide monophosphate kinases carry
out the first step of phosphorylating AMP to its diphosphate form
at the expense of ATP. In the case of adenylate kinase, this
reversible reaction is given as AMP+ATP.ident.2 ADP.
[0092] Adenylate kinases also play a role in regulating the flow of
carbon between net accumulation of glucose via the gluconeogenesis
pathway and its subsequent catabolism via the glycolytic pathway by
way of their control over the ratio of AMP to ATP. AMP is a
positive allosteric effector of the enzyme 6-phophofructo-1-kinase,
which shifts, and a negative allosteric effector for the enzyme
fructose-1,6-bisphosphatase. When the first of these enzymes is
activated, carbon flow is shifted in the direction of glycolysis;
when the second of these enzymes is activated, carbon flow shifts
in the direction of gluconeogenesis. Thus, increases in the ratio
of AMP to ATP shift carbon flow toward glycolysis, while decreases
in the ratio of AMP to ATP shift carbon flow toward glucose
formation.
[0093] These enzymes have been studied in a number of mammals,
including rat, porcine, chicken, bovine, rabbit, and humans.
Evidence from biochemical studies suggests that human tissues have
five adenylate kinase isozymes, AK1-AK5. Thus far the cDNAs of
human AK1, AK2, AK4, and AK5 have been cloned. Adenylate kinase
isoforms in humans are sequence related and also related to UMP/CMP
kinases from several species. See Rompay et al. (1999) Eur. J.
Biochem. 261:509-516, and the references cited therein.
[0094] The adenylate kinase isozymes AK1 (or myokinase), which is a
cytosolic enzyme present in brain, skeletal muscle, and
erythrocytes, and AK2, which is associated with the mitochondrial
membrane in liver, spleen, heart, and kidney, both utilize ATP as
their nucleoside triphosphate donor substrate. AK3 (or GTP:AMP
phosphotransferase) is located in the mitochondrial matrix,
primarily in heart and liver cells, and uses MgGTP instead of
MgATP. AK4 and AK5 are both localized in brain tissue.
[0095] Several regions of AK family enzymes are well conserved,
including the nucleoside triphosphate binding glycine-rich region,
the nucleoside monophosphate binding site, and the lid domain that
closes over the substrate upon binding (see Schulz (1987) Cold
Spring Harbor Symp. Quant. Biol. 52:429-439).
[0096] These enzymes assist with maintenance of energy production
and utilization within cells, particularly in cells having high
rates of growth and metabolic activity such as in heart, skeletal
muscle, and liver. In fact, adenylate kinase deficiency has been
linked to hemolytic anemia and neurological disorders such as
neurofibromatosis (Xu et al. (1992) Genomics 13:537-542. In
addition, targeting regulation of ATP synthesis has been the basis
of antiproliferative drugs for treatment of viral infections and
cancer.
[0097] Adenylate kinases are also useful for activating nucleoside
analogues used as pharmaceuticals, especially for cancer and viral
infection. Most of these analogues must be phosphorylated to the
triphosphate form in order to be pharmaceutically active. The first
phosphorylation step in the activation of nucleoside analogs is
catalyzed by deoxyribonucleoside kinases. Phosphorylation to the
di- and triphosphates are then required.
[0098] Accordingly, adenylate kinases are a major target for drug
action and development. Thus, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown adenylate kinases. The present invention advances the state
of the art by providing a previously unidentified human adenylate
kinase.
SUMMARY OF THE INVENTION
[0099] Isolated nucleic acid molecules corresponding to adenylate
kinase nucleic acid sequences are provided. Additionally, amino
acid sequences corresponding to the polynucleotides are
encompassed. In particular, the present invention provides for
isolated nucleic acid molecules comprising nucleotide sequences
encoding the amino acid sequence shown in SEQ ID NO:2 or the
nucleotide sequences encoding the DNA sequence deposited in a
bacterial host with the Patent Depository of the American Type
Culture Collection (ATCC) as Patent Deposit Number PTA-1850.
Further provided are adenylate kinase polypeptides having an amino
acid sequence encoded by a nucleic acid molecule described
herein.
[0100] The present invention also provides vectors and host cells
for recombinant expression of the nucleic acid molecules described
herein, as well as methods of making such vectors and host cells
and for using them for production of the polypeptides or peptides
of the invention by recombinant techniques.
[0101] The adenylate kinase molecules of the present invention are
useful for modulating cellular growth and/or cellular metabolic
pathways particularly for regulating one or more proteins involved
in growth and metabolism. Accordingly, in one aspect, this
invention provides isolated nucleic acid molecules encoding
adenylate kinase proteins or biologically active portions thereof,
as well as nucleic acid fragments suitable as primers or
hybridization probes for the detection of adenylate kinase-encoding
nucleic acids.
[0102] Another aspect of this invention features isolated or
recombinant adenylate kinase proteins and polypeptides. Preferred
adenylate kinase proteins and polypeptides possess at least one
biological activity possessed by naturally occurring adenylate
kinase proteins.
[0103] Variant nucleic acid molecules and polypeptides
substantially homologous to the nucleotide and amino acid sequences
set forth in the sequence listings are encompassed by the present
invention. Additionally, fragments and substantially homologous
fragments of the nucleotide and amino acid sequences are
provided.
[0104] Antibodies and antibody fragments that selectively bind the
adenylate kinase polypeptides and fragments are provided. Such
antibodies are useful in detecting the adenylate kinase
polypeptides as well as in regulating the T-cell immune response
and cellular activity, particularly growth and proliferation.
[0105] In another aspect, the present invention provides a method
for detecting the presence of adenylate kinase activity or
expression in a biological sample by contacting the biological
sample with an agent capable of detecting an indicator of adenylate
kinase activity such that the presence of adenylate kinase activity
is detected in the biological sample.
[0106] In yet another aspect, the invention provides a method for
modulating adenylate kinase activity comprising contacting a cell
with an agent that modulates (inhibits or stimulates) adenylate
kinase activity or expression such that adenylate kinase activity
or expression in the cell is modulated. In one embodiment, the
agent is an antibody that specifically binds to adenylate kinase
protein. In another embodiment, the agent modulates expression of
adenylate kinase protein by modulating transcription of an
adenylate kinase gene, splicing of an adenylate kinase mRNA, or
translation of an adenylate kinase mRNA. In yet another embodiment,
the agent is a nucleic acid molecule having a nucleotide sequence
that is antisense to the coding strand of the adenylate kinase mRNA
or the adenylate kinase gene.
[0107] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
adenylate kinase protein activity or nucleic acid expression by
administering an agent that is an adenylate kinase modulator to the
subject. In one embodiment, the adenylate kinase modulator is an
adenylate kinase protein. In another embodiment, the adenylate
kinase modulator is an adenylate kinase nucleic acid molecule. In
other embodiments, the adenylate kinase modulator is a peptide,
peptidomimetic, or other small molecule.
[0108] The present invention also provides a diagnostic assay for
identifying the presence or absence of a genetic lesion or mutation
characterized by at least one of the following: (1) aberrant
modification or mutation of a gene encoding an adenylate kinase
protein; (2) misregulation of a gene encoding an adenylate kinase
protein; and (3) aberrant post-translational modification of an
adenylate kinase protein, wherein a wild-type form of the gene
encodes a protein with an adenylate kinase activity.
[0109] In another aspect, the invention provides a method for
identifying a compound that binds to or modulates the activity of
an adenylate kinase protein. In general, such methods entail
measuring a biological activity of an adenylate kinase protein in
the presence and absence of a test compound and identifying those
compounds that alter the activity of the adenylate kinase
protein.
[0110] The invention also features methods for identifying a
compound that modulates the expression of adenylate kinase genes by
measuring the expression of the adenylate kinase sequences in the
presence and absence of the compound.
[0111] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0112] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0113] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[0114] The present invention is based, at least in part, on the
identification of novel molecules, referred to herein as adenylate
kinase nucleic acid and polypeptide molecules, which play a role
in, or function in, numerous biochemical pathways associated with
cellular growth and/or cellular metabolic activity. These growth
and metabolic pathways are described in Lodish et al. (1995)
Molecular Cell Biology (Scientific American Books Inc., New York,
N.Y.) and Devlin (1997) Textbook of Biochemistry with Clinical
Correlations (Wiley-Liss, Inc., New York, N.Y.), the contents of
which are incorporated herein by reference. In one embodiment, the
adenylate kinase molecules modulate the activity of one or more
proteins involved in cellular growth or differentiation, e.g.,
cardiac, epithelial, or neuronal cell growth or differentiation. In
another embodiment, the adenylate kinase molecules of the present
invention are capable of modulating the phosphorylation state of a
nucleoside mono-, di-, or triphosphate molecule or the
phosphorylation state of one or more proteins involved in cellular
growth or differentiation, e.g., cardiac, epithelial, or neuronal
cell growth or differentiation, as described in, for example,
Lodish et al. (1995) and Devlin (1997), supra. In addition, the
substrates of the adenylate kinases of the present invention are
targets of drugs described in Goodman and Gilman (1996), The
Pharmacological Basis of Therapeutics (9.sup.th ed.) Hartman &
Limbard Editors, the contents of which are incorporated herein by
reference. Particularly, the adenylate kinases of the invention may
modulate phosphorylation activity in tissues and cells including
lymph node, spleen, thymus, brain, lung, skeletal muscle, fetal
liver, tonsil, colon, heart, liver, immune cells, including T
cells, Th1 and Th2 cells, leukocytes, blood marrow, etc. In one
embodiment, the adenylate kinase sequences of the invention are
used to manipulate the nucleoside mono-, di-, and triphosphate pool
to alter cellular metabolic pathways, such as glycolysis and
gluconeogenesis.
[0115] Adenylate kinases play an important role in the regulation
of energy balance within cells and in energy-requiring biochemical
processes associated with cellular growth and development.
Inhibition or over-stimulation of the activity of adenylate kinases
affects the cellular equilibrium between nucleoside mono-, di-, and
triphosphates, particularly AMP, ADP, and ATP, all of which are
integrally involved in energy-requiring biochemical processes
associated with cellular growth and development. Disruption or
modulation of this equilibrium can lead to perturbed cellular
growth, which can in turn lead to cellular growth
related-disorders. As used herein, a "cellular growth-related
disorder" includes a disorder, disease, or condition characterized
by a deregulation, e.g., an upregulation or a downregulation, of
cellular growth. Cellular growth deregulation may be due to a
deregulation of cellular proliferation, cell cycle progression,
cellular differentiation and/or cellular hypertrophy. Examples of
cellular growth related disorders include cardiovascular disorders
such as heart failure, hypertension, atrial fibrillation, dilated
cardiomyopathy, idiopathic cardiomyopathy, or angina; proliferative
disorders or differentiative disorders such as cancer, e.g.,
melanoma, prostate cancer, cervical cancer, breast cancer, colon
cancer, or sarcoma. Disorders associated with the following cells
or tissues are also encompassed: lymph node, spleen, thymus, brain,
lung, skeletal muscle, fetal liver, tonsil, colon, heart, liver,
immune cells, including T cells, Th1 and Th2 cells, leukocytes,
blood marrow, etc. The compositions are also useful for the
treatment of liver fibrosis and other liver-related disorders.
[0116] Furthermore, adenylate kinase activity increases in
cerebrospinal fluid at the acute onset of ischemic brain damage and
is correlated with the severity of the lesion (Buttner et al.
(1986) J. Neurol. 233:297-303). Adenyl kinase activity also
increases in cerebrous spinal fluid in some brain tumors (Ronquist
et al. (1977) Lancet i: 1284-1286). Further, adenyl kinase may be
expressed in damaged tissue and therefore is a useful target to
measure tissue damage. Finally, deletions at 1p31 locus in many
tumors is associated with hemolytic anemia (Matsuura et al. (1989)
J. Biol. Chem. 264:10148-10155 and Mitelman et al. (1997) Nature
Genet. 15:417-474). Accordingly, the compositions are also useful
for treatment and diagnosis related to these disorders.
[0117] The disclosed invention relates to methods and compositions
for the modulation, diagnosis, and treatment of immune,
inflammatory, respiratory, and hematological disorders.
[0118] Immune disorders include, but are not limited to, chronic
inflammatory diseases and disorders, such as Crohn's disease,
reactive arthritis, including Lyme disease, insulin-dependent
diabetes, organ-specific autoimmunity, including multiple
sclerosis, Hashimoto's thyroiditis and Grave's disease, contact
dermatitis, psoriasis, graft rejection, graft versus host disease,
sarcoidosis, atopic conditions, such as asthma and allergy,
including allergic rhinitis, gastrointestinal allergies, including
food allergies, eosinophilia, conjunctivitis, glomerular nephritis,
certain pathogen susceptibilities such as helminthic (e.g.,
leishmaniasis), certain viral infections, including HIV, and
bacterial infections, including tuberculosis and lepromatous
leprosy.
[0119] Respiratory disorders include, but are not limited to,
apnea, asthma, particularly bronchial asthma, berillium disease,
bronchiectasis, bronchitis, bronchopneumonia, cystic fibrosis,
diphtheria, dyspnea, emphysema, chronic obstructive pulmonary
disease, allergic bronchopulmonary aspergillosis, pneumonia, acute
pulmonary edema, pertussis, pharyngitis, atelectasis, Wegener's
granulomatosis, Legionnaires disease, pleurisy, rheumatic fever,
and sinusitis.
[0120] Hematologic disorders include but are not limited to anemias
including sickle cell and hemolytic anemia, hemophilias including
types A and B, leukemias, thalassemias, spherocytosis, Von
Willebrand disease, chronic granulomatous disease,
glucose-6-phosphate dehydrogenase deficiency, thrombosis, clotting
factor abnormalities and deficiencies including factor VM and IX
deficiencies, hemarthrosis, hematemesis, hematomas, hematuria,
hemochromatosis, hemoglobinuria, hemolytic-uremic syndrome,
thrombocytopenias including HIV-associated thrombocytopenia,
hemorrhagic telangiectasia, idiopathic thrombocytopenic purpura,
thrombotic microangiopathy, hemosiderosis.
[0121] Liver disorders include, but are not limited to, hepatic
injury; jaundice and cholestasis, such as bilirubin and bile
formation; hepatic failure and cirrhosis, such as cirrhosis, portal
hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, including, but not limited to,
infectious hepatitis, such as viral hepatitis, including hepatitis
A-E infection and infection by other hepatitis viruses,
clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; other
forms of hepatitis; drug- and toxin-induced liver disease, such as
alcoholic liver disease; inborn errors of metabolism and pediatric
liver disease, such as hemochromatosis, Wilson disease,
.alpha..sub.1-antitrypsin deficiency, and neonatal hepatitis;
intrahepatic biliary tract disease, such as secondary biliary
cirrhosis, primary biliary cirrhosis, primary sclerosing
cholangitis, and anomalies of the biliary tree; circulatory
disorders, such as impaired blood flow into the liver, including
hepatic artery compromise and portal vein obstruction and
thrombosis, impaired blood flow through the liver, including
passive congestion and centrilobular necrosis and peliosis hepatis,
hepatic vein outflow obstruction, including hepatic vein thrombosis
(Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease
associated with pregnancy, such as preeclampsia and eclampsia,
acute fatty liver of pregnancy, and intrehepatic cholestasis of
pregnancy; hepatic complications of organ or bone marrow
transplantation, such as drug toxicity after bone marrow
transplantation, graft-versus-host disease and liver rejection, and
nonimmunologic damage to liver allografts; tumors and tumorous
conditions, such as nodular hyperplasias, adenomas, and malignant
tumors, including hepatocellular carcinoma, primary carcinoma of
the liver and metastatic tumors.
[0122] Preferred disorders include, but are not limited to
hepatitis, and especially viral hepatitis and hepatocellular
carcinoma.
[0123] The disclosed invention also relates to methods and
compositions for the modulation, diagnosis, and treatment of
disorders involving the brain, heart, lung, colon, and spleen.
[0124] Disorders involving the brain include, but are limited to,
disorders involving neurons, and disorders involving glia, such as
astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyclitis and acute
necrotizing hemorrhagic encephalomyclitis, and other diseases with
demyclination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0125] Disorders involving the heart include, but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0126] Disorders involving the lung include, but are not limited
to, congenital anomalies; atelectasis; diseases of vascular origin,
such as pulmonary congestion and edema, including hemodynamic
pulmonary edema and edema caused by microvascular injury, adult
respiratory distress syndrome (diffuse alveolar damage), pulmonary
embolism, hemorrhage, and infarction, and pulmonary hypertension
and vascular sclerosis; chronic obstructive pulmonary disease, such
as emphysema, chronic bronchitis, bronchial asthma, and
bronchiectasis; diffuse interstitial (infiltrative, restrictive)
diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity
pneumonitis, pulmonary eosinophilia (pulmonary infiltration with
eosinophilia), Bronchiolitis obliterans-organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, including Goodpasture
syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic
syndromes, pulmonary involvement in collagen vascular disorders,
and pulmonary alveolar proteinosis; complications of therapies,
such as drug-induced lung disease, radiation-induced lung disease,
and lung transplantation; tumors, such as bronchogenic carcinoma,
including paraneoplastic syndromes, bronchioloalveolar carcinoma,
neuroendocrine tumors, such as bronchial carcinoid, miscellaneous
tumors, and metastatic tumors; pathologies of the pleura, including
inflammatory pleural effusions, noninflammatory pleural effusions,
pneumothorax, and pleural tumors, including solitary fibrous tumors
(pleural fibroma) and malignant mesothelioma.
[0127] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0128] Disorders of the spleen include, but are not limited to,
splenomegaly, including nonspecific acute splenitis, congestive
spenomegaly, and spenic infarcts; neoplasms, congenital anomalies,
and rupture. Disorders associated with splenomegaly include
infections, such as nonspecific splenitis, infectious
mononucleosis, tuberculosis, typhoid fever, brucellosis,
cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis,
kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and
echinococcosis; congestive states related to partial hypertension,
such as cirrhosis of the liver, portal or splenic vein thrombosis,
and cardiac failure; lymphohematogenous disorders, such as Hodgkin
disease, non-Hodgkin lymphomas/leukemia, multiple myeloma,
myeloproliferative disorders, hemolytic anemias, and
thrombocytopenic purpura; immunologic-inflammatory conditions, such
as rheumatoid arthritis and systemic lupus erythematosus; storage
diseases such as Gaucher disease, Niemann-Pick disease, and
mucopolysaccharidoses; and other conditions, such as amyloidosis,
primary neoplasms and cysts, and secondary neoplasms.
[0129] Disorders involving the thymus include developmental
disorders, such as DiGeorge syndrome with thymic hypoplasia or
aplasia; thymic cysts; thymic hypoplasia, which involves the
appearance of lymphoid follicles within the thymus, creating thymic
follicular hyperplasia; and thymomas, including germ cell tumors,
lymphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[0130] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0131] Disorders involving T-cells include, but are not limited to,
cell-mediated hypersensitivity, such as delayed type
hypersensitivity and T-cell-mediated cytotoxicity, and transplant
rejection; autoimmune diseases, such as systemic lupus
erythematosus, Sjogren syndrome, systemic sclerosis, inflammatory
myopathies, mixed connective tissue disease, and polyarteritis
nodosa and other vasculitides; immunologic deficiency syndromes,
including but not limited to, primary immunodeficiencies, such as
thymic hypoplasia, severe combined immunodeficiency diseases, and
AIDS; leukopenia; reactive (inflammatory) proliferations of white
cells, including but not limited to, leukocytosis, acute
nonspecific lymphadenitis, and chronic nonspecific lymphadenitis;
neoplastic proliferations of white cells, including but not limited
to lymphoid neoplasms, such as precursor T-cell neoplasms, such as
acute lymphoblastic leukemia/lymphoma, peripheral T-cell and
natural killer cell neoplasms that include peripheral T-cell
lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis
fungoides and Sezary syndrome, and Hodgkin disease.
[0132] In normal bone marrow, the myelocytic series
(polymorphoneuclear cells) make up approximately 60% of the
cellular elements, and the erythrocytic series, 20-30%.
Lymphocytes, monocytes, reticular cells, plasma cells and
megakaryocytes together constitute 10-20%. Lymphocytes make up
5-15% of normal adult marrow. In the bone marrow, cell types are
add mixed so that precursors of red blood cells (erythroblasts),
macrophages (monoblasts), platelets (megakaryocytes),
polymorphoneuclear leucocytes (myeloblasts), and lymphocytes
(lymphoblasts) can be visible in one microscopic field. In
addition, stem cells exist for the different cell lineages, as well
as a precursor stem cell for the committed progenitor cells of the
different lineages. The various types of cells and stages of each
would be known to the person of ordinary skill in the art and are
found, for example, on page 42 (FIG. 2-8) of Immunology,
Immunopathology and Immunity, Fifth Edition, Sell et al. Simon and
Schuster (1996), incorporated by reference for its teaching of cell
types found in the bone marrow. According, the invention is
directed to disorders arising from these cells. These disorders
include but are not limited to the following: diseases involving
hematopoeitic stem cells; committed lymphoid progenitor cells;
lymphoid cells including B and T-cells; committed myeloid
progenitors, including monocytes, granulocytes, and megakaryocytes;
and committed erythroid progenitors. These include but are not
limited to the leukemias, including B-lymphoid leukemias,
T-lymphoid leukemias, undifferentiated leukemias; erythroleukemia,
megakaryoblastic leukemia, monocytic; [leukemias are encompassed
with and without differentiation]; chronic and acute lymphoblastic
leukemia, chronic and acute lymphocytic leukemia, chronic and acute
myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic
and acute myeloid leukemia, myelomonocytic leukemia; chronic and
acute myeloblastic leukemia, chronic and acute myelogenous
leukemia, chronic and acute promyelocytic leukemia, chronic and
acute myelocytic leukemia, hematologic malignancies of
monocyte-macrophage lineage, such as juvenile chronic myelogenous
leukemia; secondary AML, antecedent hematological disorder;
refractory anemia; aplastic anemia; reactive cutaneous
angioendotheliomatosis; fibrosing disorders involving altered
expression in dendritic cells, disorders including systemic
sclerosis, E-M syndrome, epidemic toxic oil syndrome, eosinophilic
fasciitis localized forms of scleroderma, keloid, and fibrosing
colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma,
including primary head and neck squamous cell carcinoma; sarcoma,
including kaposi's sarcoma; fibroadanoma and phyllodes tumors,
including mammary fibroadenoma; stromal tumors; phyllodes tumors,
including histiocytoma; erythroblastosis; neurofibromatosis;
diseases of the vascular endothelium; demyelinating, particularly
in old lesions; gliosis, vasogenic edema, vascular disease,
Alzheimer's and Parkinson's disease; T-cell lymphomas; B-cell
lymphomas.
[0133] Disorders involving red cells include, but are not limited
to, anemias, such as hemolytic anemias, including hereditary
spherocytosis, hemolytic disease due to erythrocyte enzyme defects:
glucose-6-phosphate dehydrogenase deficiency, sickle cell disease,
thalassemia syndromes, paroxysmal nocturnal hemoglobinuria,
immunohemolytic anemia, and hemolytic anemia resulting from trauma
to red cells; and anemias of diminished erythropoiesis, including
megaloblastic anemias, such as anemias of vitamin B12 deficiency:
pernicious anemia, and anemia of folate deficiency, iron deficiency
anemia, anemia of chronic disease, aplastic anemia, pure red cell
aplasia, and other forms of marrow failure.
[0134] Disorders involving B-cells include, but are not limited to
precursor B-cell neoplasms, such as lymphoblastic
leukemia/lymphoma. Peripheral B-cell neoplasms include, but are not
limited to, chronic lymphocytic leukemia/small lymphocytic
lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and
related entities, lymphoplasmacytic lymphoma (Waldenstrom
macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma
(MALToma), and hairy cell leukemia.
[0135] Disorders related to reduced platelet number,
thrombocytopenia, include idiopathic thrombocytopenic purpura,
including acute idiopathic thrombocytopenic purpura, drug-induced
thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic
microangiopathies: thrombotic thrombocytopenic purpura and
hemolytic-uremic syndrome.
[0136] Disorders involving precursor T-cell neoplasms include
precursor T lymphoblastic leukemia/lymphoma. Disorders involving
peripheral T-cell and natural killer cell neoplasms include T-cell
chronic lymphocytic leukemia, large granular lymphocytic leukemia,
mycosis fungoides and Sezary syndrome, peripheral T-cell lymphoma,
unspecified, angioimmunoblastic T-cell lymphoma, angiocentric
lymphoma (NK/T-cell lymphoma.sup.4a), intestinal T-cell lymphoma,
adult T-cell leukemia/lymphoma, and anaplastic large cell
lymphoma.
[0137] Specifically, the present invention provides isolated
nucleic acid molecules comprising nucleotide sequences encoding the
adenylate kinase polypeptide whose amino acid sequence is given in
SEQ ID NO:2, or a variant or fragment of the polypeptides. A
nucleotide sequence encoding an adenylate kinase polypeptide of the
invention, more particularly the polypeptide of SEQ ID NO:2, is set
forth in SEQ ID NO:1.
[0138] A novel human gene, termed clone h23552 is provided. This
sequence, and complements thereof, are referred to as "adenylate
kinase" indicating that the gene sequences share sequence
similarity to adenylate kinase genes.
[0139] The novel h23552 adenylate kinase gene encodes an
approximately 1.43 Kb mRNA transcript having the corresponding cDNA
set forth in SEQ ID NO:1. This transcript has a 634 nucleotide open
reading frame (nucleotides 200-883 of SEQ ID NO:1), which encodes a
228 amino acid protein (SEQ ID NO:2). An analysis of the
full-length h23552 polypeptide predicts that the N-terminal 47
amino acids may represent a region comprising a signal peptide.
Prosite program analysis was used to predict various sites within
the h23552 protein. An N-glycosylation site was predicted at aa
137-140. Protein kinase C phosphorylation sites were predicted at
aa 21-23, 29-31, 170-172, and 190-192. Casein kinase II
phosphorylation sites were predicted at aa 65-68 and 212-215.
Tyrosine kinase phosphorylation sites were predicted at aa 54-61
and 74-81. N-myristoylation sites were predicted at aa 42-47 and
49-54. An adenylate kinase signature sequence was predicted at aa
121-132.
[0140] The h23552 adenylate kinase protein possesses an adenylate
kinase domain sequence, from aa 40-203, as predicted by HMMer,
Version 2. This region of the protein comprises the three
functional subdomains common to nucleoside monophosphate kinases:
the nucleoside triphosphate binding glycine-rich region, the
nucleoside monophosphate binding site, and the lid domain that
closes over the substrate upon binding (see Schulz (1987) Cold
Spring Harbor Symp. Quant. Biol. 52:429-439).
[0141] The h23552 protein displays closest similarity to the
porcine UMP-CMP kinase (SP Accession Number Q29561; SEQ ID NO:3),
approximately 97.4% identity when aa 33-228 are aligned over the
full-length sequence for the porcine kinase (see FIG. 1) The
N-terminal region of the h23552 protein (aa 1-32) is novel.
Alignment of the h23552 protein with the porcine UMP-CMP kinase
indicates that the glycine-rich region corresponding to the binding
site of the nucleoside triphosphate donor resides at approximately
amino acid residues 42-50 of the h23552 protein. The region
corresponding to the nucleoside monophosphate binding site resides
at approximately amino acid residues 65-95; and the region
corresponding to the lid domain resides at approximately amino acid
residues 166-175. The similarity of the novel h23552 protein to the
porcine UMP-CMP kinase indicates the h23552 adenlyate kinase is a
member of the subclass of nucleoside monophosphate kinases referred
to as "short enzymes". Members of this subclass, which are
characterized by their short-length lid domain, include adenlyate
kinase 1 (AK1, identified in rabbit, bovine, human, pig, and
chicken), adenylate kinase 5 (AK5 identified in human), and UMP-CMP
kinases (identified in porcine, Dictyostelium discoideum,
Saccharomyces cereviseae). See Rompay et al. (1999) Eur. J.
Biochem. 261:509-516 and Fukami-Kobayashi et al. (1996) FEBS Lett.
385:214-220.
[0142] A plasmid containing the h23552 cDNA insert was deposited
with the Patent Depository of the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va., on May 29, 2000,
and assigned Patent Deposit Number PTA 1850. This deposit 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. This deposit was made merely as a
convenience for those of skill in the art and is not an admission
that a deposit is required under 35 U.S.C. .sctn. 112.
[0143] The adenylate kinase sequences of the invention are members
of a family of molecules having conserved functional features. The
term "family" when referring to the proteins and nucleic acid
molecules of the invention is intended to mean two or more proteins
or nucleic acid molecules having sufficient amino acid or
nucleotide sequence identity as defined herein. Such family members
can be naturally occurring and can be from either the same or
different species. For example, a family can contain a first
protein of murine origin and a homologue of that protein of human
origin, as well as a second, distinct protein of human origin and a
murine homologue of that protein. Members of a family may also have
common functional characteristics.
[0144] Preferred adenylate kinase polypeptides of the present
invention have an amino acid sequence sufficiently identical to the
amino acid sequence of SEQ ID NO:2. The term "sufficiently
identical" is used herein to refer to a first amino acid or
nucleotide sequence that contains a sufficient or minimum number of
identical or equivalent (e.g., with 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 have a common structural domain and/or common functional
activity. For example, amino acid or nucleotide sequences that
contain a common structural domain having at least about 45%, 55%,
or 65% identity, preferably 75% identity, more preferably 85%, 95%,
or 98% identity are defined herein as sufficiently identical.
[0145] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). 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. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are the
same length. The percent identity between two sequences can be
determined using techniques similar to those described below, with
or without allowing gaps. In calculating percent identity,
typically exact matches are counted.
[0146] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to adenylate kinase nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
adenylate kinase 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.
25:3389-3402. Alternatively, PSI-Blast can be used to perform an
iterated search which detects distant relationships between
molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.
[0147] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis
and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA
described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
85:2444-8. Within FASTA, ktup is a control option that sets the
sensitivity and speed of the search. If ktup=2, similar regions in
the two sequences being compared are found by looking at pairs of
aligned residues; if ktup=1, single aligned amino acids are
examined. ktup can be set to 2 or 1 for protein sequences, or from
1 to 6 for DNA sequences. The default if ktup is not specified is 2
for proteins and 6 for DNA. For a further description of FASTA
parameters, see bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2,
the contents of which are incorporated herein by reference.
[0148] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically exact
matches are counted.
[0149] Accordingly, another embodiment of the invention features
isolated adenylate kinase proteins and polypeptides having an
adenylate kinase protein activity. As used interchangeably herein,
a "adenylate kinase protein activity", "biological activity of an
adenylate kinase protein", or "functional activity of an adenylate
kinase protein" refers to an activity exerted by an adenylate
kinase protein, polypeptide, or nucleic acid molecule on an
adenylate kinase responsive cell as determined in vivo, or in
vitro, according to standard assay techniques. An adenylate kinase
activity can be a direct activity, such as an association with or
an enzymatic activity on a second protein, or an indirect activity,
such as a cellular activity mediated by interaction of the
adenylate kinase protein with a second protein. In a preferred
embodiment, an adenylate kinase activity includes at least one or
more of the following activities: (1) modulating (stimulating
and/or enhancing or inhibiting) cellular proliferation,
differentiation, and/or function, particularly in cells in which
the sequences are expressed, for example, cells of the lymph node,
spleen, thymus, brain, lung, skeletal muscle, fetal liver, tonsil,
colon, heart, liver, and immune cells, including Th1, Th2, T cells,
natural killer T cells, lymphocytes, leukocytes, blood marrow,
etc.); (2) modulating a target cell's energy balance, particularly
the ratio between AMP and ATP; (3) modulating the glycolytic
pathway; (4) modulating the gluconeogenesis pathway; (4) modulating
cell growth; (5) modulating the entry of cells into mitosis; (6)
modulating cellular differentiation; (7) modulating cell death; and
(8) modulating an immune response.
[0150] An "isolated" or "purified" adenylate kinase nucleic acid
molecule or protein, or biologically active portion thereof, is
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.
Preferably, an "isolated" nucleic acid is free of sequences
(preferably protein encoding sequences) that 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 purposes of the invention, "isolated"
when used to refer to nucleic acid molecules excludes isolated
chromosomes. For example, in various embodiments, the isolated
adenylate kinase 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 that naturally flank the nucleic acid molecule in genomic
DNA of the cell from which the nucleic acid is derived. An
adenylate kinase protein that is substantially free of cellular
material includes preparations of adenylate kinase protein having
less than about 30%, 20%, 10%, or 5% (by dry weight) of
non-adenylate kinase protein (also referred to herein as a
"contaminating protein"). When the adenylate kinase protein or
biologically active portion thereof is recombinantly produced,
preferably, culture medium represents less than about 30%, 20%,
10%, or 5% of the volume of the protein preparation. When adenylate
kinase protein is produced by chemical synthesis, preferably the
protein preparations have less than about 30%, 20%, 10%, or 5% (by
dry weight) of chemical precursors or non-adenylate kinase
chemicals.
[0151] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[0152] One aspect of the invention pertains to isolated nucleic
acid molecules comprising nucleotide sequences encoding adenylate
kinase proteins and polypeptides or biologically active portions
thereof, as well as nucleic acid molecules sufficient for use as
hybridization probes to identify adenylate kinase-encoding nucleic
acids (e.g., adenylate kinase mRNA) and fragments for use as PCR
primers for the amplification or mutation of adenylate kinase
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.
[0153] Nucleotide sequences encoding the adenylate kinase proteins
of the present invention include sequences set forth in SEQ ID
NO:1, the nucleotide sequence of the cDNA insert of the plasmid
deposited with the ATCC as Patent Deposit Number PTA-1850 (the
"cDNA of Patent Deposit Number PTA-1850"), and complements thereof.
By "complement" is intended a nucleotide sequence that is
sufficiently complementary to a given nucleotide sequence such that
it can hybridize to the given nucleotide sequence to thereby form a
stable duplex. The corresponding amino acid sequence for the
adenylate kinase protein encoded by these nucleotide sequences is
set forth in SEQ ID NO:2.
[0154] Nucleic acid molecules that are fragments of these adenylate
kinase nucleotide sequences are also encompassed by the present
invention. By "fragment" is intended a portion of the nucleotide
sequence encoding an adenylate kinase protein. A fragment of an
adenylate kinase nucleotide sequence may encode a biologically
active portion of an adenylate kinase protein, or it may be a
fragment that can be used as a hybridization probe or PCR primer
using methods disclosed below. A biologically active portion of an
adenylate kinase protein can be prepared by isolating a portion of
one of the adenylate kinase nucleotide sequences of the invention,
expressing the encoded portion of the adenylate kinase protein
(e.g., by recombinant expression in vitro), and assessing the
activity of the encoded portion of the adenylate kinase protein.
Nucleic acid molecules that are fragments of an adenylate kinase
nucleotide sequence comprise at least 15, 20, 50, 75, 100, 200,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400
nucleotides, or up to the number of nucleotides present in a
full-length adenylate kinase nucleotide sequence disclosed herein
(for example, 1434 nucleotides for SEQ ID NO:1) depending upon the
intended use.
[0155] It is understood that isolated fragments include any
contiguous sequence not disclosed prior to the invention as well as
sequences that are substantially the same and which are not
disclosed. Accordingly, if an isolated fragment is disclosed prior
to the present invention, that fragment is not intended to be
encompassed by the invention. When a sequence is not disclosed
prior to the present invention, an isolated nucleic acid fragment
is at least about 12, 15, 20, 25, or 30 contiguous nucleotides.
Other regions of the nucleotide sequence may comprise fragments of
various sizes, depending upon potential homology with previously
disclosed sequences.
[0156] For example, when considering the full-length, 1434
nucleotide transcript set forth in SEQ ID NO:1, the nucleotide
sequence from about nucleotide (nt) 1 to about nt 200 encompasses
isolated fragments greater than about 13, 15, or 20 nucleotides;
the nucleotide sequence from about nt 200 to about nt 1034
encompasses isolated fragments greater than about 102, 105, or 110
nucleotides; the nucleotide sequence from about nt 1034 to about nt
1434 encompasses isolated fragments greater than about 24, 25, or
28 nucleotides. The nucleotide sequence corresponding to the open
reading frame (nt 200-883 of SEQ ID NO:1) encompasses isolated
fragments greater than about 102, 105, or 110 nucleotides.
[0157] A fragment of an adenylate kinase nucleotide sequence that
encodes a biologically active portion of an adenylate kinase
protein of the invention will encode at least 15, 25, 30, 50, 75,
100, 125, 150, 175, 200, or 225 contiguous amino acids, or up to
the total number of amino acids present in a full-length adenylate
kinase protein of the invention (for example, 228 amino acids for
SEQ ID NO:2). Fragments of an adenylate kinase nucleotide sequence
that are useful as hybridization probes for PCR primers generally
need not encode a biologically active portion of an adenylate
kinase protein.
[0158] Nucleic acid molecules that are variants of the adenylate
kinase nucleotide sequences disclosed herein are also encompassed
by the present invention. "Variants" of the adenylate kinase
nucleotide sequences include those sequences that encode the
adenylate kinase proteins disclosed herein but that differ
conservatively because of the degeneracy of the genetic code. These
naturally occurring allelic variants can be identified with the use
of well-known molecular biology techniques, such as polymerase
chain reaction (PCR) and hybridization techniques as outlined
below. Variant nucleotide sequences also include synthetically
derived nucleotide sequences that have been generated, for example,
by using site-directed mutagenesis but which still encode the
adenylate kinase proteins disclosed in the present invention as
discussed below. Generally, nucleotide sequence variants of the
invention will have at least 45%, 55%, 65%, 75%, 85%, 95%, or 98%
identity to a particular nucleotide sequence disclosed herein. A
variant adenylate kinase nucleotide sequence will encode an
adenylate kinase protein that has an amino acid sequence having at
least 45%, 55%, 65%, 75%, 85%, 95%, or 98% identity to the amino
acid sequence of an adenylate kinase protein disclosed herein.
[0159] In addition to the adenylate kinase nucleotide sequences
shown in SEQ ID NOs:1 and 3, and the nucleotide sequence of the
cDNA of Patent Deposit Number PTA-1850, it will be appreciated by
those skilled in the art that DNA sequence polymorphisms that lead
to changes in the amino acid sequences of adenylate kinase proteins
may exist within a population (e.g., the human population). Such
genetic polymorphism in an adenylate kinase gene may exist among
individuals within a population due to natural allelic variation.
An allele is one of a group of genes that occur alternatively at a
given genetic locus. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame encoding an adenylate kinase protein, preferably
a mammalian adenylate kinase protein. As used herein, the phrase
"allelic variant" refers to a nucleotide sequence that occurs at an
adenylate kinase locus or to a polypeptide encoded by the
nucleotide sequence. Such natural allelic variations can typically
result in 1-5% variance in the nucleotide sequence of the adenylate
kinase gene. Any and all such nucleotide variations and resulting
amino acid polymorphisms or variations in an adenylate kinase
sequence that are the result of natural allelic variation and that
do not alter the functional activity of adenylate kinase proteins
are intended to be within the scope of the invention.
[0160] Moreover, nucleic acid molecules encoding adenylate kinase
proteins from other species (adenylate kinase homologues), which
have a nucleotide sequence differing from that of the adenylate
kinase sequences disclosed herein, are intended to be within the
scope of the invention. For example, nucleic acid molecules
corresponding to natural allelic variants and homologues of the
human adenylate kinase cDNA of the invention can be isolated based
on their identity to the human adenylate kinase nucleic acid
disclosed herein using the human cDNA, or a portion thereof, as a
hybridization probe according to standard hybridization techniques
under stringent hybridization conditions as disclosed below.
[0161] In addition to naturally-occurring allelic variants of the
adenylate kinase 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 the
invention thereby leading to changes in the amino acid sequence of
the encoded adenylate kinase proteins, without altering the
biological activity of the adenylate kinase proteins. Thus, an
isolated nucleic acid molecule encoding an adenylate kinase protein
having a sequence that differs from that of SEQ ID NO:2 can be
created by introducing one or more nucleotide substitutions,
additions, or deletions into the corresponding nucleotide sequence
disclosed herein, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Such
variant nucleotide sequences are also encompassed by the present
invention.
[0162] For example, preferably, conservative amino acid
substitutions may be made at one or more predicted, preferably
nonessential amino acid residues. A "nonessential" amino acid
residue is a residue that can be altered from the wild-type
sequence of an adenylate kinase protein (e.g., the sequence of SEQ
ID NO:2) without altering the biological activity, whereas an
"essential" amino acid residue is required for biological activity.
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). Such
substitutions would not be made for conserved amino acid residues,
or for amino acid residues residing within a conserved motif, such
as the adenylate kinase domain sequence of SEQ ID NO:2 (amino acid
residues 40-203), where such residues are essential for protein
activity.
[0163] Alternatively, variant adenylate kinase nucleotide sequences
can be made by introducing mutations randomly along all or part of
an adenylate kinase coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for
adenylate kinase biological activity to identify mutants that
retain activity. Following mutagenesis, the encoded protein can be
expressed recombinantly, and the activity of the protein can be
determined using standard assay techniques.
[0164] Thus the nucleotide sequences of the invention include the
sequences disclosed herein as well as fragments and variants
thereof. The adenylate kinase nucleotide sequences of the
invention, and fragments and variants thereof, can be used as
probes and/or primers to identify and/or clone adenylate kinase
homologues in other cell types, e.g., from other tissues, as well
as adenylate kinase homologues from other mammals. Such probes can
be used to detect transcripts or genomic sequences encoding the
same or identical proteins. These probes can be used as part of a
diagnostic test kit for identifying cells or tissues that
misexpress an adenylate kinase protein, such as by measuring levels
of an adenylate kinase-encoding nucleic acid in a sample of cells
from a subject, e.g., detecting adenylate kinase mRNA levels or
determining whether a genomic adenylate kinase gene has been
mutated or deleted.
[0165] In this manner, methods such as PCR, hybridization, and the
like can be used to identify such sequences having substantial
identity to the sequences of the invention. See, for example,
Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and
Innis, et al. (1990) PCR Protocols: A Guide to Methods and
Applications (Academic Press, NY). Adenylate kinase nucleotide
sequences isolated based on their sequence identity to the
adenylate kinase nucleotide sequences set forth herein or to
fragments and variants thereof are encompassed by the present
invention.
[0166] In a hybridization method, all or part of a known adenylate
kinase nucleotide sequence can be used to screen cDNA or genomic
libraries. Methods for construction of such cDNA and genomic
libraries are generally known in the art and are disclosed in
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The
so-called hybridization probes may be genomic DNA fragments, cDNA
fragments, RNA fragments, or other oligonucleotides, and may be
labeled with a detectable group such as .sup.32P, or any other
detectable marker, such as other radioisotopes, a fluorescent
compound, an enzyme, or an enzyme co-factor. Probes for
hybridization can be made by labeling synthetic oligonucleotides
based on the known adenylate kinase nucleotide sequence disclosed
herein. Degenerate primers designed on the basis of conserved
nucleotides or amino acid residues in a known adenylate kinase
nucleotide sequence or encoded amino acid sequence can additionally
be used. The probe typically comprises a region of nucleotide
sequence that hybridizes under stringent conditions to at least
about 12, preferably about 25, more preferably about 50, 75, 100,
125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides
of an adenylate kinase nucleotide sequence of the invention or a
fragment or variant thereof. Preparation of Probes for
Hybridization is Generally Known in the Art and is Disclosed in
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), herein
incorporated by reference.
[0167] For example, in one embodiment, a previously unidentified
adenylate kinase nucleic acid molecule hybridizes under stringent
conditions to a probe that is a nucleic acid molecule comprising
one of the adenylate kinase nucleotide sequences of the invention
or a fragment thereof. In another embodiment, the previously
unknown adenylate kinase nucleic acid molecule is at least 300,
325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900,
1000, 2,000, 3,000, 4,000 or 5,000 nucleotides in length and
hybridizes under stringent conditions to a probe that is a nucleic
acid molecule comprising one of the adenylate kinase nucleotide
sequences disclosed herein or a fragment thereof.
[0168] Accordingly, in another embodiment, an isolated previously
unknown adenylate kinase nucleic acid molecule of the invention is
at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650,
700, 800, 900, 1000, 1,100, 1,200, 1,300, or 1,400 nucleotides in
length and hybridizes under stringent conditions to a probe that is
a nucleic acid molecule comprising one of the nucleotide sequences
of the invention, preferably the coding sequence set forth in SEQ
ID NO:1, the cDNA of Patent Deposit Number PTA-1850, or a
complement, fragment, or variant thereof.
[0169] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences having at least 60%,
65%, 70%, preferably 75% identity 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, New York (1989)),
6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization conditions is 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 50-65.degree. C.
In another preferred embodiment, stringent conditions comprise
hybridization in 6.times.SSC at 42.degree. C., followed by washing
with 1.times.SSC at 55.degree. C. Preferably, an isolated nucleic
acid molecule that hybridizes under stringent conditions to an
adenylate kinase sequence of the invention 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).
[0170] Thus, in addition to the adenylate kinase nucleotide
sequences disclosed herein and fragments and variants thereof, the
isolated nucleic acid molecules of the invention also encompass
homologous DNA sequences identified and isolated from other cells
and/or organisms by hybridization with entire or partial sequences
obtained from the adenylate kinase nucleotide sequences disclosed
herein or variants and fragments thereof.
[0171] The present invention also encompasses antisense nucleic
acid molecules, i.e., molecules that are 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 adenylate kinase coding strand, or to
only a portion thereof, e.g., all or part of the protein coding
region (or open reading frame). An antisense nucleic acid molecule
can be antisense to a noncoding region of the coding strand of a
nucleotide sequence encoding an adenylate kinase protein. The
noncoding regions are the 5' and 3' sequences that flank the coding
region and are not translated into amino acids.
[0172] Given the coding-strand sequence encoding an adenylate
kinase protein disclosed herein (e.g., SEQ ID NO:1), 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
adenylate kinase mRNA, but more preferably is an oligonucleotide
that is antisense to only a portion of the coding or noncoding
region of adenylate kinase mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of adenylate kinase 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 procedures known in the art.
[0173] 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, including, but not limited to, for example
e.g., phosphorothioate derivatives and acridine substituted
nucleotides. 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).
[0174] 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 an adenylate kinase protein to thereby inhibit expression
of the protein, e.g., by inhibiting transcription and/or
translation. An example of a route of administration of antisense
nucleic acid molecules of the invention includes direct injection
at a tissue site. Alternatively, antisense nucleic acid molecules
can be modified to target selected cells and then administered
systemically. For example, antisense molecules can be linked to
peptides or antibodies to form a complex that specifically binds to
receptors or antigens expressed on a selected cell surface. 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.
[0175] An antisense nucleic acid molecule of the invention can be
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).
[0176] The invention also encompasses ribozymes, which are
catalytic RNA molecules with ribonuclease activity that are capable
of cleaving a single-stranded nucleic acid, such as an mRNA, to
which they have a complementary region. Ribozymes (e.g., hammerhead
ribozymes (described in Haselhoff and Gerlach (1988) Nature
334:585-591)) can be used to catalytically cleave adenylate kinase
mRNA transcripts to thereby inhibit translation of adenylate kinase
mRNA. A ribozyme having specificity for an adenylate
kinase-encoding nucleic acid can be designed based upon the
nucleotide sequence of an adenylate kinase cDNA disclosed herein
(e.g., SEQ ID NO:1). 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,
adenylate kinase mRNA can be used to select a catalytic RNA having
a specific ribonuclease activity from a pool of RNA molecules. See,
e.g., Bartel and Szostak (1993) Science 261:1411-1418.
[0177] The invention also encompasses nucleic acid molecules that
form triple helical structures. For example, adenylate kinase gene
expression can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the adenylate kinase
protein (e.g., the adenylate kinase promoter and/or enhancers) to
form triple helical structures that prevent transcription of the
adenylate kinase gene in target cells. See generally Helene (1991)
Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N.Y. Acad. Sci.
660:27; and Maher (1992) Bioassays 14(12):807.
[0178] In preferred embodiments, the nucleic acid molecules of the
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 acids can be modified to generate
peptide nucleic acids (see Hyrup et al. (1996) Bioorganic &
Medicinal Chemistry 4:5). 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 et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670.
[0179] PNAs of an adenylate kinase molecule 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, e.g., inducing transcription or
translation arrest or inhibiting replication. PNAs of the invention
can also be used, e.g., in the analysis of single base pair
mutations in a gene by, e.g., PNA-directed PCR clamping; as
artificial restriction enzymes when used in combination with other
enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as probes or
primers for DNA sequence and hybridization (Hyrup (1996), supra;
Perry-O'Keefe et al. (1996), supra).
[0180] In another embodiment, PNAs of an adenylate kinase molecule
can be modified, e.g., to enhance their stability, specificity, 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.
The synthesis of PNA-DNA chimeras can be performed as described in
Hyrup (1996), supra; Finn et al. (1996) Nucleic Acids Res.
24(17):3357-63; Mag et al. (1989) Nucleic Acids Res. 17:5973; and
Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.
II. Isolated Adenylate Kinase Proteins and Anti-Adenylate Kinase
Antibodies
[0181] Adenylate kinase proteins are also encompassed within the
present invention. By "adenylate kinase protein" is intended a
protein having the amino acid sequence set forth in SEQ ID NO: 2,
as well as fragments, biologically active portions, and variants
thereof.
[0182] "Fragments" or "biologically active portions" include
polypeptide fragments suitable for use as immunogens to raise
anti-adenylate kinase antibodies. Fragments include peptides
comprising amino acid sequences sufficiently identical to or
derived from the amino acid sequence of an adenylate kinase protein
of the invention and exhibiting at least one activity of an
adenylate kinase protein, but which include fewer amino acids than
the full-length (SEQ ID NO:2) adenylate kinase protein disclosed
herein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the adenylate kinase
protein. A biologically active portion of an adenylate kinase
protein can be a polypeptide that is, for example, 10, 25, 50, 100
or more amino acids in length. Such biologically active portions
can be prepared by recombinant techniques and evaluated for one or
more of the functional activities of a native adenylate kinase
protein. As used here, a fragment comprises at least 5 contiguous
amino acids of SEQ ID NO:2. The invention encompasses other
fragments, however, such as any fragment in the protein greater
than 6, 7, 8, or 9 amino acids, depending upon the intended
use.
[0183] By "variants" is intended proteins or polypeptides having an
amino acid sequence that is at least about 45%, 55%, 65%,
preferably about 75%, 85%, 95%, or 98% identical to the amino acid
sequence of SEQ ID NO:2. Variants also include polypeptides encoded
by the cDNA insert of the plasmid deposited with ATCC as Patent
Deposit Number PTA-1850, or polypeptides encoded by a nucleic acid
molecule that hybridizes to the nucleic acid molecule of SEQ ID
NO:1, or a complement thereof, under stringent conditions. Such
variants generally retain the functional activity of the adenylate
kinase proteins of the invention. Variants include polypeptides
that differ in amino acid sequence due to natural allelic variation
or mutagenesis.
[0184] The invention also provides adenylate kinase chimeric or
fusion proteins. As used herein, an adenylate kinase "chimeric
protein" or "fusion protein" comprises an adenylate kinase
polypeptide operably linked to a non-adenylate kinase polypeptide.
A "adenylate kinase polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to an adenylate kinase protein,
whereas a "non-adenylate kinase polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein that is not substantially identical to the adenylate kinase
protein, e.g., a protein that is different from the adenylate
kinase protein and which is derived from the same or a different
organism. Within an adenylate kinase fusion protein, the adenylate
kinase polypeptide can correspond to all or a portion of an
adenylate kinase protein, preferably at least one biologically
active portion of an adenylate kinase protein. Within the fusion
protein, the term "operably linked" is intended to indicate that
the adenylate kinase polypeptide and the non-adenylate kinase
polypeptide are fused in-frame to each other. The non-adenylate
kinase polypeptide can be fused to the N-terminus or C-terminus of
the adenylate kinase polypeptide.
[0185] One useful fusion protein is a GST-adenylate kinase fusion
protein in which the adenylate kinase sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant adenylate kinase
proteins.
[0186] In yet another embodiment, the fusion protein is an
adenylate kinase-immunoglobulin fusion protein in which all or part
of an adenylate kinase protein is fused to sequences derived from a
member of the immunoglobulin protein family. The adenylate
kinase-immunoglobulin fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject to inhibit an interaction between an adenylate kinase
ligand and an adenylate kinase protein on the surface of a cell,
thereby suppressing adenylate kinase-mediated signal transduction
in vivo. The adenylate kinase-immunoglobulin fusion proteins can be
used to affect the bioavailability of an adenylate kinase cognate
ligand. Inhibition of the adenylate kinase ligand/adenylate kinase
interaction may be useful therapeutically, both for treating
proliferative and differentiative disorders and for modulating
(e.g., promoting or inhibiting) cell survival. Moreover, the
adenylate kinase-immunoglobulin fusion proteins of the invention
can be used as immunogens to produce anti-adenylate kinase
antibodies in a subject, to purify adenylate kinase ligands, and in
screening assays to identify molecules that inhibit the interaction
of an adenylate kinase protein with an adenylate kinase ligand.
[0187] Preferably, an adenylate kinase chimeric or fusion protein
of the invention is produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different
polypeptide sequences may be ligated together in-frame, or the
fusion gene can be synthesized, such as with automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments,
which can subsequently be annealed and reamplified to generate a
chimeric gene sequence (see, e.g., Ausubel et al., eds. (1995)
Current Protocols in Molecular Biology) (Greene Publishing and
Wiley-Interscience, NY). Moreover, an adenylate kinase-encoding
nucleic acid can be cloned into a commercially available expression
vector such that it is linked in-frame to an existing fusion
moiety.
[0188] Variants of the adenylate kinase proteins can function as
either adenylate kinase agonists (mimetics) or as adenylate kinase
antagonists. Variants of the adenylate kinase protein can be
generated by mutagenesis, e.g., discrete point mutation or
truncation of the adenylate kinase protein. An agonist of the
adenylate kinase protein can retain substantially the same, or a
subset, of the biological activities of the naturally occurring
form of the adenylate kinase protein. An antagonist of the
adenylate kinase protein can inhibit one or more of the activities
of the naturally occurring form of the adenylate kinase protein by,
for example, competitively binding to a downstream or upstream
member of a cellular signaling cascade that includes the adenylate
kinase protein. Thus, specific biological effects can be elicited
by treatment with a variant of limited function. Treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein can have fewer side
effects in a subject relative to treatment with the naturally
occurring form of the adenylate kinase proteins.
[0189] Variants of an adenylate kinase protein that function as
either adenylate kinase agonists or as adenylate kinase antagonists
can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of an adenylate kinase protein for
adenylate kinase protein agonist or antagonist activity. In one
embodiment, a variegated library of adenylate kinase variants is
generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of adenylate kinase variants can be produced by, for example,
enzymatically ligating a mixture of synthetic oligonucleotides into
gene sequences such that a degenerate set of potential adenylate
kinase sequences is expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage
display) containing the set of adenylate kinase sequences therein.
There are a variety of methods that can be used to produce
libraries of potential adenylate kinase 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 adenylate kinase sequences. Methods for synthesizing
degenerate oligonucleotides are known in the art (see, e.g., Narang
(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).
[0190] In addition, libraries of fragments of an adenylate kinase
protein coding sequence can be used to generate a variegated
population of adenylate kinase fragments for screening and
subsequent selection of variants of an adenylate kinase protein. In
one embodiment, a library of coding sequence fragments can be
generated by treating a double-stranded PCR fragment of an
adenylate kinase 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, one can derive
an expression library that encodes N-terminal and internal
fragments of various sizes of the adenylate kinase protein.
[0191] 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 adenylate kinase 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 technique that enhances the frequency of
functional mutants in the libraries, can be used in combination
with the screening assays to identify adenylate kinase variants
(Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;
Delgrave et al. (1993) Protein Engineering 6(3):327-331).
[0192] An isolated adenylate kinase polypeptide of the invention
can be used as an immunogen to generate antibodies that bind
adenylate kinase proteins using standard techniques for polyclonal
and monoclonal antibody preparation. The full-length adenylate
kinase protein can be used or, alternatively, the invention
provides antigenic peptide fragments of adenylate kinase proteins
for use as immunogens. The antigenic peptide of an adenylate kinase
protein comprises at least 8, preferably 10, 15, 20, or 30 amino
acid residues of the amino acid sequence shown in SEQ ID NO:2 and
encompasses an epitope of an adenylate kinase protein such that an
antibody raised against the peptide forms a specific immune complex
with the adenylate kinase protein. Preferred epitopes encompassed
by the antigenic peptide are regions of a adenylate kinase protein
that are located on the surface of the protein, e.g., hydrophilic
regions.
[0193] Accordingly, another aspect of the invention pertains to
anti-adenylate kinase polyclonal and monoclonal antibodies that
bind an adenylate kinase protein. Polyclonal anti-adenylate kinase
antibodies can be prepared by immunizing a suitable subject (e.g.,
rabbit, goat, mouse, or other mammal) with an adenylate kinase
immunogen. The anti-adenylate kinase 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 adenylate kinase protein. At an
appropriate time after immunization, e.g., when the anti-adenylate
kinase 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, the
human B cell hybridoma technique (Kozbor et al. (1983) Immunol.
Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) in
Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell
(Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or trioma
techniques. The technology for producing hybridomas is well known
(see generally Coligan et al., eds. (1994) Current Protocols in
Immunology (John Wiley & Sons, Inc., New York, N.Y.); Galfre et
al. (1977) Nature 266:55052; Kenneth (1980) in Monoclonal
Antibodies: A New Dimension In Biological Analyses (Plenum
Publishing Corp., NY; and Lerner (1981) Yale J. Biol. Med.,
54:387-402).
[0194] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-adenylate kinase antibody can be
identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with an adenylate kinase protein to thereby isolate immunoglobulin
library members that bind the adenylate kinase protein. 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.theta. 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,
U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO
91/17271; WO 92/20791; WO 92/15679; 93/01288; WO 92/01047;
92/09690; and 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.
[0195] Additionally, recombinant anti-adenylate kinase antibodies,
such as chimeric and humanized monoclonal antibodies, comprising
both human and nonhuman 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 PCT Publication Nos. WO 86101533 and WO
87/02671; European Patent Application Nos. 184,187, 171,496,
125,023, and 173,494; U.S. Pat. Nos. 4,816,567 and 5,225,539;
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; Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al.
(1986) Bio/Techniques 4:214; 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.
[0196] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice that are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. See, for example,
Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93); and U.S.
Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and
5,545,806. In addition, companies such as Abgenix, Inc. (Freemont,
Calif.), can be engaged to provide human antibodies directed
against a selected antigen using technology similar to that
described above.
[0197] Completely human antibodies that recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide the selection
of a completely human antibody recognizing the same epitope. This
technology is described by Jespers et al. (1994) Bio/Technology
12:899-903).
[0198] An anti-adenylate kinase antibody (e.g., monoclonal
antibody) can be used to isolate adenylate kinase proteins by
standard techniques, such as affinity chromatography or
immunoprecipitation. An anti-adenylate kinase antibody can
facilitate the purification of natural adenylate kinase protein
from cells and of recombinantly produced adenylate kinase protein
expressed in host cells. Moreover, an anti-adenylate kinase
antibody can be used to detect adenylate kinase protein (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the adenylate kinase
protein. Anti-adenylate kinase 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 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.
[0199] 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,l-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). 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.
[0200] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al. (1985)
"Monoclonal Antibodies for Immunotargeting of Drugs in Cancer
Therapy," in Monoclonal Antibodies And Cancer Therapy, ed. Reisfeld
et al. (Alan R. Liss, Inc.), pp. 243-56); Hellstrom et al. (1987)
"Antibodies for Drug Delivery," in Controlled Drug Delivery, ed.
Robinson et al. (2d ed., Marcel Dekker, Inc.), pp. 623-53; Thorpe
(1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A
Review", in Monoclonal Antibodies '84:Biological And Clinical
Applications, ed. Pinchera et al., pp. 475-506; "Analysis, Results,
and Future Prospective of the Therapeutic Use of Radiolabeled
Antibody in Cancer Therapy," in Monoclonal Antibodies For Cancer
Detection And Therapy, ed. Baldwin et al. (Academic Press, NY), pp.
303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-58.
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.
III. Recombinant Expression Vectors and Host Cells
[0201] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an adenylate kinase protein (or a portion thereof). "Vector" refers
to a nucleic acid molecule capable of transporting another nucleic
acid to which it has been linked, such as a "plasmid", a circular
double-stranded DNA loop into which additional DNA segments can be
ligated, or a viral vector, where additional DNA segments can be
ligated into the viral genome. The vectors are useful for
autonomous replication in a host cell or may be integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome (e.g.,
nonepisomal mammalian vectors). Expression vectors are capable of
directing the expression of genes to which they are operably
linked. In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids (vectors).
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), that serve equivalent functions.
[0202] 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. This 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,
operably linked to the nucleic acid sequence to be expressed.
"Operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that 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). See,
for example, Goeddel (1990) in Gene Expression Technology: Methods
in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory
sequences include those that direct constitutive expression of a
nucleotide sequence in many types of host cell and those that
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, etc. 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., adenylate kinase proteins, mutant forms of
adenylate kinase proteins, fusion proteins, etc.).
[0203] The recombinant expression vectors of the invention can be
designed for expression of adenylate kinase protein in prokaryotic
or eukaryotic host cells. 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 nonfusion proteins. Fusion vectors add a number of
amino acids to a protein encoded therein, usually to the amino
terminus of the recombinant protein. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson
(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. Examples of
suitable inducible nonfusion E. coli expression vectors include
pTrc (Amann et al. (1988) Gene 69:301-315) and pET Id (Studier et
al. (1990) in Gene Expression Technology: Methods in Enzymology 185
(Academic Press, San Diego, Calif.), pp. 60-89). Strategies to
maximize recombinant protein expression in E. coli can be found in
Gottesman (1990) in Gene Expression Technology: Methods in
Enzymology 185 (Academic Press, CA), pp. 119-128 and Wada et al.
(1992) Nucleic Acids Res. 20:2111-2118. Target gene expression from
the pTrc vector relies on host RNA polymerase transcription from a
hybrid trp-lac fusion promoter.
[0204] Suitable eukaryotic host cells include insect cells
(examples of Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf9 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)); yeast
cells (examples of vectors for expression in yeast S. cereivisiae
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 pPicZ (Invitrogen Corporation, San Diego,
Calif.)); or mammalian cells (mammalian expression vectors include
pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al.
(1987) EMBO J. 6:187:195)). Suitable mammalian cells include
Chinese hamster ovary cells (CHO) or COS cells. 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 et al. (1989) Molecular cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See,
Goeddel (1990) in Gene Expression Technology: Methods in Enzymology
185 (Academic Press, San Diego, Calif.). Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0205] 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.
[0206] In one embodiment, the expression vector is a recombinant
mammalian expression vector that comprises tissue-specific
regulatory elements that direct expression of the nucleic acid
preferentially in a particular cell type. 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 (Baneji 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 Patent
Publication No. 264,166). Developmentally-regulated promoters are
also encompassed, for example the murine hox promoters (Kessel and
Gruss (1990) Science 249:374-379), the .alpha.-fetoprotein promoter
(Campes and Tilghman (1989) Genes Dev. 3:537-546), and the
like.
[0207] 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 operably linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to adenylate kinase mRNA.
Regulatory sequences operably linked to a nucleic acid cloned in
the antisense orientation can be chosen to 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 to 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 et al. (1986)
Reviews--Trends in Genetics, Vol. 1(1).
[0208] 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. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.) and other laboratory manuals.
[0209] 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.,
for 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 an adenylate kinase 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).
[0210] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) adenylate kinase protein. Accordingly, the invention
further provides methods for producing adenylate kinase 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 an adenylate kinase
protein has been introduced, in a suitable medium such that
adenylate kinase protein is produced. In another embodiment, the
method further comprises isolating adenylate kinase protein from
the medium or the host cell.
[0211] The host cells of the invention can also be used to produce
nonhuman transgenic animals. In general, methods for producing
transgenic animals include introducing a nucleic acid sequence
according to the present invention, the nucleic acid sequence
capable of expressing the receptor protein in a transgenic animal,
into a cell in culture or in vivo. When introduced in vivo, the
nucleic acid is introduced into an intact organism such that one or
more cell types and, accordingly, one or more tissue types, express
the nucleic acid encoding the receptor protein. Alternatively, the
nucleic acid can be introduced into virtually all cells in an
organism by transfecting a cell in culture, such as an embryonic
stem cell, as described herein for the production of transgenic
animals, and this cell can be used to produce an entire transgenic
organism. As described, in a further embodiment, the host cell can
be a fertilized oocyte. Such cells are then allowed to develop in a
female foster animal to produce the transgenic organism.
[0212] For example, in one embodiment, a host cell of the invention
is a fertilized oocyte or an embryonic stem cell into which
adenylate kinase-coding sequences have been introduced. Such host
cells can then be used to create nonhuman transgenic animals in
which exogenous adenylate kinase sequences have been introduced
into their genome or homologous recombinant animals in which
endogenous adenylate kinase sequences have been altered. Such
animals are useful for studying the function and/or activity of
adenylate kinase genes and proteins and for identifying and/or
evaluating modulators of adenylate kinase activity. As used herein,
a "transgenic animal" is a nonhuman 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 nonhuman primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A transgene is
exogenous DNA that 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 nonhuman animal, preferably a mammal, more preferably
a mouse, in which an endogenous adenylate kinase 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.
[0213] A transgenic animal of the invention can be created by
introducing adenylate kinase-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 adenylate kinase cDNA
sequence can be introduced as a transgene into the genome of a
nonhuman animal. Alternatively, a homologue of the mouse adenylate
kinase gene can be isolated based on hybridization 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 the adenylate kinase
transgene to direct expression of adenylate kinase 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, 4,870,009, and 4,873,191 and
in Hogan (1986) 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 the
adenylate kinase transgene in its genome and/or expression of
adenylate kinase 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 adenylate kinase gene can further be
bred to other transgenic animals carrying other transgenes.
[0214] To create a homologous recombinant animal, one prepares a
vector containing at least a portion of an adenylate kinase gene or
a homolog of the gene into which a deletion, addition, or
substitution has been introduced to thereby alter, e.g.,
functionally disrupt, the adenylate kinase gene. In a preferred
embodiment, the vector is designed such that, upon homologous
recombination, the endogenous adenylate kinase gene is functionally
disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock out" vector). Alternatively, the vector can
be designed such that, upon homologous recombination, the
endogenous adenylate kinase 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 adenylate kinase protein). In the homologous
recombination vector, the altered portion of the adenylate kinase
gene is flanked at its 5N and 3N ends by additional nucleic acid of
the adenylate kinase gene to allow for homologous recombination to
occur between the exogenous adenylate kinase gene carried by the
vector and an endogenous adenylate kinase gene in an embryonic stem
cell. The additional flanking adenylate kinase nucleic acid 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 vector (see, e.g.,
Thomas and Capecchi (1987) Cell 51:503 for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation), and cells in
which the introduced adenylate kinase gene has homologously
recombined with the endogenous adenylate kinase gene are selected
(see, e.g., Li et al. (1992) Cell 69:915). The selected cells are
then injected into a blastocyst of an animal (e.g., a mouse) to
form aggregation chimeras (see, e.g., Bradley (1987) in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,
ed. Robertson (IRL, Oxford), 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
vectors and homologous recombinant animals are described further in
Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in
PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169.
[0215] In another embodiment, transgenic nonhuman animals
containing selected systems that allow for regulated expression of
the transgene can be produced. 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.
[0216] Clones of the nonhuman transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813 and PCT Publication Nos. WO
97/07668 and WO 97/07669.
IV. Pharmaceutical Compositions
[0217] The adenylate kinase nucleic acid molecules, adenylate
kinase proteins, and anti-adenylate kinase 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.
[0218] The compositions of the invention are useful to treat any of
the disorders discussed herein. The compositions are provided in
therapeutically effective amounts. By "therapeutically effective
amounts" is intended an amount sufficient to modulate the desired
response. 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.
[0219] 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. 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.
[0220] The present invention encompasses agents that 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.
[0221] 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. 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.
[0222] 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.
[0223] 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 dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.RTM. (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 polyethylene 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 mannitol, sorbitol, sodium chloride, in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and
gelatin.
[0224] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an adenylate kinase
protein or anti-adenylate kinase 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 that 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.
[0225] 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. For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser that contains a suitable propellant, e.g., a
gas such as carbon dioxide, or a nebulizer.
[0226] 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. 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.
[0227] 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.
[0228] 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 with each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. Depending on the type and severity of the
disease, about 1 .mu.g/kg to about 15 mg/kg (e.g., 0.1 to 20 mg/kg)
of antibody is an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to about 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. However, other dosage regimens may be
useful. The progress of this therapy is easily monitored by
conventional techniques and assays. An exemplary dosing regimen is
disclosed in WO 94/04188. 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.
[0229] 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 (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.
[0230] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0231] 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.
[0232] 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. 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.
[0233] 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.
[0234] 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. 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.
Computer Readable Means
[0235] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0236] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0237] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0238] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and MicroSoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0239] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0240] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0241] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0242] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software includes, but is not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0243] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
V. Uses and Methods of the Invention
[0244] 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) detection assays
(e.g., chromosomal mapping, tissue typing, forensic biology); (c)
predictive medicine (e.g., diagnostic assays, prognostic assays,
monitoring clinical trials, and pharmacogenomics); and (d) methods
of treatment (e.g., therapeutic and prophylactic). The isolated
nucleic acid molecules of the invention can be used to express
adenylate kinase protein (e.g., via a recombinant expression vector
in a host cell in gene therapy applications), to detect adenylate
kinase mRNA (e.g., in a biological sample) or a genetic lesion in
an adenylate kinase gene, and to modulate adenylate kinase
activity. In addition, the adenylate kinase proteins can be used to
screen drugs or compounds that modulate the immune response as well
as to treat disorders characterized by insufficient or excessive
production of adenylate kinase protein or production of adenylate
kinase protein forms that have decreased or aberrant activity
compared to adenylate kinase wild type protein. In addition, the
anti-adenylate kinase antibodies of the invention can be used to
detect and isolate adenylate kinase proteins and modulate adenylate
kinase activity.
[0245] A. Screening Assays
[0246] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules, or other drugs) that bind to adenylate kinase proteins
or have a stimulatory or inhibitory effect on, for example,
adenylate kinase expression or adenylate kinase activity.
[0247] 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, nonpeptide oligomer, or small molecule
libraries of compounds (Lam (1997) Anticancer Drug Des.
12:145).
[0248] 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. USA 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 Gallop et al. (1994) J. Med. Chem.
37:1233.
[0249] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos.
5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992)
Proc. Natl. Acad. Sci. USA 89:1865-1869), or phage (Scott and Smith
(1990) Science 249:386-390; Devlin (1990) Science 249:404-406;
Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and
Felici (1991) J. Mol. Biol. 222:301-310).
[0250] Determining the ability of the test compound to bind to the
adenylate kinase protein can be accomplished, for example, by
coupling the test compound with a radioisotope or enzymatic label
such that binding of the test compound to the adenylate kinase
protein or biologically active portion thereof can be determined by
detecting the labeled compound in a complex. For example, test
compounds 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, test 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.
[0251] In a similar manner, one may determine the ability of the
adenylate kinase protein to bind to or interact with an adenylate
kinase target molecule. By "target molecule" is intended a molecule
with which an adenylate kinase protein binds or interacts in
nature. In a preferred embodiment, the ability of the adenylate
kinase protein to bind to or interact with an adenylate kinase
target molecule can be determined by monitoring the activity of the
target molecule. For example, the activity of the target molecule
can be monitored by detecting induction of a cellular second
messenger of the target (e.g., intracellular Ca.sup.2+,
diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity
of the target on an appropriate substrate, detecting the induction
of a reporter gene (e.g., an adenylate kinase-responsive regulatory
element operably linked to a nucleic acid encoding a detectable
marker, e.g. luciferase), or detecting a cellular response, for
example, cellular differentiation or cell proliferation.
[0252] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting an adenylate kinase
protein or biologically active portion thereof with a test compound
and determining the ability of the test compound to bind to the
adenylate kinase protein or biologically active portion thereof.
Binding of the test compound to the adenylate kinase protein can be
determined either directly or indirectly as described above. In a
preferred embodiment, the assay includes contacting the adenylate
kinase protein or biologically active portion thereof with a known
compound that binds adenylate kinase protein to form an assay
mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to preferentially bind
to adenylate kinase protein or biologically active portion thereof
as compared to the known compound.
[0253] In another embodiment, an assay is a cell-free assay
comprising contacting adenylate kinase protein or biologically
active portion thereof with a test compound and determining the
ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the adenylate kinase protein or
biologically active portion thereof. Determining the ability of the
test compound to modulate the activity of an adenylate kinase
protein can be accomplished, for example, by determining the
ability of the adenylate kinase protein to bind to an adenylate
kinase target molecule as described above for determining direct
binding. In an alternative embodiment, determining the ability of
the test compound to modulate the activity of an adenylate kinase
protein can be accomplished by determining the ability of the
adenylate kinase protein to further modulate an adenylate kinase
target molecule. For example, the catalytic/enzymatic activity of
the target molecule on an appropriate substrate can be determined
as previously described.
[0254] In yet another embodiment, the cell-free assay comprises
contacting the adenylate kinase protein or biologically active
portion thereof with a known compound that binds an adenylate
kinase protein to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the
test compound to preferentially bind to or modulate the activity of
an adenylate kinase target molecule.
[0255] In the above-mentioned assays, it may be desirable to
immobilize either an adenylate kinase protein 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. In one embodiment, a fusion protein can be
provided that adds a domain that allows one or both of the proteins
to be bound to a matrix. For example,
glutathione-S-transferase/adenylate kinase fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione-derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
nonadsorbed target protein or adenylate kinase 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 and complex formation is measured
either directly or indirectly, for example, as described above.
Alternatively, the complexes can be dissociated from the matrix,
and the level of adenylate kinase binding or activity determined
using standard techniques.
[0256] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either adenylate kinase protein or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated adenylate kinase molecules or target molecules can be
prepared from biotin-NHS (N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96-well plates (Pierce Chemicals).
Alternatively, antibodies reactive with an adenylate kinase protein
or target molecules but which do not interfere with binding of the
adenylate kinase protein to its target molecule can be derivatized
to the wells of the plate, and unbound target or adenylate kinase
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 adenylate kinase protein or
target molecule, as well as enzyme-linked assays that rely on
detecting an enzymatic activity associated with the adenylate
kinase protein or target molecule.
[0257] In another embodiment, modulators of adenylate kinase
expression are identified in a method in which a cell is contacted
with a candidate compound and the expression of adenylate kinase
mRNA or protein in the cell is determined relative to expression of
adenylate kinase mRNA or protein in a cell in the absence of the
candidate compound. When expression 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 adenylate kinase mRNA or protein expression.
Alternatively, when expression 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
adenylate kinase mRNA or protein expression. The level of adenylate
kinase mRNA or protein expression in the cells can be determined by
methods described herein for detecting adenylate kinase mRNA or
protein.
[0258] In yet another aspect of the invention, the adenylate kinase
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) Bio/Techniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication
No. WO 94/10300), to identify other proteins, which bind to or
interact with adenylate kinase protein ("adenylate kinase-binding
proteins" or "adenylate kinase-bp") and modulate adenylate kinase
activity. Such adenylate kinase-binding proteins are also likely to
be involved in the propagation of signals by the adenylate kinase
proteins as, for example, upstream or downstream elements of the
adenylate kinase pathway.
[0259] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein. Accordingly the invention is
directed to agents that modulate the level or activity of the
polypeptide or nucleic acid of the invention, the agents being
identified by screening cells, tissues, cell extracts, or tissue
extracts with the agents. Agents that alter the level or activity
can then be tested further for clinical diagnostic or therapeutic
use. Any method of screening that allows expression to be measured,
such as those disclosed herein, are relevant to produce the
identification of these agents. Thus, the invention is directed to
agents identified by the screening processes involving measuring or
detecting expression (level or activity) of the polypeptides or
nucleic acids of the invention. It is understood that agents
affecting the ability of the protein or nucleic acid to interact
with a cellular component, as in competition binding, would be
construed as affecting expression. Accordingly, screening processes
also include assays for agents that themselves bind to the protein
or nucleic acid of the invention, such as those disclosed
herein.
[0260] B. Detection Assays
[0261] 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: (1) map their respective genes on a
chromosome; (2) identify an individual from a minute biological
sample (tissue typing); and (3) aid in forensic identification of a
biological sample. These applications are described in the
subsections below.
[0262] 1. Chromosome Mapping
[0263] The isolated complete or partial adenylate kinase gene
sequences of the invention can be used to map their respective
adenylate kinase genes on a chromosome, thereby facilitating the
location of gene regions associated with genetic disease. Computer
analysis of adenylate kinase sequences can be used to rapidly
select PCR primers (preferably 15-25 bp in length) that do not span
more than one exon in the genomic DNA, thereby simplifying 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 adenylate kinase sequences will yield an
amplified fragment.
[0264] 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 in which 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 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.
[0265] Other mapping strategies that can similarly be used to map
an adenylate kinase sequence to its chromosome include in situ
hybridization (described in Fan 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. Furthermore, fluorescence in situ
hybridization (FISH) of a DNA sequence to a metaphase chromosomal
spread can be used to provide a precise chromosomal location in one
step. For a review of this technique, see Verma eta a. (1988) Human
Chromosomes: A Manual of Basic Techniques (Pergamon Press, NY). 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 in a reasonable amount of time.
[0266] 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.
[0267] 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 genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g.,
Egeland et al. (1987) Nature 325:783-787.
[0268] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the adenylate kinase 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.
[0269] 2. Tissue Typing
[0270] The adenylate kinase 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.
The sequences of the present invention are useful as additional DNA
markers for RFLP (described in U.S. Pat. No. 5,272,057).
[0271] Furthermore, the sequences of the present invention can be
used to provide an alternative technique for determining the actual
base-by-base DNA sequence of selected portions of an individual's
genome. Thus, the adenylate kinase sequences of the invention can
be used to prepare two PCR primers from the 5N and 3N ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0272] 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 adenylate kinase
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. The noncoding
sequences of SEQ ID NO:1 can comfortably provide positive
individual identification with a panel of perhaps 10 to 1,000
primers that each yield a noncoding amplified sequence of 100
bases. If a predicted coding sequence, such as that in SEQ ID NO:1,
is used, a more appropriate number of primers for positive
individual identification would be 500 to 2,000.
[0273] 3. Use of Partial Adenylate Kinase Sequences in Forensic
Biology
[0274] DNA-based identification techniques can also be used in
forensic biology. In this manner, 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.
[0275] 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" that is unique to a
particular individual. As mentioned above, actual base sequence
information can be used for identification as an accurate
alternative to patterns formed by restriction enzyme generated
fragments. Sequences targeted to noncoding regions of SEQ ID NO:1
are particularly appropriate for this use as greater numbers of
polymorphisms occur in the noncoding regions, making it easier to
differentiate individuals using this technique. Examples of
polynucleotide reagents include the adenylate kinase sequences or
portions thereof, e.g., fragments derived from the noncoding
regions of SEQ ID NO:1 having a length of at least 20 or 30
bases.
[0276] The adenylate kinase sequences described herein can further
be used to provide polynucleotide reagents, e.g., labeled or
labelable probes that can be used in, for example, an in situ
hybridization technique, to identify a specific tissue. This can be
very useful in cases where a forensic pathologist is presented with
a tissue of unknown origin. Panels of such adenylate kinase probes,
can be used to identify tissue by species and/or by organ type.
[0277] In a similar fashion, these reagents, e.g., adenylate kinase
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).
[0278] C. Predictive Medicine
[0279] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. These applications are described in the
subsections below.
[0280] 1. Diagnostic Assays
[0281] One aspect of the present invention relates to diagnostic
assays for detecting adenylate kinase protein and/or nucleic acid
expression as well as adenylate kinase activity, in the context of
a biological sample. An exemplary method for detecting the presence
or absence of adenylate kinase proteins 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 adenylate kinase protein or nucleic acid
(e.g., mRNA, genomic DNA) that encodes adenylate kinase protein
such that the presence of adenylate kinase protein is detected in
the biological sample. Results obtained with a biological sample
from the test subject may be compared to results obtained with a
biological sample from a control subject.
[0282] A preferred agent for detecting adenylate kinase mRNA or
genomic DNA is a labeled nucleic acid probe capable of hybridizing
to adenylate kinase mRNA or genomic DNA. The nucleic acid probe can
be, for example, a full-length adenylate kinase nucleic acid, such
as the nucleic acid of SEQ ID NO:1, or a portion thereof, such as a
nucleic acid molecule of at least 15, 30, 50, 100, 250, or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to adenylate kinase mRNA or genomic DNA.
Other suitable probes for use in the diagnostic assays of the
invention are described herein.
[0283] A preferred agent for detecting adenylate kinase protein is
an antibody capable of binding to adenylate kinase 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(abN).sub.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.
[0284] 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 adenylate
kinase mRNA, protein, or genomic DNA in a biological sample in
vitro as well as in vivo. For example, in vitro techniques for
detection of adenylate kinase mRNA include Northern hybridizations
and in situ hybridizations. In vitro techniques for detection of
adenylate kinase protein include enzyme linked immunosorbent assays
(ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of adenylate
kinase genomic DNA include Southern hybridizations. Furthermore, in
vivo techniques for detection of adenylate kinase protein include
introducing into a subject a labeled anti-adenylate kinase
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.
[0285] 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 peripheral blood leukocyte sample isolated by conventional
means from a subject.
[0286] The invention also encompasses kits for detecting the
presence of adenylate kinase proteins in a biological sample (a
test sample). Such kits can be used to determine if a subject is
suffering from or is at increased risk of developing a disorder
associated with aberrant expression of adenylate kinase protein
(e.g., an immunological disorder). For example, the kit can
comprise a labeled compound or agent capable of detecting adenylate
kinase protein or mRNA in a biological sample and means for
determining the amount of an adenylate kinase protein in the sample
(e.g., an anti-adenylate kinase antibody or an oligonucleotide
probe that binds to DNA encoding an adenylate kinase protein, e.g.,
SEQ ID NO:1). Kits can also include instructions for observing that
the tested subject is suffering from or is at risk of developing a
disorder associated with aberrant expression of adenylate kinase
sequences if the amount of adenylate kinase protein or mRNA is
above or below a normal level.
[0287] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) that binds
to adenylate kinase protein; and, optionally, (2) a second,
different antibody that binds to adenylate kinase protein or the
first antibody and is conjugated to a detectable agent. For
oligonucleotide-based kits, the kit can comprise, for example: (1)
an oligonucleotide, e.g., a detectably labeled oligonucleotide,
that hybridizes to an adenylate kinase nucleic acid sequence or (2)
a pair of primers useful for amplifying an adenylate kinase nucleic
acid molecule.
[0288] The kit can also comprise, e.g., a buffering agent, a
preservative, or a protein stabilizing agent. The kit can also
comprise components necessary for detecting the detectable agent
(e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples that can be assayed
and compared to the test sample contained. Each component of the
kit is usually enclosed within an individual container, and all of
the various containers are within a single package along with
instructions for observing whether the tested subject is suffering
from or is at risk of developing a disorder associated with
aberrant expression of adenylate kinase proteins.
[0289] 2. Prognostic Assays
[0290] The methods described herein can furthermore be utilized as
diagnostic or prognostic assays to identify subjects having or at
risk of developing a disease or disorder associated with adenylate
kinase protein, adenylate kinase nucleic acid expression, or
adenylate kinase activity. Prognostic assays can be used for
prognostic or predictive purposes to thereby prophylactically treat
an individual prior to the onset of a disorder characterized by or
associated with adenylate kinase protein, adenylate kinase nucleic
acid expression, or adenylate kinase activity.
[0291] Thus, the present invention provides a method in which a
test sample is obtained from a subject, and adenylate kinase
protein or nucleic acid (e.g., mRNA, genomic DNA) is detected,
wherein the presence of adenylate kinase protein or nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant adenylate kinase 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., serum), cell sample, or
tissue.
[0292] Furthermore, using the prognostic assays described herein,
the present invention provides methods for determining whether a
subject can be administered a specific agent (e.g., an agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate) or class of agents (e.g., agents
of a type that decrease adenylate kinase activity) to effectively
treat a disease or disorder associated with aberrant adenylate
kinase expression or activity. In this manner, a test sample is
obtained and adenylate kinase protein or nucleic acid is detected.
The presence of adenylate kinase protein or nucleic acid is
diagnostic for a subject that can be administered the agent to
treat a disorder associated with aberrant adenylate kinase
expression or activity.
[0293] The methods of the invention can also be used to detect
genetic lesions or mutations in an adenylate kinase gene, thereby
determining if a subject with the lesioned gene is at risk for a
disorder characterized by aberrant cell proliferation and/or
differentiation. In preferred embodiments, the methods include
detecting, in a sample of cells from the subject, the presence or
absence of a genetic lesion or mutation characterized by at least
one of an alteration affecting the integrity of a gene encoding an
adenylate kinase-protein, or the misexpression of the adenylate
kinase gene. For example, such genetic lesions or mutations can be
detected by ascertaining the existence of at least one of: (1) a
deletion of one or more nucleotides from an adenylate kinase gene;
(2) an addition of one or more nucleotides to an adenylate kinase
gene; (3) a substitution of one or more nucleotides of an adenylate
kinase gene; (4) a chromosomal rearrangement of an adenylate kinase
gene; (5) an alteration in the level of a messenger RNA transcript
of an adenylate kinase gene; (6) an aberrant modification of an
adenylate kinase 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 an adenylate kinase gene; (8) a
non-wild-type level of an adenylate kinase-protein; (9) an allelic
loss of an adenylate kinase gene; and (10) an inappropriate
post-translational modification of an adenylate kinase-protein. As
described herein, there are a large number of assay techniques
known in the art that can be used for detecting lesions in an
adenylate kinase gene. Any cell type or tissue, preferably
peripheral blood leukocytes, in which adenylate kinase proteins are
expressed may be utilized in the prognostic assays described
herein.
[0294] In certain embodiments, detection of the lesion 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 the adenylate kinase gene (see, e.g., Abravaya et al.
(1995) Nucleic Acids Res. 23:675-682). 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.
[0295] Alternative amplification methods include self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[0296] In an alternative embodiment, mutations in an adenylate
kinase gene from a sample cell can be identified by alterations in
restriction enzyme cleavage patterns of isolated test sample and
control DNA digested with one or more restriction endonucleases.
Moreover, the use of sequence specific ribozymes (see, e.g., 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.
[0297] In other embodiments, genetic mutations in an adenylate
kinase molecule 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
et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature
Medicine 2:753-759). In yet another embodiment, any of a variety of
sequencing reactions known in the art can be used to directly
sequence the adenylate kinase gene and detect mutations by
comparing the sequence of the sample adenylate kinase gene with the
corresponding wild-type (control) sequence. Examples of sequencing
reactions include those based on techniques developed by Maxim 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) Bio/Techniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT 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).
[0298] Other methods for detecting mutations in the adenylate
kinase 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). See, also
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.
[0299] In still another embodiment, the mismatch cleavage reaction
employs one or more "DNA mismatch repair" enzymes that recognize
mismatched base pairs in double-stranded DNA in defined systems for
detecting and mapping point mutations in adenylate kinase cDNAs
obtained from samples of cells. See, e.g., Hsu et al. (1994)
Carcinogenesis 15:1657-1662. According to an exemplary embodiment,
a probe based on an adenylate kinase sequence, e.g., a wild-type
adenylate kinase 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, e.g.,
U.S. Pat. No. 5,459,039.
[0300] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in adenylate kinase
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; Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
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).
[0301] 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).
[0302] 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 that 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.
[0303] 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.
[0304] The methods described herein may be performed, for example,
by utilizing prepackaged 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 diagnosed
patients exhibiting symptoms or family history of a disease or
illness involving an adenylate kinase gene.
[0305] 3. Pharmacogenomics
[0306] Agents, or modulators that have a stimulatory or inhibitory
effect on adenylate kinase activity (e.g., adenylate kinase gene
expression) as identified by a screening assay described herein,
can be administered to individuals to treat (prophylactically or
therapeutically) disorders associated with aberrant adenylate
kinase activity as well as to modulate the phenotype of an immune
response. In conjunction with such treatment, the pharmacogenomics
(i.e., the study of the relationship between an individual's
genotype and that individual's response to a foreign compound or
drug) of the individual 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, the
pharmacogenomics of the individual permits the selection of
effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based on a consideration of the individual's genotype.
Such pharmacogenomics can further be used to determine appropriate
dosages and therapeutic regimens. Accordingly, the activity of
adenylate kinase protein, expression of adenylate kinase nucleic
acid, or mutation content of adenylate kinase genes in an
individual can be determined to thereby select appropriate agent(s)
for therapeutic or prophylactic treatment of the individual.
[0307] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, e.g.,
Linder (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 are referred to as "altered drug action." Genetic
conditions transmitted as single factors altering the way the body
acts on drugs are referred to as "altered drug metabolism". These
pharmacogenetic conditions can occur either as rare defects or as
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 (antimalarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0308] 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, a PM will 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.
[0309] Thus, the activity of adenylate kinase protein, expression
of adenylate kinase nucleic acid, or mutation content of adenylate
kinase genes in an individual can be determined to thereby select
appropriate agent(s) for therapeutic or prophylactic treatment of
the individual. In addition, pharmacogenetic studies can be used to
apply genotyping of polymorphic alleles encoding drug-metabolizing
enzymes to the identification of an individual's drug
responsiveness phenotype. 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 an adenylate kinase modulator, such as a
modulator identified by one of the exemplary screening assays
described herein.
[0310] 4. Monitoring of Effects During Clinical Trials
[0311] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of adenylate kinase genes (e.g., the
ability to modulate aberrant cell proliferation and/or
differentiation) can be applied not only in basic drug screening
but also in clinical trials. For example, the effectiveness of an
agent, as determined by a screening assay as described herein, to
increase or decrease adenylate kinase gene expression, protein
levels, or protein activity, can be monitored in clinical trials of
subjects exhibiting decreased or increased adenylate kinase gene
expression, protein levels, or protein activity. In such clinical
trials, adenylate kinase expression or activity and preferably that
of other genes that have been implicated in for example, a cellular
proliferation disorder, can be used as a marker of the immune
responsiveness of a particular cell.
[0312] For example, and not by way of limitation, genes that are
modulated in cells by treatment with an agent (e.g., compound,
drug, or small molecule) that modulates adenylate kinase activity
(e.g., as identified in a screening assay described herein) can be
identified. Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of adenylate kinase genes and other genes implicated in
the disorder. The levels of gene expression (i.e., 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 adenylate kinase
genes 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.
[0313] 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)
comprising the steps of (1) obtaining a preadministration sample
from a subject prior to administration of the agent; (2) detecting
the level of expression of an adenylate kinase protein, mRNA, or
genomic DNA in the preadministration sample; (3) obtaining one or
more postadministration samples from the subject; (4) detecting the
level of expression or activity of the adenylate kinase protein,
mRNA, or genomic DNA in the postadministration samples; (5)
comparing the level of expression or activity of the adenylate
kinase protein, mRNA, or genomic DNA in the preadministration
sample with the adenylate kinase protein, mRNA, or genomic DNA in
the postadministration sample or samples; and (vi) altering the
administration of the agent to the subject accordingly to bring
about the desired effect, i.e., for example, an increase or a
decrease in the expression or activity of an adenylate kinase
protein.
[0314] C. Methods of Treatment
[0315] 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 adenylate kinase expression or activity. Additionally, the
compositions of the invention find use in the treatment of
disorders described herein.
[0316] 1. Prophylactic Methods
[0317] In one aspect, the invention provides a method for
preventing in a subject a disease or condition associated with an
aberrant adenylate kinase expression or activity by administering
to the subject an agent that modulates adenylate kinase expression
or at least one adenylate kinase gene activity. Subjects at risk
for a disease that is caused, or contributed to, by aberrant
adenylate kinase 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
adenylate kinase aberrancy, such that a disease or disorder is
prevented or, alternatively, delayed in its progression. Depending
on the type of adenylate kinase aberrancy, for example, an
adenylate kinase agonist or adenylate kinase antagonist agent can
be used for treating the subject. The appropriate agent can be
determined based on screening assays described herein.
[0318] 2. Therapeutic Methods
[0319] Another aspect of the invention pertains to methods of
modulating adenylate kinase expression or activity for therapeutic
purposes. The modulatory method of the invention involves
contacting a cell with an agent that modulates one or more of the
activities of adenylate kinase protein activity associated with the
cell. An agent that modulates adenylate kinase protein activity can
be an agent as described herein, such as a nucleic acid or a
protein, a naturally-occurring cognate ligand of an adenylate
kinase protein, a peptide, an adenylate kinase peptidomimetic, or
other small molecule. In one embodiment, the agent stimulates one
or more of the biological activities of adenylate kinase protein.
Examples of such stimulatory agents include active adenylate kinase
protein and a nucleic acid molecule encoding an adenylate kinase
protein that has been introduced into the cell. In another
embodiment, the agent inhibits one or more of the biological
activities of adenylate kinase protein. Examples of such inhibitory
agents include antisense adenylate kinase nucleic acid molecules
and anti-adenylate kinase antibodies.
[0320] 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
expression or activity of an adenylate kinase 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 a combination of agents, that modulates (e.g.,
upregulates or downregulates) adenylate kinase expression or
activity. In another embodiment, the method involves administering
an adenylate kinase protein or nucleic acid molecule as therapy to
compensate for reduced or aberrant adenylate kinase expression or
activity.
[0321] Stimulation of adenylate kinase activity is desirable in
situations in which an adenylate kinase protein is abnormally
downregulated and/or in which increased adenylate kinase activity
is likely to have a beneficial effect. Conversely, inhibition of
adenylate kinase activity is desirable in situations in which
adenylate kinase activity is abnormally upregulated and/or in which
decreased adenylate kinase activity is likely to have a beneficial
effect.
[0322] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
[0323] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0324] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
CHAPTER 2
21612, 21615, 21620, 21676, 33756, Novel Human Alcohol
Dehydrogenases
BACKGROUND OF THE INVENTION
[0325] Alcohol dehydrogenases are ubiquitous enzymes that are and
are generally classified as members of either the MDR (medium-chain
dehydrogenase/reductase) or SDR (short-chain
dehydrogenase/reductase) protein families. Members of the SDR and
MDR families appear to have similar activities though they work via
different mechanisms and structures. The SDR superfamily comprises
isomerases, lyases and oxidoreductases. The enzymes of this family
cover a wide range of substrate specificities including steroids,
alcohols, and aromatic compounds, however, most family members are
known to be NAD.sup.+- or NADP.sup.+-dependent oxidoreductases. In
the combined SDR superfamily, only a single tyrosine residue is
strictly conserved and ascribed a critical enzymatic function.
Members of the MDR superfamily are often multimeric enzymes
associated with 0, 1, or 2 zinc atoms. Substrates of the MDR
enzymes are often alcohols and aldehydes. Six different classes of
mammalian ADH isoforms are members of the MDR family. In addition
to the MDR and SDR families, alcohol dehydrogenases have also been
associated with protein families reflecting iron-dependant enzymes,
long-chain enzymes, and several types of prokaryotic enzymes with
other cofactor requirements.
[0326] Most dehydrogenase proteins function as dimers or tetramers
and possess at least two domains: the first domain comprising the
coenzyme binding site, and the second domain comprising the
substrate binding site. This latter domain determines the substrate
specificity and contains the amino acids involved in catalysis.
ADHs have a variety of substrate specificities, but act primarily
on primary or secondary alcohols, hemiacetals, cyclic secondary
alcohols, or on the corresponding aldehydes and ketones. The
catalytic role of ADH in mammalian ethanol oxidation is well
studied. ADH catalyzes the conversion of ethanol to acetaldehyde
using NAD.sup.+ as a cofactor. Specifically, the coenzyme binds
ADH, followed by an interaction with ethanol, the ethanol is
subsequently converted to acetaldehyde and the NAD.sup.+ is
converted to NADH. Members of the mammalian ADH protein family have
varying electrophoretic mobilities, Michaelis constants (binding
affinities) for ethanol, and sensitivities to pyrazol inhibition.
For instance, class I ADHs have low K.sub.m values (less than 5 mM)
for ethanol oxidation while class II and class IV ADHs have
intermediate K.sub.m values (about 30 mM). Class III ADH enzymes
are not saturable with ethanol and virtually function exclusively
as glutathione-dependent formaldehyde dehydrogenases. Allelic
variation of the mammalian genes have been identified. The kinetic
properties of the resultant variants differ significantly owing to
single amino acid substitutions in the coenzyme binding domains of
the enzymes.
[0327] Alcohol dehydrogenases play fundamental roles in
degradative, synthetic, and detoxification pathways and have been
implicated in a variety of critical developmental processes and
pathophysiological disease states. For instance, allelic variations
of ADH2 and ADH3 appear to influence the susceptibility to
alcoholism and alcoholic liver cirrhosis in Asians (Thomasson et
al. (1991) Am. J. Hum Genet. 48:677-681, Chao et al. (1994)
Hepatology 19:360-366, and Higuchi et al. (1995) Am. J. Psychiatry
152:1219-1221). Furthermore, first-pass metabolism is the
difference between the quantity of ethanol that reaches the
systemic circulation by the intravenous route and the quantity that
entered by the oral dose. Several lines of evidence now indicate
that first-pass metabolism of alcohol in humans may occur in the
liver via the activity of members of the mammalian ADH family (Yin
et al. (1999) Enzymology and Molecular Biology of Carbonyl
Metabolism 7, Plenum Publishers, New York).
[0328] ADHs are also involved in detoxification pathways. For
instance, class III ADH is unsaturable by ethanol and mainly
functions as a glutathione-dependant formaldehyde dehydrogenase and
is therefore important for the elimination of endogenously formed
formaldehyde. ADHs are also involved in the metabolism of
nitrobenzaldehyde, a dietary carcinogen. It has been suggested that
the lack of .sigma.-ADH in Japanese patients may lead to a
decreased detoxification of the dietary carcinogen
nitrobenzaldehyde and may possible be linked to the high rate of
gastric cancer in Japanese (Baron et al. (1991) Life Sci
49:1929-34; Grab et al. (1977) Cancer Res 37:4181-90 and Seedcake
et al (1980) Rev Ed 9:346-51). ADH is also involved in the
activation of 1,2 dimethylhydrazine, an experimentally used
procarcinogen.
[0329] Retinoic acid is a ligand controlling a nuclear receptor
signaling pathway that plays a key role in the regulation of
embryonic development, spermatogenesis, and epithelial
differentiation (Chambon et al. (1996) FASEB J. 10:940-954 and
Mangelsdorf et al. (1995) Cell 83:841-850). The synthesis of
retinoic acid occurs via the oxidation of retinol to retinal
followed by the conversion of retinal to retinoic acid. Members of
the alcohol dehydrogenase and short-chain dehydrogenase/reductase
families catalyze the reversible, rate limiting conversion of
retinol to retinal, while the oxidation of retinal to retinoic acid
is catalyzed by members of the aldehyde dehydrogenase or P450
enzyme families (Deuster et al. (1996) Biochemistry
35:12221-12227). Therefore, members of the ADH family influence the
growth and developmental processes mediated by the active
metabolite retinoic acid.
[0330] ADH metabolism of retinol to retinal is inhibited by
ethanol, and this may lead to altered epithelial cell
differentiation and malignant cell transformation. Furthermore, it
has been suggested that the ability of ethanol to inhibit the
oxidation of retinol by ADH underlies the pathology of fetal
alcohol syndrome, a birth defect characterized by craniofacial,
limb, and brain malformations (Duester et al. (1991) Alcohol Clin
Exp Res 15:568-572). Retinoic acid also functions to maintain
differentiation of epithelial cells and influences spermatogenesis
in adult vertebrates (Chambon et al. (1996) FASEB J. 10:940-954).
Data suggests that retinoic acid signaling in spermatogenesis and
keratinocyte differentiation may be significantly disrupted by
ethanol through ADH pathways. It has been proposed that inhibition
of retinol metabolism by ethanol may be responsible for the
testicular atrophy and spermatogenesis commonly seen in male
chronic alcoholics. Furthermore, skin diseases such as psoriasis,
have been associated with heavy drinking.
[0331] ADH may also play a role in colorectal cancers. During
colorectal carcinogenesis, ADH activity is significantly decreased
in polyps and further decreased in cancer tissue. (Egerer et al.
(1997) Gastroenterology 112:A1260). Furthermore, epidemiological
studies have demonstrated that alcohol consumption is a risk factor
for development of oral, esophageal, colorectal, and upper
gastrointestinal cancers (Blot et al. (1992) Cancer Res
52:2119s-2123s). The role of ADH in cancers of these various
tissues may result from the production of acetaldehyde following
oxidation of ethanol by ADH, an alteration in retinol metabolism or
through the role of ADH in carcinogen metabolism.
[0332] Further functional links between disease and the
oxidative/reductive actions of various dehydrogenases are being
established. For instance, ERAB is a member of the short-chain
dehydrogenase/reductase family. Interactions between and Amyloid
.beta. peptide and ERAB have been shown to mediate neurotoxicity
and apoptosis in neuronal cell lines (Yan et al. (1997) Nature
389:689-693) and thus are being implicated in the pathogenesis of
neurodegenerative disorders like Alzheimer's disease (Oppermann et
al. (1999) Enzymology and Molecular Biology of Carbonyl Metabolism
7, Plenum Publishers, New York and Oppermann et al. (1999) FEBS
Letters 451:238-242).
[0333] Accordingly, ADHs are a major target for drug action and
development. Therefore, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown ADHs. The present invention advances the state of the art
by providing previously unidentified human alcohol
dehydrogenases.
SUMMARY OF THE INVENTION
[0334] It is an object of the invention to identify novel alcohol
dehydrogenases.
[0335] It is a further object of the invention to provide novel
alcohol dehydrogenase polypeptides that are useful as reagents or
targets in assays applicable to treatment and diagnosis of
ADH-mediated or -related disorders.
[0336] It is a further object of the invention to provide
polynucleotides corresponding to the novel ADH polypeptides that
are useful as targets and reagents in ADH assays applicable to
treatment and diagnosis of ADH-mediated or -related disorders and
useful for producing novel ADH polypeptides by recombinant
methods.
[0337] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the novel ADHs.
[0338] A further specific object of the invention is to provide
compounds that modulate expression of the alcohol dehydrogenases
for treatment and diagnosis of ADH-related disorders.
[0339] The invention is thus based on the identification of novel
human alcohol dehydrogenases. The amino acid sequence for ADH
21620, 33756, 21676, 21612, and 21615, are shown in SEQ ID NOS:5,
7, 9, 11, and 13, respectfully. The nucleotide sequence for ADH
21620, 33756, 21676, 21612, and 21615 are shown in SEQ ID NOS:6, 8,
10, 12, and 14, respectfully.
[0340] The invention provides isolated ADH polypeptides, including
a polypeptide having the amino acid sequence shown in SEQ ID NOS:5,
7, 9, 11, and 13, or the amino acid sequence encoded by the cDNA
deposited with American Type Culture Collection (ATCC), University
Boulevard, Manassas, Va. 20110-2209, as Patent Deposit No. PTA-2012
(corresponding to the 33756 nucleotide sequence) on Jun. 9, 2000;
Patent Deposit No. PTA-2170 (corresponding to the 21612 nucleotide
sequence) on Jun. 27, 2000, Patent Deposit No. PTA-2171
(corresponding to the 21620 nucleotide sequence) on Jun. 27, 2000,
as Patent Deposit No. PTA-2812 (corresponding to the 21615
nucleotide sequence) on Dec. 14, 2000, and as Patent Deposit No.
PTA-2813 (corresponding to the 21676 nucleotide sequence) on Dec.
14, 2000. ATCC Patent Deposits PTA-2012, PTA-2170, PTA-2171,
PTA-2812, and PTA-2813 are referred to collectively herein as "the
deposited cDNAs."
[0341] The invention also provides isolated ADH nucleic acid
molecules having the sequences shown in SEQ ID NOS:6, 8, 10, 12,
and 14, or in the deposited cDNAs.
[0342] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequences shown in SEQ ID NOS:5, 7, 9, 11, and 13, or encoded
by the deposited cDNAs.
[0343] The invention also provides variant nucleic acid sequences
that are substantially homologous to the nucleotide sequences shown
in SEQ ID NOS:6, 8, 10, 12, and 14, or in the deposited cDNAs.
[0344] The invention also provides fragments of the polypeptides
shown in SEQ ID NOS:5, 7, 9, 11, and 13, and nucleotide sequences
shown in SEQ ID NOS:6, 8, 10, 12, and 14, as well as substantially
homologous fragments of the polypeptides or nucleic acids.
[0345] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[0346] The invention also provides vectors and host cells for
expressing the ADH nucleic acid molecules and polypeptides, and
particularly recombinant vectors and host cells.
[0347] The invention also provides methods of making the vectors
and host cells and methods for using them to produce the ADH
nucleic acid molecules and polypeptides.
[0348] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the ADH polypeptides and
fragments.
[0349] The invention also provides methods of screening for
compounds that modulate expression or activity of the ADH
polypeptides or nucleic acid (RNA or DNA).
[0350] The invention also provides a process for modulating ADH
polypeptide or nucleic acid expression or activity, especially
using the screened compounds. Modulation may be used to treat
conditions related to aberrant activity or expression of the ADH
polypeptides or nucleic acids.
[0351] The invention also provides assays for determining the
activity of or the presence or absence of the ADH polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0352] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[0353] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the invention,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0354] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0355] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Polypeptides
[0356] The invention is based on the discovery of novel human
alcohol dehydrogenases. Specifically, an expressed sequence tag
(EST) was selected based on homology to the alcohol dehydrogenase
sequence. This EST was used to design primers based on sequences
that it contains and used to identify cDNAS from human cDNA
libraries, including primary osteoblasts. Positive clones were
sequenced and the overlapping fragments were assembled. Analysis of
each of the assembled sequences revealed that the cloned cDNA
molecules encode ADHs.
[0357] The invention thus relates to novel ADHs having the deduced
amino acid sequence shown in FIGS. 7A-B, 13, 17A-B, 21A-B, and
25A-B, or the amino acid sequences shown in SEQ ID NOS:5, 7, 9, 11,
and 13, or the amino acid sequences encoded by the deposited cDNAs
as Patent Deposit Numbers PTA-2012, PTA-2170, or PTA-2171.
[0358] The deposits will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms. The deposits are provided as a convenience to those
of skill in the art and is not an admission that a deposit is
required under 35 U.S.C. .sctn. 112. The deposited sequences, as
well as the polypeptides encoded by the sequences, are incorporated
herein by reference and controls in the event of any conflict, such
as a sequencing error, with description in this application.
[0359] "ADH polypeptide" or "ADH protein" refers to the
polypeptides in SEQ ID NOS:5, 7, 9, 11, and 13, or the polypeptides
encoded by the deposited cDNAs. The term "ADH protein" or "ADH
polypeptide", however, further includes the numerous variants
described herein, as well as fragments derived from the full-length
ADHs and variants.
[0360] Tissues and/or cells in which the 21620 ADH is found
include, but are not limited to those shown in FIGS. 11 and 12.
Tissues in which the gene is highly expressed include brain, colon,
kidney, and small intestine. Moderate expression occurs in liver,
muscle, and testes. Lower positive expression occurs in the aorta,
breast, cervix, esophagus, heart, lung, lymph, ovary, placenta,
spleen, thymus, thyroid, and vein. The 21620 ADH is also expressed
in malignant breast, lung, and colon tissue, and in liver
metastases derived from malignant colonic tissue.
[0361] The present invention thus provides isolated or purified
polypeptides of the 21620 ADH, 33756 ADH, 21676 ADH, 21612 ADH, and
21615 ADH and variants and fragments thereof.
[0362] The short-chain alcohol dehydrogenase family signature is
found in the 21620 ADH from about amino acid 166 to about amino
acid 176 and in the 21615 ADH from about amino acid 147 to about
amino acid 157.
[0363] Based on a Blast search, highest homology to the 21620 ADH
was shown to Antennal-specific Short-chain Dehydrogenase/reductase
from Drosophila melanogaster (Genbank Acc. No. AF116553) and to the
Oxidoreductase from Haloferax volcani (Genbank Acc. No.
U95375).
[0364] Based on a Blast search, highest homology to the 33756 ADH
was shown to CGI-82 from Homo sapiens (Genbank Acc. No. AF151840),
UBE-1b from Mus musculus (Genbank Acc. No. AB030504), UBE-1a from
Mus musculus (Genbank Acc. No. AB030503).
[0365] Based on a Blast search, no significant homology was found
to the 21676 ADH.
[0366] Based on a Blast search, highest homology to the 21612 ADH
was shown to a protein similar to alcohol dehydrogenase from C.
elegans (Genbank Acc. No. U28739), a protein similar to alcohol
dehydrogenase from C. elegans (Genbank Acc. No. Z74029), and to the
hypothetical protein RV3224 from Mycobacterium tuberculosis
(Genbank Acc. No. Z95120).
[0367] Based on a Blast search, highest homology to the 21615 ADH
was shown to a 3-oxoacyl-(acyl carrier protein) reductase from
Thermotoga maritima (Genbank Acc. No. AAD36790).
[0368] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[0369] The ADH polypeptides can be purified to homogeneity. It is
understood, however, that preparations in which the polypeptide is
not purified to homogeneity are useful and considered to contain an
isolated form of the polypeptide. The critical feature is that the
preparation allows for the desired function of the polypeptide,
even in the presence of considerable amounts of other components.
Thus, the invention encompasses various degrees of purity.
[0370] In one embodiment, the language "substantially free of
cellular material" includes preparations of the ADH having less
than about 30% (by dry weight) other proteins (i.e., contaminating
protein), less than about 20% other proteins, less than about 10%
other proteins, or less than about 5% other proteins. When the
polypeptide is recombinantly produced, it can also be substantially
free of culture medium, i.e., culture medium represents less than
about 20%, less than about 10%, or less than about 5% of the volume
of the protein preparation.
[0371] An ADH polypeptide is also considered to be isolated when it
is part of a membrane preparation or is purified and then
reconstituted with membrane vesicles or liposomes.
[0372] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the ADH polypeptide in
which it is separated from chemical precursors or other chemicals
that are involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the polypeptide having less than about 30%
(by dry weight) chemical precursors or other chemicals, less than
about 20% chemical precursors or other chemicals, less than about
10% chemical precursors or other chemicals, or less than about 5%
chemical precursors or other chemicals.
[0373] In one embodiment, the ADH polypeptides comprise the amino
acid sequences shown in SEQ ID NOS:5, 7, 9, 11, and 13. However,
the invention also encompasses sequence variants. Variants include
a substantially homologous protein encoded by the same genetic
locus in an organism, i.e., an allelic variant.
[0374] The 21620 ADH has been mapped to human chromosome 17
(17q12-21) with flanking markers WI-3010 (9.7cR) and WI-4251
(17.3cR). Mutations near this locus include, but are not limited
to, the following: wilms tumor 4; patella aplasia or hypoplasia;
psoriasis susceptibility 2 (psors2); malignant hyperthermia
susceptibility 2 (MSH2); pallidopontonigral degeneration (PPND);
pseudohypoaldosteronism type II locus B; and gliosis and familial
progressive subcortical. In the mouse this locus is associated with
the following: susceptibility to lung cancer (Sluc4); pulmonary
adenoma resistance (Par1); radiation-induced apoptosis 4 (Rapop4);
cocked (co); open eyelids (oe); ovum mutant (Om); rimy (rmy);
susceptibility to experimental allergic encephalomyelitis 7 (Eae7);
liver weight QTL 4 (Lwq4); alopecia (Al); spleen weight OTL 1
(Swq1); modifier of von willebrand factor (Mvwf); neuron number
control (Nnc1); recombination induced mutation 3 (rim3);
bald-arthritic (Bda); bare skin (Bsk); rex (re); alymphoplasia
(aly); cleft lip 1 (clf1); seizure susceptibility 3 (Szs3);
uncovered (Uncv). Genes near this locus include CDC18L, RARA,
PDE6G, IGFBP4, TCFL4, NAGLU, FZD2, PYY, ERBB2, RABL, SCYA11, KRT12,
NEUROD2, SLC6A4, ACACA, SCYA1, and BRCA1.
[0375] The 21612 ADH has been mapped to human chromosome 9
(9q22-33) with flanking markers WI-6207 (5.7 cR) and D9S174 (6.0
cR). Mutations near this locus include, but are not limited to, the
following: hypomagnesemia with secondary hypocalcemia (HOMG);
hemophagocytic lymphohistiocytosis, familial 1; nephronophthisis
(NPHP2), infantile; HSN1, neuropathy, hereditary sensory, type 1;
high density lipoprotein deficiency (HDLDT1), tangier type 1;
dysautonomia (dys), familial; muscular dystrophy, limb-girdle, type
2H; acrofacial dysostosis 1 (AFD1), nager type; amyotrophic lateral
sclerosis 4 (ALS4), juvenile; and multiple self-healing squamous
epithelioma (MSSE). In the mouse this locus is associated with the
following: vacillans (vc), whirler (wi), ochre (och), Hertwig's
anemia (an), b-associated fitness (baf), iris stromal atrophy (is
a), lymphoma resistance (lyr), and systemic lupus erythmatosus
susceptibility 2 (sle2). Genes near this locus include SCYA5,
ZFP37, UGCG, SLC31A2, HXB, HPRP4P, ORM1, TNFSF8, TXN, IKBAKAP,
PTPN3, EDG2, CSMF, chondrosarcoma, myxoid extraskeletal, and fused
to EWS.
[0376] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the ADHs of
SEQ ID NOS:5, 7, 9, 11, and 13. Variants also include proteins
substantially homologous to the ADHs but derived from another
organism, i.e., an ortholog. Variants also include proteins that
are substantially homologous to the ADHs that are produced by
chemical synthesis. Variants also include proteins that are
substantially homologous to the ADHs that are produced by
recombinant methods. It is understood, however, that variants
exclude any amino acid sequences disclosed prior to the
invention.
[0377] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 70-75%, typically at least about 80-85%, and most
typically at least about 90-95% or more homologous. A substantially
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence hybridizing to the
nucleic acid sequence, or portion thereof, of the sequence shown in
SEQ ID NOS:6, 8, 10, 12, and 14 under stringent conditions as more
fully described below.
[0378] 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-homologous
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 amino acid sequences herein having 502 amino acid residues, at
least 165, preferably at least 200, more preferably at least 250,
even more preferably at least 300, and even more preferably at
least 350, 400, 450, and 500 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.
[0379] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the ADH.
Similarity is determined by conserved amino acid substitution. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Conservative substitutions are likely to be phenotypically silent.
Typically seen as conservative substitutions are the replacements,
one for another, among the aliphatic amino acids Ala, Val, Leu, and
Ile; interchange of the hydroxyl residues Ser and Thr, exchange of
the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr. Guidance
concerning which amino acid changes are likely to be phenotypically
silent are found in Bowie et al., Science 247:1306-1310 (1990).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0380] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0381] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. See www.ncbi.nlm.nih.gov. In
one embodiment, parameters for sequence comparison can be set at
score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).
[0382] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (J. Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at 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 sequences is
determined using the GAP program in the GCG software package
(Devereux et al. (1984) Nucleic Acids Res. 12(1):387) (available at
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.
[0383] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[0384] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[0385] Variant polypeptides can be fully functional or can lack
function in one or more activities. For example, variants of the
ADHs can have an altered developmental expression, temporal
expression or tissue-preferred expression. ADH variants can also
have an altered interaction with cellular components, substrates,
coenzymes, metal ions, or ADH subunits. An altered interaction
comprising either a higher or lower affinity of the ADH for the
various cellular components, substrates, coenzymes, metal ions, or
ADH subunits. By "coenzyme" is intended a molecule that is
associated with the ADH and is essential for ADH activity. Some
coenzymes are covalently linked to their enzyme while others are
less tightly bound. A covalently linked coenzyme is referred to as
a prosthetic group of the enzyme. By "coenzyme" is also intended
the oxidized or reduced product of the coenzyme which is formed
following the enzymatic reaction mediated by the ADH polypeptide.
For example, in the biological oxidation of an alcohol to an
aldehyde, a hydrogen ion is transferred to the coenzyme NAD.sup.+
to form the coenzyme product NADH. Coenzymes of ADH include, but
are not limited to, NAD.sup.+ and NAD.sup.+ analogues (Plapp et al.
(1986) Biochemistry 25:5396-5402 and Yamazaki et al. (1984) J.
Biochem 95:109-115), .beta.-NAD.sup.+ (Favilla et al. (1980) Eur.
J. Biochem 104, 223-227 and Creagh et al. (1993) Biotechnol.
Bioeng. 41:156-161, benzoylpyridine adenine dinucleotide (Samama et
al. (1986) Eur. J. biochem. 159:375-380), NADH, NADP.sup.+, and
NADPH. Variants of ADH may also have altered interactions with
metal ions including, but not limited to, Zn.sup.2+, Co.sup.2+,
Mg.sup.2+, Fe.sup.2+. See, for example, Yabe et al. (1992) Biosci.
Biotechnol. Biochem. 56:338-339 and Leblov et al. (1972)
Phytochemistry 11:1345-1346. Variants of ADH can also have an
altered interaction with a substrate. Substrates of ADH include,
but are not limited to, primary or secondary alcohols or
hemiacetals, and cyclic secondary alcohols. By "substrate" is also
intended the products resulting from the oxidation of the above
mentioned substrates. Such products include, for example, various
aldehydes and ketones. Other substrates include retinol, steroids,
and carcinogens such as nitrobenzaldehyde and
1,2-dimethylhydrazine. Variants of ADH can also have an altered
subunit interaction that affects the ability of ADH to form an
active multimeric structure.
[0386] Useful variants of ADH polypeptides further include
alterations in catalytic activity. The enzymatic reaction mediated
by ADH is reversible and comprises either the oxidation, i.e.,
removal of electrons, of the above mentioned substrates or their
reduction, i.e., addition of electrons. The catalytic reaction
further comprises the oxidation or reduction of the coenzyme.
Therefore, one embodiment involves a variant that results in
binding of the substrate but results in slower oxidation/reduction
or no oxidation/reduction of the substrate. Another variation can
result in an increased rate of substrate oxidation/reduction. Other
useful variation can include an altered binding affinity for a
coenzyme or substrate. For example, an increased or decreased
binding affinity of a coenzyme can alter the binding affinity of
the ADH to the substrate and also alter the rate of substrate
oxidation/reduction. Another variation can prevent the ADH monomer
from associating with other ADH subunits to form an active
multimeric complex.
[0387] Another useful variation provides a fusion protein in which
one or more domains or subregions are operationally fused to one or
more domains or subregions from another ADH. Specifically, a domain
or subregion can be introduced that alters the coenzyme or
substrate specificities or the rate of the enzymatic reaction.
[0388] Fully functional variants typically contain only
conservative variations or variations in non-critical residues or
in non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0389] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0390] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for the ADH polypeptide. This includes
preventing immunogenicity from pharmaceutical formulations by
preventing protein aggregation.
[0391] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as the binding affinity for the coenzyme or substrate or
determining the catalytic constants for substrate
oxidation/reduction. Sites that are critical for coenzyme and
substrate binding can also be determined by structural analysis
such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al. (1992) J. Mol. Biol.
224:899-904; de Vos et al. (1992) Science 255:306-312).
[0392] The assays for ADH enzyme activity are well known in the art
and can be found for example, in Oppermann et al. (1999) FEBS
451:238-242, Thomasson et al. (1993) Behavior Genetics 23:131-136,
and Zubey (1988) Macmillan Publishing Company, New York. These
assays include, but are not limited to, determination of the
Michaelis constants (K.sub.m) or the dissociation constant for the
ADH/substrate complex. Such analysis of enzyme activity may be
performed spectrophotometrically by recording the change in
absorbance of NAD.sup.+. The catalytic efficiency or k.sub.cat can
also be measured. K.sub.cat is defined as the maximum number of
molecules of substrate converted to product per active site per
unit of time. The specificity constant (k.sub.cat/K.sub.M) can also
be used to measure the ability of the ADH to discriminate between
competing substrates. Similar assays can also be performed to
measure ADH/coenzyme interactions. In vivo measurements of ADH
activity can be determined by pharmacokinetic studies. In such
studies, an ethanol dose in administered and the blood ethanol
concentration is monitored over time. The area under the time curve
indicates the rate of ethanol elimination from the system. A larger
blood alcohol concentration time curve indicates slower ethanol
metabolism.
[0393] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[0394] The invention thus also includes polypeptide fragments of
the ADHs. Fragments can be derived from the amino acid sequences
shown in SEQ ID NOS:5, 7, 9, 11, and 13. However, the invention
also encompasses fragments of the variants of the ADHs as described
herein.
[0395] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention. Accordingly, a fragment of the
21620 ADH can comprise at least about 9, 15, 20, 25, 30, 35, 40 or
more contiguous amino acids. A fragment of the 33756 ADH can
comprise at least about 21, 25, 30, 35, 40, 45, 50, or more
contiguous amino acids. A fragment of the 21676 ADH can comprise at
least about 7, 10, 15, 20, 25, 30, 35 or more contiguous amino
acids. A fragment of the 21612 ADH can comprise at least about 14,
20, 25, 30, 35, 40 or more contiguous amino acids. A fragment of
the 21615 ADH can comprise at least about 7, 10, 15, 20, 25, 30, 35
or more contiguous amino acids. Fragments can retain one or more of
the biological activities of the protein, for example the ability
to bind a coenzyme or substrate or the ability catalyze the
oxidation/reduction of a substrate. Alternatively, fragments can be
used as an immunogen to generate ADH antibodies.
[0396] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a domain or motif,
e.g., catalytic site, substrate binding site, coenzyme binding
site, short-chain alcohol dehydrogenase signature, microbodies
C-terminal targeting signals, and sites for glycosylation, protein
kinase C phosphorylation, casein kinase II phosphorylation,
tyrosine kinase phosphorylation, and N-myristoylation. Further
possible fragments include sites important for cellular and
subcellular targeting.
[0397] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[0398] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific domains mentioned above, which sub-fragments retain
the function of the domain from which they are derived.
[0399] These regions can be identified by well-known methods
involving computerized homology analysis.
[0400] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the ADH or
ADH variants. These epitope-bearing peptides are useful to raise
antibodies that bind specifically to an ADH polypeptide or region
or fragment. These peptides can contain at least 10, 12, at least
14, or between at least about 15 to about 30 amino acids.
[0401] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular site. Regions having a high
antigenicity index are shown in FIGS. 8, 14, 18, 22, and 26, for
the 21620, 33756, 21676, 21612, and 21615 ADHs, respectfully.
However, intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular peptide
regions.
[0402] The epitope-bearing ADH polypeptides may be produced by any
conventional means (Houghten, R. A. (1985) Proc. Natl. Acad. Sci.
USA 82:5131-5135). Simultaneous multiple peptide synthesis is
described in U.S. Pat. No. 4,631,211.
[0403] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the ADH fragment and an additional
region fused to the carboxyl terminus of the fragment.
[0404] The invention thus provides chimeric or fusion proteins.
These comprise an ADH peptide sequence operatively linked to a
heterologous peptide having an amino acid sequence not
substantially homologous to the ADH. "Operatively linked" indicates
that the ADH peptide and the heterologous peptide are fused
in-frame. The heterologous peptide can be fused to the N-terminus
or C-terminus of the ADH or can be internally located.
[0405] In one embodiment the fusion protein does not affect ADH
function per se. For example, the fusion protein can be a
GST-fusion protein in which the ADH sequences are fused to the
C-terminus of the GST sequences. Other types of fusion proteins
include, but are not limited to, enzymatic fusion proteins, for
example beta-galactosidase fusions, yeast two-hybrid GAL-4 fusions,
poly-His fusions and Ig fusions. Such fusion proteins, particularly
poly-His fusions, can facilitate the purification of recombinant
ADH. In certain host cells (e.g., mammalian host cells), expression
and/or secretion of a protein can be increased by using a
heterologous signal sequence. Therefore, in another embodiment, the
fusion protein contains a heterologous signal sequence at its
N-terminus.
[0406] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson
et al. J. Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing an ADH polypeptide
and various portions of the constant regions of heavy or light
chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE).
Preferred as immunoglobulin is the constant part of the heavy chain
of human IgG, particularly IgG1, where fusion takes place at the
hinge region. For some uses it is desirable to remove the Fc after
the fusion protein has been used for its intended purpose, for
example when the fusion protein is to be used as antigen for
immunizations. In a particular embodiment, the Fc part can be
removed in a simple way by a cleavage sequence, which is also
incorporated and can be cleaved with factor Xa.
[0407] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. 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 re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). An ADH-encoding nucleic acid can be
cloned into such an expression vector such that the fusion moiety
is linked in-frame to the ADH.
[0408] Another form of fusion protein is one that directly affects
ADH functions. Accordingly, an ADH polypeptide is encompassed by
the present invention in which one or more of the ADH domains (or
parts thereof) has been replaced by homologous domains (or parts
thereof) from another ADH or a short-chain dehydrogenase/reductase
family member. Accordingly, various permutations are possible. For
example, the substrate binding domain, or subregion thereof, can be
replaced with the substrate binding domain or subregion from
another ADH or a short-chain dehydrogenase/reductase family member.
As a further example, the catalytic domain, or coenzyme binding
domains or parts thereof, can be replaced with the appropriate
domain from another ADH or SDR family member. Thus, chimeric ADHs
can be formed in which one or more of the native domains or
subregions has been replaced by another.
[0409] Additionally, chimeric ADH proteins can be produced in which
one or more functional sites is derived from a different ADH or a
short-chain dehydrogenase/reductase family member. It is understood
however that sites could be derived from the ADH or a short-chain
dehydrogenase/reductase family members that occur in the mammalian
genome but which have not yet been discovered or characterized.
Such sites include but are not limited to the catalytic site,
substrate binding site, coenzyme binding site, sites important for
targeting to subcellular and cellular locations, sites functional
for interaction with ADH subunits, protein kinase A phosphorylation
sites, glycosylation sites, and other functional sites disclosed
herein.
[0410] The isolated ADHs can be purified from cells that naturally
express it. Tissues and cells that express high levels of the 21620
ADH include, but are not limited to, brain, colon, kidney, and
small intestine. Moderate levels of expression occur in the liver,
muscle, and testes. Lower positive expression occurs in the aorta,
breast, cervix, esophagus, heart, lung, lymph, ovary, placenta,
spleen, thymus, thyroid, and vein. The 21620 ADH is also expressed
in malignant breast, lung, and colon tissue, and liver metastases
derived from colon. The ADHs of the present invention can also be
purified from cells that have been altered to express it
(recombinant), or synthesized using known protein synthesis
methods.
[0411] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
ADH polypeptide is cloned into an expression vector, the expression
vector introduced into a host cell and the protein expressed in the
host cell. The protein can then be isolated from the cells by an
appropriate purification scheme using standard protein purification
techniques. Polypeptides often contain amino acids other than the
20 amino acids commonly referred to as the 20 naturally-occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in polypeptides are described in
basic texts, detailed monographs, and the research literature, and
they are well known to those of skill in the art.
[0412] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[0413] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0414] Such modifications are well-known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T.E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990)
Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y.
Acad. Sci. 663:48-62).
[0415] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0416] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[0417] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[0418] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
Polypeptide Uses
[0419] The protein sequences of the present invention can 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 the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins 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 www.ncbi.nlm.nih.gov.
[0420] The ADH polypeptides are useful for producing antibodies
specific for the ADH, regions, or fragments. Regions having a high
antigenicity index score are shown in FIGS. 8, 14, 18, 22, and 26
for the 21620 ADH, 33756 ADH, 21676 ADH, 21612 ADH, and 21615 ADH,
respectfully.
[0421] The ADH polypeptides are useful for biological assays
related to ADHs. Such assays involve any of the known ADH functions
or activities or properties useful for diagnosis and treatment of
ADH-related conditions.
[0422] The ADH polypeptides are also useful in drug screening
assays, in cell-based or cell-free systems. Cell-based systems can
be native, i.e., cells that normally express the ADH, as a biopsy
or expanded in cell culture. In one embodiment, however, cell-based
assays involve recombinant host cells expressing the ADH.
[0423] Determining the ability of the test compound to interact
with the ADH can also comprise determining the ability of the test
compound to preferentially bind to the polypeptide as compared to
the ability of a known binding molecule (e.g. a coenzyme or
substrate) to bind to the polypeptide.
[0424] The polypeptides can be used to identify compounds that
modulate ADH activity. Such compounds, for example, can increase or
decrease the affinity of the substrate or coenzyme for ADH. Such
compound can also increase or decrease the enzymatic activity of
the ADH. Additionally, such compounds can also alter the
interaction of ADH with a metal ion or alter the ability of the ADH
polypeptide to form a multimeric structure. Compounds that modulate
ADH activity include, but are not limited to, pyrazole,
4-methylpyrazole, P-hydroxymercuribenzoate, o-Phenanthroline,
iodoacetamide, iodoacetate, imidazole, colloidal bismuth
subcitrate, cimetidine, ranitidine, and aspirin.
[0425] The ADHs of the present invention and appropriate variants
and fragments can be used in high-throughput screens to assay
candidate compounds for the ability to bind to the ADH. These
compounds can be further screened against a functional ADH to
determine the effect of the compound on the ADH activity. Compounds
can be identified that activate (agonist) or inactivate
(antagonist) the ADH to a desired degree. 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).
[0426] The ADH polypeptides can be used to screen a compound for
the ability to stimulate or inhibit interaction between the ADH
protein and a target molecule that normally interacts with the ADH
protein. The target can be a coenzyme, metal ion, ADH substrate or
another ADH subunit of the multimeric ADH enzyme. The assay
includes the steps of combining the ADH protein with a candidate
compound under conditions that allow the ADH protein or fragment to
interact with the target molecule, and to detect the formation of a
complex between the ADH protein and the target or to detect the
biochemical consequence of the interaction with the ADH and the
target, such as the oxidation/reduction of the substrate or
coenzyme.
[0427] Determining the ability of the ADH to bind to a target
molecule can also be accomplished using a technology-such as
real-time Bimolecular Interaction Analysis (BIA). Sjolander et al.
(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.TM.). Changes in the optical
phenomenon surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
[0428] 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 polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam (1997)
Anticancer Drug Des. 12:145).
[0429] 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. USA 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; Carell 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. 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. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[0430] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0431] One candidate compound is a soluble full-length ADH or
fragment that competes for substrate binding or cofactor binding,
interferes with the ADH catalyzed reaction, or interferes with ADH
subunit interactions. Other candidate compounds include mutant ADHs
or appropriate fragments containing mutations that affect ADH
function and thus compete for cofactor binding or substrate binding
or interfere with the ADH catalyzed reaction or interferes with the
ADH subunit interactions. Accordingly, a fragment that competes for
substrate or coenzyme binding, for example with a higher affinity,
or a fragment that binds substrate but does not catalyze its
oxidation/reduction is encompassed by the invention.
[0432] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) ADH activity. The
assays typically involve an assay of events that result from
substrate or coenzyme oxidation/reduction that indicate ADH
activity. Thus, the expression of genes that are up- or
down-regulated in response to the ADH enzyme can be assayed. In one
embodiment, the regulatory region of such genes can be operably
linked to a marker that is easily detectable, such as
luciferase.
[0433] Any of the biological or biochemical functions mediated by
the ADH can be used as an endpoint assay. These include all of the
biochemical or biological events described herein, in the
references cited herein and incorporated by reference, and other
ADH functions known to those of ordinary skill in the art.
[0434] In the case of ADH, specific end points can include an
altered NADH/NAD.sup.+ ratio. For instance, ethanol oxidation
results in an increased NADH/NAD.sup.+ redox potential within the
cytosol and mitochondria with subsequent alteration in several
tissue metabolites. For example, the increase in cytosolic
NADH/NAD.sup.+ ratio causes an increase in the lactate/pyruvate
ratio mediated via lactate dehydrogenase. Other consequences of
ethanol- and acetaldehyde-induced redox changes include, enhanced
triglyceride synthesis, inhibition of Krebs cycle activity, lactic
acidosis, ketoacidosis, hyperuricaemia and enhanced fibrogenesis.
See, for example, Peters et al. (1998) Novartis Foundation
Symposium 216: 19-34, herein incorporated by reference.
[0435] Furthermore, the metabolism of ethanol via ADH results in
the production of acetaldehyde, which is removed by the action of
acetaldehyde dehydrogenases. Acetaldehyde alters various cellular
function including glutathione depletion and inhibition of nuclear
repair enzymes. Acetaldehyde can also alter cellular membranes
resulting in severe cellular injury (Lieber et al. (1994)
Gastroenterology 106:1085-105). Acetaldehyde toxicity depends on
its net formation and can be increased when ADH activity is low and
acetaldehyde dehydrogenase activity is high. Additional end points
that can be assayed include biological events that are a
consequence of ADH oxidation of retinol to retinal, which include
but are not limited to differentiation of epithelium and
spermatogenesis.
[0436] Binding and/or activating compounds can also be screened by
using chimeric ADH proteins in which one or more domains, sites,
and the like, as disclosed herein, or parts thereof, can be
replaced by their heterologous counterparts derived from other ADHs
or of any other short chain dehydrogenase/reductase family member.
For example, a substrate binding region or coenzyme binding region
can be used that interacts with a different substrate or coenzyme
specificity and/or affinity than the native ADH. Accordingly, a
different set of oxidized/reduced substrates or coenzymes is
available as an end-point assay for activation. Alternatively, a
heterologous targeting sequence can replace the native targeting
sequence. This will result in different subcellular or cellular
localization. As a further alternative, sites that are responsible
for developmental, temporal, or tissue specificity can be replace
by heterologous sites such that the ADH can be detected under
conditions of specific developmental, temporal, or tissue-specific
expression.
[0437] The ADH polypeptides are also useful in competition binding
assays in methods designed to discover compounds that interact with
the ADH. Thus, a compound is exposed to an ADH polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble ADH polypeptide is also added to the
mixture. If the test compound interacts with the soluble ADH
polypeptide, it decreases the amount of complex formed or activity
from the ADH target. This type of assay is particularly useful in
cases in which compounds are sought that interact with specific
regions of the ADH. Thus, the soluble polypeptide that competes
with the target ADH region is designed to contain peptide sequences
corresponding to the region of interest.
[0438] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, a substrate, such as ethanol, and a candidate compound
can be added to a sample of the ADH. Compounds that interact with
the ADH at the same site as the ethanol will reduce the amount of
complex formed between the ADH and ethanol. Accordingly, it is
possible to discover a compound that specifically prevents
interaction between the ADH and ethanol. Another example involves
adding a candidate compound to a sample of ADH and a coenzyme, such
as NAD.sup.+. A compound that competes with NAD.sup.+ will reduce
the coenzyme interaction with ADH and thereby prevent the
subsequent interaction with a substrate or the oxidation of the
substrate. Accordingly, compounds can be discovered that directly
interact with the ADH and compete with various coenzymes and
substrates. Such assays can involve any other component that
interacts with the ADH.
[0439] To perform cell free drug screening assays, it is desirable
to immobilize either the ADH, or fragment, or its target molecule
to facilitate separation of complexes from uncomplexed forms of one
or both of the proteins, as well as to accommodate automation of
the assay.
[0440] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/ADH
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 cell lysates
(e.g., .sup.35S-labeled) and the candidate compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly, or in
the supernatant after the complexes is dissociated. Alternatively,
the complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of ADH-binding protein found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques. For example, either the polypeptide or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively,
antibodies reactive with the protein but which do not interfere
with binding of the protein to its target molecule can be
derivatized to the wells of the plate, and the protein trapped in
the wells by antibody conjugation. Preparations of an ADH-binding
target component, such as a coenzyme or a substrate, and a
candidate compound are incubated in the ADH-presenting wells and
the amount of complex trapped in the well can be quantitated.
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 ADJ
target molecule, or which are reactive with ADH and compete with
the target molecule; as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the target
molecule.
[0441] Modulators of ADH activity identified according to these
drug screening assays can be used to treat a subject with a
disorder mediated by ADH, by treating cells that express the ADH.
These methods of treatment include the steps of administering the
modulators of ADH activity in a pharmaceutical composition as
described herein, to a subject in need of such treatment.
[0442] The ADHs of the present invention are expressed in various
cell types. Tissues and/or cells in which the 21620 ADH is found
include, but are not limited to those shown in FIGS. 11 and 12.
Tissues in which the gene is highly expressed include brain, colon,
kidney, and small intestine. Moderate expression occurs in liver,
muscle, and testes. Lower positive expression occurs in the aorta,
breast, cervix, esophagus, heart, lung, lymph, ovary, placenta,
spleen, thymus, thyroid, and vein. The 21620 ADH is also expressed
in the malignant breast, lung, and colon tissue, and in colon
metastases to liver.
[0443] Hence the ADHs of the present invention are relevant to
treating disorders involving these tissues. Of particular interest
are malignant breast, liver, colon and liver metastases derived
from malignant colon tissue.
[0444] Disorders involving the spleen include, but are not limited
to, splenomegaly, including nonspecific acute splenitis, congestive
spenomegaly, and spenic infarcts; neoplasms, congenital anomalies,
and rupture. Disorders associated with splenomegaly include
infections, such as nonspecific splenitis, infectious
mononucleosis, tuberculosis, typhoid fever, brucellosis,
cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis,
kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and
echinococcosis; congestive states related to partial hypertension,
such as cirrhosis of the liver, portal or splenic vein thrombosis,
and cardiac failure; lymphohematogenous disorders, such as Hodgkin
disease, non-Hodgkin lymphomas/leukemia, multiple mycloma,
myeloproliferative disorders, hemolytic anemias, and
thrombocytopenic purpura; immunologic-inflammatory conditions, such
as rheumatoid arthritis and systemic lupus erythematosus; storage
diseases such as Gaucher disease, Niemann-Pick disease, and
mucopolysaccharidoses; and other conditions, such as amyloidosis,
primary neoplasms and cysts, and secondary neoplasms.
[0445] Disorders involving the lung include, but are not limited
to, congenital anomalies; atelectasis; diseases of vascular origin,
such as pulmonary congestion and edema, including hemodynamic
pulmonary edema and edema caused by microvascular injury, adult
respiratory distress syndrome (diffuse alveolar damage), pulmonary
embolism, hemorrhage, and infarction, and pulmonary hypertension
and vascular sclerosis; chronic obstructive pulmonary disease, such
as emphysema, chronic bronchitis, bronchial asthma, and
bronchiectasis; diffuse interstitial (infiltrative, restrictive)
diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity
pneumonitis, pulmonary eosinophilia (pulmonary infiltration with
eosinophilia), Bronchiolitis obliterans-organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, including Goodpasture
syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic
syndromes, pulmonary involvement in collagen vascular disorders,
and pulmonary alveolar proteinosis; complications of therapies,
such as drug-induced lung disease, radiation-induced lung disease,
and lung transplantation; tumors, such as bronchogenic carcinoma,
including paraneoplastic syndromes, bronchioloalveolar carcinoma,
neuroendocrine tumors, such as bronchial carcinoid, miscellaneous
tumors, and metastatic tumors; pathologies of the pleura, including
inflammatory pleural effusions, noninflammatory pleural effusions,
pneumothorax, and pleural tumors, including solitary fibrous tumors
(pleural fibroma) and malignant mesothelioma.
[0446] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0447] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
[0448] Disorders involving the uterus and endometrium include, but
are not limited to, endometrial histology in the menstrual cycle;
functional endometrial disorders, such as anovulatory cycle,
inadequate luteal phase, oral contraceptives and induced
endometrial changes, and menopausal and postmenopausal changes;
inflammations, such as chronic endometritis; adenomyosis;
endometriosis; endometrial polyps; endometrial hyperplasia;
malignant tumors, such as carcinoma of the endometrium; mixed
Mullerian and mesenchymal tumors, such as malignant mixed Mullerian
tumors; tumors of the myometrium, including leiomyomas,
leiomyosarcomas, and endometrial stromal tumors.
[0449] Disorders involving the brain include, but are not limited
to, disorders involving neurons, and disorders involving glia, such
as astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myclopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyclinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyclitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0450] Disorders involving T-cells include, but are not limited to,
cell-mediated hypersensitivity, such as delayed type
hypersensitivity and T-cell-mediated cytotoxicity, and transplant
rejection; autoimmune diseases, such as systemic lupus
erythematosus, Sjogren syndrome, systemic sclerosis, inflammatory
myopathies, mixed connective tissue disease, and polyarteritis
nodosa and other vasculitides; immunologic deficiency syndromes,
including but not limited to, primary immunodeficiencies, such as
thymic hypoplasia, severe combined immunodeficiency diseases, and
AIDS; leukopenia; reactive (inflammatory) proliferations of white
cells, including but not limited to, leukocytosis, acute
nonspecific lymphadenitis, and chronic nonspecific lymphadenitis;
neoplastic proliferations of white cells, including but not limited
to lymphoid neoplasms, such as precursor T-cell neoplasms, such as
acute lymphoblastic leukemia/lymphoma, peripheral T-cell and
natural killer cell neoplasms that include peripheral T-cell
lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis
fungoides and Sezary syndrome, and Hodgkin disease.
[0451] Diseases of the skin, include but are not limited to,
disorders of pigmentation and melanocytes, including but not
limited to, vitiligo, freckle, melasma, lentigo, nevocellular
nevus, dysplastic nevi, and malignant melanoma; benign epithelial
tumors, including but not limited to, seborrheic keratoses,
acanthosis nigricans, fibroepithelial polyp, epithelial cyst,
keratoacanthoma, and adnexal (appendage) tumors; premalignant and
malignant epidermal tumors, including but not limited to, actinic
keratosis, squamous cell carcinoma, basal cell carcinoma, and
merkel cell carcinoma; tumors of the dermis, including but not
limited to, benign fibrous histiocytoma, dermatofibrosarcoma
protuberans, xanthomas, and dermal vascular tumors; tumors of
cellular immigrants to the skin, including but not limited to,
histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and
mastocytosis; disorders of epidermal maturation, including but not
limited to, ichthyosis; acute inflammatory dermatoses, including
but not limited to, urticaria, acute eczematous dermatitis, and
erythema multiforme; chronic inflammatory dermatoses, including but
not limited to, psoriasis, lichen planus, and lupus erythematosus;
blistering (bullous) diseases, including but not limited to,
pemphigus, bullous pemphigoid, dermatitis herpetiformis, and
noninflammatory blistering diseases: epidermolysis bullosa and
porphyria; disorders of epidermal appendages, including but not
limited to, acne vulgaris; panniculitis, including but not limited
to, erythema nodosum and erythema induratum; and infection and
infestation, such as verrucae, molluscum contagiosum, impetigo,
superficial fungal infections, and arthropod bites, stings, and
infestations.
[0452] In normal bone marrow, the myelocyiic series
(polymorphoneuclear cells) make up approximately 60% of the
cellular elements, and the erythrocytic series, 20-30%.
Lymphocytes, monocytes, reticular cells, plasma cells and
megakaryocytes together constitute 10-20%. Lymphocytes make up
5-15% of normal adult marrow. In the bone marrow, cell types are
add mixed so that precursors of red blood cells (erythroblasts),
macrophages (monoblasts), platelets (megakaryocytes),
polymorphoneuclear leucocytes (myeloblasts), and lymphocytes
(lymphoblasts) can be visible in one microscopic field. In
addition, stem cells exist for the different cell lineages, as well
as a precursor stem cell for the committed progenitor cells of the
different lineages. The various types of cells and stages of each
would be known to the person of ordinary skill in the art and are
found, for example, on page 42 (FIG. 2-8) of Immunology,
Immunopathology and Immunity, Fifth Edition, Sell et al. Simon and
Schuster (1996), incorporated by reference for its teaching of cell
types found in the bone marrow. According, the invention is
directed to disorders arising from these cells. These disorders
include but are not limited to the following: diseases involving
hematopoeitic stem cells; committed lymphoid progenitor cells;
lymphoid cells including B and T-cells; committed myeloid
progenitors, including monocytes, granulocytes, and megakaryocytes;
and committed erythroid progenitors. These include but are not
limited to the leukemias, including B-lymphoid leukemias,
T-lymphoid leukemias, undifferentiated leukemias; erythroleukemia,
megakaryoblastic leukemia, monocytic; [leukemias are encompassed
with and without differentiation]; chronic and acute lymphoblastic
leukemia, chronic and acute lymphocytic leukemia, chronic and acute
myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic
and acute myeloid leukemia, myelomonocytic leukemia; chronic and
acute myeloblastic leukemia, chronic and acute myelogenous
leukemia, chronic and acute promyelocytic leukemia, chronic and
acute myelocytic leukemia, hematologic malignancies of
monocyte-macrophage lineage, such as juvenile chronic myelogenous
leukemia; secondary AML, antecedent hematological disorder;
refractory anemia; aplastic anemia; reactive cutaneous
angioendotheliomatosis; fibrosing disorders involving altered
expression in dendritic cells, disorders including systemic
sclerosis, E-M syndrome, epidemic toxic oil syndrome, eosinophilic
fasciitis localized forms of scleroderma, keloid, and fibrosing
colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma,
including primary head and neck squamous cell carcinoma; sarcoma,
including kaposi's sarcoma; fibroadanoma and phyllodes tumors,
including mammary fibroadenoma; stromal tumors; phyllodes tumors,
including histiocytoma; erythroblastosis; neurofibromatosis;
diseases of the vascular endothelium; demyelinating, particularly
in old lesions; gliosis, vasogenic edema, vascular disease,
Alzheimer's and Parkinson's disease; T-cell lymphomas; B-cell
lymphomas.
[0453] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0454] Disorders involving blood vessels include, but are not
limited to, responses of vascular cell walls to injury, such as
endothelial dysfunction and endothelial activation and intimal
thickening; vascular diseases including, but not limited to,
congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[0455] Disorders involving red cells include, but are not limited
to, anemias, such as hemolytic anemias, including hereditary
spherocytosis, hemolytic disease due to erythrocyte enzyme defects:
glucose-6-phosphate dehydrogenase deficiency, sickle cell disease,
thalassemia syndromes, paroxysmal nocturnal hemoglobinuria,
immunohemolytic anemia, and hemolytic anemia resulting from trauma
to red cells; and anemias of diminished erythropoiesis, including
megaloblastic anemias, such as anemias of vitamin B12 deficiency:
pernicious anemia, and anemia of folate deficiency, iron deficiency
anemia, anemia of chronic disease, aplastic anemia, pure red cell
aplasia, and other forms of marrow failure.
[0456] Disorders involving the thymus include developmental
disorders, such as DiGeorge syndrome with thymic hypoplasia or
aplasia; thymic cysts; thymic hypoplasia, which involves the
appearance of lymphoid follicles within the thymus, creating thymic
follicular hyperplasia; and thymomas, including germ cell tumors,
lymphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[0457] Disorders involving B-cells include, but are not limited to
precursor B-cell neoplasms, such as lymphoblastic
leukemia/lymphoma. Peripheral B-cell neoplasms include, but are not
limited to, chronic lymphocytic leukemia/small lymphocytic
lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and
related entities, lymphoplasmacytic lymphoma (Waldenstrom
macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma
(MALToma), and hairy cell leukemia.
[0458] Disorders involving the kidney include, but are not limited
to, congenital anomalies including, but not limited to, cystic
diseases of the kidney, that include but are not limited to, cystic
renal dysplasia, autosomal dominant (adult) polycystic kidney
disease, autosomal recessive (childhood) polycystic kidney disease,
and cystic diseases of renal medulla, which include, but are not
limited to, medullary sponge kidney, and nephronophthisis-uremic
medullary cystic disease complex, acquired (dialysis-associated)
cystic disease, such as simple cysts; glomerular diseases including
pathologies of glomerular injury that include, but are not limited
to, in situ immune complex deposition, that includes, but is not
limited to, anti-GBM nephritis, Heymann nephritis, and antibodies
against planted antigens, circulating immune complex nephritis,
antibodies to glomerular cells, cell-mediated immunity in
glomerulonephritis, activation of alternative complement pathway,
epithelial cell injury, and pathologies involving mediators of
glomerular injury including cellular and soluble mediators, acute
glomerulonephritis, such as acute proliferative (poststreptococcal,
postinfectious) glomerulonephritis, including but not limited to,
poststreptococcal glomerulonephritis and nonstreptococcal acute
glomerulonephritis, rapidly progressive (crescentic)
glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis (membranous nephropathy), minimal change disease
(lipoid nephrosis), focal segmental glomerulosclerosis,
membranoproliferative glomerulonephritis, IgA nephropathy (Berger
disease), focal proliferative and necrotizing glomerulonephritis
(focal glomerulonephritis), hereditary nephritis, including but not
limited to, Alport syndrome and thin membrane disease (benign
familial hematuria), chronic glomerulonephritis, glomerular lesions
associated with systemic disease, including but not limited to,
systemic lupus crythematosus, Henoch-Schonlein purpura, bacterial
endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary
and immunotactoid glomerulonephritis, and other systemic disorders;
diseases affecting tubules and interstitium, including acute
tubular necrosis and tubulointerstitial nephritis, including but
not limited to, pyelonephritis and urinary tract infection, acute
pyelonephritis, chronic pyelonephritis and reflux nephropathy, and
tubulointerstitial nephritis induced by drugs and toxins, including
but not limited to, acute drug-induced interstitial nephritis,
analgesic abuse nephropathy, nephropathy associated with
nonsteroidal anti-inflammatory drugs, and other tubulointerstitial
diseases including, but not limited to, urate nephropathy,
hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases
of blood vessels including benign nephrosclerosis, malignant
hypertension and accelerated nephrosclerosis, renal artery
stenosis, and thrombotic microangiopathies including, but not
limited to, classic (childhood) hemolytic-uremic syndrome, adult
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,
idiopathic HUS/TTP, and other vascular disorders including, but not
limited to, atherosclerotic ischemic renal disease, atheroembolic
renal disease, sickle cell disease nephropathy, diffuse cortical
necrosis, and renal infarcts; urinary tract obstruction
(obstructive uropathy); urolithiasis (renal calculi, stones); and
tumors of the kidney including, but not limited to, benign tumors,
such as renal papillary adenoma, renal fibroma or hamartoma
(renomedullary interstitial cell tumor), angiomyolipoma, and
oncocytoma, and malignant tumors, including renal cell carcinoma
(hypemephroma, adenocarcinoma of kidney), which includes urothelial
carcinomas of renal pelvis.
[0459] Disorders of the breast include, but are not limited to,
disorders of development; inflammations, including but not limited
to, acute mastitis, periductal mastitis, periductal mastitis
(recurrent subareolar abscess, squamous metaplasia of lactiferous
ducts), mammary duct ectasia, fat necrosis, granulomatous mastitis,
and pathologies associated with silicone breast implants;
fibrocystic changes; proliferative breast disease including, but
not limited to, epithelial hyperplasia, sclerosing adenosis, and
small duct papillomas; tumors including, but not limited to,
stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas,
and epithelial tumors such as large duct papilloma; carcinoma of
the breast including in situ (noninvasive) carcinoma that includes
ductal carcinoma in situ (including Paget's disease) and lobular
carcinoma in situ, and invasive (infiltrating) carcinoma including,
but not limited to, invasive ductal carcinoma, no special type,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms.
[0460] Disorders in the male breast include, but are not limited
to, gynecomastia and carcinoma.
[0461] Disorders involving the testis and epididymis include, but
are not limited to, congenital anomalies such as cryptorchidism,
regressive changes such as atrophy, inflammations such as
nonspecific epididymitis and orchitis, granulomatous (autoimmune)
orchitis, and specific inflammations including, but not limited to,
gonorrhea, mumps, tuberculosis, and syphilis, vascular disturbances
including torsion, testicular tumors including germ cell tumors
that include, but are not limited to, seminoma, spermatocytic
seminoma, embryonal carcinoma, yolk sac tumor choriocarcinoma,
teratoma, and mixed tumors, tumore of sex cord-gonadal stroma
including, but not limited to, leydig (interstitial) cell tumors
and sertoli cell tumors (androblastoma), and testicular lymphoma,
and miscellaneous lesions of tunica vaginalis.
[0462] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[0463] Disorders involving the thyroid include, but are not limited
to, hyperthyroidism; hypothyroidism including, but not limited to,
cretinism and myxedema; thyroiditis including, but not limited to,
hashimoto thyroiditis, subacute (granulomatous) thyroiditis, and
subacute lymphocytic (painless) thyroiditis; Graves disease;
diffuse and multinodular goiter including, but not limited to,
diffuse nontoxic (simple) goiter and multinodular goiter; neoplasms
of the thyroid including, but not limited to, adenomas, other
benign tumors, and carcinomas, which include, but are not limited
to, papillary carcinoma, follicular carcinoma, medullary carcinoma,
and anaplastic carcinoma; and cogenital anomalies.
[0464] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0465] Disorders involving the pancreas include those of the
exocrine pancreas such as congenital anomalies, including but not
limited to, ectopic pancreas; pancreatitis, including but not
limited to, acute pancreatitis; cysts, including but not limited
to, pseudocysts; tumors, including but not limited to, cystic
tumors and carcinoma of the pancreas; and disorders of the
endocrine pancreas such as, diabetes mellitus; islet cell tumors,
including but not limited to, insulinomas, gastrinomas, and other
rare islet cell tumors.
[0466] Disorders involving the small intestine include the
malabsorption syndromes such as, celiac sprue, tropical sprue
(postinfectious sprue), whipple disease, disaccharidase (lactase)
deficiency, abetalipoproteinemia, and tumors of the small intestine
including adenomas and adenocarcinoma.
[0467] Disorders related to reduced platelet number,
thrombocytopenia, include idiopathic thrombocytopenic purpura,
including acute idiopathic thrombocytopenic purpura, drug-induced
thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic
microangiopathies: thrombotic thrombocytopenic purpura and
hemolytic-uremic syndrome.
[0468] Disorders involving precursor T-cell neoplasms include
precursor T lymphoblastic leukemia/lymphoma. Disorders involving
peripheral T-cell and natural killer cell neoplasms include T-cell
chronic lymphocytic leukemia, large granular lymphocytic leukemia,
mycosis fingoides and Sezary syndrome, peripheral T-cell lymphoma,
unspecified, angioimmunoblastic T-cell lymphoma, angiocentric
lymphoma (NK/T-cell lymphoma.sup.4a), intestinal T-cell lymphoma,
adult T-cell leukemia/lymphoma, and anaplastic large cell
lymphoma.
[0469] Disorders involving the ovary include, for example,
polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma
peritonei and stromal hyperthecosis; ovarian tumors such as, tumors
of coelomic epithelium, serous tumors, mucinous tumors,
endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma,
brenner tumor, surface epithelial tumors; germ cell tumors such as
mature (benign) teratomas, monodermal teratomas, immature malignant
teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma;
sex cord-stomal tumors such as, granulosa-theca cell tumors,
thecoma-fibromas, androblastomas, hill cell tumors, and
gonadoblastoma; and metastatic tumors such as Krukenberg
tumors.
[0470] Bone-forming cells include the osteoprogenitor cells,
osteoblasts, and osteocytes. The disorders of the bone are complex
because they may have an impact on the skeleton during any of its
stages of development. Hence, the disorders may have variable
manifestations and may involve one, multiple or all bones of the
body. Such disorders include, congenital malformations,
achondroplasia and thanatophoric dwarfism, diseases associated with
abnormal matix such as type 1 collagen disease, osteoporosis, Paget
disease, rickets, osteomalacia, high-turnover osteodystrophy,
low-turnover of aplastic disease, osteonecrosis, pyogenic
osteomyelitis, tuberculous osteomyelitism, osteoma, osteoid
osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,
chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous
cortical defects, fibrous dysplasia, fibrosarcoma, malignant
fibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal
tumor, giant cell tumor, and metastatic tumors.
[0471] Disorders in which the ADH expression is relevant include,
but are not limited to, drug/alcohol interactions, susceptibility
to alcoholism, alcohol-induced organ injury such as alcoholic liver
cirrhosis, first-pass metabolism of alcohol, fetal alcohol
syndrome, and alcohol-related cancers including, but not limited to
cancers of the esophagus, oral cavity, upper gastrointestinal tract
and colorectum. Furthermore, ADH expression is also relevant to
alcohol-induced flushing. Alcohol-induced flushing is characterized
by the rapid onset of skin vasodilation of the face, neck and chest
regions after consumption of small amounts of alcohol. Tachycardia,
headache, nausea, hypotension, and extreme drowsiness are also
common symptoms of alcohol-induced flushing. Flush reactions have
been correlated with a deficiency or absence of the ADH2 enzyme
activity. ADH expression is also relevant in the pathogenesis of
male sterility and skin diseases, such as psoriasis.
Oxidoreductases have also been implicated in the pathophysiology of
neurodegenerative disorders and apoptotic processes related to
diseases such as Alzheimer's disease.
[0472] Treatment is defined as the application or administration of
a therapeutic agent to a patient, or application or administration
of a therapeutic agent to an isolated tissue or cell line from a
patient, who has a disease, a symptom of disease or a
predisposition toward a disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disease, the symptoms of disease or the predisposition toward
disease.
[0473] A therapeutic agent includes, but is not limited to, small
molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0474] The ADH polypeptides are thus useful for treating an
ADH-associated disorder characterized by aberrant expression or
activity of an ADH. 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) expression or activity of the
protein. In another embodiment, the method involves administering
the ADH as therapy to compensate for reduced or aberrant expression
or activity of the protein.
[0475] Methods for treatment include but are not limited to the use
of soluble ADH or fragments of the ADH protein that compete for
substrate or coenzyme binding, interfere with subunit interaction,
or interfere with the reaction mediated by the ADH polypeptide.
These ADHs or fragments can have a higher affinity for the target
so as to provide effective competition.
[0476] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
aberrant development or cellular differentiation. In another
example, the subject has a proliferative disease (e.g., cancer). In
another example, the subject has a disorder mediated by an altered
NADH/NAD.sup.+ redox potential, as described herein.
[0477] In yet another aspect of the invention, the proteins of the
invention 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 WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[0478] The ADH polypeptides also are useful to provide a target for
diagnosing a disease or predisposition to disease mediated by the
ADH, including, but not limited to, diseases involving tissues in
which the ADHs are expressed as disclosed herein, and particularly
in breast, lung, colon, and liver metastases derived from malignant
colon tissue. Accordingly, methods are provided for detecting the
presence, or levels of, the ADH in a cell, tissue, or organism. The
method involves contacting a biological sample with a compound
capable of interacting with the ADH such that the interaction can
be detected.
[0479] One agent for detecting ADH is an antibody capable of
selectively binding to ADH. A biological sample includes tissues,
cells and biological fluids isolated from a subject, as well as
tissues, cells and fluids present within a subject.
[0480] The ADH also provides a target for diagnosing active
disease, or predisposition to disease, in a patient having a
variant ADH. Thus, ADH can be isolated from a biological sample and
assayed for the presence of a genetic mutation that results in an
aberrant protein. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered ADH activity in cell-based or
cell-free assay, alteration in substrate or coenzyme binding,
altered interaction with ADH subunits, altered rate of substrate
oxidation/reduction, altered antibody-binding pattern, altered
isoelectric point, direct amino acid sequencing, and any other of
the known assay techniques useful for detecting mutations in a
protein in general or in an ADH specifically.
[0481] In vitro techniques for detection of ADH include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-ADH 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. Particularly useful are methods, which detect the
allelic variant of the ADH expressed in a subject, and methods,
which detect fragments of the ADH in a sample.
[0482] The ADH polypeptides are also useful in pharmacogenomic
analysis. Pharmacogenomics deal with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, e.g.,
Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem.
43(2):254-266. The clinical outcomes of these variations result in
severe toxicity of therapeutic drugs in certain individuals or
therapeutic failure of drugs in certain individuals as a result of
individual variation in metabolism. Thus, the genotype of the
individual can determine the way a therapeutic compound acts on the
body or the way the body metabolizes the compound. Further, the
activity of drug metabolizing enzymes affects both the intensity
and duration of drug action. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
treatment based on the individual's genotype. The discovery of
genetic polymorphisms in some drug metabolizing enzymes has
explained why some patients do not obtain the expected drug
effects, show an exaggerated drug effect, or experience serious
toxicity from standard drug dosages. Polymorphisms can be expressed
in the phenotype of the extensive metabolizer and the phenotype of
the poor metabolizer. Accordingly, genetic polymorphism may lead to
allelic protein variants of the ADH in which one or more of the ADH
functions in one population is different from those in another
population. The polypeptides thus allow a target to ascertain a
genetic predisposition that can affect treatment modality. Thus, in
an ADH-based treatment, polymorphism may give rise to catalytic
regions that are more or less active. Accordingly, dosage would
necessarily be modified to maximize the therapeutic effect within a
given population containing the polymorphism. As an alternative to
genotyping, specific polymorphic polypeptides could be
identified.
[0483] The ADH polypeptides are also useful for monitoring
therapeutic effects during clinical trials and other treatment.
Thus, the therapeutic effectiveness of an agent that is designed to
increase or decrease gene expression, protein levels or ADH
activity can be monitored over the course of treatment using the
ADH polypeptides as an end-point target. The monitoring can be, for
example, as follows: (i) obtaining a pre-administration sample from
a subject prior to administration of the agent; (ii) detecting the
level of expression or activity of the protein in the
pre-administration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the protein in the
post-administration samples; (v) comparing the level of expression
or activity of the protein in the pre-administration sample with
the protein in the post-administration sample or samples; and (vi)
increasing or decreasing the administration of the agent to the
subject accordingly.
Antibodies
[0484] The invention also provides antibodies that selectively bind
to the ADH and its variants and fragments. An antibody is
considered to selectively bind, even if it also binds to other
proteins that are not substantially homologous with the ADH. These
other proteins share homology with a fragment or domain of the ADH.
This conservation in specific regions gives rise to antibodies that
bind to both proteins by virtue of the homologous sequence. In this
case, it would be understood that antibody binding to the ADH is
still selective.
[0485] To generate antibodies, an isolated ADH polypeptide is used
as an immunogen to generate antibodies using standard techniques
for polyclonal and monoclonal antibody preparation. Either the
full-length protein or antigenic peptide fragment can be used.
Regions having a high antigenicity index are shown in FIGS. 8, 14,
18, 22, 26 and 30.
[0486] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents substrate or coenzyme binding or prevents the
oxidation of substrate. Antibodies can be developed against the
entire ADH or domains of the ADH as described herein. Antibodies
can also be developed against specific functional sites as
disclosed herein.
[0487] The antigenic peptide can comprise a contiguous sequence of
at least 12, 14, 15, or amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments, which may be
disclosed prior to the invention.
[0488] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be
used.
[0489] 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.
[0490] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
Antibody Uses
[0491] The antibodies can be used to isolate an ADH by standard
techniques, such as affinity chromatography or immunoprecipitation.
The antibodies can facilitate the purification of the natural ADH
from cells and recombinantly produced ADH expressed in host
cells.
[0492] The antibodies are useful to detect the presence of ADH in
cells or tissues to determine the pattern of expression of the ADH
among various tissues in an organism and over the course of normal
development.
[0493] The antibodies can be used to detect ADH in situ, in vitro,
or in a cell lysate or supernatant in order to evaluate the
abundance and pattern of expression.
[0494] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[0495] Antibody detection of circulating fragments of the full
length ADH can be used to identify ADH turnover.
[0496] Further, the antibodies can be used to assess ADH expression
in disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to ADH
function. When a disorder is caused by an inappropriate tissue
distribution, developmental expression, or level of expression of
the ADH protein, the antibody can be prepared against the normal
ADH protein. If a disorder is characterized by a specific mutation
in the ADH, antibodies specific for this mutant protein can be used
to assay for the presence of the specific mutant ADH. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular ADH peptide
regions.
[0497] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole ADH
or portions of the ADH.
[0498] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting ADH expression
level or the presence of aberrant ADHs and aberrant tissue
distribution or developmental expression, antibodies directed
against the ADH or relevant fragments can be used to monitor
therapeutic efficacy.
[0499] Antibodies accordingly 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.
[0500] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic ADH can be
used to identify individuals that require modified treatment
modalities.
[0501] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant ADH analyzed by electrophoretic
mobility, isoelectric point, tryptic peptide digest, and other
physical assays known to those in the art.
[0502] The antibodies are also useful for tissue typing. Thus,
where a specific ADH has been correlated with expression in a
specific tissue, antibodies that are specific for this ADH can be
used to identify a tissue type.
[0503] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[0504] The antibodies are also useful for inhibiting ADH function,
for example, blocking substrate or coenzyme binding or disrupting
the oxidation/reduction of substrate.
[0505] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting ADH function. An antibody can
be used, for example, to block coenzyme or substrate binding.
Antibodies can be prepared against specific fragments containing
sites required for function or against intact ADH associated with a
cell.
[0506] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[0507] The invention also encompasses kits for using antibodies to
detect the presence of an ADH protein in a biological sample. The
kit can comprise antibodies such as a labeled or labelable antibody
and a compound or agent for detecting ADH in a biological sample;
means for determining the amount of ADH in the sample; and means
for comparing the amount of ADH 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
ADH.
Polynucleotides
[0508] The nucleotide sequences in SEQ ID NOS:6, 8, 10, 12, and 14,
were obtained by sequencing the deposited human cDNA. Accordingly,
the sequence of the deposited clones are controlling as to any
discrepancies between the two and any reference to the sequences of
SEQ ID NOS:6, 8, 10, 12, and 14, includes reference to the
sequences of the deposited cDNAs.
[0509] The specifically disclosed cDNAs comprise the coding region
and 5' and 3' untranslated sequences in SEQ ID NOS:6, 8, 10, 12,
and 14.
[0510] The invention provides isolated polynucleotides encoding the
novel ADHs. The term "ADH polynucleotide" or "ADH nucleic acid"
refers to the sequences shown in SEQ ID NOS:6, 8, 10, 12, and 14 or
in the deposited cDNAs. The term "ADH polynucleotide" or "ADH
nucleic acid" further includes variants and fragments of the ADH
polynucleotides.
[0511] An "isolated" ADH nucleic acid is one that is separated from
other nucleic acid present in the natural source of the ADH nucleic
acid. Preferably, an "isolated" nucleic acid is free of sequences
which naturally flank the ADH 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. However, there
can be some flanking nucleotide sequences, for example up to about
5 KB. The important point is that the ADH nucleic acid is isolated
from flanking sequences such that it can be subjected to the
specific manipulations described herein, such as recombinant
expression, preparation of probes and primers, and other uses
specific to the ADH nucleic acid sequences.
[0512] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0513] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0514] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0515] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0516] The ADH polynucleotides can encode the mature protein plus
additional amino or carboxyterminal amino acids, or amino acids
interior to the mature polypeptide (when the mature form has more
than one polypeptide chain, for instance). Such sequences may play
a role in processing of a protein from precursor to a mature form,
facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0517] The ADH polynucleotides include, but are not limited to, the
sequence encoding the mature polypeptide alone, the sequence
encoding the mature polypeptide and additional coding sequences,
such as a leader or secretory sequence (e.g., a pre-pro or
pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[0518] ADH polynucleotides can be in the form of RNA, such as mRNA,
or in the form DNA, including cDNA and genomic DNA obtained by
cloning or produced by chemical synthetic techniques or by a
combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0519] ADH nucleic acid can comprise the nucleotide sequences shown
in SEQ ID NOS:6, 8, 10, 12, and 14, corresponding to human the
21620, 33756, 21676, 21612, and 21615 ADH cDNAs, respectfully.
[0520] In one embodiment, the ADH nucleic acid comprises only the
coding region.
[0521] The invention further provides variant ADH polynucleotides,
and fragments thereof, that differ from the nucleotide sequences
shown in SEQ ID NOS:6, 8, 10, 12, and 14 due to degeneracy of the
genetic code and thus encode the same protein as that encoded by
the nucleotide sequences shown in SEQ ID NOS:6, 8, 10, 12, and
14.
[0522] The invention also provides ADH nucleic acid molecules
encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[0523] Typically, variants have a substantial identity with nucleic
acid molecules of SEQ ID NOS:6, 8, 10, 12, and 14, and the
complements thereof. Variation can occur in either or both the
coding and non-coding regions. The variations can produce both
conservative and non-conservative amino acid substitutions.
[0524] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding an ADH that is at least about 60-65%,
65-70%, typically at least about 70-75%, more typically at least
about 80-85%, and most typically at least about 90-95% or more
homologous to the nucleotide sequence shown in SEQ ID NOS:6, 8, 10,
12, and 14, or a fragment of this sequence. Such nucleic acid
molecules can readily be identified as being able to hybridize
under stringent conditions, to the nucleotide sequence shown in SEQ
ID NOS:6, 8, 10, 12, and 14 or a fragment of the sequence. It is
understood that stringent hybridization does not indicate
substantial homology where it is due to general homology, such as
poly A sequences, or sequences common to all or most proteins, all
ADHs, or all short-chain dehydrogenase/reductases. Moreover, it is
understood that variants do not include any of the nucleic acid
sequences that may have been disclosed prior to the invention.
[0525] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 60-65% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95% or more
identical to each other remain hybridized to one another. 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, incorporated by reference.
One 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 50-65.degree. C. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times.SSC/0.1% SDS at room
temperature, or by one or more moderate stringency washes in
0.2.times.SSC/0.1% SDS at 42.degree. C., or washed in
0.2.times.SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:6, 8, 10, 12, and
14 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).
[0526] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[0527] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NOS:6, 8, 10, 12, and 14 or the complement of SEQ ID NOS: 6,
8, 10, 12, and 14. In one embodiment, the nucleic acid consists of
a portion of the nucleotide sequence of SEQ ID NOS: 6, 8, 10, 12,
and 14, and the complement of SEQ ID NOS: 6, 8, 10, 12, and 14.
[0528] It is understood that isolated fragments include any
contiguous sequence not disclosed prior to the invention as well as
sequences that are substantially the same and which are not
disclosed. Accordingly, if a fragment is disclosed prior to the
present invention, that fragment is not intended to be encompassed
by the invention. When a sequence is not disclosed prior to the
present invention, an isolated nucleic acid fragment is at least
about 12, preferably at least about 15, 18, 20, 23 or 25
nucleotides, and can be 30, 40, 50, 100, 200, 500 or more
nucleotides in length. Longer fragments, for example, 30 or more
nucleotides in length, which encode antigenic proteins or
polypeptides described herein are useful.
[0529] For the 21620 ADH, for example, nucleotide sequences from
about 265 to about 300, from about 782 to about 870, from about
1003 to about 1035, and from about 1096 to about 1158 are not
disclosed prior to the present invention. The nucleotide sequences
from about 1 to about 301 encompasses fragments greater than about
125, 135, 145 or 155 nucleotides; the nucleotide sequences from
about 138 to about 1159 encompasses fragments greater than 268,
280, 290, or 300 nucleotides; the nucleotide sequences from about
871 to about 1560 encompasses fragments greater than 265, 275, 285,
or 295; and the nucleotide sequences from about 1036 to about 1877
encompasses fragments greater than 266, 275, 285, or 295
nucleotides.
[0530] For the 33756ADH, for example, nucleotide sequences from
about 66 to about 242 are not disclosed prior to the present
invention. The nucleotide sequences from about 1 to about 454
encompass fragments greater than 21, 25, 30, or 35 nucleotides; the
nucleotide sequences from about 1 to about 700 encompass fragments
greater than 240, 250, 260 or 275 nucleotide; and the nucleotide
sequences from about 1 to about 1153 encompass fragments greater
than 574, 580, 590 or 600 nucleotides.
[0531] For the 21676 ADH, for example, nucleotide sequences from
about 1 to about 14, from about 69 to about 94, and from about 206
to about 1699 are not disclosed prior to the present invention. The
nucleotide sequences from about 1 to about 206 encompasses
fragments greater than 20, 25, 30, 35, 40 or 45 nucleotides.
[0532] For the 21612 ADH, for example, nucleotide sequences from
about 32 to about 51, from about 679 to about 710, and from about
1525 to about 2535 are not disclosed prior to the present
invention. The nucleotide sequences from about 1 to about 678
encompasses fragments greater than 247, 260, 270, or 280
nucleotides and the nucleotide sequences from about 147 to about
2535 encompasses fragments greater than 417, 425, 435, 445 or 455
nucleotides.
[0533] For the 21615 ADH, for example, nucleotide sequences from
about 538 to about 1615 are not disclosed prior to the present
invention. The nucleotide sequence from about nucleotide 1 to about
nucleotide 788 encompasses fragments greater than 230, 240, 250 or
260 nucleotides and the nucleotide sequence from about nucleotide
442 to about 1615 encompasses fragments greater than 670, 680, 690
or 700 nucleotides.
[0534] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length ADH polynucleotides. The
fragment can be single or double-stranded and can comprise DNA or
RNA. The fragment can be derived from either the coding or the
non-coding sequence.
[0535] In another embodiment an isolated ADH nucleic acid encodes
the entire coding region. In another embodiment the isolated ADH
nucleic acid encodes a sequence corresponding to the mature
protein. For example, the mature form of the 21676 ADH is from
about amino acid 16 to the last amino acid. Other fragments include
nucleotide sequences encoding the amino acid fragments described
herein.
[0536] Thus, ADH nucleic acid fragments further include sequences
corresponding to the domains described herein, subregions also
described, and specific functional sites. ADH nucleic acid
fragments also include combinations of the domains, segments, and
other functional sites described above. A person of ordinary skill
in the art would be aware of the many permutations that are
possible.
[0537] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary sill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[0538] However, it is understood that an ADH fragment includes any
nucleic acid sequence that does not include the entire gene.
[0539] The invention also provides ADH nucleic acid fragments that
encode epitope bearing regions of the ADH proteins described
herein.
[0540] Nucleic acid fragments, according to the present invention,
are not to be construed as encompassing those fragments that may
have been disclosed prior to the invention.
Polynucleotide Uses
[0541] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to 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 protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins 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 www.ncbi.nlm.nih.gov.
[0542] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of the nucleic
acid sequence shown in SEQ ID NOS: 6, 8, 10, 12, and 14 and the
complements thereof. More typically, the probe further comprises a
label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
[0543] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[0544] The ADH polynucleotides are thus useful for probes, primers,
and in biological assays.
[0545] Where the polynucleotides are used to assess ADH properties
or functions, such as in the assays described herein, all or less
than all of the entire cDNA can be useful. Assays specifically
directed to ADH functions, such as assessing agonist or antagonist
activity, encompass the use of known fragments. Further, diagnostic
methods for assessing ADH function can also be practiced with any
fragment, including those fragments that may have been known prior
to the invention. Similarly, in methods involving treatment of ADH
dysfunction, all fragments are encompassed including those, which
may have been known in the art.
[0546] The ADH polynucleotides are useful as a hybridization probe
for cDNA and genomic DNA to isolate a full-length cDNA and genomic
clones encoding the polypeptides described in SEQ ID NOS:5, 7, 9,
11, and 13, and to isolate cDNA and genomic clones that correspond
to variants producing the same polypeptides shown in SEQ ID NOS: 5,
7, 9, 11, and 13 or the other variants described herein. Variants
can be isolated from the same tissue and organism from which the
polypeptides shown in SEQ ID NOS: 5, 7, 9, 11, and 13, were
isolated, different tissues from the same organism, or from
different organisms. This method is useful for isolating genes and
cDNA that are developmentally-controlled and therefore may be
expressed in the same tissue or different tissues at different
points in the development of an organism.
[0547] The probe can correspond to any sequence along the entire
length of the gene encoding the ADH. Accordingly, it could be
derived from 5' noncoding regions, the coding region, and 3'
noncoding regions.
[0548] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NOS:6, 8, 10, 12, and 14, or a fragment thereof,
such as an oligonucleotide of at least 12, 15, 30, 50, 100, 250 or
500 nucleotides in length and sufficient to specifically hybridize
under stringent conditions to mRNA or DNA.
[0549] Fragments of the polynucleotides described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an mRNA and a larger or full-length
cDNA can be produced.
[0550] The fragments are also useful to synthesize antisense
molecules of desired length and sequence.
[0551] Antisense nucleic acids of the invention can be designed
using the nucleotide sequences of SEQ ID NOS:6, 8, 10, 12, and 14,
and 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, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[0552] Additionally, the nucleic acid molecules of the 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 acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). 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 et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity 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. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[0553] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell ADHs 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. WO 88/0918) or
the blood brain barrier (see, e.g., PCT Publication No. WO
89/10134). 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).
[0554] The ADH polynucleotides are also useful as primers for PCR
to amplify any given region of an ADH polynucleotide.
[0555] The ADH polynucleotides are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the ADH polypeptides. Vectors also
include insertion vectors, used to integrate into another
polynucleotide sequence, such as into the cellular genome, to alter
in situ expression of ADH genes and gene products. For example, an
endogenous ADH coding sequence can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[0556] The ADH polynucleotides are also useful for expressing
antigenic portions of the ADH proteins.
[0557] The ADH polynucleotides are also useful as probes for
determining the chromosomal positions of the ADH polynucleotides by
means of in situ hybridization methods, such as FISH. (For a review
of this technique, see Verma et al. (1988) Human Chromosomes: A
Manual of Basic Techniques (Pergamon Press, New York), and PCR
mapping of somatic cell hybrids. The mapping of the sequences to
chromosomes is an important first step in correlating these
sequences with genes associated with disease.
[0558] 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.
[0559] 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 et al. ((1987) Nature 325:783-787).
[0560] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified 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.
[0561] The ADH polynucleotide probes are also useful to determine
patterns of the presence of the gene encoding the ADHs and their
variants with respect to tissue distribution, for example, whether
gene duplication has occurred and whether the duplication occurs in
all or only a subset of tissues. The genes can be naturally
occurring or can have been introduced into a cell, tissue, or
organism exogenously.
[0562] The ADH polynucleotides are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from genes encoding the polynucleotides described herein.
[0563] The ADH polynucleotides are also useful for constructing
host cells expressing a part, or all, of the ADH polynucleotides
and polypeptides.
[0564] The ADH polynucleotides are also useful for constructing
transgenic animals expressing all, or a part, of the ADH
polynucleotides and polypeptides.
[0565] The ADH polynucleotides are also useful for making vectors
that express part, or all, of the ADH polypeptides.
[0566] The ADH polynucleotides are also useful as hybridization
probes for determining the level of ADH nucleic acid expression.
Accordingly, the probes can be used to detect the presence of, or
to determine levels of, ADH nucleic acid in cells, tissues, and in
organisms. The nucleic acid whose level is determined can be DNA or
RNA. Accordingly, probes corresponding to the polypeptides
described herein can be used to assess gene copy number in a given
cell, tissue, or organism. This is particularly relevant in cases
in which there has been an amplification of the ADH genes.
[0567] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
ADH genes, as on extrachromosomal elements or as integrated into
chromosomes in which the ADH gene is not normally found, for
example as a homogeneously staining region.
[0568] These uses are relevant for diagnosis of disorders involving
an increase or decrease in ADH expression relative to normal, such
as a proliferative disorder or a differentiative or developmental
disorder.
[0569] Tissues and/or cells in which the 21620 ADH is expressed are
shown in FIGS. 11 and 12 and are described above herein. As such,
the gene is particularly relevant for the treatment of disorders
involving these tissues.
[0570] Furthermore, disorders in which ADH expression is relevant
are disclosed herein above.
[0571] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of ADH nucleic acid, in which a test sample
is obtained from a subject and nucleic acid (e.g., mRNA, genomic
DNA) is detected, wherein the presence of the nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant expression or activity of the
nucleic acid.
[0572] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[0573] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0574] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express the ADH, such as by
measuring the level of an ADH-encoding nucleic acid in a sample of
cells from a subject e.g., mRNA or genomic DNA, or determining if
the ADH gene has been mutated.
[0575] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate ADH nucleic acid expression
(e.g., antisense, polypeptides, peptidomimetics, small molecules or
other drugs). A cell is contacted with a candidate compound and the
expression of mRNA determined. The level of expression of the mRNA
in the presence of the candidate compound is compared to the level
of expression of the mRNA in the absence of the candidate compound.
The candidate compound can then be identified as a modulator of
nucleic acid expression based on this comparison and be used, for
example to treat a disorder characterized by aberrant nucleic acid
expression. The modulator can bind to the nucleic acid or
indirectly modulate expression, such as by interacting with other
cellular components that affect nucleic acid expression.
[0576] 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 gent to a subject) in patients or in
transgenic animals.
[0577] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the ADH gene. The method typically
includes assaying the ability of the compound to modulate the
expression of the ADH nucleic acid and thus identifying a compound
that can be used to treat a disorder characterized by undesired ADH
nucleic acid expression.
[0578] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
ADH nucleic acid or recombinant cells genetically engineered to
express specific nucleic acid sequences.
[0579] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[0580] The assay for ADH nucleic acid expression can involve direct
assay of nucleic acid levels, such as mRNA levels, or on collateral
compounds involved in the ADH catalized reaction (such as
oxidized/reduced products, NAD.sup.+/NADH ratio, or components of
the retinoic and signaling pathway). Further, the expression of
genes that are up- or down-regulated in response to the ADH signal
pathway can also be assayed. In this embodiment the regulatory
regions of these genes can be operably linked to a reporter gene
such as luciferase.
[0581] Thus, modulators of ADH gene expression can be identified in
a method wherein a cell is contacted with a candidate compound and
the expression of mRNA determined. The level of expression of ADH
mRNA in the presence of the candidate compound is compared to the
level of expression of ADH mRNA in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of nucleic acid expression based on this comparison and
be used, for example to treat a disorder characterized by aberrant
nucleic acid expression. When expression of mRNA is statistically
significantly greater in the presence of the candidate compound
than in its absence, the candidate compound is identified as a
stimulator of nucleic acid expression. When nucleic acid expression
is statistically significantly less in the presence of the
candidate compound than in its absence, the candidate compound is
identified as an inhibitor of nucleic acid expression.
[0582] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate ADH nucleic
acid expression. Modulation includes both up-regulation (i.e.
activation or agonization) or down-regulation (suppression or
antagonization) or effects on nucleic acid activity (e.g. when
nucleic acid is mutated or improperly modified). Treatment is of
disorders characterized by aberrant expression or activity of the
nucleic acid. Disorders that the gene is particularly relevant for
treating have been disclosed herein above.
[0583] Alternatively, a modulator for ADH nucleic acid expression
can be a small molecule or drug identified using the screening
assays described herein as long as the drug or small molecule
inhibits the ADH nucleic acid expression.
[0584] The ADH polynucleotides are also useful for monitoring the
effectiveness of modulating compounds on the expression or activity
of the ADH gene in clinical trials or in a treatment regimen. Thus,
the gene expression pattern can serve as a barometer for the
continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance. The gene expression pattern can also serve as a marker
indicative of a physiological response of the affected cells to the
compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0585] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[0586] The ADH polynucleotides are also useful in diagnostic assays
for qualitative changes in ADH nucleic acid, and particularly in
qualitative changes that lead to pathology. The polynucleotides can
be used to detect mutations in ADH genes and gene expression
products such as mRNA. The polynucleotides can be used as
hybridization probes to detect naturally-occurring genetic
mutations in the ADH gene and thereby to determine whether a
subject with the mutation is at risk for a disorder caused by the
mutation. Mutations include deletion, addition, or substitution of
one or more nucleotides in the gene, chromosomal rearrangement,
such as inversion or transposition, modification of genomic DNA,
such as aberrant methylation patterns or changes in gene copy
number, such as amplification. Detection of a mutated form of the
ADH gene associated with a dysfunction provides a diagnostic tool
for an active disease or susceptibility to disease when the disease
results from overexpression, underexpression, or altered expression
of an ADH.
[0587] Mutations in the ADH gene can be detected at the nucleic
acid level by a variety of techniques. Genomic DNA can be analyzed
directly or can be amplified by using PCR prior to analysis. RNA or
cDNA can be used in the same way.
[0588] In certain embodiments, detection of the mutation 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) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
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 patient, 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
gene under conditions such that hybridization and amplification of
the 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.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0589] 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.
[0590] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[0591] Alternatively, mutations in an ADH gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0592] Further, sequence-specific ribozymes (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.
[0593] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0594] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[0595] Furthermore, sequence differences between a mutant ADH gene
and a wild-type gene can be determined by direct DNA sequencing. 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).
[0596] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). 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 one 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).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[0597] In other embodiments, genetic mutations 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
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin
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.
[0598] The ADH polynucleotides are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
polynucleotides can be used to study the relationship between an
individual's genotype and the individual's response to a compound
used for treatment (pharmacogenomic relationship). In the present
case, for example, a mutation in the ADH gene that results in
altered affinity for a coenzyme could result in an excessive or
decreased drug effect with standard concentrations of the coenzyme
that activates the ADH. Accordingly, the ADH polynucleotides
described herein can be used to assess the mutation content of the
gene in an individual in order to select an appropriate compound or
dosage regimen for treatment.
[0599] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[0600] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[0601] The ADH polynucleotides are also useful for chromosome
identification when the sequence is identified with an individual
chromosome and to a particular location on the chromosome. First,
the DNA sequence is matched to the chromosome by in situ or other
chromosome-specific hybridization. Sequences can also be correlated
to specific chromosomes by preparing PCR primers that can be used
for PCR screening of somatic cell hybrids containing individual
chromosomes from the desired species. Only hybrids containing the
chromosome containing the gene homologous to the primer will yield
an amplified fragment. Sublocalization can be achieved using
chromosomal fragments. Other strategies include prescreening with
labeled flow-sorted chromosomes and preselection by hybridization
to chromosome-specific libraries. Further mapping strategies
include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the 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.
[0602] The ADH polynucleotides can also be used to identify
individuals from small biological samples. This can be done for
example using restriction fragment-length polymorphism (RFLP) to
identify an individual. Thus, the polynucleotides described herein
are useful as DNA markers for RFLP (See U.S. Pat. No.
5,272,057).
[0603] Furthermore, the ADH sequence can be used to provide an
alternative technique, which determines the actual DNA sequence of
selected fragments in the genome of an individual. Thus, the ADH
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 DNA from an individual for subsequent
sequencing.
[0604] 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. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
ADH sequences can be used to obtain such identification sequences
from individuals and from tissue. The sequences represent unique
fragments of the human genome. 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.
[0605] If a panel of reagents from the sequences 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.
[0606] The ADH polynucleotides can also be used in forensic
identification procedures. PCR technology can be used to amplify
DNA sequences taken from very small biological samples, such as a
single hair follicle, body fluids (e.g. blood, saliva, or semen).
The amplified sequence can then be compared to a standard allowing
identification of the origin of the sample.
[0607] The ADH polynucleotides can thus 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 described 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 the noncoding region are
particularly useful since greater polymorphism occurs in the
noncoding regions, making it easier to differentiate individuals
using this technique.
[0608] The ADH polynucleotides 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. This is useful in cases in which a
forensic pathologist is presented with a tissue of unknown origin.
Panels of ADH probes can be used to identify tissue by species
and/or by organ type.
[0609] In a similar fashion, these primers and 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).
[0610] Alternatively, the ADH polynucleotides can be used directly
to block transcription or translation of ADH gene sequences by
means of antisense or ribozyme constructs. Thus, in a disorder
characterized by abnormally high or undesirable ADH gene
expression, nucleic acids can be directly used for treatment.
[0611] The ADH polynucleotides are thus useful as antisense
constructs to control ADH gene expression in cells, tissues, and
organisms. A DNA antisense polynucleotide is designed to be
complementary to a region of the gene involved in transcription,
preventing transcription and hence production of ADH protein. An
antisense RNA or DNA polynucleotide would hybridize to the mRNA and
thus block translation of mRNA into ADH protein.
[0612] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NOS:6, 8, 10, 12,
and 14, which also includes the start codon and antisense molecules
which are complementary to a fragment of the 3' untranslated region
of SEQ ID NOS:6, 8, 10, 12, and 14.
[0613] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of an ADH nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired ADH nucleic acid expression.
This technique involves cleavage by means of ribozymes containing
nucleotide sequences complementary to one or more regions in the
mRNA that attenuate the ability of the mRNA to be translated.
Possible regions include coding regions and particularly coding
regions corresponding to the catalytic and other functional
activities of the ADH protein.
[0614] The ADH polynucleotides also provide vectors for gene
therapy in patients containing cells that are aberrant in ADH gene
expression. Thus, recombinant cells, which include the patient's
cells that have been engineered ex vivo and returned to the
patient, are introduced into an individual where the cells produce
the desired ADH protein to treat the individual.
[0615] The invention also encompasses kits for detecting the
presence of an ADH nucleic acid in a biological sample. For
example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting ADH nucleic
acid in a biological sample; means for determining the amount of
ADH nucleic acid in the sample; and means for comparing the amount
of ADH nucleic acid 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 ADH mRNA or
DNA.
Computer Readable Means
[0616] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0617] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0618] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0619] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0620] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0621] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0622] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0623] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software includes, but is not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0624] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
Vectors/Host Cells
[0625] The invention also provides vectors containing the ADH
polynucleotides. The term "vector" refers to a vehicle, preferably
a nucleic acid molecule that can transport the ADH polynucleotides.
When the vector is a nucleic acid molecule, the ADH polynucleotides
are covalently linked to the vector nucleic acid. With this aspect
of the invention, the vector includes a plasmid, single or double
stranded phage, a single or double stranded RNA or DNA viral
vector, or artificial chromosome, such as a BAC, PAC, YAC, OR
MAC.
[0626] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the ADH polynucleotides. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the ADH polynucleotides when the host cell
replicates.
[0627] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the ADH
polynucleotides. The vectors can function in procaryotic or
eukaryotic cells or in both (shuttle vectors).
[0628] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the ADH polynucleotides
such that transcription of the polynucleotides is allowed in a host
cell. The polynucleotides can be introduced into the host cell with
a separate polynucleotide capable of affecting transcription. Thus,
the second polynucleotide may provide a trans-acting factor
interacting with the cis-regulatory control region to allow
transcription of the ADH polynucleotides from the vector.
Alternatively, a trans-acting factor may be supplied by the host
cell. Finally, a trans-acting factor can be produced from the
vector itself.
[0629] It is understood, however, that in some embodiments,
transcription and/or translation of the ADH polynucleotides can
occur in a cell-free system.
[0630] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[0631] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0632] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0633] A variety of expression vectors can be used to express an
ADH polynucleotide. Such vectors include chromosomal, episomal, and
virus-derived vectors, for example vectors derived from bacterial
plasmids, from bacteriophage, from yeast episomes, from yeast
chromosomal elements, including yeast artificial chromosomes, from
viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia
viruses, adenoviruses, poxviruses, pseudorabies viruses, and
retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0634] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e., tissue specific) or may provide
for inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0635] The ADH polynucleotides can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0636] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0637] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the ADH
polypeptides. Fusion vectors can increase the expression of a
recombinant protein, increase the solubility of the recombinant
protein, and aid in the purification of the protein by acting for
example as a ligand for affinity purification. A proteolytic
cleavage site may be introduced at the junction of the fusion
moiety so that the desired polypeptide can ultimately be separated
from the fusion moiety. Proteolytic enzymes include, but are not
limited to, factor Xa, thrombin, and enterokinase. Typical fusion
expression vectors include pGEX (Smith et al. (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. 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. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[0638] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[0639] The ADH polynucleotides can also be expressed by expression
vectors that are operative in yeast. Examples of vectors for
expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari
et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan et al. (1982) Cell
30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and
pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0640] The ADH polynucleotides can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL
series (Lucklow et al. (1989) Virology 170:31-39).
[0641] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[0642] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the ADH
polynucleotides. The person of ordinary skill in the art would be
aware of other vectors suitable for maintenance propagation or
expression of the polynucleotides described herein. These are found
for example in Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0643] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0644] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0645] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[0646] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the ADH polynucleotides can be introduced
either alone or with other polynucleotides that are not related to
the ADH polynucleotides such as those providing trans-acting
factors for expression vectors. When more than one vector is
introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the ADH polynucleotide
vector.
[0647] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0648] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0649] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0650] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the ADH polypeptides or heterologous
to these polypeptides.
[0651] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0652] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
Uses of Vectors and Host Cells
[0653] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also 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.
[0654] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing ADH proteins or
polypeptides that can be further purified to produce desired
amounts of ADH protein or fragments. Thus, host cells containing
expression vectors are useful for polypeptide production.
[0655] Host cells are also useful for conducting cell-based assays
involving the ADH or ADH fragments. Thus, a recombinant host cell
expressing a native ADH is useful to assay for compounds that
stimulate or inhibit ADH function. This includes gene expression at
the level of transcription or translation, interactions with
coenzymes, substrates or ADH subunits, and catalysis of substrate
oxidation/reduction.
[0656] Host cells are also useful for identifying ADH mutants in
which these functions are affected. If the mutants naturally occur
and give rise to a pathology, host cells containing the mutations
are useful to assay compounds that have a desired effect on the
mutant ADH (for example, stimulating or inhibiting function) which
may not be indicated by their effect on the native ADH.
[0657] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation by means of a heterologous domain,
segment, site, and the like, as disclosed herein.
[0658] Further, mutant ADHs can be designed in which one or more of
the various functions is engineered to be increased or decreased
(e.g., coenzyme, substrate, or ADH subunits) and used to augment or
replace ADH proteins in an individual. Thus, host cells can provide
a therapeutic benefit by replacing an aberrant ADH or providing an
aberrant ADH that provides a therapeutic result. In one embodiment,
the cells provide ADHs that are abnormally active.
[0659] In another embodiment, the cells provide ADH that are
abnormally inactive. These ADHs can compete with endogenous ADHs in
the individual.
[0660] In another embodiment, cells expressing ADHs that are not
catalytically active, are introduced into an individual in order to
compete with endogenous ADHs for substrate, coenzymes or ADH
subunits. For example, in the case in which excessive amounts of an
ADH substrate is part of a treatment modality, it may be necessary
to inactivate this molecule at a specific point in treatment.
Providing cells that compete for the molecule, but which cannot be
affected by ADH activation would be beneficial.
[0661] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous ADH polynucleotide
sequences in a host cell genome. The host cell includes, but is not
limited to, a stable cell line, cell in vivo, or cloned
microorganism. This technology is more fully described in WO
93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the ADH polynucleotides or sequences proximal or
distal to an ADH gene are allowed to integrate into a host cell
genome by homologous recombination where expression of the gene can
be affected. In one embodiment, regulatory sequences are introduced
that either increase or decrease expression of an endogenous
sequence. Accordingly, an ADH protein can be produced in a cell not
normally producing it. Alternatively, increased expression of ADH
protein can be effected in a cell normally producing the protein at
a specific level. Further, expression can be decreased or
eliminated by introducing a specific regulatory sequence. The
regulatory sequence can be heterologous to the ADH protein sequence
or can be a homologous sequence with a desired mutation that
affects expression. Alternatively, the entire gene can be deleted.
The regulatory sequence can be specific to the host cell or capable
of functioning in more than one cell type. Still further, specific
mutations can be introduced into any desired region of the gene to
produce mutant ADH proteins. Such mutations could be introduced,
for example, into the specific functional regions such as the
substrate-binding site.
[0662] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered ADH gene. Alternatively, the host
cell can be a stem cell or other early tissue precursor that gives
rise to a specific subset of cells and can be used to produce
transgenic tissues in an animal. See also Thomas et al., Cell
51:503 (1987) for a description of homologous recombination
vectors. The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced gene
has homologously recombined with the endogenous ADH gene is
selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected
cells are 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
vectors and 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; WO 91/01140; and
WO 93/04169.
[0663] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. 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 in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of an ADH protein and identifying and evaluating
modulators of ADH protein activity.
[0664] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0665] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which ADH polynucleotide sequences have
been introduced.
[0666] A transgenic animal can be produced by introducing 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. Any of the ADH
nucleotide sequences can be introduced as a transgene into the
genome of a non-human animal, such as a mouse.
[0667] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the ADH
protein to particular cells.
[0668] 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 the transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0669] 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)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. 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 is
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.
[0670] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
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 blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0671] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect substrate and coenzyme binding, and oxidation of the
substrate may not be evident from in vitro cell-free or cell-based
assays. Accordingly, it is useful to provide non-human transgenic
animals to assay in vivo ADH function, including substrate and
coenzyme interactions and substrate oxidation. Similar methods
could be used to determine the effect of specific mutant ADHs and
the effect of chimeric ADHs on such enzyme functions. It is also
possible to assess the effect of null mutations, that is mutations
that substantially or completely eliminate one or more ADH
functions.
[0672] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the ADH
protein in a transgenic animal, into a cell in culture or in vivo.
When introduced in vivo, the nucleic acid is introduced into an
intact organism such that one or more cell types and, accordingly,
one or more tissue types, express the nucleic acid encoding the ADH
protein. Alternatively, the nucleic acid can be introduced into
virtually all cells in an organism by transfecting a cell in
culture, such as an embryonic stem cell, as described herein for
the production of transgenic animals, and this cell can be used to
produce an entire transgenic organism. As described, in a further
embodiment, the host cell can be a fertilized oocyte. Such cells
are then allowed to develop in a female foster animal to produce
the transgenic organism.
Pharmaceutical Compositions
[0673] The ADH nucleic acid molecules, protein (such as an
extracellular loop), modulators of the protein, and antibodies
(also referred to herein as "active compounds") can be incorporated
into pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically comprise the
nucleic acid molecule, protein, modulator, or antibody and a
pharmaceutically acceptable carrier.
[0674] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation, in
vivo, of polynucleotides that have been exogenously introduced into
a subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[0675] 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, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions.
[0676] 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
ampules, disposable syringes or multiple dose vials made of glass
or plastic.
[0677] 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 polyethylene 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 mannitol, 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.
[0678] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an ADH protein or anti-ADH
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.
[0679] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. 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.
[0680] 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.
[0681] 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.
[0682] 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.
[0683] 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.
[0684] 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.
[0685] 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 (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
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.
[0686] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0687] 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.
[0688] 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. 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.
[0689] 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.
[0690] 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. 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.
[0691] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
CHAPTER 3
23484, a Novel Human Ubiquitin Protease
BACKGROUND OF THE INVENTION
The Ubiquitin System
[0692] Several biological processes are controlled by the
ubiquitination of cellular protein. Cellular processes that are
affected by ubiquitin modification include the regulation of gene
expression, regulation of the cell cycle and cell division,
cellular housekeeping, cell-specific metabolic pathways, disposal
of mutated or post-translationally damaged proteins, the cellular
stress response, modification of cell surface receptors, DNA
repair, import of proteins into mitochondria, uptake of precursors
into neurons, biogenesis of mitochondria, ribosomes, and
peroxisomes, apoptosis, and growth factor-mediated signal
transduction.
[0693] For some protein substrates ubiquitination leads to protein
degradation by the 26S proteasomal complex. A wide variety of
protein substrates are degraded by the 26S proteasomal complex
following ubiquitination of the substrate. Degradation of a protein
by the ubiquitin system involves two steps. The first involves the
covalent attachment of multiple ubiquitin molecules to the
substrate protein. The second involves degradation of the
ubiquitinated protein by the 26S proteasome. In some cases,
degradation of the ubiquitinated protein can occur by means of the
lysosomal pathway.
[0694] The 26S proteasome comprises a 20S core catalytic complex
which is flanked by two 19S regulatory complexes. The 26S complex
recognizes ubiquitinated proteins. Substrate recognition by the 26S
proteasome, however, may be mediated by the interaction of specific
subunits of the 19S complex with the ubiquitin chain. The
ubiquitinated protein is degraded by specific and energy-dependent
proteases into free amino acids and free and reutilizable
ubiquitin.
[0695] The 19S regulatory complex consists of many subunits that
can be classified into ATPases and non-ATPases. This complex is
thought to act in recognition, unfolding, and translocation of the
substrates into the 20S proteasome for proteolysis. The regulatory
complex contains isopeptidases capable of deubiquitinating
substrates (Spataro et al. (1998) British Journal of Cancer
77:448-455).
[0696] The ubiquitin proteasome pathway functions to degrade
abnormal proteins, short-lived normal proteins, long-lived normal
proteins, and proteins of the endoplasmic recticulum. Important
regulatory proteins rapidly inactivated by proteolysis include
c-JUN, c-FOS, and p53 (Lecker et al. (1999) Journa of Nutrition
129:227 S-237S). Conditions that stimulate protein degradation by
the ubiquitin proteasome pathway include eating disorders, renal
tubular defects, diabetes, uremia, neuromuscular disease,
immobilization, burn injuries, sepsis, cancer, cachexia,
hyperadrenocortisolism and hyperthyroidism.
[0697] Cellular proteins degraded by the ubiquitin system include
cell cycle regulators, including mitotic cyclins, G1 cyclins, CDK
inhibitors, anaphase inhibitors, transcription factors, tumor
suppressors, and oncoproteins such as NF-.kappa.B and
I.kappa.B.alpha., p53, JUN, .beta.-catenin, E2F-1, and membrane
proteins such as Step 2p, GH receptor, T-cell receptor,
platelet-derived growth factor, lymphocyte homing receptor, MET
tyrosine kinase receptor, hepatocyte growth factor-scatter factor,
connexin 43, the high affinity IgE receptor, the prolactin
receptor, and the EGF receptor (Hershko et al. (1998) Annual Review
of Biochemistry 67:425-479).
[0698] Ubiquitination does not only result in proteolytic
degradation. For some protein substrates, ubiquitination is a
reversible post-translational modification that can regulate
cellular targeting and enzymatic activity. This includes targeting
to the vacuole, activation of enzyme activity, such as Ik.beta.
kinase activation, and activation of cytokine receptor-mediated
signal transduction (D'Andrea et al. (1998) Critical Reviews In
Biochemistry and Molecular Biology 33:337-352). The T-cell receptor
undergoes ubiquitination in response to receptor engagement.
Platelet derived growth factor undergoes multiple ubiquitination
following ligand binding. Soluble steel factor has been shown to
stimulate rapid polyubiquitination of the c-KIT receptor.
[0699] It has been shown that protein degradation accounts for
regulation of proteins such as cyclins, cyclin-dependent kinase
inhibitors, p53, c-JUN and c-FOS (Spitaro et al. above). The
ubiquitin system has also shown to be involved in antigen
presentation. The 26S proteasome is responsible for processing
MHC-restricted class I antigens (Spitaro et al. above).
[0700] The ubiquitin system has been implicated in various
diseases. One group includes pathology that results from loss of
function, a mutation in an enzyme or substrate that leads to
stabilization of the protein and consequent build up of a protein
to abnormally high levels. The second involves pathologies that
result from a gain of function that produces increased protein
degradation.
[0701] The ubiquitin system has been implicated in various
malignancies. In cervical carcinoma, low levels of p53 have been
found. This protein is targeted for degradation by HPV
E6-associated protein. Removal of the suppressor by this
oncoprotein may be a mechanism utilized by the virus to transform
cells. Other results have shown that c-JUN, but not the
transforming counterpart, v-JUN, is ubiquitinated and subsequently
degraded. Other studies show that low levels of p27, a cell
division kinase inhibitor whose degradation is necessary for proper
cell cycle progression, is correlated with colorectal, and breast
carcinomas. The low level of this enzyme is due to activation of
the ubiquitin system.
[0702] Human genetic diseases involving aberrant proteolysis have
been reviewed (Kato (1999) Human Mutation 13:87-98). Cystic
fibrosis has been correlated with the ubiquitin system. The cystic
fibrosis transmembrane regulator in cystic fibrosis patients is
almost completely degraded by the ubiquitin system so that an
abnormally low amount of the wild type protein is found on the cell
surface. In Angelman's syndrome, one of the enzymes involved in
ubiquitination (E3) is affected. In Liddle syndrome, the E3 enzyme
is also affected.
[0703] The ubiquitin system can also affect the immune and
inflammatory response. The persistence of EBNA-1 contributes to
some virus related pathologies. A sequence on this protein was
found to inhibit degradation by the ubiquitin system. This
inhibited processing and subsequent presentation of viral epitopes
by MHC protein.
[0704] The ubiquitin system has also been implicated in
neurodegenerative diseases. Ubiquitin immunohistochemistry has
shown enrichment of ubiquitin conjugates in senile plaques,
lysosomes, endosomes, and a variety of inclusion bodies and
degenerative fibers in many neurodegenerative diseases, such as
Alzheimer's, Parkinson's and Lewy body diseases, amyotrophic
lateral sclerosis, and Creutzfeld-Jakob disease. Further, in
Huntington disease and spinocerebellar ataxias, the proteins
encoded by the affected genes aggregate in ubiquitin- and
proteasome-positive intranuclear inclusion bodies.
[0705] The ubiquitin system has been associated with muscle wasting
(Mitch et al. (1999) American Journal of Physiology 276:C1132-C1138
and Lecker et al. above) and muscle-wasting diseases and in such
pathological states as fasting, starvation, sepsis, and
denervation, all of which result from accelerated
ubiquitin-mediated proteolysis (see Ciechanover, EMBO Journal
17:7151-7160 (1998)).
[0706] The ubiquitin system is also involved in development. The
involvement in human brain development is indicated by the fact
that a mutation in an E3 enzyme is implicated as the cause of
Angelman's syndrome, a disorder characterized by mental
retardation, seizures, and abnormal gait (Hershko et al.
above).
[0707] The ubiquitin system is also associated with apoptosis.
Ubiquitin-proteasome-mediated proteolysis is reported to play an
important role in apoptosis of nerve growth factor-deprived neurons
(Sadoul et al. (1996) EMBO Journal 15:3845-3852). One of the first
genes shown to be involved in programmed cell death is the
polyubiquitin gene that is regulated during metamorphosis of
Manduca sexta. Radiation-induced apoptosis in human lymphocytes has
been shown to be accompanied by increased ubiquitin mRNA and
ubiquitinylated nuclear proteins. Further, drugs that interfere
with proteasome function, such as lactacystin, prevent
radiation-induced cell death of thymocytes (Hershko et al.
above).
Deubiquitinating Enzymes
[0708] Deubiquitinating enzymes are cysteine proteases that
specifically cleave ubiquitin conjugates at the ubiquitin carboxy
terminus. These enzymes are responsible for processing linear
polyubiquitin chains to generate free ubiquitin from precursor
fusion proteins. They also affect pools of free ubiquitin by
recycling branched chain ubiquitin. These enzymes also remove
ubiquitin from ubiquitin- and polyubiquitin-conjugated target
protein, thereby regulating localization or activity of the target.
Further, these enzymes can remove ubiquitin from a ubiquitinated
tagged protein and thereby rescue the protein from degradation by
the 26S proteasome. The end result of each of these activities, is
to affect the level of free intracellular ubiquitin (D'Andrea et
al., above) and the level of specific proteins.
[0709] Ubiquitin is synthesized in a variety of
functionally-distinct forms. One of these is a linear head-to-tail
polyubiquitin precursor. Release of the free molecules involves
specific enzymatic cleavage between the fused residues. The last
ubiquitin moiety in many of these precursors is encoded with an
extra C-terminal residue that must be removed to expose the active
C-terminal Gly. In general, the recycling enzymes are thiol
proteases that recognize the C-terminal domain/residue of
ubiquitin. These are divided into two classes. The first is
designated ubiquitin C-terminal hydrolase (UCH) and the second is
designated ubiquitin-specific protease (UBP; isopeptidases)
(Ciechanover, above). These enzymes have been reviewed in detail in
D'Andrea, above.
[0710] UBPs contain six conserved regions. One surrounds the
conserved cysteine, one surrounds the aspartic acid, one surrounds
the histidine, and three additional regions of unknown function
have been identified. These six domains provide a molecular
signature for the UBP family. Short sequences surrounding the
cysteine residue and histidine residue are highly conserved among
all UBPs. Sequence comparison of several UBP family members reveals
that there are various subfamilies. One subfamily, designated DUB,
contains enzymes that are transcriptionally induced in response to
cytokines. The UBP family contains enzymes whose members have
multiple ubiquitin binding sites. Identified members of this family
include DUB1, isoT, UBP3, Doa4, Tre2, and FAF (D'Andrea et al.
above).
[0711] The UCH family is distinct from the UBP family. These
enzymes are cysteine proteases but do not contain the six homology
domains characteristic of the UBP family. Further, there is only
one binding site for ubiquitin. With respect to substrate
specificity, the UCH family preferentially cleaves ubiquitin from
small molecules, such as peptides and amino acids. Further, the two
families share little sequence homology with each other, although
the UCH signature can be found in some UBPs.
[0712] The deubiquitinating enzymes can promote either degradation
or stabilization of a given substrate. One of the best
characterized deubiquitinating enzymes is the yeast UBP14p enzyme
which has a human homolog designated isopeptidase-T. Isopeptidase-T
hydrolyzes free polyubiquitin chains and stimulates degradation of
polyubiquitinated protein substrates by the 26S proteasome. In
vitro data suggest that the cellular role of isopeptidase-T is to
dissemble unanchored polyubiquitin chains. The isopeptidase-T then
sequentially degrades these polyubiquitin chains into ubiquitin
monomers.
[0713] The yeast Doa4 promotes ubiquitin-mediated proteolysis of
cellular substrates. The primary function appears to be the
hydrolysis of isopeptide-linked ubiquitin chains from peptides that
are the by-products of proteasome degradation. The function appears
to be the clipping of polymeric ubiquitin from peptide degradation
products. In summary, with respect to a degradation function,
isopeptidases can produce free ubiquitin monomers from straight
chain polyubiquitin, branched chain polyubiquitin, ubiquitin or
polyubiquitin attached to substrate proteins, and ubiquitin or
polyubiquitin attached to substrate remnants, such as peptides or
amino acids.
[0714] Deubiquitinating enzymes that promote stabilization of
substrates include the FAF protein. Results show that the FAF
protein deubiquitinates and rescues a ubiquitin-conjugated target,
preventing its degradation by the proteasome. Another
deubiquitinating enzyme, designated PA700 isospeptidase, also
prevents proteasome degradation. This enzyme has been isolated from
the 19S regulatory complex. This enzyme appears to remove one
ubiquitin at a time starting from the distal end of a polyubiquitin
chain.
[0715] The enzymes have been associated with growth control. The
mammalian oncoprotein Tre-2 is a member of the UBP superfamily. The
transforming isoform of the Tre-2 oncoprotein is a truncated UPB
lacking the histidine domain and lacking deubiquitinating activity.
The full length Tre-2 protein has deubiquitinating activity but no
transforming activity. Accordingly, it has been suggested that this
protein acts as a growth suppressor within the cell.
[0716] Another UBP that regulates cellular function is designated
DUB. DUB-1 was originally shown to be induced by interleukin-3
stimulation. It has been postulated that the DUB protein family is
generally responsive to cytokines. It has also been shown that
another family member, DUB-2, is induced by interleukin-2. Zhu et
al. (1997) Journal of Biological Chemistry 272:51-57.
[0717] The enzymes may deubiquitinate cell surface growth factor
receptors thereby prolonging receptor half life and amplifying
growth signals. They may also deubiquitinate proteins involved in
signal transduction and deubiquitinate cell cycle regulators such
as cyclins or cyclin-CDK inhibitors. See D'Andrea above.
[0718] UBPs have also been linked to the chromatin regulatory
process, transcriptional silencing. UBP-3 has been reported to
complex with SIR-4, a trans-acting factor that is required for
establishment and maintenance of silencing. Accordingly, UBP-3 may
act as an inhibitor of silencing by either stabilizing an inhibitor
or by removing a positive regulator.
[0719] The murine UNP protooncogene has been shown to encode a
nuclear ubiquitin protease whose overexpression leads to oncogenic
transformation in NIH3T3 cells. A cDNA was cloned corresponding to
the human homolog of this gene. It was shown to map to a region
frequently rearranged in human tumor cells. Further, it was shown
that levels of this gene are elevated in small cell tumors and
adenocarcinomas of the lung, suggesting a causative role of the
gene in the neoplastic process (Gray et al. (1995) Oncogene
10:2179-2183).
[0720] A novel ubiquitin-specific protease, designated UBP-43, was
cloned from a leukemia fusion protein in AML1-ETO Knockin mice.
This protease was shown to function in hematopoitic cell
differentiation. The overexpression of this gene was shown to block
cytokine-induced terminal differentiation of monocytic cells (Liu
et al. (1999) Molecular and Cellular Biology 19:3029-3038).
[0721] In summary, deubiquitinating enzymes are potentially
powerful targets for modulating ubiquitination. Modulation of
ubiquitination can increase or decrease the proteolysis of specific
proteins, particularly key proteins in cellular processes, can
increase or decrease levels of general proteolysis, thus affecting
the basic metabolic state, and may increase or decrease the pool of
free ubiquitin monomers available for ubiquitination.
[0722] Accordingly, ubiquitin proteases are a major target for drug
action and development. Thus, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown ubiquitin proteases. The present invention advances the
state of the art by providing a previously unidentified human
deubiquitinating enzyme.
SUMMARY OF THE INVENTION
[0723] It is an object of the invention to identify novel ubiquitin
proteases.
[0724] It is a further object of the invention to provide novel
ubiquitin protease polypeptides that are useful as reagents or
targets in assays applicable to treatment and diagnosis of
ubiquitin-mediated or -related disorders, especially disorders
mediated by or related to deubiquitinating enzymes.
[0725] It is a further object of the invention to provide
polynucleotides corresponding to the novel ubiquitin protease
polypeptides that are useful as targets and reagents in assays
applicable to treatment and diagnosis of ubiquitin or ubiquitin
protease-mediated or -related disorders and useful for producing
novel ubiquitin protease polypeptides by recombinant methods.
[0726] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the novel ubiquitin protease.
[0727] A further specific object of the invention is to provide
compounds that modulate expression of the ubiquitin protease for
treatment and diagnosis of ubiquitin and ubiquitin protease-related
disorders.
[0728] The invention is thus based on the identification of a novel
human ubiquitin protease. The amino acid sequence is shown in SEQ
ID NO:15. The nucleotide sequence is shown in SEQ ID NO:16.
[0729] The invention provides isolated ubiquitin protease
polypeptides, including a polypeptide having the amino acid
sequence shown in SEQ ID NO:15 or the amino acid sequence encoded
by the cDNA deposited as ATCC No. PTA-1849 on May 9, 2000 ("the
deposited cDNA").
[0730] The invention also provides isolated ubiquitin protease
nucleic acid molecules having the sequence shown in SEQ ID NO:16 or
in the deposited cDNA.
[0731] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO:15 or encoded by the deposited
cDNA.
[0732] The invention also provides variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO:16 or in the deposited cDNA.
[0733] The invention also provides fragments of the polypeptide
shown in SEQ ID NO:15 and nucleotide sequence shown in SEQ ID
NO:16, as well as substantially homologous fragments of the
polypeptide or nucleic acid.
[0734] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[0735] The invention also provides vectors and host cells for
expressing the ubiquitin protease nucleic acid molecules and
polypeptides, and particularly recombinant vectors and host
cells.
[0736] The invention also provides methods of making the vectors
and host cells and methods for using them to produce the ubiquitin
protease nucleic acid molecules and polypeptides.
[0737] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the ubiquitin protease
polypeptides and fragments.
[0738] The invention also provides methods of screening for
compounds that modulate expression or activity of the ubiquitin
protease polypeptides or nucleic acid (RNA or DNA).
[0739] The invention also provides a process for modulating
ubiquitin protease polypeptide or nucleic acid expression or
activity, especially using the screened compounds. Modulation may
be used to treat conditions related to aberrant activity or
expression of the ubiquitin protease polypeptides or nucleic acids
or of the ubiquitin system. In addition, modulation may be used to
treat conditions, such as viral infection, that are affected by the
ubiquitin protease.
[0740] The invention also provides assays for determining the
activity of or the presence or absence of the ubiquitin protease
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0741] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[0742] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the invention,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0743] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0744] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Polypeptides
[0745] The invention is based on the identification of a novel
human ubiquitin protease. Specifically, an expressed sequence tag
(EST) was selected based on homology to ubiquitin protease
sequences. This EST was used to design primers based on sequences
that it contains and used to identify a cDNA from a human prostate
library. Positive clones were sequenced and the overlapping
fragments were assembled. Analysis of the assembled sequence
revealed that the cloned cDNA molecule encodes a ubiquitin protease
containing the conserved amino acid residues found in UBP and UCH
thiol proteases.
[0746] The invention thus relates to a novel ubiquitin protease
having the deduced amino acid sequence shown in FIGS. 33A-33D (SEQ
ID NO:15) or having the amino acid sequence encoded by the
deposited cDNA, ATCC No. PTA-1849 on May 9, 2000.
[0747] The deposits will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms. The deposits are provided as a convenience to those
of skill in the art and are not an admission that a deposit is
required under 35 U.S.C. .sctn. 112. The deposited sequences, as
well as the polypeptides encoded by the sequences, are incorporated
herein by reference and controls in the event of any conflict, such
as a sequencing error, with description in this application.
[0748] "Ubiquitin protease polypeptide" or "ubiquitin protease
protein" refers to the polypeptide in SEQ ID NO:15 or encoded by
the deposited cDNA. The term "ubiquitin protease protein" or
"ubiquitin protease polypeptide", however, further includes the
numerous variants described herein, as well as fragments derived
from the full-length ubiquitin proteases and variants.
[0749] Tissues and/or cells in which the ubiquitin protease is
expressed include, but are not limited to those shown in FIGS. 37
and 38. Tissues in which the gene is highly expressed include fetal
kidney, testes, fetal liver, ovary, and fetal heart. Expression is
also seen in the kidney, thyroid, undifferentiated osteoblasts and
skeletal muscle. The ubiquitin protease is also expressed in normal
liver and in normal and malignant breast, lung, and colon tissue
and in liver metastases derived from malignant colonic tissues.
Hence, the ubiquitin protease is relevant to disorders involving
the tissues in which it is expressed, especially in breast, lung,
colon, and colon metastases to liver. Expression has been confirmed
by Northern blot analysis.
[0750] The present invention thus provides an isolated or purified
ubiquitin protease polypeptide and variants and fragments
thereof.
[0751] Based on a BLAST search, highest homology was shown to
Ubiquitin Carboxyl-terminal hydrolase (AL031525) from S. pombe.
[0752] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[0753] The ubiquitin protease polypeptides can be purified to
homogeneity. It is understood, however, that preparations in which
the polypeptide is not purified to homogeneity are useful and
considered to contain an isolated form of the polypeptide. The
critical feature is that the preparation allows for the desired
function of the polypeptide, even in the presence of considerable
amounts of other components. Thus, the invention encompasses
various degrees of purity.
[0754] In one embodiment, the language "substantially free of
cellular material" includes preparations of the ubiquitin protease
having less than about 30% (by dry weight) other proteins (i.e.,
contaminating protein), less than about 20% other proteins, less
than about 10% other proteins, or less than about 5% other
proteins. When the polypeptide is recombinantly produced, it can
also be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation.
[0755] An ubiquitin protease polypeptide is also considered to be
isolated when it is part of a membrane preparation or is purified
and then reconstituted with membrane vesicles or liposomes.
[0756] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the ubiquitin protease
polypeptide in which it is separated from chemical precursors or
other chemicals that are involved in its synthesis. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of the polypeptide having
less than about 30% (by dry weight) chemical precursors or other
chemicals, less than about 20% chemical precursors or other
chemicals, less than about 10% chemical precursors or other
chemicals, or less than about 5% chemical precursors or other
chemicals.
[0757] In one embodiment, the ubiquitin protease polypeptide
comprises the amino acid sequence shown in SEQ ID NO:15. However,
the invention also encompasses sequence variants. Variants include
a substantially homologous protein encoded by the same genetic
locus in an organism, i.e., an allelic variant.
[0758] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the
ubiquitin protease of SEQ ID NO:15. Variants also include proteins
substantially homologous to the ubiquitin protease but derived from
another organism, i.e., an ortholog. Variants also include proteins
that are substantially homologous to the ubiquitin protease that
are produced by chemical synthesis. Variants also include proteins
that are substantially homologous to the ubiquitin protease that
are produced by recombinant methods. It is understood, however,
that variants exclude any amino acid sequences disclosed prior to
the invention.
[0759] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 70-75%, typically at least about 80-85%, and most
typically at least about 90-95% or more homologous. A substantially
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence hybridizing to the
nucleic acid sequence, or portion thereof, of the sequence shown in
SEQ ID NO:16 under stringent conditions as more fully described
below.
[0760] 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-homologous
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 (i.e., 100%=the entire coding sequence). 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.
[0761] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the
ubiquitin protease. Similarity is determined by conserved amino
acid substitution. Such substitutions are those that substitute a
given amino acid in a polypeptide by another amino acid of like
characteristics. Conservative substitutions are likely to be
phenotypically silent. Typically seen as conservative substitutions
are the replacements, one for another, among the aliphatic amino
acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues
Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution between the amide residues Asn and Gln, exchange of
the basic residues Lys and Arg and replacements among the aromatic
residues Phe, Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
TABLE-US-00002 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small.quadrature.
Alanine Serine Threonine Methionine Glycine
[0762] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0763] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. In one embodiment, parameters
for sequence comparison can be set at score=100, wordlength=12, or
can be varied (e.g., W=5 or W=20).
[0764] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (J. Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package,
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 (Devereux et al. (1984)
Nucleic Acids Res. 12(1):387), 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.
[0765] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[0766] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[0767] Variant polypeptides can be fully functional or can lack
function in one or more activities. Thus, in the present case,
variations can affect the function, for example, of ubiquitin
binding, ubiquitin recognition, interaction with ubiquitinated
substrate protein, such as binding or proteolysis, subunit
interaction, particularly within the proteasome, activation or
binding by ATP, developmental expression, temporal expression,
tissue-specific expression, interacting with cellular components,
such as transcriptional regulatory factors, and particularly
trans-acting transcriptional regulatory factors, proteolytic
cleavage of peptide bonds in polyubiquitin and peptide bonds
between ubiquitin or polyubiquitin and substrate protein, and
proteolytic cleavage of peptide bonds between ubiquitin or
polyubiquitin and a peptide or amino acid.
[0768] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0769] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0770] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for the ubiquitin protease polypeptide.
This includes preventing immunogenicity from pharmaceutical
formulations by preventing protein aggregation.
[0771] Useful variations further include alteration of catalytic
activity. For example, one embodiment involves a variation at the
binding site that results in binding but not hydrolysis, or slower
hydrolysis, of the peptide bond. A further useful variation results
in an increased rate of hydrolysis of the peptide bond. A further
useful variation at the same site can result in higher or lower
affinity for substrate. Useful variations also include changes that
provide for affinity for a different ubiquitinated substrate
protein than that normally recognized. Other useful variations
involving altered recognition affect recognition of the type of
substrate normally recognized. For example, one variation could
result in recognition of ubiquitinated intact substrate but not of
substrate remnants, such as ubiquitinated amino acid or peptide
that are proteolysis products that result from the hydrolysis of
the intact ubiquitinated substrate. Alternatively, the protease
could be varied so that one or more of the remnant products is
recognized but not the intact protein substrate. Another variation
would affect the ability of the protease to rescue a ubiquitinated
protein. Thus, protein substrates that are normally rescued from
proteolysis would be subject to degradation. Further useful
variations affect the ability of the protease to be induced by
activators, such as cytokines, including but not limited to, those
disclosed herein. Another useful variation would affect the
recognition of ubiquitin substrate so that the enzyme could not
recognize one or more of a linear polyubiquitin, branched chain
polyubiquitin, linear polyubiquitinated substrate, or branched
chain polyubiquitin substrate. Specific variations include
truncation in which, for example, a HIS domain is deleted, the
variation resulting in decrease or loss of deubiquitination
activity. Another useful variation includes one that prevents
activation by ATP. Another useful variation provides a fusion
protein in which one or more domains or subregions are
operationally fused to one or more domains or subregions from
another UBP or from a UCH. Specifically, a domain or subregion can
be introduced that provides a rescue function to an enzyme not
normally having this function or for recognition of a specific
substrate wherein recognition is not available to the original
enzyme. Other variations include those that affect ubiquitin
recognition or recognition of a ubiquitinated substrate protein.
Further variations could affect specific subunit interaction,
particularly in the proteasome. Other variations would affect
developmental, temporal, or tissue-specific expression. Other
variations would affect the interaction with cellular components,
such as transcriptional regulatory factors.
[0772] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as peptide hydrolysis in vitro or ubiquitin-dependent in vitro
activity, such as proliferative activity, receptor-mediated signal
transduction, and other cellular processes including, but not
limited, those disclosed herein that are a function of the
ubiquitin system. Sites that are critical for binding or
recognition can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et
al. (1992) Science 255:306-312).
[0773] The assays for deubiquitinating enzyme activity are well
known in the art and can be found, for example, in Zhu et al.
(1997) Journal of Biological Chemistry 272:51-57, Mitch et al.
(1999) American Journal of Physiology 276:C1132-C1138, Liu et al.
(1999) Molecular and Cell Biology 19:3029-3038, and such as those
cited in various reviews, for example, Ciechanover et al. (1994)
The FASEB Journal 8:182-192, Chiechanover (1994) Biol. Chem.
Hoppe-Seyler 375:565-581, Hershko et al. (1998) Annual Review of
Biochemistry 67:425-479, Swartz (1999) Annual Review of Medicine
50:57-74, Ciechanover (1998) EMBO Journal 17:7151-7160, and
D'Andrea et al. (1998) Critical Reviews in Biochemistry and
Molecular Biology 33:337-352. These assays include, but are not
limited to, the disappearance of substrate, including decrease in
the amount of polyubiquitin or ubiquitinated substrate protein or
protein remnant, appearance of intermediate and end products, such
as appearance of free ubiquitin monomers, general protein turnover,
specific protein turnover, ubiquitin binding, binding to
ubiquitinated substrate protein, subunit interaction, interaction
with ATP, interaction with cellular components such as trans-acting
regulatory factors, stabilization of specific proteins, and the
like.
[0774] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[0775] The invention thus also includes polypeptide fragments of
the ubiquitin protease. Fragments can be derived from the amino
acid sequence shown in SEQ ID NO:15. However, the invention also
encompasses fragments of the variants of the ubiquitin proteases as
described herein.
[0776] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention.
[0777] Accordingly, a fragment can comprise at least about 11, 15,
20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids.
Fragments can retain one or more of the biological activities of
the protein, for example the ability to bind to ubiquitin or
hydrolyze peptide bonds, as well as fragments that can be used as
an immunogen to generate ubiquitin protease antibodies.
[0778] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a domain or motif,
e.g., catalytic site, UCH family 2 signature, signature for the
immunoglobulin and major histocompatibility complex proteins, and
sites for glycosylation, cAMP and cGMP-dependent protein kinase
phosphorylation, protein kinase C phosphorylation, casein kinase II
phosphorylation, tyrosine kinase phosphorylation, N-myristoylation,
and amidation. Further possible fragments include the catalytic
site or domain including conserved amino acid residues found in UBP
and UCH thiol proteases. Such regions include, for example, about
amino acids 123 to 138 of SEQ ID NO:15 or the UCH2 family 2
signature found from about amino acids 365 to about 383 of SEQ ID
NO:15. Additional domains include ubiquitin recognition sites,
ubiquitin binding sites, sites important for subunit interaction,
and sites important for carrying out the other functions of the
protease as described herein.
[0779] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[0780] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific domains mentioned above, which sub-fragments retain
the function of the domain from which they are derived.
[0781] These regions can be identified by well-known methods
involving computerized homology analysis.
[0782] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the
ubiquitin protease and variants. These epitope-bearing peptides are
useful to raise antibodies that bind specifically to a ubiquitin
protease polypeptide or region or fragment. These peptides can
contain at least 11, 12, at least 14, or between at least about 15
to about 30 amino acids.
[0783] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular site. Regions having a high
antigenicity index are shown in FIG. 34. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular peptide
regions.
[0784] The epitope-bearing ubiquitin protease polypeptides may be
produced by any conventional means (Houghten, R. A. (1985) Proc.
Natl. Acad. Sci. USA 82:5131-5135). Simultaneous multiple peptide
synthesis is described in U.S. Pat. No. 4,631,211.
[0785] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the ubiquitin protease fragment and
an additional region fused to the carboxyl terminus of the
fragment.
[0786] The invention thus provides chimeric or fusion proteins.
These comprise a ubiquitin protease peptide sequence operatively
linked to a heterologous peptide having an amino acid sequence not
substantially homologous to the ubiquitin protease. "Operatively
linked" indicates that the ubiquitin protease peptide and the
heterologous peptide are fused in-frame. The heterologous peptide
can be fused to the N-terminus or C-terminus of the ubiquitin
protease or can be internally located.
[0787] In one embodiment the fusion protein does not affect
ubiquitin protease function per se. For example, the fusion protein
can be a GST-fusion protein in which the ubiquitin protease
sequences are fused to the C-terminus of the GST sequences. Other
types of fusion proteins include, but are not limited to, enzymatic
fusion proteins, for example beta-galactosidase fusions, yeast
two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions. Such
fusion proteins, particularly poly-His fusions, can facilitate the
purification of recombinant ubiquitin protease. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a protein can be increased by using a heterologous signal sequence.
Therefore, in another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus.
[0788] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson
et al. J. Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing a ubiquitin protease
polypeptide and various portions of the constant regions of heavy
or light chains of immunoglobulins of various subclass (IgG, IgM,
IgA, IgE). Preferred as immunoglobulin is the constant part of the
heavy chain of human IgG, particularly IgG1, where fusion takes
place at the hinge region. For some uses it is desirable to remove
the Fc after the fusion protein has been used for its intended
purpose, for example when the fusion protein is to be used as
antigen for immunizations. In a particular embodiment, the Fc part
can be removed in a simple way by a cleavage sequence, which is
also incorporated and can be cleaved with factor Xa.
[0789] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. 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 re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A ubiquitin protease-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the ubiquitin protease.
[0790] Another form of fusion protein is one that directly affects
ubiquitin protease functions. Accordingly, a ubiquitin protease
polypeptide is encompassed by the present invention in which one or
more of the ubiquitin protease domains (or parts thereof) has been
replaced by homologous domains (or parts thereof) from another UBP
or UCH species. Accordingly, various permutations are possible. One
or more functional sites as disclosed herein from the specifically
disclosed protease can be replaced by one or more functional sites
from a corresponding UBP family member or from a UCH family member.
Thus, chimeric ubiquitin proteases can be formed in which one or
more of the native domains or subregions has been replaced by
another.
[0791] Additionally, chimeric ubiquitin protease proteins can be
produced in which one or more functional sites is derived from a
different ubiquitin protease family. It is understood however that
sites could be derived from ubiquitin protease families that occur
in the mammalian genome but which have not yet been discovered or
characterized. Such sites include but are not limited to any of the
functional sites disclosed herein.
[0792] The isolated ubiquitin proteases can be purified from any of
the cells that naturally express it, such as, fetal kidney, testes,
fetal liver, ovary, fetal heart, kidney, thyroid, undifferentiated
osteoblasts, skeletal muscle, malignant breast tissue, primary lung
tumors and liver metastases derived from colon. Alternatively, the
ubiquitin protease may be purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods.
[0793] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
ubiquitin protease polypeptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Polypeptides often contain amino
acids other than the 20 amino acids commonly referred to as the 20
naturally-occurring amino acids. Further, many amino acids,
including the terminal amino acids, may be modified by natural
processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Common modifications that occur naturally in polypeptides
are described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the
art.
[0794] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[0795] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0796] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T.E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990)
Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y.
Acad. Sci. 663:48-62).
[0797] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0798] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[0799] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[0800] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
Polypeptide Uses
[0801] The protein sequences of the present invention can 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 the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins 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.
[0802] The ubiquitin protease polypeptides are useful for producing
antibodies specific for the ubiquitin protease, regions, or
fragments. Regions having a high antigenicity index score are shown
in FIG. 34.
[0803] The ubiquitin protease polypeptides are useful for
biological assays related to ubiquitin protease function. Such
assays involve any of the known functions or activities or
properties useful for diagnosis and treatment of ubiquitin- or
ubiquitin protease-related conditions or conditions in which
expression of the protease is relevant, such as in viral
infections. Potential assays have been disclosed herein and
generically include disappearance of substrate, appearance of end
product, and general or specific protein turnover.
[0804] The ubiquitin protease polypeptides are also useful in drug
screening assays, in cell-based or cell-free systems. Cell-based
systems can be native, i.e., cells that normally express the
ubiquitin protease, as a biopsy or expanded in cell culture. In one
embodiment, however, cell-based assays involve recombinant host
cells expressing the ubiquitin protease.
[0805] Determining the ability of the test compound to interact
with the ubiquitin protease can also comprise determining the
ability of the test compound to preferentially bind to the
polypeptide as compared to the ability of a known binding molecule
(e.g., ubiquitin) to bind to the polypeptide.
[0806] The polypeptides can be used to identify compounds that
modulate ubiquitin protease activity. Such compounds, for example,
can increase or decrease affinity for polyubiquitin, either linear
or branched chain, ubiquitinated protein substrate, or
ubiquitinated protein substrate remnants. Such compounds could
also, for example, increase or decrease the rate of binding to
these components. Such compounds could also compete with these
components for binding to the ubiquitin protease or displace these
components bound to the ubiquitin protease. Such compounds could
also affect interaction with other components, such as ATP, other
subunits, for example, in the 19S complex, and transcriptional
regulatory factors. It is understood, therefore, that such
compounds can be identified not only by means of ubiquitin, but by
means of any of the components that functionally interact with the
disclosed protease. This includes, but is not limited to, any of
those components disclosed herein.
[0807] Both ubiquitin protease and appropriate variants and
fragments can be used in high-throughput screens to assay candidate
compounds for the ability to bind to the ubiquitin protease. These
compounds can be further screened against a functional ubiquitin
protease to determine the effect of the compound on the ubiquitin
protease activity. Compounds can be identified that activate
(agonist) or inactivate (antagonist) the ubiquitin protease to a
desired degree. 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.
[0808] The ubiquitin protease polypeptides can be used to screen a
compound for the ability to stimulate or inhibit interaction
between the ubiquitin protease protein and a target molecule that
normally interacts with the ubiquitin protease protein. The target
can be ubiquitin, ubiquitinated substrate, or polyubiquitin or
another component of the pathway with which the ubiquitin protease
protein normally interacts (for example, ATP). The assay includes
the steps of combining the ubiquitin protease protein with a
candidate compound under conditions that allow the ubiquitin
protease protein or fragment to interact with the target molecule,
and to detect the formation of a complex between the ubiquitin
protease protein and the target or to detect the biochemical
consequence of the interaction with the ubiquitin protease and the
target. Any of the associated effects of protease function can be
assayed. This includes the production of hydrolysis products, such
as free terminal peptide substrate, free terminal amino acid from
the hydrolyzed substrate, free ubiquitin, lower molecular weight
species of hydrolyzed polyubiquitin, released intact substrate
protein resulting from rescue from proteolysis, free polyubiquitin
formed from hydrolysis of the polyubiquitin from intact substrate,
and substrate remnants, such as amino acids and peptides produced
from proteolysis of the substrate protein, and biological endpoints
of the pathway.
[0809] Determining the ability of the ubiquitin protease to bind to
a target molecule can also be accomplished using a technology such
as real-time Bimolecular Interaction Analysis (BIA). Sjolander et
al. (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.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR)
can be used as an indication of real-time reactions between
biological molecules.
[0810] 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 polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[0811] 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. USA 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; Carell 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. 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. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[0812] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0813] One candidate compound is a soluble full-length ubiquitin
protease or fragment that competes for substrate binding. Other
candidate compounds include mutant ubiquitin proteases or
appropriate fragments containing mutations that affect ubiquitin
protease function and compete for substrate. Accordingly, a
fragment that competes for substrate, for example with a higher
affinity, or a fragment that binds substrate but does not hydrolyze
the peptide bond, is encompassed by the invention.
[0814] Other candidate compounds include ubiquitinated protein or
protein analog that binds to the protease but is not released or
released slowly. Other candidate compounds include analogs of the
other natural substrates, such as substrate remnants that bind to
but are not released or released more slowly. Further candidate
compounds include activators of the proteases such as cytokines,
including but not limited to, those disclosed herein.
[0815] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) ubiquitin protease
activity. The assays typically involve an assay of events in the
pathway that indicate ubiquitin protease activity. This can include
cellular events that result from deubiquitination, such as cell
cycle progression, programmed cell death, growth factor-mediated
signal transduction, or any of the cellular processes including,
but not limited to, those disclosed herein as resulting from
deubiquitination. Specific phenotypes include changes in stress
response, DNA replication, receptor internalization, cellular
transformation or reversal of transformation, and transcriptional
silencing.
[0816] Assays are based on the multiple cellular functions of
deubiquitinating enzymes. These enzymes act at various different
levels in the regulation of protein ubiquitination. A
deubiquitinating enzyme can degrade a linear polyubiquitin chain
into monomeric ubiquitin molecules. Deubiquitinating enzymes, such
as isopeptidase-T, can degrade a branched multiubiquitin chain into
monomeric ubiquitin molecules. Deubiquitinating enzymes can remove
ubiquitin from a ubiquitin-conjugated target protein. The
deubiquitinating enzyme, such as FAF or PA700 isopeptidase, can
remove polyubiquitin from a ubiquitinated target protein, and
thereby rescue the target from degradation by the 26S proteasome.
Deubiquitinating enzymes such as Doa-4 can remove polyubiquitin
from proteasome degradation products. UCH family members tend to
hydrolyze monoubiquitinated substrate (Larsen et al. (1998)
Biochemistry 10:3358-68). The UCH deubiquitinating enzyme AP-UCH
enhances proteolytic activity of Protein Kinase A (PKA) through the
ubiquitin-proteosome pathway. Furthermore, BAP1 has been identified
as a new member of the UCH family and interacts with BRAC1, thereby
enhancing BRCA1 mediated cell growth suppression (Jensen et al.
(1998) Oncogene 16: 1097-1112). The end result of all of the
deubiquitinating enzymes is to regulate the cellular pool of free
monomeric ubiquitin. Accordingly, assays can be based on detection
of any of the products produced by hydrolysis/deubiquitination.
[0817] Further, the expression of genes that are up- or
down-regulated by action of the ubiquitin protease can be assayed.
In one embodiment, the regulatory region of such genes can be
operably linked to a marker that is easily detectable, such as
luciferase.
[0818] Accordingly, any of the biological or biochemical functions
mediated by the ubiquitin protease can be used as an endpoint
assay. These include all of the biochemical or
biochemical/biological events described herein, in the references
cited herein, incorporated by reference for these endpoint assay
targets, and other functions known to those of ordinary skill in
the art.
[0819] Binding and/or activating compounds can also be screened by
using chimeric ubiquitin protease proteins in which one or more
domains, sites, and the like, as disclosed herein, or parts
thereof, can be replaced by their heterologous counterparts derived
from other ubiquitin proteases. For example, a recognition or
binding region can be used that interacts with different substrate
specificity and/or affinity than the native ubiquitin protease.
Accordingly, a different set of pathway components is available as
an end-point assay for activation. Further, sites that are
responsible for developmental, temporal, or tissue specificity can
be replaced by heterologous sites such that the protease can be
detected under conditions of specific developmental, temporal, or
tissue-specific expression.
[0820] The ubiquitin protease polypeptides are also useful in
competition binding assays in methods designed to discover
compounds that interact with the ubiquitin protease. Thus, a
compound is exposed to a ubiquitin protease polypeptide under
conditions that allow the compound to bind to or to otherwise
interact with the polypeptide. Soluble ubiquitin protease
polypeptide is also added to the mixture. If the test compound
interacts with the soluble ubiquitin protease polypeptide, it
decreases the amount of complex formed or activity from the
ubiquitin protease target. This type of assay is particularly
useful in cases in which compounds are sought that interact with
specific regions of the ubiquitin protease. Thus, the soluble
polypeptide that competes with the target ubiquitin protease region
is designed to contain peptide sequences corresponding to the
region of interest.
[0821] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, ubiquitin and a candidate compound can be added to a
sample of the ubiquitin protease. Compounds that interact with the
ubiquitin protease at the same site as ubiquitin will reduce the
amount of complex formed between the ubiquitin protease and
ubiquitin. Accordingly, it is possible to discover a compound that
specifically prevents interaction between the ubiquitin protease
and ubiquitin. Another example involves adding a candidate compound
to a sample of ubiquitin protease and polyubiquitin. A compound
that competes with polyubiquitin will reduce the amount of
hydrolysis or binding of the polyubiquitin to the ubiquitin
protease. Accordingly, compounds can be discovered that directly
interact with the ubiquitin protease and compete with
polyubiquitin. Such assays can involve any other component that
interacts with the ubiquitin protease, such as ubiquitinated
substrate protein, ubiquitinated substrate remnants, and cellular
components with which the protease interacts such as
transcriptional regulatory factors.
[0822] To perform cell free drug screening assays, it is desirable
to immobilize either the ubiquitin protease, or fragment, or its
target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0823] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/ubiquitin
protease 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 cell lysates
(e.g., .sup.35S-labeled) and the candidate compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly, or in
the supernatant after the complexes is dissociated. Alternatively,
the complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of ubiquitin protease-binding protein found
in the bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do
not interfere with binding of the protein to its target molecule
can be derivatized to the wells of the plate, and the protein
trapped in the wells by antibody conjugation. Preparations of a
ubiquitin protease-binding target component, such as ubiquitin,
polyubiquitin, ubiquitinated substrate protein, ubiquitinated
substrate protein remnant, or ubiquitinated remnant amino acid, and
a candidate compound are incubated in the ubiquitin
protease-presenting wells and the amount of complex trapped in the
well can be quantitated. 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 ubiquitin protease target molecule, or which are
reactive with ubiquitin protease and compete with the target
molecule; as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the target molecule.
[0824] Modulators of ubiquitin protease activity identified
according to these drug screening assays can be used to treat a
subject with a disorder mediated or affected by the ubiquitin
protease pathway, by treating cells that express the ubiquitin
protease or cells in which protease expression is desirable (such
as virus-infected cells). Such cells include, for example, fetal
kidney, testes, fetal liver, ovary, fetal heart, kidney, thyroid,
undifferentiated osteoblasts, skeletal muscle, and malignant
breast, lung and colon tissue, as well as liver metastases derived
from malignant colonic tissue. These methods of treatment include
the steps of administering the modulators of ubiquitin protease
activity in a pharmaceutical composition as described herein, to a
subject in need of such treatment.
[0825] Tissues and/or cells in which the ubiquitin protease is
expressed include, but are not limited to those shown in FIGS. 37
and 38. Tissues in which the gene is highly expressed include fetal
kidney, testes, fetal liver, ovary, and fetal heart. Expression is
also seen in the kidney, thyroid, undifferentiated osteoblasts and
skeletal muscle. The ubiquitin protease is also expressed in normal
liver and in normal and malignant breast, lung, and colon tissue
and in liver metastases derived from malignant colonic tissues.
Hence, the ubiquitin protease is relevant to treating disorders
involving these tissues, breast, lung, colon carcinoma, and colon
metastases to liver.
[0826] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
[0827] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0828] Disorders involving the kidney include, but are not limited
to, congenital anomalies including, but not limited to, cystic
diseases of the kidney, that include but are not limited to, cystic
renal dysplasia, autosomal dominant (adult) polycystic kidney
disease, autosomal recessive (childhood) polycystic kidney disease,
and cystic diseases of renal medulla, which include, but are not
limited to, medullary sponge kidney and nephronophthisis-uremic
medullary cystic disease complex, acquired (dialysis-associated)
cystic disease and simple cysts; glomerular diseases including
pathologies of glomerular injury that include, but are not limited
to, in situ immune complex deposition, that includes, but is not
limited to, anti-GBM nephritis, Heymann nephritis, and antibodies
against planted antigens, circulating immune complex nephritis,
antibodies to glomerular cells, cell-mediated immunity in
glomerulonephritis, activation of alternative complement pathway,
epithelial cell injury, and pathologies involving mediators of
glomerular injury including cellular and soluble mediators, acute
glomerulonephritis, such as acute proliferative (poststreptococcal,
postinfectious) glomerulonephritis, including but not limited to,
poststreptococcal glomerulonephritis and nonstreptococcal acute
glomerulonephritis, rapidly progressive (crescentic)
glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis (membranous nephropathy), minimal change disease
(lipoid nephrosis), focal segmental glomerulosclerosis,
membranoproliferative glomerulonephritis, IgA nephropathy (Berger
disease), focal proliferative and necrotizing glomerulonephritis
(focal glomerulonephritis), hereditary nephritis, including but not
limited to, Alport syndrome and thin membrane disease (benign
familial hematuria), chronic glomerulonephritis, glomerular lesions
associated with systemic disease, including but not limited to,
systemic lupus crythematosus, Henoch-Schonlein purpura, bacterial
endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary
and immunotactoid glomerulonephritis, and other systemic disorders;
diseases affecting tubules and interstitium, including, but not
limited to, acute tubular necrosis and tubulointerstitial
nephritis, including but not limited to, pyelonephritis and urinary
tract infection, acute pyelonephritis, chronic pyelonephritis and
reflux nephropathy, tubulointerstitial nephritis induced by drugs
and toxins, including but not limited to, acute drug-induced
interstitial nephritis, analgesic abuse nephropathy, and
nephropathy associated with nonsteroidal anti-inflammatory drugs,
and other tubulointerstitial diseases including, but not limited
to, urate nephropathy, hypercalcemia and nephrocalcinosis, and
multiple myeloma; diseases of blood vessels including, including
but not limited to, benign nephrosclerosis, malignant hypertension
and accelerated nephrosclerosis, renal artery stenosis, and
thrombotic microangiopathies, including, but not limited to,
classic (childhood) hemolytic-uremic syndrome, adult
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,
idiopathic HUS/TTP, and other vascular disorders including, but not
limited to, atherosclerotic ischemic renal disease, atheroembolic
renal disease, sickle cell disease nephropathy, diffuse cortical
necrosis, and renal infarcts; urinary tract obstruction
(obstructive uropathy); urolithiasis (renal calculi, stones); and
tumors of the kidney including, but not limited to, benign tumors,
such as renal papillary adenoma, renal fibroma or hamartoma
(renomedullary interstitial cell tumor), angiomyolipoma, and
oncocytoma, and malignant tumors, including renal cell carcinoma
(hypemephroma, adenocarcinoma of kidney), which includes urothelial
carcinomas of renal pelvis.
[0829] Disorders of the breast include, but are not limited to,
disorders of development; inflammations, including but not limited
to, acute mastitis, periductal mastitis (recurrent subareolar
abscess, squamous metaplasia of lactiferous ducts), mammary duct
ectasia, fat necrosis, granulomatous mastitis, and pathologies
associated with silicone breast implants; fibrocystic changes;
proliferative breast disease including, but not limited to,
epithelial hyperplasia, sclerosing adenosis, and small duct
papillomas; tumors including, but not limited to, stromal tumors
such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial
tumors, such as large duct papilloma; carcinoma of the breast
including in situ (noninvasive) carcinoma that includes ductal
carcinoma in situ (including Paget's disease) and lobular carcinoma
in situ, and invasive (infiltrating) carcinoma including, but not
limited to, invasive ductal carcinoma, no special type, invasive
lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms. Disorders in the male breast
include, but are not limited to, gynecomastia and carcinoma.
[0830] Disorders involving the testis and epididymis include, but
are not limited to, congenital anomalies such as cryptorchidism,
regressive changes such as atrophy, inflammations such as
nonspecific epididymitis and orchitis, granulomatous (autoimmune)
orchitis, and specific inflammations including, but not limited to,
gonorrhea, mumps, tuberculosis, and syphilis, vascular disturbances
including torsion, testicular tumors including germ cell tumors
that include, but are not limited to, seminoma, spermatocytic
seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma,
teratoma, and mixed tumors, tumors of sex cord-gonadal stroma
including, but not limited to, Leydig (interstitial) cell tumors
and Sertoli cell tumors (androblastoma), and testicular lymphoma,
and miscellaneous lesions of tunica vaginalis.
[0831] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[0832] Disorders involving the thyroid include, but are not limited
to, hyperthyroidism; hypothyroidism including, but not limited to,
cretinism and myxedema; thyroiditis including, but not limited to,
hashimoto thyroiditis, subacute (granulomatous) thyroiditis, and
subacute lymphocytic (painless) thyroiditis; Graves disease;
diffuse and multinodular goiter including, but not limited to,
diffuse nontoxic (simple) goiter and multinodular goiter; neoplasms
of the thyroid including, but not limited to, adenomas, other
benign tumors, and carcinomas, which include, but are not limited
to, papillary carcinoma, follicular carcinoma, medullary carcinoma,
and anaplastic carcinoma; and cogenital anomalies.
[0833] Disorders involving the skeletal muscle include tumors, such
as rhabdomyosarcoma.
[0834] Disorders involving the lung include, but are not limited
to, congenital anomalies; atelectasis; diseases of vascular origin,
such as pulmonary congestion and edema, including hemodynamic
pulmonary edema and edema caused by microvascular injury, adult
respiratory distress syndrome (diffuse alveolar damage), pulmonary
embolism, hemorrhage, and infarction, and pulmonary hypertension
and vascular sclerosis; chronic obstructive pulmonary disease, such
as emphysema, chronic bronchitis, bronchial asthma, and
bronchiectasis; diffuse interstitial (infiltrative, restrictive)
diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity
pneumonitis, pulmonary eosinophilia (pulmonary infiltration with
eosinophilia), Bronchiolitis obliterans-organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, including Goodpasture
syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic
syndromes, pulmonary involvement in collagen vascular disorders,
and pulmonary alveolar proteinosis; complications of therapies,
such as drug-induced lung disease, radiation-induced lung disease,
and lung transplantation; tumors, such as bronchogenic carcinoma,
including paraneoplastic syndromes, bronchioloalveolar carcinoma,
neuroendocrine tumors, such as bronchial carcinoid, miscellaneous
tumors, and metastatic tumors; pathologies of the pleura, including
inflammatory pleural effusions, noninflammatory pleural effusions,
pneumothorax, and pleural tumors, including solitary fibrous tumors
(pleural fibroma) and malignant mesothelioma.
[0835] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0836] Disorders involving the ovary include, for example,
polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma
peritonei and stromal hyperthecosis; ovarian tumors such as, tumors
of coelomic epithelium, serous tumors, mucinous tumors,
endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma,
brenner tumor, surface epithelial tumors; germ cell tumors such as
mature (benign) teratomas, monodermal teratomas, immature malignant
teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma;
sex cord-stomal tumors such as, granulosa-theca cell tumors,
thecoma-fibromas, androblastomas, hill cell tumors, and
gonadoblastoma; and metastatic tumors such as Krukenberg
tumors.
[0837] Bone-forming cells include the osteoprogenitor cells,
osteoblasts, and osteocytes. The disorders of the bone are complex
because they may have an impact on the skeleton during any of its
stages of development. Hence, the disorders may have variable
manifestations and may involve one, multiple or all bones of the
body. Such disorders include, congenital malformations,
achondroplasia and thanatophoric dwarfism, diseases associated with
abnormal matix such as type 1 collagen disease, osteoporois, paget
disease, rickets, osteomalacia, high-turnover osteodystrophy,
low-turnover of aplastic disease, osteonecrosis, pyogenic
osteomyelitis, tuberculous osteomyelitism, osteoma, osteoid
osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,
chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous
cortical defects, fibrous dysplasia, fibrosarcoma, malignant
fibrous histiocytoma, ewing sarcoma, primitive neuroectodermal
tumor, giant cell tumor, and metastatic tumors.
[0838] The ubiquitin-proteasome pathway has been implicated in the
regulation of viral infection. Recent studies have shown that
ubiquitination of the herpes simplex virus type I (HSV-1)
transactivator protein ICP0 and the hepatitis B virus X protein
(HBX) are influenced by the ubiquitin-proteasome pathway during
viral infection (Weber et al. (1999) Virology 253:288-98 and Hu et
al. (1999) J Virol 73:7231-40). In addition, inactivation of the
ubiquitin-proteasome pathway inhibits Vmw110, an immediate early
protein of HSV-1, from stimulating lytic infection. (Everett et al.
(1998) EMBO J. 17:7161-9). Furthermore, a cellular deubiquitinating
enzyme, Herpes-virus associated ubiquitin specific protease, HAUSP,
has also been implicated in the regulation of HSV infection
(Everett et al. (1997) EMBO J. 16:1519-1530). Hence, the ubiquitin
protease find use in the treatment of disorders resulting from
viral infection.
[0839] Transcriptional profiling and Taqman profiling techniques
showed that the expression of the ubiquitin protease of the present
invention was upregulated in HSV-infected human ganglia cells
compared to uninfected ganglia. Furthermore, cell lines that
express a hepatitis B virus (HepG2.215) showed higher expression
levels of the ubiquitin protease 23484 when compared to the
parental HepG2 control cell line. The ubiquitin protease 23484 is
therefore an important host gene for HSV and HVB pathogenesis and
finds use in the treatment of disorders resulting from herpes
simplex virus and hepatitis B infection.
[0840] Additional disorders in which the ubiquitin protease
expression is relevant include, but are not limited to the
following:
[0841] Respiratory viral pathogens and their associated disorders
include, for example, adenovirus, resulting in upper and lower
respiratory tract infections; conjuctivitis and diarrhea;
echovirus, resulting in upper respiratory tract infections,
pharyngitis and rash; rhinovirus, resulting in upper respiratory
tract infections; cosackievirus, resulting in Pleurodynia,
herpangia, hand-foot-mouth disease; coronavirus, resulting in upper
respiratory tract infections; influenza A and B viruses, resulting
in influenza; parainfluenza virus 1-4, resulting in upper and lower
respiratory tract infections and croup; respiratory syncytial
virus, resulting in bronchiolitis and pneumonia.
[0842] Digestive viral pathogens and their associated disorders
include, for example, mumps virus, resulting in mumps,
pancreatitis, and orchitis; rotavirus, resulting in childhood
diarrhea; Norwalk Agent, resulting in gastroenteritis; hepatitis A
virus, resulting in acute viral hepatitis; hepatitis B virus,
hepatitis D virus and hepatitis C virus, resulting in acute or
chronic hepatitis; hepatitis E virus, resulting in enterically
transmitted hepatitis.
[0843] Systemic viral pathogens associated with disorders involving
skin eruptions include, for example, measles virus, resulting in
measles (rubeola); rubella virus, resulting in German measles
(rubella); parvovirus, resulting in erythema infectiosum and
aplastic anemia; varicella-zoster virus, resulting in chicken pox
and shingles; herpes simplex virus 1-associated, resulting in cold
sores; and herpes simplex virus 2, resulting in genital herpes.
[0844] Systemic viral pathogens associated with hematopoietic
disorders include, for example, cytomegalovirus, resulting in
cytomegalic inclusion disease; Epstein-Barr virus, resulting in
mononucleosis; HTLV-1, resulting in adult T-cell leukemia and
tropical spastic paraparesis; HTLV-II; and HIV 1 and HIV 2,
resulting in AIDS.
[0845] Arboviral pathogens associated with hemorrhagic fevers
include, for example, dengue virus 1-4, resulting in dengue and
hemorrhagic fever; yellow fever virus, resulting in yellow fever;
Colorado tick fever virus, resulting in Colorado tick fever; and
regional hemorrhagic fever viruses, resulting in Bolivian,
Argentinian, Lassa fever.
[0846] Viral pathogens associated with warty growths and other
hyperplasias include, for example, papillomavirus, resulting in
condyloma and cervical carcinoma; and molluscum virus, resulting in
molluscum contagiosum.
[0847] Viral pathogens associated with central nervous system
disorders include, for example, poliovirus, resulting in
poliomyelitis; rabiesvirus, associated with rabies; JC virus,
associated with progressive multifocal leukoencephalophathy; and
arboviral encephalitis viruses, resulting in Eastern, Western,
Venezuelan, St. Louis, or California group encephalitis.
[0848] Viral pathogens associated with cancer include, for example,
human papillomaviruses, implicated in the genesis of several
cancers including squamous cell carcinoma of the cervix and
anogenital region, oral cancer and laryngeal cancers; Epstein-Barr
virus, implicated in pathogenesis of the African form of Burkitt
lymphoma, B-cell lymphomas, Hodgkin disease, and nasopharyngeal
carcinomas; hepatitis B virus, implicated in liver cancer; human
T-cell leukemia virus type 1 (HTLV-1), associated with T-cell
leukemia/lymphoma; and the Kaposi sarcoma herpesvirus (KSHV).
[0849] The ubiquitin protease polypeptides are thus useful for
treating a ubiquitin protease-associated disorder characterized by
aberrant expression or activity of a ubiquitin protease. The
polypeptides can also be useful for treating a disorder
characterized by excessive amounts of polyubiquitin or
ubiquitinated substrate/remnant/amino acid. 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) expression or
activity of the protein. In another embodiment, the method involves
administering the ubiquitin protease as therapy to compensate for
reduced or aberrant expression or activity of the protein.
[0850] Methods for treatment include but are not limited to the use
of soluble ubiquitin protease or fragments of the ubiquitin
protease protein that compete for substrates including those
disclosed herein. These ubiquitin proteases or fragments can have a
higher affinity for the target so as to provide effective
competition.
[0851] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect, such as
virally-infected cells. Likewise, inhibition of activity is
desirable in situations in which the protein is abnormally
upregulated and/or in which decreased activity is likely to have a
beneficial effect. In one example of such a situation, a subject
has a disorder characterized by aberrant development or cellular
differentiation. In another example, the subject has a
proliferative disease (e.g., cancer) or a disorder characterized by
an aberrant hematopoietic response. In another example, it is
desirable to achieve tissue regeneration in a subject (e.g., where
a subject has undergone brain or spinal cord injury and it is
desirable to regenerate neuronal tissue in a regulated manner).
[0852] In yet another aspect of the invention, the proteins of the
invention 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 WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[0853] The ubiquitin protease polypeptides also are useful to
provide a target for diagnosing a disease or predisposition to
disease mediated by the ubiquitin protease, including, but not
limited to, diseases involving tissues in which the ubiquitin
proteases are expressed as disclosed herein, such as breast, lung,
and liver cancer (colon metastases). Accordingly, methods are
provided for detecting the presence, or levels of, the ubiquitin
protease in a cell, tissue, or organism. The method involves
contacting a biological sample with a compound capable of
interacting with the ubiquitin protease such that the interaction
can be detected.
[0854] The polypeptides are also useful for treating a disorder
characterized by reduced amounts of these components. Thus,
increasing or decreasing the activity of the protease is beneficial
to treatment. The polypeptides are also useful to provide a target
for diagnosing a disease characterized by excessive substrate or
reduced levels of substrate. Accordingly, where substrate is
excessive, use of the protease polypeptides can provide a
diagnostic assay. Furthermore, for example, proteases having
reduced activity can be used to diagnose conditions in which
reduced substrate is responsible for the disorder.
[0855] One agent for detecting ubiquitin protease is an antibody
capable of selectively binding to ubiquitin protease. A biological
sample includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject.
[0856] The ubiquitin protease also provides a target for diagnosing
active disease, or predisposition to disease, in a patient having a
variant ubiquitin protease. Thus, ubiquitin protease can be
isolated from a biological sample and assayed for the presence of a
genetic mutation that results in an aberrant protein. This includes
amino acid substitution, deletion, insertion, rearrangement, (as
the result of aberrant splicing events), and inappropriate
post-translational modification. Analytic methods include altered
electrophoretic mobility, altered tryptic peptide digest, altered
ubiquitin protease activity in cell-based or cell-free assay,
alteration in binding to or hydrolysis of polyubiquitin, binding to
ubiquitinated substrate protein or hydrolysis of the ubiquitin from
the protein, binding to ubiquitinated protein remnant, including
peptide or amino acid, and hydrolysis of the ubiquitin from the
remnant, general protein turnover, specific protein turnover,
antibody-binding pattern, altered isoelectric point, direct amino
acid sequencing, and any other of the known assay techniques useful
for detecting mutations in a protein in general or in a ubiquitin
protease specifically, including assays discussed herein.
[0857] In vitro techniques for detection of ubiquitin protease
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-ubiquitin protease 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. Particularly useful are methods, which
detect the allelic variant of the ubiquitin protease expressed in a
subject, and methods, which detect fragments of the ubiquitin
protease in a sample.
[0858] The ubiquitin protease polypeptides are also useful in
pharmacogenomic analysis. Pharmacogenomics deal with clinically
significant hereditary variations in the response to drugs due to
altered drug disposition and abnormal action in affected persons.
See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem.
43(2):254-266. The clinical outcomes of these variations result in
severe toxicity of therapeutic drugs in certain individuals or
therapeutic failure of drugs in certain individuals as a result of
individual variation in metabolism. Thus, the genotype of the
individual can determine the way a therapeutic compound acts on the
body or the way the body metabolizes the compound. Further, the
activity of drug metabolizing enzymes affects both the intensity
and duration of drug action. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
treatment based on the individual's genotype. The discovery of
genetic polymorphisms in some drug metabolizing enzymes has
explained why some patients do not obtain the expected drug
effects, show an exaggerated drug effect, or experience serious
toxicity from standard drug dosages. Polymorphisms can be expressed
in the phenotype of the extensive metabolizer and the phenotype of
the poor metabolizer. Accordingly, genetic polymorphism may lead to
allelic protein variants of the ubiquitin protease in which one or
more of the ubiquitin protease functions in one population is
different from those in another population. The polypeptides thus
allow a target to ascertain a genetic predisposition that can
affect treatment modality. Thus, in a ubiquitin-based treatment,
polymorphism may give rise to catalytic regions that are more or
less active. Accordingly, dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing the polymorphism. As an alternative to genotyping,
specific polymorphic polypeptides could be identified.
[0859] The ubiquitin protease polypeptides are also useful for
monitoring therapeutic effects during clinical trials and other
treatment. Thus, the therapeutic effectiveness of an agent that is
designed to increase or decrease gene expression, protein levels or
ubiquitin protease activity can be monitored over the course of
treatment using the ubiquitin protease polypeptides as an end-point
target. The monitoring can be, for example, as follows: (i)
obtaining a pre-administration sample from a subject prior to
administration of the agent; (ii) detecting the level of expression
or activity of the protein in the pre-administration sample; (iii)
obtaining one or more post-administration samples from the subject;
(iv) detecting the level of expression or activity of the protein
in the post-administration samples; (v) comparing the level of
expression or activity of the protein in the pre-administration
sample with the protein in the post-administration sample or
samples; and (vi) increasing or decreasing the administration of
the agent to the subject accordingly.
Antibodies
[0860] The invention also provides antibodies that selectively bind
to the ubiquitin protease and its variants and fragments. An
antibody is considered to selectively bind, even if it also binds
to other proteins that are not substantially homologous with the
ubiquitin protease. These other proteins share homology with a
fragment or domain of the ubiquitin protease. This conservation in
specific regions gives rise to antibodies that bind to both
proteins by virtue of the homologous sequence. In this case, it
would be understood that antibody binding to the ubiquitin protease
is still selective.
[0861] To generate antibodies, an isolated ubiquitin protease
polypeptide is used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. Either the full-length protein or antigenic peptide
fragment can be used. Regions having a high antigenicity index are
shown in FIG. 34.
[0862] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents substrate hydrolysis or binding. Antibodies can
be developed against the entire ubiquitin protease or domains of
the ubiquitin protease as described herein. Antibodies can also be
developed against specific functional sites as disclosed
herein.
[0863] The antigenic peptide can comprise a contiguous sequence of
at least 12, 14, 15, or amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments, which may be
disclosed prior to the invention.
[0864] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be
used.
[0865] 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.
[0866] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
Antibody Uses
[0867] The antibodies can be used to isolate a ubiquitin protease
by standard techniques, such as affinity chromatography or
immunoprecipitation. The antibodies can facilitate the purification
of the natural ubiquitin protease from cells and recombinantly
produced ubiquitin protease expressed in host cells.
[0868] The antibodies are useful to detect the presence of
ubiquitin protease in cells or tissues to determine the pattern of
expression of the ubiquitin protease among various tissues in an
organism and over the course of normal development.
[0869] The antibodies can be used to detect ubiquitin protease in
situ, in vitro, or in a cell lysate or supernatant in order to
evaluate the abundance and pattern of expression.
[0870] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[0871] Antibody detection of circulating fragments of the
full-length ubiquitin protease can be used to identify ubiquitin
protease turnover.
[0872] Further, the antibodies can be used to assess ubiquitin
protease expression in disease states such as in active stages of
the disease or in an individual with a predisposition toward
disease related to ubiquitin or ubiquitin protease function. When a
disorder is caused by an inappropriate tissue distribution,
developmental expression, or level of expression of the ubiquitin
protease protein, the antibody can be prepared against the normal
ubiquitin protease protein. If a disorder is characterized by a
specific mutation in the ubiquitin protease, antibodies specific
for this mutant protein can be used to assay for the presence of
the specific mutant ubiquitin protease. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular ubiquitin protease
peptide regions.
[0873] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole
ubiquitin protease or portions of the ubiquitin protease.
[0874] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting ubiquitin
protease expression level or the presence of aberrant ubiquitin
proteases and aberrant tissue distribution or developmental
expression, antibodies directed against the ubiquitin protease or
relevant fragments can be used to monitor therapeutic efficacy.
[0875] Antibodies accordingly 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.
[0876] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic ubiquitin
protease can be used to identify individuals that require modified
treatment modalities.
[0877] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant ubiquitin protease analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0878] The antibodies are also useful for tissue typing. Thus,
where a specific ubiquitin protease has been correlated with
expression in a specific tissue, antibodies that are specific for
this ubiquitin protease can be used to identify a tissue type.
[0879] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[0880] The antibodies are also useful for inhibiting ubiquitin
protease function, for example, blocking ubiquitin or polyubiquitin
binding, or binding to ubiquitinated substrate or substrate
remnants.
[0881] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting ubiquitin protease function. An
antibody can be used, for example, to block ubiquitin binding.
Antibodies can be prepared against specific fragments containing
sites required for function or against intact ubiquitin protease
associated with a cell.
[0882] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[0883] The invention also encompasses kits for using antibodies to
detect the presence of a ubiquitin protease protein in a biological
sample. The kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting ubiquitin
protease in a biological sample; means for determining the amount
of ubiquitin protease in the sample; and means for comparing the
amount of ubiquitin protease 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
ubiquitin protease.
Polynucleotides
[0884] The nucleotide sequence in SEQ ID NO:16 was obtained by
sequencing the deposited human cDNA. Accordingly, the sequence of
the deposited clone is controlling as to any discrepancies between
the two and any reference to the sequence of SEQ ID NO:16 includes
reference to the sequence of the deposited cDNA.
[0885] The specifically disclosed cDNA comprises the coding region
and 5' and 3' untranslated sequences in SEQ ID NO:16.
[0886] The invention provides isolated polynucleotides encoding the
novel ubiquitin protease. The term "ubiquitin protease
polynucleotide" or "ubiquitin protease nucleic acid" refers to the
sequence shown in SEQ ID NO:16 or in the deposited cDNA. The term
"ubiquitin protease polynucleotide" or "ubiquitin protease nucleic
acid" further includes variants and fragments of the ubiquitin
protease polynucleotide.
[0887] An "isolated" ubiquitin protease nucleic acid is one that is
separated from other nucleic acid present in the natural source of
the ubiquitin protease nucleic acid. Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the
ubiquitin protease 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. However, there can be some
flanking nucleotide sequences, for example up to about 5 KB. The
important point is that the ubiquitin protease nucleic acid is
isolated from flanking sequences such that it can be subjected to
the specific manipulations described herein, such as recombinant
expression, preparation of probes and primers, and other uses
specific to the ubiquitin protease nucleic acid sequences.
[0888] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0889] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0890] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0891] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0892] The ubiquitin protease polynucleotides can encode the mature
protein plus additional amino or carboxyterminal amino acids, or
amino acids interior to the mature polypeptide (when the mature
form has more than one polypeptide chain, for instance). Such
sequences may play a role in processing of a protein from precursor
to a mature form, facilitate protein trafficking, prolong or
shorten protein half-life or facilitate manipulation of a protein
for assay or production, among other things. As generally is the
case in situ, the additional amino acids may be processed away from
the mature protein by cellular enzymes.
[0893] The ubiquitin protease polynucleotides include, but are not
limited to, the sequence encoding the mature polypeptide alone, the
sequence encoding the mature polypeptide and additional coding
sequences, such as a leader or secretory sequence (e.g., a pre-pro
or pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[0894] Ubiquitin protease polynucleotides can be in the form of
RNA, such as mRNA, or in the form DNA, including cDNA and genomic
DNA obtained by cloning or produced by chemical synthetic
techniques or by a combination thereof. The nucleic acid,
especially DNA, can be double-stranded or single-stranded.
Single-stranded nucleic acid can be the coding strand (sense
strand) or the non-coding strand (anti-sense strand).
[0895] Ubiquitin protease nucleic acid can comprise the nucleotide
sequence shown in SEQ ID NO:16, corresponding to human cDNA.
[0896] In one embodiment, the ubiquitin protease nucleic acid
comprises only the coding region.
[0897] The invention further provides variant ubiquitin protease
polynucleotides, and fragments thereof, that differ from the
nucleotide sequence shown in SEQ ID NO:16 due to degeneracy of the
genetic code and thus encode the same protein as that encoded by
the nucleotide sequence shown in SEQ ID NO:16.
[0898] The invention also provides ubiquitin protease nucleic acid
molecules encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[0899] Typically, variants have a substantial identity with a
nucleic acid molecule of SEQ ID NO:16 and the complements thereof.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[0900] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a ubiquitin protease that is at least
about 60-65%, 65-70%, typically at least about 70-75%, more
typically at least about 80-85%, and most typically at least about
90-95% or more homologous to the nucleotide sequence shown in SEQ
ID NO:16. Such nucleic acid molecules can readily be identified as
being able to hybridize under stringent conditions, to the
nucleotide sequence shown in SEQ ID NO:16 or a fragment of the
sequence. It is understood that stringent hybridization does not
indicate substantial homology where it is due to general homology,
such as poly A sequences, or sequences common to all or most
proteins or all deubiquitinating enzymes. Moreover, it is
understood that variants do not include any of the nucleic acid
sequences that may have been disclosed prior to the invention.
[0901] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 60-65% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95% or more
identical to each other remain hybridized to one another. 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, incorporated by reference.
One 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 50-65.degree. C. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times.SSC/0.1% SDS at room
temperature, or by one or more moderate stringency washes in
0.2.times.SSC/0.1% SDS at 42.degree. C., or washed in
0.2.times.SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:15 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).
[0902] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[0903] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:16 or the complement of SEQ ID NO:16. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO:16 or the complement of SEQ ID NO:16.
[0904] It is understood that isolated fragments include any
contiguous sequence not disclosed prior to the invention as well as
sequences that are substantially the same and which are not
disclosed. Accordingly, if a fragment is disclosed prior to the
present invention, that fragment is not intended to be encompassed
by the invention. When a sequence is not disclosed prior to the
present invention, an isolated nucleic acid fragment is at least
about 15, preferably at least about 18, 20, 23 or 25 nucleotides,
and can be 30, 40, 50, 100, 200, 500 or more nucleotides in
length.
[0905] For example, nucleotide sequences 1 to about 269, about 761
to about 817, about 994 to about 1554, and about 1735 to about 2314
are not disclosed prior to the invention. The nucleotide sequence
from about 269 to 761 encompasses fragments greater than 14, 18,
20, 23 or 25 nucleotides; the nucleotide sequence from about 817 to
about 994 encompasses fragments greater than 6, 10, 15, 20, or 25
nucleotides; the nucleotide sequences from about 1154 to 1735
encompasses fragments greater than 13, 18, 20, 23 or 25
nucleotides; and the nucleotide sequence from about 2314 to about
2520 encompasses fragments greater than 33, 40, 45, or 50
nucleotides. Longer fragments, for example, 30 or more nucleotides
in length, which encode antigenic proteins or polypeptides
described herein are useful.
[0906] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length ubiquitin protease
polynucleotides. The fragment can be single or double-stranded and
can comprise DNA or RNA. The fragment can be derived from either
the coding or the non-coding sequence.
[0907] In another embodiment an isolated ubiquitin protease nucleic
acid encodes the entire coding region. Other fragments include
nucleotide sequences encoding the amino acid fragments described
herein.
[0908] Thus, ubiquitin protease nucleic acid fragments further
include sequences corresponding to the domains described herein,
subregions also described, and specific functional sites. Ubiquitin
protease nucleic acid fragments also include combinations of the
domains, segments, and other functional sites described above. A
person of ordinary skill in the art would be aware of the many
permutations that are possible.
[0909] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary sill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[0910] However, it is understood that a ubiquitin protease fragment
includes any nucleic acid sequence that does not include the entire
gene.
[0911] The invention also provides ubiquitin protease nucleic acid
fragments that encode epitope bearing regions of the ubiquitin
protease proteins described herein.
[0912] Nucleic acid fragments, according to the present invention,
are not to be construed as encompassing those fragments that may
have been disclosed prior to the invention.
Polynucleotide Uses
[0913] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to 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 protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins 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.
[0914] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of the nucleic
acid sequence shown in SEQ ID NO:16 and the complements thereof.
More typically, the probe further comprises a label, e.g.,
radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
[0915] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[0916] The ubiquitin protease polynucleotides are thus useful for
probes, primers, and in biological assays.
[0917] Where the polynucleotides are used to assess ubiquitin
protease properties or functions, such as in the assays described
herein, all or less than all of the entire cDNA can be useful.
Assays specifically directed to ubiquitin protease functions, such
as assessing agonist or antagonist activity, encompass the use of
known fragments. Further, diagnostic methods for assessing
ubiquitin protease function can also be practiced with any
fragment, including those fragments that may have been known prior
to the invention. Similarly, in methods involving treatment of
ubiquitin protease dysfunction, all fragments are encompassed
including those, which may have been known in the art.
[0918] The ubiquitin protease polynucleotides are useful as a
hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding the polypeptide
described in SEQ ID NO:15 and to isolate cDNA and genomic clones
that correspond to variants producing the same polypeptide shown in
SEQ ID NO:15 or the other variants described herein. Variants can
be isolated from the same tissue and organism from which the
polypeptides shown in SEQ ID NO:15 were isolated, different tissues
from the same organism, or from different organisms. This method is
useful for isolating genes and cDNA that are
developmentally-controlled and therefore may be expressed in the
same tissue or different tissues at different points in the
development of an organism.
[0919] The probe can correspond to any sequence along the entire
length of the gene encoding the ubiquitin protease. Accordingly, it
could be derived from 5' noncoding regions, the coding region, and
3' noncoding regions.
[0920] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:16 or a fragment thereof that is sufficient to
specifically hybridize under stringent conditions to mRNA or
DNA.
[0921] Fragments of the polynucleotides described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an mRNA and a larger or full-length
cDNA can be produced.
[0922] The fragments are also useful to synthesize antisense
molecules of desired length and sequence.
[0923] Antisense nucleic acids of the invention can be designed
using the nucleotide sequence of SEQ ID NO:16, and 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,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N2-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).
[0924] Additionally, the nucleic acid molecules of the 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 acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). 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 et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity 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. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[0925] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell ubiquitin proteases 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. WO 88/0918) or the blood brain barrier (see, e.g.,
PCT Publication No. WO 89/10134). 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).
[0926] The ubiquitin protease polynucleotides are also useful as
primers for PCR to amplify any given region of a ubiquitin protease
polynucleotide.
[0927] The ubiquitin protease polynucleotides are also useful for
constructing recombinant vectors. Such vectors include expression
vectors that express a portion of, or all of, the ubiquitin
protease polypeptides. Vectors also include insertion vectors, used
to integrate into another polynucleotide sequence, such as into the
cellular genome, to alter in situ expression of ubiquitin protease
genes and gene products. For example, an endogenous ubiquitin
protease coding sequence can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[0928] The ubiquitin protease polynucleotides are also useful for
expressing antigenic portions of the ubiquitin protease
proteins.
[0929] The ubiquitin protease polynucleotides are also useful as
probes for determining the chromosomal positions of the ubiquitin
protease polynucleotides by means of in situ hybridization methods,
such as FISH. (For a review of this technique, see Verma et al.
(1988) Human Chromosomes: A Manual of Basic Techniques (Pergamon
Press, New York), and PCR mapping of somatic cell hybrids. The
mapping of the sequences to chromosomes is an important first step
in correlating these sequences with genes associated with
disease.
[0930] 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.
[0931] 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 et al. ((1987) Nature 325:783-787).
[0932] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified 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.
[0933] The ubiquitin protease polynucleotide probes are also useful
to determine patterns of the presence of the gene encoding the
ubiquitin proteases and their variants with respect to tissue
distribution, for example, whether gene duplication has occurred
and whether the duplication occurs in all or only a subset of
tissues. The genes can be naturally occurring or can have been
introduced into a cell, tissue, or organism exogenously.
[0934] The ubiquitin protease polynucleotides are also useful for
designing ribozymes corresponding to all, or a part, of the mRNA
produced from genes encoding the polynucleotides described
herein.
[0935] The ubiquitin protease polynucleotides are also useful for
constructing host cells expressing a part, or all, of the ubiquitin
protease polynucleotides and polypeptides.
[0936] The ubiquitin protease polynucleotides are also useful for
constructing transgenic animals expressing all, or a part, of the
ubiquitin protease polynucleotides and polypeptides.
[0937] The ubiquitin protease polynucleotides are also useful for
making vectors that express part, or all, of the ubiquitin protease
polypeptides.
[0938] The ubiquitin protease polynucleotides are also useful as
hybridization probes for determining the level of ubiquitin
protease nucleic acid expression. Accordingly, the probes can be
used to detect the presence of, or to determine levels of,
ubiquitin protease nucleic acid in cells, tissues, and in
organisms. The nucleic acid whose level is determined can be DNA or
RNA. Accordingly, probes corresponding to the polypeptides
described herein can be used to assess gene copy number in a given
cell, tissue, or organism. This is particularly relevant in cases
in which there has been an amplification of the ubiquitin protease
genes.
[0939] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
ubiquitin protease genes, as on extrachromosomal elements or as
integrated into chromosomes in which the ubiquitin protease gene is
not normally found, for example as a homogeneously staining
region.
[0940] These uses are relevant for diagnosis of disorders involving
an increase or decrease in ubiquitin protease expression relative
to normal, such as a proliferative disorder, a differentiative or
developmental disorder, or a hematopoietic disorder.
[0941] Tissues and/or cells in which the ubiquitin protease is
expressed include, but are not limited to those shown in FIGS. 37
and 38. Tissues in which the gene is highly expressed include fetal
kidney, testes, fetal liver, ovary, and fetal heart. Expression is
also seen in the kidney, thyroid, undifferentiated osteoblasts and
skeletal muscle. The ubiquitin protease is also expressed in normal
liver and in normal and malignant breast, lung, and colon tissue
and in liver metastases derived from malignant colonic tissues. The
ubiquitin proteases are thus specifically involved in breast, lung,
and liver cancer.
[0942] As such, the gene is particularly relevant for the treatment
of disorders involving these tissues. Disorders in which the
ubiquitin protease expression is relevant are disclosed herein
above.
[0943] Furthermore, the ubiquitin protease is useful to treat viral
infections and disorders resulting from viral infections. Such
disorders are discussed above.
[0944] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of ubiquitin protease nucleic acid, in which
a test sample is obtained from a subject and nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of the nucleic
acid is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the nucleic acid.
[0945] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[0946] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0947] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express the ubiquitin protease,
such as by measuring the level of a ubiquitin protease-encoding
nucleic acid in a sample of cells from a subject e.g., mRNA or
genomic DNA, or determining if the ubiquitin protease gene has been
mutated.
[0948] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate ubiquitin protease nucleic acid
expression (e.g., antisense, polypeptides, peptidomimetics, small
molecules or other drugs). A cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of the mRNA in the presence of the candidate compound is
compared to the level of expression of the mRNA in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of nucleic acid expression based on this
comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. The modulator
can bind to the nucleic acid or indirectly modulate expression,
such as by interacting with other cellular components that affect
nucleic acid expression.
[0949] 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 gent to a subject) in patients or in
transgenic animals.
[0950] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the ubiquitin protease gene. The method
typically includes assaying the ability of the compound to modulate
the expression of the ubiquitin protease nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by undesired ubiquitin protease nucleic acid
expression.
[0951] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
ubiquitin protease nucleic acid or recombinant cells genetically
engineered to express specific nucleic acid sequences.
[0952] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[0953] The assay for ubiquitin protease nucleic acid expression can
involve direct assay of nucleic acid levels, such as mRNA levels,
or on collateral compounds involved in the pathway (such as free
ubiquitin pool or protein turnover). Further, the expression of
genes that are up- or down-regulated in response to the ubiquitin
protease activity can also be assayed. In this embodiment the
regulatory regions of these genes can be operably linked to a
reporter gene such as luciferase.
[0954] Thus, modulators of ubiquitin protease gene expression can
be identified in a method wherein a cell is contacted with a
candidate compound and the expression of mRNA determined. The level
of expression of ubiquitin protease mRNA in the presence of the
candidate compound is compared to the level of expression of
ubiquitin protease mRNA in the absence of the candidate compound.
The candidate compound can then be identified as a modulator of
nucleic acid expression based on this comparison and be used, for
example to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[0955] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate ubiquitin
protease nucleic acid expression. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or effects on nucleic acid activity
(e.g., when nucleic acid is mutated or improperly modified).
Treatment includes disorders characterized by aberrant expression
or activity of the nucleic acid. In addition, disorders that are
influenced by the ubiquitin protease may also be treated. Examples
of such disorders are disclosed herein.
[0956] Alternatively, a modulator for ubiquitin protease nucleic
acid expression can be a small molecule or drug identified using
the screening assays described herein as long as the drug or small
molecule inhibits the ubiquitin protease nucleic acid
expression.
[0957] The ubiquitin protease polynucleotides are also useful for
monitoring the effectiveness of modulating compounds on the
expression or activity of the ubiquitin protease gene in clinical
trials or in a treatment regimen. Thus, the gene expression pattern
can serve as a barometer for the continuing effectiveness of
treatment with the compound, particularly with compounds to which a
patient can develop resistance. The gene expression pattern can
also serve as a marker indicative of a physiological response of
the affected cells to the compound. Accordingly, such monitoring
would allow either increased administration of the compound or the
administration of alternative compounds to which the patient has
not become resistant. Similarly, if the level of nucleic acid
expression falls below a desirable level, administration of the
compound could be commensurately decreased.
[0958] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[0959] The ubiquitin protease polynucleotides are also useful in
diagnostic assays for qualitative changes in ubiquitin protease
nucleic acid, and particularly in qualitative changes that lead to
pathology. The polynucleotides can be used to detect mutations in
ubiquitin protease genes and gene expression products such as mRNA.
The polynucleotides can be used as hybridization probes to detect
naturally-occurring genetic mutations in the ubiquitin protease
gene and thereby to determine whether a subject with the mutation
is at risk for a disorder caused by the mutation. Mutations include
deletion, addition, or substitution of one or more nucleotides in
the gene, chromosomal rearrangement, such as inversion or
transposition, modification of genomic DNA, such as aberrant
methylation patterns or changes in gene copy number, such as
amplification. Detection of a mutated form of the ubiquitin
protease gene associated with a dysfunction provides a diagnostic
tool for an active disease or susceptibility to disease when the
disease results from overexpression, underexpression, or altered
expression of a ubiquitin protease.
[0960] Mutations in the ubiquitin protease gene can be detected at
the nucleic acid level by a variety of techniques. Genomic DNA can
be analyzed directly or can be amplified by using PCR prior to
analysis. RNA or cDNA can be used in the same way.
[0961] In certain embodiments, detection of the mutation 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) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
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 patient, 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
gene under conditions such that hybridization and amplification of
the 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.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0962] 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.
[0963] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[0964] Alternatively, mutations in a ubiquitin protease gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0965] Further, sequence-specific ribozymes (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.
[0966] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0967] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and SI protection or
the chemical cleavage method.
[0968] Furthermore, sequence differences between a mutant ubiquitin
protease gene and a wild-type gene can be determined by direct DNA
sequencing. 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).
[0969] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). 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 one 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).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[0970] In other embodiments, genetic mutations 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
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two-dimensional
arrays containing light-generated DNA probes as described in Cronin
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.
[0971] The ubiquitin protease polynucleotides are also useful for
testing an individual for a genotype that while not necessarily
causing the disease, nevertheless affects the treatment modality.
Thus, the polynucleotides can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship). In the
present case, for example, a mutation in the ubiquitin protease
gene that results in altered affinity for ubiquitin could result in
an excessive or decreased drug effect with standard concentrations
of ubiquitin or analog. Accordingly, the ubiquitin protease
polynucleotides described herein can be used to assess the mutation
content of the gene in an individual in order to select an
appropriate compound or dosage regimen for treatment.
[0972] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[0973] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[0974] The ubiquitin protease polynucleotides are also useful for
chromosome identification when the sequence is identified with an
individual chromosome and to a particular location on the
chromosome. First, the DNA sequence is matched to the chromosome by
in situ or other chromosome-specific hybridization. Sequences can
also be correlated to specific chromosomes by preparing PCR primers
that can be used for PCR screening of somatic cell hybrids
containing individual chromosomes from the desired species. Only
hybrids containing the chromosome containing the gene homologous to
the primer will yield an amplified fragment. Sublocalization can be
achieved using chromosomal fragments. Other strategies include
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to chromosome-specific libraries. Further mapping
strategies include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the 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.
[0975] The ubiquitin protease polynucleotides can also be used to
identify individuals based on small biological samples. This can be
done for example using restriction fragment-length polymorphism
(RFLP) to identify an individual. Thus, the polynucleotides
described herein are useful as DNA markers for RFLP (See U.S. Pat.
No. 5,272,057).
[0976] Furthermore, the ubiquitin protease sequence can be used to
provide an alternative technique, which determines the actual DNA
sequence of selected fragments in the genome of an individual.
Thus, the ubiquitin protease 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 DNA from an
individual for subsequent sequencing.
[0977] 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. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
ubiquitin protease sequences can be used to obtain such
identification sequences from individuals and from tissue. The
sequences represent unique fragments of the human genome. 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.
[0978] If a panel of reagents from the sequences 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.
[0979] The ubiquitin protease polynucleotides can also be used in
forensic identification procedures. PCR technology can be used to
amplify DNA sequences taken from very small biological samples,
such as a single hair follicle, body fluids (e.g., blood, saliva,
or semen). The amplified sequence can then be compared to a
standard allowing identification of the origin of the sample.
[0980] The ubiquitin protease polynucleotides can thus 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 described
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 the
noncoding region are particularly useful since greater polymorphism
occurs in the noncoding regions, making it easier to differentiate
individuals using this technique.
[0981] The ubiquitin protease polynucleotides 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. This is useful in cases
in which a forensic pathologist is presented with a tissue of
unknown origin. Panels of ubiquitin protease probes can be used to
identify tissue by species and/or by organ type.
[0982] In a similar fashion, these primers and 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).
[0983] Alternatively, the ubiquitin protease polynucleotides can be
used directly to block transcription or translation of ubiquitin
protease gene sequences by means of antisense or ribozyme
constructs. Thus, in a disorder characterized by abnormally high or
undesirable ubiquitin protease gene expression, nucleic acids can
be directly used for treatment.
[0984] The ubiquitin protease polynucleotides are thus useful as
antisense constructs to control ubiquitin protease gene expression
in cells, tissues, and organisms. A DNA antisense polynucleotide is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
ubiquitin protease protein. An antisense RNA or DNA polynucleotide
would hybridize to the mRNA and thus block translation of mRNA into
ubiquitin protease protein.
[0985] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:16 which also
includes the start codon and antisense molecules which are
complementary to a fragment of the 3' untranslated region of SEQ ID
NO:16.
[0986] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of ubiquitin
protease nucleic acid. Accordingly, these molecules can treat a
disorder characterized by abnormal or undesired ubiquitin protease
nucleic acid expression. This technique involves cleavage by means
of ribozymes containing nucleotide sequences complementary to one
or more regions in the mRNA that attenuate the ability of the mRNA
to be translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the ubiquitin protease protein.
[0987] The ubiquitin protease polynucleotides also provide vectors
for gene therapy in patients containing cells that are aberrant in
ubiquitin protease gene expression. Thus, recombinant cells, which
include the patient's cells that have been engineered ex vivo and
returned to the patient, are introduced into an individual where
the cells produce the desired ubiquitin protease protein to treat
the individual.
[0988] The invention also encompasses kits for detecting the
presence of a ubiquitin protease nucleic acid in a biological
sample. For example, the kit can comprise reagents such as a
labeled or labelable nucleic acid or agent capable of detecting
ubiquitin protease nucleic acid in a biological sample; means for
determining the amount of ubiquitin protease nucleic acid in the
sample; and means for comparing the amount of ubiquitin protease
nucleic acid 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 ubiquitin
protease mRNA or DNA.
Computer Readable Means
[0989] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0990] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0991] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0992] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0993] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0994] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0995] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0996] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0997] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
Vectors/Host Cells
[0998] The invention also provides vectors containing the ubiquitin
protease polynucleotides. The term "vector" refers to a vehicle,
preferably a nucleic acid molecule that can transport the ubiquitin
protease polynucleotides. When the vector is a nucleic acid
molecule, the ubiquitin protease polynucleotides are covalently
linked to the vector nucleic acid. With this aspect of the
invention, the vector includes a plasmid, single or double stranded
phage, a single or double stranded RNA or DNA viral vector, or
artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
[0999] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the ubiquitin protease polynucleotides.
Alternatively, the vector may integrate into the host cell genome
and produce additional copies of the ubiquitin protease
polynucleotides when the host cell replicates.
[1000] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
ubiquitin protease polynucleotides. The vectors can function in
procaryotic or eukaryotic cells or in both (shuttle vectors).
[1001] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the ubiquitin protease
polynucleotides such that transcription of the polynucleotides is
allowed in a host cell. The polynucleotides can be introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus, the second polynucleotide may provide a
trans-acting factor interacting with the cis-regulatory control
region to allow transcription of the ubiquitin protease
polynucleotides from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself.
[1002] It is understood, however, that in some embodiments,
transcription and/or translation of the ubiquitin protease
polynucleotides can occur in a cell-free system.
[1003] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[1004] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[1005] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[1006] A variety of expression vectors can be used to express a
ubiquitin protease polynucleotide. Such vectors include
chromosomal, episomal, and virus-derived vectors, for example
vectors derived from bacterial plasmids, from bacteriophage, from
yeast episomes, from yeast chromosomal elements, including yeast
artificial chromosomes, from viruses such as baculoviruses,
papovaviruses such as SV40, Vaccinia viruses, adenoviruses,
poxviruses, pseudorabies viruses, and retroviruses. Vectors may
also be derived from combinations of these sources such as those
derived from plasmid and bacteriophage genetic elements, e.g.,
cosmids and phagemids. Appropriate cloning and expression vectors
for prokaryotic and eukaryotic hosts are described in Sambrook et
al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[1007] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e., tissue specific) or may provide
for inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[1008] The ubiquitin protease polynucleotides can be inserted into
the vector nucleic acid by well-known methodology. Generally, the
DNA sequence that will ultimately be expressed is joined to an
expression vector by cleaving the DNA sequence and the expression
vector with one or more restriction enzymes and then ligating the
fragments together. Procedures for restriction enzyme digestion and
ligation are well known to those of ordinary skill in the art.
[1009] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[1010] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the
ubiquitin protease polypeptides. Fusion vectors can increase the
expression of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting for example as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired polypeptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enterokinase. Typical
fusion expression vectors include pGEX (Smith et al. (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. 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. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[1011] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[1012] The ubiquitin protease polynucleotides can also be expressed
by expression vectors that are operative in yeast. Examples of
vectors for expression in yeast e.g., S. cerevisiae include
pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan et
al. (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene
54:113-123), and pYES2 (Invitrogen Corporation, San Diego,
Calif.).
[1013] The ubiquitin protease polynucleotides can also be expressed
in insect cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL
series (Lucklow et al. (1989) Virology 170:31-39).
[1014] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[1015] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
ubiquitin protease polynucleotides. The person of ordinary skill in
the art would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[1016] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[1017] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[1018] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[1019] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the ubiquitin protease polynucleotides can be
introduced either alone or with other polynucleotides that are not
related to the ubiquitin protease polynucleotides such as those
providing trans-acting factors for expression vectors. When more
than one vector is introduced into a cell, the vectors can be
introduced independently, co-introduced or joined to the ubiquitin
protease polynucleotide vector.
[1020] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[1021] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[1022] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[1023] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the ubiquitin protease polypeptides
or heterologous to these polypeptides.
[1024] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[1025] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
Uses of Vectors and Host Cells
[1026] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also 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.
[1027] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing ubiquitin protease
proteins or polypeptides that can be further purified to produce
desired amounts of ubiquitin protease protein or fragments. Thus,
host cells containing expression vectors are useful for polypeptide
production.
[1028] Host cells are also useful for conducting cell-based assays
involving the ubiquitin protease or ubiquitin protease fragments.
Thus, a recombinant host cell expressing a native ubiquitin
protease is useful to assay for compounds that stimulate or inhibit
ubiquitin protease function. This includes disappearance of
substrate (polyubiquitin, ubiquitinated substrate protein,
ubiquitinated substrate remnants), appearance of end product
(ubiquitin monomers, polyubiquitin hydrolyzed from substrate or
substrate remnant, free substrate that has been rescued by
hydrolysis of ubiquitin), general or specific protein turnover, and
the various other molecular functions described herein that
include, but are not limited to, substrate recognition, substrate
binding, subunit association, and interaction with other cellular
components. Modulation of gene expression can occur at the level of
transcription or translation.
[1029] Host cells are also useful for identifying ubiquitin
protease mutants in which these functions are affected. If the
mutants naturally occur and give rise to a pathology, host cells
containing the mutations are useful to assay compounds that have a
desired effect on the mutant ubiquitin protease (for example,
stimulating or inhibiting function) which may not be indicated by
their effect on the native ubiquitin protease.
[1030] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation or alter specific function by means
of a heterologous domain, segment, site, and the like, as disclosed
herein.
[1031] Further, mutant ubiquitin proteases can be designed in which
one or more of the various functions is engineered to be increased
or decreased (e.g., binding to ubiquitin, polyubiquitin, or
ubiquitinated protein substrate) and used to augment or replace
ubiquitin protease proteins in an individual. Thus, host cells can
provide a therapeutic benefit by replacing an aberrant ubiquitin
protease or providing an aberrant ubiquitin protease that provides
a therapeutic result. In one embodiment, the cells provide
ubiquitin proteases that are abnormally active.
[1032] In another embodiment, the cells provide ubiquitin proteases
that are abnormally inactive. These ubiquitin proteases can compete
with endogenous ubiquitin proteases in the individual.
[1033] In another embodiment, cells expressing ubiquitin proteases
that cannot be activated, are introduced into an individual in
order to compete with endogenous ubiquitin proteases for ubiquitin
substrates. For example, in the case in which excessive ubiquitin
substrate or analog is part of a treatment modality, it may be
necessary to inactivate this molecule at a specific point in
treatment. Providing cells that compete for the molecule, but which
cannot be affected by ubiquitin protease activation would be
beneficial.
[1034] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous ubiquitin protease
polynucleotide sequences in a host cell genome. The host cell
includes, but is not limited to, a stable cell line, cell in vivo,
or cloned microorganism. This technology is more fully described in
WO 93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the ubiquitin protease polynucleotides or
sequences proximal or distal to a ubiquitin protease gene are
allowed to integrate into a host cell genome by homologous
recombination where expression of the gene can be affected. In one
embodiment, regulatory sequences are introduced that either
increase or decrease expression of an endogenous sequence.
Accordingly, a ubiquitin protease can be produced in a cell not
normally producing it. Alternatively, increased expression of
ubiquitin protease can be effected in a cell normally producing the
protein at a specific level. Further, expression can be decreased
or eliminated by introducing a specific regulatory sequence. The
regulatory sequence can be heterologous to the ubiquitin protease
protein sequence or can be a homologous sequence with a desired
mutation that affects expression. Alternatively, the entire gene
can be deleted. The regulatory sequence can be specific to the host
cell or capable of functioning in more than one cell type. Still
further, specific mutations can be introduced into any desired
region of the gene to produce mutant ubiquitin protease proteins.
Such mutations could be introduced, for example, into the specific
functional regions such as the ligand-binding site.
[1035] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered ubiquitin protease gene.
Alternatively, the host cell can be a stem cell or other early
tissue precursor that gives rise to a specific subset of cells and
can be used to produce transgenic tissues in an animal. See also
Thomas et al., Cell 51:503 (1987) for a description of homologous
recombination vectors. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced gene has homologously recombined with the endogenous
ubiquitin protease gene is selected (see e.g., Li, E. et al. (1992)
Cell 69:915). The selected cells are 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 vectors and 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; WO 91/01140; and WO 93/04169.
[1036] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. 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 in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of a ubiquitin protease protein and identifying and
evaluating modulators of ubiquitin protease protein activity.
[1037] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[1038] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which ubiquitin protease polynucleotide
sequences have been introduced.
[1039] A transgenic animal can be produced by introducing 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. Any of the
ubiquitin protease nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[1040] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
ubiquitin protease protein to particular cells.
[1041] 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 the transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[1042] 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 PI. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. 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 is
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.
[1043] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
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 blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[1044] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect, for example, binding, activation, and protein turnover, may
not be evident from in vitro cell-free or cell-based assays.
Accordingly, it is useful to provide non-human transgenic animals
to assay in vivo ubiquitin protease function, including substrate
interaction, the effect of specific mutant ubiquitin proteases on
ubiquitin protease function and substrate interaction, and the
effect of chimeric ubiquitin proteases. It is also possible to
assess the effect of null mutations, that is mutations that
substantially or completely eliminate one or more ubiquitin
protease functions.
[1045] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the
receptor protein in a transgenic animal, into a cell in culture or
in vivo. When introduced in vivo, the nucleic acid is introduced
into an intact organism such that one or more cell types and,
accordingly, one or more tissue types, express the nucleic acid
encoding the receptor protein. Alternatively, the nucleic acid can
be introduced into virtually all cells in an organism by
transfecting a cell in culture, such as an embryonic stem cell, as
described herein for the production of transgenic animals, and this
cell can be used to produce an entire transgenic organism. As
described, in a further embodiment, the host cell can be a
fertilized oocyte. Such cells are then allowed to develop in a
female foster animal to produce the transgenic organism.
Pharmaceutical Compositions
[1046] The ubiquitin protease nucleic acid molecules, protein
modulators of the protein, and antibodies (also referred to herein
as "active compounds") can be incorporated into pharmaceutical
compositions suitable for administration to a subject, e.g., a
human. Such compositions typically comprise the nucleic acid
molecule, protein, modulator, or antibody and a pharmaceutically
acceptable carrier.
[1047] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation, in
vivo, of polynucleotides that have been exogenously introduced into
a subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[1048] 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, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. 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 ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[1049] 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 polyethylene 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 mannitol, 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.
[1050] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a ubiquitin protease
protein or anti-ubiquitin protease 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.
[1051] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. 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.
[1052] 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.
[1053] 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.
[1054] 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.
[1055] 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.
[1056] 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.
[1057] 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 (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
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.
[1058] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[1059] 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.
[1060] 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. 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.
[1061] 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.
[1062] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the purview 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. 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.
[1063] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
CHAPTER 4
18891, a Novel Human Lipase
BACKGROUND OF THE INVENTION
[1064] Lipases are indispensable for the bioconversion of lipids
within an organism through the catalysis of a variety of reactions
that include hydrolysis, alcoholysis, acidolysis, esterfication and
aminolysis. In humans, several lipases have been identified which
possess lipolytic activities that regulate levels of triglycerides
and cholesterol in the body. Enzymes from this superfamily include
lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic
lipase (PL). While all three enzymes hydrolyze lipid emulsions and
have similar aqueous-lipid interfacial catalytic activities, they
each possess unique properties and physiological functions. All
three enzymes act preferentially on the sn-1 and sn-3 bonds of
triglycerides, to release fatty acids from the glycerol backbone
(Dolphin et al. (1992) Structure and Function of Apolipoproteins,
Rosseneu, M. (ed) CRC Press, Inc, Boca Ratan, 295-362). However,
while PL completes the hydrolysis of alimentary triglycerides, the
LPL and HL enzymes hydrolyze triglycerides found in circulating
lipoproteins.
[1065] Due to the insolubility of lipids in water, the plasma
transports complex lipids among various tissues as components of
lipoproteins. Each lipoprotein contains a neutral lipid core
composed of triacylglycerol and/or a cholesterol ester. Surrounding
the core is a layer of proteins, phospholipids, and cholesterol.
The proteins associated with the lipoprotein comprise a class of
proteins referred to as apoproteins (apo). Based on apoprotein
composition and density, lipoproteins have been classified into
five major types that include chylomicrons, high-density
lipoproteins (HDL), intermediate-density lipoproteins (IDL),
low-density lipoproteins (LDL), and very-low density lipoproteins
(VLDL).
[1066] Lipoprotein lipase (LPL) is the major enzyme responsible for
the hydrolysis of triglyceride molecules present in circulating
lipoproteins. LPL is associated with the luminal side of
capillaries and arteries through an interaction with
heparin-sulfate chains of proteoglycans and/or by glycerol
phosphatidylinostintol. With the help of the activator apo CII, LPL
hydrolyzes triglycerides of lipoproteins to produce free fatty
acids. Muscle and adipose tissue assimilate these fatty acids.
Alternatively, the fatty acids can be bound to albumin and
transported to other tissues. As the lipase hydrolyzes the
triglycerides of the lipoprotein, the particles become smaller and
are often referred to as lipoprotein remnants. Within the plasma
compartment, LPL converts chylomicrons to remnants and begins the
cascade requirements for conversion of VLDL to LDL.
[1067] In its active form, LPL is a glycosylated non-covalent
homodimer, with each subunit containing a binding site for heparin
and apolipoprotein (apo) CII, an activator protein required for LPL
activity. In addition to hydrolysis of triglycerides, LPL can
hydrolyze a variety of other substrates, for example, long and
short chain glycerides, phospholipids and various synthetic
substrates (Olivecrona et al. (1987) Lipoprotein Lipase
Borensztajn, J. (ed) Evener Publisher, Inc., pages 15-58).
[1068] In addition to the lypolytic activity of LPL described
above, LPL plays additional roles in lipid metabolism. After
sufficient hydrolysis, lipoprotein lipase is released from
proteoglycans and travels with the remnants of the chylomicrons or
VLDL. In the plasma LPL may then act to sequester the remnant
particles on surface proteoglycans. Subsequently LPL can act as a
ligand for receptors such as the LDL receptor, LDL-receptor related
protein, gp330, or the VLDL receptor. This interaction with the
cell surface receptor facilitates the uptake and degradation of
plasma lipoproteins by cells (Williams et al. (1992) J. Biol. Chem.
267:13284-13292 and Nykjaer et al. (1993) J. Biol. Chem.
268:15048-15055).
[1069] Furthermore, LPL expressed in macrophages has been
implicated in the cellular uptake of lipoprotein lipids and fat
soluble vitamins, the degradation of lipid-containing pathogens and
cell debris, and the creation of fatty acids for the energy
requirements of the cell.
[1070] Disruption of LPL activity has also been implicated in other
biological functions including, for example, enhanced oxidative
stress in blood cells, increased fluidity of the membrane
components of these cells and increases the susceptibility of their
mitochondrial DNA to structural alterations (Ven Murthy et al.
(1996) Acta Biochimica Polonica 43:227-40).
[1071] Hepatic lipase (HL) has functions in lipid metabolism
similar to those of LPL. HL is located on the surface of liver
sinusoids through glycosaminoglycan links where it interacts with
lipoproteins and hydrolyzes triglycerides into free fatty acids.
Unlike LPL, the activity of HL does not require an activator, but
its activity may be stimulated by apo E. Thus, the preferred
substrates of HL are the triglycerides of apo E-containing
lipoproteins, such as chylomicron remnants, IDL, and HDL.
Furthermore, the actions of HL on HDL are important in the reverse
cholesterol transport process, a mechanism thought to reduce excess
accumulation of cholesterol in hepatic tissue.
[1072] Like LPL, hepatic lipase has also been implicated in the
uptake and degradation of lipoprotein in the hepatic tissue.
Evidence suggests that HL may interact with cell surface receptors,
such as those described above, and direct hepatic cellular uptake
of lipoproteins and lipoprotein remnants. (Chappell et al. (1998)
Progress in Lipid Research 37: 363-422).
[1073] In its active form, HL exists as a monomer comprising both
triglyceride lipase activity and phospholipase activity. As with
LPL, treatment with heparin, results in the release of HL from the
cell surfaces. While glycosylation plays an important role in
secretion and affinity of LPL, it does not seem to be crucial for
HL activity.
[1074] Pancreatic lipase (PL) is synthesized in acinar cells of the
exocrine pancreas along with its protein activator, colipase. The
pancreatic duct transports glycosylated PL and colipase into the
duodenum. PL does not become anchored to membrane surfaces like LPL
or HL. Instead, the free monomer of PL interacts with colipase
which helps to anchor the PL to the lipid-water interface where the
enzyme completes the hydrolysis of alimentary triglycerides.
[1075] In summary, lipases play a key role in lipid metabolism by
regulating levels of cholesterol and triglycerides and therefore
influence major metabolic processes including effects on lipid and
lipoprotein concentrations, energy homeostasis, body weight, and
body composition-parameters. Each of these metabolic consequences
has been associated with common diseases, such as,
hypertriglyceridemia, atherosclerosis, obesity and various other
disease states described further below.
[1076] Accordingly, lipases are a major target for drug action and
development. Thus, it is valuable to the field of pharmaceutical
development to identify and characterize previously unknown
lipases. The present invention advances the state of the art by
providing a previously unidentified human lipase enzyme.
SUMMARY OF THE INVENTION
[1077] It is an object of the invention to identify novel
lipases.
[1078] It is a further object of the invention to provide novel
lipase polypeptides that are useful as reagents or targets in
assays applicable to treatment and diagnosis of lipase-mediated or
-related disorders, especially disorders mediated by or related to
lipase enzymes.
[1079] It is a further object of the invention to provide
polynucleotides corresponding to the novel lipase polypeptides that
are useful as targets and reagents in assays applicable to
treatment and diagnosis of lipase or lipase-mediated or -related
disorders and useful for producing novel lipase polypeptides by
recombinant methods.
[1080] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the novel lipase.
[1081] A further specific object of the invention is to provide
compounds that modulate expression of the lipase for treatment and
diagnosis of lipase and lipase-related disorders.
[1082] The invention is thus based on the identification of a novel
human lipase. The amino acid sequence is shown in SEQ ID NO:17. The
nucleotide sequence is shown in SEQ ID NO:18.
[1083] The invention provides isolated lipase polypeptides,
including a polypeptide having the amino acid sequence shown in SEQ
ID NO:17 or the amino acid sequence encoded by the cDNA deposited
as ATCC Patent Deposit No. PTA-1915 on May 24, 2000 ("the deposited
cDNA").
[1084] The invention also provides isolated lipase nucleic acid
molecules having the sequence shown in SEQ ID NO:18 or in the
deposited cDNA.
[1085] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO:17 or encoded by the deposited
cDNA.
[1086] The invention also provides variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO:18 or in the deposited cDNA.
[1087] The invention also provides fragments of the polypeptide
shown in SEQ ID NO:17 and nucleotide sequence shown in SEQ ID
NO:18, as well as substantially homologous fragments of the
polypeptide or nucleic acid.
[1088] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[1089] The invention also provides vectors and host cells for
expressing the lipase nucleic acid molecules and polypeptides, and
particularly recombinant vectors and host cells.
[1090] The invention also provides methods of making the vectors
and host cells and methods for using them to produce the lipase
nucleic acid molecules and polypeptides.
[1091] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the lipase polypeptides and
fragments.
[1092] The invention also provides methods of screening for
compounds that modulate expression or activity of the lipase
polypeptides or nucleic acid (RNA or DNA).
[1093] The invention also provides a process for modulating lipase
polypeptide or nucleic acid expression or activity, especially
using the screened compounds. Modulation may be used to treat
conditions related to aberrant activity or expression of the lipase
polypeptides or nucleic acids or aberrant activity resulting in the
altered accumulation/degradation of lipids.
[1094] The invention also provides assays for determining the
activity of or the presence or absence of the lipase polypeptides
or nucleic acid molecules in a biological sample, including for
disease diagnosis.
[1095] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[1096] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the invention,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[1097] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[1098] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Polypeptides
[1099] The invention is based on the identification of a novel
human lipase. Specifically, an expressed sequence tag (EST) was
selected based on homology to lipase sequences. This EST was used
to design primers based on sequences that it contains and used to
identify a cDNA from a brain library. Positive clones were
sequenced and the overlapping fragments were assembled. Analysis of
the assembled sequence revealed that the cloned cDNA molecule
encodes a lipase.
[1100] The invention thus relates to a novel lipase having the
deduced amino acid sequence shown in FIGS. 39A-39B (SEQ ID NO:17)
or having the amino acid sequence encoded by the cDNA insert of the
plasmid deposited with American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209, on May 24,
2000 and assigned Patent Deposit Number PTA-1915.
[1101] The deposits will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms. The deposits are provided as a convenience to those
of skill in the art and are not an admission that a deposit is
required under 35 U.S.C. .sctn.112. The deposited sequences, as
well as the polypeptides encoded by the sequences, are incorporated
herein by reference and controls in the event of any conflict, such
as a sequencing error, with description in this application.
[1102] "Lipase polypeptide" or "lipase protein" refers to the
polypeptide in SEQ ID NO:17 or encoded by the deposited cDNA. The
term "lipase protein" or "lipase polypeptide", however, further
includes the numerous variants described herein, as well as
fragments derived from the full-length lipase and variants.
[1103] Tissues and/or cells in which the lipase is expressed
include, but are not limited to those shown in FIGS. 43, 44, and
45. Tissues in which the gene is highly expressed include liver,
fetal liver, breast, brain, fetal kidney, and testis. Moderate
expression occurs in prostate, skeletal muscle, colon, kidney, and
thyroid. Lower positive expression occurs in heart, fetal heart,
small intestine, spleen, lung, ovary, vein, aorta, placenta,
osteoblasts, cervix, esophagus, thymus, tonsil, and lymph node. The
lipase is also expressed in malignant breast, lung, and colon
tissue and in liver metastases derived from malignant colonic
tissues. Hence, the lipase is relevant to disorders involving the
tissues in which it is expressed.
[1104] The present invention thus provides an isolated or purified
lipase polypeptide and variants and fragments thereof.
[1105] Based on Clustal W sequence alignment, highest homology was
shown to lipase 1 precursor (triacylglycerol lipase) from
Psychrobacter immobilis (Ace. No. Q02104).
[1106] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[1107] The lipase polypeptides can be purified to homogeneity. It
is understood, however, that preparations in which the polypeptide
is not purified to homogeneity are useful and considered to contain
an isolated form of the polypeptide. The critical feature is that
the preparation allows for the desired function of the polypeptide,
even in the presence of considerable amounts of other components.
Thus, the invention encompasses various degrees of purity.
[1108] In one embodiment, the language "substantially free of
cellular material" includes preparations of the lipase having less
than about 30% (by dry weight) other proteins (i.e., contaminating
protein), less than about 20% other proteins, less than about 10%
other proteins, or less than about 5% other proteins. When the
polypeptide is recombinantly produced, it can also be substantially
free of culture medium, i.e., culture medium represents less than
about 20%, less than about 10%, or less than about 5% of the volume
of the protein preparation.
[1109] A lipase polypeptide is also considered to be isolated when
it is part of a membrane preparation or is purified and then
reconstituted with membrane vesicles or liposomes.
[1110] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the lipase polypeptide in
which it is separated from chemical precursors or other chemicals
that are involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the polypeptide having less than about 30%
(by dry weight) chemical precursors or other chemicals, less than
about 20% chemical precursors or other chemicals, less than about
10% chemical precursors or other chemicals, or less than about 5%
chemical precursors or other chemicals.
[1111] In one embodiment, the lipase polypeptide comprises the
amino acid sequence shown in SEQ ID NO:17 or the mature form of the
polypeptide. However, the invention also encompasses sequence
variants. Variants include a substantially homologous protein
encoded by the same genetic locus in an organism, i.e., an allelic
variant.
[1112] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the lipase
of SEQ ID NO:17. Variants also include proteins substantially
homologous to the lipase but derived from another organism, i.e.,
an ortholog. Variants also include proteins that are substantially
homologous to the lipase that are produced by chemical synthesis.
Variants also include proteins that are substantially homologous to
the lipase that are produced by recombinant methods. It is
understood, however, that variants exclude any amino acid sequences
disclosed prior to the invention.
[1113] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 70-75%, typically at least about 80-85%, and most
typically at least about 90-95% or more homologous. A substantially
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence hybridizing to the
nucleic acid sequence, or portion thereof, of the sequence shown in
SEQ ID NO:18 under stringent conditions as more fully described
below.
[1114] 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-homologous
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 (i.e., 100%=the entire coding sequence). 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.
[1115] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the lipase.
Similarity is determined by conserved amino acid substitution. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Conservative substitutions are likely to be phenotypically silent.
Typically seen as conservative substitutions are the replacements,
one for another, among the aliphatic amino acids Ala, Val, Leu, and
Ile; interchange of the hydroxyl residues Ser and Thr, exchange of
the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr. Guidance
concerning which amino acid changes are likely to be phenotypically
silent are found in Bowie et al., Science 247:1306-1310 (1990).
TABLE-US-00003 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[1116] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[1117] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. See www.ncbi.nlm.nih.gov. In
one embodiment, parameters for sequence comparison can be set at
score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).
[1118] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (J. Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at 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
(Devereux et al. (1984) Nucleic Acids Res. 12(1):387) (available at
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.
[1119] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[1120] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[1121] Variant polypeptides can be fully functional or can lack
function in one or more activities. Thus, in the present case,
variations can affect the function of the lipase at a variety of
biological levels, including, disrupting interactions with the
proteoglycans, such as CSPG, HSPG, DSPG, disrupting interaction
with cell surface receptors, such as the LDL receptor, LDL-related
receptor protein, gp330, or the VLDL receptor, disrupting
interactions with heparin, disrupting interactions with apoproteins
or lipoproteins, disrupting interactions with activator molecules,
such as apo CII or colipase, disrupting triglyceride lipase
activity or phospholipase activity, or disrupting homodimer
formation. Variant polypeptides having such defects have been
identified for LPL and are described in, for example, Murthy et al.
(1996) Pharmacol. Ther. 70: 101-135, incorporated herein by
reference for teaching these variations.
[1122] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[1123] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[1124] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for the lipase polypeptide. This includes
preventing immunogenicity from pharmaceutical formulations by
preventing protein aggregation.
[1125] Useful variations further include alteration of catalytic
activity. For example, one embodiment involves a variation at the
binding site that results in binding but not hydrolysis, or slower
hydrolysis, of the triglyceride or phospholipid. A further useful
variation results in an increased rate of hydrolysis of the
triglycerides or phospholipids. Additional variations include
altered affinity for co-activator proteins, cell surface receptors,
proteoglycans, heparin, triglycerides, phospholipids, lipoproteins
or apoproteins. A further useful variation at the same site can
result in higher or lower affinity for substrates. Useful
variations also include changes that result in affinity to a
different lipoprotein or lipoprotein remnant than that normally
recognized. Other variations could result in altered recognition of
apoproteins thereby changing the preferred lipoproteins hydrolyzed
by the lipase. Further useful variations affect the ability of the
lipase to be induced by various activators, including, but not
limited to, those disclosed herein. Specific variations include
truncations in which a catalytic domain or substrate binding domain
is deleted. This variation results in a decrease or loss of lipid
hydrolytic activity or substrate binding. Another useful variation
includes one that prevents glycosylation. Further useful variations
provide a fusion protein in which one or more domains or subregions
are operationally fused to one or more domains or subregions from
another lipase. Specifically, a domain or subregion can be
introduced that provides a rescue function to an enzyme not
normally having this function or for recognition of a specific
substrate wherein recognition is not available to the original
enzyme. Further variations could affect specific subunit
interaction, particularly required for homodimerization or
interaction with activator proteins. Other variations would affect
developmental, temporal, or tissue-specific expression. Other
variations would affect the interaction with cellular components,
such as transcriptional regulatory factors.
[1126] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as the ability to hydrolyze triglycerides or phospholipids in
vitro. Alternatively, in vitro activity may be measured by the
ability to interact with various molecules, including but not
limited to, heparin, proteoglycans, cell surface receptors,
lipoproteins, apoproteins or activator proteins. Sites that are
critical for binding or recognition can also be determined by
structural analysis such as crystallization, nuclear magnetic
resonance or photoaffinity labeling (Smith et al. (1992) J. Mol.
Biol. 224:899-904; de Vos et al. (1992) Science 255:306-312).
[1127] The assays for lipase enzyme activity are well known in the
art and can be found, for example, in Brun et al. (1989) Metabolism
38:1005-1009, Brunzell et al. (1992) Atherosclerosis IX, Stein
(eds.) R&L Creative Communications Ltd., Tel Aviv 271-273,
Peeva et al. (1992) Int. J. Obes. Relat. Metab. Disord. 16:
737-744, Ma et al. (1991) N. Engl. J. Med. 324: 1761-1766, Ma et
al. (1992) J. Biol. Chem. 267: 1918-1923, Connelly et al. (1987) J.
Clin. Invest. 80: 1597-1606, Huff et al. (1990) J. Lipid Res. 31:
385-396, and Hixson et al. (1990) J Lipid Res. 31: 545-548. These
assays include measurements of triglyceride or lipoprotein
concentrations in the blood stream. For lipases associated with
proteoglycans, plasma lipolytic activity may be determined
following heparin treatment. In this protocol, lipase activity is
measured with a synthetic triglyceride substrate using plasma
samples obtained following heparin administration. Post-heparin
plasma may also be used to measure the lipase mass by immunoassay
to determine if a catalytically defective lipase enzyme is released
into the plasma. Lipase activity can also be determined in s.c.
biopsies of adipose tissue and through the detection of lipase gene
mutations. Additional assays include measuring lipase activation by
the co-activator molecules.
[1128] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[1129] The invention thus also includes polypeptide fragments of
the lipase. Fragments can be derived from the amino acid sequence
shown in SEQ ID NO:17. However, the invention also encompasses
fragments of the variants of the lipase as described herein.
[1130] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention.
[1131] Accordingly, a fragment can comprise at least about 8, 13,
15, 20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids.
Fragments can retain one or more of the biological activities of
the protein, for example the ability to bind to polyglycan,
interact with cell surface receptors, interact with activator
molecules, catalyze triglyceride hydrolysis, or retain
phospholipase activity. Fragments can be used as an immunogen to
generate lipase antibodies.
[1132] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a domain or motif,
e.g., catalytic sites, signal peptides, transmembrane segments, and
sites for protein kinase C phosphorylation, casein kinase II
phosphorylation, and N-myristoylation. Additional domains include
catalytic domains involved in triglyceride hydrolysis and
phospholipase activity, heparin binding sites, cell-surface
receptor binding sites, triglyceride binding sites, sites important
for homodimerization or activator interaction, and sites important
for carrying out the other functions of the lipase as described
herein.
[1133] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[1134] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific domains mentioned above, which sub-fragments retain
the function of the domain from which they are derived.
[1135] These regions can be identified by well-known methods
involving computerized homology analysis.
[1136] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the lipase
and variants. These epitope-bearing peptides are useful to raise
antibodies that bind specifically to a lipase polypeptide or region
or fragment. These peptides can contain at least 8, at least 10,
13, 15, or between at least about 16 to about 30 amino acids.
[1137] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular site. Regions having a high
antigenicity index are shown in FIG. 40. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular peptide
regions.
[1138] The epitope-bearing lipase polypeptides may be produced by
any conventional means (Houghten, R. A. (1985) Proc. Natl. Acad.
Sci. USA 82:5131-5135). Simultaneous multiple peptide synthesis is
described in U.S. Pat. No. 4,631,211.
[1139] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the lipase fragment and an
additional region fused to the carboxyl terminus of the
fragment.
[1140] The invention thus provides chimeric or fusion proteins.
These comprise a lipase peptide sequence operatively linked to a
heterologous peptide having an amino acid sequence not
substantially homologous to the lipase. "Operatively linked"
indicates that the lipase peptide and the heterologous peptide are
fused in-frame. The heterologous peptide can be fused to the
N-terminus or C-terminus of the lipase or can be internally
located.
[1141] In one embodiment the fusion protein does not affect lipase
function per se. For example, the fusion protein can be a
GST-fusion protein in which the lipase sequences are fused to the
N- or C-terminus of the GST sequences. Other types of fusion
proteins include, but are not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL-4 fusions, poly-His fusions and Ig fusions. Such fusion
proteins, particularly poly-His fusions, can facilitate the
purification of a recombinant lipase protein. In certain host cells
(e.g., mammalian host cells), expression and/or secretion of a
protein can be increased by using a heterologous signal sequence.
Therefore, in another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus.
[1142] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson
et al. J. Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing a lipase polypeptide
and various portions of the constant regions of heavy or light
chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE).
Preferred as immunoglobulin is the constant part of the heavy chain
of human IgG, particularly IgG1, where fusion takes place at the
hinge region. For some uses it is desirable to remove the Fc after
the fusion protein has been used for its intended purpose, for
example when the fusion protein is to be used as antigen for
immunizations. In a particular embodiment, the Fc part can be
removed in a simple way by a cleavage sequence, which is also
incorporated and can be cleaved with factor Xa.
[1143] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. 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 re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A lipase-encoding nucleic acid can be
cloned into such an expression vector such that the fusion moiety
is linked in-frame to the lipase.
[1144] Another form of fusion protein is one that directly affects
lipase functions. Accordingly, a lipase polypeptide is encompassed
by the present invention in which one or more of the lipase domains
(or parts thereof) has been replaced by homologous lipase domains
(or parts thereof) from another species. Accordingly, various
permutations are possible. One or more functional sites as
disclosed herein from the specifically disclosed lipase can be
replaced by one or more functional sites from a corresponding
lipase of another species. Thus, chimeric lipases can be formed in
which one or more of the native domains or subregions has been
replaced by another. For example, the catalytic domain of the
lipase of the present invention may be replaced by the catalytic
domain of a different lipase polypeptide. Alternatively, protein
domains that mediate the interaction with lipoproteins or domains
that mediated the uptake of lipoproteins by cell surface receptors
can be used to replace homologous domains of the lipase of the
present invention. In doing so the binding affinity to various
substrates and/or the rate of catalysis is altered.
[1145] Additionally, chimeric lipase proteins can be produced in
which one or more functional sites is derived from a different
member of the lipase superfamily. It is understood however that
sites could be derived from lipase families that occur in the
mammalian genome but which have not yet been discovered or
characterized. Such sites include but are not limited to any of the
functional sites disclosed herein.
[1146] The isolated lipase can be purified from any of the cells
that naturally express it, including, but not limited to those
shown in FIGS. 43, 44, and 45. Tissues in which the gene is highly
expressed include liver, fetal liver, breast, brain, fetal kidney,
and testis. Moderate expression occurs in prostate, skeletal
muscle, colon, kidney, and thyroid. Lower positive expression
occurs in heart, fetal heart, small intestine, spleen, lung, ovary,
vein, aorta, placenta, osteoblasts, cervix, esophagus, thymus,
tonsil, and lymph node. The lipase is also expressed in normal
liver and in normal and malignant breast, lung, and colon tissue
and in liver metastases derived from malignant colonic tissues.
Alternatively, the lipase may be purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods.
[1147] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
lipase polypeptide is cloned into an expression vector, the
expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques.
[1148] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally-occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in polypeptides are described in
basic texts, detailed monographs, and the research literature, and
they are well known to those of skill in the art.
[1149] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[1150] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[1151] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T.E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990)
Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y.
Acad. Sci. 663:48-62).
[1152] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
lipase, and they may be circular, with or without branching,
generally as a result of post-translation events, including natural
processing events and events brought about by human manipulation
which do not occur naturally. Circular, branched and branched
circular polypeptides may be synthesized by non-translational
natural processes and by synthetic methods.
[1153] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[1154] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[1155] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
Polypeptide Uses
[1156] The protein sequences of the present invention can 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 the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins 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 www.ncbi.nlm.nih.gov.
[1157] The lipase polypeptides are useful for producing antibodies
specific for the lipase protein, regions, or fragments. Regions
having a high antigenicity index score are shown in FIG. 40.
[1158] The lipase polypeptides are useful for biological assays
related to lipase function. Such assays involve any of the known
functions or activities or properties useful for diagnosis and
treatment of lipase- or lipase-related conditions or conditions in
which expression of the lipase is relevant, such as in
hypertriacylglycerolaemia, obesity, atherogenesis, and the various
other conditions described herein. Potential assays have been
disclosed herein.
[1159] The lipase polypeptides are also useful in drug screening
assays, in cell-based or cell-free systems. Cell-based systems can
be native, i.e., cells that normally express the lipase, as a
biopsy or expanded in cell culture. In one embodiment, however,
cell-based assays involve recombinant host cells expressing the
lipase.
[1160] Determining the ability of the test compound to interact
with the lipase can also comprise determining the ability of the
test compound to preferentially bind to the polypeptide as compared
to the ability of a known binding molecule (e.g., an activator
(such as colipase, apo CII), cell surface receptor, heparin,
proteoglycan, triglyceride, or phospholipid, or lipoprotein) to
bind to the polypeptide.
[1161] The polypeptides can be used to identify compounds that
modulate lipase activity. Modulators of lipase activity comprise
agents that influence the enzyme at a variety of biological levels,
including, but not limited to agents that disrupt the interaction
with the proteoglycans of the cell wall, such as HSPG-degrading
enzymes, heparin, chlorate, or APOE; agents that disrupt the
interaction with cell surface receptors; agents which disrupt the
interaction with activator molecules or homodimer formation; agents
that disrupt interaction with lipoproteins; or agents that disrupt
triglyceride hydrolysis or phospholipase activity.
[1162] The tissue specific regulation of lipase is complex with
identical modulators regulating activity differently under various
metabolic conditions. While specific modulators of lipase activity
have been described above, additional modulators include, but are
not limited to, apoproteins and a non-proteoglycan LPL-binding
protein having sequence homology to apo B and apo B (Sivaram et al.
(1992) J. Biol. Chem. 267:16517-16552; Sivaram et al. (1994) J.
Biol. Chem. 269:9409-9412). It has also been postulated that the
lipolysis-stimulated receptor (LSR) plays a role in LPL activation
(Yen et al. (1994) Biochemistry 33:1172-1180). Additional
modulators of lipase activity include, fasting, feeding, growth
hormone, insulin, exercise, estrogen, thyroid hormone,
catecholamines, hormones of the adrenergic system, vitamin D
derivatives, glucagon, catecholamines, glucocorticoids, and 1, 25
dihydroxy-vitamin D. Further modulators comprise inflammatory
mediators such as cytokines, interleukins, and interferons.
[1163] Modulators associated with an increase activity of lipase
activity include, but are not limited to various apoproteins, such
as apo CII, and glycosylation. Furthermore, lipase enzymatic
activity is stabilized in the presence of lipids or by binding to
lipid-water interfaces and detergents, such as deoxycholate.
Modulators associated with a decrease in lipase activity include,
but are not limited to, increased concentrations of apo CII or apo
cm (Shirari et al. (1981) Biochim. Biophys. Acta 665:504-510), TNF
(Kern et al. (1997) Journal of Nutrition 127:1917 S-1922S), fatty
acids, high salt concentrations, and Orlistar (La Roche,
Basele).
[1164] Both transcription and post-transcriptional levels of lipase
expression are regulated by various dietary, environmental, and
developmental factors and include, for example, hormones, such as
insulin, thyroid hormone, and glucocorticoids (Pykalisto et al.
(1976) J. clin. Endocronol. Metab. 43:591-600; Nillson-Ehle et al.
(1980) Annual Rev Biochem 49:667-693; and Cryer et al. (1981) Int.
J. Biochem 13:525-541). Various transcriptional factors such as
CEBP, ADD-1, SREBP-1 and PPAR .delta. also regulate expression of
specific lipases. It is understood, therefore, that such compounds
can be identified not only by means of direct interaction with the
lipase, but by means of any of the components that functionally
interact with the disclosed lipase. This includes, but is not
limited to, any of those components disclosed herein.
[1165] Both lipase and appropriate variants and fragments can be
used in high-throughput screens to assay candidate compounds for
the ability to bind to the lipase. These compounds can be further
screened against a functional lipase to determine the effect of the
compound on the lipase activity. Compounds can be identified that
activate (agonist) or inactivate (antagonist) the lipase to a
desired degree. 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).
[1166] The lipase polypeptides can be used to screen a compound for
the ability to stimulate or inhibit interaction between the lipase
protein and a target molecule that normally interacts with the
lipase protein. The target can be a lipoprotein, lipoprotein
remnant, apoprotein, cell surface receptors, heparin, proteoglycan,
triglyceride, phospholipid or another component of the pathway with
which the lipase protein normally interacts. The assay includes the
steps of combining the lipase protein with a candidate compound
under conditions that allow the lipase protein or fragment to
interact with the target molecule, and to detect the formation of a
complex between the lipase protein and the target or to detect the
biochemical consequence of the interaction with the lipase and the
target. Any of the associated effects of triglyceride hydrolysis or
phospholipase function can be assayed. This includes the production
of fatty acids from triglycerides and phospholipids.
[1167] Determining the ability of the lipase to bind to a target
molecule can also be accomplished using a technology such as
real-time Bimolecular Interaction Analysis (BIA). Sjolander et al.
(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.TM.). Changes in the optical
phenomenon surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
[1168] 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 polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[1169] 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. USA 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; Carell 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. 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. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[1170] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[1171] One candidate compound is a soluble full-length lipase or
fragment that competes for substrate binding. Other candidate
compounds include mutant lipases or appropriate fragments
containing mutations that affect lipase function and compete for
substrate. Accordingly, a fragment that competes for substrate, for
example with a higher affinity, or a fragment that binds substrate
but does not hydrolyze the triglyceride or phospholipid, is
encompassed by the invention.
[1172] Other candidate compounds include lipase protein or protein
analog that binds to the lipid, lipoprotein, proteoglycan, cell
surface receptor, or other substrates identified herein but is not
released or released slowly. Other candidate compounds include
analogs of the other natural substrates, such as substrates that
bind to but are not released or released more slowly. Further
candidate compounds include activators of the lipases, including
but not limited to, those disclosed herein.
[1173] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) lipase activity. The
assays typically involve an assay of events in the pathway that
indicate lipase activity. This can include cellular events that are
influenced by lipid metabolism, such as but not limited to, lipid
or lipoprotein concentrations. Specific phenotypes include
metabolic consequences including effects on energy homeostasis,
body weight and body composition-parameters.
[1174] Assays are based on the multiple cellular functions of
lipase enzymes. As described herein, these enzymes act at various
levels in the regulation of lipid metabolism. Accordingly, assays
can be based on detection of any of the products produced by the
lipase enzyme.
[1175] Further, the expression of genes that are up- or
down-regulated by action of the lipase can be assayed. In one
embodiment, the regulatory region of such genes can be operably
linked to a marker that is easily detectable, such as
luciferase.
[1176] Accordingly, any of the biological or biochemical functions
mediated by the lipase can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art.
[1177] Binding and/or activating compounds can also be screened by
using chimeric lipase proteins in which one or more domains, sites,
and the like, as disclosed herein, or parts thereof, can be
replaced by their heterologous counterparts derived from other
lipase protein. For example, a recognition or binding region can be
used that interacts with different substrate specificity and/or
affinity than the native lipase. Accordingly, a different set of
pathway components is available as an end-point assay for
activation. Further, sites that are responsible for developmental,
temporal, or tissue specificity can be replaced by heterologous
sites such that the lipase can be detected under conditions of
specific developmental, temporal, or tissue-specific
expression.
[1178] The lipase polypeptides are also useful in competition
binding assays in methods designed to discover compounds that
interact with the lipase. Thus, a compound is exposed to a lipase
polypeptide under conditions that allow the compound to bind to or
to otherwise interact with the polypeptide. A lipase target,
comprising a polypeptide or agent which is known to interact with
lipase, is also added to the mixture. If the test compound
interacts with the soluble lipase polypeptide, it decreases the
amount of complex formed or the activity from the lipase target.
This type of assay is particularly useful in cases in which
compounds are sought that interact with specific regions of the
lipase. Thus, the soluble polypeptide that competes with the target
lipase region is designed to contain peptide sequences
corresponding to the region of interest.
[1179] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, a candidate compound can be added to a sample of the
lipase. Compounds that interact with the lipase at the same site as
a lipase substrate disclosed herein will reduce the amount of
complex formed between the lipase and substrate. Accordingly, it is
possible to discover a compound that specifically prevents
interaction between the lipase and it various substrates. A
compound that competes with lipase catalytic activity will reduce
the rate of triglyceride or phospholipid hydrolysis. Alternatively,
a compound may also compete at the level of substrate interaction.
Accordingly, compounds can be discovered that directly interact
with the lipase and interfere with its function. Such assays can
involve any other component that interacts with the lipase such as
heparin, proteoglycans, lipoproteins, lipoprotein remnants, cell
surface receptors, triglycerides, phospholipids, activator
proteins, and other compounds described herein.
[1180] To perform cell free drug screening assays, it is desirable
to immobilize either the lipase, or fragment, or its target
molecule to facilitate separation of complexes from uncomplexed
forms of one or both of the proteins, as well as to accommodate
automation of the assay.
[1181] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/lipase
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 cell lysates
(e.g., .sup.35S-labeled) and the candidate compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly, or in
the supernatant after the complexes is dissociated. Alternatively,
the complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of lipase-binding protein found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques. For example, either the polypeptide or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively,
antibodies reactive with the protein but which do not interfere
with binding of the protein to its target molecule can be
derivatized to the wells of the plate, and the protein trapped in
the wells by antibody conjugation. Preparations of a lipase-binding
target component, such as, activator proteins, cell surface
receptors, lipoproteins, apoproteins, triglycerides or
phospholipids and a candidate compound are incubated in the
lipase-presenting wells and the amount of complex trapped in the
well can be quantitated. 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 lipase target molecule, or which are reactive
with lipase and compete with the target molecule; as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the target molecule.
[1182] Modulators of lipase activity identified according to these
drug screening assays can be used to treat a subject with a
disorder mediated or affected by a lipase, by treating cells that
express the lipase or cells in which lipase expression is
desirable. These methods of treatment include the steps of
administering the modulators of lipase activity in a pharmaceutical
composition as described herein, to a subject in need of such
treatment.
[1183] Tissues and/or cells in which the lipase is expressed
include, but are not limited to those shown in FIGS. 43, 44, and
45. Tissues in which the gene is highly expressed include liver,
fetal liver, breast, brain, fetal kidney, and testis. Moderate
expression occurs in prostate, skeletal muscle, colon, kidney, and
thyroid. Lower positive expression occurs in heart, fetal heart,
small intestine, spleen, lung, ovary, vein, aorta, placenta,
osteoblasts, cervix, esophagus, thymus, tonsil, and lymph node. The
lipase is also expressed in malignant breast, lung, and colon
tissue and in liver metastases derived from malignant colonic
tissues. Hence, the lipase is relevant to disorders involving the
tissues in which it is expressed.
[1184] Disorders involving the spleen include, but are not limited
to, splenomegaly, including nonspecific acute splenitis, congestive
spenomegaly, and spenic infarcts; neoplasms, congenital anomalies,
and rupture. Disorders associated with splenomegaly include
infections, such as nonspecific splenitis, infectious
mononucleosis, tuberculosis, typhoid fever, brucellosis,
cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis,
kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and
echinococcosis; congestive states related to partial hypertension,
such as cirrhosis of the liver, portal or splenic vein thrombosis,
and cardiac failure; lymphohematogenous disorders, such as Hodgkin
disease, non-Hodgkin lymphomas/leukemia, multiple myeloma,
myeloproliferative disorders, hemolytic anemias, and
thrombocytopenic purpura; immunologic-inflammatory conditions, such
as rheumatoid arthritis and systemic lupus erythematosus; storage
diseases such as Gaucher disease, Niemann-Pick disease, and
mucopolysaccharidoses; and other conditions, such as amyloidosis,
primary neoplasms and cysts, and secondary neoplasms.
[1185] Disorders involving the lung include, but are not limited
to, congenital anomalies; atelectasis; diseases of vascular origin,
such as pulmonary congestion and edema, including hemodynamic
pulmonary edema and edema caused by microvascular injury, adult
respiratory distress syndrome (diffuse alveolar damage), pulmonary
embolism, hemorrhage, and infarction, and pulmonary hypertension
and vascular sclerosis; chronic obstructive pulmonary disease, such
as emphysema, chronic bronchitis, bronchial asthma, and
bronchiectasis; diffuse interstitial (infiltrative, restrictive)
diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity
pneumonitis, pulmonary eosinophilia (pulmonary infiltration with
eosinophilia), Bronchiolitis obliterans-organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, including Goodpasture
syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic
syndromes, pulmonary involvement in collagen vascular disorders,
and pulmonary alveolar proteinosis; complications of therapies,
such as drug-induced lung disease, radiation-induced lung disease,
and lung transplantation; tumors, such as bronchogenic carcinoma,
including paraneoplastic syndromes, bronchioloalveolar carcinoma,
neuroendocrine tumors, such as bronchial carcinoid, miscellaneous
tumors, and metastatic tumors; pathologies of the pleura, including
inflammatory pleural effusions, noninflammatory pleural effusions,
pneumothorax, and pleural tumors, including solitary fibrous tumors
(pleural fibroma) and malignant mesothelioma.
[1186] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[1187] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
[1188] Disorders involving the brain include, but are not limited
to, disorders involving neurons, and disorders involving glia, such
as astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyclia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[1189] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[1190] Disorders involving the kidney include, but are not limited
to, congenital anomalies including, but not limited to, cystic
diseases of the kidney, that include but are not limited to, cystic
renal dysplasia, autosomal dominant (adult) polycystic kidney
disease, autosomal recessive (childhood) polycystic kidney disease,
and cystic diseases of renal medulla, which include, but are not
limited to, medullary sponge kidney, and nephronophthisis-uremic
medullary cystic disease complex, acquired (dialysis-associated)
cystic disease, such as simple cysts; glomerular diseases including
pathologies of glomerular injury that include, but are not limited
to, in situ immune complex deposition, that includes, but is not
limited to, anti-GBM nephritis, Heymann nephritis, and antibodies
against planted antigens, circulating immune complex nephritis,
antibodies to glomerular cells, cell-mediated immunity in
glomerulonephritis, activation of alternative complement pathway,
epithelial cell injury, and pathologies involving mediators of
glomerular injury including cellular and soluble mediators, acute
glomerulonephritis, such as acute proliferative (poststreptococcal,
postinfectious) glomerulonephritis, including but not limited to,
poststreptococcal glomerulonephritis and nonstreptococcal acute
glomerulonephritis, rapidly progressive (crescentic)
glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis (membranous nephropathy), minimal change disease
(lipoid nephrosis), focal segmental glomerulosclerosis,
membranoproliferative glomerulonephritis, IgA nephropathy (Berger
disease), focal proliferative and necrotizing glomerulonephritis
(focal glomerulonephritis), hereditary nephritis, including but not
limited to, Alport syndrome and thin membrane disease (benign
familial hematuria), chronic glomerulonephritis, glomerular lesions
associated with systemic disease, including but not limited to,
systemic lupus erythematosus, Henoch-Schonlein purpura, bacterial
endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary
and immunotactoid glomerulonephritis, and other systemic disorders;
diseases affecting tubules and interstitium, including acute
tubular necrosis and tubulointerstitial nephritis, including but
not limited to, pyelonephritis and urinary tract infection, acute
pyelonephritis, chronic pyelonephritis and reflux nephropathy, and
tubulointerstitial nephritis induced by drugs and toxins, including
but not limited to, acute drug-induced interstitial nephritis,
analgesic abuse nephropathy, nephropathy associated with
nonsteroidal anti-inflammatory drugs, and other tubulointerstitial
diseases including, but not limited to, urate nephropathy,
hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases
of blood vessels including benign nephrosclerosis, malignant
hypertension and accelerated nephrosclerosis, renal artery
stenosis, and thrombotic microangiopathies including, but not
limited to, classic (childhood) hemolytic-uremic syndrome, adult
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,
idiopathic HUS/TTP, and other vascular disorders including, but not
limited to, atherosclerotic ischemic renal disease, atheroembolic
renal disease, sickle cell disease nephropathy, diffuse cortical
necrosis, and renal infarcts; urinary tract obstruction
(obstructive uropathy); urolithiasis (renal calculi, stones); and
tumors of the kidney including, but not limited to, benign tumors,
such as renal papillary adenoma, renal fibroma or hamartoma
(renomedullary interstitial cell tumor), angiomyolipoma, and
oncocytoma, and malignant tumors, including renal cell carcinoma
(hypemephroma, adenocarcinoma of kidney), which includes urothelial
carcinomas of renal pelvis.
[1191] Disorders of the breast include, but are not limited to,
disorders of development; inflammations, including but not limited
to, acute mastitis, periductal mastitis, periductal mastitis
(recurrent subareolar abscess, squamous metaplasia of lactiferous
ducts), mammary duct ectasia, fat necrosis, granulomatous mastitis,
and pathologies associated with silicone breast implants;
fibrocystic changes; proliferative breast disease including, but
not limited to, epithelial hyperplasia, sclerosing adenosis, and
small duct papillomas; tumors including, but not limited to,
stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas,
and epithelial tumors such as large duct papilloma; carcinoma of
the breast including in situ (noninvasive) carcinoma that includes
ductal carcinoma in situ (including Paget's disease) and lobular
carcinoma in situ, and invasive (infiltrating) carcinoma including,
but not limited to, invasive ductal carcinoma, no special type,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms.
[1192] Disorders in the male breast include, but are not limited
to, gynecomastia and carcinoma.
[1193] Disorders involving the testis and epididymis include, but
are not limited to, congenital anomalies such as cryptorchidism,
regressive changes such as atrophy, inflammations such as
nonspecific epididymitis and orchitis, granulomatous (autoimmune)
orchitis, and specific inflammations including, but not limited to,
gonorrhea, mumps, tuberculosis, and syphilis, vascular disturbances
including torsion, testicular tumors including germ cell tumors
that include, but are not limited to, seminoma, spermatocytic
seminoma, embryonal carcinoma, yolk sac tumor choriocarcinoma,
teratoma, and mixed tumors, tumore of sex cord-gonadal stroma
including, but not limited to, leydig (interstitial) cell tumors
and sertoli cell tumors (androblastoma), and testicular lymphoma,
and miscellaneous lesions of tunica vaginalis.
[1194] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[1195] Disorders involving the thyroid include, but are not limited
to, hyperthyroidism; hypothyroidism including, but not limited to,
cretinism and myxedema; thyroiditis including, but not limited to,
hashimoto thyroiditis, subacute (granulomatous) thyroiditis, and
subacute lymphocytic (painless) thyroiditis; Graves disease;
diffuse and multinodular goiter including, but not limited to,
diffuse nontoxic (simple) goiter and multinodular goiter; neoplasms
of the thyroid including, but not limited to, adenomas, other
benign tumors, and carcinomas, which include, but are not limited
to, papillary carcinoma, follicular carcinoma, medullary carcinoma,
and anaplastic carcinoma; and cogenital anomalies.
[1196] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[1197] Disorders involving the small intestine include the
malabsorption syndromes such as, celiac sprue, tropical sprue
(postinfectious sprue), whipple disease, disaccharidase (lactase)
deficiency, abetalipoproteinemia, and tumors of the small intestine
including adenomas and adenocarcinoma.
[1198] Disorders involving blood vessels include, but are not
limited to, responses of vascular cell walls to injury, such as
endothelial dysfunction and endothelial activation and intimal
thickening; vascular diseases including, but not limited to,
congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[1199] Disorders involving the thymus include developmental
disorders, such as DiGeorge syndrome with thymic hypoplasia or
aplasia; thymic cysts; thymic hypoplasia, which involves the
appearance of lymphoid follicles within the thymus, creating thymic
follicular hyperplasia; and thymomas, including germ cell tumors,
lymphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[1200] Disorders involving the ovary include, for example,
polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma
peritonei and stromal hyperthecosis; ovarian tumors such as, tumors
of coelomic epithelium, serous tumors, mucinous tumors,
endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma,
brenner tumor, surface epithelial tumors; germ cell tumors such as
mature (benign) teratomas, monodermal teratomas, immature malignant
teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma;
sex cord-stomal tumors such as, granulosa-theca cell tumors,
thecoma-fibromas, androblastomas, hill cell tumors, and
gonadoblastoma; and metastatic tumors such as Krukenberg
tumors.
[1201] Bone-forming cells include the osteoprogenitor cells,
osteoblasts, and osteocytes. The disorders of the bone are complex
because they may have an impact on the skeleton during any of its
stages of development. Hence, the disorders may have variable
manifestations and may involve one, multiple or all bones of the
body. Such disorders include, congenital malformations,
achondroplasia and thanatophoric dwarfism, diseases associated with
abnormal matix such as type 1 collagen disease, osteoporois, paget
disease, rickets, osteomalacia, high-turnover osteodystrophy,
low-turnover of aplastic disease, osteonecrosis, pyogenic
osteomyelitis, tuberculous osteomyelitism, osteoma, osteoid
osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,
chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous
cortical defects, fibrous dysplasia, fibrosarcoma, malignant
fibrous histiocytoma, ewing sarcoma, primitive neuroectodermal
tumor, giant cell tumor, and metastatic tumors.
[1202] In addition, lipases influence a number of processes which
affect the biology of both blood vessel walls and the pancreas.
Therefore, lipases find use in the treatment of disorders of blood
vessels, which include, but are not limited to, responses of
vascular cell walls to injury, such as endothelial dysfunction and
endothelial activation and intimal thickening; vascular diseases
including, but not limited to, congenital anomalies, such as
arteriovenous fistula, atherosclerosis, and hypertensive vascular
disease, such as hypertension; inflammatory disease--the
vasculitides, such as giant cell (temporal) arteritis, Takayasu
arteritis, polyarteritis nodosa (classic), Kawasaki syndrome
(mucocutaneous lymph node syndrome), microscopic polyanglitis
(microscopic polyarteritis, hypersensitivity or leukocytoclastic
anglitis), Wegener granulomatosis, thromboanglitis obliterans
(Buerger disease), vasculitis associated with other disorders, and
infectious arteritis; Raynaud disease; aneurysms and dissection,
such as abdominal aortic aneurysms, syphilitic (luetic) aneurysms,
and aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[1203] Disorders involving the pancreas include those of the
exocrine pancreas such as congenital anomalies, including but not
limited to, ectopic pancreas; pancreatitis, including but not
limited to, acute pancreatitis; cysts, including but not limited
to, pseudocysts; tumors, including but not limited to, cystic
tumors and carcinoma of the pancreas; and disorders of the
endocrine pancreas such as, diabetes mellitus; islet cell tumors,
including but not limited to, insulinomas, gastrinomas, and other
rare islet cell tumors.
[1204] Lipases play critical roles in lipid metabolism and are
associated with various lipid-related pathologies in humans such
as, but not limited to, Wolman's disease, hypertension, Type II
diabetes, retinopathy and cholesterol ester storage disease.
Furthermore, a decrease in LPL activity impairs the catabolism of
chylomicrons and VLDL resulting in massive hypertriglyceridemia.
Decreased LPL activity has been also associated with many
disorders, including for example, chylomicronemia syndrome. This
syndrome has multiple clinical symptoms and manifestations review
by Murthy et al. (1996) Pharmacol. Ther. 70:101-135. Additional
disorders resulting from defective LPL activity include, familial
lipoprotein lipase deficiency with fasting chylomicronemia (type I
hyperlipidemia) (Santamarina et al. (1992) Curr Opin Lipidology
3:186), LPL deficiency, familial combined hyperlipidaemia (FCHL)
(Babirak et al. (1992) Arteriosclerosis thromb. 12:1176; Seed et
al. (1994) Clin Invest 72: 100), hypertriglyceridemia, pancreatitis
and abnormalities in post prandial lipemia. In addition, LPL
activity is abnormally regulated in obesity (Kern et al. (1997) J.
Nut. 127: 1917S-1922S) and is also affected by alcohol and several
hormones (Taskinen et al. (1987) Lipoprotein Lipase, Borensztajn J.
(ed) Evener Chicago). Furthermore, changes in circulating
lipoprotein and creation of lipolytic products have been implicated
in a number of processes that affect the biology of vessel walls.
For example, atherogenesis is associated with increased LPL
activity. In addition, autoantibodies against LPL have been
reported in patients with idiopathic thrombocytopenic purpura and
Grave's disease (Kihara et al. (1989) N. Engl. J. Med.
320:1255-1259) and heparin resistance was noted in a case of
disseminated lupus erythematosus (Glueck et al. (1969) Am. J. Med.
47:318-324). Polymorphisms in LDL gene have also been associated
with altered levels of total and HDL cholesterol (Mitchell et al.
(1994) Hum. Biol. 66:383-397), coronary heart disease (Mattu et al.
(1994) Arterioscler. Thromb. 14:1090-1097), and insulin resistance
(Cole et al. (1993) Genet. Epidemiol. 10:177-188).
[1205] The hydrolysis of HDL by hepatic lipase regulates
cholesterol levels in hepatic tissue. Pathologies associated with
cholesterol include, but are not limited to, atherosclerosis,
xanthomas, inflammation and necrosis, cholesterolosis and gall
stone formation.
[1206] The lipase polypeptides are thus useful for treating a
lipase-associated disorder characterized by aberrant expression or
activity of a lipase. The polypeptides can also be useful for
treating a disorder characterized by excessive amounts of
lipoproteins, triglycerides or cholesterol. 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) expression or
activity of the protein. In another embodiment, the method involves
administering the lipase as therapy to compensate for reduced or
aberrant expression or activity of the protein. In another
embodiment, the lipase polypeptides are useful for treating breast,
lung, colon, and liver cancers.
[1207] Methods for treatment include but are not limited to the use
of soluble lipase or fragments of the lipase protein that compete
for substrates including those disclosed herein. These lipases or
fragments can have a higher affinity for the target so as to
provide effective competition.
[1208] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
aberrant metabolism of lipids resulting in altered lipoprotein
concentrations, energy homeostasis, body weight, artherosclerosis,
and body weight parameters.
[1209] In yet another aspect of the invention, the proteins of the
invention 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 WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[1210] The lipase polypeptides also are useful to provide a target
for diagnosing a disease or predisposition to disease mediated by
the lipase, including, but not limited to, diseases involving
tissues in which the lipase are expressed as disclosed herein.
Accordingly, methods are provided for detecting the presence, or
levels of, the lipase in a cell, tissue, or organism. The method
involves contacting a biological sample with a compound capable of
interacting with the lipase such that the interaction can be
detected.
[1211] The polypeptides are also useful for treating a disorder
characterized by reduced amounts of these components. Thus,
increasing or decreasing the activity of the lipase is beneficial
to treatment. The polypeptides are also useful to provide a target
for diagnosing a disease characterized by excessive substrate or
reduced levels of substrate. Accordingly, where substrate is
excessive, use of the lipase polypeptides can provide a diagnostic
assay. Furthermore, for example, lipases having reduced activity
can be used to diagnose conditions in which reduced substrate is
responsible for the disorder.
[1212] One agent for detecting lipase is an antibody capable of
selectively binding to the lipase polypeptide. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject.
[1213] The lipase also provides a target for diagnosing active
disease, or predisposition to disease, in a patient having a
variant lipase. Thus, lipase can be isolated from a biological
sample and assayed for the presence of a genetic mutation that
results in an aberrant protein. This includes amino acid
substitution, deletion, insertion, rearrangement, (as the result of
aberrant splicing events), and inappropriate post-translational
modification. Analytic methods include altered electrophoretic
mobility, altered tryptic peptide digest, altered lipase activity
in cell-based or cell-free assay, alteration in binding to or
hydrolysis of lipids, binding to activator proteins, cell surface
receptors, apoproteins, lipoproteins, proteoglycans, heparin, or
antibody-binding pattern, altered isoelectric point, direct amino
acid sequencing, and any other of the known assay techniques useful
for detecting mutations in a protein in general or in a lipase
specifically, including assays discussed herein.
[1214] In vitro techniques for detection of lipase include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-lipase 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. Particularly useful are methods, which detect the
allelic variant of the lipase expressed in a subject, and methods,
which detect fragments of the lipase in a sample.
[1215] The lipase polypeptides are also useful in pharmacogenomic
analysis. Pharmacogenomics deal with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, e.g.,
Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem.
43(2):254-266. The clinical outcomes of these variations result in
severe toxicity of therapeutic drugs in certain individuals or
therapeutic failure of drugs in certain individuals as a result of
individual variation in metabolism. Thus, the genotype of the
individual can determine the way a therapeutic compound acts on the
body or the way the body metabolizes the compound. Further, the
activity of drug metabolizing enzymes affects both the intensity
and duration of drug action. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
treatment based on the individual's genotype. The discovery of
genetic polymorphisms in some drug metabolizing enzymes has
explained why some patients do not obtain the expected drug
effects, show an exaggerated drug effect, or experience serious
toxicity from standard drug dosages. Polymorphisms can be expressed
in the phenotype of the extensive metabolizer and the phenotype of
the poor metabolizer. Accordingly, genetic polymorphism may lead to
allelic protein variants of the lipase in which one or more of the
lipase functions in one population is different from those in
another population. The polypeptides thus allow a target to
ascertain a genetic predisposition that can affect treatment
modality. Thus, in a lipase-based treatment, polymorphism may give
rise to catalytic regions that are more or less active.
Accordingly, dosage would necessarily be modified to maximize the
therapeutic effect within a given population containing the
polymorphism. As an alternative to genotyping, specific polymorphic
polypeptides could be identified.
[1216] The lipase polypeptides are also useful for monitoring
therapeutic effects during clinical trials and other treatment.
Thus, the therapeutic effectiveness of an agent that is designed to
increase or decrease gene expression, protein levels or lipase
activity can be monitored over the course of treatment using the
lipase polypeptides as an end-point target. The monitoring can be,
for example, as follows: (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression or activity of the protein in the
pre-administration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the protein in the
post-administration samples; (v) comparing the level of expression
or activity of the protein in the pre-administration sample with
the protein in the post-administration sample or samples; and (vi)
increasing or decreasing the administration of the agent to the
subject accordingly.
Antibodies
[1217] The invention also provides antibodies that selectively bind
to the lipase and its variants and fragments. An antibody is
considered to selectively bind, even if it also binds to other
proteins that are not substantially homologous with the lipase.
These other proteins share homology with a fragment or domain of
the lipase polypeptide. This conservation in specific regions gives
rise to antibodies that bind to both proteins by virtue of the
homologous sequence. In this case, it would be understood that
antibody binding to the lipase is still selective.
[1218] To generate antibodies, an isolated lipase polypeptide is
used as an immunogen to generate antibodies using standard
techniques for polyclonal and monoclonal antibody preparation.
Either the full-length protein or antigenic peptide fragment can be
used. Regions having a high antigenicity index are shown in FIG.
40.
[1219] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents substrate hydrolysis or binding. Antibodies can
be developed against the entire lipase protein or domains of the
lipase as described herein. Antibodies can also be developed
against specific functional sites as disclosed herein.
[1220] The antigenic peptide can comprise a contiguous sequence of
at least 8, 13, 14, 15, or 30 amino acid residues. In one
embodiment, fragments correspond to regions that are located on the
surface of the protein, e.g., hydrophilic regions. These fragments
are not to be construed, however, as encompassing any fragments,
which may be disclosed prior to the invention.
[1221] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be
used.
[1222] 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.
[1223] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
Antibody Uses
[1224] The antibodies can be used to isolate a lipase by standard
techniques, such as affinity chromatography or immunoprecipitation.
The antibodies can facilitate the purification of the natural
lipase from cells and recombinantly produced lipase expressed in
host cells.
[1225] The antibodies are useful to detect the presence of lipase
in cells or tissues to determine the pattern of expression of the
lipase among various tissues in an organism and over the course of
normal development.
[1226] The antibodies can be used to detect lipase in situ, in
vitro, or in a cell lysate or supernatant in order to evaluate the
abundance and pattern of expression.
[1227] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[1228] Antibody detection of circulating fragments of the full
length lipase can be used to identify lipase turnover.
[1229] Further, the antibodies can be used to assess lipase
expression in disease states such as in active stages of the
disease or in an individual with a predisposition toward disease
related to lipid metabolism. When a disorder is caused by an
inappropriate tissue distribution, developmental expression, or
level of expression of the lipase protein, the antibody can be
prepared against the normal lipase protein. If a disorder is
characterized by a specific mutation in the lipase, antibodies
specific for this mutant protein can be used to assay for the
presence of the specific mutant lipase polypeptides. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular lipase-peptide
regions.
[1230] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole
lipase or portions of the lipase.
[1231] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting lipase expression
level or the presence of aberrant lipase proteins and aberrant
tissue distribution or developmental expression, antibodies
directed against the lipase or relevant fragments can be used to
monitor therapeutic efficacy.
[1232] Antibodies accordingly 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.
[1233] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic lipases can
be used to identify individuals that require modified treatment
modalities.
[1234] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant lipase analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[1235] The antibodies are also useful for tissue typing. Thus,
where a specific lipase has been correlated with expression in a
specific tissue, antibodies that are specific for this lipase can
be used to identify a tissue type.
[1236] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[1237] The antibodies are also useful for inhibiting the various
lipase functions as described herein.
[1238] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting lipase function. Antibodies can
be prepared against specific fragments containing sites required
for function or against intact lipase associated with a cell.
[1239] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[1240] The invention also encompasses kits for using antibodies to
detect the presence of a lipase protein in a biological sample. The
kit can comprise antibodies such as a labeled or labelable antibody
and a compound or agent for detecting lipase in a biological
sample; means for determining the amount of lipase in the sample;
and means for comparing the amount of lipase 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 lipase.
Polynucleotides
[1241] The nucleotide sequence in SEQ ID NO:18 was obtained by
sequencing the deposited human cDNA. Accordingly, the sequence of
the deposited clone is controlling as to any discrepancies between
the two and any reference to the sequence of SEQ ID NO:18 includes
reference to the sequence of the deposited cDNA.
[1242] The specifically disclosed cDNA comprises the coding region
and 5' and 3' untranslated sequences in SEQ ID NO:18.
[1243] The invention provides isolated polynucleotides encoding the
novel lipase. The term "lipase polynucleotide" or "lipase nucleic
acid" refers to the sequence shown in SEQ ID NO:18 or in the
deposited cDNA. The term "lipase polynucleotide" or "lipase nucleic
acid" further includes variants and fragments of the lipase
polynucleotide.
[1244] An "isolated" lipase nucleic acid is one that is separated
from other nucleic acid present in the natural source of the lipase
nucleic acid. Preferably, an "isolated" nucleic acid is free of
sequences which naturally flank the lipase 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.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB. The important point is that the lipase
nucleic acid is isolated from flanking sequences such that it can
be subjected to the specific manipulations described herein, such
as recombinant expression, preparation of probes and primers, and
other uses specific to the lipase nucleic acid sequences.
[1245] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[1246] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[1247] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[1248] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[1249] The lipase polynucleotides can encode the mature protein
plus additional amino or carboxyterminal amino acids, or amino
acids interior to the mature polypeptide (when the mature form has
more than one polypeptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[1250] The lipase polynucleotides include, but are not limited to,
the sequence encoding the mature polypeptide alone, the sequence
encoding the mature polypeptide and additional coding sequences,
such as a leader or secretory sequence (e.g., a pre-pro or
pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[1251] Lipase polynucleotides can be in the form of RNA, such as
mRNA, or in the form DNA, including cDNA and genomic DNA obtained
by cloning or produced by chemical synthetic techniques or by a
combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[1252] Lipase nucleic acid can comprise the nucleotide sequence
shown in SEQ ID NO:18, corresponding to human cDNA.
[1253] In one embodiment, the lipase nucleic acid comprises only
the coding region.
[1254] The invention further provides variant lipase
polynucleotides, and fragments thereof, that differ from the
nucleotide sequence shown in SEQ ID NO:18 due to degeneracy of the
genetic code and thus encode the same protein as that encoded by
the nucleotide sequence shown in SEQ ID NO:18.
[1255] The invention also provides lipase nucleic acid molecules
encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[1256] Typically, variants have a substantial identity with a
nucleic acid molecule of SEQ ID NO:18 and the complements thereof.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[1257] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a lipase that is at least about
60-65%, 65-70%, typically at least about 70-75%, more typically at
least about 80-85%, and most typically at least about 90-95% or
more homologous to the nucleotide sequence shown in SEQ ID NO:18.
Such nucleic acid molecules can readily be identified as being able
to hybridize under stringent conditions, to the nucleotide sequence
shown in SEQ ID NO:18 or a fragment of the sequence. It is
understood that stringent hybridization does not indicate
substantial homology where it is due to general homology, such as
poly A sequences, or sequences common to all or most proteins or
all lipase enzymes. Moreover, it is understood that variants do not
include any of the nucleic acid sequences that may have been
disclosed prior to the invention.
[1258] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 60-65% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95% or more
identical to each other remain hybridized to one another. 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, incorporated by reference.
One 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 50-65.degree. C. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times.SSC/0.1% SDS at room
temperature, or by one or more moderate stringency washes in
0.2.times.SSC/0.1% SDS at 42.degree. C., or washed in
0.2.times.SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:17 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).
[1259] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[1260] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:18 or the complement of SEQ ID NO:18. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO:18 or the complement of SEQ ID NO:18.
[1261] It is understood that isolated fragments include any
contiguous sequence not disclosed prior to the invention as well as
sequences that are substantially the same and which are not
disclosed. Accordingly, if a fragment is disclosed prior to the
present invention, that fragment is not intended to be encompassed
by the invention. When a sequence is not disclosed prior to the
present invention, an isolated nucleic acid fragment is at least
about 6, preferably at least about 10, 13, 18, 20, 23 or 25
nucleotides, and can be 30, 40, 50, 100, 200, 500 or more
nucleotides in length. Nucleotide sequences from about 1517 to 1964
are not disclosed prior to the invention. Longer fragments, for
example, 30 or more nucleotides in length, which encode antigenic
proteins or polypeptides described herein are useful.
[1262] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length lipase polynucleotides. The
fragment can be single or double-stranded and can comprise DNA or
RNA. The fragment can be derived from either the coding or the
non-coding sequence.
[1263] In another embodiment an isolated lipase nucleic acid
encodes the entire coding region. Other fragments include
nucleotide sequences encoding the amino acid fragments described
herein.
[1264] Thus, lipase nucleic acid fragments further include
sequences corresponding to the domains described herein, subregions
also described, and specific functional sites. Lipase nucleic acid
fragments also include combinations of the domains, segments, and
other functional sites described above. A person of ordinary skill
in the art would be aware of the many permutations that are
possible.
[1265] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary sill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[1266] However, it is understood that a lipase fragment includes
any nucleic acid sequence that does not include the entire
gene.
[1267] The invention also provides lipase nucleic acid fragments
that encode epitope bearing regions of the lipase proteins
described herein.
[1268] Nucleic acid fragments, according to the present invention,
are not to be construed as encompassing those fragments that may
have been disclosed prior to the invention.
Polynucleotide Uses
[1269] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to 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 protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins 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 www.ncbi.nlm.nih.gov.
[1270] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of the nucleic
acid sequence shown in SEQ ID NO:18 and the complements thereof.
More typically, the probe further comprises a label, e.g.,
radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
[1271] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[1272] The lipase polynucleotides are thus useful for probes,
primers, and in biological assays.
[1273] Where the polynucleotides are used to assess lipase
properties or functions, such as in the assays described herein,
all or less than all of the entire cDNA can be useful. Assays
specifically directed to lipase functions, such as assessing
agonist or antagonist activity, encompass the use of known
fragments. Further, diagnostic methods for assessing lipase
function can also be practiced with any fragment, including those
fragments that may have been known prior to the invention.
Similarly, in methods involving treatment of lipase dysfunction,
all fragments are encompassed including those, which may have been
known in the art.
[1274] The lipase polynucleotides are useful as a hybridization
probe for cDNA and genomic DNA to isolate a full-length cDNA and
genomic clones encoding the polypeptide described in SEQ ID NO:17
and to isolate cDNA and genomic clones that correspond to variants
producing the same polypeptide shown in SEQ ID NO:17 or the other
variants described herein. Variants can be isolated from the same
tissue and organism from which the polypeptides shown in SEQ ID
NO:17 were isolated, different tissues from the same organism, or
from different organisms. This method is useful for isolating genes
and cDNA that are developmentally-controlled and therefore may be
expressed in the same tissue or different tissues at different
points in the development of an organism.
[1275] The probe can correspond to any sequence along the entire
length of the gene encoding the lipase. Accordingly, it could be
derived from 5' noncoding regions, the coding region, and 3'
noncoding regions.
[1276] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:18 or a fragment thereof that is sufficient to
specifically hybridize under stringent conditions to mRNA or
DNA.
[1277] Fragments of the polynucleotides described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an mRNA and a larger or full-length
cDNA can be produced.
[1278] The fragments are also useful to synthesize antisense
molecules of desired length and sequence.
[1279] Antisense nucleic acids of the invention can be designed
using the nucleotide sequence of SEQ ID NO:18, and 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,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N2-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).
[1280] Additionally, the nucleic acid molecules of the 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 acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). 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 et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity 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. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[1281] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell lipases 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.
WO 88/0918) or the blood brain barrier (see, e.g., PCT Publication
No. WO 89/10134). 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).
[1282] The lipase polynucleotides are also useful as primers for
PCR to amplify any given region of a lipase polynucleotide.
[1283] The lipase polynucleotides are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the lipase polypeptides. Vectors
also include insertion vectors, used to integrate into another
polynucleotide sequence, such as into the cellular genome, to alter
in situ expression of lipase genes and gene products. For example,
an endogenous lipase coding sequence can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[1284] The lipase polynucleotides are also useful for expressing
antigenic portions of the lipase proteins.
[1285] The lipase polynucleotides are also useful as probes for
determining the chromosomal positions of the lipase polynucleotides
by means of in situ hybridization methods, such as FISH. (For a
review of this technique, see Verma et al. (1988) Human
Chromosomes: A Manual of Basic Techniques (Pergamon Press, New
York), and PCR mapping of somatic cell hybrids. The mapping of the
sequences to chromosomes is an important first step in correlating
these sequences with genes associated with disease.
[1286] 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.
[1287] 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 et al. ((1987) Nature 325:783-787).
[1288] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified 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.
[1289] The lipase polynucleotide probes are also useful to
determine patterns of the presence of the gene encoding the lipase
and their variants with respect to tissue distribution, for
example, whether gene duplication has occurred and whether the
duplication occurs in all or only a subset of tissues. The genes
can be naturally occurring or can have been introduced into a cell,
tissue, or organism exogenously.
[1290] The lipase polynucleotides are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from genes encoding the polynucleotides described herein.
[1291] The lipase polynucleotides are also useful for constructing
host cells expressing a part, or all, of the lipase polynucleotides
and polypeptides.
[1292] The lipase polynucleotides are also useful for constructing
transgenic animals expressing all, or a part, of the lipase
polynucleotides and polypeptides.
[1293] The lipase polynucleotides are also useful for making
vectors that express part, or all, of the lipase polypeptides.
[1294] The lipase polynucleotides are also useful as hybridization
probes for determining the level of lipase nucleic acid expression.
Accordingly, the probes can be used to detect the presence of, or
to determine levels of, lipase nucleic acid in cells, tissues, and
in organisms. The nucleic acid whose level is determined can be DNA
or RNA. Accordingly, probes corresponding to the polypeptides
described herein can be used to assess gene copy number in a given
cell, tissue, or organism. This is particularly relevant in cases
in which there has been an amplification of the lipase genes.
[1295] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
lipase genes, as on extrachromosomal elements or as integrated into
chromosomes in which the lipase gene is not normally found, for
example as a homogeneously staining region.
[1296] These uses are relevant for diagnosis of disorders involving
an increase or decrease in lipase expression relative to normal,
such as a developmental or a metabolic disorder.
[1297] Tissues and/or cells in which the lipase is expressed
include, but are not limited to those shown in FIGS. 43, 44, and
45. Such tissues/cells include liver, fetal liver, breast, brain,
fetal kidney, and testis. Moderate expression occurs in prostate,
skeletal muscle, colon, kidney, and thyroid. Lower positive
expression occurs in heart, fetal heart, small intestine, spleen,
lung, ovary, vein, aorta, placenta, osteoblasts, cervix, esophagus,
thymus, tonsil, and lymph node. The lipase is also expressed in
malignant breast, lung, and colon tissue and in liver metastases
derived from malignant colonic tissues. Hence, the lipase is
relevant to disorders involving the tissues in which it is
expressed. As such, the gene is particularly relevant for the
treatment of disorders involving breast, lung, liver, and colon
cancer. Disorders in which the lipase expression is relevant
include, but are not limited to those disclosed herein above.
[1298] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of lipase nucleic acid, in which a test
sample is obtained from a subject and nucleic acid (e.g., mRNA,
genomic DNA) is detected, wherein the presence of the nucleic acid
is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the nucleic acid.
[1299] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[1300] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[1301] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express the lipase, such as by
measuring the level of a lipase-encoding nucleic acid in a sample
of cells from a subject e.g., mRNA or genomic DNA, or determining
if the lipase gene has been mutated.
[1302] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate lipase nucleic acid expression
(e.g., antisense, polypeptides, peptidomimetics, small molecules or
other drugs). A cell is contacted with a candidate compound and the
expression of mRNA determined. The level of expression of the mRNA
in the presence of the candidate compound is compared to the level
of expression of the mRNA in the absence of the candidate compound.
The candidate compound can then be identified as a modulator of
nucleic acid expression based on this comparison and be used, for
example to treat a disorder characterized by aberrant nucleic acid
expression. The modulator can bind to the nucleic acid or
indirectly modulate expression, such as by interacting with other
cellular components that affect nucleic acid expression.
[1303] 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 gent to a subject) in patients or in
transgenic animals.
[1304] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the lipase gene. The method typically
includes assaying the ability of the compound to modulate the
expression of the lipase nucleic acid and thus identifying a
compound that can be used to treat a disorder characterized by
undesired lipase nucleic acid expression.
[1305] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
lipase nucleic acid or recombinant cells genetically engineered to
express specific nucleic acid sequences.
[1306] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[1307] The assay for lipase nucleic acid expression can involve
direct assay of nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in the pathway. Further, the
expression of genes that are up- or down-regulated in response to
the lipase activity can also be assayed. In this embodiment the
regulatory regions of these genes can be operably linked to a
reporter gene such as luciferase.
[1308] Thus, modulators of lipase gene expression can be identified
in a method wherein a cell is contacted with a candidate compound
and the expression of mRNA determined. The level of expression of
lipase mRNA in the presence of the candidate compound is compared
to the level of expression of lipase mRNA in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of nucleic acid expression based on this comparison
and be used, for example to treat a disorder characterized by
aberrant nucleic acid expression. When expression of mRNA is
statistically significantly greater in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of nucleic acid expression. When nucleic
acid expression is statistically significantly less in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of nucleic acid
expression.
[1309] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate lipase
nucleic acid expression. Modulation includes both up-regulation
(i.e. activation or agonization) or down-regulation (suppression or
antagonization) or effects on nucleic acid activity (e.g. when
nucleic acid is mutated or improperly modified). Treatment includes
disorders characterized by aberrant expression or activity of the
nucleic acid. In addition, disorders that are influenced by the
lipase may also be treated. Examples of such disorders are
disclosed herein.
[1310] Alternatively, a modulator for lipase nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the lipase nucleic acid expression.
[1311] The lipase polynucleotides are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the lipase gene in clinical trials or in a treatment
regimen. Thus, the gene expression pattern can serve as a barometer
for the continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance. The gene expression pattern can also serve as a marker
indicative of a physiological response of the affected cells to the
compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[1312] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[1313] The lipase polynucleotides are also useful in diagnostic
assays for qualitative changes in lipase nucleic acid, and
particularly in qualitative changes that lead to pathology. The
polynucleotides can be used to detect mutations in lipase genes and
gene expression products such as mRNA. The polynucleotides can be
used as hybridization probes to detect naturally-occurring genetic
mutations in the lipase gene and thereby to determine whether a
subject with the mutation is at risk for a disorder caused by the
mutation. Mutations include deletion, addition, or substitution of
one or more nucleotides in the gene, chromosomal rearrangement,
such as inversion or transposition, modification of genomic DNA,
such as aberrant methylation patterns or changes in gene copy
number, such as amplification. Detection of a mutated form of the
lipase gene associated with a dysfunction provides a diagnostic
tool for an active disease or susceptibility to disease when the
disease results from overexpression, underexpression, or altered
expression of a lipase.
[1314] Mutations in the lipase gene can be detected at the nucleic
acid level by a variety of techniques. Genomic DNA can be analyzed
directly or can be amplified by using PCR prior to analysis. RNA or
cDNA can be used in the same way.
[1315] In certain embodiments, detection of the mutation 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) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
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 patient, 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
gene under conditions such that hybridization and amplification of
the 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.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[1316] 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.
[1317] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[1318] Alternatively, mutations in a lipase gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[1319] Further, sequence-specific ribozymes (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.
[1320] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[1321] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[1322] Furthermore, sequence differences between a mutant lipase
gene and a wild-type gene can be determined by direct DNA
sequencing. 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).
[1323] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). 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 one 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).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[1324] In other embodiments, genetic mutations 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
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two-dimensional
arrays containing light-generated DNA probes as described in Cronin
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.
[1325] The lipase polynucleotides are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
polynucleotides can be used to study the relationship between an
individual's genotype and the individual's response to a compound
used for treatment (pharmacogenomic relationship). Accordingly, the
lipase polynucleotides described herein can be used to assess the
mutation content of the gene in an individual in order to select an
appropriate compound or dosage regimen for treatment.
[1326] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[1327] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[1328] The lipase polynucleotides are also useful for chromosome
identification when the sequence is identified with an individual
chromosome and to a particular location on the chromosome. First,
the DNA sequence is matched to the chromosome by in situ or other
chromosome-specific hybridization. Sequences can also be correlated
to specific chromosomes by preparing PCR primers that can be used
for PCR screening of somatic cell hybrids containing individual
chromosomes from the desired species. Only hybrids containing the
chromosome containing the gene homologous to the primer will yield
an amplified fragment. Sublocalization can be achieved using
chromosomal fragments. Other strategies include prescreening with
labeled flow-sorted chromosomes and preselection by hybridization
to chromosome-specific libraries. Further mapping strategies
include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the 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.
[1329] The lipase polynucleotides can also be used to identify
individuals based on small biological samples. This can be done for
example using restriction fragment-length polymorphism (RFLP) to
identify an individual. Thus, the polynucleotides described herein
are useful as DNA markers for RFLP (See U.S. Pat. No.
5,272,057).
[1330] Furthermore, the lipase sequence can be used to provide an
alternative technique, which determines the actual DNA sequence of
selected fragments in the genome of an individual. Thus, the lipase
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 DNA from an individual for subsequent
sequencing.
[1331] 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. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
lipase sequences can be used to obtain such identification
sequences from individuals and from tissue. The sequences represent
unique fragments of the human genome. 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.
[1332] If a panel of reagents from the sequences 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.
[1333] The lipase polynucleotides can also be used in forensic
identification procedures. PCR technology can be used to amplify
DNA sequences taken from very small biological samples, such as a
single hair follicle, body fluids (e.g. blood, saliva, or semen).
The amplified sequence can then be compared to a standard allowing
identification of the origin of the sample.
[1334] The lipase polynucleotides can thus 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 described 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 the noncoding region are
particularly useful since greater polymorphism occurs in the
noncoding regions, making it easier to differentiate individuals
using this technique.
[1335] The lipase polynucleotides 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. This is useful in cases in which a
forensic pathologist is presented with a tissue of unknown origin.
Panels of lipase probes can be used to identify tissue by species
and/or by organ type.
[1336] In a similar fashion, these primers and 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).
[1337] Alternatively, the lipase polynucleotides can be used
directly to block transcription or translation of lipase gene
sequences by means of antisense or ribozyme constructs. Thus, in a
disorder characterized by abnormally high or undesirable lipase
gene expression, nucleic acids can be directly used for
treatment.
[1338] The lipase polynucleotides are thus useful as antisense
constructs to control lipase gene expression in cells, tissues, and
organisms. A DNA antisense polynucleotide is designed to be
complementary to a region of the gene involved in transcription,
preventing transcription and hence production of lipase protein. An
antisense RNA or DNA polynucleotide would hybridize to the mRNA and
thus block translation of mRNA into lipase protein.
[1339] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:18 which also
includes the start codon and antisense molecules which are
complementary to a fragment of the 3' untranslated region of SEQ ID
NO:18.
[1340] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of lipase nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired lipase nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the lipase protein.
[1341] The lipase polynucleotides also provide vectors for gene
therapy in patients containing cells that are aberrant in lipase
gene expression. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired lipase protein to treat the individual.
[1342] The invention also encompasses kits for detecting the
presence of a lipase nucleic acid in a biological sample. For
example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting lipase nucleic
acid in a biological sample; means for determining the amount of
lipase nucleic acid in the sample; and means for comparing the
amount of lipase nucleic acid 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
lipase mRNA or DNA.
Computer Readable Means
[1343] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[1344] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[1345] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[1346] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[1347] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[1348] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[1349] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[1350] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[1351] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
Vectors/Host Cells
[1352] The invention also provides vectors containing the lipase
polynucleotides. The term "vector" refers to a vehicle, preferably
a nucleic acid molecule that can transport the lipase
polynucleotides. When the vector is a nucleic acid molecule, the
lipase polynucleotides are covalently linked to the vector nucleic
acid. With this aspect of the invention, the vector includes a
plasmid, single or double stranded phage, a single or double
stranded RNA or DNA viral vector, or artificial chromosome, such as
a BAC, PAC, YAC, OR MAC.
[1353] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the lipase polynucleotides. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the lipase polynucleotides when the host cell
replicates.
[1354] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
lipase polynucleotides. The vectors can function in procaryotic or
eukaryotic cells or in both (shuttle vectors).
[1355] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the lipase
polynucleotides such that transcription of the polynucleotides is
allowed in a host cell. The polynucleotides can be introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus, the second polynucleotide may provide a
trans-acting factor interacting with the cis-regulatory control
region to allow transcription of the lipase polynucleotides from
the vector. Alternatively, a trans-acting factor may be supplied by
the host cell. Finally, a trans-acting factor can be produced from
the vector itself.
[1356] It is understood, however, that in some embodiments,
transcription and/or translation of the lipase polynucleotides can
occur in a cell-free system.
[1357] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[1358] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[1359] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[1360] A variety of expression vectors can be used to express a
lipase polynucleotide. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[1361] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e., tissue specific) or may provide
for inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[1362] The lipase polynucleotides can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[1363] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[1364] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the lipase
polypeptides. Fusion vectors can increase the expression of a
recombinant protein, increase the solubility of the recombinant
protein, and aid in the purification of the protein by acting for
example as a ligand for affinity purification. A proteolytic
cleavage site may be introduced at the junction of the fusion
moiety so that the desired polypeptide can ultimately be separated
from the fusion moiety. Proteolytic enzymes include, but are not
limited to, factor Xa, thrombin, and enterokinase. Typical fusion
expression vectors include pGEX (Smith et al. (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. 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. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[1365] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[1366] The lipase polynucleotides can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan et al.
(1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene
54:113-123), and pYES2 (Invitrogen Corporation, San Diego,
Calif.).
[1367] The lipase polynucleotides can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL
series (Lucklow et al. (1989) Virology 170:31-39).
[1368] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[1369] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
lipase polynucleotides. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[1370] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[1371] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[1372] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[1373] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the lipase polynucleotides can be introduced
either alone or with other polynucleotides that are not related to
the lipase polynucleotides such as those providing trans-acting
factors for expression vectors. When more than one vector is
introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the lipase polynucleotide
vector.
[1374] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[1375] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[1376] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[1377] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the lipase polypeptides or
heterologous to these polypeptides.
[1378] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[1379] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
Uses of Vectors and Host Cells
[1380] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also 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.
[1381] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing lipase proteins or
polypeptides that can be further purified to produce desired
amounts of lipase protein or fragments. Thus, host cells containing
expression vectors are useful for polypeptide production.
[1382] Host cells are also useful for conducting cell-based assays
involving the lipase or lipase fragments. Thus, a recombinant host
cell expressing a native lipase is useful to assay for compounds
that stimulate or inhibit lipase function. This includes
disappearance of substrate (triglycerides, phospholipids,
lipoproteins), appearance of end product (fatty acids), and the
various other molecular functions described herein that include,
but are not limited to, substrate recognition, substrate binding,
subunit association, and interaction with other cellular
components. Modulation of gene expression can occur at the level of
transcription or translation.
[1383] Host cells are also useful for identifying lipase mutants in
which these functions are affected. If the mutants naturally occur
and give rise to a pathology, host cells containing the mutations
are useful to assay compounds that have a desired effect on the
mutant lipase (for example, stimulating or inhibiting function)
which may not be indicated by their effect on the native
lipase.
[1384] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation or alter specific function by means
of a heterologous domain, segment, site, and the like, as disclosed
herein.
[1385] Further, mutant lipase can be designed in which one or more
of the various functions is engineered to be increased or
decreased, for example, substrate binding activity or the catalytic
activity of the lipase, and used to augment or replace lipase
proteins in an individual. Thus, host cells can provide a
therapeutic benefit by replacing an aberrant lipase or providing an
aberrant lipase that provides a therapeutic result. In one
embodiment, the cells provide lipase that are abnormally
active.
[1386] In another embodiment, the cells provide lipase that are
abnormally inactive. These lipases can compete with endogenous
lipase polypeptides in the individual.
[1387] In another embodiment, cells expressing lipase that cannot
be activated, are introduced into an individual in order to compete
with endogenous lipases for its various substrates. For example, in
the case in which excessive lipase or analog is part of a treatment
modality, it may be necessary to inactivate this molecule at a
specific point in treatment. Providing cells that compete for the
molecule, but which cannot be affected by lipase activation would
be beneficial.
[1388] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous lipase
polynucleotide sequences in a host cell genome. The host cell
includes, but is not limited to, a stable cell line, cell in vivo,
or cloned microorganism. This technology is more fully described in
WO 93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the lipase polynucleotides or sequences proximal
or distal to a lipase gene are allowed to integrate into a host
cell genome by homologous recombination where expression of the
gene can be affected. In one embodiment, regulatory sequences are
introduced that either increase or decrease expression of an
endogenous sequence. Accordingly, a lipase can be produced in a
cell not normally producing it. Alternatively, increased expression
of lipase can be effected in a cell normally producing the protein
at a specific level. Further, expression can be decreased or
eliminated by introducing a specific regulatory sequence. The
regulatory sequence can be heterologous to the lipase protein
sequence or can be a homologous sequence with a desired mutation
that affects expression. Alternatively, the entire gene can be
deleted. The regulatory sequence can be specific to the host cell
or capable of functioning in more than one cell type. Still
further, specific mutations can be introduced into any desired
region of the gene to produce mutant lipase proteins. Such
mutations could be introduced, for example, into specific
functional regions such as the triglyceride or phospholipid-binding
site.
[1389] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered lipase gene. Alternatively, the host
cell can be a stem cell or other early tissue precursor that gives
rise to a specific subset of cells and can be used to produce
transgenic tissues in an animal. See also Thomas et al., Cell
51:503 (1987) for a description of homologous recombination
vectors. The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced gene
has homologously recombined with the endogenous lipase gene is
selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected
cells are 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
vectors and 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; WO 91/01140; and
WO 93/04169.
[1390] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. 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 in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of a lipase protein and identifying and evaluating
modulators of lipase protein activity.
[1391] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[1392] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which a lipase polynucleotide sequences
have been introduced.
[1393] A transgenic animal can be produced by introducing 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. Any of the lipase
nucleotide sequences can be introduced as a transgene into the
genome of a non-human animal, such as a mouse.
[1394] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the lipase
protein to particular cells.
[1395] 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 the transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[1396] 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)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. 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 is
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.
[1397] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
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 blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[1398] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect, for example, binding, activation, and protein turnover, may
not be evident from in vitro cell-free or cell-based assays.
Accordingly, it is useful to provide non-human transgenic animals
to assay in vivo lipase function, including substrate interaction,
the effect of specific mutant on lipase function and substrate
interaction, and the effect of chimeric lipases. It is also
possible to assess the effect of null mutations, that is mutations
that substantially or completely eliminate one or more lipase
functions.
[1399] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the
lipase in a transgenic animal, into a cell in culture or in vivo.
When introduced in vivo, the nucleic acid is introduced into an
intact organism such that one or more cell types and, accordingly,
one or more tissue types, express the nucleic acid encoding the
lipase. Alternatively, the nucleic acid can be introduced into
virtually all cells in an organism by transfecting a cell in
culture, such as an embryonic stem cell, as described herein for
the production of transgenic animals, and this cell can be used to
produce an entire transgenic organism. As described, in a further
embodiment, the host cell can be a fertilized oocyte. Such cells
are then allowed to develop in a female foster animal to produce
the transgenic organism.
Pharmaceutical Compositions
[1400] The lipase nucleic acid molecules, protein modulators of the
protein, and antibodies (also referred to herein as "active
compounds") can be incorporated into pharmaceutical compositions
suitable for administration to a subject, e.g., a human. Such
compositions typically comprise the nucleic acid molecule, protein,
modulator, or antibody and a pharmaceutically acceptable
carrier.
[1401] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation, in
vivo, of polynucleotides that have been exogenously introduced into
a subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[1402] 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, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. 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 ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[1403] 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 polyethylene 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 mannitol, 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.
[1404] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a lipase protein or
anti-lipase 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.
[1405] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. 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.
[1406] 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.
[1407] 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.
[1408] 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.
[1409] 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.
[1410] 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.
[1411] 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 (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
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.
[1412] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[1413] 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.
[1414] 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. 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.
[1415] 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.
[1416] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the purview 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. 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.
[1417] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
CHAPTER 5
25678, a Novel Human Adenylate Cyclase
BACKGROUND OF THE INVENTION
[1418] Adenylate cyclase is a membrane-bound enzyme that acts as an
effector protein in a receptor-effector system referred to as the
cAMP signal transduction pathway. As such, it plays a key
intermediate role in the conversion of extracellular signals,
perceived by various receptors following binding of a particular
ligand, into intracellular signals that, in turn, generate specific
cellular responses.
[1419] A variety of hormones, neurotransmitters, and olfactants
regulate the synthesis of cAMP by adenylate cyclases. In most
tissues, regulation of cAMP synthesis is accomplished through three
plasma membrane-associated components: G-protein-coupled receptors
(GPCRs), which interact with regulatory hormones and
neurotransmitters; heterotrimeric G proteins that either stimulate
or inhibit the catalytic subunit of adenylate cyclase in response
to interaction of ligands with appropriate GPCRs; and the catalytic
entity, adenylate cyclase. Each G protein contains a guanine
nucleotide-binding alpha subunit and a complex of tightly
associated .beta.- and .gamma.-subunits. When a G protein is
activated following binding of a ligand to a GPCR, GDP is released
from the .alpha.-subunit in exchange for GTP. Binding of the GTP
results in conformational changes that yield dissociation of the
GTP-bound .alpha.-subunit from the .beta.-.gamma.-subunit complex.
The resulting macromolecular complexes regulate catalytic activity
of adenylate cyclase. Where the receptor is a stimulatory receptor
(R.sub.s), interaction with a stimulatory G-protein, termed
G.sub.s, results in activation of the adenylate cyclase catalytic
subunit by the GTP-bound form of the G.sub.s, .alpha.-subunit. In
contrast, where the receptor is an inhibitory receptor (R.sub.1),
interaction with an inhibitory G-protein (one of several known
G.sub.1s) results in inhibition of the adenylate cyclase catalytic
subunit by the GTP-bound form of the G.sub.s .alpha.-subunit. In
addition, the G-protein .beta.-.gamma.-subunit complex may interact
with and influence adenylate cyclase activity independent of or in
parallel with the GTP-bound .alpha.-subunit, depending upon the
adenylate cyclase isoform involved. See Taussig and Gilman (1995)
J. Biol. Chem. 6:1-4; Hardman et al., eds. (1996) Goodman and
Gilman's Pharmacological Basis of Therapeutics (McGraw-Hill
Company, New York, N.Y.).
[1420] When activated, the catalytic subunit of adenylate cyclase
converts intracellular ATP into cAMP. This second messenger then
activates protein kinases, particularly protein kinase A.
Activation of this protein kinase causes the phosphorylation of
downstream target proteins involved in a number of metabolic
pathways, thus initiating a signal transduction cascade.
[1421] The extent to which adenylate cyclase converts ATP to cAMP
is highly dependent on the state of phosphorylation of the various
components of the hormone-sensitive adenylate cyclase system. For
example, stimulatory and inhibitory receptors are desensitized and
down-regulated following phosphorylation by various kinases,
particularly cAMP-dependent protein kinases, protein kinase C, and
other receptor-specific kinases that preferentially use
agonist-bound forms of receptors as substrates. In this manner,
tight regulation of the cellular cAMP concentration, and hence
regulation of the cAMP signal transduction pathway, is achieved
(Taussig and Gilman (1995) J. Biol. Chem. 270:1-4).
[1422] Adenylate cyclase activation may also occur through
increased intracellular calcium concentration, especially in
nervous system and cardiovascular tissues. After depolarization,
the influx of calcium elicits the activation of calmodulin, an
intracellular calcium-binding protein. In the cardiovascular
system, this effect gives rise to the contraction of the blood
vessels or cardiac myocytes. The activated calmodulin has been
shown to bind and activate some isoforms of adenylate cyclase.
[1423] Several novel isoforms of mammalian adenylate cyclase have
been identified through molecular cloning. Type I adenylate cyclase
(CYA1) is primarily localized in brain tissues (see Krupinski et al
(1989) Science 244:1558-1564; Gilman (1987) Ann. Rev. Biochem.
56:615-649, citing Salter et al. (1981) J. Biol. Chem.
256:9830-9833; Andreasen et al. (1983) Biochemistry 22:2757-2762;
and Smigel et al (1986) J. Biol. Chem. 261:1976-1982 for bovine
CYA1; and Villacres et al. (1993) Genomics 16:473-478 for human
CYA1). The type II adenylate cyclase (CYA2) is localized in brain
and lung tissues (see Feinstein et al. (1991) Proc. Natl. Acad.
Sci. USA 88:10173-10177 for rat CYA2; and Stengel et al. (1992)
Hum. Genet. 90:126-130 for human CYA2). Type III adenylate cyclase
(CYA3) is primarily localized in olfactory neuroepithelium and is
thought to mediate olfactory receptor responses (Bakalyar and Reed
(1990) Science 250:1403-1406; Glatt and Snyder (1993) Nature
361:536-538; and Xia (1992) Neurosci. Lett. 144:169-173). Type IV
adenylate cyclase (CYA4) most resembles type II, but is expressed
in a variety of peripheral tissues and in the central nervous
system (Gao and Gilman (1991) Proc. Natl. Acad. Sci. USA
88:10178-10182, for rat CYA4). Type V adenylate cyclase (CYA5)
(Ishikawa et al. (1992) J. Biol. Chem. 267:13553-13557; Premont et
al. (1992) Proc. Natl. Acad. Sci. USA 89:9809-9813; and Glatt and
Snyder (1993) Nature 361:536-538; Krupinski et al. (1992) J. Biol.
Chem. 267:24858-24862) and type VI adenylate cyclase (CYA6)
(Premont et al. (1992) Proc. Natl. Acad. Sci. USA 89:9808-9813;
Yoshimura and Cooper (1992) Proc. Natl. Acad. Sci. USA
89:6716-6720; Katsushika et al. (1992) Proc. Natl. Acad. Sci. USA
89:8774-8778; and Krupinski et al. (1992) J. Biol. Chem.
267:24858-24862) both exhibit a widely distributed expression
pattern, with type V having high expression in heart and striatum,
and type VI having high expression in heart and brain. Type VII
adenylate cyclase (CYA7) is widely distributed, though may be
absent from brain tissues (Krupinski et al (1992) J. Biol. Chem.
267:24858-24862). Type VIII adenylate cyclase (CYA8) is abundant in
brain tissues (Krupinski et al. (1992) J. Biol. Chem.
267:24858-24862; and Parma et al. (1991) Biochem. Biophys. Res.
Commun. 179:455-462). Type IX adenylate cyclase (CYA9) is widely
expressed, at high levels in skeletal muscle and brain (Premont et
al. (1996) J. Biol. Chem. 271:13900-13907).
[1424] The different isoforms of adenylate cyclase exhibit unique
patterns of regulatory responses (see Sunahara et al. (1996) Annu.
Tev. Pharmacol. Toxicol 36:461-480). For example, all of these
isoforms are activated by the .alpha.-subunit of a particular G
protein, termed Gs, which couples the stimulatory action of the
ligand-bound receptor to activation of adenylate cyclase. The
adenylate cyclases designated type I, III, and VIII are also
stimulated by Ca.sup.2+/calmodulin in vitro, while type II, IV, V,
VI, VII, and IX are not. Type I is inhibited by G protein
.beta.-.gamma.-subunit complex, independently of G.sub.s
activation, while Type II is highly stimulated by G protein
.beta.-.gamma.-subunit complex when simultaneously activated by Gs
alpha subunit. Type III, in contrast, is not affected by G protein
.beta.-.gamma.-subunit complex. Type V and type VI are both are
inhibited by low levels of Ca.sup.2+, but appear to be unaffected
by G protein .beta.-.gamma.-subunit complex. Type IX is unique in
that it is stimulated by Mg.sup.2+, but is not affected by G
protein .beta.-.gamma.-subunit complex.
[1425] The genes for these adenylate cyclases all encode proteins
having molecular weights of approximately 120,000 and which range
from 1064 to 1353 amino acid residues. These proteins are predicted
to have a short cytoplasmic amino terminus followed by a first
motif consisting of six transmembrane spans and a cytoplasmic
(domain C.sub.1), and then a second motif, also consisting of six
transmembrane spans and a second cytoplasmic domain (domain
C.sub.2). The two cytoplasmic domains are approximately 40 kDa each
and contain a region of homology (designated C.sub.1a and C.sub.2a)
with each other and with the catalytic domains of membrane-bound
guanylate cyclases. Based on this similarity, these domains are
considered to be nucleotide binding domains, and together have been
shown to be sufficient to confer enzymatic activity (Tang and
Gilman (1995) Science 268:1769-1772).
[1426] Alterations in the cAMP signal transduction pathway have
been associated with diseases such as asthma, cancer, inflammation,
hypertension, atherosclerosis, and heart failure. Antihypertensive
drug therapy involves modulation of adenylate cyclase levels
(Marcil et al. (1996) Hypertension 28:83-90). In addition, studies
of heart in human and animal models indicate that adenylate cyclase
has a function in cardiomyopathy (Michael et al. (1995)
Hypertension 25:962-970, Roth et al (1999) Circulation
99:3099-3102), ischemia (Sandhu et al. (1996) Circulation Research
78:137-147), myocardial infarction (Espinasse et al. (1999)
Cardiovascular Research 42:87-98) and congestive heart failure
(Kawahira et al. (1998) Circulation 98:262-267, Panza et al. (1995)
Circulation 91:1732-1738). The enzyme is also related to some
mental disorders. Studies of learning and memory in animal models
indicate a likely role for calmodulin-activated adenylate cyclases
in conditioning (Abrams and Kandel (1988) Trends Neurosci.
11:128-135), learning (Livingstone et al. (1984) Cell 37:205-215),
and long-term potentiation (Frey et al. (1993) Science
260:161-1664). Furthermore, the cAMP signaling pathway plays an
important role in cardiovascular physiology. For instance, cAMP
activates protein kinase A (PKA). The activated subunits of PKA
initiate a series of enzymatic reactions that ultimately activate
multiple proteins that regulate both the rate and force of cardiac
contraction.
[1427] Accordingly, adenylate cyclases are a major target for drug
action and development. Accordingly, it is valuable to the field of
pharmaceutical development to identify and characterize novel
adenylate cyclases and tissues and disorders in which adenylate
cyclases are differentially expressed. The present invention
advances the state of the art by providing a novel human adenylate
cyclase and tissues and disorders in which expression of a human
adenylate cyclase is relevant. Accordingly, the invention provides
methods directed to expression of the adenylate cyclase.
SUMMARY OF THE INVENTION
[1428] It is an object of the invention to identify novel adenylate
cyclases and tissues and disorders in which expression of the
adenylate cyclase is relevant.
[1429] It is a further object of the invention to provide novel
adenylate cyclase polypeptides that are useful as reagents or
targets in adenylate cyclase assays applicable to treatment and
diagnosis of disorders mediated by or related to the adenylate
cyclase.
[1430] It is a further object of the invention to provide
polynucleotides corresponding to the adenylate cyclase polypeptides
that are useful as targets or reagents in adenylate cyclase assays
applicable to treatment and diagnosis of disorders mediated by or
related to the adenylate cyclase and useful for producing novel
adenylate cyclase polypeptides by recombinant methods.
[1431] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the adenylate cyclase in specific tissues and disorders.
[1432] A further specific object of the invention is to provide
compounds that modulate expression of the adenylate cyclase for
treatment and diagnosis of adenylate cyclase-mediated or related
disorders.
[1433] The invention is thus based on the identification and
expression of a human adenylate cyclase, especially in specific
tissues and disorders.
[1434] The invention provides methods of screening for compounds
that modulate expression or activity of the adenylate cyclase
polypeptides or nucleic acid (RNA or DNA) in the specific tissues
or disorders.
[1435] The invention also provides a process for modulating
adenylate cyclase polypeptide or nucleic acid expression or
activity, especially using the screened compounds.
[1436] Modulation may be used to treat conditions related to
aberrant activity or expression of the adenylate cyclase
polypeptides or nucleic acids.
[1437] The invention also provides assays for determining the
activity of or the presence or absence of the adenylate cyclase
polypeptides or nucleic acid molecules in specific biological
samples, including for disease diagnosis.
[1438] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[1439] The invention provides isolated adenylate cyclase
polypeptides, including a polypeptide having the amino acid
sequence shown in SEQ ID NO:19 or the amino acid sequence encoded
by the cDNA insert of the plasmid deposited as ATCC Patent Deposit
PTA-1871 on May 12, 2000 ("the deposited cDNA").
[1440] The invention also provides an isolated adenylate cyclase
nucleic acid molecule having the sequence shown in SEQ ID NO:20 or
encoded by the deposited cDNA.
[1441] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO:19 or encoded by the deposited
cDNA.
[1442] The invention also provides variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO:20 or in the deposited cDNA.
[1443] The invention also provides fragments of the polypeptide
shown in SEQ ID NO:19 and nucleotide sequence shown in SEQ ID
NO:20, as well as substantially homologous fragments of the
polypeptide or nucleic acid.
[1444] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[1445] The invention also provides vectors and host cells that
express the adenylate cyclase and provides methods for expressing
the adenylate cyclase nucleic acid molecules and polypeptides in
specific cell types and disorders, and particularly recombinant
vectors and host cells.
[1446] The invention also provides methods of making the vectors
and host cells and provides methods for using them to produce
adenylate cyclase nucleic acid molecules and polypeptides and to
assay expression and cellular effects of expression of the
adenylate cyclase nucleic acid molecules and polypeptides in
specific cell types and disorders.
[1447] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the adenylate cyclase
polypeptides and fragments.
[1448] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[1449] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[1450] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[1451] The present invention is based, at least in part, on the
identification of novel molecules, referred to herein as adenylate
cyclase nucleic acid and polypeptide molecules, which play a key
role in regulation of the cyclic AMP (cAMP) signal transduction
pathway by virtue of their conversion of intracellular ATP into
cAMP. In one embodiment, the adenylate cyclase molecules modulate
the activity of one or more proteins involved in cellular
metabolism associated with cell maintenance, growth, or
differentiation, e.g., cardiac, epithelial, or neuronal cell
maintenance, growth, or differentiation. In another embodiment, the
adenylate cyclase molecules of the present invention are capable of
modulating the phosphorylation state of one or more proteins
involved in cellular metabolism associated with cell maintenance,
growth, or differentiation, e.g., cardiac, epithelial, or neuronal
cell maintenance, growth or differentiation, via their indirect
effect on cAMP-dependent protein kinases, particularly protein
kinase A, as described in, for example, Devlin (1997) Textbook of
Biochemistry with Clinical Correlations (Wiley-Liss, Inc., New
York, N.Y.). In addition, the receptors which trigger activity of
the adenylate cyclases of the present invention are targets of
drugs as described in Goodman and Gilman (1996), The
Pharmacological Basis of Therapeutics (9.sup.th ed.) Hartman &
Limbard Editors, the contents of which are incorporated herein by
reference.
[1452] As used herein, a "signaling pathway" refers to the
modulation (e.g., stimulation or inhibition) of a cellular
function/activity upon the binding of a ligand to a receptor.
Examples of such functions include mobilization of intracellular
molecules that participate in a signal transduction pathway, e.g.,
phosphatidylinositol 4,5-bisphosphate (PIP.sub.2), inositol
1,4,5-triphosphate (IP3) and adenylate cyclase; polarization of the
plasma membrane; production or secretion of molecules; alteration
in the structure of a cellular component; cell proliferation, e.g.,
synthesis of DNA; cell migration; cell differentiation; and cell
survival.
[1453] The response depends on the type of cell. In some cells,
binding of a ligand to the receptor may stimulate an activity such
as release of compounds, gating of a channel, cellular adhesion,
migration, differentiation, etc., through phosphatidylinositol or
cyclic AMP metabolism and turnover while in other cells, binding
will produce a different result.
[1454] The cAMP turnover pathway is a signaling pathway. As used
herein, "cyclic AMP turnover and metabolism" refers to the
molecules involved in the turnover and metabolism of cAMP as well
as to the activities of these molecules. Cyclic AMP is a second
messenger produced in response to ligand-induced stimulation of
certain receptors. In the cAMP signaling pathway, binding of a
ligand can lead to the activation of the enzyme adenyl cyclase,
which catalyzes the synthesis of cAMP. The newly synthesized cAMP
can in turn activate a cAMP-dependent protein kinase. This
activated kinase can phosphorylate a voltage-gated potassium
channel protein, or an associated protein, and lead to the
inability of the potassium channel to open during an action
potential. The inability of the potassium channel to open results
in a decrease in the outward flow of potassium, which normally
repolarizes the membrane of a neuron, leading to prolonged membrane
depolarization.
[1455] The cGMP turnover pathway is also a signaling pathway. As
used herein, "cyclic GMP turnover and metabolism" refers to the
molecules involved in the turnover and metabolism of cGMP as well
as to the activities of these molecules. Cyclic GMP is a second
messenger produced in response to ligand-induced stimulation of
certain receptors. In the cGMP signaling pathway, binding of a
ligand can lead to the activation of the enzyme guanyl cyclase,
which catalyzes the synthesis of cGMP. Synthesized cGMP can in turn
activate a cGMP-dependent protein kinase.
[1456] The invention is directed to methods, uses and reagents
applicable to methods and uses that are applied to cells, tissues
and disorders of these cells and tissues wherein adenylate cyclase
expression is relevant. The adenylate cyclase is expressed in a
variety of tissues as shown in FIGS. 50 and 51. Accordingly, the
methods and uses of the invention as disclosed in greater detail
below apply to these tissues, disorders involving these tissues,
and particularly to the disorders with which gene expression is
associated, as shown in these figures and as disclosed herein.
Accordingly, the methods, uses and reagents disclosed in greater
detail below especially apply to prostate, skeletal muscle, brain,
and testis. In addition, low positive expression is also observed
in aorta with lower relative expression in the aorta with intimal
proliferations, and internal mammary artery. In addition, using
heart as a reference, low positive expression is seen in ischemic
and myopathic hearts. Accordingly, the uses, reagents and methods
disclosed in detail herein below apply especially to these tissues,
cell types, and disorders.
Methods Using the Polypeptides
[1457] The protein sequences of the present invention can 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 the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins 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):3889-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See www.ncbi.nlm.nih.gov.
[1458] The adenylate cyclase polypeptides are useful for producing
antibodies specific for the adenylate cyclase, regions, or
fragments. Regions having a high antigenicity index score are shown
in FIG. 47.
[1459] The invention provides methods using the adenylate cyclase,
variants, or fragments, including but not limited to use in the
cells, tissues, and disorders as disclosed herein.
[1460] The invention provides biological assays related to
adenylate cyclases. Such assays involve any of the known functions
or activities or properties useful for diagnosis and treatment of
cyclic adenylate cyclase-related conditions. These include, but are
not limited to, binding and/or activation by G-protein subunits,
alpha, beta or gamma, hydrolysis of ATP or GTP and consequent
modulation of cAMP and/or cGMP intracellular concentration, ability
to be bound by specific antibody, GTP or ATP binding, and protein
kinase A phosphorylation, as well as the various other properties
and functions disclosed herein and disclosed in the references
cited herein.
[1461] The invention provides drug screening assays, in cell-based
or cell-free systems. Cell-based systems can be native, i.e., cells
that normally express the adenylate cyclase, as a biopsy, or
expanded in cell culture. In one embodiment, cell-based assays
involve recombinant host cells expressing the adenylate cyclase.
Accordingly, cells that are useful in this regard include, but are
not limited to, those disclosed herein as expressing or
differentially expressing the adenylate cyclase, such as those
shown in FIGS. 50 and 51. These include, but are not limited to,
cells or tissues derived from prostate, skeletal muscle, brain,
colon, ovary, aorta, testis, placenta, fetal heart, aorta with
intimal proliferations, internal mammary artery, kidney, and
saphenous vein. Such cells can naturally express the gene or can be
recombinant, containing one or more copies of
exogenously-introduced adenylate cyclase sequences or genetically
modified to modulate expression of the endogenous adenylate cyclase
sequence.
[1462] This aspect of the invention particularly relates to cells
derived from subjects with disorders involving the tissues in which
the adenylate cyclase is expressed or derived from tissues subject
to disorders including, but not limited to, those disclosed herein.
These disorders may naturally occur, as in populations of human
subjects, or may occur in model systems such as in vitro systems or
in vivo, such as in non-human transgenic organisms, particularly in
non-human transgenic animals.
[1463] Such assays can involve the identification of agents that
interact with the adenylate cyclase protein. This interaction can
be detected by functional assays, such as the ability to be
affected by an effector molecule, such as binding and/or activation
by G-protein subunits or hydrolysis of ATP and/or GTP to modulate
intracellular cAMP/cGMP concentrations. Such interaction can also
be measured by ultimate biological effects, such as phosphorylation
of protein kinases, for example protein kinase A, and other
downstream effectors in the signal transduction pathway, having
biological effects on immunity/inflammation or cell proliferation,
i.e., any of the effects of modulating the intracellular levels of
the second messengers cAMP and cGMP.
[1464] Determining the ability of the test compound to interact
with the adenylate cyclase can also comprise determining the
ability of the test compound to preferentially bind to the
polypeptide as compared to the ability of a known binding molecule
(e.g., G-protein, calmodulin, GTP or ATP) to bind to the
polypeptide.
[1465] In yet another aspect of the invention, the invention
provides methods to identify proteins that interact with the
adenylate cyclase in the tissues and disorders disclosed. The
proteins of the invention 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 WO 94/10300), to identify other proteins
(captured proteins) which bind to or interact with the proteins of
the invention and modulate their activity.
[1466] The invention provides methods to identify compounds that
modulate adenylate cyclase activity. Such compounds, for example,
can increase or decrease affinity or rate of binding to GTP or ATP,
compete with GTP or ATP for binding to the adenylate cyclase, or
displace GTP or ATP bound to the adenylate cyclase. Such compounds
can also increase or decrease affinity or rate of binding to
calmodulin, compete with calmodulin for binding to the adenyl
cyclase, or displace calmodulin bound to the adenyl cyclase. Such
compounds can also, for example, increase or decrease the affinity
or rate of binding of one or more G-protein subunits, compete with
the subunits for binding, or displace the subunits bound to the
adenyl cyclase. Both adenylate cyclase and appropriate variants and
fragments can be used in high-throughput screens to assay candidate
compounds for the ability to bind to the adenylate cyclase. These
compounds can be further screened against a functional adenylate
cyclase to determine the effect of the compound on the adenylate
cyclase activity. Compounds can be identified that activate
(agonist) or inactivate (antagonist) the adenylate cyclase to a
desired degree. 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. The subject can be
a human subject, for example, a subject in a clinical trial or
undergoing treatment or diagnosis, or a non-human transgenic
subject, such as a transgenic animal model for disease.
[1467] The invention provides methods to screen a compound for the
ability to stimulate or inhibit interaction between the adenylate
cyclase protein and a target molecule that normally interacts with
the adenylate cyclase protein. The target can be an ATP or GTP, or
another component of the signal pathway with which the adenylate
cyclase protein normally interacts, including but not limited to,
calmodulin, or a G-protein subunit (one or more of alpha, beta, or
gamma). The assay includes the steps of combining the adenylate
cyclase protein with a candidate compound under conditions that
allow the adenylate cyclase protein or fragment to interact with
the target molecule, and to detect the formation of a complex
between the adenylate cyclase protein and the target, or to detect
the biochemical consequence of the interaction with the adenylate
cyclase and the target, such as any of the associated effects of
signal transduction such as protein kinase A phosphorylation, cAMP
or cGMP turnover, and biological endpoints of the pathway.
[1468] Determining the ability of the adenylate cyclase to bind to
a target molecule can also be accomplished using a technology such
as real-time Bimolecular Interaction Analysis (BIA). Sjolander et
al. (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.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR)
can be used as an indication of real-time reactions between
biological molecules.
[1469] 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 polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[1470] 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. USA 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; Carell 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. 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. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[1471] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[1472] One candidate compound is a soluble full-length adenylate
cyclase or fragment that competes for GTP or ATP binding. Other
candidate compounds include mutant adenylate cyclases or
appropriate fragments containing mutations that affect adenylate
cyclase function and thus compete for GTP or ATP. Accordingly, a
fragment that competes for ATP or GTP, for example with a higher
affinity, or a fragment that binds ATP or GTP but does not cyclize
it, is encompassed by the invention. Other fragments that are
encompassed include, but are not limited to, those that will bind
but not be activated by G-protein subunits, or bind but not be
activated by calmodulin.
[1473] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) adenylate cyclase
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate adenylate cyclase
activity. Thus, the expression of genes that are up- or
down-regulated in response to the adenylate cyclase dependent
signal cascade can be assayed. In one embodiment, the regulatory
region of such genes can be operably linked to a marker that is
easily detectable, such as luciferase.
[1474] Any of the biological or biochemical functions mediated by
the adenylate cyclase can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art.
[1475] In the case of the adenylate cyclase, specific end points
can include ATP and GTP cyclization and a decrease or increase in
intracellular cAMP or cGMP concentrations or in protein kinase A
activation.
[1476] Assays for adenylate cyclase function include, but are not
limited to, those that are well known in the art and available to
the person of ordinary skill in the art, for example, G-protein
subunit binding and activation of adenyl cyclase such as that
disclosed in Taussig et al. (1995), or Sunahara et al., herein
above, effect on cAMP- or cGMP-dependent kinases, as described for
example in Devlin, herein above, changes in intracellular cAMP
and/or cGMP concentration, as described in Sunahara et al., herein
above, and stimulation by calmodulin in vitro, as disclosed in
Sunahara et al., herein above. Further, nucleotide triphosphate
binding domains (e.g., for ATP and GTP) can be assayed according to
Tang et al. (1995), herein above. All of these references are
incorporated herein by reference for these assays.
[1477] Binding and/or activating compounds can also be screened by
using chimeric adenylate cyclase proteins in which one or more
domains, sites, and the like, as disclosed herein, or parts
thereof, can be replaced by their heterologous counterparts derived
from other adenylate cyclase isoforms of the same family or from
adenylate cyclase isoforms of any other adenylate cyclase family.
For example, a catalytic region can be used that interacts with a
different cyclic nucleotide specificity and/or affinity than the
native adenylate cyclase. Accordingly, a different set of signal
transduction components is available as an end-point assay for
activation. Alternatively, a heterologous effector protein
binding/activation sequence can replace the native sequence. For
example, a different G-protein subunit can be bound or interact
with the modified adenyl cyclase. Accordingly, the adenyl cyclase
is subject to different modulation by different stimulatory or
inhibitory G-protein subunits based on inhibitory or stimulatory
receptor interaction with the G-protein. As a further alternative,
the site of modification by an effector protein, for example
phosphorylation by a protein kinase can be replaced with the site
from a different effector protein. This could also provide the use
of a different signal transduction pathway for endpoint
determination. Activation can also be detected by a reporter gene
containing an easily detectable coding region operably linked to a
transcriptional regulatory sequence that is part of the native
signal transduction pathway.
[1478] The invention provides competition binding assays designed
to discover compounds that interact with the adenylate cyclase.
Thus, a compound is exposed to a adenylate cyclase polypeptide
under conditions that allow the compound to bind or to otherwise
interact with the polypeptide. Soluble adenylate cyclase
polypeptide is also added to the mixture. If the test compound
interacts with the soluble adenylate cyclase polypeptide, it
decreases the amount of complex formed or activity from the
adenylate cyclase target. This type of assay is particularly useful
in cases in which compounds are sought that interact with specific
regions of the adenylate cyclase. Thus, the soluble polypeptide
that competes with the target adenylate cyclase region is designed
to contain peptide sequences corresponding to the region of
interest.
[1479] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, calmodulin or one or more G-protein subunits and a
candidate compound can be added to a sample of the adenylate
cyclase. Compounds that interact with the adenylate cyclase at the
same site as these components will reduce the amount of complex
formed between the adenylate cyclase and these components.
Accordingly, it is possible to discover a compound that
specifically prevents interaction between the adenylate cyclase and
these components. Another example involves adding a candidate
compound to a sample of adenylate cyclase and ATP or GTP. A
compound that competes with ATP or GTP will reduce the amount of
cyclization or binding of the ATP or GTP to the adenylate cyclase.
Accordingly, compounds can be discovered that directly interact
with the adenylate cyclase and compete with ATP or GTP. Such assays
can involve any other component that interacts with the adenylate
cyclase.
[1480] To perform cell-free drug screening assays, it is desirable
to immobilize either the adenylate cyclase, or fragment, or its
target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[1481] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/adenylate
cyclase 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 cell lysates
(e.g., .sup.35S-labeled) and the candidate compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly, or in
the supernatant after the complexes is dissociated. Alternatively,
the complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of adenylate cyclase-binding protein found
in the bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do
not interfere with binding of the protein to its target molecule
can be derivatized to the wells of the plate, and the protein
trapped in the wells by antibody conjugation. Preparations of a
adenylate cyclase-binding component, such as ATP or G-protein
subunit, and a candidate compound are incubated in the adenylate
cyclase-presenting wells and the amount of complex trapped in the
well can be quantitated. 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 adenylate cyclase target molecule, or which are
reactive with adenylate cyclase and compete with the target
molecule; as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the target molecule.
[1482] Modulators of adenylate cyclase level or activity identified
according to these assays can be used to test the effects of
modulation of expression of the enzyme on the outcome of clinically
relevant disorders. This can be accomplished in vitro, in vivo,
such as in human clinical trials, and in test models derived from
other organisms, such as non-human transgenic subjects. Modulation
in such subjects includes, but is not limited to, modulation of the
cells, tissues, and disorders particularly disclosed herein.
Modulators of adenylate cyclase activity identified according to
these drug screening assays can be used to treat a subject with a
disorder mediated by the adenylate cyclase pathway, by treating
cells that express the adenylate cyclase, such as those disclosed
herein, especially in FIGS. 50 and 51, as well as those disorders
disclosed in the references cited herein above. In one embodiment,
the cells that are treated are derived from prostate, skeletal
muscle, brain, testis and aorta, and as such, modulation is
particularly relevant to disorders involving these tissues. In
another embodiment, modulation is in aortic tissue with intimal
proliferations or in ischemic or myopathic heart tissue.
Accordingly, disorders in which modulation is particularly relevant
can include these tissues. These methods of treatment include the
steps of administering the modulators of adenylate cyclase activity
in a pharmaceutical composition as described herein, to a subject
in need of such treatment.
[1483] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[1484] Disorders involving the brain include, but are not limited
to, disorders involving neurons, and disorders involving glia, such
as astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal
degeneration, multiple system atrophy, including striatonigral
degeneration, Shy-Drager syndrome, and olivopontocerebellar
atrophy, and Huntington disease; spinocerebellar degenerations,
including spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[1485] Diseases of the skin, include but are not limited to,
disorders of pigmentation and melanocytes, including but not
limited to, vitiligo, freckle, melasma, lentigo, nevocellular
nevus, dysplastic nevi, and malignant melanoma; benign epithelial
tumors, including but not limited to, seborrheic keratoses,
acanthosis nigricans, fibroepithelial polyp, epithelial cyst,
keratoacanthoma, and adnexal (appendage) tumors; premalignant and
malignant epidermal tumors, including but not limited to, actinic
keratosis, squamous cell carcinoma, basal cell carcinoma, and
merkel cell carcinoma; tumors of the dermis, including but not
limited to, benign fibrous histiocytoma, dermatofibrosarcoma
protuberans, xanthomas, and dermal vascular tumors; tumors of
cellular immigrants to the skin, including but not limited to,
histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and
mastocytosis; disorders of epidermal maturation, including but not
limited to, ichthyosis; acute inflammatory dermatoses, including
but not limited to, urticaria, acute eczematous dermatitis, and
erythema multiforme; chronic inflammatory dermatoses, including but
not limited to, psoriasis, lichen planus, and lupus erythematosus;
blistering (bullous) diseases, including but not limited to,
pemphigus, bullous pemphigoid, dermatitis herpetiformis, and
noninflammatory blistering diseases: epidermolysis bullosa and
porphyria; disorders of epidermal appendages, including but not
limited to, acne vulgaris; panniculitis, including but not limited
to, erythema nodosum and erythema induratum; and infection and
infestation, such as verrucae, molluscum contagiosum, impetigo,
superficial fungal infections, and arthropod bites, stings, and
infestations.
[1486] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[1487] Disorders involving blood vessels include, but are not
limited to, responses of vascular cell walls to injury, such as
endothelial dysfunction and endothelial activation and intimal
thickening; vascular diseases including, but not limited to,
congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[1488] Disorders involving the kidney include, but are not limited
to, congenital anomalies including, but not limited to, cystic
diseases of the kidney, that include but are not limited to, cystic
renal dysplasia, autosomal dominant (adult) polycystic kidney
disease, autosomal recessive (childhood) polycystic kidney disease,
and cystic diseases of renal medulla, which include, but are not
limited to, medullary sponge kidney, and nephronophthisis-uremic
medullary cystic disease complex, acquired (dialysis-associated)
cystic disease, such as simple cysts; glomerular diseases including
pathologies of glomerular injury that include, but are not limited
to, in situ immune complex deposition, that includes, but is not
limited to, anti-GBM nephritis, Heymann nephritis, and antibodies
against planted antigens, circulating immune complex nephritis,
antibodies to glomerular cells, cell-mediated immunity in
glomerulonephritis, activation of alternative complement pathway,
epithelial cell injury, and pathologies involving mediators of
glomerular injury including cellular and soluble mediators, acute
glomerulonephritis, such as acute proliferative (poststreptococcal,
postinfectious) glomerulonephritis, including but not limited to,
poststreptococcal glomerulonephritis and nonstreptococcal acute
glomerulonephritis, rapidly progressive (crescentic)
glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis (membranous nephropathy), minimal change disease
(lipoid nephrosis), focal segmental glomerulosclerosis,
membranoproliferative glomerulonephritis, IgA nephropathy (Berger
disease), focal proliferative and necrotizing glomerulonephritis
(focal glomerulonephritis), hereditary nephritis, including but not
limited to, Alport syndrome and thin membrane disease (benign
familial hematuria), chronic glomerulonephritis, glomerular lesions
associated with systemic disease, including but not limited to,
systemic lupus erythematosus, Henoch-Schonlein purpura, bacterial
endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary
and immunotactoid glomerulonephritis, and other systemic disorders;
diseases affecting tubules and interstitium, including acute
tubular necrosis and tubulointerstitial nephritis, including but
not limited to, pyelonephritis and urinary tract infection, acute
pyelonephritis, chronic pyelonephritis and reflux nephropathy, and
tubulointerstitial nephritis induced by drugs and toxins, including
but not limited to, acute drug-induced interstitial nephritis,
analgesic abuse nephropathy, nephropathy associated with
nonsteroidal anti-inflammatory drugs, and other tubulointerstitial
diseases including, but not limited to, urate nephropathy,
hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases
of blood vessels including benign nephrosclerosis, malignant
hypertension and accelerated nephrosclerosis, renal artery
stenosis, and thrombotic microangiopathies including, but not
limited to, classic (childhood) hemolytic-uremic syndrome, adult
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,
idiopathic HUS/TTP, and other vascular disorders including, but not
limited to, atherosclerotic ischemic renal disease, atheroembolic
renal disease, sickle cell disease nephropathy, diffuse cortical
necrosis, and renal infarcts; urinary tract obstruction
(obstructive uropathy); urolithiasis (renal calculi, stones); and
tumors of the kidney including, but not limited to, benign tumors,
such as renal papillary adenoma, renal fibroma or hamartoma
(renomedullary interstitial cell tumor), angiomyolipoma, and
oncocytoma, and malignant tumors, including renal cell carcinoma
(hypemephroma, adenocarcinoma of kidney), which includes urothelial
carcinomas of renal pelvis.
[1489] Disorders involving the testis and epididymis include, but
are not limited to, congenital anomalies such as cryptorchidism,
regressive changes such as atrophy, inflammations such as
nonspecific epididymitis and orchitis, granulomatous (autoimmune)
orchitis, and specific inflammations including, but not limited to,
gonorrhea, mumps, tuberculosis, and syphilis, vascular disturbances
including torsion, testicular tumors including germ cell tumors
that include, but are not limited to, seminoma, spermatocytic
seminoma, embryonal carcinoma, yolk sac tumor choriocarcinoma,
teratoma, and mixed tumors, tumore of sex cord-gonadal stroma
including, but not limited to, leydig (interstitial) cell tumors
and sertoli cell tumors (androblastoma), and testicular lymphoma,
and miscellaneous lesions of tunica vaginalis.
[1490] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[1491] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[1492] Disorders involving the ovary include, for example,
polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma
peritonei and stromal hyperthecosis; ovarian tumors such as, tumors
of coelomic epithelium, serous tumors, mucinous tumors,
endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma,
brenner tumor, surface epithelial tumors; germ cell tumors such as
mature (benign) teratomas, monodermal teratomas, immature malignant
teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma;
sex cord-stomal tumors such as, granulosa-theca cell tumors,
thecoma-fibromas, androblastomas, hill cell tumors, and
gonadoblastoma; and metastatic tumors such as Krukenberg
tumors.
[1493] The invention thus provides methods for treating a disorder
characterized by aberrant expression or activity of a adenylate
cyclase. 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 down-regulates) expression or activity of the protein. In
another embodiment, the method involves administering the adenylate
cyclase as therapy to compensate for reduced or aberrant expression
or activity of the protein.
[1494] Methods for treatment include but are not limited to the use
of soluble adenylate cyclase or fragments of the adenylate cyclase
protein that compete for ATP or GTP or G-protein. These adenylate
cyclases or fragments can have a higher affinity for the target so
as to provide effective competition.
[1495] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
aberrant development or cellular differentiation. In another
example, the subject has a proliferative disease (e.g., cancer) or
a disorder characterized by an aberrant hematopoictic response. In
another example, it is desirable to achieve tissue regeneration in
a subject (e.g., where a subject has undergone brain or spinal cord
injury and it is desirable to regenerate neuronal tissue in a
regulated manner).
[1496] The invention also provides methods for diagnosing a disease
or predisposition to disease mediated by the adenylate cyclase,
including, but not limited to, diseases involving tissues in which
the adenylate cyclases are expressed, as disclosed herein, and
particularly in prostate, skeletal muscle, brain, testes, as well
as aorta, aorta with intimal proliferations, internal mammary
artery, kidney, and saphenous vein. In addition, as indicated in
FIG. 51, positive differential expression occurs in diseased heart
tissue from patients with myopathy and ischemia. In view of these
results, in one embodiment of the invention, these disorders are
treated by modulating the level or activity of the adenylate
cyclase gene in diseased hearts. Treatment is therefore especially
directed to these tissues and cells thereof. Likewise, in one
embodiment, diagnosis is directed to cells and tissues involved in
these disorders. As mentioned above, treatment and diagnosis can be
in human subjects in which the disease normally occurs and in model
systems, both in vitro and in vivo, such as in transgenic
animals.
[1497] Accordingly, methods are directed to detecting the presence,
or levels of, the adenylate cyclase in a cell, tissue, or organism.
The methods involve contacting a biological sample with a compound
capable of interacting with the adenylate cyclase such that the
interaction can be detected.
[1498] One agent for detecting adenylate cyclase is an antibody
capable of selectively binding to adenylate cyclase. A biological
sample includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject.
[1499] The invention also provides methods for diagnosing active
disease, or predisposition to disease, in a patient having a
variant adenylate cyclase. Thus, adenylate cyclase can be isolated
from a biological sample and assayed for the presence of a genetic
mutation that results in an aberrant protein. This includes amino
acid substitution, deletion, insertion, rearrangement, (as the
result of aberrant splicing events), and inappropriate
post-translational modification. Analytic methods include altered
electrophoretic mobility, altered tryptic peptide digest, altered
adenylate cyclase activity in cell-based or cell-free assay,
alteration in ATP or GTP binding or cyclization, G-protein subunit
binding or calmodulin or antibody-binding pattern, altered
isoelectric point, direct amino acid sequencing, and any other of
the known assay techniques useful for detecting mutations in a
protein in general or in a adenylate cyclase specifically.
[1500] In vitro techniques for detection of adenylate cyclase
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-adenylate cyclase 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. Particularly useful are methods, which detect
the allelic variant of the adenylate cyclase expressed in a
subject, and methods, which detect fragments of the adenylate
cyclase in a sample.
[1501] The invention also provides methods of pharmacogenomic
analysis including, but not limited to, in the cells, tissues and
disorders disclosed herein in which expression of the adenylate
cyclase either occurs or shows differential expression.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985, and
Linder, M. W. (1997) Clin. Chem. 43(2):254-266. The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes affects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
adenylate cyclase in which one or more of the adenylate cyclase
functions in one population is different from those in another
population. The polypeptides can be used as a target to ascertain a
genetic predisposition that can affect treatment modality. Thus, in
a GTP- or ATP-based treatment, polymorphism may give rise to
catalytic regions that are more or less active. Accordingly, dosage
would necessarily be modified to maximize the therapeutic effect
within a given population containing the polymorphism. As an
alternative to genotyping, specific polymorphic polypeptides could
be identified.
[1502] The invention also provides for monitoring therapeutic
effects during clinical trials and other treatment. Thus, the
therapeutic effectiveness of an agent that is designed to increase
or decrease gene expression, protein levels or adenylate cyclase
activity can be monitored over the course of treatment using the
adenylate cyclase polypeptides as an end-point target. The
monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression or activity of
the protein in the pre-administration sample; (iii) obtaining one
or more post-administration samples from the subject; (iv)
detecting the level of expression or activity of the protein in the
post-administration samples; (v) comparing the level of expression
or activity of the protein in the pre-administration sample with
the protein in the post-administration sample or samples; and (vi)
increasing or decreasing the administration of the agent to the
subject accordingly.
Polypeptides
[1503] The methods and uses herein disclosed can be based on
polypeptide reagents and targets. The invention is thus based on
the discovery of a novel human adenylate cyclase. Specifically, an
expressed sequence tag (EST) was selected based on homology to
adenylate cyclase sequences. This EST was used to design primers
based on sequences that it contains and used to identify a cDNA
from a fetal testis cDNA library. Positive clones were sequenced
and the overlapping fragments were assembled. Analysis of the
assembled sequence revealed that the cloned cDNA molecule encodes
an adenylate cyclase similar to a rat adenylate cyclase.
[1504] The invention thus relates to a novel human adenylate
cyclase and to the expression of the adenylate cyclase having the
deduced amino acid sequence shown in FIGS. 46A-46D (SEQ ID NO:19)
or having the amino acid sequence encoded by the cDNA insert of the
plasmid deposited with the ATCC as Patent Deposit Number
PTA-1871.
[1505] The deposits will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms. The deposits are provided as a convenience to those
of skill in the art and are not an admission that a deposit is
required under 35 U.S.C. .sctn. 112. The deposited sequences as
well as the polypeptides encoded by the sequences, are incorporated
herein by reference and control in the event of any conflict, such
as a sequencing error, with description in this application.
[1506] "Adenylate cyclase polypeptide" or "adenylate cyclase
protein" refers to the polypeptide in SEQ ID NO:19 or encoded by
the deposited cDNA. The term "adenylate cyclase protein" or
"adenylate cyclase polypeptide", however, further includes the
numerous variants described herein, as well as fragments derived
from the full-length adenylate cyclases and variants.
[1507] Tissues and/or cells in which the adenylate cyclase is found
include, but are not limited to those shown in FIGS. 50 and 51, and
particularly in prostate, skeletal muscle, brain, testis and aorta.
In addition, the adenylate cyclase is expressed in diseased
tissues, including but limited to, heart tissue derived from
patients with myopathy or ischemia.
[1508] The present invention thus provides an isolated or purified
adenylate cyclase polypeptide and variants and fragments
thereof.
[1509] Based on a BLAST search, high homology was shown to adenyl
cyclase from rat, CYA2 Type II (EC 4.6.1.1) (ATP
pyrophosphate-lyase), SwissProt Acc. No. P26769, and a rat adenyl
cyclase, PATENT Acc. No. R94560.
[1510] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material, when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[1511] The adenylate cyclase polypeptides can be purified to
homogeneity. It is understood, however, that preparations in which
the polypeptide is not purified to homogeneity are useful and
considered to contain an isolated form of the polypeptide. The
critical feature is that the preparation allows for the desired
function of the polypeptide, even in the presence of considerable
amounts of other components. Thus, the invention encompasses
various degrees of purity.
[1512] In one embodiment, the language "substantially free of
cellular material" includes preparations of the adenylate cyclase
having less than about 30% (by dry weight) other proteins (i.e.,
contaminating protein), less than about 20% other proteins, less
than about 10% other proteins, or less than about 5% other
proteins. When the polypeptide is recombinantly produced, it can
also be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation.
[1513] An adenylate cyclase polypeptide is also considered to be
isolated when it is part of a membrane preparation or is purified
and then reconstituted with membrane vesicles or liposomes.
[1514] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the adenylate cyclase
polypeptide in which it is separated from chemical precursors or
other chemicals that are involved in its synthesis. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of the polypeptide having
less than about 30% (by dry weight) chemical precursors or other
chemicals, less than about 20% chemical precursors or other
chemicals, less than about 10% chemical precursors or other
chemicals, or less than about 5% chemical precursors or other
chemicals.
[1515] In one embodiment, the adenylate cyclase polypeptide
comprises the amino acid sequence shown in SEQ ID NO:19. However,
the invention also encompasses sequence variants. Variants include
a substantially homologous protein encoded by the same genetic
locus in an organism, i.e., an allelic variant.
[1516] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the
adenylate cyclase of SEQ ID NO:19. Variants also include proteins
substantially homologous to the adenylate cyclase but derived from
another organism, i.e., an ortholog. Variants also include proteins
that are substantially homologous to the adenylate cyclase that are
produced by chemical synthesis. Variants also include proteins that
are substantially homologous to the adenylate cyclase that are
produced by recombinant methods. It is understood, however, that
variants exclude any amino acid sequences disclosed prior to the
invention.
[1517] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 70-75%, typically at least about 80-85%, and most
typically at least about 90-95% or more homologous. A substantially
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence hybridizing to the
nucleic acid sequence, or portion thereof, of the sequence shown in
SEQ ID NO:20 under stringent conditions as more fully described
below.
[1518] 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-homologous
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% or more of the
length of the reference sequence. 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.
[1519] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the
adenylate cyclase. Similarity is determined by conserved amino acid
substitution. Such substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Conservative substitutions are likely to be
phenotypically silent. Typically seen as conservative substitutions
are the replacements, one for another, among the aliphatic amino
acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues
Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution between the amide residues Asn and Gln, exchange of
the basic residues Lys and Arg and replacements among the aromatic
residues Phe, Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
TABLE-US-00004 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[1520] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[1521] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. See www.ncbi.nlm.nih.gov. In
one embodiment, parameters for sequence comparison can be set at
score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).
[1522] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (J. Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at 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
(Devereux et al. (1984) Nucleic Acids Res. 12(1):387) (available at
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.
[1523] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[1524] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[1525] Variant polypeptides can be fully functional or can lack
function in one or more activities. Thus, in the present case,
variations can affect the function, for example, of one or more of
the regions corresponding to a catalytic region, regulatory region,
targeting region, regions involved in membrane association, regions
involved in enzyme activation, for example, by phosphorylation, and
regions involved in interaction with components of the cyclic
nucleotide-dependent signal transduction pathways, (e.g., ATP, GTP,
G-protein, or calmodulin).
[1526] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[1527] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[1528] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for the adenylate cyclase polypeptide.
This includes preventing immunogenicity from pharmaceutical
formulations by preventing protein aggregation.
[1529] Useful variations further include alteration of catalytic
activity. For example, one embodiment involves a variation at the
binding site that results in binding but not cyclization, or slower
cyclization, of ATP or GTP. A further useful variation at the same
site can result in altered affinity for ATP or GTP. Useful
variation includes one that prevents activation by G-protein.
Another useful variation provides a fusion protein in which one or
more domains or subregions are operationally fused to one or more
domains or subregions from another adenylate cyclase isoform or
family.
[1530] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as ATP or GTP cyclization in vitro or cGMP- or cAMP-dependent
in vitro activity, such as proliferative activity. Sites that are
critical for GTP or ATP or G-protein binding can also be determined
by structural analysis such as crystallization, nuclear magnetic
resonance or photoaffinity labeling (Smith et al. (1992) J. Mol.
Biol. 224:899-904; de Vos et al. (1992) Science 255:306-312).
[1531] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[1532] The invention thus also includes polypeptide fragments of
the adenylate cyclase. Fragments can be derived from the amino acid
sequence shown in SEQ ID NO:19. However, the invention also
encompasses fragments of the variants of the adenylate cyclase as
described herein.
[1533] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments per se that may have
been disclosed prior to the invention (although the methods herein
can pertain to known fragments).
[1534] Accordingly, a fragment can comprise at least about 10, 15,
20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids.
Fragments can retain one or more of the biological activities of
the protein, for example the ability to bind to or cyclize GTP or
ATP, as well as fragments that can be used as an immunogen to
generate adenylate cyclase antibodies.
[1535] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a domain or motif,
e.g., catalytic site, adenylate cyclase signature, and sites for
glycosylation, protein kinase C phosphorylation, casein kinase II
phosphorylation, tyrosine kinase phosphorylation, and
N-myristoylation. Further possible fragments include the catalytic
site, sites important for cellular and subcellular targeting, sites
functional for interacting with components of other cGMP or
cAMP-dependent signal transduction pathways, and regulatory
sites.
[1536] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[1537] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific domains mentioned above, which sub-fragments retain
the function of the domain from which they are derived.
[1538] These regions can be identified by well-known methods
involving computerized homology analysis.
[1539] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the
adenylate cyclase and variants. These epitope-bearing peptides are
useful to raise antibodies that bind specifically to a adenylate
cyclase polypeptide or region or fragment. These peptides can
contain at least 10, 12, at least 14, or between at least about 15
to about 30 amino acids.
[1540] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular site. Regions having a high
antigenicity index are shown in FIG. 48. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular peptide
regions.
[1541] The epitope-bearing adenylate cyclase polypeptides may be
produced by any conventional means (Houghten, R. A. (1985) Proc.
Natl. Acad. Sci. USA 82:5131-5135). Simultaneous multiple peptide
synthesis is described in U.S. Pat. No. 4,631,211.
[1542] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the adenylate cyclase fragment and
an additional region fused to the carboxyl terminus of the
fragment.
[1543] The invention thus provides chimeric or fusion proteins.
These comprise a adenylate cyclase peptide sequence operatively
linked to a heterologous peptide having an amino acid sequence not
substantially homologous to the adenylate cyclase. "Operatively
linked" indicates that the adenylate cyclase peptide and the
heterologous peptide are fused in-frame. The heterologous peptide
can be fused to the N-terminus or C-terminus of the adenylate
cyclase or can be internally located.
[1544] In one embodiment the fusion protein does not affect
adenylate cyclase function per se. For example, the fusion protein
can be a GST-fusion protein in which the adenylate cyclase
sequences are fused to the N- or C-terminus of the GST sequences.
Other types of fusion proteins include, but are not limited to,
enzymatic fusion proteins, for example beta-galactosidase fusions,
yeast two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions.
Such fusion proteins, particularly poly-His fusions, can facilitate
the purification of recombinant adenylate cyclase. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a protein can be increased by using a heterologous signal sequence.
Therefore, in another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus.
[1545] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson
et al. J. Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing a adenylate cyclase
polypeptide and various portions of the constant regions of heavy
or light chains of immunoglobulins of various subclass (IgG, IgM,
IgA, IgE). Preferred as immunoglobulin is the constant part of the
heavy chain of human IgG, particularly IgG1, where fusion takes
place at the hinge region. For some uses it is desirable to remove
the Fc after the fusion protein has been used for its intended
purpose, for example when the fusion protein is to be used as
antigen for immunizations. In a particular embodiment, the Fc part
can be removed in a simple way by a cleavage sequence, which is
also incorporated and can be cleaved with factor Xa.
[1546] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. 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 re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). An adenylate cyclase-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the adenylate cyclase.
[1547] Another form of fusion protein is one that directly affects
adenylate cyclase functions. Accordingly, a adenylate cyclase
polypeptide is encompassed by the present invention in which one or
more of the adenylate cyclase domains (or parts thereof) has been
replaced by homologous domains (or parts thereof) from another
adenylate cyclase family. Accordingly, various permutations are
possible. For example, the aminoterminal regulatory domain, or
subregion thereof, can be replaced with the domain or subregion
from another isoform or adenylate cyclase family. As a further
example, the catalytic domain or parts thereof, can be replaced;
the carboxyterminal domain or subregion can be replaced. Thus,
chimeric adenylate cyclases can be formed in which one or more of
the native domains or subregions has been replaced by another.
[1548] Additionally, chimeric adenylate cyclase proteins can be
produced in which one or more functional sites is derived from a
different isoform, or from another adenylate cyclase family. It is
understood, however, that sites could be derived from adenylate
cyclase families that occur in the mammalian genome but which have
not yet been discovered or characterized. Such sites include but
are not limited to a catalytic site, regulatory site, sites
important for targeting to subcellular and cellular locations,
sites functional for interaction with components of cyclic AMP- and
cyclic GMP-dependent signal transduction pathways, phosphorylation
sites, glycosylation sites, and other functional sites disclosed
herein.
[1549] The isolated adenylate cyclase can be purified from cells
that naturally express it, such as from those shown in FIGS. 50 and
51 and/or specifically disclosed herein above, among others,
especially purified from cells that have been altered to express it
(recombinant), or synthesized using known protein synthesis
methods.
[1550] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
adenylate cyclase polypeptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques.
[1551] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally-occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in polypeptides are described in
basic texts, detailed monographs, and the research literature, and
they are well known to those of skill in the art.
[1552] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[1553] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[1554] Such modifications are well-known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T.E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990)
Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y.
Acad. Sci. 663:48-62).
[1555] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[1556] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[1557] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[1558] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
Methods Using the Antibodies
[1559] Methods for using antibodies as disclosed herein are
particularly applicable to the cells, tissues and disorders shown
in FIGS. 50 and 51 and as otherwise discussed herein above.
[1560] The invention provides methods using antibodies that
selectively bind to the adenylate cyclase and its variants and
fragments. An antibody is considered to selectively bind, even if
it also binds to other proteins that are not substantially
homologous with the adenylate cyclase. These other proteins share
homology with a fragment or domain of the adenylate cyclase. This
conservation in specific regions gives rise to antibodies that bind
to both proteins by virtue of the homologous sequence. In this
case, it would be understood that antibody binding to the adenylate
cyclase is still selective.
[1561] The invention provides methods of using antibodies to
isolate a adenylate cyclase by standard techniques, such as
affinity chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the adenylate cyclase from cells
naturally expressing it and cells recombinantly producing it.
[1562] The antibodies can be used to detect the presence of
adenylate cyclase in cells or tissues to determine the pattern of
expression of the adenylate cyclase among various tissues in an
organism and over the course of normal development.
[1563] The antibodies can be used to detect adenylate cyclase in
situ, in vitro, or in a cell lysate or supernatant in order to
evaluate the abundance and pattern of expression.
[1564] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[1565] Antibody detection of circulating fragments of the
full-length adenylate cyclase can be used to identify adenylate
cyclase turnover.
[1566] Further, the antibodies can be used to assess adenylate
cyclase expression in disease states such as in active stages of
the disease or in an individual with a predisposition toward
disease related to adenylate cyclase function. When a disorder is
caused by an inappropriate tissue distribution, developmental
expression, or level of expression of the adenylate cyclase
protein, the antibody can be prepared against the normal adenylate
cyclase protein. If a disorder is characterized by a specific
mutation in the adenylate cyclase, antibodies specific for this
mutant protein can be used to assay for the presence of the
specific mutant adenylate cyclase. However, intracellularly-made
antibodies ("intrabodies") are also encompassed, which would
recognize intracellular adenylate cyclase peptide regions.
[1567] The antibodies can also be used to assess normal and
aberrant subcellular localization in cells in the various tissues
in an organism. Antibodies can be developed against the whole
adenylate cyclase or portions of the adenylate cyclase.
[1568] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting adenylate cyclase
expression level or the presence of aberrant adenylate cyclases and
aberrant tissue distribution or developmental expression,
antibodies directed against the adenylate cyclase or relevant
fragments can be used to monitor therapeutic efficacy.
[1569] Antibodies accordingly 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.
[1570] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic adenylate
cyclase can be used to identify individuals that require modified
treatment modalities.
[1571] Antibodies can also be used in diagnostic procedures as an
immunological marker for aberrant adenylate cyclase analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[1572] The antibodies are also useful for tissue typing. Thus,
where the adenylate cyclase is expressed in a specific tissue,
antibodies that are specific for this adenylate cyclase can be used
to identify the tissue type.
[1573] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[1574] The antibodies are also useful for inhibiting adenylate
cyclase function, for example, blocking binding of GTP or ATP,
G-protein, or the catalytic site.
[1575] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting adenylate cyclase function. An
antibody can be used, for example, to block ATP or GTP binding.
Antibodies can be prepared against specific fragments containing
sites required for function or against intact adenylate
cyclase.
[1576] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.,
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[1577] The invention also encompasses kits for using antibodies to
detect the presence of a adenylate cyclase protein in a biological
sample. The kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting adenylate
cyclase in a biological sample; means for determining the amount of
adenylate cyclase in the sample; and means for comparing the amount
of adenylate cyclase 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 adenylate
cyclase.
Antibodies
[1578] The methods for using antibodies described above are based
on the generation of antibodies that specifically bind to the
adenylate cyclase or its variants or fragments.
[1579] To generate antibodies, an isolated adenylate cyclase
polypeptide is used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. Either the full-length protein or antigenic peptide
fragment can be used. Regions having a high antigenicity index are
shown in FIG. 48.
[1580] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents G-protein ATP or GTP binding. Antibodies can be
developed against the entire adenylate cyclase or domains of the
adenylate cyclase as described herein. Antibodies can also be
developed against specific functional sites as disclosed
herein.
[1581] The antigenic peptide can comprise a contiguous sequence of
at least 12, 14, 15, or 30 amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments, which may be
disclosed prior to the invention.
[1582] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be
used.
[1583] 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.
[1584] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
Methods Using the Polynucleotides
[1585] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to 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 protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins 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 www.ncbi.nlm.nih.gov.
[1586] The methods and uses described herein below for the
adenylate cyclase polynucleotide are particularly applicable to the
cells, tissues, and disorders shown in FIGS. 50 and 51, and
specifically discussed herein above.
[1587] The nucleic acid fragments useful to practice the invention
provide probes or primers in assays, such as those described
herein. "Probes" are oligonucleotides that hybridize in a
base-specific manner to a complementary strand of nucleic acid.
Such probes include polypeptide nucleic acids, as described in
Nielsen et al. (1991) Science 254:1497-1500. Typically, a probe
comprises a region of nucleotide sequence that hybridizes under
highly stringent conditions to at least about 15, typically about
20-25, and more typically about 40, 50 or 75 consecutive
nucleotides of the nucleic acid sequence shown in SEQ ID NO:20 and
the complements thereof. More typically, the probe further
comprises a label, e.g., radioisotope, fluorescent compound,
enzyme, or enzyme co-factor.
[1588] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[1589] The adenylate cyclase polynucleotides can be utilized as
probes and primers in biological assays.
[1590] Where the polynucleotides are used to assess adenylate
cyclase properties or functions, such as in the assays described
herein, all or less than all of the entire cDNA can be useful.
Assays specifically directed to adenylate cyclase functions, such
as assessing agonist or antagonist activity, encompass the use of
known fragments. Further, diagnostic methods for assessing
adenylate cyclase function can also be practiced with any fragment,
including those fragments that may have been known prior to the
invention. Similarly, in methods involving treatment of adenylate
cyclase dysfunction, all fragments are encompassed including those,
which may have been known in the art.
[1591] The invention utilizes the adenylate cyclase polynucleotides
as a hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding variant polypeptides
and to isolate cDNA and genomic clones that correspond to variants
producing the same polypeptides shown in SEQ ID NO:19 or the other
variants described herein. Variants can be isolated from the same
tissue and organism from which the polypeptide shown in SEQ ID
NO:19 was isolated, different tissues from the same organism, or
from different organisms. This method is useful for isolating
variant genes and cDNA that are developmentally controlled and
therefore may be expressed in the same tissue or different tissues
at different points in the development of an organism. This method
is useful for isolating variant genes and cDNA that are expressed
in the cells, tissues, and disorders disclosed herein.
[1592] The probe can correspond to any sequence along the entire
length of the gene encoding the adenylate cyclase. Accordingly, it
could be derived from 5' noncoding regions, the coding region, and
3' noncoding regions.
[1593] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:20, or a fragment thereof, such as an
oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to mRNA or DNA.
[1594] Fragments of the polynucleotides described herein can also
be used to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an mRNA and a larger or full-length
cDNA can be produced.
[1595] Fragments can also be used to synthesize antisense molecules
of desired length and sequence.
[1596] Antisense nucleic acids, useful in treatment and diagnosis,
can be designed using the nucleotide sequences of SEQ ID NO:20, and
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, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[1597] Additionally, the nucleic acid molecules useful to practice
the 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 acids can be modified to generate
peptide nucleic acids (see Hyrup et al. (1996) Bioorganic &
Medicinal Chemistry 4:5). 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 et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity 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. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[1598] The nucleic acid molecules and fragments useful to practice
the invention can also include other appended groups such as
peptides (e.g., for targeting host cell adenylate cyclases 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. WO 88/0918) or the blood brain
barrier (see, e.g., PCT Publication No. WO 89/10134). 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).
[1599] The adenylate cyclase polynucleotides can also be used as
primers for PCR to amplify any given region of a adenylate cyclase
polynucleotide.
[1600] The adenylate cyclase polynucleotides can also be used to
construct recombinant vectors. Such vectors include expression
vectors that express a portion of, or all of, the adenylate cyclase
polypeptides. Vectors also include insertion vectors, used to
integrate into another polynucleotide sequence, such as into the
cellular genome, to alter in situ expression of adenylate cyclase
genes and gene products. For example, an endogenous adenylate
cyclase coding sequence can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[1601] The adenylate cyclase polynucleotides can also be used to
express antigenic portions of the adenylate cyclase protein.
[1602] The adenylate cyclase polynucleotides can also be used as
probes for determining the chromosomal positions of the adenylate
cyclase polynucleotides by means of in situ hybridization methods,
such as FISH. (For a review of this technique, see Verma et al.
(1988) Human Chromosomes: A Manual of Basic Techniques (Pergamon
Press, New York), and PCR mapping of somatic cell hybrids. The
mapping of the sequence to chromosomes is important in correlating
these sequences with genes associated with disease, especially
where translocations and/or amplification have occurred.
[1603] 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.
[1604] 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 et al. ((1987) Nature 325:783-787).
[1605] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified 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.
[1606] The adenylate cyclase polynucleotide probes can also be used
to determine patterns of the presence of the gene encoding the
adenylate cyclase with respect to tissue distribution, for example,
whether gene duplication has occurred and whether the duplication
occurs in all or only a subset of cells in a tissue. The genes can
be naturally occurring or can have been introduced into a cell,
tissue, or organism exogenously.
[1607] The adenylate cyclase polynucleotides can also be used to
design ribozymes corresponding to all, or a part, of the mRNA
produced from genes encoding the polynucleotides described herein,
the ribozymes being useful to treat or diagnose a disorder or
otherwise modulate expression of the nucleic acid.
[1608] The adenylate cyclase polynucleotides can also be used to
make vectors that express part, or all, of the adenylate cyclase
polypeptides.
[1609] The adenylate cyclase polynucleotides can also be used to
construct host cells expressing a part, or all, of the adenylate
cyclase polynucleotides and polypeptides.
[1610] The adenylate cyclase polynucleotides can also be used to
construct transgenic animals expressing all, or a part, of the
adenylate cyclase polynucleotides and polypeptides.
[1611] The adenylate cyclase polynucleotides can also be used as
hybridization probes to determine the level of adenylate cyclase
nucleic acid expression. Accordingly, the probes can be used to
detect the presence of, or to determine levels of, adenylate
cyclase nucleic acid in cells, tissues, and in organisms. DNA or
RNA level can be determined. Probes can be used to assess gene copy
number in a given cell, tissue, or organism. This is particularly
relevant in cases in which there has been an amplification of the
adenylate cyclase gene.
[1612] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
adenylate cyclase gene, as on extrachromosomal elements or as
integrated into chromosomes in which the adenylate cyclase gene is
not normally found, for example, as a homogeneously staining
region.
[1613] These uses are relevant for diagnosis of disorders involving
an increase or decrease in adenylate cyclase expression relative to
normal, such as a proliferative disorder, a differentiative or
developmental disorder, or a hematopoietic disorder, such as in the
cells and tissues shown in FIGS. 50 and 51 and otherwise
specifically discussed herein. Thus in one embodiment, disorders
include diseases of the heart, such as myopathy and ischemia.
[1614] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of adenylate cyclase nucleic acid, in which
a test sample is obtained from a subject and nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of the nucleic
acid is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the nucleic acid.
[1615] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[1616] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[1617] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express the adenylate cyclase,
such as by measuring the level of a adenylate cyclase-encoding
nucleic acid in a sample of cells from a subject e.g., mRNA or
genomic DNA, or determining if the adenylate cyclase gene has been
mutated.
[1618] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate adenylate cyclase nucleic acid
expression (e.g., antisense, polypeptides, peptidomimetics, small
molecules or other drugs). A cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of the mRNA in the presence of the candidate compound is
compared to the level of expression of the mRNA in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of nucleic acid expression based on this
comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. The modulator
can bind to the nucleic acid or indirectly modulate expression,
such as by interacting with other cellular components that affect
nucleic acid expression.
[1619] 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 gene to a subject) in patients or in
transgenic animals.
[1620] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
expression of the adenylate cyclase gene. The method typically
includes assaying the ability of the compound to modulate the
expression of the adenylate cyclase nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by excessive or deficient adenylate cyclase nucleic
acid expression.
[1621] The assays can be performed in cell-based and cell-free
systems, such as systems using the tissues described herein, in
which the gene is expressed or in model systems for the disorders
to which the invention pertains. Cell-based assays include cells
naturally expressing the adenylate cyclase nucleic acid or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[1622] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[1623] The assay for adenylate cyclase nucleic acid expression can
involve direct assay of nucleic acid levels, such as mRNA levels,
or on collateral compounds involved in the signal pathway (such as
cAMP or cGMP turnover). Further, the expression of genes that are
up- or down-regulated in response to the adenylate cyclase signal
pathway can also be assayed. In this embodiment the regulatory
regions of these genes can be operably linked to a reporter gene
such as luciferase.
[1624] Thus, modulators of adenylate cyclase gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of adenylate cyclase mRNA in the presence of the
candidate compound is compared to the level of expression of
adenylate cyclase mRNA in the absence of the candidate compound.
The candidate compound can then be identified as a modulator of
nucleic acid expression based on this comparison and be used, for
example to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[1625] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate adenylate
cyclase nucleic acid expression. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or effects on nucleic acid activity
(e.g., when nucleic acid is mutated or improperly modified).
Treatment is of disorders characterized by aberrant expression or
activity of the nucleic acid.
[1626] The gene is particularly relevant for the treatment of
disorders involving the tissues shown in FIGS. 50 and 51,
particularly in prostate, skeletal muscle, brain, and testes, as
well as tissues and cells involved in myopathy and ischemia.
[1627] Alternatively, a modulator for adenylate cyclase nucleic
acid expression can be a small molecule or drug identified using
the screening assays described herein as long as the drug or small
molecule inhibits the adenylate cyclase nucleic acid
expression.
[1628] The adenylate cyclase polynucleotides are also useful for
monitoring the effectiveness of modulating compounds on the
expression or activity of the adenylate cyclase gene in clinical
trials or in a treatment regimen. Thus, the gene expression pattern
can serve as a barometer for the continuing effectiveness of
treatment with the compound, particularly with compounds to which a
patient can develop resistance. The gene expression pattern can
also serve as a marker indicative of a physiological response of
the affected cells to the compound. Accordingly, such monitoring
would allow either increased administration of the compound or the
administration of alternative compounds to which the patient has
not become resistant. Similarly, if the level of nucleic acid
expression falls below a desirable level, administration of the
compound could be commensurately decreased.
[1629] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[1630] The adenylate cyclase polynucleotides can be used in
diagnostic assays for qualitative changes in adenylate cyclase
nucleic acid, and particularly in qualitative changes that lead to
pathology. The polynucleotides can be used to detect mutations in
adenylate cyclase genes and gene expression products such as mRNA.
The polynucleotides can be used as hybridization probes to detect
naturally-occurring genetic mutations in the adenylate cyclase gene
and thereby to determine whether a subject with the mutation is at
risk for a disorder caused by the mutation. Mutations include
deletion, addition, or substitution of one or more nucleotides in
the gene, chromosomal rearrangement, such as inversion or
transposition, modification of genomic DNA, such as aberrant
methylation patterns or changes in gene copy number, such as
amplification. Detection of a mutated form of the adenylate cyclase
gene associated with a dysfunction provides a diagnostic tool for
an active disease or susceptibility to disease when the disease
results from overexpression, underexpression, or altered expression
of a adenylate cyclase.
[1631] Mutations in the adenylate cyclase gene can be detected at
the nucleic acid level by a variety of techniques. Genomic DNA can
be analyzed directly or can be amplified by using PCR prior to
analysis. RNA or cDNA can be used in the same way.
[1632] In certain embodiments, detection of the mutation 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) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
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 patient, 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
gene under conditions such that hybridization and amplification of
the 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.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[1633] 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.
[1634] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[1635] Alternatively, mutations in a adenylate cyclase gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[1636] Further, sequence-specific ribozymes (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.
[1637] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[1638] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[1639] Furthermore, sequence differences between a mutant adenylate
cyclase gene and a wild-type gene can be determined by direct DNA
sequencing. 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).
[1640] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). 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 one 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).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[1641] In other embodiments, genetic mutations 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
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two-dimensional
arrays containing light-generated DNA probes as described in Cronin
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.
[1642] The adenylate cyclase polynucleotides can also be used for
testing an individual for a genotype that while not necessarily
causing the disease, nevertheless affects the treatment modality.
Thus, the polynucleotides can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship). In the
present case, for example, a mutation in the adenylate cyclase gene
that results in altered affinity for ATP or GTP could result in an
excessive or decreased drug effect with standard concentrations of
ATP or GTP. Accordingly, the adenylate cyclase polynucleotides
described herein can be used to assess the mutation content of the
gene in an individual in order to select an appropriate compound or
dosage regimen for treatment.
[1643] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[1644] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[1645] The adenylate cyclase polynucleotides are also useful for
chromosome identification when the sequence is identified with an
individual chromosome and to a particular location on the
chromosome. First, the DNA sequence is matched to the chromosome by
in situ or other chromosome-specific hybridization. Sequences can
also be correlated to specific chromosomes by preparing PCR primers
that can be used for PCR screening of somatic cell hybrids
containing individual chromosomes from the desired species. Only
hybrids containing the chromosome containing the gene homologous to
the primer will yield an amplified fragment. Sublocalization can be
achieved using chromosomal fragments. Other strategies include
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to chromosome-specific libraries. Further mapping
strategies include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the 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.
[1646] The adenylate cyclase polynucleotides can also be used to
identify individuals from small biological samples. This can be
done for example using restriction fragment-length polymorphism
(RFLP) to identify an individual. Thus, the polynucleotides
described herein are useful as DNA markers for RFLP (See U.S. Pat.
No. 5,272,057).
[1647] Furthermore, the adenylate cyclase sequence can be used to
provide an alternative technique, which determines the actual DNA
sequence of selected fragments in the genome of an individual.
Thus, the adenylate cyclase 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 DNA from an
individual for subsequent sequencing.
[1648] 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. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
adenylate cyclase sequences can be used to obtain such
identification sequences from individuals and from tissue. The
sequences represent unique fragments of the human genome. 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.
[1649] If a panel of reagents from the sequences 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.
[1650] The adenylate cyclase polynucleotides can also be used in
forensic identification procedures. PCR technology can be used to
amplify DNA sequences taken from very small biological samples,
such as a single hair follicle, body fluids (e.g. blood, saliva, or
semen). The amplified sequence can then be compared to a standard
allowing identification of the origin of the sample.
[1651] The adenylate cyclase polynucleotides can thus 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 described
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 the
noncoding region are particularly useful since greater polymorphism
occurs in the noncoding regions, making it easier to differentiate
individuals using this technique.
[1652] The adenylate cyclase polynucleotides 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. This is useful in cases
in which a forensic pathologist is presented with a tissue of
unknown origin. Panels of adenylate cyclase probes can be used to
identify tissue by species and/or by organ type.
[1653] In a similar fashion, these primers and 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).
[1654] Alternatively, the adenylate cyclase polynucleotides can be
used directly to block transcription or translation of adenylate
cyclase gene sequences by means of antisense or ribozyme
constructs. Thus, in a disorder characterized by abnormally high or
undesirable adenylate cyclase gene expression, nucleic acids can be
directly used for treatment.
[1655] The adenylate cyclase polynucleotides are thus useful as
antisense constructs to control adenylate cyclase gene expression
in cells, tissues, and organisms. A DNA antisense polynucleotide is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
adenylate cyclase protein. An antisense RNA or DNA polynucleotide
would hybridize to the mRNA and thus block translation of mRNA into
adenylate cyclase protein.
[1656] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:20 which also
includes the start codon and antisense molecules which are
complementary to a fragment of the 3' untranslated region of SEQ ID
NO:20.
[1657] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of adenylate
cyclase nucleic acid. Accordingly, these molecules can treat a
disorder characterized by abnormal or undesired adenylate cyclase
nucleic acid expression. This technique involves cleavage by means
of ribozymes containing nucleotide sequences complementary to one
or more regions in the mRNA that attenuate the ability of the mRNA
to be translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the adenylate cyclase protein.
[1658] The adenylate cyclase polynucleotides also provide vectors
for gene therapy in patients containing cells that are aberrant in
adenylate cyclase gene expression. Thus, recombinant cells, which
include the patient's cells that have been engineered ex vivo and
returned to the patient, are introduced into an individual where
the cells produce the desired adenylate cyclase protein to treat
the individual.
[1659] The invention also encompasses kits for detecting the
presence of a adenylate cyclase nucleic acid in a biological
sample. For example, the kit can comprise reagents such as a
labeled or labelable nucleic acid or agent capable of detecting
adenylate cyclase nucleic acid in a biological sample; means for
determining the amount of adenylate cyclase nucleic acid in the
sample; and means for comparing the amount of adenylate cyclase
nucleic acid 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 adenylate cyclase
mRNA or DNA.
Polynucleotides
[1660] The nucleotide sequence in SEQ ID NO:20 was obtained by
sequencing the deposited human cDNA. Accordingly, the sequence of
the deposited clone is controlling as to any discrepancies between
the two and any reference to the sequence of SEQ ID NO:20 includes
reference to the sequence of the deposited cDNA.
[1661] The specifically disclosed cDNA comprises the coding region
and 5' and 3' untranslated sequences in SEQ ID NO:20.
[1662] The invention provides isolated polynucleotides encoding the
adenylate cyclase. The term "adenylate cyclase polynucleotide" or
"adenylate cyclase nucleic acid" refers to the sequence shown in
SEQ ID NO:20 or in the deposited cDNA. The term "adenylate cyclase
polynucleotide" or "adenylate cyclase nucleic acid" further
includes variants and fragments of the adenylate cyclase
polynucleotides.
[1663] The methods and uses described herein can be based on the
adenylate cyclase polynucleotide as a reagent or as a target.
[1664] The invention thus provides methods and uses for the
nucleotide sequence in SEQ ID NO:20.
[1665] An "isolated" adenylate cyclase nucleic acid is one that is
separated from other nucleic acid present in the natural source of
the adenylate cyclase nucleic acid. Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the
adenylate cyclase 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. However, there can be some
flanking nucleotide sequences, for example up to about 5 KB. The
important point is that the adenylate cyclase nucleic acid is
isolated from flanking sequences such that it can be subjected to
the specific manipulations described herein, such as recombinant
expression, preparation of probes and primers, and other uses
specific to the adenylate cyclase nucleic acid sequences.
[1666] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic-acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[1667] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[1668] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[1669] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[1670] The adenylate cyclase polynucleotides can encode the mature
protein plus additional amino or carboxyterminal amino acids, or
amino acids interior to the mature polypeptide (when the mature
form has more than one polypeptide chain, for instance). Such
sequences may play a role in processing of a protein from precursor
to a mature form, facilitate protein trafficking, prolong or
shorten protein half-life or facilitate manipulation of a protein
for assay or production, among other things. As generally is the
case in situ, the additional amino acids may be processed away from
the mature protein by cellular enzymes.
[1671] The adenylate cyclase polynucleotides include, but are not
limited to, the sequence encoding the mature polypeptide alone, the
sequence encoding the mature polypeptide and additional coding
sequences, such as a leader or secretory sequence (e.g., a pre-pro
or pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[1672] Adenylate cyclase polynucleotides can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[1673] In one embodiment, the adenylate cyclase nucleic acid
comprises only the coding region.
[1674] The invention further provides variant adenylate cyclase
polynucleotides, and fragments thereof, that differ from the
nucleotide sequence shown in SEQ ID NO:20 due to degeneracy of the
genetic code and thus encode the same protein as that encoded by
the nucleotide sequence shown in SEQ ID NO:20.
[1675] The invention also provides adenylate cyclase nucleic acid
molecules encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[1676] Typically, variants have a substantial identity with a
nucleic acid molecule of SEQ ID NO:20 and the complements thereof.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[1677] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a adenylate cyclase that is at least
about 60-65%, 65-70%, typically at least about 70-75%, more
typically at least about 80-85%, and most typically at least about
90-95% or more homologous to the nucleotide sequence shown in SEQ
ID NO:20 or a fragment of this sequence. Such nucleic acid
molecules can readily be identified as being able to hybridize
under stringent conditions, to the nucleotide sequence shown in SEQ
ID NO:20 or a fragment of the sequence. It is understood that
stringent hybridization does not indicate substantial homology
where it is due to general homology, such as poly A sequences, or
sequences common to all or most proteins, or all adenylate
cyclases. Moreover, variants per se do not include any nucleic acid
(or amino acid) sequence disclosed prior to the present invention,
although the methods herein can encompass such variants.
[1678] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 60-65% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95% or more
identical to each other remain hybridized to one another. 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, incorporated by reference.
One 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 50-65.degree. C. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times.SSC/0.1% SDS at room
temperature, or by one or more moderate stringency washes in
0.2.times.SSC/0.1% SDS at 42.degree. C., or washed in
0.2.times.SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:19 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).
[1679] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[1680] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:20 or the complement of SEQ ID NO:20. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO:20 and the complement of SEQ ID NO:20. The nucleic
acid fragments of the invention are at least about 15, preferably
at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50,
100, 200, 500 or more nucleotides in length. Longer fragments, for
example, 30 or more nucleotides in length, which encode antigenic
proteins or polypeptides described herein are useful.
[1681] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length adenylate cyclase
polynucleotide. The fragment can be single or double-stranded and
can comprise DNA or RNA. The fragment can be derived from either
the coding or the non-coding sequence.
[1682] In another embodiment an isolated adenylate cyclase nucleic
acid encodes the entire coding region. In another embodiment the
isolated adenylate cyclase nucleic acid encodes a sequence
corresponding to the mature protein that may be from about amino
acid 6 to the last amino acid. Other fragments include nucleotide
sequences encoding the amino acid fragments described herein.
[1683] Thus, adenylate cyclase nucleic acid fragments further
include sequences corresponding to the domains described herein,
subregions also described, and specific functional sites. Adenylate
cyclase nucleic acid fragments also include combinations of the
domains, segments, and other functional sites described above. A
person of ordinary skill in the art would be aware of the many
permutations that are possible.
[1684] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary skill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[1685] However, it is understood that a adenylate cyclase fragment
includes any nucleic acid sequence that does not include the entire
gene.
[1686] The invention also provides adenylate cyclase nucleic acid
fragments that encode epitope bearing regions of the adenylate
cyclase proteins described herein.
Computer Readable Means
[1687] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[1688] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[1689] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[1690] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[1691] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[1692] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[1693] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[1694] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[1695] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
Methods Using Vectors and Host Cells
[1696] The methods using vectors and host cells are particularly
relevant where vectors are expressed in the cells, tissues, and
disorders shown in FIGS. 50 and 51, and otherwise discussed herein,
or where the host cells are those that naturally express the gene,
as shown in these figures and which may be the native or a
recombinant cell expressing the gene.
[1697] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also 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.
[1698] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing adenylate cyclase
proteins or polypeptides that can be further purified to produce
desired amounts of adenylate cyclase protein or fragments. Thus,
host cells containing expression vectors are useful for polypeptide
production, as well as cells producing significant amounts of the
polypeptide, for example, the high-expressers shown in FIG. 51, in
other words, testes, prostate, skeletal muscle and brain.
[1699] Host cells are also useful for conducting cell-based assays
involving the adenylate cyclase or adenylate cyclase fragments.
Thus, a recombinant host cell expressing a native adenylate cyclase
is useful to assay for compounds that stimulate or inhibit
adenylate cyclase function. This includes ATP or GTP binding, gene
expression at the level of transcription or translation, G-protein
interaction, and components of the signal transduction pathway.
[1700] Host cells are also useful for identifying adenylate cyclase
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant adenylate cyclase (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native adenylate cyclase.
[1701] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation by means of a heterologous domain,
segment, site, and the like, as disclosed herein.
[1702] Further, mutant adenylate cyclases can be designed in which
one or more of the various functions is engineered to be increased
or decreased (e.g., ATP binding or G-protein binding) and used to
augment or replace adenylate cyclase proteins in an individual.
Thus, host cells can provide a therapeutic benefit by replacing an
aberrant adenylate cyclase or providing an aberrant adenylate
cyclase that provides a therapeutic result. In one embodiment, the
cells provide adenylate cyclases that are abnormally active.
[1703] In another embodiment, the cells provide a adenylate cyclase
that is abnormally inactive. This adenylate cyclase can compete
with endogenous adenylate cyclase in the individual.
[1704] In another embodiment, cells expressing adenylate cyclases
that cannot be activated are introduced into an individual in order
to compete with endogenous adenylate cyclase for ATP. For example,
in the case in which excessive ATP is part of a treatment modality,
it may be necessary to inactivate this molecule at a specific point
in treatment. Providing cells that compete for the molecule, but
which cannot be affected by adenylate cyclase activation would be
beneficial.
[1705] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous adenylate cyclase
polynucleotide sequences in a host cell genome. The host cell
includes, but is not limited to, a stable cell line, cell in vivo,
or cloned microorganism. This technology is more fully described in
WO 93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the adenylate cyclase polynucleotides or sequences
proximal or distal to a adenylate cyclase gene are allowed to
integrate into a host cell genome by homologous recombination where
expression of the gene can be affected. In one embodiment,
regulatory sequences are introduced that either increase or
decrease expression of an endogenous sequence. Accordingly, a
adenylate cyclase protein can be produced in a cell not normally
producing it. Alternatively, increased expression of adenylate
cyclase protein can be effected in a cell normally producing the
protein at a specific level. Further, expression can be decreased
or eliminated by introducing a specific regulatory sequence. The
regulatory sequence can be heterologous to the adenylate cyclase
protein sequence or can be a homologous sequence with a desired
mutation that affects expression. Alternatively, the entire gene
can be deleted. The regulatory sequence can be specific to the host
cell or capable of functioning in more than one cell type. Still
further, specific mutations can be introduced into any desired
region of the gene to produce mutant adenylate cyclase proteins.
Such mutations could be introduced, for example, into the specific
functional regions such as the nucleotide triphosphate site.
[1706] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered adenylate cyclase gene.
Alternatively, the host cell can be a stem cell or other early
tissue precursor that gives rise to a specific subset of cells and
can be used to produce transgenic tissues in an animal. See also
Thomas et al., Cell 51:503 (1987) for a description of homologous
recombination vectors. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced gene has homologously recombined with the endogenous
adenylate cyclase gene is selected (see e.g., Li, E. et al. (1992)
Cell 69:915). The selected cells are 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 vectors and homologous recombinant animals are
described further in Bradley, A. (1991) Current Opinions in
Biotechnology 2:823-829 and in PCT International Publication Nos.
WO 90/11354; WO 91/01140; and WO 93/04169.
[1707] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. 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 in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of an adenylate cyclase protein and identifying and
evaluating modulators of adenylate cyclase protein activity.
[1708] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[1709] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which adenylate cyclase polynucleotide
sequences have been introduced.
[1710] A transgenic animal can be produced by introducing 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. Any of the
adenylate cyclase nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[1711] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
adenylate cyclase protein to particular cells.
[1712] 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 the transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[1713] 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 PI. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. 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 is
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.
[1714] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
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 blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[1715] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect cAMP binding, adenylate cyclase activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo adenylate cyclase function,
including ATP interaction, the effect of specific mutant adenylate
cyclases on adenylate cyclase function and ATP interaction, and the
effect of chimeric adenylate cyclases. It is also possible to
assess the effect of null mutations, that is mutations that
substantially or completely eliminate one or more adenylate cyclase
functions.
[1716] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the
protein in a transgenic animal, into a cell in culture or in vivo.
When introduced in vivo, the nucleic acid is introduced into an
intact organism such that one or more cell types and, accordingly,
one or more tissue types, express the nucleic acid encoding the
protein. Alternatively, the nucleic acid can be introduced into
virtually all cells in an organism by transfecting a cell in
culture, such as an embryonic stem cell, as described herein for
the production of transgenic animals, and this cell can be used to
produce an entire transgenic organism. As described, in a further
embodiment, the host cell can be a fertilized oocyte. Such cells
are then allowed to develop in a female foster animal to produce
the transgenic organism.
Vectors/Host Cells
[1717] The methods using the vectors and host cells discussed above
are based on the vectors and host cells including, but not limited
to, those described below.
[1718] The invention also provides methods using vectors containing
the adenylate cyclase polynucleotides. The term "vector" refers to
a vehicle, preferably a nucleic acid molecule that can transport
the adenylate cyclase polynucleotides. When the vector is a nucleic
acid molecule, the adenylate cyclase polynucleotides are covalently
linked to the vector nucleic acid. With this aspect of the
invention, the vector includes a plasmid, single or double stranded
phage, a single or double stranded RNA or DNA viral vector, or
artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
[1719] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the adenylate cyclase polynucleotides.
Alternatively, the vector may integrate into the host cell genome
and produce additional copies of the adenylate cyclase
polynucleotides when the host cell replicates.
[1720] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
adenylate cyclase polynucleotides. The vectors can function in
procaryotic or eukaryotic cells or in both (shuttle vectors).
[1721] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the adenylate cyclase
polynucleotides such that transcription of the polynucleotides is
allowed in a host cell. The polynucleotides can be introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus, the second polynucleotide may provide a
trans-acting factor interacting with the cis-regulatory control
region to allow transcription of the adenylate cyclase
polynucleotides from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself.
[1722] It is understood, however, that in some embodiments,
transcription and/or translation of the adenylate cyclase
polynucleotides can occur in a cell-free system.
[1723] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[1724] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[1725] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[1726] A variety of expression vectors can be used to express a
adenylate cyclase polynucleotide. Such vectors include chromosomal,
episomal, and virus-derived vectors, for example vectors derived
from bacterial plasmids, from bacteriophage, from yeast episomes,
from yeast chromosomal elements, including yeast artificial
chromosomes, from viruses such as baculoviruses, papovaviruses such
as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies
viruses, and retroviruses. Vectors may also be derived from
combinations of these sources such as those derived from plasmid
and bacteriophage genetic elements, e.g., cosmids and phagemids.
Appropriate cloning and expression vectors for prokaryotic and
eukaryotic hosts are described in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
[1727] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e., tissue specific) or may provide
for inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[1728] The adenylate cyclase polynucleotides can be inserted into
the vector nucleic acid by well-known methodology. Generally, the
DNA sequence that will ultimately be expressed is joined to an
expression vector by cleaving the DNA sequence and the expression
vector with one or more restriction enzymes and then ligating the
fragments together. Procedures for restriction enzyme digestion and
ligation are well known to those of ordinary skill in the art.
[1729] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[1730] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the
adenylate cyclase polypeptides. Fusion vectors can increase the
expression of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting for example as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired polypeptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enterokinase. Typical
fusion expression vectors include pGEX (Smith et al. (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. 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. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[1731] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[1732] The adenylate cyclase polynucleotides can also be expressed
by expression vectors that are operative in yeast. Examples of
vectors for expression in yeast e.g., S. cerevisiae include
pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan et
al. (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene
54:113-123), and pYES2 (invitrogen Corporation, San Diego,
Calif.).
[1733] The adenylate cyclase polynucleotides can also be expressed
in insect cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL
series (Lucklow et al. (1989) Virology 170:31-39).
[1734] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[1735] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
adenylate cyclase polynucleotides. The person of ordinary skill in
the art would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[1736] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[1737] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as, mammalian cells.
[1738] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[1739] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the adenylate cyclase polynucleotides can be
introduced either alone or with other polynucleotides that are not
related to the adenylate cyclase polynucleotides such as those
providing trans-acting factors for expression vectors. When more
than one vector is introduced into a cell, the vectors can be
introduced independently, co-introduced or joined to the adenylate
cyclase polynucleotide vector.
[1740] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[1741] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[1742] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[1743] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the adenylate cyclase polypeptides or
heterologous to these polypeptides.
[1744] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[1745] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
Pharmaceutical Compositions
[1746] The invention encompasses use of the polypeptides, nucleic
acids, and other agents in pharmaceutical compositions to
administer to the cells in which expression of the adenylate
cyclase is relevant and in disorders as disclosed herein. Uses are
both diagnostic and therapeutic. The adenylate cyclase nucleic acid
molecules, protein, modulators of the protein, and antibodies (also
referred to herein as "active compounds") can be incorporated into
pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically comprise the
nucleic acid molecule, protein, modulator, or antibody and a
pharmaceutically acceptable carrier. It is understood however, that
administration can also be to cells in vitro as well as to in vivo
model systems such as non-human transgenic animals.
[1747] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation, in
vivo, of polynucleotides that have been exogenously introduced into
a subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[1748] 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, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. 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 ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[1749] 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 polyethylene 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 mannitol, 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.
[1750] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a adenylate cyclase
protein or anti-adenylate cyclase 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.
[1751] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. 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.
[1752] 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.
[1753] 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.
[1754] 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.
[1755] 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.
[1756] 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.
[1757] 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 (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
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.
[1758] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[1759] 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.
[1760] 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. 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.
[1761] 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.
[1762] 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. 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.
[1763] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
CHAPTER 6
Novel Human GTPase Activator Proteins
BACKGROUND OF THE INVENTION
The Ras Superfamily of GTPases
[1764] Proteins regulating Ras and its relatives have been reviewed
in Boguski et al. (Nature 366:643-654 (1993)), summarized below.
Ras proteins and their relatives are key in the control of normal
and transformed cell growth. Small GTPases related to Ras control a
wide variety of cellular processes which include aspects of growth
and differentiation, control of the cytoskeleton and regulation of
cellular traffic between membrane bound compartments. These
proteins cycle between active and inactive states bound to GTP and
GDP. This cycling is influenced by three classes of proteins that
switch the GTPase on, switch it off, and prevent it from switching.
Further, the intracellular location of the GTPase can be controlled
by another class of regulatory protein. The GTP-bound form of the
GTPase is converted to the GDP-bound form by an intrinsic capacity
to hydrolyze GTP. This process is accelerated by a
GTPase-activating protein (GAP). Activation involves the
replacement of GDP with GTP. This event is mediated by proteins
designated guanine nucleotide exchange factors (GEF) or guanine
nucleotide releasing protein (GNRP) and guanine nucleotide
dissociation stimulator (GDS). The process is inhibited by guanine
nucleotide dissociation inhibitors (GDI). Further, membrane
anchoring of the GTPase is critical for proper function and is
regulated, among other enzymes, by prenyltransferases.
[1765] The Ras superfamily of GTPases can be roughly divided into
three main families. The first family is the "true" Ras protein,
each of which has the ability to function as an oncogene following
mutational activation. These proteins transmit signals from
tyrosine kinases at the plasma membrane to a cascade of
serine/threonine kinases, which deliver signals to the cell
nucleus. Constitutive activation of the pathway contributes to
malignant transformation. The second group is the Rho/Rac protein
subgroup, involved in organizing the cytoskeleton. Rac is required
for membrane ruffling induced by growth factors and the formation
of actin stress fibers requires Rho. In yeast, the CDC42 product
controls cell polarity, another process in which actin is involved.
In addition, Rac proteins are components of the NADPH oxidase
system that generates superoxide in phagocytes. A third family is
the Rab protein family. Members of this group regulate membrane
trafficking, i.e., transport of vesicles between different
intracellular compartments.
[1766] In addition to the three major families, further subgroups
exist, exemplified by Ran and Arf. Ran proteins are nuclear GTPases
involved in mitosis. Arf (ADP-ribosylation factor) proteins are
necessary for ADP-ribosylation of G.sub.sa (the GTPase subunit of
s-type heterotrimeric G-proteins) by cholera toxin and are thought
to be involved in membrane vesicle fusion and transport.
[1767] Ras GEFs are proteins that activate Ras proteins by
exchanging bound GDP for free GTP. These include Ras GRF, MmSosI,
DnSoS, Step 6, Cdc25, Scd25, Lte1, and BUD5. The loss of GEF
function can be complemented by mutations that constitutively
activate the Ras proteins or, in some cases, by a loss of GAP
activity. GEFs first associate with the GDP-bound form of the
GTPase. GDP dissociates from this complex at an increased rate
leaving the GEF bound to the empty GTPase. GTP then binds
immediately, effecting GEF dissociation and leaving the GTPase in
active form. Accordingly, a stable complex can exist between GEF
and GTPase in the absence of nucleotide. Thus, GEFs recognize both
GDP and GTP-bound forms of Ras in vitro and in vivo.
[1768] Dominant negative Ras mutants exist that block normal Ras
activation. These have reduced affinity for GTP and may be
defective in the final step of the exchange process, i.e.
displacement of GEF by GTP. Accordingly, these mutants sequester
GEF into a dead-end complex and are useful to remove GEF activity
from cells so that activation of endogenous Ras proteins cannot
occur. However, Ras may also be activated by inhibiting GAP
activity without the need for GEF.
[1769] GEFs also include ral GEF. It is 20-fold more active on Ral
A and Ral B than on members of the Ras, Rho/Rac and Rab GTPase
families.
[1770] GEFs also include rap GEF. Cell polarity and budding in
yeast involve GTPases of the Rap and Rho subgroup. A GEF specific
for mammalian Rap proteins remains to be identified. Rap has the
ability to interfere with Ras signaling by blocking activation of
RAF and the serine/threonine kinase cascade.
[1771] GEFs also include Rho/Rac GEFs. GEFs specific for Rac and
Rho proteins include, but are not limited to, Cdc24, Dbl, Vav, Bcr,
Ras GRF, and ect 2. The human Dbl has been shown to act as a GEF
for CDC42Hs (the human homolog of CDC42 is known as G25K) and on
Rho. Further, Dbl binds several Rac/Rho-like proteins in vitro.
[1772] smg GDS (small GTP-binding protein) was originally described
as a GEF for mammalian Rap proteins. It also promotes nucleotide
exchange on Rho and Rac proteins. The protein works efficiently
only on isoprenylated proteins. Ras and Rho/Rac proteins are
modified by different isoprenoid moieties. Rho/Rac proteins receive
20-carbon geranylgeranyl groups.
[1773] Guanine nucleotide dissociation inhibitors (GDIs) include
rab GDI. The protein affects the rate of GDP dissociation from Rab
proteins. It inhibits GDP/GTP exchange and prevents the GDP-bound
form from binding to membranes. These activities depend on the
C-terminal geranylgeranyl group, at least of Rab3A.
[1774] Rho GDI was first identified as a factor capable of
inhibiting dissociation of GDP from post-translationally modified
Rho proteins. It has the ability to remove Rho proteins from
cellular membranes in cell-free systems. This indicates that it
could regulate the available Rho proteins associated with membranes
or facilitate movement of Rho from one membrane compartment to
another. Rac proteins bound to Rho GDI have also been identified as
components of the NADPH oxidase system that generates oxygen
radicals in activated phagocytes. Rac and Rho GDI form a
heterodimer required for oxidase stimulation in vitro. Along with
two other cytosolic factors, the components assemble into a
membrane-bound complex which uses electrons from NADPH to generate
superoxide anions. Recombinant Rac proteins in their GDP-bound
state can replace the requirement for Rac and Rho GDI in this
system. This indicates that Rho GDI can recognize the GTP-bound
form of Rac and protect it from Rac GAPs.
[1775] GTPase-activating proteins are disclosed in Table 1 in
Boguski et al., above. These include Ras GAP proteins. These
proteins have low intrinsic GTPase activity and their inactivation
is dependent on GAP in vivo. Of the Ras GAPs, neurofibromin, p120
GAP, Ira1, and Ira2 also have specificity for Rac. Of the rap GAP
family, Rap1GAP also has specificity for Rac. Rho/Rac GAPs with
specificity for Rac include Bcr, N-chimerin, rotund, p190,
GRB-1/p85a, and 3BP-1.
[1776] Ras-like GTPases are targeted to membranes where they act by
the post-translational attachment of isoprenoid lipids (or prenyl
groups). Prenylation involves the covalent thioether linkage of
farnesyl (15-carbon) or geranylgeranyl (20-carbon) groups to
cysteine residues near the C-terminus. These reactions are
catalyzed by prenyltransferases that differ in their isoprenoid
substrates and protein targets. Type 1 geranylgeranyl transferase
recognizes a CAAX motif but prefers a leucine residue in the
X-position. Substrates include members of Rho/Rac families.
[1777] p21-activated protein kinases (PAKs) are activated through
direct interaction with the GTPases Rac and Cdc42Hs. These GTPases
are implicated in the control of mitogen-activated protein kinase
(MAP) kinase c-Jun N-terminal kinase (JNK) and the reorganization
of the actin cytoskeleton. Recently, Aronheim et al. (Current
Biology 8:1125-1128 (1998)) reported on the biological role of PAK2
and identified its molecular targets. A two-hybrid system, "the Ras
recruitment system" was used to detect protein-protein interactions
at the inner surface of the plasma membranes. The PAK2 regulatory
domain was fused at the carboxy terminus of a Ras mutant protein
and screened against a cDNA library. Four clones were identified
that interacted specifically with PAK regulatory region and were
shown to encode a homolog of the GTPase Cdc42Hs. This protein,
designated Chp, showed an overall sequence identity to Cdc42Hs of
approximately 52%. Results from microinjection of this protein into
cells implicated it in the induction of lamellipodia and showed
that it activates the JNK MAP kinase cascade.
[1778] Proteins regulating Ras and its relatives have been reviewed
in Boguski et al., Nature 366: 643-654 (1993), summarized below. As
indicated above, GTPases cycle between inactive and active states
bound to GDP and GTP respectively. As indicated above, cycling can
be influenced by three different classes of proteins that switch
the GTPase on, switch it off, and protect it from switching.
Classes of regulatory proteins of Ras-like GTPases include GEF,
GDI, and GAP. GEFs catalyze exchange of GDP for GTP. GAPs catalyze
conversion of GTP-bound forms back to their inactive GDP states.
GDI proteins for Rab and Rho affect nucleotide dissociation and GAP
attack and may also be involved in membrane localization and
solubility. The intracellular location of the GTPase can be
controlled by a fourth class of regulatory protein affecting the
regulators with which the GTPase can interact.
[1779] Table 1 of Boguski et al. lists various GAPs, the organisms
from which they are derived, substrate specificity, and other
characterization. These include (in the Table) the following GAPs:
RasGAP; Neurofibromin (NF1) with a positive specificity for H-ras,
N-ras, K-ras, RAS1 and RAS2 and a negative specificity for Rho,
Rac, and Rab; p120GAP with a positive specificity for H-ras, N-ras,
K-ras, R-ras, RAS1 and RAS2 and a negative specificity for Rho, Rac
and Rab; Gap1 with a positive specificity for Ras1; Ira1 with a
positive specificity for RAS and RAS2 and a negative specificity
for Rho, Rac and Rab and potentially H-ras; Ira2 with a positive
specificity for RAS and RAS2 and a negative specificity Rho, Rac
and Rab and potentially H-ras; Sar1/gap1 with a positive
specificity for Ras1, RAS1 and Ras2; Bud2 with a positive
specificity for Bud1; RapGAP and Rap1GAP with a positive
specificity for Rap1A and Rap2 and a negative specificity for Ras,
Rho and Rac; Rho/racGAP and Bar with a positive specificity for Rac
and CDC42Hs and a negative specificity for Rho and Ras; n-Chimaerin
with a positive specificity for Rac and a negative specificity for
Rho, CDC42Hs and Ras; rotund locus and p 190 with a positive
specificity for Rac, Rho and CDC42Hs and a negative specificity for
Ras, GRB-1/p85a and 3BP-1.
[1780] RasGAP is one class of GAP. Ras proteins have a very low
intrinsic GTPase activity and their inactivation is dependent on
GAPs in vivo. For example, some oncogenic mutants of Ras proteins
are resistant to GAP-mediated GTPase stimulation and are
constitutively blocked in their active GTP-bound states. Yeast
contains two RasGAP proteins, IRA1 and IRA2 which contain domains
homologous to the human and other mammalian p120-GAPs. In the
absence of IRA gene product, yeast RAS proteins accumulate in their
GTP-bound state, becoming hyperactive and leading to overproduction
of cAMP. In yeast, therefore, RasGAPs are not effectors but serve
as negative regulators. NF1 is a human protein defective in von
Recklinghausen neurofibromatosis. This protein contains a domain
homologous to the catalytic domains of p120-GAP IRA1 and IRA2. It
may, in fact, be the mammalian homolog of IRA1 and IRA2. Mutant NF1
alleles are associated with sporadic cancers unrelated to
neurofibromatosis or to neural crest tissues. Drosophila contains a
protein, 70% identical to neurofibromin. It also contains a
distinct RasGAP (referred to as GAP1) that is a component of the
Sos tyrosine kinase/Ras1 signalling pathway. Loss of GAP1
stimulates Ras1 function, indicating that it is a negative
regulator.
[1781] RapGAP is another GAP class. Rap1A is around 50% identical
to Ras and, like Ras, binds to p120-GAP and to raf1 by its effector
binding domain. Rap1A binds p120-GAP but its GTPase activity is not
enhanced by this interaction. Another protein, rap1GAP, is
responsible for the Rap1A GTPase activation. Rap1GAP is unrelated
to rasGAP but contains several sites for phosphorylation by Cdc2
and cAMP-dependent kinases. Ras proteins, and most GTPases, depend
on a glutamine residue at position 61 (or equivalent) for intrinsic
or GAP-mediated GTP hydrolysis. Rap1, however, has a threonine at
this position.
[1782] Rho/Rac GAP is another class of GAP. A mammalian GAP
specific for Rho has been purified and shown to contain a region
related to the C-terminal domain of Bcr and to a human brain
protein, n-chimaerin. Bcr is a putative RhoGEF. Bcr and n-chimaerin
stimulate GTP hydrolysis by the Rho-like proteins Rac1 and Rac2,
but not by Rho proteins themselves. This activity is mediated by
the C-terminal 401 amino acids of Bcr. This domain does not
resemble RasGAP or Rap1GAP. Chimaerin also contains an N-terminal
DAG binding motif. Further, a multidomain protein, p90, that binds
to p120-GAP and regulates its activity contains a central domain
related to a putative DNA binding transcriptional repressor. At the
C-terminus, there is a 145 residue region that is related to
RhoGAPs.
[1783] GTPase activators (GAPs) are a major target for drug action
and development. Accordingly, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown GAPs. The present invention advances the state of the art
by providing previously unidentified human GAPs.
SUMMARY OF THE INVENTION
[1784] It is an object of the invention to identify novel GAPs.
[1785] It is a further object of the invention to provide novel GAP
polypeptides that are useful as reagents or targets in assays
applicable to treatment and diagnosis of GAP-mediated
disorders.
[1786] It is a further object of the invention to provide
polynucleotides corresponding to the novel polypeptides that are
useful as targets and reagents in assays applicable to treatment
and diagnosis of GAP-mediated disorders and useful for producing
novel GAP polypeptides by recombinant methods.
[1787] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression or
activity of the novel GAP.
[1788] A further specific object of the invention is to provide
compounds that modulate expression of the GAP for treatment and
diagnosis of GAP-related disorders.
[1789] The invention is thus based on the identification of two
novel GAPs, designated herein 26651 and 26138.
[1790] The invention provides isolated GAP polypeptides including a
polypeptide having an amino acid sequence shown in SEQ ID NO:22,
SEQ ID NO:25, or an amino acid sequence encoded by the cDNA
deposited with the ATCC as PTA-1918 on May 25, 2000 ("the deposited
cDNA").
[1791] The invention also provides isolated GAP nucleic acid
molecules having a sequence shown in SEQ ID NO:21, 23, 24, or 26,
or in the deposited cDNA.
[1792] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to an amino
acid sequence shown in SEQ ID NO:22, SEQ ID NO:25, or encoded by
the deposited cDNA.
[1793] The invention also provides variant nucleic acid sequences
that are substantially homologous to a nucleotide sequence shown in
SEQ ID NO:21, 23, 24, or 26, or in the deposited cDNA.
[1794] The invention also provides fragments of polypeptides shown
in SEQ ID NO:22 or SEQ ID NO:25 and polynucleotides shown in SEQ ID
NO:21, 23, 24, or 26, as well as substantially homologous fragments
of the polypeptide or nucleic acid.
[1795] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described above. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[1796] The invention also provides vectors and host cells for
expressing the GAP nucleic acid molecules and polypeptides and
particularly recombinant vectors and host cells.
[1797] The invention also provides methods of making the vectors
and host cells and methods for using them to produce the GAP
nucleic acid molecules and polypeptides.
[1798] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the GAP polypeptides and
fragments.
[1799] The invention also provides methods of screening for
compounds that modulate expression or activity of the GAP
polypeptides or nucleic acid (RNA or DNA).
[1800] The invention also provides a process for modulating the GAP
polypeptide or nucleic acid expression or activity, especially
using the screened compounds. Modulation may be used to treat
conditions related to aberrant activity or expression of the GAP
polypeptides or nucleic acids.
[1801] The invention also provides assays for determining the
presence or absence of and level of the GAP polypeptides or nucleic
acid molecules in a biological sample, including for disease
diagnosis.
[1802] The invention also provides assays for determining the
presence of a mutation in the GAP polypeptides or nucleic acid
molecules, including for disease diagnosis.
[1803] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the invention,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[1804] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[1805] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Receptor Function/Signal Pathway
[1806] As used herein, a "signaling pathway" refers to the
modulation (e.g., stimulation or inhibition) of a cellular
function/activity upon the binding of a ligand to a GPCR. Examples
of such functions include mobilization of intracellular molecules
that participate in a signal transduction pathway, e.g.,
phosphatidylinositol 4,5-bisphosphate (PIP.sub.2), inositol
1,4,5-triphosphate (IP3) and adenylate cyclase; polarization of the
plasma membrane; production or secretion of molecules; alteration
in the structure of a cellular component; cell proliferation, e.g.,
synthesis of DNA; cell migration; cell differentiation; and cell
survival.
[1807] Since the 22651 GAP is expressed in tissues that include,
but are not limited to, adrenal gland, pituitary, skin and spinal
cord, cells participating in a receptor protein signaling pathway
in which this protein is involved may include, but are not limited
to cells derived from these tissues.
[1808] Since the 26138 GAP is expressed in tonsil, spleen, fetal
liver, adult liver, fibrotic liver, granulocytes, neutrophils,
erythroid cells, adipose tissue, bone marrow, colon, lung, kidney,
heart, lymphocyte, megakaryocytes and T-cells, among others, cells
participating in a receptor protein signaling pathway in which this
protein is involved may include, but are not limited to cells
derived from these tissues as well as those tissues and cell lines
shown in FIGS. 64A-64B.
[1809] The response mediated by a receptor protein depends on the
type of cell. For example, in some cells, binding of a ligand to
the receptor protein may stimulate an activity such as release of
compounds, gating of a channel, cellular adhesion, migration,
differentiation, etc., through phosphatidylinositol or cyclic AMP
metabolism and turnover while in other cells, the binding of the
ligand will produce a different result. Regardless of the cellular
activity/response modulated by the receptor protein, the protein,
as a GPCR, would interact with G proteins to produce one or more
secondary signals, in a variety of intracellular signal
transduction pathways, e.g., through phosphatidylinositol or cyclic
AMP metabolism and turnover, in a cell.
[1810] As used herein, "phosphatidylinositol turnover and
metabolism" refers to the molecules involved in the turnover and
metabolism of phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) as
well as to the activities of these molecules. PIP.sub.2 is a
phospholipid found in the cytosolic leaflet of the plasma membrane.
Binding of ligand to the receptor activates, in some cells, the
plasma-membrane enzyme phospholipase C that in turn can hydrolyze
PIP2 to produce 1,2-diacylglycerol (DAG) and inositol
1,4,5-triphosphate (IP3). Once formed IP3 can diffuse to the
endoplasmic reticulum surface where it can bind an IP3 receptor,
e.g., a calcium channel protein containing an IP3 binding site. IP3
binding can induce opening of the channel, allowing calcium ions to
be released into the cytoplasm. IP3 can also be phosphorylated by a
specific kinase to form inositol 1,3,4,5-tetraphosphate (IP4), a
molecule which can cause calcium entry into the cytoplasm from the
extracellular medium. IP3 and IP4 can subsequently be hydrolyzed
very rapidly to the inactive products inositol 1,4-biphosphate
(IP2) and inositol 1,3,4-triphosphate, respectively. These inactive
products can be recycled by the cell to synthesize PIP.sub.2. The
other second messenger produced by the hydrolysis of PIP.sub.2,
namely 1,2-diacylglycerol (DAG), remains in the cell membrane where
it can serve to activate the enzyme protein kinase C. Protein
kinase C is usually found soluble in the cytoplasm of the cell, but
upon an increase in the intracellular calcium concentration, this
enzyme can move to the plasma membrane where it can be activated by
DAG. The activation of protein kinase C in different cells results
in various cellular responses such as the phosphorylation of
glycogen synthase, or the phosphorylation of various transcription
factors, e.g., NF-kB. The language "phosphatidylinositol activity",
as used herein, refers to an activity of PIP.sub.2 or one of its
metabolites.
[1811] Another signaling pathway in which a receptor may
participate is the cAMP turnover pathway. As used herein, "cyclic
AMP turnover and metabolism" refers to the molecules involved in
the turnover and metabolism of cyclic AMP (cAMP) as well as to the
activities of these molecules. Cyclic AMP is a second messenger
produced in response to ligand-induced stimulation of certain G
protein coupled receptors. In the cAMP signaling pathway, binding
of a ligand to a GPCR can lead to the activation of the enzyme
adenyl cyclase, which catalyzes the synthesis of cAMP. The newly
synthesized cAMP can in turn activate a cAMP-dependent protein
kinase. This activated kinase can phosphorylate a voltage-gated
potassium channel protein, or an associated protein, and lead to
the inability of the potassium channel to open during an action
potential. The inability of the potassium channel to open results
in a decrease in the outward flow of potassium, which normally
repolarizes the membrane of a neuron, leading to prolonged membrane
depolarization.
Polypeptides
[1812] The invention is based on the identification of novel human
GAPs. Specifically, an expressed sequence tag (EST) was selected
based on homology to GAP sequences. This EST was used to design
primers based on primary sequences that it contains and used to
identify a cDNA from human cDNA libraries. Positive clones were
sequenced and the overlapping fragments were assembled. Analysis of
the assembled sequence revealed that the cloned cDNA molecule
encodes a GAP.
[1813] The invention thus relates to novel GAPs having the deduced
amino acid sequence shown in FIGS. 52A-52B and 57A-57C (SEQ ID
NO:22 and SEQ ID NO:25) or having the amino acid sequence encoded
by the deposited cDNA, ATCC Patent Deposit No. PTA-1918.
[1814] Plasmids containing the 26651 sequences of the invention
were deposited with the Patent Depository of the American Type
Culture Collection (ATCC), Manassas, Va., on May 25, 2000 and
assigned Patent Deposit No. PTA-1918. The deposit will be
maintained under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms. The
deposit is provided as a convenience to those of skill in the art
and is not an admission that a deposit is required under 35 U.S.C.
.sctn. 112. The deposited sequence, as well as the polypeptide
encoded by the sequence, is incorporated herein by reference and
controls in the event of any conflict, such as a sequencing error,
with description in this application.
[1815] "GAP", "GAP polypeptide" or "GAP protein" refer to a
polypeptide set forth in SEQ ID NO:22, SEQ ID NO:25, or encoded by
the deposited cDNA. The terms, however, further include the
numerous variants described herein, as well as fragments derived
from the full-length GAP polypeptide and variants.
[1816] The present invention thus provides an isolated or purified
GAP polypeptide and variants and fragments thereof. By "variants"
is intended proteins or polypeptides having an amino acid sequence
that is at least about 60%, 65%, or 70%, preferably about 75%, 85%,
95%, or 98% identical to the amino acid sequence of SEQ ID NO:22 or
SEQ ID NO:25. Variants also include polypeptides encoded by the
cDNA insert of the plasmid deposited with ATCC as Patent Deposit
No. PTA-1918, or polypeptides encoded by a nucleic acid molecule
that hybridizes to the nucleic acid molecule of SEQ ID NO:21, 23,
24 or 26, or a complement thereof, under stringent conditions. In
another embodiment, a variant of an isolated polypeptide of the
present invention differs, by at least 1, but less than 5, 10, 20,
50, or 100 amino acid residues from the sequence shown in SEQ ID
NO:22 or SEQ ID NO:25. If alignment is needed for this comparison
the sequences should be aligned for maximum identity. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences. Such variants retain the functional
activity of the polypeptide set forth in SEQ ID NO:22 or SEQ ID
NO:25. Variants include polypeptides that differ in amino acid
sequence due to natural allelic variation or mutagenesis.
[1817] Based on a BLAST search of the 26651 sequence, homology was
shown to human and other mammalian Rho-GTPase activators. A search
for complete domains in PFAM showed a classification in the RhoGAP
family. PRODOM analysis also shows a relationship with Rho-type
GTPase activating proteins.
[1818] A search for complete domains in PFAM with the 26138
sequence showed classification in the rasGAP family,
GTPase-activator protein for Ras-like GTPase.
[1819] 26651 nucleic acid is expressed in tissues that include, but
are not limited to, adrenal gland, pituitary, skin and spinal cord.
Chromosome mapping with STS using WI-13730 shows that the gene is
located on the X chromosome between DXS 994 and DXS 1062 (143.2-145
cM).
[1820] The 26138 nucleic acid is expressed in tissues that include,
but are not limited to, tonsil, spleen, fetal liver, adult liver,
fibrotic liver, granulocytes, neutrophils, erythroid cells, adipose
tissue, bone marrow, colon, lung, kidney, heart, lymphocyte,
megakaryocytes and T-cells, as well as the tissues and cell lines
shown in FIGS. 64A-64B. Chromosome mapping information for this
gene is shown in FIG. 63.
[1821] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[1822] The GAP polypeptides can be purified to homogeneity. It is
understood, however, that preparations in which the polypeptide is
not purified to homogeneity are useful and considered to contain an
isolated form of the polypeptide. The critical feature is that the
preparation allows for the desired function of the polypeptide,
even in the presence of considerable amounts of other components.
Thus, the invention encompasses various degrees of purity.
[1823] In one embodiment, the language "substantially free of
cellular material" includes preparations of the GAP polypeptide
having less than about 30% (by dry weight) other proteins (i.e.,
contaminating protein), less than about 20% other proteins, less
than about 10% other proteins, or less than about 5% other
proteins. When the polypeptide is recombinantly produced, it can
also be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation.
[1824] A polypeptide is also considered to be isolated when it is
part of a membrane preparation or is purified and then
reconstituted with membrane vesicles or liposomes.
[1825] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the polypeptide in which
it is separated from chemical precursors or other chemicals that
are involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the polypeptide having less than about 30%
(by dry weight) chemical precursors or other chemicals, less than
about 20% chemical precursors or other chemicals, less than about
10% chemical precursors or other chemicals, or less than about 5%
chemical precursors or other chemicals.
[1826] In one embodiment, the polypeptide comprises an amino acid
sequence shown in SEQ ID NO:22 or SEQ ID NO:25. However, the
invention also encompasses sequence variants. Variants include a
substantially homologous protein encoded by the same genetic locus
in an organism, i.e., an allelic variant. Variants also encompass
proteins derived from other genetic loci in an organism, but having
substantial homology to a GAP of SEQ ID NO:22 or SEQ ID NO:25.
Variants also include proteins substantially homologous to the GAP
but derived from another organism, i.e., an ortholog. Variants also
include proteins that are substantially homologous to GAP
polypeptides of the invention that are produced by chemical
synthesis. Variants also include proteins that are substantially
homologous to the GAP that are produced by recombinant methods.
Variants retain the GAP activity of the polypeptides set forth in
SEQ ID NO:22 or SEQ ID NO:25. It is understood, however, that
variants exclude any amino acid sequences disclosed prior to the
invention.
[1827] As used herein, two amino acid or nucleotide sequences are
substantially homologous when the sequences have at least about
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity. A substantially homologous amino acid
sequence, according to the present invention, will be encoded by a
nucleic acid sequence hybridizing to the nucleic acid sequence, or
portion thereof, of the sequence shown in SEQ ID NO:21, 23, 24, or
26 under stringent conditions as more fully described below.
[1828] 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-homologous
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. 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.
[1829] 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 (1970) J. Mol. Biol. 48:444-453 algorithm
which has been incorporated into the GAP program in the GCG
software package (available at www.gcg.com), using either a Blossum
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 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. A particularly preferred set of
parameters (and the one that should be used if the practitioner is
uncertain about what parameters should be applied to determine if a
molecule is within a sequence identity or homology limitation of
the invention) is using a Blossum 62 scoring matrix with a gap open
penalty of 12, a gap extend penalty of 4, and a frameshift gap
penalty of 5.
[1830] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller (1989) CABIOS 4:11-17 which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4.
[1831] The nucleic acid and protein sequences described herein can
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 the 26651 or 26138 nucleic acid molecules
of the invention. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the 26651 or 26138 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
www.ncbi.nlm.nih.gov.
[1832] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by a
polypeptide of the invention. Similarity is determined by conserved
amino acid substitution. Such substitutions are those that
substitute a given amino acid in a polypeptide by another amino
acid of like characteristics. Conservative substitutions are likely
to be phenotypically silent. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr, exchange of the acidic residues Asp
and Glu, substitution between the amide residues Asn and Gln,
exchange of the basic residues Lys and Arg and replacements among
the aromatic residues Phe, Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al., Science 247:1306-1310 (1990).
TABLE-US-00005 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[1833] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[1834] Variant polypeptides can be fully functional or can lack
function in one or more activities. Thus, in the present case,
variations can affect the function, for example, of one or more
regions corresponding to, membrane association, GTPase binding,
interaction with regulatory proteins such as those in the
background above.
[1835] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids which result in no change or an
insignificant change in function. Alternatively, such substitutions
may positively or negatively affect function to some degree.
[1836] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[1837] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for a polypeptide of the invention. This
includes preventing immunogenicity from pharmaceutical formulations
by preventing protein aggregation.
[1838] Useful variations further include alteration of binding
characteristics. For example, one embodiment involves a variation
at the binding site that results in binding but not release, or
slower release of a binding molecule. A further useful variation at
the same sites can result in a higher affinity. Useful variations
also include changes that provide for affinity for another binding
molecule. Another useful variation includes one that allows binding
but which prevents activation by an effector. A useful variation
affects binding to the GTPase, e.g., Ras or Rho. Binding can be
with greater affinity, with less tendency to dissociate or lesser
affinity with a higher tendency to dissociate. Alternatively, a
variation can affect interaction with any of the regulatory
proteins which in turn affects association with the GTPase. A
further useful variation affects interaction with the regulatory
protein responsible for subcellular localization of the GAP.
[1839] Another useful variation provides a fusion protein in which
one or more domains or subregions is operationally fused to one or
more domains or subregions from another GAP, including, but not
limited to, subfamilies discussed above in the background in the
families of GTPase activators.
[1840] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity
such as receptor binding or in vitro, or in vivo proliferative
activity. Sites that are critical for substrate or effector binding
can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et
al. Science 255:306-312 (1992)).
[1841] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[1842] The invention thus also includes polypeptide fragments of
the GAPs. Fragments can be derived from an amino acid sequence
shown in SEQ ID NO:22 or SEQ ID NO:25. However, the invention also
encompasses fragments of the variants of the proteins of the
invention as described herein.
[1843] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention.
[1844] As used herein, a fragment comprises at least 5 contiguous
amino acids. Fragments can retain one or more of the biological
activities of the protein, for example the ability to bind to a
GTPase, as well as fragments that can be used as an immunogen to
generate antibodies.
[1845] Biologically active fragments (peptides which are about, for
example, 5-10, 10-15, 15-20, 25-30, 35-40, 40-50, 50-60, 60-70,
70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-150, 150-200,
200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-547 or up
to the number of amino acids in the full length sequence) can
comprise a domain or motif, e.g., a GTPase binding site, a
regulatory site for interaction with any of the regulatory proteins
affecting GAP activity, membrane anchoring site, or glycosylation
sites, phosphorylation sites, and myristoylation sites. Such
domains or motifs can be identified by means of routine
computerized homology searching procedures. Domains/motifs include,
but are not limited to, those shown in the figures.
[1846] Fragments also include combinations of domains or motifs
including, but not limited to, those mentioned above. Fragments,
for example, can extend in one or both directions from the
functional site to encompass 5, 10, 15, 20, 30, 40, 50, 100, 150,
200, 250, 300, 350, 400, 450, 500, 547, or up to the number of
amino acids disclosed in SEQ ID NO:22 and SEQ ID NO:25. Further,
fragments can include sub-fragments of the specific domains
mentioned above, which sub-fragments retain the function of the
domain from which they are derived.
[1847] These regions can be identified by well-known methods
involving computerized homology analysis.
[1848] Fragments also include antigenic fragments and specifically
those shown to have a high antigenic index in FIGS. 54 and 58.
[1849] Further possible fragments include but are not limited to
fragments defining a GTPase binding site, regulatory protein
binding, or membrane association. By this is intended a discrete
fragment that provides the relevant function or allows the relevant
function to be identified. In a preferred embodiment, the fragment
contains a GTPase-binding site.
[1850] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of a protein
of the invention and variants. These epitope-bearing peptides are
useful to raise antibodies that bind specifically to a polypeptide
of the invention or region or fragment. These peptides can contain
at least 6, 10, 12, at least 14, or between at least about 15 to
about 30 amino acids.
[1851] A polypeptide of the invention (including variants and
fragments which may have been disclosed prior to the present
invention) are useful for biological assays related to GAPs,
especially of the RasGAP or RhoGAP family. Such assays involve any
of the known GAP functions or activities or properties useful for
diagnosis and treatment of GAP-related conditions. They include,
especially, diseases involving the tissues in which a protein of
the invention is expressed as disclosed herein. For GAP activity,
assays include but are not limited to those disclosed herein,
including those in references cited in the background herein, which
are incorporated herein by reference for teaching these assays.
Such assays include but are not limited to GTPase binding or
activation, binding to GAP regulatory proteins, complex formation
with any of the regulatory proteins, and biological effects such as
those disclosed in the Background above. These include but are not
limited to reorganization the actin cytoskeleton, transformation,
growth, effects on differentiation, membrane ruffling induced by
growth factors, formation of actin stress fibers, and generation of
superoxide in phagocytes.
[1852] Disorders involving T-cells include, but are not limited to,
cell-mediated hypersensitivity, such as delayed type
hypersensitivity and T-cell-mediated cytotoxicity, and transplant
rejection; autoimmune diseases, such as systemic lupus
erythematosus, Sjogren syndrome, systemic sclerosis, inflammatory
myopathies, mixed connective tissue disease, and polyarteritis
nodosa and other vasculitides; immunologic deficiency syndromes,
including but not limited to, primary immunodeficiencies, such as
thymic hypoplasia, severe combined immunodeficiency diseases, and
AIDS; leukopenia; reactive (inflammatory) proliferations of white
cells, including but not limited to, leukocytosis, acute
nonspecific lymphadenitis, and chronic nonspecific lymphadenitis;
neoplastic proliferations of white cells, including but not limited
to lymphoid neoplasms, such as precursor T-cell neoplasms, such as
acute lymphoblastic leukemia/lymphoma, peripheral T-cell and
natural killer cell neoplasms that include peripheral T-cell
lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis
fungoides and Sezary syndrome, and Hodgkin disease.
[1853] Diseases of the skin, include but are not limited to,
disorders of pigmentation and melanocytes, including but not
limited to, vitiligo, freckle, melasma, lentigo, nevocellular
nevus, dysplastic nevi, and malignant melanoma; benign epithelial
tumors, including but not limited to, seborrheic keratoses,
acanthosis nigricans, fibroepithelial polyp, epithelial cyst,
keratoacanthoma, and adnexal (appendage) tumors; premalignant and
malignant epidermal tumors, including but not limited to, actinic
keratosis, squamous cell carcinoma, basal cell carcinoma, and
merkel cell carcinoma; tumors of the dermis, including but not
limited to, benign fibrous histiocytoma, dermatofibrosarcoma
protuberans, xanthomas, and dermal vascular tumors; tumors of
cellular immigrants to the skin, including but not limited to,
histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and
mastocytosis; disorders of epidermal maturation, including but not
limited to, ichthyosis; acute inflammatory dermatoses, including
but not limited to, urticaria, acute eczematous dermatitis, and
erythema multiforme; chronic inflammatory dermatoses, including but
not limited to, psoriasis, lichen planus, and lupus erythematosus;
blistering (bullous) diseases, including but not limited to,
pemphigus, bullous pemphigoid, dermatitis herpetiformis, and
noninflammatory blistering diseases: epidermolysis bullosa and
porphyria; disorders of epidermal appendages, including but not
limited to, acne vulgaris; panniculitis, including but not limited
to, erythema nodosum and erythema induratum; and infection and
infestation, such as verrucae, molluscum contagiosum, impetigo,
superficial fungal infections, and arthropod bites, stings, and
infestations.
[1854] In normal bone marrow, the myelocytic series
(polymorphoneuclear cells) make up approximately 60% of the
cellular elements, and the erythrocytic series, 20-30%.
Lymphocytes, monocytes, reticular cells, plasma cells and
megakaryocytes together constitute 10-20%. Lymphocytes make up
5-15% of normal adult marrow. In the bone marrow, cell types are
add mixed so that precursors of red blood cells (erythroblasts),
macrophages (monoblasts), platelets (megakaryocytes),
polymorphoneuclear leukocytes (myeloblasts), and lymphocytes
(lymphoblasts) can be visible in one microscopic field. In
addition, stem cells exist for the different cell lineages, as well
as a precursor stem cell for the committed progenitor cells of the
different lineages. The various types of cells and stages of each
would be known to the person of ordinary skill in the art and are
found, for example, on page 42 (FIG. 2-8) of Immunology,
Immunopathology and Immunity, Fifth Edition, Sell et al. Simon and
Schuster (1996), incorporated by reference for its teaching of cell
types found in the bone marrow. According, the invention is
directed to disorders arising from these cells. These disorders
include but are not limited to the following: diseases involving
hematopoietic stem cells; committed lymphoid progenitor cells;
lymphoid cells including B and T-cells; committed myeloid
progenitors, including monocytes, granulocytes, and megakaryocytes;
and committed erythroid progenitors. These include but are not
limited to the leukemias, including B-lymphoid leukemias,
T-lymphoid leukemias, undifferentiated leukemias; erythroleukemia,
megakaryoblastic leukemia, monocytic; [leukemias are encompassed
with and without differentiation]; chronic and acute lymphoblastic
leukemia, chronic and acute lymphocytic leukemia, chronic and acute
myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic
and acute myeloid leukemia, myelomonocytic leukemia; chronic and
acute myeloblastic leukemia, chronic and acute myelogenous
leukemia, chronic and acute promyclocytic leukemia, chronic and
acute myelocytic leukemia, hematologic malignancies of
monocyte-macrophage lineage, such as juvenile chronic myelogenous
leukemia; secondary AML, antecedent hematological disorder;
refractory anemia; aplastic anemia; reactive cutaneous
angioendotheliomatosis; fibrosing disorders involving altered
expression in dendritic cells, disorders including systemic
sclerosis, E-M syndrome, epidemic toxic oil syndrome, cosinophilic
fasciitis localized forms of scleroderma, keloid, and fibrosing
colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma,
including primary head and neck squamous cell carcinoma; sarcoma,
including kaposi's sarcoma; fibroadenoma and phyllodes tumors,
including mammary fibroadenoma; stromal tumors; phyllodes tumors,
including histiocytoma; erythroblastosis; neurofibromatosis;
diseases of the vascular endothelium; demyelinating, particularly
in old lesions; gliosis, vasogenic edema, vascular disease,
Alzheimer's and Parkinson's disease; T-cell lymphomas; B-cell
lymphomas.
[1855] Disorders involving the spleen include, but are not limited
to, splenomegaly, including nonspecific acute splenitis, congestive
spenomegaly, and spenic infarcts; neoplasms, congenital anomalies,
and rupture. Disorders associated with splenomegaly include
infections, such as nonspecific splenitis, infectious
mononucleosis, tuberculosis, typhoid fever, brucellosis,
cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis,
kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and
echinococcosis; congestive states related to partial hypertension,
such as cirrhosis of the liver, portal or splenic vein thrombosis,
and cardiac failure; lymphohematogenous disorders, such as Hodgkin
disease, non-Hodgkin lymphomas/leukemia, multiple myeloma,
mycloproliferative disorders, hemolytic anemias, and
thrombocytopenic purpura; immunologic-inflammatory conditions, such
as rheumatoid arthritis and systemic lupus erythematosus; storage
diseases such as Gaucher disease, Niemann-Pick disease, and
mucopolysaccharidoses; and other conditions, such as amyloidosis,
primary neoplasms and cysts, and secondary neoplasms.
[1856] Disorders involving blood vessels include, but are not
limited to, responses of vascular cell walls to injury, such as
endothelial dysfunction and endothelial activation and intimal
thickening; vascular diseases including, but not limited to,
congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[1857] Disorders involving red cells include, but are not limited
to, anemias, such as hemolytic anemias, including hereditary
spherocytosis, hemolytic disease due to erythrocyte enzyme defects:
glucose-6-phosphate dehydrogenase deficiency, sickle cell disease,
thalassemia syndromes, paroxysmal nocturnal hemoglobinuria,
immunohemolytic anemia, and hemolytic anemia resulting from trauma
to red cells; and anemias of diminished erythropoiesis, including
megaloblastic anemias, such as anemias of vitamin B12 deficiency:
pernicious anemia, and anemia of folate deficiency, iron deficiency
anemia, anemia of chronic disease, aplastic anemia, pure red cell
aplasia, and other forms of marrow failure.
[1858] Disorders involving B-cells include, but are not limited to
precursor B-cell neoplasms, such as lymphoblastic
leukemia/lymphoma. Peripheral B-cell neoplasms include, but are not
limited to, chronic lymphocytic leukemia/small lymphocytic
lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and
related entities, lymphoplasmacytic lymphoma (Waldenstrom
macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma
(MALToma), and hairy cell leukemia.
[1859] Disorders related to reduced platelet number,
thrombocytopenia, include idiopathic thrombocytopenic purpura,
including acute idiopathic thrombocytopenic purpura, drug-induced
thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic
microangiopathies: thrombotic thrombocytopenic purpura and
hemolytic-uremic syndrome.
[1860] Disorders involving precursor T-cell neoplasms include
precursor T lymphoblastic leukemia/lymphoma. Disorders involving
peripheral T-cell and natural killer cell neoplasms include T-cell
chronic lymphocytic leukemia, large granular lymphocytic leukemia,
mycosis fungoides and Sezary syndrome, peripheral T-cell lymphoma,
unspecified, angioimmunoblastic T-cell lymphoma, angiocentric
lymphoma (NK/T-cell lymphoma.sup.4a), intestinal T-cell lymphoma,
adult T-cell leukemia/lymphoma, and anaplastic large cell
lymphoma.
[1861] Bone-forming cells include the osteoprogenitor cells,
osteoblasts, and osteocytes. The disorders of the bone are complex
because they may have an impact on the skeleton during any of its
stages of development. Hence, the disorders may have variable
manifestations and may involve one, multiple or all bones of the
body. Such disorders include, congenital malformations,
achondroplasia and thanatophoric dwarfism, diseases associated with
abnormal matrix such as type 1 collagen disease, osteoporosis,
Paget disease, rickets, osteomalacia, high-turnover osteodystrophy,
low-turnover of aplastic disease, osteonecrosis, pyogenic
osteomyelitis, tuberculous osteomyclitism, osteoma, osteoid
osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,
chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous
cortical defects, fibrous dysplasia, fibrosarcoma, malignant
fibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal
tumor, giant cell tumor, and metastatic tumors.
[1862] Disorders involving the tonsils include, but are not limited
to, tonsillitis, Peritonsillar abscess, squamous cell carcinoma,
dyspnea, hyperplasia, follicular hyperplasia, reactive lymphoid
hyperplasia, non-Hodgkin's lymphoma and B-cell lymphoma.
[1863] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
[1864] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[1865] Disorders involving the lung include, but are not limited
to, congenital anomalies; atelectasis; diseases of vascular origin,
such as pulmonary congestion and edema, including hemodynamic
pulmonary edema and edema caused by microvascular injury, adult
respiratory distress syndrome (diffuse alveolar damage), pulmonary
embolism, hemorrhage, and infarction, and pulmonary hypertension
and vascular sclerosis; chronic obstructive pulmonary disease, such
as emphysema, chronic bronchitis, bronchial asthma, and
bronchiectasis; diffuse interstitial (infiltrative, restrictive)
diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity
pneumonitis, pulmonary eosinophilia (pulmonary infiltration with
eosinophilia), Bronchiolitis obliterans-organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, including Goodpasture
syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic
syndromes, pulmonary involvement in collagen vascular disorders,
and pulmonary alveolar proteinosis; complications of therapies,
such as drug-induced lung disease, radiation-induced lung disease,
and lung transplantation; tumors, such as bronchogenic carcinoma,
including paraneoplastic syndromes, bronchioloalveolar carcinoma,
neuroendocrine tumors, such as bronchial carcinoid, miscellaneous
tumors, and metastatic tumors; pathologies of the pleura, including
inflammatory pleural effusions, noninflammatory pleural effusions,
pneumothorax, and pleural tumors, including solitary fibrous tumors
(pleural fibroma) and malignant mesothelioma.
[1866] The epitope-bearing polypeptides may be produced by any
conventional means (Houghten, R. A., Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985)). Simultaneous multiple peptide synthesis is
described in U.S. Pat. No. 4,631,211.
[1867] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the polypeptide fragment and an
additional region fused to the carboxyl terminus of the
fragment.
[1868] The invention thus provides chimeric or fusion proteins.
These comprise a protein of the invention operatively linked to a
heterologous protein having an amino acid sequence not
substantially homologous to the protein of the invention. In the
case where an expression cassette contains two protein coding
regions joined in a contiguous manner in the same reading frame,
the encoded polypeptide is herein defined as a "heterologous
polypeptide" or a "chimeric polypeptide" or a "fusion polypeptide".
As used herein, a GAP "heterologous protein" or "chimeric protein"
or "fusion protein" comprises a GAP polypeptide operably linked to
a non-GAP polypeptide. The heterologous protein can be fused to the
N-terminus or C-terminus of the protein of the invention.
"Operatively linked" indicates that the protein of the invention
and the heterologous protein are fused in-frame.
[1869] In one embodiment the fusion protein does not affect GAP
function per se. For example, the fusion protein can be a
GST-fusion protein in which the sequences of the invention are
fused to the N- or C-terminus of the GST sequences. Other types of
fusion proteins include, but are not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL-4 fusions, poly-His fusions and Ig fusions. Such fusion
proteins, particularly poly-His fusions, can facilitate the
purification of a recombinant protein of the invention. In certain
host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased by using a heterologous
signal sequence. Therefore, in another embodiment, the fusion
protein contains a heterologous signal sequence at its C- or
N-terminus.
[1870] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify
antagonists. Bennett et al. (J. Mol. Recog. 8:52-58 (1995)) and
Johanson et al. (J. Biol. Chem. 270, 16:9459-9471 (1995)). Thus,
this invention also encompasses soluble fusion proteins containing
a polypeptide of the invention and various portions of the constant
regions of heavy or light chains of immunoglobulins of various
subclass (IgG, IgM, IgA, IgE). Preferred as immunoglobulin is the
constant part of the heavy chain of human IgG, particularly IgG1,
where fusion takes place at the hinge region. For some uses it is
desirable to remove the Fc after the fusion protein has been used
for its intended purpose, for example when the fusion protein is to
be used as antigen for immunizations. In a particular embodiment,
the Fc part can be removed in a simple way by a cleavage sequence
which is also incorporated and can be cleaved with factor Xa.
[1871] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. 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 re-amplified to
generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A GAP-encoding nucleic acid of the
invention can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the GAP.
[1872] Another form of fusion protein is one that directly affects
the GAP functions. Accordingly, a polypeptide is encompassed by the
present invention in which one or more of the domains (or parts
thereof) has been replaced by homologous domains (or parts thereof)
from another GAP. Various permutations are possible. Thus, chimeric
proteins can be formed in which one or more of the native domains,
subregions, or motifs has been replaced. A form of fusion protein
is that in which GAP activator or regulatory domains are derived
from a different GAP family, including but not limited to those
described in the background herein above, such as RabGAP.
[1873] The isolated protein of the invention can be purified from
cells that naturally express it, including but not limited to,
those described herein above, purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods.
[1874] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding a
polypeptide of the invention is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Polypeptides often contain amino
acids other than the 20 amino acids commonly referred to as the 20
naturally-occurring amino acids. Further, many amino acids,
including the terminal amino acids, may be modified by natural
processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Common modifications that occur naturally in polypeptides
are described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the
art.
[1875] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[1876] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cysteine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[1877] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T.E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth.
Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad.
Sci. 663:48-62 (1992)).
[1878] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing event and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[1879] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
amino terminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[1880] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[1881] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
Polypeptide Uses
[1882] The polypeptides of the invention are useful for producing
antibodies specific for the protein, regions, or fragments. Regions
having a high antigenicity index score are shown in FIGS. 54 and
58.
[1883] The polypeptides (including variants and fragments which may
have been disclosed prior to the present invention) are useful for
biological assays related to GAPs. Such assays involve any of the
known GAP functions or activities such as those described herein,
such functions or activities or properties being useful for
diagnosis and treatment of GAP-related conditions. Treatment is
defined as the application or administration of a therapeutic agent
to a patient, or application or administration of a therapeutic
agent to an isolated tissue or cell line from a patient, who has a
disease, a symptom of disease or a predisposition toward a disease,
with the purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve or affect the disease, the symptoms of disease
or the predisposition toward disease. "Subject", as used herein,
can refer to a mammal, e.g. a human, or to an experimental or
animal or disease model. The subject can also be a non-human
animal, e.g. a horse, cow, goat, or other domestic animal. A
therapeutic agent includes, but is not limited to, small molecules,
peptides, antibodies, ribozymes and antisense oligonucleotides.
[1884] The polypeptides of the invention are also useful in drug
screening assays, in cell-based or cell-free systems. Cell-based
systems can be native, i.e., cells that normally express the
protein, as a biopsy or expanded in cell culture. For the various
biological assays described herein, these cells included but are
not limited to, those disclosed above. In one embodiment, however,
cell-based assays involve recombinant host cells expressing the
protein.
[1885] Determining the ability of the test compound to interact
with the polypeptide can also comprise determining the ability of
the test compound to preferentially bind to the polypeptide as
compared to the ability of the substrate (i.e., GTPase) or effector
(i.e., regulatory molecule), or a biologically active portion
thereof, to bind to the polypeptide.
[1886] The polypeptides can be used to identify compounds that
modulate peptide, e.g., GAP activity. Such compounds, for example,
can increase or decrease affinity or rate of binding to a known
substrate or effector, compete with substrate or effector for
binding, or displace bound substrate or effector. Both a protein of
the invention and appropriate variants and fragments can be used in
high-throughput screens to assay candidate compounds for the
ability to bind to a protein of the invention (i.e., 26651 GAP or
26138 GAP). These compounds can be further screened against a
functional polypeptide of the invention to determine the effect of
the compound on the protein activity. Compounds can be identified
that activate (agonist) or inactivate (antagonist) the protein to a
desired degree. 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).
[1887] The polypeptides can be used to screen a compound for the
ability to stimulate or inhibit interaction between the protein and
a target molecule that normally interacts with the GAP. The target
can be a GTPase, regulatory protein, or other regulatory molecule
or a component of the signal pathway with which the GAP normally
interacts. The assay includes the steps of combining the protein of
the invention with a candidate compound under conditions that allow
the protein or fragment to interact with the target molecule, and
to detect the formation of a complex between the protein and the
target or to detect the biochemical consequence of the interaction
with the protein and the target. When a protein of the invention is
involved in a specific signal pathway, the biological consequence
can include any of the associated effects of signal transduction
such as G-protein phosphorylation, cyclic AMP or
phosphatidylinositol turnover, and adenylate cyclase or
phospholipase C activation, or any of the associated effects of
GTPase activity including, but not limited to, programmed cell
death (apoptosis), membrane trafficking, organization of the actin
cytoskeleton, activation of protein kinases activated by direct
interaction with GTPases, and in particular, with Rho and Ras,
membrane ruffling, formation of actin stress fibers, or generalized
cellular effects such as transformation, and effects on growth and
differentiation.
[1888] Determining the ability of the protein to bind to a target
molecule can also be accomplished using a technology such as
real-time Bimolecular 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.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR)
can be used as an indication of real-time reactions between
biological molecules.
[1889] 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 polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[1890] 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. USA 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; Carell 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. 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. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[1891] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[1892] One candidate compound is a soluble full-length protein of
the invention or fragment that competes for substrate or effector
binding. Other candidate compounds include mutant proteins of the
invention or appropriate fragments containing mutations that affect
protein function and thus compete for substrate or effector.
Accordingly, a fragment that competes for substrate or effector,
for example with a higher affinity, or a fragment that binds but
does not allow release, is encompassed by the invention. A
candidate compound includes, but is not limited to, a GTPase analog
that competes for native GTPase binding.
[1893] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) protein activity.
When the function of a protein of the invention is related to a
signal transduction pathway, the assays typically involve an assay
of events in the signal transduction pathway that indicate GAP
activity. Thus, the expression of genes that are up- or
down-regulated in response to the receptor protein dependent signal
cascade can be assayed. For GAP function, assays typically involve
an assay of events in the pathway for example, GTPase activation or
inhibition, GTP or GDP binding to a GTPase, and end points such as
membrane ruffling and effects on cytoskeletal organization, actin
organization, and the like. In one embodiment, the regulatory
region of such genes can be operably linked to a marker that is
easily detectable, such as luciferase. Alternatively,
phosphorylation of a protein of the invention, or a G-protein
target, could also be measured.
[1894] Any of the biological or biochemical functions mediated by a
protein of the invention can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art.
[1895] Binding and/or activating regions, or domains, such as
compounds can also be screened by using chimeric proteins of the
invention in which regions or domains, such as the GTPase binding
regions, catalytic (i.e., activation or inhibition) regions,
regions interacting with regulatory proteins of GAP, or parts
thereof, can be replaced by heterologous domains or regions.
Activation can also be detected by a reporter gene containing an
easily detectable coding region operably linked to a
transcriptional regulatory sequence that is part of a signal
transduction pathway in which a GAP of the invention is
involved.
[1896] The polypeptides of the invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the polypeptide. Thus, a compound is
exposed to the polypeptide under conditions that allow the compound
to bind or to otherwise interact with the polypeptide. Soluble
polypeptide of the invention is also added to the mixture. If the
test compound interacts with the soluble polypeptide, it decreases
the amount of complex formed or activity from the target. This type
of assay is particularly useful in cases in which compounds are
sought that interact with specific regions of the polypeptide.
Thus, the soluble polypeptide that competes with the target region
is designed to contain peptide sequences corresponding to the
region of interest.
[1897] To perform cell free drug screening assays, it is desirable
to immobilize either the protein, or fragment, or its target
molecule to facilitate separation of complexes from uncomplexed
forms of one or both of the proteins, as well as to accommodate
automation of the assay.
[1898] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/GAP
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 cell lysates
(e.g., .sup.35S-labeled) and the candidate compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly, or in
the supernatant after the complexes are dissociated. Alternatively,
the complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of GAP-binding protein found in the bead
fraction quantified from the gel using standard electrophoretic
techniques. For example, either the polypeptide or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively,
antibodies reactive with the protein but which do not interfere
with binding of the protein to its target molecule can be
derivatized to the wells of the plate, and the protein trapped in
the wells by antibody conjugation. Preparations of a GAP binding
protein and a candidate compound are incubated in the GAP
presenting wells and the amount of complex trapped in the well can
be quantified. 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 GAP
target molecule, or which are reactive with GAP and compete with
the target molecule; as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the target
molecule.
[1899] Modulators of GAP activity identified according to these
drug screening assays can be used to treat a subject with a
disorder mediated by a protein of the invention, by treating cells
that express a protein of the invention, such as those disclosed
herein.
[1900] These methods of treatment include the steps of
administering the modulators of protein activity in a
pharmaceutical composition as described herein, to a subject in
need of such treatment.
[1901] The polypeptides of the invention are thus useful for
treating a GAP-associated disorder characterized by aberrant
expression or activity of a GAP. 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) expression or
activity of the protein. In another embodiment, the method involves
administering a protein as therapy to compensate for reduced or
aberrant expression or activity of the protein.
[1902] Stimulation of protein activity is desirable in situations
in which the protein is abnormally downregulated and/or in which
increased protein activity is likely to have a beneficial effect.
Likewise, inhibition of protein activity is desirable in situations
in which the protein is abnormally upregulated and/or in which
decreased protein activity is likely to have a beneficial effect.
An example of such a situation occurs when the GAP is inactivating
a protein and inhibition of the GAP allows activation of the
protein (Chen et al. (1998) Neuron 20:895-904). In one example of
such a situation, a subject has a disorder characterized by
aberrant development or cellular differentiation. In another
example of such a situation, the subject has a proliferative
disease (e.g., cancer) or a disorder characterized by an aberrant
hematopoietic response. In another example of such a situation, it
is desirable to achieve tissue regeneration in a subject (e.g.,
where a subject has undergone brain or spinal cord injury and it is
desirable to regenerate neuronal tissue in a regulated manner).
[1903] In yet another aspect of the invention, the proteins of the
invention 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 WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[1904] The polypeptides of the invention also are useful to provide
a target for diagnosing a disease or predisposition to disease
mediated by a GAP, especially in diseases involving the tissues in
which a protein of the invention is expressed such as are disclosed
herein. Accordingly, methods are provided for detecting the
presence, or levels of, a protein of the invention in a cell,
tissue, or organism. The method involves contacting a biological
sample with a compound capable of interacting with the protein such
that the interaction can be detected.
[1905] One agent for detecting the protein is an antibody capable
of selectively binding to the protein. A biological sample includes
tissues, cells and biological fluids isolated from a subject, as
well as tissues, cells and fluids present within a subject.
[1906] The protein of the invention also provides a target for
diagnosing active disease, or predisposition to disease, in a
patient having a variant protein of the invention. Thus, the
protein can be isolated from a biological sample, assayed for the
presence of a genetic mutation that results in an aberrant protein.
This includes amino acid substitution, deletion, insertion,
rearrangement, (as the result of aberrant splicing events), and
inappropriate post-translational modification. Analytic methods
include altered electrophoretic mobility, altered tryptic peptide
digest, altered GAP activity in cell-based or cell-free assays,
altered antibody-binding pattern, altered isoelectric point, direct
amino acid sequencing, and any other of the known assay techniques
useful for detecting mutations in a protein.
[1907] In vitro techniques for detection of protein of the
invention include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations and immunofluorescence.
Alternatively, the protein can be detected in vivo in a subject by
introducing into the subject a labeled anti-GAP 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. Particularly useful are methods which
detect the allelic variant of the protein expressed in a subject
and methods which detect fragments of the protein in a sample.
[1908] The polypeptides are also useful in pharmacogenomic
analysis. Pharmacogenomics deal with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, e.g.,
Eichelbaum, M., Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985
(1996), and Linder, M. W., Clin. Chem. 43(2):254-266 (1997). The
clinical outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants in which
one or more functions in one population are different from those in
another population. The polypeptides thus allow a target to
ascertain a genetic predisposition that can affect treatment
modality. Thus, in a substrate or effector-based treatment,
polymorphism may give rise to domains and/or other binding regions
that are more or less active in binding and/or activation.
Accordingly, dosage would necessarily be modified to maximize the
therapeutic effect within a given population containing a
polymorphism. As an alternative to genotyping, specific polymorphic
polypeptides could be identified.
[1909] The polypeptides are also useful for monitoring therapeutic
effects during clinical trials and other treatment. Thus, the
therapeutic effectiveness of an agent that is designed to increase
or decrease gene expression, protein levels or activity can be
monitored over the course of treatment using the polypeptides as an
end-point target. The monitoring can be, for example, as follows:
(i) obtaining a pre-administration sample from a subject prior to
administration of the agent; (ii) detecting the level of expression
or activity of a specified protein in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the protein in the post-administration samples; (v)
comparing the level of expression or activity of the protein in the
pre-administration sample with the protein in the
post-administration sample or samples; and (vi) increasing or
decreasing the administration of the agent to the subject
accordingly.
[1910] The polypeptides are also useful for treating a
GAP-associated disorder. Accordingly, methods for treatment include
the use of soluble protein or fragments of the protein that compete
for GTPase binding. These proteins or fragments can have a higher
affinity for the GTPase so as to provide effective competition.
Antibodies
[1911] The invention also provides antibodies that selectively bind
to a protein of the invention and its variants and fragments. An
antibody is considered to selectively bind, even if it also binds
to other proteins that are not substantially homologous with the
protein. These other proteins share homology with a fragment or
domain of the protein. This conservation in specific regions gives
rise to antibodies that bind to both proteins by virtue of the
homologous sequence. In this case, it would be understood that
antibody binding to the protein is still selective.
[1912] To generate antibodies, an isolated polypeptide is used as
an immunogen to generate antibodies using standard techniques for
polyclonal and monoclonal antibody preparation. Either the
full-length protein or antigenic peptide fragment can be used.
Regions having a high antigenicity index are shown in FIGS. 54 and
58.
[1913] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents GTPase binding. Antibodies can be developed
against the entire protein or portions of the protein. Antibodies
may also be developed against specific functional sites, such as
the site of GTPase binding, or sites that are phosphorylated,
myristoylated, or glycosylated.
[1914] An antigenic fragment will typically comprise at least 6
contiguous amino acid residues. The antigenic peptide can comprise
a contiguous sequence of at least 12, at least 14 amino acid
residues, at least 15 amino acid residues, at least 20 amino acid
residues, or at least 30 amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments which may be
disclosed prior to the invention.
[1915] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be
used.
[1916] 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.
[1917] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, protein or chemically synthesized
peptides.
Antibody Uses
[1918] The antibodies can be used to isolate a protein by standard
techniques, such as affinity chromatography or immunoprecipitation.
The antibodies can facilitate the purification of the natural
protein from cells and recombinantly produced protein expressed in
host cells.
[1919] The antibodies are useful to detect the presence of the
protein in cells or tissues to determine the pattern of expression
among various tissues in an organism and over the course of normal
development.
[1920] The antibodies can be used to detect the protein in situ, in
vitro, or in a cell lysate or supernatant in order to evaluate the
abundance and pattern of expression.
[1921] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[1922] Antibody detection of circulating fragments of a full-length
protein of the invention can be used to identify protein
turnover.
[1923] Further, the antibodies can be used to assess the GAP
expression in disease states such as in active stages of the
disease or in an individual with a predisposition toward disease
related to the GAP function. When a disorder is caused by an
inappropriate tissue distribution, developmental expression, or
level of expression of the protein, the antibody can be prepared
against the normal protein. If a disorder is characterized by a
specific mutation in the protein, antibodies specific for this
mutant protein can be used to assay for the presence of the
specific mutant protein. However, intracellularly-made antibodies
("intrabodies") are also encompassed, which would recognize
intracellular peptide regions.
[1924] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole
protein or portions, such as those discussed herein.
[1925] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting the expression
level or the presence of an aberrant protein of the invention and
aberrant tissue distribution or developmental expression,
antibodies directed against the protein or relevant fragments can
be used to monitor therapeutic efficacy. Antibodies accordingly 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.
[1926] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins of
the invention can be used to identify individuals that require
modified treatment modalities.
[1927] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[1928] The antibodies are also useful for tissue typing. Thus,
where a specific GAP of the invention has been correlated with
expression in a specific tissue, antibodies that are specific for
this protein can be used to identify a tissue type.
[1929] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[1930] The antibodies are also useful for inhibiting protein
function, for example, blocking GTPase or regulatory molecule,
e.g., protein, binding.
[1931] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting a function. An antibody can be
used, for example, to block GTPase binding. Antibodies can be
prepared against specific fragments containing sites required for
function or against an intact protein of the invention associated
with a cell.
[1932] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[1933] The invention also encompasses kits for using antibodies to
detect the presence of a protein of the invention in a biological
sample. The kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting the
protein in a biological sample; means for determining the amount of
the protein in the sample; and means for comparing the amount of
the protein 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 the protein.
Polynucleotides
[1934] The nucleotide sequences in SEQ ID NO:21, 23, 24, and 26
were obtained by sequencing the deposited human full length cDNAs.
Accordingly, the sequence of the deposited clone is controlling as
to any discrepancies between the two and any reference to a
sequence of SEQ ID NO:21, 23, 24, or 26 includes reference to a
sequence of the deposited cDNA.
[1935] The specifically disclosed cDNAs comprise the coding region
and 5' and 3' untranslated sequences (SEQ ID NO:21 or SEQ ID
NO:24).
[1936] The invention provides isolated polynucleotides encoding a
protein of the invention. The term "GAP polynucleotide," "GAP
nucleic acid," "polynucleotide of the invention" or "nucleic acid
of the invention" refers to a sequence shown in SEQ ID NO:21, 23,
24, 26, or in the deposited cDNA. The terms further include
variants and fragments of a polynucleotide of the invention.
[1937] An "isolated" nucleic acid is one that is separated from
other nucleic acid present in the natural source of the nucleic
acid. 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. However, there can
be some flanking nucleotide sequences, for example up to about 5
KB. The important point is that the nucleic acid is isolated from
flanking sequences such that it can be subjected to the specific
manipulations described herein such as recombinant expression,
preparation of probes and primers, and other uses specific to GAP
nucleic acid sequences.
[1938] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[1939] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[1940] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[1941] The GAP polynucleotides can encode the mature protein plus
additional amino or carboxyl-terminal amino acids, or amino acids
interior to the mature polypeptide (when the mature form has more
than one polypeptide chain, for instance). Such sequences may play
a role in processing of a protein from precursor to a mature form,
facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[1942] The GAP polynucleotides include, but are not limited to, the
sequence encoding the mature polypeptide alone, the sequence
encoding the mature polypeptide and additional coding sequences,
such as a leader or secretory sequence (e.g., a pre-pro or
pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[1943] Polynucleotides can be in the form of RNA, such as mRNA, or
in the form of DNA, including cDNA and genomic DNA obtained by
cloning or produced by chemical synthetic techniques or by a
combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[1944] One nucleic acid comprises a nucleotide sequence shown in
SEQ ID NO:21, 23, 24, or 26, corresponding to human cDNA.
[1945] In one embodiment, the nucleic acid comprises the coding
regions set forth in SEQ ID NO:23 or 26.
[1946] The invention further provides variant polynucleotides, and
fragments thereof, that differ from a nucleotide sequence shown in
SEQ ID NO:21, 23, 24, or 26 due to degeneracy of the genetic code
and thus encode the same polypeptides as those set forth in SEQ ID
NO:22 or 25.
[1947] The invention also provides nucleic acid molecules encoding
the variant polypeptides described herein. Generally, nucleotide
sequence variants of the invention will have at least 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity to the nucleotide sequences disclosed herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[1948] Variation can occur in either or both the coding and
non-coding regions. The variations can produce both conservative
and non-conservative amino acid substitutions.
[1949] Typically, variants have a substantial identity with a
nucleic acid molecule of SEQ ID NO:21, 23, 24, or 26, or the
complements thereof.
[1950] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a protein that is at least about 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more homologous to a nucleotide sequence shown in SEQ
ID NO:21, 23, 24, 26, or a fragment of this sequence. Such nucleic
acid molecules can readily be identified as being able to hybridize
under stringent conditions, to a nucleotide sequence shown in SEQ
ID NO:21, 23, 24, 26, or a fragment of the sequence. It is
understood that stringent hybridization does not indicate
substantial homology where it is due to general homology, such as
poly A sequences, or sequences common to all or most proteins, all
or most GAPs, or all or most RasGAPs or RhoGAPs. Moreover, it is
understood that variants do not include any of the nucleic acid
sequences that may have been disclosed prior to the invention.
[1951] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing.
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. Aqueous and nonaqueous methods are
described in that reference and either can be used. A preferred,
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. Another 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 55.degree. C. A further 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
60.degree. C. Preferably, 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 65.degree. C. Particularly preferred
stringency conditions (and the conditions that should be used if
the practitioner is uncertain about what conditions should be
applied to determine if a molecule is within a hybridization
limitation of the invention) are 0.5M Sodium Phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C. Preferably, an isolated nucleic acid molecule
of the invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:21, 23, 24, or 26, corresponds to a
naturally-occurring nucleic acid molecule.
[1952] 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).
[1953] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[1954] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to a nucleotide sequence of
SEQ ID NO:21, 23, 24, 26, or the complements thereof. In one
embodiment, the nucleic acid consists of a portion of a nucleotide
sequence of SEQ ID NO:21, 23, 24, 26 or complements thereof. The
nucleic acid fragments of the invention are at least about 10, 15,
preferably at least about 20 or 25 nucleotides, and can be 30, 38,
40, 50, 68, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,
2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, or 2847
nucleotides for SEQ ID NO:21. Alternatively, a nucleic acid
molecule that is a fragment of a 26651-like nucleotide sequence of
the present invention comprises a nucleotide sequence consisting of
nucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600,
600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200,
1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,
1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400,
2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2847 of SEQ ID
NO:21. The nucleic acid fragments of the invention are at least
about 10, 15, 20, 25, 30, 38, 40, 50, 68, 75, 100, 150, 200, 250,
300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,
1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,
2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, or 3391 nucleotides
for SEQ ID NO:24. Alternatively, a nucleic acid molecule that is a
fragment of a 26138-like nucleotide sequence of the present
invention comprises a nucleotide sequence consisting of nucleotides
1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,
700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300,
1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900,
1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500,
2500-2600, 2600-2700, 2700-2800, 2800-2900, 2900-3000, 3000-3100,
3100-3200, 3200-3300, 3300-3391 of SEQ ID NO:24. Longer fragments,
for example, 30 or more nucleotides in length, which encode
antigenic proteins or polypeptides described herein are useful.
[1955] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length GAP polynucleotides. The
fragment can be single or double stranded and can comprise DNA or
RNA. The fragment can be derived from either the coding or the
non-coding sequence.
[1956] In another embodiment an isolated nucleic acid encodes the
entire coding region. Other fragments include nucleotide sequences
encoding the amino acid fragments described herein. Further
fragments can include subfragments of the specific domains or sites
described herein. Fragments also include nucleic acid sequences
corresponding to specific amino acid sequences described above or
fragments thereof. Nucleic acid fragments, according to the present
invention, are not to be construed as encompassing those fragments
that may have been disclosed prior to the invention.
[1957] Nucleic acid fragments further include sequences
corresponding to the domains described herein, subregions also
described, and specific functional sites. Nucleic acid fragments
also include combinations of the domains, segments, loops, and
other functional sites described above. A person of ordinary skill
in the art would be aware of the many permutations that are
possible.
[1958] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary skill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[1959] However, it is understood that a fragment includes any
nucleic acid sequence that does not include the entire gene.
[1960] The invention also provides nucleic acid fragments that
encode epitope bearing regions of the proteins described
herein.
[1961] The isolated polynucleotide sequences, and especially
fragments, are useful as DNA probes and primers.
[1962] For example, the coding region of a gene of the invention
can be isolated using the known nucleotide sequence to synthesize
an oligonucleotide probe. A labeled probe can then be used to
screen a cDNA library, genomic DNA library, or mRNA to isolate
nucleic acid corresponding to the coding region. Further, primers
can be used in PCR reactions to clone specific regions of these
genes.
[1963] A 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 5, 10, 12, typically about 25, more typically
about 40, 50 or 75 consecutive nucleotides of the sense or
antisense strand of SEQ ID NO:21, 23, 24, 26, or other GAP
polynucleotides. A probe further comprises a label, e.g.,
radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
Polynucleotide Uses
[1964] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of a nucleic
acid of SEQ ID NO:21, 23, 24, 26, or the complements thereof. More
typically, the probe further comprises a label, e.g., radioisotope,
fluorescent compound, enzyme, or enzyme co-factor.
[1965] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[1966] The polynucleotides are useful for probes, primers, and in
biological assays, including, but not limited to, methods using the
cells and tissues in which the gene is expressed, particularly in
which the gene is significantly expressed, and involving disorders
including, but not limited to, those also discussed herein above
with respect to biological methods and assays involving the GAP
polypeptides of the invention.
[1967] Where the polynucleotides are used to assess or GAP
properties or functions, such as in the assays described herein,
all or less than all of the entire cDNA can be useful. In this
case, even fragments that may have been known prior to the
invention are encompassed. Thus, for example, assays specifically
directed to GAPs, and especially RasGAP or RhoGAP functions, such
as assessing agonist or antagonist activity, encompass the use of
known fragments. Further, diagnostic methods for assessing function
can also be practiced with any fragment, including those fragments
that may have been known prior to the invention. Similarly, in
methods involving modulation or treatment of GAP-related
dysfunction, all fragments are encompassed including those which
may have been known in the art.
[1968] The polynucleotides are useful as a hybridization probe for
cDNA and genomic DNA to isolate a full-length cDNA and genomic
clones encoding a polypeptide described in SEQ ID NO:22 or SEQ ID
NO:25 and to isolate cDNA and genomic clones that correspond to
variants producing one of the same polypeptides shown in SEQ ID
NO:22, SEQ ID NO:25, or the other variants described herein.
Variants can be isolated from the same tissue and organism from
which a polypeptide shown in SEQ ID NO:22 or SEQ ID NO:25 was
isolated, different tissues from the same organism, or from
different organisms. This method is useful for isolating genes and
cDNA that are developmentally-controlled and therefore may be
expressed in the same tissue or different tissues at different
points in the development of an organism.
[1969] The probe can correspond to any sequence along the entire
length of the gene encoding a protein of the invention.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. It is understood, however,
as discussed herein, that fragments corresponding to the probe do
not include those fragments that may have been disclosed prior to
the present invention.
[1970] The nucleic acid probe can be, for example, a full-length
cDNA that encodes a polypeptide set forth in SEQ ID NO:22 or a
fragment thereof, such as an oligonucleotide of at least 5, 10, 12,
15, 30, 38, 50, 68, 100, 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,
1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300,
2400, 2500, 2600, 2700, 2800, 2847 nucleotides in length for SEQ ID
NO:21 and sufficient to specifically hybridize under stringent
conditions to mRNA or DNA. The nucleic acid probe can be, for
example, a full-length cDNA that encodes a polypeptide set forth in
SEQ ID NO:25 or a fragment thereof, such as an oligonucleotide of
at least 5, 10, 12, 15, 30, 38, 50, 68, 100, 150, 200, 250, 300,
350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,
2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100,
3200, 3300, or 3391 nucleotides in length for SEQ ID NO:24 and
sufficient to specifically hybridize under stringent conditions to
mRNA or DNA.
[1971] Fragments of the polynucleotides' described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an mRNA and a larger or full-length
cDNA can be produced.
[1972] The fragments are also useful to synthesize antisense
molecules of desired length and sequence.
[1973] Antisense nucleic acids of the invention can be designed
using a nucleotide sequence of SEQ ID NO:21, 23, 24 or 26, and
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, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest.
[1974] Additionally, the nucleic acid molecules of the 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 acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). 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 et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity 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. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[1975] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell 26651 or 26138 proteins 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. WO 88/0918) or the blood brain barrier (see, e.g.,
PCT Publication No. WO 89/10134). 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).
[1976] The polynucleotides are also useful as primers for PCR to
amplify any given region of a polynucleotide of the invention.
[1977] The polynucleotides are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the GAP polypeptides of the
invention. Vectors also include insertion vectors, used to
integrate into another polynucleotide sequence, such as into the
cellular genome, to alter in situ expression of genes and gene
products of the invention. For example, an endogenous coding
sequence can be replaced via homologous recombination with all or
part of the coding region containing one or more specifically
introduced mutations.
[1978] The polynucleotides are also useful for expressing antigenic
portions of the proteins of the invention.
[1979] The polynucleotides are also useful as probes for
determining the chromosomal positions of the polynucleotides of the
invention by means of in situ hybridization methods, such as FISH
(For a review of this technique, see Verma et al. (1988) Human
Chromosomes: A Manual of Basic Techniques (Pergamon Press, New
York)), and PCR mapping of somatic cell hybrids. The mapping of the
sequences to chromosomes is an important first step in correlating
these sequences with genes associated with disease.
[1980] 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.
[1981] 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 Mendelian Inheritance in Man, V. McKusick, 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 et al. (1987) Nature 325:783-787. The chromosomal
location of 26138 on human chromosome 19 is indicated in FIG.
63.
[1982] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified 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 form 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.
[1983] The polynucleotide probes are also useful to determine
patterns of the presence of the gene encoding the proteins and
their variants with respect to tissue distribution, for example,
whether gene duplication has occurred and whether the duplication
occurs in all or only a subset of tissues. The genes can be
naturally occurring or can have been introduced into a cell,
tissue, or organism exogenously.
[1984] The polynucleotides are also useful for designing ribozymes
corresponding to all, or a part, of the mRNA produced from genes
encoding the polynucleotides described herein.
[1985] The polynucleotides are also useful for constructing host
cells expressing a part, or all, of the polynucleotides and
polypeptides.
[1986] The polynucleotides are also useful for constructing
transgenic animals expressing all, or a part, of the
polynucleotides and polypeptides.
[1987] The polynucleotides are also useful for making vectors that
express part, or all, of the polypeptides.
[1988] The polynucleotides are also useful as hybridization probes
for determining the level of nucleic acid expression of a sequence
of the invention. Accordingly, the probes can be used to detect the
presence of, or to determine levels of, a nucleic acid molecule of
the invention in cells, tissues, and in organisms. The nucleic acid
whose level is determined can be DNA or RNA. Accordingly, probes
corresponding to the polypeptides described herein can be used to
assess gene copy number in a given cell, tissue, or organism. This
is particularly relevant in cases in which there has been an
amplification of a gene of the invention.
[1989] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of a
gene of the invention, as on extrachromosomal elements or as
integrated into chromosomes in which the gene is not normally
found, for example as a homogeneously staining region.
[1990] These uses are relevant for diagnosis of disorders involving
an increase or decrease in expression relative to normal, such as a
proliferative disorder, a differentiative or developmental
disorder, a hematopoietic disorder or a viral disorder, especially
as disclosed herein.
[1991] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of a nucleic acid of the invention, in which
a test sample is obtained from a subject and nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of the nucleic
acid is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the nucleic acid. "Misexpression or aberrant expression", as
used herein, refers to a non-wild type pattern of gene expression,
at the RNA or protein level. It includes: expression at non-wild
type levels, i.e., over or under expression; a pattern of
expression that differs from wild type in terms of the time or
stage at which the gene is expressed, e.g., increased or decreased
expression (as compared with wild type) at a predetermined
developmental period or stage; a pattern of expression that differs
from wild type in terms of decreased expression (as compared with
wild type) in a predetermined cell type or tissue type; a pattern
of expression that differs from wild type in terms of the splicing
size, amino acid sequence, post-transitional modification, or
biological activity of the expressed polypeptide; a pattern of
expression that differs from wild type in terms of the effect of an
environmental stimulus or extracellular stimulus on expression of
the gene, e.g., a pattern of increased or decreased expression (as
compared with wild type) in the presence of an increase or decrease
in the strength of the stimulus.
[1992] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[1993] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[1994] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a protein of the
invention, such as by measuring the level of a nucleic acid
encoding the protein in a sample of cells from a subject e.g., mRNA
or genomic DNA, or determining if the gene has been mutated.
[1995] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate expression of a nucleic acid of
the invention (e.g., antisense, polypeptides, peptidomimetics,
small molecules or other drugs). A cell is contacted with a
candidate compound and the expression of mRNA determined. The level
of expression of an mRNA of the invention in the presence of the
candidate compound is compared to the level of expression of the
mRNA in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of nucleic acid
expression based on this comparison and be used, for example to
treat a disorder characterized by aberrant nucleic acid expression.
The modulator can bind to the nucleic acid or indirectly modulate
expression, such as by interacting with other cellular components
that affect nucleic acid expression.
[1996] 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) in patients or in
transgenic animals.
[1997] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of a gene of the invention. The method
typically includes assaying the ability of the compound to modulate
the expression of a nucleic acid of the invention and thus
identifying a compound that can be used to treat a disorder
characterized by undesired expression of a nucleic acid of the
invention.
[1998] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing a
nucleic acid of the invention, such as discussed herein above, or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[1999] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[2000] The assay for expression of a nucleic acid of the invention
can involve direct assay of nucleic acid levels, such as mRNA
levels, or on collateral compounds involved in GAP function.
Further, the expression of genes that are up- or down-regulated in
response to GAP activity, as in a signal pathway (such as cyclic
AMP or phosphatidylinositol turnover) can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[2001] Thus, modulators of gene expression can be identified in a
method wherein a cell is contacted with a candidate compound and
the expression of mRNA determined. The level of expression of mRNA
in the presence of the candidate compound is compared to the level
of expression of mRNA in the absence of the candidate compound. The
candidate compound can then be identified as a modulator of nucleic
acid expression based on this comparison and be used, for example
to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[2002] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate nucleic acid
expression. Modulation includes both up-regulation (i.e. activation
or agonization) or down-regulation (suppression or antagonization)
or effects on nucleic acid activity (e.g. when nucleic acid is
mutated or improperly modified) Treatment is of disorders
characterized by aberrant expression or activity of the nucleic
acid.
[2003] Alternatively, a modulator for nucleic acid expression can
be a small molecule or drug identified using the screening assays
described herein as long as the drug or small molecule inhibits the
nucleic acid expression.
[2004] The polynucleotides are also useful for monitoring the
effectiveness of modulating compounds on the expression or activity
of the gene in clinical trials or in a treatment regimen. Thus, the
gene expression pattern can serve as a barometer for the continuing
effectiveness of treatment with the compound, particularly with
compounds to which a patient can develop resistance. The gene
expression pattern can also serve as a marker indicative of a
physiological response of the affected cells to the compound.
Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[2005] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[2006] The polynucleotides are also useful in diagnostic assays for
qualitative changes in a nucleic acid of the invention, and
particularly in qualitative changes that lead to pathology. The
polynucleotides can be used to detect mutations in genes of the
invention and gene expression products such as mRNA. The
polynucleotides can be used as hybridization probes to detect
naturally-occurring genetic mutations in a gene of the invention
and thereby to determine whether a subject with the mutation is at
risk for a disorder caused by the mutation. Mutations include
deletion, addition, or substitution of one or more nucleotides in
the gene, chromosomal rearrangement, such as inversion or
transposition, modification of genomic DNA, such as aberrant
methylation patterns or changes in gene copy number, such as
amplification. Detection of a mutated form of the gene associated
with a dysfunction provides a diagnostic tool for an active disease
or susceptibility to disease when the disease results from
overexpression, underexpression, or altered expression of a protein
of the invention.
[2007] Mutations in the gene can be detected at the nucleic acid
level by a variety of techniques. Genomic DNA can be analyzed
directly or can be amplified by using PCR prior to analysis. RNA or
cDNA can be used in the same way.
[2008] In certain embodiments, detection of the mutation 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., Science 241:1077-1080 (1988);
and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which
can be particularly useful for detecting point mutations in the
gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).
This method can include the steps of collecting a sample of cells
from a patient, 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
gene under conditions such that hybridization and amplification of
the 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.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[2009] 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.
[2010] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[2011] Alternatively, mutations in a gene of the invention can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[2012] Further, sequence-specific ribozymes (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.
[2013] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[2014] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[2015] Furthermore, sequence differences between a mutant gene of
the invention and the wild-type gene can be determined by direct
DNA sequencing. 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., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl.
Biochem. Biotechnol. 38:147-159 (1993)).
[2016] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al, Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). 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 one 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).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[2017] In other embodiments, genetic mutations 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
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin
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.
[2018] The polynucleotides are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
polynucleotides can be used to study the relationship between an
individual's genotype and the individual's response to a compound
used for treatment (pharmacogenomic relationship). In the present
case, for example, a mutation in the gene that results in altered
affinity for a GTPase or an effector molecule (or analog) could
result in an excessive or decreased drug effect with standard
concentrations of GTPase, or effector (or analog). Accordingly, the
polynucleotides described herein can be used to assess the mutation
content of the gene in an individual in order to select an
appropriate compound or dosage regimen for treatment.
[2019] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[2020] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control-sample with the presence of mRNA or genomic DNA
in the test sample.
[2021] The polynucleotides are also useful for chromosome
identification when the sequence is identified with an individual
chromosome and to a particular location on the chromosome. First,
the DNA sequence is matched to the chromosome by in situ or other
chromosome-specific hybridization. Sequences can also be correlated
to specific chromosomes by preparing PCR primers that can be used
for PCR screening of somatic cell hybrids containing individual
chromosomes from the desired species. Only hybrids containing the
chromosome containing the gene homologous to the primer will yield
an amplified fragment. Sublocalization can be achieved using
chromosomal fragments. Other strategies include prescreening with
labeled flow-sorted chromosomes and preselection by hybridization
to chromosome-specific libraries. Further mapping strategies
include fluorescence in situ hybridization which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the 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.
[2022] The polynucleotides can also be used to identify individuals
from small biological samples. This can be done for example using
restriction fragment-length polymorphism (RFLP) to identify an
individual. Thus, the polynucleotides described herein are useful
as DNA markers for RFLP (See U.S. Pat. No. 5,272,057).
[2023] Furthermore, the sequence can be used to provide an
alternative technique which determines the actual DNA sequence of
selected fragments in the genome of an individual. Thus, the
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 DNA from an individual for subsequent
sequencing.
[2024] 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. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
sequences can be used to obtain such identification sequences from
individuals and from tissue. The sequences represent unique
fragments of the human genome. 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.
[2025] If a panel of reagents from the sequences 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.
[2026] The polynucleotides can also be used in forensic
identification procedures. PCR technology can be used to amplify
DNA sequences taken from very small biological samples, such as a
single hair follicle, body fluids (e.g. blood, saliva, or semen).
The amplified sequence can then be compared to a standard allowing
identification of the origin of the sample.
[2027] The polynucleotides can thus 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 described 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 the noncoding region are
particularly useful since greater polymorphism occurs in the
noncoding regions, making it easier to differentiate individuals
using this technique.
[2028] The polynucleotides 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. This is useful in cases in which a
forensic pathologist is presented with a tissue of unknown origin.
Panels of probes can be used to identify tissue by species and/or
by organ type.
[2029] In a similar fashion, these primers and 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).
[2030] Alternatively, the polynucleotides can be used directly to
block transcription or translation of nucleic acid sequences of the
invention by means of antisense or ribozyme constructs. Thus, in a
disorder characterized by abnormally high or undesirable expression
of a gene of the invention, nucleic acids can be directly used for
treatment.
[2031] The polynucleotides are thus useful as antisense constructs
to control expression of a gene of the invention in cells, tissues,
and organisms. A DNA antisense polynucleotide is designed to be
complementary to a region of the gene involved in transcription,
preventing transcription and hence production of protein. An
antisense RNA or DNA polynucleotide would hybridize to the mRNA and
thus block translation of mRNA into protein.
[2032] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of a sequence of SEQ ID
NO:21 or SEQ ID NO:24 which also includes the start codon and
antisense molecules which are complementary to a fragment of the 3'
untranslated region of the sequence.
[2033] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of a nucleic acid
of the invention. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired expression of a nucleic acid
of the invention. This technique involves cleavage by means of
ribozymes containing nucleotide sequences complementary to one or
more regions in the mRNA that attenuate the ability of the mRNA to
be translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the protein, such as GTPase binding.
It is understood that these regions include any of those specific
domains, sites, segments, motifs, and the like that are disclosed
as specific regions or sites herein.
[2034] The polynucleotides also provide vectors for gene therapy in
patients containing cells that are aberrant in expression of a gene
of the invention. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired protein to treat the individual.
[2035] The invention also encompasses kits for detecting the
presence of a nucleic acid of the invention in a biological sample.
For example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting the nucleic
acid in a biological sample; means for determining the amount of
the nucleic acid in the sample; and means for comparing the amount
of the nucleic acid 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 a mRNA or DNA of
the invention.
Computer Readable Means
[2036] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[2037] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[2038] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[2039] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and MicroSoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[2040] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[2041] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[2042] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[2043] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[2044] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
Vectors/Host Cells
[2045] The invention also provides vectors containing the
polynucleotides of the invention. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, that can transport the
polynucleotides. When the vector is a nucleic acid molecule, the
polynucleotides are covalently linked to the vector nucleic acid.
With this aspect of the invention, the vector includes a plasmid,
single or double stranded phage, a single or double stranded RNA or
DNA viral vector, or artificial chromosome, such as a BAC, PAC,
YAC, OR MAC.
[2046] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the polynucleotides. Alternatively, the vector
may integrate into the host cell genome and produce additional
copies of the polynucleotides when the host cell replicates.
[2047] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
polynucleotides. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[2048] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the polynucleotides such
that transcription of the polynucleotides is allowed in a host
cell. The polynucleotides can be introduced into the host cell with
a separate polynucleotide capable of affecting transcription. Thus,
the second polynucleotide may provide a trans-acting factor
interacting with the cis-regulatory control region to allow
transcription of the polynucleotides from the vector.
Alternatively, a trans-acting factor may be supplied by the host
cell. Finally, a trans-acting factor can be produced from the
vector itself.
[2049] It is understood, however, that in some embodiments,
transcription and/or translation of the polynucleotides can occur
in a cell-free system.
[2050] The regulatory sequences to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[2051] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[2052] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[2053] A variety of expression vectors can be used to express a
polynucleotide of the invention. Such vectors include chromosomal,
episomal, and virus-derived vectors, for example vectors derived
from bacterial plasmids, from bacteriophage, from yeast episomes,
from yeast chromosomal elements, including yeast artificial
chromosomes, from viruses such as baculoviruses, papovaviruses such
as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies
viruses, and retroviruses. Vectors may also be derived from
combinations of these sources such as those derived from plasmid
and bacteriophage genetic elements, e.g., cosmids and phagemids.
Appropriate cloning and expression vectors for prokaryotic and
eukaryotic hosts are described in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[2054] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e., tissue specific) or may provide
for inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[2055] The polynucleotides can be inserted into the vector nucleic
acid by well-known methodology. Generally, the DNA sequence that
will ultimately be expressed is joined to an expression vector by
cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[2056] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[2057] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the
polypeptides. Fusion vectors can increase the expression of a
recombinant protein, increase the solubility of the recombinant
protein, and aid in the purification of the protein by acting for
example as a ligand for affinity purification. A proteolytic
cleavage site may be introduced at the junction of the fusion
moiety so that the desired polypeptide can ultimately be separated
from the fusion moiety. Proteolytic enzymes include, but are not
limited to, factor Xa, thrombin, and enterokinase. Typical fusion
expression vectors include pGEX (Smith et al., Gene 67:31-40
(1988)), 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. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amann et al.,
Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185:60-89 (1990)).
[2058] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has 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).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al., Nucleic Acids Res.
20:2111-2118 (1992)).
[2059] The polynucleotides can also be expressed by expression
vectors that are operative in yeast. Examples of vectors for
expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari,
et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell
30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)),
and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[2060] The polynucleotides can also be expressed in insect cells
using, for example, baculovirus expression vectors. Baculovirus
vectors available for expression of proteins in cultured insect
cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol.
Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al.,
Virology 170:31-39 (1989)).
[2061] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. Nature 329:840 (1987)) and pMT2PC (Kaufman
et al., EMBO J. 6:187-195 (1987)).
[2062] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the 26651
or 26138 polynucleotides. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example 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.
[2063] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[2064] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[2065] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those 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).
[2066] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the polynucleotides can be introduced either
alone or with other polynucleotides that are not related to the
polynucleotides such as those providing trans-acting factors for
expression vectors. When more than one vector is introduced into a
cell, the vectors can be introduced independently, co-introduced or
joined to the polynucleotide vector.
[2067] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[2068] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[2069] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[2070] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the polypeptides or heterologous to
these polypeptides.
[2071] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[2072] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
[2073] Host cells of particular interest include those derived from
the tissues in which the 26651 polypeptides of the invention are
expressed, including tonsil, spleen, fetal liver, adult liver,
fibrotic liver, granulocytes, neutrophils, erythroid cells, adipose
tissue, bone marrow, colon, lung, kidney, heart, lymphocyte,
megakaryocytes, T-cells, and the tissues and cell lines shown in
FIGS. 64A-64B.
[2074] Host cells of particular interest include those derived from
the tissues in which the 26138 polypeptides of the invention are
expressed, including tonsil, spleen, fetal liver, adult liver,
fibrotic liver, granulocytes, neutrophils, erythroid cells, adipose
tissue, bone marrow, colon, lung, kidney, heart, lymphocyte,
megakaryocytes, T-cells, and the tissues and cell lines shown in
FIGS. 64A-64B.
Uses of Vectors and Host Cells
[2075] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also 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. A "purified
preparation of cells", as used herein, refers to, in the case of
plant or animal cells, an in vitro preparation of cells and not an
entire intact plant or animal. In the case of cultured cells or
microbial cells, it consists of a preparation of at least 10% and
more preferably 50% of the subject cells.
[2076] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing proteins or polypeptides
of the invention that can be further purified to produce desired
amounts of the proteins or polypeptides, including fusion proteins
or polypeptides, encoded by nucleic acids as described herein (e.g.
26651-like or 26138-like polypeptides, mutant forms of 26651-like
or 26138-like polypeptides, fusion proteins, etc.). It is further
recognized that the nucleic acid sequences of the invention can be
altered to contain codons, which are preferred, or non-preferred,
for a particular expression system. For example, the nucleic acid
can be one in which at least one altered codon, and preferably at
least 10% or 20% of the codons have been altered such that the
sequence is optimized for expression in E. coli, yeast, human,
insect, or CHO cells. Methods for determining codon usage are well
known in the art. Thus, host cells containing expression vectors
are useful for polypeptide production.
[2077] Host cells are also useful for conducting cell-based assays
involving the protein of the invention or fragments. Thus, a
recombinant host cell expressing the native protein is useful to
assay for compounds that stimulate or inhibit protein function.
This can include GTPase binding, gene expression at the level of
transcription or translation, effector interaction, and components
of a signal transduction or other pathway.
[2078] Cells of particular relevance are those in which the protein
is expressed as disclosed herein.
[2079] Host cells are also useful for identifying mutants in which
these functions are affected. If the mutants naturally occur and
give rise to a pathology, host cells containing the mutations are
useful to assay compounds that have a desired effect on the mutant
protein (for example, stimulating or inhibiting function) which may
not be indicated by their effect on the native protein.
[2080] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation by means of heterologous sites or
domains, for example, a binding region, on any given host cell.
[2081] Further, mutant proteins of the invention can be designed in
which one or more of the various functions is engineered to be
increased or decreased (e.g., GTPase binding) and used to augment
or replace proteins of the invention in an individual. Thus, host
cells can provide a therapeutic benefit by replacing an aberrant
protein or providing an aberrant protein that provides a
therapeutic result. In one embodiment, the cells provide proteins
that are abnormally active.
[2082] In another embodiment, the cells provide proteins that are
abnormally inactive. These can compete with the endogenous protein
in the individual.
[2083] In another embodiment, cells expressing the proteins that
cannot be activated, are introduced into an individual in order to
compete with the endogenous protein for GTPase. For example, in the
case in which excessive GTPase (or analog) is part of a treatment
modality, it may be necessary to inactivate the compound at a
specific point in treatment. Providing cells that compete for the
compound, but which cannot be affected by protein activation would
be beneficial.
[2084] Homologously recombinant host cells can also be produced
that allow the in situ alteration of the endogenous polynucleotide
sequences in a host cell genome. The host cell includes, but is not
limited to, a stable cell line, cell in vivo, or cloned
microorganism. This technology is more fully described in WO
93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the GAP polynucleotides or sequences proximal or
distal to a GAP gene are allowed to integrate into a host cell
genome by homologous recombination where expression of the gene can
be affected. In one embodiment, regulatory sequences are introduced
that either increase or decrease expression of an endogenous
sequence. Accordingly, a GAP protein can be produced in a cell not
normally producing it. Alternatively, increased expression of GAP
protein can be effected in a cell normally producing the protein at
a specific level. Further, expression can be decreased or
eliminated by introducing a specific regulatory sequence. The
regulatory sequence can be heterologous to the protein sequence or
can be a homologous sequence with a desired mutation that affects
expression. Alternatively, the entire-gene can be deleted. The
regulatory sequence can be specific to the host cell or capable of
functioning in more than one cell type. Still further, specific
mutations can be introduced into any desired region of the gene to
produce mutant GAP proteins. Such mutations could be introduced,
for example, into the specific functional regions such as the
ligand-binding site.
[2085] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered gene of the invention. Alternatively,
the host cell can be a stem cell or other early tissue precursor
that gives rise to a specific subset of cells and can be used to
produce transgenic tissues in an animal. See also Thomas et al.,
Cell 51:503 (1987) for a description of homologous recombination
vectors. The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced gene
has homologously recombined with the endogenous gene is selected
(see e.g., Li, E. et al., Cell 69:915 (1992)). The selected cells
are 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
vectors and homologous recombinant animals are described further in
Bradley, A. (1991) Current Opinions in Biotechnology 2:823-829 and
in PCT International Publication Nos. WO 90/11354; WO 91/01140; and
WO 93/04169.
[2086] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. 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 in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of a protein of the invention and identifying and
evaluating modulators of the protein activity.
[2087] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[2088] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which polynucleotide sequences of the
invention have been introduced.
[2089] A transgenic animal can be produced by introducing 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. Any of the
nucleotide sequences of the invention can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[2090] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
protein to particular cells.
[2091] 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 the transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[2092] 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. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). 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 is
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.
[2093] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) 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 blastocyst and then transferred to a pseudopregnant
female foster animal. The offspring born of this female foster
animal will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[2094] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect GTPase binding or activation, and signal transduction, may
not be evident from in vitro cell-free or cell-based assays.
Accordingly, it is useful to provide non-human transgenic animals
to assay in vivo GAP function, including GTPase interaction, the
effect of specific mutant proteins on GAP function and GTPase
interaction, and the effect of chimeric proteins. It is also
possible to assess the effect of null mutations, that is mutations
that substantially or completely eliminate one or more protein
functions.
[2095] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the
protein in a transgenic animal, into a cell in culture or in vivo.
When introduced in vivo, the nucleic acid is introduced into an
intact organism such that one or more cell types and, accordingly,
one or more tissue types, express the nucleic acid encoding the
protein. Alternatively, the nucleic acid can be introduced into
virtually all cells in an organism by transfecting a cell in
culture, such as an embryonic stem cell, as described herein for
the production of transgenic animals, and this cell can be used to
produce an entire transgenic organism. As described, in a further
embodiment, the host cell can be a fertilized oocyte. Such cells
are then allowed to develop in a female foster animal to produce
the transgenic organism.
Pharmaceutical Compositions
[2096] The nucleic acid molecules, proteins, modulators of the
protein, and antibodies (also referred to herein as "active
compounds") can be incorporated into pharmaceutical compositions
suitable for administration to a subject, e.g., a human. Such
compositions typically comprise the nucleic acid molecule, protein,
modulator, or antibody and a pharmaceutically acceptable
carrier.
[2097] 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, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. 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.
[2098] 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 polyethylene 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 mannitol, 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.
[2099] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a protein of the invention
or 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.
[2100] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. 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.
[2101] 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.
[2102] 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.
[2103] 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.
[2104] 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.
[2105] 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.
[2106] 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 (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al., PNAS 91:3054-3057
(1994)). 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.
[2107] 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.
[2108] 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. 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.
[2109] 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.
[2110] 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. 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.
[2111] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
OTHER EMBODIMENTS
[2112] In another aspect, the invention features, a method of
analyzing a plurality of capture probes. The method can be used,
e.g., to analyze gene expression. The method includes: providing a
two dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence;
contacting the array with a 26651 or 26138, preferably purified,
nucleic acid, preferably purified, polypeptide, preferably
purified, or antibody, and thereby evaluating the plurality of
capture probes. Binding, e.g., in the case of a nucleic acid,
hybridization with a capture probe at an address of the plurality,
is detected, e.g., by signal generated from a label attached to the
26651 or 26138 nucleic acid, polypeptide, or antibody.
[2113] The capture probes can be a set of nucleic acids from a
selected sample, e.g., a sample of nucleic acids derived from a
control or non-stimulated tissue or cell.
[2114] The method can include contacting the 26651 or 26138 nucleic
acid, polypeptide, or antibody with a first array having a
plurality of capture probes and a second array having a different
plurality of capture probes. The results of each hybridization can
be compared, e.g., to analyze differences in expression between a
first and second sample. The first plurality of capture probes can
be from a control sample, e.g., a wild type, normal, or
non-diseased, non-stimulated, sample, e.g., a biological fluid,
tissue, or cell sample. The second plurality of capture probes can
be from an experimental sample, e.g., a mutant type, at risk,
disease-state or disorder-state, or stimulated, sample, e.g., a
biological fluid, tissue, or cell sample.
[2115] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of 26651 or 26138. Such methods can be used to diagnose a
subject, e.g., to evaluate risk for a disease or disorder, to
evaluate suitability of a selected treatment for a subject, to
evaluate whether a subject has a disease or disorder. 26651 or
26138 is associated with GAP activity, thus it is useful for
disorders associated with abnormal GTPase signaling, GTPase release
of substrates, GTPase activation, or other GAP regulated
processes.
[2116] The method can be used to detect SNPs.
[2117] In another aspect, the invention features, a method of
analyzing a plurality of probes. The method is useful, e.g., for
analyzing gene expression. The method includes: providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality having a unique capture probe, e.g.,
wherein the capture probes are from a cell or subject which express
or mis express 26651 or 26138 or from a cell or subject in which a
26651 or 26138 mediated response has been elicited, e.g., by
contact of the cell with 26651 or 26138 nucleic acid or protein, or
administration to the cell or subject 26651 or 26138 nucleic acid
or protein; contacting the array with one or more inquiry probe,
wherein an inquiry probe can be a nucleic acid, polypeptide, or
antibody (which is preferably other than 26651 or 26138 nucleic
acid, polypeptide, or antibody); providing a two dimensional array
having a plurality of addresses, each address of the plurality
being positionally distinguishable from each other address of the
plurality, and each address of the plurality having a unique
capture probe, e.g., wherein the capture probes are from a cell or
subject which does not express 26651 or 26138 (or does not express
as highly as in the case of the 26651 or 26138 positive plurality
of capture probes) or from a cell or subject which in which a 26651
or 26138 mediated response has not been elicited (or has been
elicited to a lesser extent than in the first sample); contacting
the array with one or more inquiry probes (which is preferably
other than a 26651 or 26138 nucleic acid, polypeptide, or
antibody), and thereby evaluating the plurality of capture probes.
Binding, e.g., in the case of a nucleic acid, hybridization with a
capture probe at an address of the plurality, is detected, e.g., by
signal generated from a label attached to the nucleic acid,
polypeptide, or antibody.
[2118] In another aspect, the invention features, a method of
analyzing 26651 or 26138, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a 26651 or 26138 nucleic acid or amino
acid sequence; comparing the 26651 or 26138 sequence with one or
more preferably a plurality of sequences from a collection of
sequences, e.g., a nucleic acid or protein sequence database; to
thereby analyze 26651 or 26138.
[2119] Preferred databases include GenBank.TM.. The method can
include evaluating the sequence identity between a 26651 or 26138
sequence and a database sequence. The method can be performed by
accessing the database at a second site, e.g., over the
internet.
[2120] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of 26651 or 26138. The set includes a
plurality of oligonucleotides, each of which has a different
nucleotide at an interrogation position, e.g., an SNP or the site
of a mutation. In a preferred embodiment, the oligonucleotides of
the plurality are identical in sequence with one another (except
for differences in length). The oligonucleotides can be provided
with different labels, such that an oligonucleotide that hybridizes
to one allele provides a signal that is distinguishable from an
oligonucleotide which hybridizes to a second allele.
[2121] 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 are incorporated herein by
reference.
EXPERIMENTAL
Example 1
Identification and Characterization of Human 26651 GAP
[2122] The human 26651 GAP sequence (FIGS. 52A-52B), which is
approximately 2847 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
547 amino acids (nucleotides 60-1703 of SEQ ID NO:21; SEQ ID
NO:23). The coding sequence encodes a 547 amino acid protein (SEQ
ID NO:22).
[2123] PFAM analysis indicates that the 26651 polypeptide shares a
high degree of sequence similarity with GAPs. Further, PFAM
analysis indicates that the 26651 shares a high degree of sequence
similarity with the Rho-GAP subclass. For general information
regarding PFAM identifiers, PS prefix and PF prefix domain
identification numbers, refer to Sonnhammer et al (1997) Protein
28:405-420 and
www.psc.edu/general/software/packages/pfam/pfam.html.
[2124] As used herein, the term "Rho-GAP domain" includes an amino
acid sequence of about 80-300 amino acid residues in length and
having a bit score for the alignment of the sequence to the Rho-GAP
domain (HMM) of at least 8. Preferably, a Rho-GAP domain includes
at least about 100-250 amino acids, more preferably about 120-200
amino acid residues, or about 120-180 amino acids and has a bit
score for the alignment of the sequence to the Rho-GAP domain (HMM)
of at least 16 or greater. The Rho-GAP domain (HMM) has been
assigned the PFAM Accession PF00620 (pfam.wustl.edu/). An alignment
of the Rho-GAP domain (amino acids 236 to 397 of SEQ ID NO:22) of
human 26651-like polypeptides with a consensus amino acid sequence
derived from a hidden Markov model (SEQ ID NO:27) is depicted in
FIGS. 56A-B.
[2125] In a preferred embodiment a 26651-like polypeptide or
protein has a "Rho-GAP domain" or a region which includes at least
about 100-250, more preferably about 120-200, or 120-180 amino acid
residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or
100% sequence identity with an "Rho-GAP domain," e.g., the Rho-GAP
domain of human 26651 (e.g., amino acid residues 236-397 of SEQ ID
NO:22).
[2126] PFAM analysis indicates that the 26651 polypeptide shares a
high degree of sequence similarity with dockerin. The dockerin
domain (HMM) has been assigned the PFAM Accession PF00404
(pfam.wustl.edu/). The dockerin domain of 26651 falls between amino
acids 278 to 298 of SEQ ID NO:22.
[2127] To identify the presence of a "Rho-GAP domain" in a
26651-like protein sequence, and make the determination that a
polypeptide or protein of interest has a particular profile, the
amino acid sequence of the protein can be searched against a
database of HMMs (e.g., the Pfam database, release 2.1) using the
default parameters (www.sanger.ac.uk/Software/Pfam/HMM_search). For
example, the hmmsf program, which is available as part of the HMMER
package of search programs, is a family specific default program
for MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonhammer et al. (1997)
Proteins 28(3):405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al. (1990) Meth. Enzymol.
183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference.
[2128] ProDom analysis of 26651 revealed 35% identity to a protein
GTPase domain (p99.2 (80) P85A(4) P85B(4)CHIN(2)//PROTEIN GTPASE
DOMAIN SH2 ACTIVATION ZINC 3-KINASE SH3 PHOSPHATIDYLINOSITOL
REGULATORY). ProDom analysis further revealed regions having 27%
and 37% identity with the long isoform of RhoGapX-1 (P99.2 (2)
043182(1) 043437(1)//RHO-TYPE GTPASE ACTIVATING PROTEIN RHOGAPX-1
LONG ISOFORM). A region having 28% identity to a trithorax
transcription regulation protein (p99.2 (3) Q24742(1) Q27255(1)
TRX(1)//TRITHORAX PROTEIN PREDICTED TRX TRANSCRIPTION REGULATION
ZINC-FINGER METAL-BINDING DNA-BINDING NUCLEAR) was identified by
ProDom analysis. The ProDom analysis also identified regions of
26651 with 29%, 26%, and 23% identity to the T-DNA region of a TI
plasmid (p99.2 (1) Q44390-AGRTU//T1 PLASMID PT11 5955 T-DNA REGION
PLASMID), cosmid (p99.2 (1) Q20299_CAELL//COSMID F41H10), and a
hypothetical protein (p99.2 (1) O26888-METTH//HYPOTHETICAL 21.6 KD
PROTEIN HYPOTHETICAL PROTEIN), respectively.
Example 2
Identification and Characterization of Human 26138 GAP
[2129] The human 26138 GAP sequence (FIGS. 57A-57C), which is
approximately 3391 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
3018 nucleotides (nucleotides 78-3095 of SEQ ID NO:24: SEQ ID
NO:26). The coding sequence encodes a 1005 amino acid protein (SEQ
ID NO:25).
[2130] PFAM analysis indicates that the 26138 polypeptide shares a
high degree of sequence similarity with GAPs. Further, PFAM
analysis indicates that the 26138 polypeptide shares a high degree
of sequence similarity with the Ras-GAP subclass. For general
information regarding PFAM identifiers, PS prefix and PF prefix
domain identification numbers, refer to Sonnhammer et al. (1997)
Protein 28:405-420 and
www.psc.edu/general/software/packages/pfam/pfam.html.
[2131] As used herein, the term "Ras-GAP domain" includes an amino
acid sequence of about 80-300 amino acid residues in length and
having a bit score for the alignment of the sequence to the Ras-GAP
domain (HMM) of at least 8. Preferably, a Ras-GAP domain includes
at least about 100-250 amino acids, more preferably about 130-200
amino acid residues, or about 160-200 amino acids and has a bit
score for the alignment of the sequence to the Ras-GAP domain (HMM)
of at least 16 or greater. Ras-GAP domain (HMM) has been assigned
the PFAM Accession PF 00616 (pfam.wustl.edu/). An alignment of the
Ras-GAP domain (amino acids 473 to 645 of SEQ ID NO:25) of human
26138 with a consensus amino acid sequence derived from a hidden
Markov model (SEQ ID NO:29) is depicted in FIGS. 61A-61B.
[2132] In a preferred embodiment a 26138-like polypeptide or
protein has a "Ras-GAP domain" or a region which includes at least
about 100-250 more preferably about 130-200 or 160-200 amino acid
residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or
100% sequence identity with an "Ras-GAP domain," e.g., the Ras-GAP
domain of human 26138 (e.g., amino acid residues 473 to 645 of SEQ
ID NO:25).
[2133] PFAM analysis indicates that the 26138 polypeptide shares a
high degree of sequence similarity with the gntR family of
bacterial regulatory proteins. The gntR domain (HMM) has been
assigned the PFAM Accession Number PF00392 (pfam.wustl.edu/). The
gntR domain of 26138 falls between amino acids 405 to 433 of SEQ ID
NO:25. PFAM analysis indicates that the 26138 polypeptide shares a
region (amino acids 253 to 287 of SEQ ID NO:25) with similarity to
the pleckstrin homology domain. The pleckstrin homology domain has
been assigned the PFAM Accession Number PF00169.
[2134] To identify the presence of a "Ras-GAP domain" in a
26138-like protein sequence, and make the determination that a
polypeptide or protein of interest has a particular profile, the
amino acid sequence of the protein can be searched against a
database of HMMs (e.g., the Pfam database, release 2.1) using the
default parameters (www.sanger.ac.uk/Software/Pfam/HMM_search). For
example, the hmmsf program, which is available as part of the HMMER
package of search programs, is a family specific default program
for MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonhammer et al. (1997)
Proteins 28(3):405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al (1990) Meth. Enzymol.
183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference.
[2135] ProDom analysis of 2613 revealed 25% identity to p99.2 (1)
O44242_CAEEL//GAP 2-4. Further analysis revealed 38% identity to a
ras-GAP inhibitory regulator (p99.2 (29) GTPA(3) NF1(2) GAP
1(2)//PROTEIN GTPASE ACTIVATION GTPASE-ACTIVATING RAS NEUROFIBROMIN
P21 ACTIVATOR INHIBITORY REGULATOR). A region having 31% identity
to a filament intermediate repeat heptad (p99.2 (314) LAMA (10)
DESM (8) LAM1(8)//FILAMENT INTERMEDIATE REPEAT HEPTAD PATTERN
COILED COIL KERATIN PROTEIN TYPE) was identified by ProDom
analysis. 26138 is 26%, 30%, and 26% identical to several
hypothetical proteins: p99.2 (1) YWKC_BACSU//HYPOTHETICAL 21.1 KD
PROTEIN IN TDK-PRFA INTERGENIC REGION; p99.2 (1)
YBYO_YEAST//HYPOTHETICAL 47.4 KD PROTEIN IN OPY1-AGP2 INTERGENIC
REGION; and p99.2 (1) YFHG_ECOLI//HYPOTHETICAL 27.3 KD PROTEIN IN
GLNB-PURL INTERGENIC REGION ORF-1 F239, respectively. 26138 also
shares 23% identity to GAG GAG-POL polypeptides (p99.2 (2) Q88284
(1) Q88285(1)//POLYPROTEIN GAG GAG-POL).
Example 3
Tissue Distribution of 26138 mRNA
[2136] Expression levels of 26138 in various tissue and cell types
were determined by quantitative RT-PCR (Reverse Transcriptase
Polymerase Chain Reaction; Taqman.RTM. brand PCR kit, Applied
Biosystems). The quantitative RT-PCR reactions were performed
according to the kit manufacturer's instructions. The results of
the Taqman.RTM. analysis are shown in FIGS. 64A-64B.
[2137] 26138 was expressed in a variety of human tissues including
tonsil, spleen, fetal liver, adult liver, fibrotic liver,
granulocytes, neutrophils, erythroid cells, adipose tissue, bone
marrow, colon, lung, kidney, heart, lymphocyte, megakaryocytes and
T-cells.
Example 4
Tissue Distribution of 26651 or 26138 mRNA
[2138] Northern blot hybridizations with various RNA samples are
performed under standard conditions and washed under stringent
conditions, i.e., 0.2.times.SSC at 65.degree. C. A DNA probe
corresponding to all or a portion of the 26651 or 26138 cDNA (SEQ
ID NO:21 or 24) can be used. The DNA is radioactively labeled with
.sup.32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.)
according to the instructions of the supplier. Filters containing
mRNA from mouse hematopoietic and endocrine tissues, and cancer
cell lines (Clontech, Palo Alto, Calif.) are probed in ExpressHyb
hybridization solution (Clontech) and washed at high stringency
according to manufacturer's recommendations.
Example 5
Recombinant Expression of 26651 or 26138 in Bacterial Cells
[2139] In this example, 26651 or 26138 is expressed as a
recombinant glutathione-S-transferase (GST) fusion polypeptide in
E. coli and the fusion polypeptide is isolated and characterized.
Specifically, 26651 or 26138 is fused to GST and this fusion
polypeptide is expressed in E. coli, e.g., strain PEB199.
Expression of the GST-26651 or 26138 fusion protein in PEB199 is
induced with FPTG. 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 6
Expression of Recombinant 26651 or 26138 Protein in COS Cells
[2140] To express the 26651 or 26138 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 26651 or
26138 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.
[2141] To construct the plasmid, the 26651 or 26138 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 26651 or 26138 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 26651 or 26138 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 26651 or 26138
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.
[2142] COS cells are subsequently transfected with the 26651 or
26138-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 26651 or 26138 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.
[2143] Alternatively, DNA containing the 26651 or 26138 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 26651 or 26138 polypeptide is
detected by radiolabelling and immunoprecipitation using a 26651 or
26138 specific monoclonal antibody.
[2144] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
Sequence CWU 1
1
3011434DNAHomo sapiensCDS(200)...(886) 1cgtgggcgga cgcgtgggtg
cgtgtgtggc cttttttatt tgagagagca agaggcgccg 60cggacgcctg ggcagccacg
gcggcggggc cgcggtgggc gccggctcag cccgcccctt 120tctcccgccg
cctccccgcc ccgccccgcg ccgcgccggc cgctgtcagc tccctcagcg
180tccggccgag gcgcggtgt atg ctg agc cgc tgc cgc agc cgg ctg ctc cac
232 Met Leu Ser Arg Cys Arg Ser Arg Leu Leu His 1 5 10gtc ctg ggc
ctt agc ttc ctg ctg cag acc cgc cgg ccg att ctc ctc 280Val Leu Gly
Leu Ser Phe Leu Leu Gln Thr Arg Arg Pro Ile Leu Leu 15 20 25tgc tct
cca cgt ctc atg aag ccg ctg gtc gtg ttc gtc ctc ggc ggc 328Cys Ser
Pro Arg Leu Met Lys Pro Leu Val Val Phe Val Leu Gly Gly 30 35 40ccc
ggc gcc ggc aag ggg acc cag tgc gcc cgc atc gtc gag aaa tat 376Pro
Gly Ala Gly Lys Gly Thr Gln Cys Ala Arg Ile Val Glu Lys Tyr 45 50
55ggc tac aca cac ctt tct gca gga gag ctg ctt cgt gat gaa agg aag
424Gly Tyr Thr His Leu Ser Ala Gly Glu Leu Leu Arg Asp Glu Arg Lys
60 65 70 75aac cca gat tca cag tat ggt gaa ctt att gaa aag tac att
aaa gaa 472Asn Pro Asp Ser Gln Tyr Gly Glu Leu Ile Glu Lys Tyr Ile
Lys Glu 80 85 90gga aag att gta cca gtt gag ata acc atc agt tta tta
aag agg gaa 520Gly Lys Ile Val Pro Val Glu Ile Thr Ile Ser Leu Leu
Lys Arg Glu 95 100 105atg gat cag aca atg gct gcc aat gct cag aag
aat aaa ttc ttg att 568Met Asp Gln Thr Met Ala Ala Asn Ala Gln Lys
Asn Lys Phe Leu Ile 110 115 120gat ggg ttt cca aga aat caa gac aac
ctt caa gga tgg aac aag acc 616Asp Gly Phe Pro Arg Asn Gln Asp Asn
Leu Gln Gly Trp Asn Lys Thr 125 130 135atg gat ggg aag gca gat gta
tct ttc gtt ctc ttt ttt gac tgt aat 664Met Asp Gly Lys Ala Asp Val
Ser Phe Val Leu Phe Phe Asp Cys Asn140 145 150 155aat gag att tgt
att gaa cga tgt ctt gag agg gga aag agt agt ggt 712Asn Glu Ile Cys
Ile Glu Arg Cys Leu Glu Arg Gly Lys Ser Ser Gly 160 165 170agg agt
gat gac aac aga gag agc ttg gaa aag aga att cag acc tac 760Arg Ser
Asp Asp Asn Arg Glu Ser Leu Glu Lys Arg Ile Gln Thr Tyr 175 180
185ctt cag tca aca aag cca att att gac tta tat gaa gaa atg ggg aaa
808Leu Gln Ser Thr Lys Pro Ile Ile Asp Leu Tyr Glu Glu Met Gly Lys
190 195 200gtc aag aaa ata gat gct tct aaa tct gtt gat gaa gtt ttt
gat gaa 856Val Lys Lys Ile Asp Ala Ser Lys Ser Val Asp Glu Val Phe
Asp Glu 205 210 215gtt gtg cag att ttt gac aag gaa ggc taa
ttctaaacct gaaagcatcc 906Val Val Gln Ile Phe Asp Lys Glu Gly *220
225ttgaaatcat gcttgaatat tgctttgata gctgctatca tgaccccttt
ttaaggcaat 966tctaatcttt cataactaca tctcaattag tggctggaaa
gtacatggta aaacaaagta 1026aattttttta tgttcttttt tttggtcaca
ggagtagaca gtgaattcag gtttaacttc 1086accttagtta tggtgctcac
caaacgaagg gtatcagcta ttttttttta aattcaaaaa 1146gaatatccct
tttatagttt gtgccttctg tgagcaaaac tttttagtac gcgtatatat
1206ccctctagta atcacaacat tttaggattt agggatcccg cttcctcttt
ttcttgcaag 1266ttttaaattt ccaaccttaa gtgaatttgt ggaccaaatt
tcaaaggaac tttttgtgta 1326gtcagttctt gcacatgtgt ttggtaaaca
aactcaaaat ggattcttag gagcatttaa 1386gtggttatta aatactgacc
atttgctgta aaaagatgaa aaaactta 14342228PRTHomo sapiens 2Met Leu Ser
Arg Cys Arg Ser Arg Leu Leu His Val Leu Gly Leu Ser 1 5 10 15Phe
Leu Leu Gln Thr Arg Arg Pro Ile Leu Leu Cys Ser Pro Arg Leu 20 25
30Met Lys Pro Leu Val Val Phe Val Leu Gly Gly Pro Gly Ala Gly Lys
35 40 45Gly Thr Gln Cys Ala Arg Ile Val Glu Lys Tyr Gly Tyr Thr His
Leu 50 55 60Ser Ala Gly Glu Leu Leu Arg Asp Glu Arg Lys Asn Pro Asp
Ser Gln65 70 75 80Tyr Gly Glu Leu Ile Glu Lys Tyr Ile Lys Glu Gly
Lys Ile Val Pro 85 90 95Val Glu Ile Thr Ile Ser Leu Leu Lys Arg Glu
Met Asp Gln Thr Met 100 105 110Ala Ala Asn Ala Gln Lys Asn Lys Phe
Leu Ile Asp Gly Phe Pro Arg 115 120 125Asn Gln Asp Asn Leu Gln Gly
Trp Asn Lys Thr Met Asp Gly Lys Ala 130 135 140Asp Val Ser Phe Val
Leu Phe Phe Asp Cys Asn Asn Glu Ile Cys Ile145 150 155 160Glu Arg
Cys Leu Glu Arg Gly Lys Ser Ser Gly Arg Ser Asp Asp Asn 165 170
175Arg Glu Ser Leu Glu Lys Arg Ile Gln Thr Tyr Leu Gln Ser Thr Lys
180 185 190Pro Ile Ile Asp Leu Tyr Glu Glu Met Gly Lys Val Lys Lys
Ile Asp 195 200 205Ala Ser Lys Ser Val Asp Glu Val Phe Asp Glu Val
Val Gln Ile Phe 210 215 220Asp Lys Glu Gly2253196PRTSus scrofa 3Met
Arg Pro Lys Val Val Phe Val Leu Gly Gly Pro Gly Ala Gly Lys 1 5 10
15Gly Thr Gln Cys Ala Arg Ile Val Glu Lys Tyr Gly Tyr Thr His Leu
20 25 30Ser Ala Gly Glu Leu Leu Arg Asp Glu Arg Lys Asn Pro Asp Ser
Gln 35 40 45Tyr Gly Glu Leu Ile Glu Lys Tyr Ile Lys Asp Gly Lys Ile
Val Pro 50 55 60Val Glu Ile Thr Ile Ser Leu Leu Arg Arg Glu Met Asp
Gln Thr Met65 70 75 80Ala Ala Asn Ala Gln Lys Asn Lys Phe Leu Ile
Asp Gly Phe Pro Arg 85 90 95Asn Gln Asp Asn Leu Gln Gly Trp Asn Lys
Thr Met Asp Gly Lys Ala 100 105 110Asp Val Ser Phe Val Leu Phe Phe
Asp Cys Asn Asn Glu Ile Cys Ile 115 120 125Glu Arg Cys Leu Glu Arg
Gly Lys Ser Ser Gly Arg Ser Asp Asp Asn 130 135 140Arg Glu Ser Leu
Glu Lys Arg Ile Gln Thr Tyr Leu Gln Ser Thr Lys145 150 155 160Pro
Ile Ile Asp Leu Tyr Glu Glu Met Gly Lys Val Lys Lys Ile Asp 165 170
175Ala Ser Lys Ser Val Asp Glu Val Phe Asp Glu Val Val Lys Ile Phe
180 185 190Asp Lys Glu Gly 1954190PRTArtificial SequenceConsensus
sequence for adenylate kinase 4Leu Leu Gly Pro Pro Gly Ala Gly Lys
Gly Thr Gln Ala Glu Arg Ile 1 5 10 15Val Lys Lys Tyr Gly Ile Pro
His Leu Ser Thr Gly Asp Leu Leu Arg 20 25 30Ala Glu Val Lys Ser Gly
Thr Glu Leu Gly Lys Glu Ala Lys Glu Tyr 35 40 45Met Asp Lys Gly Glu
Leu Val Pro Asp Glu Val Val Ile Gly Leu Val 50 55 60Lys Glu Arg Leu
Glu Gln Asn Val Asp Ala Lys Lys Asn Gly Phe Leu65 70 75 80Leu Asp
Gly Phe Pro Arg Thr Val Pro Gln Ala Glu Ala Leu Glu Glu 85 90 95Met
Leu Glu Glu Ala Gly Ile Lys Leu Asp Ala Val Ile Glu Leu Asp 100 105
110Val Pro Asp Glu Val Leu Val Glu Arg Leu Thr Gly Arg Arg Ile His
115 120 125Pro Thr Ser Gly Arg Ser Tyr His Leu Glu Phe Asn Pro Pro
Lys Val 130 135 140Glu Gly Lys Asp Asp Val Thr Gly Glu Pro Leu Leu
Gln Arg Arg Ala145 150 155 160Asp Asp Asn Glu Glu Thr Val Lys Lys
Arg Leu Glu Thr Tyr His Lys 165 170 175Gln Thr Glu Pro Val Ile Asp
Tyr Tyr Lys Lys Lys Gly Lys 180 185 1905260PRTHomo sapiens 5Met Ala
Arg Pro Gly Met Glu Arg Trp Arg Asp Arg Leu Ala Leu Val 1 5 10
15Thr Gly Ala Ser Gly Gly Ile Gly Ala Ala Val Ala Arg Ala Leu Val
20 25 30Gln Gln Gly Leu Lys Val Val Gly Cys Ala Arg Thr Val Gly Asn
Ile 35 40 45Glu Glu Leu Ala Ala Glu Cys Lys Ser Ala Gly Tyr Pro Gly
Thr Leu 50 55 60Ile Pro Tyr Arg Cys Asp Leu Ser Asn Glu Glu Asp Ile
Leu Ser Met65 70 75 80Phe Ser Ala Ile Arg Ser Gln His Ser Gly Val
Asp Ile Cys Ile Asn 85 90 95Asn Ala Gly Leu Ala Arg Pro Asp Thr Leu
Leu Ser Gly Ser Thr Ser 100 105 110Gly Trp Lys Asp Met Phe Asn Val
Asn Val Leu Ala Leu Ser Ile Cys 115 120 125Thr Arg Glu Ala Tyr Gln
Ser Met Lys Glu Arg Asn Val Asp Asp Gly 130 135 140His Ile Ile Asn
Ile Asn Ser Met Ser Gly His Arg Val Leu Pro Leu145 150 155 160Ser
Val Thr His Phe Tyr Ser Ala Thr Lys Tyr Ala Val Thr Ala Leu 165 170
175Thr Glu Gly Leu Arg Gln Glu Leu Arg Glu Ala Gln Thr His Ile Arg
180 185 190Ala Thr Cys Ile Ser Pro Gly Val Val Glu Thr Gln Phe Ala
Phe Lys 195 200 205Leu His Asp Lys Asp Pro Glu Lys Ala Ala Ala Thr
Tyr Glu Gln Met 210 215 220Lys Cys Leu Lys Pro Glu Asp Val Ala Glu
Ala Val Ile Tyr Val Leu225 230 235 240Ser Thr Pro Ala His Ile Gln
Ile Gly Asp Ile Gln Met Arg Pro Thr 245 250 255Glu Gln Val Thr
26061909DNAHomo sapiensCDS(421)...(1203)misc_feature1552, 1883,
1888, 1890n = A,T,C or G 6tacttagact cagccggctt ttccacgctt
tgcctgaccc tgctttgctc aactgtacgt 60cttgtttcgt tttctgttct gcgccgttac
agatccaagc tctgaaaaac cagaaagtta 120actggtaagt ttagtctttt
tgtcttttat ttcaggtccc ggatccggtg atccaaatct 180aagaactgct
cctcagtgag tgttgccttt acttctaggc ctgtacggaa gtgttacttc
240tgctctaaaa gctgcggaat tctaatacga ctcactatag ggagtcgacc
cacgcgtccg 300gggtctaggc gcggatcgga cccaagcagg tcggcggcgg
cggcaggaga gcggccgggc 360gtcagctcct cgacccccgt gtcgggctag
tccagcgagg cggacgggcg gcgtgggccc 420atg gcc agg ccc ggc atg gag cgg
tgg cgc gac cgg ctg gcg ctg gtg 468Met Ala Arg Pro Gly Met Glu Arg
Trp Arg Asp Arg Leu Ala Leu Val 1 5 10 15acg ggg gcc tcg ggg ggc
atc ggc gcg gcc gtg gcc cgg gcc ctg gtc 516Thr Gly Ala Ser Gly Gly
Ile Gly Ala Ala Val Ala Arg Ala Leu Val 20 25 30cag cag gga ctg aag
gtg gtg ggc tgc gcc cgc act gtg ggc aac atc 564Gln Gln Gly Leu Lys
Val Val Gly Cys Ala Arg Thr Val Gly Asn Ile 35 40 45gag gag ctg gct
gct gaa tgt aag agt gca ggc tac ccc ggg act ttg 612Glu Glu Leu Ala
Ala Glu Cys Lys Ser Ala Gly Tyr Pro Gly Thr Leu 50 55 60atc ccc tac
aga tgt gac cta tca aat gaa gag gac atc ctc tcc atg 660Ile Pro Tyr
Arg Cys Asp Leu Ser Asn Glu Glu Asp Ile Leu Ser Met 65 70 75 80ttc
tca gct atc cgt tct cag cac agc ggt gta gac atc tgc atc aac 708Phe
Ser Ala Ile Arg Ser Gln His Ser Gly Val Asp Ile Cys Ile Asn 85 90
95aat gct ggc ttg gcc cgg cct gac acc ctg ctc tca ggc agc acc agt
756Asn Ala Gly Leu Ala Arg Pro Asp Thr Leu Leu Ser Gly Ser Thr Ser
100 105 110ggt tgg aag gac atg ttc aat gtg aac gtg ctg gcc ctc agc
atc tgc 804Gly Trp Lys Asp Met Phe Asn Val Asn Val Leu Ala Leu Ser
Ile Cys 115 120 125aca cgg gaa gcc tac cag tcc atg aag gag cgg aat
gtg gac gat ggg 852Thr Arg Glu Ala Tyr Gln Ser Met Lys Glu Arg Asn
Val Asp Asp Gly 130 135 140cac atc att aac atc aat agc atg tct ggc
cac cga gtg tta ccc ctg 900His Ile Ile Asn Ile Asn Ser Met Ser Gly
His Arg Val Leu Pro Leu145 150 155 160tct gtg acc cac ttc tat agt
gcc acc aag tat gcc gtc act gcg ctg 948Ser Val Thr His Phe Tyr Ser
Ala Thr Lys Tyr Ala Val Thr Ala Leu 165 170 175aca gag gga ctg agg
caa gag ctt cgg gag gcc cag acc cac atc cga 996Thr Glu Gly Leu Arg
Gln Glu Leu Arg Glu Ala Gln Thr His Ile Arg 180 185 190gcc acg tgc
atc tct cca ggt gtg gtg gag aca caa ttc gcc ttc aaa 1044Ala Thr Cys
Ile Ser Pro Gly Val Val Glu Thr Gln Phe Ala Phe Lys 195 200 205ctc
cac gac aag gac cct gag aag gca gct gcc acc tat gag caa atg 1092Leu
His Asp Lys Asp Pro Glu Lys Ala Ala Ala Thr Tyr Glu Gln Met 210 215
220aag tgt ctc aaa ccc gag gat gtg gcc gag gct gtt atc tac gtc ctc
1140Lys Cys Leu Lys Pro Glu Asp Val Ala Glu Ala Val Ile Tyr Val
Leu225 230 235 240agc act ccc gca cac atc cag att gga gac atc cag
atg agg ccc acg 1188Ser Thr Pro Ala His Ile Gln Ile Gly Asp Ile Gln
Met Arg Pro Thr 245 250 255gag cag gtg acc tag tgactgtggg
agctcctcct tccctcccca cccttcatgg 1243Glu Gln Val Thr *
260cttgcctcct gcctctggat tttaggtgtt gatttctgga tcacgggata
ccacttcctg 1303tccacacccc gaccaggggc tagaaaattt gtttgagatt
tttatatcat cttgtcaaat 1363tgcttcagtt gtaaatgtga aaaatgggct
ggggaaagga ggtggtgtcc ctaattgttt 1423tacttgttaa cttgttcttg
tgcccctggg cacttggcct ttgtctgctc tcagtgtctt 1483ccctttgaca
tgggaaagga gttgtggcca aaatccccat cttcttgcac ctcaacgtct
1543gtggctyang ggctggggtg gcagagggag gccttcacct tatatctgtg
ttgttatcca 1603gggctccaga cttcctcctc tgcctgcccc actgcaccct
ctccccctta tctatctcct 1663tctcggctcc ccagcccagt cttggcttct
tgtcccctcc tggggtcatc cctccactct 1723gactctgact atggcagcag
aacaccaggg cctggcccag tggatttcat ggtgatcatt 1783aaaaaagaaa
aatcgcaacc aaaaaaaaaa aaaaaagggc gggccgctag actagtytag
1843agaaaaaacc tcccacacct ccccybdamm ytkacgccgn acgcnanggg
ggcaatcaag 1903gacgct 19097260PRTHomo sapiens 7Met Glu Lys Cys Glu
Ala Ala Ala Lys Asp Ile Arg Gly Glu Thr Leu 1 5 10 15Asn His His
Val Asn Ala Arg His Leu Asp Leu Ala Ser Leu Lys Ser 20 25 30Ile Arg
Glu Phe Ala Ala Lys Ile Ile Glu Glu Glu Glu Arg Val Asp 35 40 45Ile
Leu Ile Asn Asn Ala Gly Val Met Arg Cys Pro His Trp Thr Thr 50 55
60Glu Asp Gly Phe Glu Met Gln Phe Gly Val Asn His Leu Gly His Phe65
70 75 80Leu Leu Thr Asn Leu Leu Leu Asp Lys Leu Lys Ala Ser Ala Pro
Ser 85 90 95Arg Ile Ile Asn Leu Ser Ser Leu Ala His Val Ala Gly His
Ile Asp 100 105 110Phe Asp Asp Leu Asn Trp Gln Thr Arg Lys Tyr Asn
Thr Lys Ala Ala 115 120 125Tyr Cys Gln Ser Lys Leu Ala Ile Val Leu
Phe Thr Lys Glu Leu Ser 130 135 140Arg Arg Leu Gln Gly Ser Gly Val
Thr Val Asn Ala Leu His Pro Gly145 150 155 160Val Ala Arg Thr Glu
Leu Gly Arg His Thr Gly Ile His Gly Ser Thr 165 170 175Phe Ser Ser
Thr Thr Leu Gly Pro Ile Phe Trp Leu Leu Val Lys Ser 180 185 190Pro
Glu Leu Ala Ala Gln Pro Ser Thr Tyr Leu Ala Val Ala Glu Glu 195 200
205Leu Ala Asp Val Ser Gly Lys Tyr Phe Asp Gly Leu Lys Gln Lys Ala
210 215 220Pro Ala Pro Glu Ala Glu Asp Glu Glu Val Ala Arg Arg Leu
Trp Ala225 230 235 240Glu Ser Ala Arg Leu Val Gly Leu Glu Ala Pro
Ser Val Arg Glu Gln 245 250 255Pro Leu Pro Arg 26081153DNAHomo
sapiensCDS(265)...(1047)misc_feature24, 25, 27, 77, 78n = A,T,C or
G 8ccgcgccccg ccctcgcagc ccanntncgg acgcgggccc agccgcgcct
gcgcttccgc 60tcgcctgtgg ctgcaannag cgcgctcttc ctcggagcta cccaggcggc
tggtgtagca 120gcaagctccg cgccgacccc tgacgcctga cgcctgtccc
cggcccggca tgagccgcta 180cctgctgccg ctgtcggcgc tgggcacggt
agcaggcgct cgccgtgctg ctcaagaggc 240aacatcatcc tggcctgccg agac atg
gag aag tgt gag gcg gca gca aag 291 Met Glu Lys Cys Glu Ala Ala Ala
Lys 1 5gac atc cgc ggg gag acc ctc aat cac cat gtc aac gcc cgg cac
ctg 339Asp Ile Arg Gly Glu Thr Leu Asn His His Val Asn Ala Arg His
Leu 10 15 20 25gac ttg gct tcc ctc aag tct atc cga gag ttt gca gca
aag atc att 387Asp Leu Ala Ser Leu Lys Ser Ile Arg Glu Phe Ala Ala
Lys Ile Ile 30 35 40gaa gag gag gag cga gtg gac att cta atc aac aac
gcg ggt gtg atg 435Glu Glu Glu Glu Arg Val Asp Ile Leu Ile Asn Asn
Ala Gly Val Met 45 50 55cgg tgc ccc cac tgg acc acc gag gac ggc ttc
gag atg cag ttt ggc 483Arg Cys Pro His Trp Thr Thr Glu Asp Gly Phe
Glu Met Gln Phe Gly 60 65 70gtt aac cac ctg ggt cac ttt ctc ttg aca
aac ttg ctg ctg gac aag 531Val Asn His Leu Gly His Phe Leu Leu Thr
Asn Leu Leu Leu
Asp Lys 75 80 85ctg aaa gcc tca gcc cct tcg cgg atc atc aac ctc tcg
tcc ctg gcc 579Leu Lys Ala Ser Ala Pro Ser Arg Ile Ile Asn Leu Ser
Ser Leu Ala 90 95 100 105cat gtt gct ggg cac ata gac ttt gac gac
ttg aac tgg cag acg agg 627His Val Ala Gly His Ile Asp Phe Asp Asp
Leu Asn Trp Gln Thr Arg 110 115 120aag tat aac acc aaa gcc gcc tac
tgc cag agc aag ctc gcc atc gtc 675Lys Tyr Asn Thr Lys Ala Ala Tyr
Cys Gln Ser Lys Leu Ala Ile Val 125 130 135ctc ttc acc aag gag ttg
agc cgg cgg ctg caa ggc tct ggt gtg act 723Leu Phe Thr Lys Glu Leu
Ser Arg Arg Leu Gln Gly Ser Gly Val Thr 140 145 150gtc aac gcc ctg
cac ccc ggc gtg gcc agg aca gag ctg ggc aga cac 771Val Asn Ala Leu
His Pro Gly Val Ala Arg Thr Glu Leu Gly Arg His 155 160 165acg ggc
atc cat ggc tcc acc ttc tcc agc acc aca ctc ggg ccc atc 819Thr Gly
Ile His Gly Ser Thr Phe Ser Ser Thr Thr Leu Gly Pro Ile170 175 180
185ttc tgg ctg ctg gtc aag agc ccc gag ctg gcc gcc cag ccc agc aca
867Phe Trp Leu Leu Val Lys Ser Pro Glu Leu Ala Ala Gln Pro Ser Thr
190 195 200tac ctg gcc gtg gcg gag gaa ctg gcg gat gtt tcc gga aag
tac ttc 915Tyr Leu Ala Val Ala Glu Glu Leu Ala Asp Val Ser Gly Lys
Tyr Phe 205 210 215gat gga ctc aaa cag aag gcc ccg gcc ccc gag gct
gag gat gag gag 963Asp Gly Leu Lys Gln Lys Ala Pro Ala Pro Glu Ala
Glu Asp Glu Glu 220 225 230gtg gcc cgg agg ctt tgg gct gaa agt gcc
cgc ctg gtg ggc tta gag 1011Val Ala Arg Arg Leu Trp Ala Glu Ser Ala
Arg Leu Val Gly Leu Glu 235 240 245gct ccc tct gtg agg gag cag ccc
ctc ccc aga taa cctctggagc 1057Ala Pro Ser Val Arg Glu Gln Pro Leu
Pro Arg *250 255 260agatttgaaa gccaggatgg cgcctccaga ccgaggacag
ctgtccgcca tgcccgcagc 1117ttcctggcac tacctgagcc gggagaccca ggactg
11539331PRTHomo sapiens 9Met Ser Arg Tyr Leu Leu Pro Leu Ser Ala
Leu Gly Thr Val Ala Gly 1 5 10 15Ala Ala Val Leu Leu Lys Asp Tyr
Val Thr Gly Gly Ala Cys Pro Ser 20 25 30Lys Ala Thr Ile Pro Gly Lys
Thr Val Ile Val Thr Gly Ala Asn Thr 35 40 45Gly Ile Gly Lys Gln Thr
Ala Leu Glu Leu Ala Arg Arg Gly Gly Asn 50 55 60Ile Ile Leu Ala Cys
Arg Asp Met Glu Lys Cys Glu Ala Ala Ala Lys65 70 75 80Asp Ile Arg
Gly Glu Thr Leu Asn His His Val Asn Ala Arg His Leu 85 90 95Asp Leu
Ala Ser Leu Lys Ser Ile Arg Glu Phe Ala Ala Lys Ile Ile 100 105
110Glu Glu Glu Glu Arg Val Asp Ile Leu Ile Asn Asn Ala Gly Val Met
115 120 125Arg Cys Pro His Trp Thr Thr Glu Asp Gly Phe Glu Met Gln
Phe Gly 130 135 140Val Asn His Leu Gly His Phe Leu Leu Thr Asn Leu
Leu Leu Asp Lys145 150 155 160Leu Lys Ala Ser Ala Pro Ser Arg Ile
Ile Asn Leu Ser Ser Leu Ala 165 170 175His Val Ala Gly His Ile Asp
Phe Asp Asp Leu Asn Trp Gln Thr Arg 180 185 190Lys Tyr Asn Thr Lys
Ala Ala Tyr Cys Gln Ser Lys Leu Ala Ile Val 195 200 205Leu Phe Thr
Lys Glu Leu Ser Arg Arg Leu Gln Gly Ser Gly Val Thr 210 215 220Val
Asn Ala Leu His Pro Gly Val Ala Arg Thr Glu Leu Gly Arg His225 230
235 240Thr Gly Ile His Gly Ser Thr Phe Ser Ser Thr Thr Leu Gly Pro
Ile 245 250 255Phe Trp Leu Leu Val Lys Ser Pro Glu Leu Val Ala Gln
Pro Ser Thr 260 265 270Tyr Leu Ala Val Ala Glu Glu Leu Ala Asp Val
Ser Gly Lys Tyr Phe 275 280 285Asp Gly Leu Lys Gln Lys Ala Pro Ala
Pro Glu Ala Glu Asp Glu Glu 290 295 300Val Ala Arg Arg Leu Trp Ala
Glu Ser Ala Arg Leu Val Gly Leu Glu305 310 315 320Ala Pro Ser Val
Arg Glu Gln Pro Leu Pro Arg 325 330101699DNAHomo
sapiensCDS(538)...(1533)misc_feature3, 19, 71, 74, 111, 138, 160n =
A,T,C or G 10gcntgtgggt cccttcttna aattgggtcc ccccgtttta ggtaagttta
aaagctcaag 60gttcaaagac nggncctttt gtcgggggct ccttgaagcc tactagatca
ncggctctca 120gctttttttt ttgggggncc cccccctttg ggaacccccn
tggctttgct tcaaacttct 180aaggtctttt gtttcgtttt ctgttcctgc
gccgttacag atccaagytc tgaaaaacca 240gaaagttaac tggtaagttt
agtctttttg tcttttattt caggtcccgg atccggtggt 300ggtgcaaatc
aaagaactgc tcctcagtgg atgttgcctt tacttctagg cctgtacgaa
360gtgttacttc tgctctaaaa gctgcggaat tctaatacga ctcactatag
ggagtcgacc 420cacgcgtccg cggacgcgtg ggcggacgcg tgggcggagc
tacccaggcg gctggtgtgc 480agcaagctcc gcgccgactc cggacgcctg
acgcctgacg cctgtccccg gcccggc atg 540 Met 1agc cgc tac ctg ctg ccg
ctg tcg gcg ctg ggc acg gta gca ggc gcc 588Ser Arg Tyr Leu Leu Pro
Leu Ser Ala Leu Gly Thr Val Ala Gly Ala 5 10 15gcc gtg ctg ctc aag
gac tat gtc acc ggt ggg gct tgc ccc agc aag 636Ala Val Leu Leu Lys
Asp Tyr Val Thr Gly Gly Ala Cys Pro Ser Lys 20 25 30gcc acc atc cct
ggg aag acg gtc atc gtg acg ggc gcc aac aca ggc 684Ala Thr Ile Pro
Gly Lys Thr Val Ile Val Thr Gly Ala Asn Thr Gly 35 40 45atc ggg aag
cag acc gcc ttg gaa ctg gcc agg aga gga ggc aac atc 732Ile Gly Lys
Gln Thr Ala Leu Glu Leu Ala Arg Arg Gly Gly Asn Ile 50 55 60 65atc
ctg gcc tgc cga gac atg gag aag tgt gag gcg gca gca aag gac 780Ile
Leu Ala Cys Arg Asp Met Glu Lys Cys Glu Ala Ala Ala Lys Asp 70 75
80atc cgc ggg gag acc ctc aat cac cat gtc aac gcc cgg cac ctg gac
828Ile Arg Gly Glu Thr Leu Asn His His Val Asn Ala Arg His Leu Asp
85 90 95ttg gct tcc ctc aag tct atc cga gag ttt gca gca aag atc att
gaa 876Leu Ala Ser Leu Lys Ser Ile Arg Glu Phe Ala Ala Lys Ile Ile
Glu 100 105 110gag gag gag cga gtg gac att cta atc aac aac gcg ggt
gtg atg cgg 924Glu Glu Glu Arg Val Asp Ile Leu Ile Asn Asn Ala Gly
Val Met Arg 115 120 125tgc ccc cac tgg acc acc gag gac ggc ttc gag
atg cag ttt ggc gtt 972Cys Pro His Trp Thr Thr Glu Asp Gly Phe Glu
Met Gln Phe Gly Val130 135 140 145aac cac ctg ggt cac ttt ctc ttg
aca aac ttg ctg ctg gac aag ctg 1020Asn His Leu Gly His Phe Leu Leu
Thr Asn Leu Leu Leu Asp Lys Leu 150 155 160aaa gcc tca gcc cct tcg
cgg atc atc aac ctc tcg tcc ctg gcc cat 1068Lys Ala Ser Ala Pro Ser
Arg Ile Ile Asn Leu Ser Ser Leu Ala His 165 170 175gtt gct ggg cac
ata gac ttt gac gac ttg aac tgg cag acg agg aag 1116Val Ala Gly His
Ile Asp Phe Asp Asp Leu Asn Trp Gln Thr Arg Lys 180 185 190tat aac
acc aaa gcc gcc tac tgc cag agc aag ctc gcc atc gtc ctc 1164Tyr Asn
Thr Lys Ala Ala Tyr Cys Gln Ser Lys Leu Ala Ile Val Leu 195 200
205ttc acc aag gag ctg agc cgg cgg ctg caa ggc tct ggt gtg act gtc
1212Phe Thr Lys Glu Leu Ser Arg Arg Leu Gln Gly Ser Gly Val Thr
Val210 215 220 225aac gcc ctg cac ccc ggc gtg gcc agg aca gag ctg
ggc aga cac acg 1260Asn Ala Leu His Pro Gly Val Ala Arg Thr Glu Leu
Gly Arg His Thr 230 235 240ggc atc cat ggc tcc acc ttc tcc agc acc
aca ctc ggg ccc atc ttc 1308Gly Ile His Gly Ser Thr Phe Ser Ser Thr
Thr Leu Gly Pro Ile Phe 245 250 255tgg ctg ctg gtc aag agc ccc gag
ctg gtc gcc cag ccc agc aca tac 1356Trp Leu Leu Val Lys Ser Pro Glu
Leu Val Ala Gln Pro Ser Thr Tyr 260 265 270ctg gcc gtg gcg gag gaa
ctg gcg gat gtt tcc gga aag tac ttc gat 1404Leu Ala Val Ala Glu Glu
Leu Ala Asp Val Ser Gly Lys Tyr Phe Asp 275 280 285gga ctc aaa cag
aag gcc ccg gcc ccc gag gct gag gat gag gag gtg 1452Gly Leu Lys Gln
Lys Ala Pro Ala Pro Glu Ala Glu Asp Glu Glu Val290 295 300 305gcc
cgg agg ctt tgg gct gaa agt gcc cgc ctg gtg ggc tta gag gct 1500Ala
Arg Arg Leu Trp Ala Glu Ser Ala Arg Leu Val Gly Leu Glu Ala 310 315
320ccc tct gtg agg gag cag ccc ctc ccc aga taa cctctggagc
agatttgaaa 1553Pro Ser Val Arg Glu Gln Pro Leu Pro Arg * 325
330gccaggatgg cgcctccaga ccgaggacag ctgtccgcca tgcccgcagc
ttcctggcac 1613tacctgagcc gggagaccca ggactggcgg ccgctagact
agtctagaga aaaaacctcc 1673cacacctccc cctgaacctg aaacat
169911418PRTHomo sapiens 11Met Leu Pro Asn Thr Gly Arg Leu Ala Gly
Cys Thr Val Phe Ile Thr 1 5 10 15Gly Ala Ser Arg Gly Ile Gly Lys
Ala Ile Ala Leu Lys Ala Ala Lys 20 25 30Asp Gly Ala Asn Ile Val Ile
Ala Ala Lys Thr Ala Gln Pro His Pro 35 40 45Lys Leu Leu Gly Thr Ile
Tyr Thr Ala Ala Glu Glu Ile Glu Ala Val 50 55 60Gly Gly Lys Ala Leu
Pro Cys Ile Val Asp Val Arg Asp Glu Gln Gln65 70 75 80Ile Ser Ala
Ala Val Glu Lys Ala Ile Lys Lys Phe Gly Gly Ile Asp 85 90 95Ile Leu
Val Asn Asn Ala Ser Ala Ile Ser Leu Thr Asn Thr Leu Asp 100 105
110Thr Pro Thr Lys Arg Leu Asp Leu Met Met Asn Val Asn Thr Arg Gly
115 120 125Thr Tyr Leu Ala Ser Lys Ala Cys Ile Pro Tyr Leu Lys Lys
Ser Lys 130 135 140Val Ala His Ile Leu Asn Ile Ser Pro Pro Leu Asn
Leu Asn Pro Val145 150 155 160Trp Phe Lys Gln His Cys Ala Tyr Thr
Ile Ala Lys Tyr Gly Met Ser 165 170 175Met Tyr Val Leu Gly Met Ala
Glu Glu Phe Lys Gly Glu Ile Ala Val 180 185 190Asn Ala Leu Trp Pro
Lys Thr Ala Ile His Thr Ala Ala Met Asp Met 195 200 205Leu Gly Gly
Pro Gly Ile Glu Ser Gln Cys Arg Lys Val Asp Ile Ile 210 215 220Ala
Asp Ala Ala Tyr Ser Ile Phe Gln Lys Pro Lys Ser Phe Thr Gly225 230
235 240Asn Phe Val Ile Asp Glu Asn Ile Leu Lys Glu Glu Gly Ile Glu
Asn 245 250 255Phe Asp Val Tyr Ala Ile Lys Pro Gly His Pro Leu Gln
Pro Asp Phe 260 265 270Phe Leu Asp Glu Tyr Pro Glu Ala Val Ser Lys
Lys Val Glu Ser Thr 275 280 285Gly Ala Val Pro Glu Phe Lys Glu Glu
Lys Leu Gln Leu Gln Pro Lys 290 295 300Pro Arg Ser Gly Ala Val Glu
Glu Thr Phe Arg Ile Val Lys Asp Ser305 310 315 320Leu Ser Asp Asp
Val Val Lys Ala Thr Gln Ala Ile Tyr Leu Phe Glu 325 330 335Leu Ser
Gly Glu Asp Gly Gly Thr Trp Phe Leu Asp Leu Lys Ser Lys 340 345
350Gly Gly Asn Val Gly Tyr Gly Glu Pro Ser Asp Gln Ala Asp Val Val
355 360 365Met Ser Met Thr Thr Asp Asp Phe Val Lys Met Phe Ser Gly
Lys Leu 370 375 380Lys Pro Thr Met Ala Phe Met Ser Gly Lys Leu Lys
Ile Lys Gly Asn385 390 395 400Met Ala Leu Ala Ile Lys Leu Glu Lys
Leu Met Asn Gln Met Asn Ala 405 410 415Arg Leu122535DNAHomo
sapiensCDS(762)...(2018)misc_feature89n = A,T,C or G 12aggcagaagt
atgcaaagca tgcatctcaa attagtcagc aaaccatagt cccggcccct 60aactccgccc
atcccgcccc taactccgnc ccagttccgg cccattctcc gccccatggc
120tgactaattt tttttattta tgcagagccg aggccgcctc ggcctctgag
ctattccaga 180agtagtgagg aggctttttt ggaggcctag gcttttgcaa
aaagctcctc gatcgagggg 240ctcgcatctc tccttcacgc gcccgccgcc
ctacctgagg ccgccatcca cgccggttga 300gtcgcgttct gccgcctccc
gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa 360gtttaaagct
caggtcgaga ccgggccttt gtccggcgct cccttggagc ctacctagac
420tcagccggct ctccacgctt tgcctgaccc tgcttgctca actctacgtc
tttgtttcag 480ttttctgttc tgcgccgtta cagatccaag ctctgaaaaa
ccagaaagtt aactggtaag 540tttagtcttt ttgtctttta tttcaggtcc
cggatccggt ggtggtgcaa atcaaagaac 600tgctcctcag tggatgttgc
ctttacttct aggcctgtac ggaagtgtta cttctgctct 660aaaagctgcg
gaattctaat acgactcact atagggwgtc gacccacgcg tccgctcgcc
720gccgccgctg tcgccgccac ctcctctgat ctacgaaagt c atg tta ccc aac
acc 776 Met Leu Pro Asn Thr 1 5ggg agg ctg gca gga tgt aca gtt ttt
atc aca ggt gca agc cgt ggc 824Gly Arg Leu Ala Gly Cys Thr Val Phe
Ile Thr Gly Ala Ser Arg Gly 10 15 20att ggc aaa gct att gca ttg aaa
gca gca aag gat gga gca aat att 872Ile Gly Lys Ala Ile Ala Leu Lys
Ala Ala Lys Asp Gly Ala Asn Ile 25 30 35gtt att gct gca aag acc gcc
cag cca cat cca aaa ctt cta ggc aca 920Val Ile Ala Ala Lys Thr Ala
Gln Pro His Pro Lys Leu Leu Gly Thr 40 45 50atc tat act gct gct gaa
gaa att gaa gca gtt gga gga aag gcc ttg 968Ile Tyr Thr Ala Ala Glu
Glu Ile Glu Ala Val Gly Gly Lys Ala Leu 55 60 65cca tgt att gtt gat
gtg aga gat gaa cag cag atc agt gct gca gtg 1016Pro Cys Ile Val Asp
Val Arg Asp Glu Gln Gln Ile Ser Ala Ala Val 70 75 80 85gag aaa gcc
atc aag aaa ttt gga gga att gat att ctg gta aat aat 1064Glu Lys Ala
Ile Lys Lys Phe Gly Gly Ile Asp Ile Leu Val Asn Asn 90 95 100gcc
agt gcc att agt ttg acc aat aca ttg gac aca cct acc aag aga 1112Ala
Ser Ala Ile Ser Leu Thr Asn Thr Leu Asp Thr Pro Thr Lys Arg 105 110
115ttg gat ctg atg atg aac gtg aac acc aga ggc acc tac ctt gca tct
1160Leu Asp Leu Met Met Asn Val Asn Thr Arg Gly Thr Tyr Leu Ala Ser
120 125 130aaa gca tgt att cct tat ttg aaa aag agc aaa gtt gct cat
atc ctc 1208Lys Ala Cys Ile Pro Tyr Leu Lys Lys Ser Lys Val Ala His
Ile Leu 135 140 145aat atc agt cca cca ctg aac cta aat cca gtt tgg
ttc aaa cag cac 1256Asn Ile Ser Pro Pro Leu Asn Leu Asn Pro Val Trp
Phe Lys Gln His150 155 160 165tgt gct tat acc att gct aag tat ggt
atg tct atg tat gtg ctt gga 1304Cys Ala Tyr Thr Ile Ala Lys Tyr Gly
Met Ser Met Tyr Val Leu Gly 170 175 180atg gca gaa gaa ttt aaa ggt
gaa att gca gtc aat gca tta tgg cct 1352Met Ala Glu Glu Phe Lys Gly
Glu Ile Ala Val Asn Ala Leu Trp Pro 185 190 195aaa aca gcc ata cac
act gct gct atg gat atg ctg gga gga cct ggt 1400Lys Thr Ala Ile His
Thr Ala Ala Met Asp Met Leu Gly Gly Pro Gly 200 205 210atc gaa agc
cag tgt aga aaa gtt gat atc att gca gat gca gca tat 1448Ile Glu Ser
Gln Cys Arg Lys Val Asp Ile Ile Ala Asp Ala Ala Tyr 215 220 225tcc
att ttc caa aag cca aaa agt ttt act ggc aac ttt gtc att gat 1496Ser
Ile Phe Gln Lys Pro Lys Ser Phe Thr Gly Asn Phe Val Ile Asp230 235
240 245gaa aat atc tta aaa gaa gaa gga ata gaa aat ttt gac gtt tat
gca 1544Glu Asn Ile Leu Lys Glu Glu Gly Ile Glu Asn Phe Asp Val Tyr
Ala 250 255 260att aaa cca ggt cat cct ttg caa cca gat ttc ttc tta
gat gaa tac 1592Ile Lys Pro Gly His Pro Leu Gln Pro Asp Phe Phe Leu
Asp Glu Tyr 265 270 275cca gaa gca gtt agc aag aaa gtg gaa tca act
ggt gct gtt cca gaa 1640Pro Glu Ala Val Ser Lys Lys Val Glu Ser Thr
Gly Ala Val Pro Glu 280 285 290ttc aaa gaa gag aaa ctg cag ctg caa
cca aaa cca cgt tct gga gct 1688Phe Lys Glu Glu Lys Leu Gln Leu Gln
Pro Lys Pro Arg Ser Gly Ala 295 300 305gtg gaa gaa aca ttt aga att
gtt aag gac tct ctc agt gat gat gtt 1736Val Glu Glu Thr Phe Arg Ile
Val Lys Asp Ser Leu Ser Asp Asp Val310 315 320 325gtt aaa gcc act
caa gca atc tat ctg ttt gaa ctc tcc ggt gaa gat 1784Val Lys Ala Thr
Gln Ala Ile Tyr Leu Phe Glu Leu Ser Gly Glu Asp 330 335 340ggt ggc
acg tgg ttt ctt gat ctg aaa agc aag ggt ggg aat gtc gga 1832Gly Gly
Thr Trp Phe Leu Asp Leu Lys Ser Lys Gly Gly Asn Val Gly 345 350
355tat gga gag cct tct gat cag gca gat gtg gtg atg agt atg act
act
1880Tyr Gly Glu Pro Ser Asp Gln Ala Asp Val Val Met Ser Met Thr Thr
360 365 370gat gac ttt gta aaa atg ttt tca ggg aaa cta aaa cca aca
atg gca 1928Asp Asp Phe Val Lys Met Phe Ser Gly Lys Leu Lys Pro Thr
Met Ala 375 380 385ttc atg tca ggg aaa ttg aag att aaa ggt aac atg
gcc cta gca atc 1976Phe Met Ser Gly Lys Leu Lys Ile Lys Gly Asn Met
Ala Leu Ala Ile390 395 400 405aaa ttg gag aag cta atg aat cag atg
aat gcc aga ctg tga 2018Lys Leu Glu Lys Leu Met Asn Gln Met Asn Ala
Arg Leu * 410 415aggaaaatat aaaaaaaaag tcgactgcta tgctcaaaaa
gtaaaaaaag ctcaacagtt 2078aaaatctaat gtttgttttc tttcctgtta
tattataagg atatgcacgt ttgttctgga 2138aaagatagaa tttgtctcta
aaagacttga aattgtaatt aaaatggcaa gctaatcaaa 2198cataagcttc
attaagtggg attctaagac agtctgtgtt tttatatttc aagggtttaa
2258ccctttgagc cttacatctc attcactgtc tttctccaag aaaagtattt
tgggcggaca 2318gtcagatcaa gcagtaaaat tagctctttc aaatcttctt
gtcatgtaaa atgaagctag 2378tctgttttaa aatttttagt tttggattgt
atactaatga aaatcttaat gatgttttkr 2438wtttttatat acytawtttw
aarraaawyy twwwwwrkwc mttttwmcaa aaawtwttaa 2498aaawkrrwww
kwrytskgsg mgraswmwaw rwrammc 253513245PRTHomo sapiens 13Met Gly
Arg Leu Asp Gly Lys Val Ile Ile Leu Thr Ala Ala Ala Gln 1 5 10
15Gly Ile Gly Gln Ala Ala Ala Leu Ala Phe Ala Arg Glu Gly Ala Lys
20 25 30Val Ile Ala Thr Asp Ile Asn Glu Ser Lys Leu Gln Glu Leu Glu
Lys 35 40 45Tyr Pro Gly Ile Gln Thr Arg Val Leu Asp Val Thr Lys Lys
Lys Gln 50 55 60Ile Asp Gln Phe Ala Asn Glu Val Glu Arg Leu Asp Val
Leu Phe Asn65 70 75 80Val Ala Gly Phe Val His His Gly Thr Val Leu
Asp Cys Glu Glu Lys 85 90 95Asp Trp Asp Phe Ser Met Asn Leu Asn Val
Arg Ser Met Tyr Leu Met 100 105 110Ile Lys Ala Phe Leu Pro Lys Met
Leu Ala Gln Lys Ser Gly Asn Ile 115 120 125Ile Asn Met Ser Ser Val
Ala Ser Ser Val Lys Gly Val Val Asn Arg 130 135 140Cys Val Tyr Ser
Thr Thr Lys Ala Ala Val Ile Gly Leu Thr Lys Ser145 150 155 160Val
Ala Ala Asp Phe Ile Gln Gln Gly Ile Arg Cys Asn Cys Val Cys 165 170
175Pro Gly Thr Val Asp Thr Pro Ser Leu Gln Glu Arg Ile Gln Ala Arg
180 185 190Gly Asn Pro Glu Glu Ala Arg Asn Asp Phe Leu Lys Arg Gln
Lys Thr 195 200 205Gly Arg Phe Ala Thr Ala Glu Glu Ile Ala Met Leu
Cys Val Tyr Leu 210 215 220Ala Ser Asp Glu Ser Ala Tyr Val Thr Gly
Asn Pro Val Ile Ile Asp225 230 235 240Gly Gly Trp Ser Leu
245141716DNAHomo sapiensCDS(637)...(1374)misc_feature15n = A,T,C or
G 14atgcaaaagc cgagnccgcc tcggcctcta agctattcca gaagtagtaa
gaaggctttt 60ttgaaggcct aggcttttgc aaaaagctcc tcgatcgagg ggctcgcatc
tctccttcac 120ggggccgccg ccctacctga ggccgccatc cacgccggtt
gagtcgcgtt ctgccgcctc 180ccgcctgtgg tgcctcctga actgcgtccg
ccgtytaggt aagtttaaag ctcaggtcga 240gaccgggcct ttgtccggcg
ctcccttgga gcctacctag actcagccgg ctctccacgc 300tttgcctgac
cctgcttgct caactctacg tctttgtttc gttttctgtt ctgcgccgtt
360acagatccaa gctctgaaaa accagaaagt taactggtaa gtttagtctt
tttgtctttt 420atttcaggtc ccggatccgg tggtggtgca aatcaaagaa
ctgctcctca gtggatgttg 480cctttacttc taggcctgta cggaagtgtt
acttctgctc taaaagctgc ggaattctaa 540tacgactcac tatagggagt
cgacccacgc gtccgcaaac cgagttctgg agaacgccat 600cagctcgctg
cttaaaatta aaccacaggt tccatt atg ggt cga ctt gat ggg 654 Met Gly
Arg Leu Asp Gly 1 5aaa gtc atc atc ctg acg gcc gct gct cag ggg att
ggc caa gca gct 702Lys Val Ile Ile Leu Thr Ala Ala Ala Gln Gly Ile
Gly Gln Ala Ala 10 15 20gcc tta gct ttt gca aga gaa ggt gcc aaa gtc
ata gcc aca gac att 750Ala Leu Ala Phe Ala Arg Glu Gly Ala Lys Val
Ile Ala Thr Asp Ile 25 30 35aat gag tcc aaa ctt cag gaa ctg gaa aag
tac ccg ggt att caa act 798Asn Glu Ser Lys Leu Gln Glu Leu Glu Lys
Tyr Pro Gly Ile Gln Thr 40 45 50cgt gtc ctt gat gtc aca aag aag aaa
caa att gat cag ttt gcc aat 846Arg Val Leu Asp Val Thr Lys Lys Lys
Gln Ile Asp Gln Phe Ala Asn 55 60 65 70gaa gtt gag aga ctt gat gtt
ctc ttt aat gtt gct ggt ttt gtc cat 894Glu Val Glu Arg Leu Asp Val
Leu Phe Asn Val Ala Gly Phe Val His 75 80 85cat gga act gtc ctg gat
tgt gag gag aaa gac tgg gac ttc tcg atg 942His Gly Thr Val Leu Asp
Cys Glu Glu Lys Asp Trp Asp Phe Ser Met 90 95 100aat ctc aat gtg
cgc agc atg tac ctg atg atc aag gca ttc ctt cct 990Asn Leu Asn Val
Arg Ser Met Tyr Leu Met Ile Lys Ala Phe Leu Pro 105 110 115aaa atg
ctt gct cag aaa tct ggc aat att atc aac atg tct tct gtg 1038Lys Met
Leu Ala Gln Lys Ser Gly Asn Ile Ile Asn Met Ser Ser Val 120 125
130gct tcc agc gtc aaa gga gtt gtg aac aga tgt gtg tac agc aca acc
1086Ala Ser Ser Val Lys Gly Val Val Asn Arg Cys Val Tyr Ser Thr
Thr135 140 145 150aag gca gcc gtg att ggc ctc aca aaa tct gtg gct
gca gat ttc atc 1134Lys Ala Ala Val Ile Gly Leu Thr Lys Ser Val Ala
Ala Asp Phe Ile 155 160 165cag cag ggc atc agg tgc aac tgt gtg tgc
cca gga aca gtt gat acg 1182Gln Gln Gly Ile Arg Cys Asn Cys Val Cys
Pro Gly Thr Val Asp Thr 170 175 180cca tct cta caa gaa aga ata caa
gcc aga gga aat cct gaa gag gca 1230Pro Ser Leu Gln Glu Arg Ile Gln
Ala Arg Gly Asn Pro Glu Glu Ala 185 190 195cgg aat gat ttc ctg aag
aga caa aag acg gga aga ttc gca act gca 1278Arg Asn Asp Phe Leu Lys
Arg Gln Lys Thr Gly Arg Phe Ala Thr Ala 200 205 210gaa gaa ata gcc
atg ctc tgc gtg tat ttg gct tct gat gaa tct gct 1326Glu Glu Ile Ala
Met Leu Cys Val Tyr Leu Ala Ser Asp Glu Ser Ala215 220 225 230tat
gta act ggt aac cct gtc atc att gat gga ggc tgg agc ttg tga 1374Tyr
Val Thr Gly Asn Pro Val Ile Ile Asp Gly Gly Trp Ser Leu * 235 240
245ttttaggatc tccatggtgg gaaggaaggc aggcccttcc tatccacagt
gaacctggtt 1434acgaagaaaa ctcaccaatc atctccttcc tgttaatcac
atgttaatga aaataagctc 1494tttttaatga tgtcactgtt tgcaagagtc
tgattcttta agtatattaa tctctttgta 1554atctcttctg aaatcattgt
aaagaaataa aaatattgaa ctcaaaaaaa aaaaaaaaaa 1614aagggcggcc
gctagactag tctagagaaa aaacctccca cacctccccc tgaacctgaa
1674acataaaatg aatgcmattg ttgktggtaa cttgttattg ca
1716151123PRTHomo sapiens 15Met Pro Ile Val Asp Lys Leu Lys Glu Ala
Leu Lys Pro Gly Arg Lys 1 5 10 15Asp Ser Ala Asp Asp Gly Glu Leu
Gly Lys Leu Leu Ala Ser Ser Ala 20 25 30Lys Lys Val Leu Leu Gln Lys
Ile Glu Phe Glu Pro Ala Ser Lys Ser 35 40 45Phe Ser Tyr Gln Leu Glu
Ala Leu Lys Ser Lys Tyr Val Leu Leu Asn 50 55 60Pro Lys Thr Glu Gly
Ala Ser Arg His Lys Ser Gly Asp Asp Pro Pro65 70 75 80Ala Arg Arg
Gln Gly Ser Glu His Thr Tyr Glu Ser Cys Gly Asp Gly 85 90 95Val Pro
Ala Pro Gln Lys Val Leu Phe Pro Thr Glu Arg Leu Ser Leu 100 105
110Arg Trp Glu Arg Val Phe Arg Val Gly Ala Gly Leu His Asn Leu Gly
115 120 125Asn Thr Cys Phe Leu Asn Ala Thr Ile Gln Cys Leu Thr Tyr
Thr Pro 130 135 140Pro Leu Ala Asn Tyr Leu Leu Ser Lys Glu His Ala
Arg Ser Cys His145 150 155 160Gln Gly Ser Phe Cys Met Leu Cys Val
Met Gln Asn His Ile Val Gln 165 170 175Ala Phe Ala Asn Ser Gly Asn
Ala Ile Lys Pro Val Ser Phe Ile Arg 180 185 190Asp Leu Lys Lys Ile
Ala Arg His Phe Arg Phe Gly Asn Gln Glu Asp 195 200 205Ala His Glu
Phe Leu Arg Tyr Thr Ile Asp Ala Met Gln Lys Ala Cys 210 215 220Leu
Asn Gly Cys Ala Lys Leu Asp Arg Gln Thr Gln Ala Thr Thr Leu225 230
235 240Val His Gln Ile Phe Gly Gly Tyr Leu Arg Ser Arg Val Lys Cys
Ser 245 250 255Val Cys Lys Ser Val Ser Asp Thr Tyr Asp Pro Tyr Leu
Asp Val Ala 260 265 270Leu Glu Ile Arg Gln Ala Ala Asn Ile Val Arg
Ala Leu Glu Leu Phe 275 280 285Val Lys Ala Asp Val Leu Ser Gly Glu
Asn Ala Tyr Met Cys Ala Lys 290 295 300Cys Lys Lys Lys Val Pro Ala
Ser Lys Arg Phe Thr Ile His Arg Thr305 310 315 320Ser Asn Val Leu
Thr Leu Ser Leu Lys Arg Phe Ala Asn Phe Ser Gly 325 330 335Gly Lys
Ile Thr Lys Asp Val Gly Tyr Pro Glu Phe Leu Asn Ile Arg 340 345
350Pro Tyr Met Ser Gln Asn Asn Gly Asp Pro Val Met Tyr Gly Leu Tyr
355 360 365Ala Val Leu Val His Ser Gly Tyr Ser Cys His Ala Gly His
Tyr Tyr 370 375 380Cys Tyr Val Lys Ala Ser Asn Gly Gln Trp Tyr Gln
Met Asn Asp Ser385 390 395 400Leu Val His Ser Ser Asn Val Lys Val
Val Leu Asn Gln Gln Ala Tyr 405 410 415Val Leu Phe Tyr Leu Arg Ile
Pro Gly Ser Lys Lys Ser Pro Glu Gly 420 425 430Leu Ile Ser Arg Thr
Gly Ser Ser Ser Leu Pro Gly Arg Pro Ser Val 435 440 445Ile Pro Asp
His Ser Lys Lys Asn Ile Gly Asn Gly Ile Ile Ser Ser 450 455 460Pro
Leu Thr Gly Lys Arg Gln Asp Ser Gly Thr Met Lys Lys Pro His465 470
475 480Thr Thr Glu Glu Ile Gly Val Pro Ile Ser Arg Asn Gly Ser Thr
Leu 485 490 495Gly Leu Lys Ser Gln Asn Gly Cys Ile Pro Pro Lys Leu
Pro Ser Gly 500 505 510Ser Pro Ser Pro Lys Leu Ser Gln Thr Pro Thr
His Met Pro Thr Ile 515 520 525Leu Asp Asp Pro Gly Lys Lys Val Lys
Lys Pro Ala Pro Pro Gln His 530 535 540Phe Ser Pro Arg Thr Ala Gln
Gly Leu Pro Gly Thr Ser Asn Ser Asn545 550 555 560Ser Ser Arg Ser
Gly Ser Gln Arg Gln Gly Ser Trp Asp Ser Arg Asp 565 570 575Val Val
Leu Ser Thr Ser Pro Lys Leu Leu Ala Thr Ala Thr Ala Asn 580 585
590Gly His Gly Leu Lys Gly Asn Asp Glu Ser Ala Gly Leu Asp Arg Arg
595 600 605Gly Ser Ser Ser Ser Ser Pro Glu His Ser Ala Ser Ser Asp
Ser Thr 610 615 620Lys Ala Pro Gln Thr Pro Arg Ser Gly Ala Ala His
Leu Cys Asp Ser625 630 635 640Gln Glu Thr Asn Cys Ser Thr Ala Gly
His Ser Lys Thr Pro Pro Ser 645 650 655Gly Ala Asp Ser Lys Thr Val
Lys Leu Lys Ser Pro Val Leu Ser Asn 660 665 670Thr Thr Thr Glu Pro
Ala Ser Thr Met Ser Pro Pro Pro Ala Lys Lys 675 680 685Leu Ala Leu
Ser Ala Lys Lys Ala Ser Thr Leu Trp Arg Ala Thr Gly 690 695 700Asn
Asp Leu Arg Pro Pro Pro Pro Ser Pro Ser Ser Asp Leu Thr His705 710
715 720Pro Met Lys Thr Ser His Pro Val Val Ala Ser Thr Trp Pro Val
His 725 730 735Arg Ala Arg Ala Val Ser Pro Ala Pro Gln Ser Ser Ser
Arg Leu Gln 740 745 750Pro Pro Phe Ser Pro His Pro Thr Leu Leu Ser
Ser Thr Pro Lys Pro 755 760 765Pro Gly Thr Ser Glu Pro Arg Ser Cys
Ser Ser Ile Ser Thr Ala Leu 770 775 780Pro Gln Val Asn Glu Asp Leu
Val Ser Leu Pro His Gln Leu Pro Glu785 790 795 800Ala Ser Glu Pro
Pro Gln Ser Pro Ser Glu Lys Arg Lys Lys Thr Phe 805 810 815Val Gly
Glu Pro Gln Arg Leu Gly Ser Glu Thr Arg Leu Pro Gln His 820 825
830Ile Arg Glu Ala Thr Ala Ala Pro His Gly Lys Arg Lys Arg Lys Lys
835 840 845Lys Lys Arg Pro Glu Asp Thr Ala Ala Ser Ala Leu Gln Glu
Gly Gln 850 855 860Thr Gln Arg Gln Pro Gly Ser Pro Met Tyr Arg Arg
Glu Gly Gln Ala865 870 875 880Gln Leu Pro Ala Val Arg Arg Gln Glu
Asp Gly Thr Gln Pro Gln Val 885 890 895Asn Gly Gln Gln Val Gly Cys
Val Thr Asp Gly His His Ala Ser Ser 900 905 910Arg Lys Arg Arg Arg
Lys Gly Ala Glu Gly Leu Gly Glu Glu Gly Gly 915 920 925Leu His Gln
Asp Pro Leu Arg His Ser Cys Ser Pro Met Gly Asp Gly 930 935 940Asp
Pro Glu Ala Met Glu Glu Ser Pro Arg Lys Lys Lys Lys Lys Lys945 950
955 960Arg Lys Gln Glu Thr Gln Arg Ala Val Glu Glu Asp Gly His Leu
Lys 965 970 975Cys Pro Arg Ser Ala Lys Pro Gln Asp Ala Val Val Pro
Glu Ser Ser 980 985 990Ser Cys Ala Pro Ser Ala Asn Gly Trp Cys Pro
Gly Asp Arg Met Gly 995 1000 1005Leu Ser Gln Ala Pro Pro Val Ser
Trp Asn Gly Glu Arg Glu Ser Asp 1010 1015 1020Val Val Gln Glu Leu
Leu Lys Tyr Ser Ser Asp Lys Ala Tyr Gly Arg1025 1030 1035 1040Lys
Val Leu Thr Trp Asp Gly Lys Met Ser Ala Val Ser Gln Asp Ala 1045
1050 1055Ile Glu Asp Ser Arg Gln Ala Arg Thr Glu Thr Val Val Asp
Asp Trp 1060 1065 1070Asp Glu Glu Phe Asp Arg Gly Lys Glu Lys Lys
Ile Lys Lys Phe Lys 1075 1080 1085Arg Glu Lys Arg Arg Asn Phe Asn
Ala Phe Gln Lys Leu Gln Thr Arg 1090 1095 1100Arg Asn Phe Trp Ser
Val Thr His Pro Ala Lys Ala Ala Ser Leu Ser1105 1110 1115 1120Tyr
Arg Arg163941DNAHomo sapiensCDS(279)...(3650) 16cacgcgtccg
ggcgccggag gcccggatgg tgcgcgtgct gggccgcggg ccgaaggagt 60cgccagggct
gcgtaggctt gtggcgcgcc cgcggagagg ccggggctct gacgcccgct
120ctgcggcttc ggtgtttgaa caggccacag tccaggagcg cttacattca
ggagctccgc 180gtagcacctg cccaaccaaa ctcagccctc cgttaagatc
ctggttccat gccgcagtag 240gacagcaggc ccaagtctgc acatcccagt gatgcacc
atg cca ata gtg gat aag 296 Met Pro Ile Val Asp Lys 1 5ttg aag gag
gcc ctg aaa ccc ggc cgc aag gac tcg gct gat gat gga 344Leu Lys Glu
Ala Leu Lys Pro Gly Arg Lys Asp Ser Ala Asp Asp Gly 10 15 20gaa ctg
ggg aag ctt ctt gcc tcc tct gcc aag aag gtc ctt tta cag 392Glu Leu
Gly Lys Leu Leu Ala Ser Ser Ala Lys Lys Val Leu Leu Gln 25 30 35aaa
atc gag ttc gag cca gcc agc aag agc ttc tcc tac cag ctg gag 440Lys
Ile Glu Phe Glu Pro Ala Ser Lys Ser Phe Ser Tyr Gln Leu Glu 40 45
50gcc tta aag agc aaa tat gtg ttg ctc aac ccc aaa aca gag gga gct
488Ala Leu Lys Ser Lys Tyr Val Leu Leu Asn Pro Lys Thr Glu Gly Ala
55 60 65 70agt cgc cac aag agt gga gat gac cca ccg gcc agg aga cag
ggc agt 536Ser Arg His Lys Ser Gly Asp Asp Pro Pro Ala Arg Arg Gln
Gly Ser 75 80 85gag cac acg tat gag agc tgt ggt gac gga gtc cca gcc
ccg cag aaa 584Glu His Thr Tyr Glu Ser Cys Gly Asp Gly Val Pro Ala
Pro Gln Lys 90 95 100gtg ctt ttc ccc acg gag cga ctg tct ctg agg
tgg gag cgg gtc ttc 632Val Leu Phe Pro Thr Glu Arg Leu Ser Leu Arg
Trp Glu Arg Val Phe 105 110 115cgc gtg ggc gca gga ctc cac aac ctt
ggc aac acc tgc ttt ctc aat 680Arg Val Gly Ala Gly Leu His Asn Leu
Gly Asn Thr Cys Phe Leu Asn 120 125 130gcc acc atc cag tgc ttg acc
tac aca cca cct cta gcc aac tac ctg 728Ala Thr Ile Gln Cys Leu Thr
Tyr Thr Pro Pro Leu Ala Asn Tyr Leu135 140 145 150ctc tcc aag gag
cat gct cgc agc tgc cac cag gga agc ttc tgc atg 776Leu Ser Lys Glu
His Ala Arg Ser Cys His Gln Gly Ser Phe Cys Met 155 160 165ctg tgt
gtc atg cag aac cac att gtc cag gcc ttc gcc aac agc ggc 824Leu Cys
Val Met Gln Asn His Ile Val Gln
Ala Phe Ala Asn Ser Gly 170 175 180aac gcc atc aag ccc gtc tcc ttc
atc cga gac ctg aaa aag atc gcc 872Asn Ala Ile Lys Pro Val Ser Phe
Ile Arg Asp Leu Lys Lys Ile Ala 185 190 195cga cac ttc cgc ttt ggg
aac cag gag gac gcg cat gag ttc ctg cgg 920Arg His Phe Arg Phe Gly
Asn Gln Glu Asp Ala His Glu Phe Leu Arg 200 205 210tac acc atc gac
gcc atg cag aaa gcc tgc ctg aat ggc tgt gcc aag 968Tyr Thr Ile Asp
Ala Met Gln Lys Ala Cys Leu Asn Gly Cys Ala Lys215 220 225 230ttg
gat cgt caa acg cag gct act acc ttg gtc cat caa att ttt gga 1016Leu
Asp Arg Gln Thr Gln Ala Thr Thr Leu Val His Gln Ile Phe Gly 235 240
245ggg tat ctc aga tca cgc gtg aag tgc tcc gtg tgc aag agc gtc tcg
1064Gly Tyr Leu Arg Ser Arg Val Lys Cys Ser Val Cys Lys Ser Val Ser
250 255 260gac acc tac gac ccc tac ttg gac gtc gcg ctg gag atc cgg
caa gct 1112Asp Thr Tyr Asp Pro Tyr Leu Asp Val Ala Leu Glu Ile Arg
Gln Ala 265 270 275gcg aat att gtg cgt gct ctg gaa ctt ttt gtg aaa
gca gat gtc ctg 1160Ala Asn Ile Val Arg Ala Leu Glu Leu Phe Val Lys
Ala Asp Val Leu 280 285 290agt gga gag aat gcc tac atg tgt gct aaa
tgc aag aag aag gtt cca 1208Ser Gly Glu Asn Ala Tyr Met Cys Ala Lys
Cys Lys Lys Lys Val Pro295 300 305 310gcc agc aag cgc ttc acc atc
cac aga aca tcc aac gtc tta acc ctt 1256Ala Ser Lys Arg Phe Thr Ile
His Arg Thr Ser Asn Val Leu Thr Leu 315 320 325tcc ctc aag cgc ttt
gcc aac ttc agc ggg ggg aag atc acc aag gat 1304Ser Leu Lys Arg Phe
Ala Asn Phe Ser Gly Gly Lys Ile Thr Lys Asp 330 335 340gta ggc tat
ccg gaa ttc ctc aac ata cgt ccg tat atg tcc cag aat 1352Val Gly Tyr
Pro Glu Phe Leu Asn Ile Arg Pro Tyr Met Ser Gln Asn 345 350 355aat
ggt gat cct gtc atg tat gga ctc tat gct gtc ctg gtg cac tcg 1400Asn
Gly Asp Pro Val Met Tyr Gly Leu Tyr Ala Val Leu Val His Ser 360 365
370ggc tac agc tgc cat gcc ggg cac tat tac tgc tac gtg aag gca agc
1448Gly Tyr Ser Cys His Ala Gly His Tyr Tyr Cys Tyr Val Lys Ala
Ser375 380 385 390aat gga cag tgg tac cag atg aat gat tcc ttg gtc
cat tcc agc aac 1496Asn Gly Gln Trp Tyr Gln Met Asn Asp Ser Leu Val
His Ser Ser Asn 395 400 405gtc aag gtg gtt ctg aac cag cag gcc tac
gtg ctg ttc tat ctg cga 1544Val Lys Val Val Leu Asn Gln Gln Ala Tyr
Val Leu Phe Tyr Leu Arg 410 415 420att cca ggc tct aag aaa agt ccc
gag ggc ctc atc tcc agg aca ggc 1592Ile Pro Gly Ser Lys Lys Ser Pro
Glu Gly Leu Ile Ser Arg Thr Gly 425 430 435tcc tcc tcc ctt ccc ggc
cgc ccg agt gtg att cca gat cac tcc aag 1640Ser Ser Ser Leu Pro Gly
Arg Pro Ser Val Ile Pro Asp His Ser Lys 440 445 450aag aac atc ggc
aat ggg att att tcc tcc cca ctg act gga aag cga 1688Lys Asn Ile Gly
Asn Gly Ile Ile Ser Ser Pro Leu Thr Gly Lys Arg455 460 465 470caa
gac tct ggg acg atg aag aag ccg cac acc act gaa gag att ggt 1736Gln
Asp Ser Gly Thr Met Lys Lys Pro His Thr Thr Glu Glu Ile Gly 475 480
485gtg ccc ata tcc agg aat ggc tcc acc ctg ggc ctg aag tcc cag aac
1784Val Pro Ile Ser Arg Asn Gly Ser Thr Leu Gly Leu Lys Ser Gln Asn
490 495 500ggc tgc att cct cca aag ctg ccc tcg ggg tcc cct tcc ccc
aaa ctc 1832Gly Cys Ile Pro Pro Lys Leu Pro Ser Gly Ser Pro Ser Pro
Lys Leu 505 510 515tcc cag aca ccc aca cac atg cca acc atc cta gac
gac cct gga aag 1880Ser Gln Thr Pro Thr His Met Pro Thr Ile Leu Asp
Asp Pro Gly Lys 520 525 530aag gtg aag aag cca gct cct cca cag cac
ttt tcc ccc aga act gct 1928Lys Val Lys Lys Pro Ala Pro Pro Gln His
Phe Ser Pro Arg Thr Ala535 540 545 550cag ggg ctg cct ggg acc agc
aac tcg aat agc agc aga tct ggg agc 1976Gln Gly Leu Pro Gly Thr Ser
Asn Ser Asn Ser Ser Arg Ser Gly Ser 555 560 565caa agg cag ggc tcc
tgg gac agc agg gat gtt gtc ctc tct acc tca 2024Gln Arg Gln Gly Ser
Trp Asp Ser Arg Asp Val Val Leu Ser Thr Ser 570 575 580cct aag ctc
ctg gct aca gcc act gcc aac ggg cat ggg ctg aag ggg 2072Pro Lys Leu
Leu Ala Thr Ala Thr Ala Asn Gly His Gly Leu Lys Gly 585 590 595aac
gac gag agc gct ggc ctc gac agg agg ggc tcc agc agc tcc agc 2120Asn
Asp Glu Ser Ala Gly Leu Asp Arg Arg Gly Ser Ser Ser Ser Ser 600 605
610cca gag cac tcg gcc agc agc gac tcc acc aag gcc ccc cag acc ccc
2168Pro Glu His Ser Ala Ser Ser Asp Ser Thr Lys Ala Pro Gln Thr
Pro615 620 625 630agg agt gga gcg gcc cat ctc tgc gat tct cag gaa
acg aac tgt tcc 2216Arg Ser Gly Ala Ala His Leu Cys Asp Ser Gln Glu
Thr Asn Cys Ser 635 640 645acc gct ggc cac tcc aaa acg ccg cca agt
gga gca gat tct aag acg 2264Thr Ala Gly His Ser Lys Thr Pro Pro Ser
Gly Ala Asp Ser Lys Thr 650 655 660gtg aag ctg aag tcc cct gtc ctg
agc aac acc acc act gag cct gca 2312Val Lys Leu Lys Ser Pro Val Leu
Ser Asn Thr Thr Thr Glu Pro Ala 665 670 675agc acc atg tct cct cca
cca gcc aaa aaa ctg gcc ctt tct gcc aag 2360Ser Thr Met Ser Pro Pro
Pro Ala Lys Lys Leu Ala Leu Ser Ala Lys 680 685 690aag gcc agc acc
ctg tgg agg gcg acc ggc aat gac ctc cgt cca cct 2408Lys Ala Ser Thr
Leu Trp Arg Ala Thr Gly Asn Asp Leu Arg Pro Pro695 700 705 710ccc
ccc tca cca tcc tcc gac ctc acc cac ccc atg aaa acc tct cac 2456Pro
Pro Ser Pro Ser Ser Asp Leu Thr His Pro Met Lys Thr Ser His 715 720
725ccc gtc gtt gcc tcc act tgg ccc gtc cat aga gcc agg gct gtg tca
2504Pro Val Val Ala Ser Thr Trp Pro Val His Arg Ala Arg Ala Val Ser
730 735 740cct gct ccc caa tca tcc agc cgc ctg caa ccc ccc ttc agc
ccc cac 2552Pro Ala Pro Gln Ser Ser Ser Arg Leu Gln Pro Pro Phe Ser
Pro His 745 750 755ccc aca ttg ctg tcc agt acc ccc aag ccc cca ggg
acg tca gaa cca 2600Pro Thr Leu Leu Ser Ser Thr Pro Lys Pro Pro Gly
Thr Ser Glu Pro 760 765 770cgg agc tgc tcc tcc atc tcg acg gcg ctg
cct cag gtc aac gag gac 2648Arg Ser Cys Ser Ser Ile Ser Thr Ala Leu
Pro Gln Val Asn Glu Asp775 780 785 790ctt gtg tct ctt cca cac cag
ttg cca gag gcc agt gag ccc ccc cag 2696Leu Val Ser Leu Pro His Gln
Leu Pro Glu Ala Ser Glu Pro Pro Gln 795 800 805agc ccc tct gag aag
agg aaa aag acc ttt gtg gga gag ccg cag agg 2744Ser Pro Ser Glu Lys
Arg Lys Lys Thr Phe Val Gly Glu Pro Gln Arg 810 815 820ctg ggc tca
gag acg cgc ctc cca cag cac atc agg gag gcc act gcg 2792Leu Gly Ser
Glu Thr Arg Leu Pro Gln His Ile Arg Glu Ala Thr Ala 825 830 835gct
ccc cac ggg aag agg aag agg aag aag aag aag cgc ccg gag gac 2840Ala
Pro His Gly Lys Arg Lys Arg Lys Lys Lys Lys Arg Pro Glu Asp 840 845
850aca gct gcc agc gcc ctg cag gag ggg cag aca cag aga cag cct ggg
2888Thr Ala Ala Ser Ala Leu Gln Glu Gly Gln Thr Gln Arg Gln Pro
Gly855 860 865 870agc ccc atg tac agg agg gag ggc cag gca cag ctg
ccc gct gtc aga 2936Ser Pro Met Tyr Arg Arg Glu Gly Gln Ala Gln Leu
Pro Ala Val Arg 875 880 885cgg cag gaa gat ggc aca cag cca cag gtg
aat ggc cag cag gtg gga 2984Arg Gln Glu Asp Gly Thr Gln Pro Gln Val
Asn Gly Gln Gln Val Gly 890 895 900tgt gtt acg gac ggc cac cac gcg
agc agc agg aag cgg agg agg aaa 3032Cys Val Thr Asp Gly His His Ala
Ser Ser Arg Lys Arg Arg Arg Lys 905 910 915gga gca gaa ggt ctt ggt
gaa gaa ggc ggc ctg cac cag gac cca ctt 3080Gly Ala Glu Gly Leu Gly
Glu Glu Gly Gly Leu His Gln Asp Pro Leu 920 925 930cgg cac agc tgc
tct ccc atg ggt gat ggt gat cca gag gcc atg gaa 3128Arg His Ser Cys
Ser Pro Met Gly Asp Gly Asp Pro Glu Ala Met Glu935 940 945 950gag
tct cca agg aaa aag aaa aag aaa aaa aga aag cag gag aca cag 3176Glu
Ser Pro Arg Lys Lys Lys Lys Lys Lys Arg Lys Gln Glu Thr Gln 955 960
965cgg gca gta gaa gag gat ggg cat ctc aaa tgc cca agg agt gcc aag
3224Arg Ala Val Glu Glu Asp Gly His Leu Lys Cys Pro Arg Ser Ala Lys
970 975 980ccc caa gat gct gtt gtc ccc gag tcc agc agc tgc gca cca
tcc gcg 3272Pro Gln Asp Ala Val Val Pro Glu Ser Ser Ser Cys Ala Pro
Ser Ala 985 990 995aat ggc tgg tgt cct ggg gac cgc atg ggg ctg agc
cag gcc cct cct 3320Asn Gly Trp Cys Pro Gly Asp Arg Met Gly Leu Ser
Gln Ala Pro Pro 1000 1005 1010gtg tct tgg aat gga gag cgg gag tct
gat gtg gtc cag gaa ctg ctc 3368Val Ser Trp Asn Gly Glu Arg Glu Ser
Asp Val Val Gln Glu Leu Leu1015 1020 1025 1030aaa tac tca tct gat
aaa gct tac ggg aga aaa gtt ctg acc tgg gat 3416Lys Tyr Ser Ser Asp
Lys Ala Tyr Gly Arg Lys Val Leu Thr Trp Asp 1035 1040 1045ggc aag
atg tcg gcg gtc agt cag gat gct att gaa gac agc aga cag 3464Gly Lys
Met Ser Ala Val Ser Gln Asp Ala Ile Glu Asp Ser Arg Gln 1050 1055
1060gcc cgg act gag acc gtg gtt gat gac tgg gac gaa gag ttt gac cga
3512Ala Arg Thr Glu Thr Val Val Asp Asp Trp Asp Glu Glu Phe Asp Arg
1065 1070 1075ggg aag gaa aag aaa att aaa aaa ttt aag aga gag aag
agg aga aac 3560Gly Lys Glu Lys Lys Ile Lys Lys Phe Lys Arg Glu Lys
Arg Arg Asn 1080 1085 1090ttc aac gcc ttc cag aaa ctt cag act cga
cgg aac ttc tgg tct gtg 3608Phe Asn Ala Phe Gln Lys Leu Gln Thr Arg
Arg Asn Phe Trp Ser Val1095 1100 1105 1110act cac cca gca aag gct
gcc agc ctc agc tat cgc cgc tga 3650Thr His Pro Ala Lys Ala Ala Ser
Leu Ser Tyr Arg Arg * 1115 1120ctgtgcccct gtggaaggag atgatcaagc
ggtgctgatt cacgagagtg gaagcctcca 3710gagcttgggg ctttctggct
gctcttcatt gacctgtgtg ttcccagcac acgaacagcg 3770cccctaacgg
agatttgttc agcgactgaa tatacacctg taaacgagta gcatgtatac
3830attgattttg attacaaatg gttctgtatt atataccacc gttctgactg
cttttttcac 3890ttatagcttg gaaattgtct tctgttggta atacagaaat
ctgtttcagt c 394117337PRTHomo sapiens 17Met Asp Leu Asp Val Val Asn
Met Phe Val Ile Ala Gly Gly Thr Leu 1 5 10 15Ala Ile Pro Ile Leu
Ala Phe Val Ala Ser Phe Leu Leu Trp Pro Ser 20 25 30Ala Leu Ile Arg
Ile Tyr Tyr Trp Tyr Trp Arg Arg Thr Leu Gly Met 35 40 45Gln Val Arg
Tyr Val His His Glu Asp Tyr Gln Phe Cys Tyr Ser Phe 50 55 60Arg Gly
Arg Pro Gly His Lys Pro Ser Ile Leu Met Leu His Gly Phe65 70 75
80Ser Ala His Lys Asp Met Trp Leu Ser Val Val Lys Phe Leu Pro Lys
85 90 95Asn Leu His Leu Val Cys Val Asp Met Pro Gly His Glu Gly Thr
Thr 100 105 110Arg Ser Ser Leu Asp Asp Leu Ser Ile Asp Gly Gln Val
Lys Arg Ile 115 120 125His Gln Phe Val Glu Cys Leu Lys Leu Asn Lys
Lys Pro Phe His Leu 130 135 140Val Gly Thr Ser Met Gly Gly Gln Val
Ala Gly Val Tyr Ala Ala Tyr145 150 155 160Tyr Pro Ser Asp Val Ser
Ser Leu Cys Leu Val Cys Pro Ala Gly Leu 165 170 175Gln Tyr Ser Thr
Asp Asn Gln Phe Val Gln Arg Leu Lys Glu Leu Gln 180 185 190Gly Ser
Ala Ala Val Glu Lys Ile Pro Leu Ile Pro Ser Thr Pro Glu 195 200
205Glu Met Ser Glu Met Leu Gln Leu Cys Ser Tyr Val Arg Phe Lys Val
210 215 220Pro Gln Gln Ile Leu Gln Gly Leu Val Asp Val Arg Ile Pro
His Asn225 230 235 240Asn Phe Tyr Arg Lys Leu Phe Leu Glu Ile Val
Ser Glu Lys Ser Arg 245 250 255Tyr Ser Leu His Gln Asn Met Asp Lys
Ile Lys Val Pro Thr Gln Ile 260 265 270Ile Trp Gly Lys Gln Asp Gln
Val Leu Asp Val Ser Gly Ala Asp Met 275 280 285Leu Ala Lys Ser Ile
Ala Asn Cys Gln Val Glu Leu Leu Glu Asn Cys 290 295 300Gly His Ser
Val Val Met Glu Arg Pro Arg Lys Thr Ala Lys Leu Ile305 310 315
320Ile Asp Phe Leu Ala Ser Val His Asn Thr Asp Asn Asn Lys Lys Leu
325 330 335Asp181964DNAHomo sapiensCDS(164)...(1174) 18cacgcgtccg
gctgggctgg gcgccggagc tgggagcggc gcgggtagga gcccggcggc 60aggtcccagc
ccggggctag agaccgaggg ccggggtccg ggcccggcgg cgggacccag
120gcggttgagg ctggtcagga gtcagccagc ctgaaagagc agg atg gat ctt gat
175 Met Asp Leu Asp 1gtg gtt aac atg ttt gtg att gcg ggc ggc acg
ctg gcc atc cca atc 223Val Val Asn Met Phe Val Ile Ala Gly Gly Thr
Leu Ala Ile Pro Ile 5 10 15 20ctg gca ttt gtg gct tca ttt ctt ctg
tgg cct tca gca ctg ata aga 271Leu Ala Phe Val Ala Ser Phe Leu Leu
Trp Pro Ser Ala Leu Ile Arg 25 30 35atc tat tat tgg tac tgg cgg agg
aca ttg ggc atg caa gtc cgc tat 319Ile Tyr Tyr Trp Tyr Trp Arg Arg
Thr Leu Gly Met Gln Val Arg Tyr 40 45 50gtt cac cat gaa gac tat cag
ttc tgt tat tcc ttc cgg ggc agg cct 367Val His His Glu Asp Tyr Gln
Phe Cys Tyr Ser Phe Arg Gly Arg Pro 55 60 65ggg cac aaa ccc tcc atc
ctc atg ctc cac gga ttc tct gcc cac aag 415Gly His Lys Pro Ser Ile
Leu Met Leu His Gly Phe Ser Ala His Lys 70 75 80gat atg tgg ctc agt
gtg gtc aag ttc ctt cca aag aac ctg cac ttg 463Asp Met Trp Leu Ser
Val Val Lys Phe Leu Pro Lys Asn Leu His Leu 85 90 95 100gtc tgc gtg
gac atg cca gga cat gag ggc acc acc cgc tcc tcc ctg 511Val Cys Val
Asp Met Pro Gly His Glu Gly Thr Thr Arg Ser Ser Leu 105 110 115gat
gac ctg tcc ata gat ggg caa gtt aag agg ata cac cag ttt gta 559Asp
Asp Leu Ser Ile Asp Gly Gln Val Lys Arg Ile His Gln Phe Val 120 125
130gaa tgc ctg aag ctg aac aaa aaa cct ttc cac ctg gta ggc acc tcc
607Glu Cys Leu Lys Leu Asn Lys Lys Pro Phe His Leu Val Gly Thr Ser
135 140 145atg ggt ggc cag gtg gct ggg gtg tat gct gct tac tac cca
tcg gat 655Met Gly Gly Gln Val Ala Gly Val Tyr Ala Ala Tyr Tyr Pro
Ser Asp 150 155 160gtc tcc agc ctg tgt ctc gtg tgt cct gct ggc ctg
cag tac tca act 703Val Ser Ser Leu Cys Leu Val Cys Pro Ala Gly Leu
Gln Tyr Ser Thr165 170 175 180gac aat caa ttt gta caa cgg ctc aaa
gaa ctg cag ggc tct gcc gcc 751Asp Asn Gln Phe Val Gln Arg Leu Lys
Glu Leu Gln Gly Ser Ala Ala 185 190 195gtg gag aag att ccc ttg atc
ccg tct acc cca gaa gag atg agt gaa 799Val Glu Lys Ile Pro Leu Ile
Pro Ser Thr Pro Glu Glu Met Ser Glu 200 205 210atg ctt cag ctc tgc
tcc tat gtc cgc ttc aag gtg ccc cag cag atc 847Met Leu Gln Leu Cys
Ser Tyr Val Arg Phe Lys Val Pro Gln Gln Ile 215 220 225ctg caa ggc
ctt gtc gat gtc cgc atc cct cat aac aac ttc tac cga 895Leu Gln Gly
Leu Val Asp Val Arg Ile Pro His Asn Asn Phe Tyr Arg 230 235 240aag
ttg ttt ttg gaa atc gtc agt gag aag tcc aga tac tct ctc cat 943Lys
Leu Phe Leu Glu Ile Val Ser Glu Lys Ser Arg Tyr Ser Leu His245 250
255 260cag aac atg gac aag atc aag gtt ccg acg cag atc atc tgg ggg
aaa 991Gln Asn Met Asp Lys Ile Lys Val Pro Thr Gln Ile Ile Trp Gly
Lys 265 270 275caa gac cag gtg ctg gat gtg tct ggg gca gac atg ttg
gcc aag tca 1039Gln Asp Gln Val Leu Asp Val Ser Gly Ala Asp Met Leu
Ala Lys Ser 280 285 290att gcc aac tgc cag gtg gag ctt ctg gaa aac
tgt ggg cac tca gta 1087Ile Ala Asn Cys Gln Val Glu Leu Leu Glu Asn
Cys Gly His Ser Val 295 300 305gtg atg gaa aga ccc agg aag aca gcc
aag ctc ata atc gac ttt tta 1135Val Met Glu Arg Pro Arg Lys Thr Ala
Lys Leu Ile Ile Asp Phe Leu 310 315
320gct tct gtg cac aac aca gac aac aac aag aag ctg gac tgaggccccg
1184Ala Ser Val His Asn Thr Asp Asn Asn Lys Lys Leu Asp325 330
335actgcagcct gcattctgca cacagcatct gctcccatcc cccaagtctg
acgcagccac 1244cactctcagg gatcctgccc caaatgcggt cggagcgcca
gtgaccctga ggaagcccgt 1304cccttatccc tggtatccac ggttccccag
agctttgggg accacgcgaa aacctccaag 1364atatttttca caaaatagaa
actcatatgg aacaaaataa gaaaccccag ccatgaaatc 1424taccatgaag
tcttcaagtt catgtcactg agaagcttgt gcaaagcagc caccttggac
1484cataattaaa tcaaggacat tttctttgag acattcctta tagttggaga
ctcaagatat 1544ttttgttgca tcaggtgtat tcccttgcat gggcagtggc
ttttatagga gcattagtcc 1604tcattcgctg aaccctgttg tttaggtcta
atttaagttt tacatagaga cccatgtatg 1664actgcagccc attggctgca
agaccaggga ggaaagtggc aagctgtaga aaatgtttac 1724acgcatggag
gggcattgct ctagccctca gagcgtccgg agcagcaggg tacatgggtg
1784ggaggttcat tcagcaccca ccagtcaggt atgttctgag tgaacccaca
gcagtcgcag 1844aatgagcacc tggcagggtg ggtttcctag gaataattta
ttatttttaa aaataggcct 1904aataaagcaa taatgttcta gaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1964191086PRTHomo sapiens 19Met
Arg Arg Arg Arg Tyr Leu Arg Asp Arg Ser Glu Glu Ala Ala Gly 1 5 10
15Gly Gly Asp Gly Leu Pro Arg Ser Arg Asp Trp Leu Tyr Glu Ser Tyr
20 25 30Tyr Cys Met Ser Gln Gln His Pro Leu Ile Val Phe Leu Leu Leu
Ile 35 40 45Val Met Gly Ser Cys Leu Ala Leu Leu Ala Val Phe Phe Ala
Leu Gly 50 55 60Leu Glu Val Glu Asp His Val Ala Phe Leu Ile Thr Val
Pro Thr Ala65 70 75 80Leu Ala Ile Phe Phe Ala Ile Phe Ile Leu Val
Cys Ile Glu Ser Val 85 90 95Phe Lys Lys Leu Leu Arg Leu Phe Ser Leu
Val Ile Trp Ile Cys Leu 100 105 110Val Ala Met Gly Tyr Leu Phe Met
Cys Phe Gly Gly Thr Val Ser Pro 115 120 125Trp Asp Gln Val Ser Phe
Phe Leu Phe Ile Ile Phe Val Val Tyr Thr 130 135 140Met Leu Pro Phe
Asn Met Arg Asp Ala Ile Ile Ala Ser Val Leu Thr145 150 155 160Ser
Ser Ser His Thr Ile Val Leu Ser Val Cys Leu Ser Ala Thr Pro 165 170
175Gly Gly Lys Glu His Leu Val Trp Gln Ile Leu Ala Asn Val Ile Ile
180 185 190Phe Ile Cys Gly Asn Leu Ala Gly Ala Tyr His Lys His Leu
Met Glu 195 200 205Leu Ala Leu Gln Gln Thr Tyr Gln Asp Thr Cys Asn
Cys Ile Lys Ser 210 215 220Arg Ile Lys Leu Glu Phe Glu Lys Arg Gln
Gln Glu Arg Leu Leu Leu225 230 235 240Ser Leu Leu Pro Ala His Ile
Ala Met Glu Met Lys Ala Glu Ile Ile 245 250 255Gln Arg Leu Gln Gly
Pro Lys Ala Gly Gln Met Glu Asn Thr Asn Asn 260 265 270Phe His Asn
Leu Tyr Val Lys Arg His Thr Asn Val Ser Ile Leu Tyr 275 280 285Ala
Asp Ile Val Gly Phe Thr Arg Leu Ala Ser Asp Cys Ser Pro Gly 290 295
300Glu Leu Val His Met Leu Asn Glu Leu Phe Gly Lys Phe Asp Gln
Ile305 310 315 320Ala Lys Glu Asn Glu Cys Met Arg Ile Lys Ile Leu
Gly Asp Cys Tyr 325 330 335Tyr Cys Val Ser Gly Leu Pro Ile Ser Leu
Pro Asn His Ala Lys Asn 340 345 350Cys Val Lys Met Gly Leu Asp Met
Cys Glu Ala Ile Lys Lys Val Arg 355 360 365Asp Ala Thr Gly Val Asp
Ile Asn Met Arg Val Gly Val His Ser Gly 370 375 380Asn Val Leu Cys
Gly Val Ile Gly Leu Gln Lys Trp Gln Tyr Asp Val385 390 395 400Trp
Ser His Asp Val Thr Leu Ala Asn His Met Glu Ala Gly Gly Val 405 410
415Pro Gly Arg Val His Ile Ser Ser Val Thr Leu Glu His Leu Asn Gly
420 425 430Ala Tyr Lys Val Glu Glu Gly Asp Gly Asp Ile Arg Asp Pro
Tyr Leu 435 440 445Lys Gln His Leu Val Lys Thr Tyr Phe Val Ile Asn
Pro Lys Gly Glu 450 455 460Arg Arg Ser Pro Gln His Leu Phe Arg Pro
Arg His Thr Leu Asp Gly465 470 475 480Ala Lys Met Arg Ala Ser Val
Arg Met Thr Arg Tyr Leu Glu Ser Trp 485 490 495Gly Ala Ala Lys Pro
Phe Ala His Leu His His Arg Asp Ser Met Thr 500 505 510Thr Glu Asn
Gly Lys Ile Ser Thr Thr Asp Val Pro Met Gly Gln His 515 520 525Asn
Phe Gln Asn Arg Thr Leu Arg Thr Lys Ser Gln Lys Lys Arg Phe 530 535
540Glu Glu Glu Leu Asn Glu Arg Met Ile Gln Ala Ile Asp Gly Ile
Asn545 550 555 560Ala Gln Lys Gln Trp Leu Lys Ser Glu Asp Ile Gln
Arg Ile Ser Leu 565 570 575Leu Phe Tyr Asn Lys Val Leu Glu Lys Glu
Tyr Arg Ala Thr Ala Leu 580 585 590Pro Ala Phe Lys Tyr Tyr Val Thr
Cys Ala Cys Leu Ile Phe Phe Cys 595 600 605Ile Phe Ile Val Gln Ile
Leu Val Leu Pro Lys Thr Ser Val Leu Gly 610 615 620Ile Ser Phe Gly
Ala Ala Phe Leu Leu Leu Ala Phe Ile Leu Phe Val625 630 635 640Cys
Phe Ala Gly Gln Leu Leu Gln Cys Ser Lys Lys Ala Ser Pro Leu 645 650
655Leu Met Trp Leu Leu Lys Ser Ser Gly Ile Ile Ala Asn Arg Pro Trp
660 665 670Pro Arg Ile Ser Leu Thr Ile Ile Thr Thr Ala Ile Ile Leu
Met Met 675 680 685Ala Val Phe Asn Met Phe Phe Leu Ser Asp Ser Glu
Glu Thr Ile Pro 690 695 700Pro Thr Ala Asn Thr Thr Asn Thr Ser Phe
Ser Ala Ser Asn Asn Gln705 710 715 720Val Ala Ile Leu Arg Ala Gln
Asn Leu Phe Phe Leu Pro Tyr Phe Ile 725 730 735Tyr Ser Cys Ile Leu
Gly Leu Ile Ser Cys Ser Val Phe Leu Arg Val 740 745 750Asn Tyr Glu
Leu Lys Met Leu Ile Met Met Val Ala Leu Val Gly Tyr 755 760 765Asn
Thr Ile Leu Leu His Thr His Ala His Val Leu Gly Asp Tyr Ser 770 775
780Gln Val Leu Phe Glu Arg Pro Gly Ile Trp Lys Asp Leu Lys Thr
Met785 790 795 800Gly Ser Val Ser Leu Ser Ile Phe Phe Ile Thr Leu
Leu Val Leu Gly 805 810 815Arg Gln Asn Glu Tyr Tyr Cys Arg Leu Asp
Phe Leu Trp Lys Asn Lys 820 825 830Phe Lys Lys Glu Arg Glu Glu Ile
Glu Thr Met Glu Asn Leu Asn Arg 835 840 845Val Leu Leu Glu Asn Val
Leu Pro Ala His Val Ala Glu His Phe Leu 850 855 860Ala Arg Ser Leu
Lys Asn Glu Glu Leu Tyr His Gln Ser Tyr Asp Cys865 870 875 880Val
Cys Val Met Phe Ala Ser Ile Pro Asp Phe Lys Glu Phe Tyr Thr 885 890
895Glu Ser Asp Val Asn Lys Glu Gly Leu Glu Cys Leu Arg Leu Leu Asn
900 905 910Glu Ile Ile Ala Asp Phe Asp Asp Leu Leu Ser Lys Pro Lys
Phe Ser 915 920 925Gly Val Glu Lys Ile Lys Thr Ile Gly Ser Thr Tyr
Met Ala Ala Thr 930 935 940Gly Leu Ser Ala Val Pro Ser Gln Glu His
Ser Gln Glu Pro Glu Arg945 950 955 960Gln Tyr Met His Ile Gly Thr
Met Val Glu Phe Ala Phe Ala Leu Val 965 970 975Gly Lys Leu Asp Ala
Ile Asn Lys His Ser Phe Asn Asp Phe Lys Leu 980 985 990Arg Val Gly
Ile Asn His Gly Pro Val Ile Ala Gly Val Ile Gly Ala 995 1000
1005Gln Lys Pro Gln Tyr Asp Ile Trp Gly Asn Thr Val Asn Val Ala Ser
1010 1015 1020Arg Met Asp Ser Thr Gly Val Leu Asp Lys Ile Gln Val
Thr Glu Glu1025 1030 1035 1040Thr Ser Leu Val Leu Gln Thr Leu Gly
Tyr Thr Cys Thr Cys Arg Gly 1045 1050 1055Ile Ile Asn Val Lys Gly
Lys Gly Asp Leu Lys Thr Tyr Phe Val Asn 1060 1065 1070Thr Glu Met
Ser Arg Ser Leu Ser Gln Ser Asn Val Ala Ser 1075 1080
1085204011DNAHomo sapiensCDS(71)...(3331) 20cacgcgtccg cccggccccc
gcccgcgcac ggcgggcgcc ctgtgagcgg ccccgatgtg 60gcaggaggcg atg cgg
cgc cgc cgc tac ctg cgg gac cgc tcc gag gag 109 Met Arg Arg Arg Arg
Tyr Leu Arg Asp Arg Ser Glu Glu 1 5 10gcg gcg ggc ggc gga gac ggg
ctg ccg cgg tcc cgg gac tgg ctc tac 157Ala Ala Gly Gly Gly Asp Gly
Leu Pro Arg Ser Arg Asp Trp Leu Tyr 15 20 25gag tcc tac tac tgc atg
agc cag cag cac ccg ctc atc gtc ttc ctg 205Glu Ser Tyr Tyr Cys Met
Ser Gln Gln His Pro Leu Ile Val Phe Leu 30 35 40 45ctg ctc atc gtc
atg ggc tcc tgc ctc gcc ctg ctc gcc gtc ttc ttc 253Leu Leu Ile Val
Met Gly Ser Cys Leu Ala Leu Leu Ala Val Phe Phe 50 55 60gcg ctc ggg
ctg gaa gtt gaa gac cat gtg gcg ttt cta ata aca gtt 301Ala Leu Gly
Leu Glu Val Glu Asp His Val Ala Phe Leu Ile Thr Val 65 70 75cca act
gcc ctg gcg att ttc ttt gcg ata ttt atc ctg gtc tgc atc 349Pro Thr
Ala Leu Ala Ile Phe Phe Ala Ile Phe Ile Leu Val Cys Ile 80 85 90gag
tct gtg ttt aag aag ctg ctg cgc ctc ttc tcg ttg gtg ata tgg 397Glu
Ser Val Phe Lys Lys Leu Leu Arg Leu Phe Ser Leu Val Ile Trp 95 100
105ata tgc ctt gtt gcc atg gga tac ctg ttc atg tgt ttt gga ggc acc
445Ile Cys Leu Val Ala Met Gly Tyr Leu Phe Met Cys Phe Gly Gly
Thr110 115 120 125gtc tct ccc tgg gac cag gta tcg ttc ttc ctc ttc
atc atc ttc gtg 493Val Ser Pro Trp Asp Gln Val Ser Phe Phe Leu Phe
Ile Ile Phe Val 130 135 140gtg tac acc atg ctg ccc ttc aac atg cga
gac gcc atc att gcc agc 541Val Tyr Thr Met Leu Pro Phe Asn Met Arg
Asp Ala Ile Ile Ala Ser 145 150 155gtc ctc acc tcc tcc tcc cac acc
atc gtg ctt agc gtc tgc ctg tct 589Val Leu Thr Ser Ser Ser His Thr
Ile Val Leu Ser Val Cys Leu Ser 160 165 170gca aca ccg gga ggc aag
gag cac ctg gtc tgg cag atc ctg gcc aat 637Ala Thr Pro Gly Gly Lys
Glu His Leu Val Trp Gln Ile Leu Ala Asn 175 180 185gtg atc att ttc
atc tgt ggg aac ctg gcg gga gcc tac cat aag cac 685Val Ile Ile Phe
Ile Cys Gly Asn Leu Ala Gly Ala Tyr His Lys His190 195 200 205ctc
atg gaa ctc gct ctt cag caa aca tat cag gac acc tgt aat tgc 733Leu
Met Glu Leu Ala Leu Gln Gln Thr Tyr Gln Asp Thr Cys Asn Cys 210 215
220atc aag tcg cgg atc aag ttg gaa ttt gaa aaa cgt caa cag gag cgg
781Ile Lys Ser Arg Ile Lys Leu Glu Phe Glu Lys Arg Gln Gln Glu Arg
225 230 235ctt ctg ctc tcc ctg ctg ccg gcc cac atc gcc atg gag atg
aaa gcg 829Leu Leu Leu Ser Leu Leu Pro Ala His Ile Ala Met Glu Met
Lys Ala 240 245 250gag atc atc cag agg ctg cag ggc ccc aag gcg ggc
cag atg gag aac 877Glu Ile Ile Gln Arg Leu Gln Gly Pro Lys Ala Gly
Gln Met Glu Asn 255 260 265aca aat aac ttc cac aac ctg tat gtg aag
cgg cat aca aac gtg agc 925Thr Asn Asn Phe His Asn Leu Tyr Val Lys
Arg His Thr Asn Val Ser270 275 280 285atc tta tac gct gac atc gtt
ggc ttt acc cgg ctg gca agt gac tgc 973Ile Leu Tyr Ala Asp Ile Val
Gly Phe Thr Arg Leu Ala Ser Asp Cys 290 295 300tcc ccg gga gaa cta
gtc cac atg ctg aat gag ctc ttt gga aag ttt 1021Ser Pro Gly Glu Leu
Val His Met Leu Asn Glu Leu Phe Gly Lys Phe 305 310 315gat caa att
gca aag gag aat gaa tgc atg aga att aaa att tta gga 1069Asp Gln Ile
Ala Lys Glu Asn Glu Cys Met Arg Ile Lys Ile Leu Gly 320 325 330gac
tgc tac tac tgt gta tct gga ctc cct ata tct ctc cct aac cat 1117Asp
Cys Tyr Tyr Cys Val Ser Gly Leu Pro Ile Ser Leu Pro Asn His 335 340
345gcc aag aac tgt gtg aaa atg ggg ctg gac atg tgt gaa gcc ata aag
1165Ala Lys Asn Cys Val Lys Met Gly Leu Asp Met Cys Glu Ala Ile
Lys350 355 360 365aaa gtg agg gat gct act gga gtt gat atc aac atg
cgc gtg ggc gtg 1213Lys Val Arg Asp Ala Thr Gly Val Asp Ile Asn Met
Arg Val Gly Val 370 375 380cat tct ggg aat gtc ctg tgt ggc gtg att
ggt ctg cag aag tgg caa 1261His Ser Gly Asn Val Leu Cys Gly Val Ile
Gly Leu Gln Lys Trp Gln 385 390 395tat gat gtg tgg tca cat gat gtg
acc ttg gcc aac cac atg gaa gct 1309Tyr Asp Val Trp Ser His Asp Val
Thr Leu Ala Asn His Met Glu Ala 400 405 410gga ggg gtc cct gga cgt
gtt cac att tct tct gtc acc ctg gag cac 1357Gly Gly Val Pro Gly Arg
Val His Ile Ser Ser Val Thr Leu Glu His 415 420 425ttg aat ggc gct
tat aaa gtg gag gag gga gat ggt gac att agg gac 1405Leu Asn Gly Ala
Tyr Lys Val Glu Glu Gly Asp Gly Asp Ile Arg Asp430 435 440 445cca
tat tta aaa cag cac ctg gtg aaa acc tac ttt gtg atc aac ccc 1453Pro
Tyr Leu Lys Gln His Leu Val Lys Thr Tyr Phe Val Ile Asn Pro 450 455
460aag gga gaa cga cgg agc ccc cag cat ctc ttc aga cct cgc cac acc
1501Lys Gly Glu Arg Arg Ser Pro Gln His Leu Phe Arg Pro Arg His Thr
465 470 475ctt gat gga gcc aaa atg agg gcc tcg gtc cgc atg acc cgg
tac ttg 1549Leu Asp Gly Ala Lys Met Arg Ala Ser Val Arg Met Thr Arg
Tyr Leu 480 485 490gag tcc tgg ggg gca gcc aag ccc ttt gca cac cta
cat cac agg gac 1597Glu Ser Trp Gly Ala Ala Lys Pro Phe Ala His Leu
His His Arg Asp 495 500 505agc atg acc aca gag aac ggc aag atc agc
acc acg gat gta ccc atg 1645Ser Met Thr Thr Glu Asn Gly Lys Ile Ser
Thr Thr Asp Val Pro Met510 515 520 525ggt cag cat aat ttt caa aat
cgc acc tta aga acc aag tca caa aag 1693Gly Gln His Asn Phe Gln Asn
Arg Thr Leu Arg Thr Lys Ser Gln Lys 530 535 540aag aga ttt gaa gaa
gaa ttg aat gaa agg atg att caa gca att gat 1741Lys Arg Phe Glu Glu
Glu Leu Asn Glu Arg Met Ile Gln Ala Ile Asp 545 550 555ggg att aat
gca cag aag caa tgg ctc aag tct gaa gac att cag aga 1789Gly Ile Asn
Ala Gln Lys Gln Trp Leu Lys Ser Glu Asp Ile Gln Arg 560 565 570atc
tca ctg ctt ttc tat aac aaa gta cta gaa aaa gag tac cgg gcc 1837Ile
Ser Leu Leu Phe Tyr Asn Lys Val Leu Glu Lys Glu Tyr Arg Ala 575 580
585acg gca ctg cca gcg ttc aag tat tat gtg act tgt gcc tgt ctc ata
1885Thr Ala Leu Pro Ala Phe Lys Tyr Tyr Val Thr Cys Ala Cys Leu
Ile590 595 600 605ttc ttc tgc atc ttc att gtg cag att ctc gtg ctg
cca aaa acg tct 1933Phe Phe Cys Ile Phe Ile Val Gln Ile Leu Val Leu
Pro Lys Thr Ser 610 615 620gtc ctg ggc atc tcc ttt ggg gct gcg ttt
ctc ttg ctg gcc ttc atc 1981Val Leu Gly Ile Ser Phe Gly Ala Ala Phe
Leu Leu Leu Ala Phe Ile 625 630 635ctc ttc gtc tgc ttt gct gga cag
ctt ctg caa tgc agc aaa aaa gcc 2029Leu Phe Val Cys Phe Ala Gly Gln
Leu Leu Gln Cys Ser Lys Lys Ala 640 645 650tct ccc ctg ctc atg tgg
ctt ttg aag tcc tcg ggc atc att gcc aac 2077Ser Pro Leu Leu Met Trp
Leu Leu Lys Ser Ser Gly Ile Ile Ala Asn 655 660 665cgc ccc tgg cca
cgg atc tct ctc acg atc atc acc aca gcc atc ata 2125Arg Pro Trp Pro
Arg Ile Ser Leu Thr Ile Ile Thr Thr Ala Ile Ile670 675 680 685tta
atg atg gcc gtg ttc aac atg ttt ttc ctg agt gac tca gag gaa 2173Leu
Met Met Ala Val Phe Asn Met Phe Phe Leu Ser Asp Ser Glu Glu 690 695
700aca atc cct cca act gcc aac aca aca aac aca agc ttt tca gcc tca
2221Thr Ile Pro Pro Thr Ala Asn Thr Thr Asn Thr Ser Phe Ser Ala Ser
705 710 715aat aat cag gtg gcg att ctg cgt gcg cag aat tta ttt ttc
ctc ccg 2269Asn Asn Gln Val Ala Ile Leu Arg Ala Gln Asn Leu Phe Phe
Leu Pro 720 725 730tac ttt atc tac agc tgc att ctg gga ctg ata tcc
tgt tcc gtg ttc 2317Tyr Phe Ile Tyr Ser Cys Ile Leu Gly Leu Ile Ser
Cys Ser Val Phe 735 740 745ctg cgg gta aac tat gag ctg aag atg ttg
atc atg atg gtg gcc ttg 2365Leu Arg Val Asn Tyr
Glu Leu Lys Met Leu Ile Met Met Val Ala Leu750 755 760 765gtg ggc
tac aac acc atc cta ctc cac acc cac gcc cac gtc ctg ggc 2413Val Gly
Tyr Asn Thr Ile Leu Leu His Thr His Ala His Val Leu Gly 770 775
780gac tac agc cag gtc tta ttt gag aga cca ggc att tgg aaa gac ctg
2461Asp Tyr Ser Gln Val Leu Phe Glu Arg Pro Gly Ile Trp Lys Asp Leu
785 790 795aag acc atg ggc tct gtg tct ctc tct ata ttc ttc atc aca
ctg ctt 2509Lys Thr Met Gly Ser Val Ser Leu Ser Ile Phe Phe Ile Thr
Leu Leu 800 805 810gtt ctg ggt aga cag aat gaa tat tac tgt agg tta
gac ttc tta tgg 2557Val Leu Gly Arg Gln Asn Glu Tyr Tyr Cys Arg Leu
Asp Phe Leu Trp 815 820 825aag aac aaa ttc aaa aaa gag cgg gag gag
ata gag acc atg gag aac 2605Lys Asn Lys Phe Lys Lys Glu Arg Glu Glu
Ile Glu Thr Met Glu Asn830 835 840 845ctg aac cgc gtg ctg ctg gag
aac gtg ctt ccc gcg cac gtg gct gag 2653Leu Asn Arg Val Leu Leu Glu
Asn Val Leu Pro Ala His Val Ala Glu 850 855 860cac ttc ctg gcc agg
agc ctg aag aat gag gag cta tac cac cag tcc 2701His Phe Leu Ala Arg
Ser Leu Lys Asn Glu Glu Leu Tyr His Gln Ser 865 870 875tat gac tgc
gtc tgc gtc atg ttt gcc tcc att ccg gat ttc aaa gaa 2749Tyr Asp Cys
Val Cys Val Met Phe Ala Ser Ile Pro Asp Phe Lys Glu 880 885 890ttt
tat aca gaa tcc gac gtg aac aag gag ggc ttg gaa tgc ctt cgg 2797Phe
Tyr Thr Glu Ser Asp Val Asn Lys Glu Gly Leu Glu Cys Leu Arg 895 900
905ctc ctg aac gag atc atc gct gac ttt gat gat ctt ctt tcc aag cca
2845Leu Leu Asn Glu Ile Ile Ala Asp Phe Asp Asp Leu Leu Ser Lys
Pro910 915 920 925aaa ttc agt gga gtt gaa aag att aag acc att ggc
agc aca tac atg 2893Lys Phe Ser Gly Val Glu Lys Ile Lys Thr Ile Gly
Ser Thr Tyr Met 930 935 940gca gca aca ggt ctg agc gct gtg ccc agc
cag gag cac tcc cag gag 2941Ala Ala Thr Gly Leu Ser Ala Val Pro Ser
Gln Glu His Ser Gln Glu 945 950 955ccc gag cgg cag tac atg cac att
ggc acc atg gtg gag ttt gct ttt 2989Pro Glu Arg Gln Tyr Met His Ile
Gly Thr Met Val Glu Phe Ala Phe 960 965 970gcc ctg gta ggg aag ctg
gat gcc atc aac aag cac tcc ttc aac gac 3037Ala Leu Val Gly Lys Leu
Asp Ala Ile Asn Lys His Ser Phe Asn Asp 975 980 985ttc aaa ttg cga
gtg ggt att aac cat gga cct gtg ata gct ggt gtg 3085Phe Lys Leu Arg
Val Gly Ile Asn His Gly Pro Val Ile Ala Gly Val 990 995 1000
1005att gga gct cag aag cca caa tat gat atc tgg ggc aac act gtc aat
3133Ile Gly Ala Gln Lys Pro Gln Tyr Asp Ile Trp Gly Asn Thr Val Asn
1010 1015 1020gtg gcc agt agg atg gac agc acc gga gtc ctg gac aaa
ata cag gtt 3181Val Ala Ser Arg Met Asp Ser Thr Gly Val Leu Asp Lys
Ile Gln Val 1025 1030 1035acc gag gag acg agc ctc gtc ctg cag acc
ctc gga tac acg tgc acc 3229Thr Glu Glu Thr Ser Leu Val Leu Gln Thr
Leu Gly Tyr Thr Cys Thr 1040 1045 1050tgt cga gga ata atc aac gtg
aaa gga aag ggg gac ctg aag acg tac 3277Cys Arg Gly Ile Ile Asn Val
Lys Gly Lys Gly Asp Leu Lys Thr Tyr 1055 1060 1065ttt gta aac aca
gaa atg tca agg tcc ctt tcc cag agc aac gtg gca 3325Phe Val Asn Thr
Glu Met Ser Arg Ser Leu Ser Gln Ser Asn Val Ala1070 1075 1080
1085tcc tga agagtcacct tcattttggc aagaagactg tattttcagg aaggtatcac
3381Ser *acactttctg actgcaactt ctgtcccttg tttttgatgt gcgtgctgtc
tgtcctatgg 3441agcctctgca gactcgttct cgtgacccag tggcataccg
tttggtgtct gatgtgtgcc 3501cagatcgttc tgccacttgc actgtgcttg
ctcctaagca aaagggaaaa ggagcgcgcg 3561tgatagaaga aaagcactgg
gagaactaac agaggagaaa ggtgaaacac acacacattc 3621ttaaggcaat
aaaactaggg ggtgtatatt atcttctggg gcatgttctt ttctggaaaa
3681tatggtagct cgccaaccgc atctgctcat ctgatattca aacacacagt
attcgtgaat 3741aagttgattc tgtcccccac gtggactctg tgctcaccca
ttgtctcatt gccagtggtg 3801tccaagggcc cccgttggga cccacggctc
tcgtccctct gctccgtgtg tctcatgcca 3861gcagcacgtc gccatccgtc
accagaatta gtcctcacag cctaggacca gttttgtatc 3921aaactcgtct
gatgttttga tgccatttgt cttttgtaaa gttaattcat taaaagtttt
3981atgtactttg aaaaaaaaaa aaaaagagcg 4011212847DNAHomo
sapiensCDS(60)...(1703) 21tcactatagg gagtcgaccc acgcgtccga
tgcagccttg ataatcatcc gattccaga 59atg ggt ggc tgc att cct ttt ctg
aag gca gca agg gca ctg tgc ccc 107Met Gly Gly Cys Ile Pro Phe Leu
Lys Ala Ala Arg Ala Leu Cys Pro 1 5 10 15aga atc atg ccc cct ttg
ctg ttg ttg tcc gcc ttc att ttt tta gtg 155Arg Ile Met Pro Pro Leu
Leu Leu Leu Ser Ala Phe Ile Phe Leu Val 20 25 30agt gtc ttg gga gga
gcc cca gga cac aac ccc gac cgc agg acg aag 203Ser Val Leu Gly Gly
Ala Pro Gly His Asn Pro Asp Arg Arg Thr Lys 35 40 45atg gta tcg ata
cac agc ctc tct gag ctg gag cgt ctg aag ctg caa 251Met Val Ser Ile
His Ser Leu Ser Glu Leu Glu Arg Leu Lys Leu Gln 50 55 60gag act gct
tac cac gaa ctc gtg gcc aga cat ttc ctc tcc gaa ttc 299Glu Thr Ala
Tyr His Glu Leu Val Ala Arg His Phe Leu Ser Glu Phe 65 70 75 80aaa
cct gac aga gct ctg cct att gac cgt ccg aac acc ttg gat aag 347Lys
Pro Asp Arg Ala Leu Pro Ile Asp Arg Pro Asn Thr Leu Asp Lys 85 90
95tgg ttt ctg att ttg aga gga cag cag agg gct gta tca cac aag aca
395Trp Phe Leu Ile Leu Arg Gly Gln Gln Arg Ala Val Ser His Lys Thr
100 105 110ttt ggc att agc ctg gaa gag gtc ctg gtg aac gag ttt acc
cgc cgc 443Phe Gly Ile Ser Leu Glu Glu Val Leu Val Asn Glu Phe Thr
Arg Arg 115 120 125aag cat ctt gaa ctg aca gcc acg atg cag gtt gaa
gaa gcc acc ggt 491Lys His Leu Glu Leu Thr Ala Thr Met Gln Val Glu
Glu Ala Thr Gly 130 135 140cag gct gcg ggc cgt cgt cgg gga aac gtg
gtg cga agg gtg ttt ggc 539Gln Ala Ala Gly Arg Arg Arg Gly Asn Val
Val Arg Arg Val Phe Gly145 150 155 160cgc atc cgg cgc ttt ttc agt
cgc agg cgg aat gag ccc acc ttg ccc 587Arg Ile Arg Arg Phe Phe Ser
Arg Arg Arg Asn Glu Pro Thr Leu Pro 165 170 175cgg gag ttc act cgc
cgt ggg cgt cga ggt gca gtg tct gtg gat agt 635Arg Glu Phe Thr Arg
Arg Gly Arg Arg Gly Ala Val Ser Val Asp Ser 180 185 190ctg gct gag
ctg gaa gac gga gcc ctg ctg ctg cag acc ctg cag ctt 683Leu Ala Glu
Leu Glu Asp Gly Ala Leu Leu Leu Gln Thr Leu Gln Leu 195 200 205tca
aaa att tcc ttt cca att ggc caa cga ctt ctg gga tcc aaa agg 731Ser
Lys Ile Ser Phe Pro Ile Gly Gln Arg Leu Leu Gly Ser Lys Arg 210 215
220aag atg agt ctc aat ccg att gcg aaa caa atc ccc cag gtt gtt gag
779Lys Met Ser Leu Asn Pro Ile Ala Lys Gln Ile Pro Gln Val Val
Glu225 230 235 240gct tgc tgc caa ttc att gaa aaa cat ggc tta agc
gca gtg ggg att 827Ala Cys Cys Gln Phe Ile Glu Lys His Gly Leu Ser
Ala Val Gly Ile 245 250 255ttt acc ctt gaa tac tcc gtg cag cga gtg
cgt cag ctc cgt gaa gaa 875Phe Thr Leu Glu Tyr Ser Val Gln Arg Val
Arg Gln Leu Arg Glu Glu 260 265 270ttt gat caa ggt ctg gat gta gtg
ctg gat gac aat cag aat gtg cat 923Phe Asp Gln Gly Leu Asp Val Val
Leu Asp Asp Asn Gln Asn Val His 275 280 285gat gtg gct gca ctc ctc
aag gag ttt ttc cgt gac atg aag gat tct 971Asp Val Ala Ala Leu Leu
Lys Glu Phe Phe Arg Asp Met Lys Asp Ser 290 295 300ctg ctg cca gat
gat ctg tac atg tca ttc ctc ctg aca gca act tta 1019Leu Leu Pro Asp
Asp Leu Tyr Met Ser Phe Leu Leu Thr Ala Thr Leu305 310 315 320aag
ccc cag gat cag ctt tct gcc ctg cag ttg ctg gtc tac ctg atg 1067Lys
Pro Gln Asp Gln Leu Ser Ala Leu Gln Leu Leu Val Tyr Leu Met 325 330
335cca ccc tgc cac agt gat acc ctg gag cgt ctg ctg aag gcc ctg cat
1115Pro Pro Cys His Ser Asp Thr Leu Glu Arg Leu Leu Lys Ala Leu His
340 345 350aaa atc act gag aac tgc gag gac tca att ggc att gat gga
cag ttg 1163Lys Ile Thr Glu Asn Cys Glu Asp Ser Ile Gly Ile Asp Gly
Gln Leu 355 360 365gtc cca ggc aac cgt atg act tcc act aac ttg gcc
ttg gtg ttt gga 1211Val Pro Gly Asn Arg Met Thr Ser Thr Asn Leu Ala
Leu Val Phe Gly 370 375 380tct gct ctc ctg aaa aaa gga aag ttt ggc
aag aga gag tcc agg aaa 1259Ser Ala Leu Leu Lys Lys Gly Lys Phe Gly
Lys Arg Glu Ser Arg Lys385 390 395 400aca aag ctg ggg att gat cac
tat gtt gct tct gtc aat gtg gtc cgt 1307Thr Lys Leu Gly Ile Asp His
Tyr Val Ala Ser Val Asn Val Val Arg 405 410 415gcc atg att gat aac
tgg gat gtc ctc ttc cag gtg cct ccc cat att 1355Ala Met Ile Asp Asn
Trp Asp Val Leu Phe Gln Val Pro Pro His Ile 420 425 430cag agg cag
gtt gct aag cgc gtg tgg aag tcc agc ccg gaa gca ctt 1403Gln Arg Gln
Val Ala Lys Arg Val Trp Lys Ser Ser Pro Glu Ala Leu 435 440 445gat
ttt atc aga cgc agg aac ttg agg aag atc cag agt gca cgc ata 1451Asp
Phe Ile Arg Arg Arg Asn Leu Arg Lys Ile Gln Ser Ala Arg Ile 450 455
460aag atg gaa gag gat gca cta ctt tct gat cca gtg gaa acc tct gct
1499Lys Met Glu Glu Asp Ala Leu Leu Ser Asp Pro Val Glu Thr Ser
Ala465 470 475 480gaa gcc cgg gct gct gtc ctt gct caa agc aag cct
tct gat gaa ggt 1547Glu Ala Arg Ala Ala Val Leu Ala Gln Ser Lys Pro
Ser Asp Glu Gly 485 490 495tcc tct gag gag cca gct gtg cct tcc ggc
act gcc cgt tcc cat gac 1595Ser Ser Glu Glu Pro Ala Val Pro Ser Gly
Thr Ala Arg Ser His Asp 500 505 510gat gag gaa gga gcg ggt aac cct
ccc att ccg gag caa gac cgc cca 1643Asp Glu Glu Gly Ala Gly Asn Pro
Pro Ile Pro Glu Gln Asp Arg Pro 515 520 525ttg ctc cgt gtg ccc cgg
gag aag gag gcc aaa act ggc gtc agc tac 1691Leu Leu Arg Val Pro Arg
Glu Lys Glu Ala Lys Thr Gly Val Ser Tyr 530 535 540ttc ttt cct tag
atgtttttcc ttctataagg tgccagacag gggaaaaggg 1743Phe Phe Pro
*545tgggggtaca tctgggatgt cacaggaaac attaaggaga gagttgaagg
taaagatctg 1803aaggtaagaa ggagttccac ctgatgctcg ggtcaggatg
agaattccaa acacactgcc 1863agccccttca ctggggatgc ttggkctctt
ctgctggtaa aagcagagat gttttctgtg 1923tcatgcccaa gctccccggt
gctaccttgc ctttctcttt tacccctgat cttggctttc 1983tctctctctc
tgcagacttt cctttaattg atgtgacatt tgtggtaaac acctttccca
2043gggaacctca caaatcttga gatgctttcc cttccccaaa tgggattgca
tgatttccct 2103gactttccta ccctcctcca gagagctcag ttggaaaggc
cctcaagagg catgctagaa 2163cgttaggtca gcctactgac agctgacaaa
caattaatgc gaaatcatgt cacaccaacc 2223catagccgtg tccacgcagc
aactccacca ccttaggatt tccccctcca aattattcag 2283accaatggct
tgccaaatgg cctctcccaa aattctgtac agttttgctc aggtcacgcc
2343aacagggaaa cctcaagtgt aggtctaatt agtgtttctg ggatccaaag
ttagaggaaa 2403atttagattt tattgcctgg atctgcttta aagacaattg
gtgtttacac cctcttgtca 2463gcaaaacagc tagttaggta aggacatata
gttccaagta ggtaaagtca cttgattaca 2523aatgttctta actatcgtct
ctgtaattcc tttatacagg acagtacaaa attgtgggac 2583atgctctggt
aacacacaga tatgggttgc atatgatcca gaattacagc tgatattatg
2643gatgacaact gctaaggtcc ataaaatgaa gactgtattg tattgaggga
tagaaattga 2703tcatttaatg ggtaacaact gctgagctca aagatttgtg
attgttaaaa cttctctggc 2763atttaatcat taataaacat ctgtattgtg
ccaccagcat aaaaaaaaaa aaaaaaaaaa 2823aaaaaaaaaa aaaaaaaaaa aagg
284722547PRTHomo sapiens 22Met Gly Gly Cys Ile Pro Phe Leu Lys Ala
Ala Arg Ala Leu Cys Pro 1 5 10 15Arg Ile Met Pro Pro Leu Leu Leu
Leu Ser Ala Phe Ile Phe Leu Val 20 25 30Ser Val Leu Gly Gly Ala Pro
Gly His Asn Pro Asp Arg Arg Thr Lys 35 40 45Met Val Ser Ile His Ser
Leu Ser Glu Leu Glu Arg Leu Lys Leu Gln 50 55 60Glu Thr Ala Tyr His
Glu Leu Val Ala Arg His Phe Leu Ser Glu Phe65 70 75 80Lys Pro Asp
Arg Ala Leu Pro Ile Asp Arg Pro Asn Thr Leu Asp Lys 85 90 95Trp Phe
Leu Ile Leu Arg Gly Gln Gln Arg Ala Val Ser His Lys Thr 100 105
110Phe Gly Ile Ser Leu Glu Glu Val Leu Val Asn Glu Phe Thr Arg Arg
115 120 125Lys His Leu Glu Leu Thr Ala Thr Met Gln Val Glu Glu Ala
Thr Gly 130 135 140Gln Ala Ala Gly Arg Arg Arg Gly Asn Val Val Arg
Arg Val Phe Gly145 150 155 160Arg Ile Arg Arg Phe Phe Ser Arg Arg
Arg Asn Glu Pro Thr Leu Pro 165 170 175Arg Glu Phe Thr Arg Arg Gly
Arg Arg Gly Ala Val Ser Val Asp Ser 180 185 190Leu Ala Glu Leu Glu
Asp Gly Ala Leu Leu Leu Gln Thr Leu Gln Leu 195 200 205Ser Lys Ile
Ser Phe Pro Ile Gly Gln Arg Leu Leu Gly Ser Lys Arg 210 215 220Lys
Met Ser Leu Asn Pro Ile Ala Lys Gln Ile Pro Gln Val Val Glu225 230
235 240Ala Cys Cys Gln Phe Ile Glu Lys His Gly Leu Ser Ala Val Gly
Ile 245 250 255Phe Thr Leu Glu Tyr Ser Val Gln Arg Val Arg Gln Leu
Arg Glu Glu 260 265 270Phe Asp Gln Gly Leu Asp Val Val Leu Asp Asp
Asn Gln Asn Val His 275 280 285Asp Val Ala Ala Leu Leu Lys Glu Phe
Phe Arg Asp Met Lys Asp Ser 290 295 300Leu Leu Pro Asp Asp Leu Tyr
Met Ser Phe Leu Leu Thr Ala Thr Leu305 310 315 320Lys Pro Gln Asp
Gln Leu Ser Ala Leu Gln Leu Leu Val Tyr Leu Met 325 330 335Pro Pro
Cys His Ser Asp Thr Leu Glu Arg Leu Leu Lys Ala Leu His 340 345
350Lys Ile Thr Glu Asn Cys Glu Asp Ser Ile Gly Ile Asp Gly Gln Leu
355 360 365Val Pro Gly Asn Arg Met Thr Ser Thr Asn Leu Ala Leu Val
Phe Gly 370 375 380Ser Ala Leu Leu Lys Lys Gly Lys Phe Gly Lys Arg
Glu Ser Arg Lys385 390 395 400Thr Lys Leu Gly Ile Asp His Tyr Val
Ala Ser Val Asn Val Val Arg 405 410 415Ala Met Ile Asp Asn Trp Asp
Val Leu Phe Gln Val Pro Pro His Ile 420 425 430Gln Arg Gln Val Ala
Lys Arg Val Trp Lys Ser Ser Pro Glu Ala Leu 435 440 445Asp Phe Ile
Arg Arg Arg Asn Leu Arg Lys Ile Gln Ser Ala Arg Ile 450 455 460Lys
Met Glu Glu Asp Ala Leu Leu Ser Asp Pro Val Glu Thr Ser Ala465 470
475 480Glu Ala Arg Ala Ala Val Leu Ala Gln Ser Lys Pro Ser Asp Glu
Gly 485 490 495Ser Ser Glu Glu Pro Ala Val Pro Ser Gly Thr Ala Arg
Ser His Asp 500 505 510Asp Glu Glu Gly Ala Gly Asn Pro Pro Ile Pro
Glu Gln Asp Arg Pro 515 520 525Leu Leu Arg Val Pro Arg Glu Lys Glu
Ala Lys Thr Gly Val Ser Tyr 530 535 540Phe Phe Pro545231644DNAHomo
sapiens 23atgggtggct gcattccttt tctgaaggca gcaagggcac tgtgccccag
aatcatgccc 60cctttgctgt tgttgtccgc cttcattttt ttagtgagtg tcttgggagg
agccccagga 120cacaaccccg accgcaggac gaagatggta tcgatacaca
gcctctctga gctggagcgt 180ctgaagctgc aagagactgc ttaccacgaa
ctcgtggcca gacatttcct ctccgaattc 240aaacctgaca gagctctgcc
tattgaccgt ccgaacacct tggataagtg gtttctgatt 300ttgagaggac
agcagagggc tgtatcacac aagacatttg gcattagcct ggaagaggtc
360ctggtgaacg agtttacccg ccgcaagcat cttgaactga cagccacgat
gcaggttgaa 420gaagccaccg gtcaggctgc gggccgtcgt cggggaaacg
tggtgcgaag ggtgtttggc 480cgcatccggc gctttttcag tcgcaggcgg
aatgagccca ccttgccccg ggagttcact 540cgccgtgggc gtcgaggtgc
agtgtctgtg gatagtctgg ctgagctgga agacggagcc 600ctgctgctgc
agaccctgca gctttcaaaa atttcctttc caattggcca acgacttctg
660ggatccaaaa ggaagatgag tctcaatccg attgcgaaac aaatccccca
ggttgttgag 720gcttgctgcc aattcattga aaaacatggc ttaagcgcag
tggggatttt tacccttgaa 780tactccgtgc agcgagtgcg tcagctccgt
gaagaatttg atcaaggtct ggatgtagtg 840ctggatgaca atcagaatgt
gcatgatgtg gctgcactcc tcaaggagtt tttccgtgac 900atgaaggatt
ctctgctgcc agatgatctg tacatgtcat tcctcctgac agcaacttta
960aagccccagg atcagctttc tgccctgcag ttgctggtct acctgatgcc
accctgccac 1020agtgataccc tggagcgtct gctgaaggcc ctgcataaaa
tcactgagaa ctgcgaggac 1080tcaattggca ttgatggaca gttggtccca
ggcaaccgta
tgacttccac taacttggcc 1140ttggtgtttg gatctgctct cctgaaaaaa
ggaaagtttg gcaagagaga gtccaggaaa 1200acaaagctgg ggattgatca
ctatgttgct tctgtcaatg tggtccgtgc catgattgat 1260aactgggatg
tcctcttcca ggtgcctccc catattcaga ggcaggttgc taagcgcgtg
1320tggaagtcca gcccggaagc acttgatttt atcagacgca ggaacttgag
gaagatccag 1380agtgcacgca taaagatgga agaggatgca ctactttctg
atccagtgga aacctctgct 1440gaagcccggg ctgctgtcct tgctcaaagc
aagccttctg atgaaggttc ctctgaggag 1500ccagctgtgc cttccggcac
tgcccgttcc catgacgatg aggaaggagc gggtaaccct 1560cccattccgg
agcaagaccg cccattgctc cgtgtgcccc gggagaagga ggccaaaact
1620ggcgtcagct acttctttcc ttag 1644243391DNAHomo
sapiensCDS(78)...(3095) 24gaggaagcca ggcggggtgc agacggctgc
tgattctggg gctggtcagg aaaccaagga 60gacccccccc cccaacc atg gac cca
ccg tcg cca agc cgg acc tcc caa 110 Met Asp Pro Pro Ser Pro Ser Arg
Thr Ser Gln 1 5 10acc cag ccc aca gcc acc tct ccg ctg act tcc tac
cgc tgg cac aca 158Thr Gln Pro Thr Ala Thr Ser Pro Leu Thr Ser Tyr
Arg Trp His Thr 15 20 25ggg ggc ggt ggg gag aag gcg gct gga ggg ttc
cgc tgg ggc cgc ttt 206Gly Gly Gly Gly Glu Lys Ala Ala Gly Gly Phe
Arg Trp Gly Arg Phe 30 35 40gct ggc tgg ggc agg gcc ctg agc cac cag
gag ccc atg gtc agc acc 254Ala Gly Trp Gly Arg Ala Leu Ser His Gln
Glu Pro Met Val Ser Thr 45 50 55cag cca gcc cct cgc tcg ata ttc cgt
cgg gtc cta tct gcg cct ccc 302Gln Pro Ala Pro Arg Ser Ile Phe Arg
Arg Val Leu Ser Ala Pro Pro 60 65 70 75aag gag tca cgg acc agt cgc
ctt cga ctc tcc aag gcc ctc tgg ggg 350Lys Glu Ser Arg Thr Ser Arg
Leu Arg Leu Ser Lys Ala Leu Trp Gly 80 85 90agg cat aag aac cca ccg
ccg gag cca gac ccg gag ccg gag cag gag 398Arg His Lys Asn Pro Pro
Pro Glu Pro Asp Pro Glu Pro Glu Gln Glu 95 100 105gcc cca gag ctg
gag ccg gag cca gag ctg gag ccc cct acc cca cag 446Ala Pro Glu Leu
Glu Pro Glu Pro Glu Leu Glu Pro Pro Thr Pro Gln 110 115 120atc cct
gag gcc ccc aca ccc aac gtg cct gtc tgg gac att ggg ggc 494Ile Pro
Glu Ala Pro Thr Pro Asn Val Pro Val Trp Asp Ile Gly Gly 125 130
135ttc acc ctg ctt gat ggg aag ctg gtg ctg ctt gga gga gag gag gag
542Phe Thr Leu Leu Asp Gly Lys Leu Val Leu Leu Gly Gly Glu Glu
Glu140 145 150 155ggt cct cga agg ccc cgg gtg gga agt gct agc tcc
gag ggc agc atc 590Gly Pro Arg Arg Pro Arg Val Gly Ser Ala Ser Ser
Glu Gly Ser Ile 160 165 170cac gtg gcc atg ggg aac ttc agg gat cca
gat cgg atg cct gga aaa 638His Val Ala Met Gly Asn Phe Arg Asp Pro
Asp Arg Met Pro Gly Lys 175 180 185aca gaa ccg gag act gct ggt ccc
aac cag gtc cac aac gtt cgg ggg 686Thr Glu Pro Glu Thr Ala Gly Pro
Asn Gln Val His Asn Val Arg Gly 190 195 200ttg ctc aag agg ctg aaa
gag aag aaa aag gcc aga ccc ccc agt gct 734Leu Leu Lys Arg Leu Lys
Glu Lys Lys Lys Ala Arg Pro Pro Ser Ala 205 210 215ctg ggc tct agg
gag tcg ctg gcc aca ctc tct gaa ctg gac ctg ggt 782Leu Gly Ser Arg
Glu Ser Leu Ala Thr Leu Ser Glu Leu Asp Leu Gly220 225 230 235gcc
gag cgg gat gtg cgg atc tgg cca ctg cac ccc agc ctc ctg ggg 830Ala
Glu Arg Asp Val Arg Ile Trp Pro Leu His Pro Ser Leu Leu Gly 240 245
250gag ccc cac tgc ttt cag gta acc tgg acg ggt gga agc cgc tgc ttc
878Glu Pro His Cys Phe Gln Val Thr Trp Thr Gly Gly Ser Arg Cys Phe
255 260 265tct tgt cgc tcg gcc gct gag aga gac cgc tgg atc gag gac
ctt cgt 926Ser Cys Arg Ser Ala Ala Glu Arg Asp Arg Trp Ile Glu Asp
Leu Arg 270 275 280cgc caa ttc cag ccc acc cag gac aac gtg gag cgg
gaa gag aca tgg 974Arg Gln Phe Gln Pro Thr Gln Asp Asn Val Glu Arg
Glu Glu Thr Trp 285 290 295ctg agc gtg tgg gtg cac gaa gcg aag ggg
ctt ccc cga gca gcg gcg 1022Leu Ser Val Trp Val His Glu Ala Lys Gly
Leu Pro Arg Ala Ala Ala300 305 310 315ggg gca ccc ggc gtg cgc gcc
gag ctg tgg ctg gat ggc gcg ctg ctg 1070Gly Ala Pro Gly Val Arg Ala
Glu Leu Trp Leu Asp Gly Ala Leu Leu 320 325 330gca cgc acg gcg cct
cgg gcc ggc cca ggc cag ctc ttc tgg gcc gag 1118Ala Arg Thr Ala Pro
Arg Ala Gly Pro Gly Gln Leu Phe Trp Ala Glu 335 340 345cgc ttc cac
ttc gag gcg ctg cca ccg gca cgt cgc ctg tcg ctg cgg 1166Arg Phe His
Phe Glu Ala Leu Pro Pro Ala Arg Arg Leu Ser Leu Arg 350 355 360ctg
cgc ggc ttg ggc ccg gga agc gcg gtg ctg ggc cgc gtg gcc ctg 1214Leu
Arg Gly Leu Gly Pro Gly Ser Ala Val Leu Gly Arg Val Ala Leu 365 370
375gcg ctg gag gag ctg gac gcc cca cgc gcg cct gcc gcc ggt ctg gag
1262Ala Leu Glu Glu Leu Asp Ala Pro Arg Ala Pro Ala Ala Gly Leu
Glu380 385 390 395cgc tgg ttc ccg ctg ctc ggg gcg ccg gcg ggc gca
gcg ctg cgg gcg 1310Arg Trp Phe Pro Leu Leu Gly Ala Pro Ala Gly Ala
Ala Leu Arg Ala 400 405 410cgg att cgg gcg cgt cgc ctg cgc gtg ctg
ccg tcc gag cgc tac aag 1358Arg Ile Arg Ala Arg Arg Leu Arg Val Leu
Pro Ser Glu Arg Tyr Lys 415 420 425gag ctg gcg gag ttc ctc acc ttc
cac tat gcg cgc ctc tgc ggg gcc 1406Glu Leu Ala Glu Phe Leu Thr Phe
His Tyr Ala Arg Leu Cys Gly Ala 430 435 440ctg gag ccc gcg ctg cct
gcg cag gcc aag gag gag ctg gcg gca gcc 1454Leu Glu Pro Ala Leu Pro
Ala Gln Ala Lys Glu Glu Leu Ala Ala Ala 445 450 455atg gtg cgc gtg
ctg cgg gcc acc ggc cgg gcg cag gcg ctg gtg act 1502Met Val Arg Val
Leu Arg Ala Thr Gly Arg Ala Gln Ala Leu Val Thr460 465 470 475gac
ctg ggc act gcg gag ctg gcg cgc tgt gga ggc cgt gag gcg ctg 1550Asp
Leu Gly Thr Ala Glu Leu Ala Arg Cys Gly Gly Arg Glu Ala Leu 480 485
490ctg ttc cgg gaa aac aca ttg gcc acc aag gct atc gat gag tac atg
1598Leu Phe Arg Glu Asn Thr Leu Ala Thr Lys Ala Ile Asp Glu Tyr Met
495 500 505aag ctc gtg gca cag gat tac ctc cag gag acc ctg gga cag
gtt gtg 1646Lys Leu Val Ala Gln Asp Tyr Leu Gln Glu Thr Leu Gly Gln
Val Val 510 515 520cgg cgt ctc tgt gct tct act gag gac tgt gaa gtg
gac ccc agc aaa 1694Arg Arg Leu Cys Ala Ser Thr Glu Asp Cys Glu Val
Asp Pro Ser Lys 525 530 535tgt cca gcc tcg gag ctg cca gag cac cag
gcc aga ctt cgg aac agc 1742Cys Pro Ala Ser Glu Leu Pro Glu His Gln
Ala Arg Leu Arg Asn Ser540 545 550 555tgc gag gag gtc ttc gaa acc
att atc cat tcc tac gac tgg ttc cct 1790Cys Glu Glu Val Phe Glu Thr
Ile Ile His Ser Tyr Asp Trp Phe Pro 560 565 570gcg gag ctg ggc atc
gtg ttc tca agc tgg cga gaa gca tgt aaa gaa 1838Ala Glu Leu Gly Ile
Val Phe Ser Ser Trp Arg Glu Ala Cys Lys Glu 575 580 585cgt ggc tct
gag gtg ctg ggc ccc cga ctg gtg tgc gcc tcc ctc ttc 1886Arg Gly Ser
Glu Val Leu Gly Pro Arg Leu Val Cys Ala Ser Leu Phe 590 595 600ctg
cgg ctc ctg tgc cct gcc atc ctg gca ccc agc ctc ttt ggt ttg 1934Leu
Arg Leu Leu Cys Pro Ala Ile Leu Ala Pro Ser Leu Phe Gly Leu 605 610
615gca cca gac cat cca gca ccc ggc cca gcc cgc acc ctc aca ctg att
1982Ala Pro Asp His Pro Ala Pro Gly Pro Ala Arg Thr Leu Thr Leu
Ile620 625 630 635gcc aag gtc atc cag aac ctc gcc aac cgt gcc ccg
ttc ggt gag aag 2030Ala Lys Val Ile Gln Asn Leu Ala Asn Arg Ala Pro
Phe Gly Glu Lys 640 645 650gag gcc tac atg ggc ttc atg aat agc ttc
ctg gag gaa cat gga cca 2078Glu Ala Tyr Met Gly Phe Met Asn Ser Phe
Leu Glu Glu His Gly Pro 655 660 665gcc atg caa tgc ttc ctg gac cag
gta gcc atg gtg gat gtg gat gct 2126Ala Met Gln Cys Phe Leu Asp Gln
Val Ala Met Val Asp Val Asp Ala 670 675 680gcc ccc agt ggt tac cag
ggc agt ggt gat ctg gcc ctc cag tta gct 2174Ala Pro Ser Gly Tyr Gln
Gly Ser Gly Asp Leu Ala Leu Gln Leu Ala 685 690 695gtc ctg cat gcc
cag ctc tgt aca att ttt gct gag ctt gac cag aca 2222Val Leu His Ala
Gln Leu Cys Thr Ile Phe Ala Glu Leu Asp Gln Thr700 705 710 715acc
cga gac acc ctg gaa cca ctg ccc acc atc ctg cga gcc att gag 2270Thr
Arg Asp Thr Leu Glu Pro Leu Pro Thr Ile Leu Arg Ala Ile Glu 720 725
730gag ggc cag cct gtg ctt gtg tca gtg cca atg cgt ctc cca ctg ccc
2318Glu Gly Gln Pro Val Leu Val Ser Val Pro Met Arg Leu Pro Leu Pro
735 740 745ccg gcc cag gtc cac tcc agc ctc tcc gca ggg gag aag ccc
ggc ttc 2366Pro Ala Gln Val His Ser Ser Leu Ser Ala Gly Glu Lys Pro
Gly Phe 750 755 760ctg gcc ccc cgg gac ctc ccc aag cac acc cct ctc
atc tcc aag agc 2414Leu Ala Pro Arg Asp Leu Pro Lys His Thr Pro Leu
Ile Ser Lys Ser 765 770 775cag tct ctg cgc agc gtt cgc cgc tca gag
agt tgg gcc cgg cca cgg 2462Gln Ser Leu Arg Ser Val Arg Arg Ser Glu
Ser Trp Ala Arg Pro Arg780 785 790 795ccg gac gaa gag cgg ccc ctg
cgg cgg ccc cgg ccg gtg cag cgc acg 2510Pro Asp Glu Glu Arg Pro Leu
Arg Arg Pro Arg Pro Val Gln Arg Thr 800 805 810cag agt gtc ccg gtc
cgg cgt cct gcc cgc cgc cgc caa tct gcg ggg 2558Gln Ser Val Pro Val
Arg Arg Pro Ala Arg Arg Arg Gln Ser Ala Gly 815 820 825ccc tgg ccg
cga ccc aaa ggc tcc ctg agc atg gga cca gcg ccc cgc 2606Pro Trp Pro
Arg Pro Lys Gly Ser Leu Ser Met Gly Pro Ala Pro Arg 830 835 840gcc
cgg cct tgg acc cgg gac tcc gcc tcg ctg cct cgg aag ccg tcg 2654Ala
Arg Pro Trp Thr Arg Asp Ser Ala Ser Leu Pro Arg Lys Pro Ser 845 850
855gta ccc tgg cag cgc caa atg gac cag ccg caa gac cga aac cag gca
2702Val Pro Trp Gln Arg Gln Met Asp Gln Pro Gln Asp Arg Asn Gln
Ala860 865 870 875ctg ggc acg cac cga cct gtg aac aag ttg gca gag
ctg cag tgc gag 2750Leu Gly Thr His Arg Pro Val Asn Lys Leu Ala Glu
Leu Gln Cys Glu 880 885 890gtg gcc gct ctg cgt gag gag cag aaa gtg
ctg tcc cgc ctc gtg gag 2798Val Ala Ala Leu Arg Glu Glu Gln Lys Val
Leu Ser Arg Leu Val Glu 895 900 905tcg ctg agc acc caa atc cgg gcc
ttg acg gag cag cag gag cag ctg 2846Ser Leu Ser Thr Gln Ile Arg Ala
Leu Thr Glu Gln Gln Glu Gln Leu 910 915 920cgg ggc cag ctg cag gat
ctg gac tcc agg ctc cgt gct ggg agc tca 2894Arg Gly Gln Leu Gln Asp
Leu Asp Ser Arg Leu Arg Ala Gly Ser Ser 925 930 935gag ttt gat tca
gag cac aac cta aca agc aat gaa ggg cac agt ctg 2942Glu Phe Asp Ser
Glu His Asn Leu Thr Ser Asn Glu Gly His Ser Leu940 945 950 955aaa
aac ctg gag cac cgc cta aat gag atg gag aga act cag gct cag 2990Lys
Asn Leu Glu His Arg Leu Asn Glu Met Glu Arg Thr Gln Ala Gln 960 965
970ctg agg gat gct gtc cag agc ctg cag ctt tct cca agg acg cgg ggg
3038Leu Arg Asp Ala Val Gln Ser Leu Gln Leu Ser Pro Arg Thr Arg Gly
975 980 985tct tgg agt caa ccc cag ccc ctc aaa gca ccc tgc ctc aat
gga gac 3086Ser Trp Ser Gln Pro Gln Pro Leu Lys Ala Pro Cys Leu Asn
Gly Asp 990 995 1000acc acc tga gctgcccatc ctgcctcatc acacgtggtc
tgggagcaga 3135Thr Thr * 1005gagatagcca tcttaggggg ggtgtctgac
tttgccttag ccctacttgg cctacagtgg 3195ggagtggagc tgctggtccc
aaccactctg gcagtatgaa gttgcccagt aaaatcttga 3255tttcagtgaa
aaaaaaaaaa aaaagggcgg rccgctagac twagtctaga gaaaaaacct
3315cccacacctc cccctgaacc traaacathc cammacctcc ccctsawsmw
smwrmawmaw 3375araawksaaw gcaatg 3391251005PRTHomo sapiens 25Met
Asp Pro Pro Ser Pro Ser Arg Thr Ser Gln Thr Gln Pro Thr Ala 1 5 10
15Thr Ser Pro Leu Thr Ser Tyr Arg Trp His Thr Gly Gly Gly Gly Glu
20 25 30Lys Ala Ala Gly Gly Phe Arg Trp Gly Arg Phe Ala Gly Trp Gly
Arg 35 40 45Ala Leu Ser His Gln Glu Pro Met Val Ser Thr Gln Pro Ala
Pro Arg 50 55 60Ser Ile Phe Arg Arg Val Leu Ser Ala Pro Pro Lys Glu
Ser Arg Thr65 70 75 80Ser Arg Leu Arg Leu Ser Lys Ala Leu Trp Gly
Arg His Lys Asn Pro 85 90 95Pro Pro Glu Pro Asp Pro Glu Pro Glu Gln
Glu Ala Pro Glu Leu Glu 100 105 110Pro Glu Pro Glu Leu Glu Pro Pro
Thr Pro Gln Ile Pro Glu Ala Pro 115 120 125Thr Pro Asn Val Pro Val
Trp Asp Ile Gly Gly Phe Thr Leu Leu Asp 130 135 140Gly Lys Leu Val
Leu Leu Gly Gly Glu Glu Glu Gly Pro Arg Arg Pro145 150 155 160Arg
Val Gly Ser Ala Ser Ser Glu Gly Ser Ile His Val Ala Met Gly 165 170
175Asn Phe Arg Asp Pro Asp Arg Met Pro Gly Lys Thr Glu Pro Glu Thr
180 185 190Ala Gly Pro Asn Gln Val His Asn Val Arg Gly Leu Leu Lys
Arg Leu 195 200 205Lys Glu Lys Lys Lys Ala Arg Pro Pro Ser Ala Leu
Gly Ser Arg Glu 210 215 220Ser Leu Ala Thr Leu Ser Glu Leu Asp Leu
Gly Ala Glu Arg Asp Val225 230 235 240Arg Ile Trp Pro Leu His Pro
Ser Leu Leu Gly Glu Pro His Cys Phe 245 250 255Gln Val Thr Trp Thr
Gly Gly Ser Arg Cys Phe Ser Cys Arg Ser Ala 260 265 270Ala Glu Arg
Asp Arg Trp Ile Glu Asp Leu Arg Arg Gln Phe Gln Pro 275 280 285Thr
Gln Asp Asn Val Glu Arg Glu Glu Thr Trp Leu Ser Val Trp Val 290 295
300His Glu Ala Lys Gly Leu Pro Arg Ala Ala Ala Gly Ala Pro Gly
Val305 310 315 320Arg Ala Glu Leu Trp Leu Asp Gly Ala Leu Leu Ala
Arg Thr Ala Pro 325 330 335Arg Ala Gly Pro Gly Gln Leu Phe Trp Ala
Glu Arg Phe His Phe Glu 340 345 350Ala Leu Pro Pro Ala Arg Arg Leu
Ser Leu Arg Leu Arg Gly Leu Gly 355 360 365Pro Gly Ser Ala Val Leu
Gly Arg Val Ala Leu Ala Leu Glu Glu Leu 370 375 380Asp Ala Pro Arg
Ala Pro Ala Ala Gly Leu Glu Arg Trp Phe Pro Leu385 390 395 400Leu
Gly Ala Pro Ala Gly Ala Ala Leu Arg Ala Arg Ile Arg Ala Arg 405 410
415Arg Leu Arg Val Leu Pro Ser Glu Arg Tyr Lys Glu Leu Ala Glu Phe
420 425 430Leu Thr Phe His Tyr Ala Arg Leu Cys Gly Ala Leu Glu Pro
Ala Leu 435 440 445Pro Ala Gln Ala Lys Glu Glu Leu Ala Ala Ala Met
Val Arg Val Leu 450 455 460Arg Ala Thr Gly Arg Ala Gln Ala Leu Val
Thr Asp Leu Gly Thr Ala465 470 475 480Glu Leu Ala Arg Cys Gly Gly
Arg Glu Ala Leu Leu Phe Arg Glu Asn 485 490 495Thr Leu Ala Thr Lys
Ala Ile Asp Glu Tyr Met Lys Leu Val Ala Gln 500 505 510Asp Tyr Leu
Gln Glu Thr Leu Gly Gln Val Val Arg Arg Leu Cys Ala 515 520 525Ser
Thr Glu Asp Cys Glu Val Asp Pro Ser Lys Cys Pro Ala Ser Glu 530 535
540Leu Pro Glu His Gln Ala Arg Leu Arg Asn Ser Cys Glu Glu Val
Phe545 550 555 560Glu Thr Ile Ile His Ser Tyr Asp Trp Phe Pro Ala
Glu Leu Gly Ile 565 570 575Val Phe Ser Ser Trp Arg Glu Ala Cys Lys
Glu Arg Gly Ser Glu Val 580 585 590Leu Gly Pro Arg Leu Val Cys Ala
Ser Leu Phe Leu Arg Leu Leu Cys 595 600 605Pro Ala Ile Leu Ala Pro
Ser Leu Phe Gly Leu Ala Pro Asp His Pro 610 615 620Ala Pro Gly Pro
Ala Arg Thr Leu Thr Leu Ile Ala Lys Val Ile Gln625 630 635 640Asn
Leu Ala Asn Arg Ala Pro Phe Gly Glu Lys Glu Ala Tyr Met Gly 645 650
655Phe Met Asn Ser Phe Leu Glu Glu His Gly Pro Ala Met Gln Cys Phe
660 665 670Leu Asp Gln Val Ala Met Val Asp Val Asp Ala Ala Pro Ser
Gly Tyr 675 680
685Gln Gly Ser Gly Asp Leu Ala Leu Gln Leu Ala Val Leu His Ala Gln
690 695 700Leu Cys Thr Ile Phe Ala Glu Leu Asp Gln Thr Thr Arg Asp
Thr Leu705 710 715 720Glu Pro Leu Pro Thr Ile Leu Arg Ala Ile Glu
Glu Gly Gln Pro Val 725 730 735Leu Val Ser Val Pro Met Arg Leu Pro
Leu Pro Pro Ala Gln Val His 740 745 750Ser Ser Leu Ser Ala Gly Glu
Lys Pro Gly Phe Leu Ala Pro Arg Asp 755 760 765Leu Pro Lys His Thr
Pro Leu Ile Ser Lys Ser Gln Ser Leu Arg Ser 770 775 780Val Arg Arg
Ser Glu Ser Trp Ala Arg Pro Arg Pro Asp Glu Glu Arg785 790 795
800Pro Leu Arg Arg Pro Arg Pro Val Gln Arg Thr Gln Ser Val Pro Val
805 810 815Arg Arg Pro Ala Arg Arg Arg Gln Ser Ala Gly Pro Trp Pro
Arg Pro 820 825 830Lys Gly Ser Leu Ser Met Gly Pro Ala Pro Arg Ala
Arg Pro Trp Thr 835 840 845Arg Asp Ser Ala Ser Leu Pro Arg Lys Pro
Ser Val Pro Trp Gln Arg 850 855 860Gln Met Asp Gln Pro Gln Asp Arg
Asn Gln Ala Leu Gly Thr His Arg865 870 875 880Pro Val Asn Lys Leu
Ala Glu Leu Gln Cys Glu Val Ala Ala Leu Arg 885 890 895Glu Glu Gln
Lys Val Leu Ser Arg Leu Val Glu Ser Leu Ser Thr Gln 900 905 910Ile
Arg Ala Leu Thr Glu Gln Gln Glu Gln Leu Arg Gly Gln Leu Gln 915 920
925Asp Leu Asp Ser Arg Leu Arg Ala Gly Ser Ser Glu Phe Asp Ser Glu
930 935 940His Asn Leu Thr Ser Asn Glu Gly His Ser Leu Lys Asn Leu
Glu His945 950 955 960Arg Leu Asn Glu Met Glu Arg Thr Gln Ala Gln
Leu Arg Asp Ala Val 965 970 975Gln Ser Leu Gln Leu Ser Pro Arg Thr
Arg Gly Ser Trp Ser Gln Pro 980 985 990Gln Pro Leu Lys Ala Pro Cys
Leu Asn Gly Asp Thr Thr 995 1000 1005263018DNAHomo sapiens
26atggacccac cgtcgccaag ccggacctcc caaacccagc ccacagccac ctctccgctg
60acttcctacc gctggcacac agggggcggt ggggagaagg cggctggagg gttccgctgg
120ggccgctttg ctggctgggg cagggccctg agccaccagg agcccatggt
cagcacccag 180ccagcccctc gctcgatatt ccgtcgggtc ctatctgcgc
ctcccaagga gtcacggacc 240agtcgccttc gactctccaa ggccctctgg
gggaggcata agaacccacc gccggagcca 300gacccggagc cggagcagga
ggccccagag ctggagccgg agccagagct ggagccccct 360accccacaga
tccctgaggc ccccacaccc aacgtgcctg tctgggacat tgggggcttc
420accctgcttg atgggaagct ggtgctgctt ggaggagagg aggagggtcc
tcgaaggccc 480cgggtgggaa gtgctagctc cgagggcagc atccacgtgg
ccatggggaa cttcagggat 540ccagatcgga tgcctggaaa aacagaaccg
gagactgctg gtcccaacca ggtccacaac 600gttcgggggt tgctcaagag
gctgaaagag aagaaaaagg ccagaccccc cagtgctctg 660ggctctaggg
agtcgctggc cacactctct gaactggacc tgggtgccga gcgggatgtg
720cggatctggc cactgcaccc cagcctcctg ggggagcccc actgctttca
ggtaacctgg 780acgggtggaa gccgctgctt ctcttgtcgc tcggccgctg
agagagaccg ctggatcgag 840gaccttcgtc gccaattcca gcccacccag
gacaacgtgg agcgggaaga gacatggctg 900agcgtgtggg tgcacgaagc
gaaggggctt ccccgagcag cggcgggggc acccggcgtg 960cgcgccgagc
tgtggctgga tggcgcgctg ctggcacgca cggcgcctcg ggccggccca
1020ggccagctct tctgggccga gcgcttccac ttcgaggcgc tgccaccggc
acgtcgcctg 1080tcgctgcggc tgcgcggctt gggcccggga agcgcggtgc
tgggccgcgt ggccctggcg 1140ctggaggagc tggacgcccc acgcgcgcct
gccgccggtc tggagcgctg gttcccgctg 1200ctcggggcgc cggcgggcgc
agcgctgcgg gcgcggattc gggcgcgtcg cctgcgcgtg 1260ctgccgtccg
agcgctacaa ggagctggcg gagttcctca ccttccacta tgcgcgcctc
1320tgcggggccc tggagcccgc gctgcctgcg caggccaagg aggagctggc
ggcagccatg 1380gtgcgcgtgc tgcgggccac cggccgggcg caggcgctgg
tgactgacct gggcactgcg 1440gagctggcgc gctgtggagg ccgtgaggcg
ctgctgttcc gggaaaacac attggccacc 1500aaggctatcg atgagtacat
gaagctcgtg gcacaggatt acctccagga gaccctggga 1560caggttgtgc
ggcgtctctg tgcttctact gaggactgtg aagtggaccc cagcaaatgt
1620ccagcctcgg agctgccaga gcaccaggcc agacttcgga acagctgcga
ggaggtcttc 1680gaaaccatta tccattccta cgactggttc cctgcggagc
tgggcatcgt gttctcaagc 1740tggcgagaag catgtaaaga acgtggctct
gaggtgctgg gcccccgact ggtgtgcgcc 1800tccctcttcc tgcggctcct
gtgccctgcc atcctggcac ccagcctctt tggtttggca 1860ccagaccatc
cagcacccgg cccagcccgc accctcacac tgattgccaa ggtcatccag
1920aacctcgcca accgtgcccc gttcggtgag aaggaggcct acatgggctt
catgaatagc 1980ttcctggagg aacatggacc agccatgcaa tgcttcctgg
accaggtagc catggtggat 2040gtggatgctg cccccagtgg ttaccagggc
agtggtgatc tggccctcca gttagctgtc 2100ctgcatgccc agctctgtac
aatttttgct gagcttgacc agacaacccg agacaccctg 2160gaaccactgc
ccaccatcct gcgagccatt gaggagggcc agcctgtgct tgtgtcagtg
2220ccaatgcgtc tcccactgcc cccggcccag gtccactcca gcctctccgc
aggggagaag 2280cccggcttcc tggccccccg ggacctcccc aagcacaccc
ctctcatctc caagagccag 2340tctctgcgca gcgttcgccg ctcagagagt
tgggcccggc cacggccgga cgaagagcgg 2400cccctgcggc ggccccggcc
ggtgcagcgc acgcagagtg tcccggtccg gcgtcctgcc 2460cgccgccgcc
aatctgcggg gccctggccg cgacccaaag gctccctgag catgggacca
2520gcgccccgcg cccggccttg gacccgggac tccgcctcgc tgcctcggaa
gccgtcggta 2580ccctggcagc gccaaatgga ccagccgcaa gaccgaaacc
aggcactggg cacgcaccga 2640cctgtgaaca agttggcaga gctgcagtgc
gaggtggccg ctctgcgtga ggagcagaaa 2700gtgctgtccc gcctcgtgga
gtcgctgagc acccaaatcc gggccttgac ggagcagcag 2760gagcagctgc
ggggccagct gcaggatctg gactccaggc tccgtgctgg gagctcagag
2820tttgattcag agcacaacct aacaagcaat gaagggcaca gtctgaaaaa
cctggagcac 2880cgcctaaatg agatggagag aactcaggct cagctgaggg
atgctgtcca gagcctgcag 2940ctttctccaa ggacgcgggg gtcttggagt
caaccccagc ccctcaaagc accctgcctc 3000aatggagaca ccacctga
301827170PRTArtificial SequenceRho-Gap Consensus sequence 27Pro Ile
Ile Val Glu Lys Cys Val Glu Tyr Ile Glu Lys Leu Tyr Pro 1 5 10
15Leu Ala Glu Arg Gly Leu Gln Glu Glu Gly Ile Tyr Arg Val Ser Gly
20 25 30Ser Ala Ser Arg Val Lys Glu Leu Arg Glu Ala Phe Asp Lys Asp
Gly 35 40 45Ala Pro Asp Ser Leu Glu Leu Ser Glu Lys Glu Trp Phe Asp
Val His 50 55 60Val Val Ala Gly Leu Leu Lys Leu Tyr Leu Arg Glu Leu
Pro Glu Pro65 70 75 80Leu Ile Pro Tyr Asp Leu Tyr Glu Glu Phe Ile
Arg Ala Ala Lys Glu 85 90 95Gln Ile Glu Asp Pro Asp Glu Arg Leu Arg
Ala Leu Lys Glu Leu Leu 100 105 110Ser Ser Lys Leu Pro Arg Ala His
Tyr Asn Thr Leu Arg Tyr Leu Leu 115 120 125Thr His Leu Asn Arg Val
Ala Glu Ile Tyr Ile Glu Asn Ser Ala Val 130 135 140Asn Lys Met Asn
Ala Arg Asn Leu Ala Ile Val Phe Gly Pro Thr Leu145 150 155 160Leu
Arg Pro Pro Asp Lys Glu Ser Asn Asp 165 17028193PRTArtificial
SequenceRho-Gap3 Consensus sequence 28Ser Pro Ile Pro Ile Ile Val
Glu Lys Cys Ile Glu Tyr Leu Glu Lys 1 5 10 15Arg Gly Leu Asp Thr
Glu Gly Ile Tyr Arg Val Ser Gly Ser Lys Ser 20 25 30Arg Val Lys Glu
Leu Arg Glu Ala Phe Asp Ser Gly Glu Asp Asp Leu 35 40 45Asp Ser Leu
Asp Glu Ser Ile Thr Glu Glu Ser Glu Asp Leu Glu Glu 50 55 60Tyr Asp
Val His Asp Val Ala Gly Leu Leu Lys Leu Tyr Leu Arg Glu65 70 75
80Leu Pro Glu Pro Leu Leu Thr Phe Glu Leu Tyr Glu Glu Phe Ile Glu
85 90 95Ala Ala Lys Leu Tyr Gln Ile Glu Ala Thr Ser Arg Lys Gln Ser
Glu 100 105 110Lys Ser Glu Asp Glu Glu Glu Arg Leu Arg Ala Leu Arg
Glu Leu Leu 115 120 125Ser Leu Leu Pro Pro Ala Asn Arg Ala Thr Leu
Arg Tyr Leu Leu His 130 135 140Leu Asn Arg Val Ala Glu His Ser Glu
Val Asn Lys Met Thr Ala Arg145 150 155 160Asn Leu Ala Ile Val Phe
Gly Pro Thr Leu Leu Arg Pro Pro Leu Thr 165 170 175Asp Ile Lys His
Gln Asn Lys Val Val Glu Thr Leu Ile Glu Asn Ala 180 185
190Asp29231PRTArtificial SequenceRas-Gap Consensus sequence 29Leu
Val Lys Thr Leu Leu Gln Lys Glu Ile Glu Ser Lys Ala Asp Asp 1 5 10
15Pro Thr Thr Leu Phe Arg Gly Asn Ser Leu Ala Ser Lys Met Leu Glu
20 25 30Gln Tyr Phe Arg Arg Ala Arg Gly Asn Glu Tyr Leu Arg Lys Thr
Leu 35 40 45Arg Pro Val Leu Lys Glu Ile Ile Glu Ser Lys Asp Trp Gln
His Leu 50 55 60Ser Cys Glu Ile Asp Pro Leu Lys Val Tyr Lys Lys Leu
Val Asn Gln65 70 75 80Gly Glu Leu Ser Thr Ser Glu Leu Asp Tyr Asp
Leu Thr Asn Glu Glu 85 90 95Val Leu Asp Glu Glu Glu Lys Ser Glu Ala
Ile Glu Glu Asn Leu Arg 100 105 110Asn Leu Leu Lys Tyr Thr Glu Lys
Leu Leu Glu Ala Ile Thr Ser Ser 115 120 125Ser Asp Glu Phe Pro Pro
Glu Leu Arg Tyr Ile Cys Lys Cys Leu Arg 130 135 140Gln Ser Ala Cys
Glu Lys Phe Pro Asp Asn Ala Thr Val Lys Glu Lys145 150 155 160Lys
Glu Asn Lys Lys Ser Val Val Ser Gln Arg Phe Glu Gln Val Ile 165 170
175Leu Ser Ala Val Gly Gly Phe Val Phe Leu Arg Phe Ile Asn Pro Ala
180 185 190Ile Val Ser Pro Asp Leu Phe Asn Ile Ile Asp Lys Ser Pro
Ser Ala 195 200 205Gln Ala Thr Thr Asp Gln Arg Arg Thr Leu Thr Leu
Ile Ala Lys Val 210 215 220Ile Gln Ser Leu Ala Asn Gly225
23030390PRTArtificial SequenceRas-Gap2 Consensus sequence 30Leu Lys
Gln Gly Glu Leu Gly Ser Leu Arg Leu Lys Thr Val Tyr Thr 1 5 10
15Thr Asp Phe Ile Leu Pro Ser Glu Ala Tyr Glu Glu Leu Leu Glu Leu
20 25 30Leu Leu Glu Ser Val Asp Val Glu Pro Leu Thr Ala Ser Leu Ala
Ser 35 40 45Ala Leu Glu Glu Val Cys Ser Val Leu Asp Lys Asp Glu Leu
Ala Thr 50 55 60Lys Leu Val Arg Leu Phe Leu Arg Arg Gly Arg Gly Lys
Pro Phe Leu65 70 75 80Arg Ala Leu Ile Asp Lys Glu Val Glu Arg Thr
Asp Asp Pro Val Asn 85 90 95Thr Leu Phe Arg Gly Asn Ser Leu Ala Thr
Lys Ser Met Glu Val Tyr 100 105 110Met Lys Leu Val Gly Asn Gln Tyr
Leu His Thr Thr Leu Lys Pro Val 115 120 125Leu Lys Lys Ile Val Glu
Glu Lys Lys Glu Ser Cys Glu Val Asp Pro 130 135 140Ser Lys Leu Glu
Val Asn Asp Val Ile Ser Phe Gly Asp Pro Val Glu145 150 155 160Gly
Glu Asp Leu Glu Thr Asn Leu Glu Asn Leu Leu Gln Tyr Val Glu 165 170
175Arg Leu Phe Asp Ala Ile Ile Asn Ser Ser Asp Arg Leu Pro Tyr Gly
180 185 190Leu Arg Asp Ile Cys Lys Gln Leu Arg Gln Ala Ala Glu Lys
Arg Phe 195 200 205Pro Ser Ala Thr Gln Asp Val Arg Tyr Lys Ala Val
Ser Ser Phe Val 210 215 220Phe Leu Arg Phe Phe Cys Pro Ala Ile Leu
Ser Pro Lys Leu Phe Asn225 230 235 240Leu Val Asp Glu His Pro Asp
Pro Thr Thr Arg Arg Thr Leu Thr Leu 245 250 255Ile Ala Lys Val Leu
Gln Asn Leu Ala Asn Leu Ser Glu Ser Lys Ser 260 265 270Lys Leu Phe
Gly Ser Lys Glu Pro Trp Met Glu Pro Leu Phe Lys Asn 275 280 285Asp
Phe Leu Lys Gln His Lys Asp Arg Val Lys Asp Phe Leu Asp Glu 290 295
300Leu Ser Ser Val Asp Glu Pro Ser Glu Ser Leu Val Asp Lys Val
Glu305 310 315 320Glu Leu Pro Thr Lys Ser Lys Pro Val Ser Thr Ile
Ser Gly Arg Glu 325 330 335Leu Ser Leu Leu His Ser Leu Leu Leu Glu
Asn Gly Asp Ala Leu Lys 340 345 350Arg Lys Lys Asn Asn Asn Arg Asp
His Lys Ala Leu Gly Glu Asp Pro 355 360 365Leu Asp Lys Leu Leu Phe
Lys Leu Arg Tyr Phe Arg Leu Thr Thr His 370 375 380Lys Leu Thr Asn
Gly Lys385 390
* * * * *
References