U.S. patent application number 11/040595 was filed with the patent office on 2006-01-19 for novel nucleic acid sequences encoding adenylate kinase, phospholipid scramblase-like, dna fragmentation factor-like, phosphatidylserine synthase-like, and atpase-like molecules and uses therefor.
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 | 20060014244 11/040595 |
Document ID | / |
Family ID | 27581113 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060014244 |
Kind Code |
A1 |
Chun; Miyoung ; et
al. |
January 19, 2006 |
Novel nucleic acid sequences encoding adenylate kinase,
phospholipid scramblase-like, DNA fragmentation factor-like,
phosphatidylserine synthase-like, and ATPase-like molecules and
uses therefor
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: |
Chun; Miyoung; (Belmont,
MA) ; Glucksmann; Maria Alexandra; (Lexington,
MA) ; Kapeller-Libermann; Rosana; (Chestnut Hill,
MA) ; Meyers; Rachel E.; (Newton, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
27581113 |
Appl. No.: |
11/040595 |
Filed: |
January 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10165800 |
Jun 7, 2002 |
6953682 |
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11040595 |
Jan 21, 2005 |
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09781677 |
Feb 12, 2001 |
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10165800 |
Jun 7, 2002 |
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09795038 |
Feb 26, 2001 |
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11040595 |
Jan 21, 2005 |
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09790180 |
Feb 21, 2001 |
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11040595 |
Jan 21, 2005 |
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09790838 |
Feb 22, 2001 |
6489152 |
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11040595 |
Jan 21, 2005 |
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09790179 |
Feb 21, 2001 |
6479268 |
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11040595 |
Jan 21, 2005 |
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60181705 |
Feb 10, 2000 |
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60186234 |
Feb 29, 2000 |
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60185947 |
Feb 29, 2000 |
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60185946 |
Feb 29, 2000 |
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60185609 |
Feb 29, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/194; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
C07K 14/47 20130101;
C12N 9/1229 20130101; G01N 33/6896 20130101; G01N 33/574 20130101;
C12N 9/1288 20130101; C12N 9/14 20130101 |
Class at
Publication: |
435/069.1 ;
536/023.2; 435/194; 435/320.1; 435/325 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C12N 9/12 20060101
C12N009/12 |
Claims
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence which is at least 60% identical to the nucleotide sequence
of SEQ ID NO:1, 3, 5, 7, 10, 12, 16, 18, 21, or 23, or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession No. ______; b) a nucleic acid molecule comprising
a fragment of at least 300 nucleotides of the nucleotide sequence
of SEQ ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21, or 23, or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession No. ______; c) a nucleic acid molecule which
encodes a polypeptide comprising the amino acid sequence of SEQ ID
NO:2, 6, 11, 17 or 22, or the amino acid sequence encoded by the
cDNA insert of the plasmid deposited with the ATCC as Accession No.
______; d) a nucleic acid molecule which encodes a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, 6,
11, 17, 22, or the amino acid sequence encoded by the cDNA insert
of the plasmid deposited with the ATCC as Accession No. ______,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:2, 6, 11, 17, 22, or the amino acid sequence encoded
by the cDNA insert of the plasmid deposited with the ATCC as
Accession Number ______; and e) a nucleic acid molecule which
encodes a naturally occurring allelic variant of a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, 6, 11, 17, 22,
or the amino acid sequence encoded by the cDNA insert of the
plasmid deposited with the ATCC as Accession No. ______, wherein
the nucleic acid molecule hybridizes to a nucleic acid molecule
comprising the complement of SEQ ID NO:1, 3, 5, 7, 10, 12, 16, 18,
21, or 23 under stringent conditions.
2. The isolated nucleic acid molecule of claim 1, which is selected
from the group consisting of: a) a nucleic acid comprising the
nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21,
or 23, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession No. ______; and b) a nucleic acid
molecule which encodes a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, 6, 11, 17, or 22, or the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
the ATCC as Accession No. ______.
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 which contains the nucleic acid molecule of claim
1.
6. The host cell of claim 5 which is a mammalian host cell.
7. A non-human mammalian host cell containing the nucleic acid
molecule of claim 1.
8. An isolated polypeptide selected from the group consisting of:
a) a polypeptide which is encoded by a nucleic acid molecule
comprising a nucleotide sequence which is at least 60% identical to
a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1,
3, 5, 7, 10, 12, 16, 18, 21, or 23, or the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC as Accession No.
______, or a complement thereof; b) a naturally occurring allelic
variant of a polypeptide comprising the amino acid sequence of SEQ
ID NO:2, 6, 11, 17, or 22, or the amino acid sequence encoded by
the cDNA insert of the plasmid deposited with the ATCC as Accession
No. ______, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21, or 23, or a complement
thereof under stringent conditions; and c) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, 6,
11, 17, or 22, or the amino acid sequence encoded by the cDNA
insert of the plasmid deposited with the ATCC as Accession No.
______, wherein the fragment comprises at least 15 contiguous amino
acids of SEQ ID NO:2, 6, 11, 17, or 22.
9. The isolated polypeptide of claim 8 comprising the amino acid
sequence of SEQ ID NO:2, 6, 11, 17, 22.
10. The polypeptide of claim 8 further comprising heterologous
amino acid sequences.
11. An antibody which selectively binds to a polypeptide of claim
8.
12. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2, 6, 11, 17, or 22, or the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with the ATCC
as Accession No. ______; b) a polypeptide comprising a fragment of
the amino acid sequence of SEQ ID NO:2, 6, 11, 17, or 22, or the
amino acid sequence encoded by the cDNA insert of the plasmid
deposited with the ATCC as Accession No. ______, wherein the
fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2, 6, 11, 17, or 22, or the amino acid sequence encoded by the
cDNA insert of the plasmid deposited with the ATCC as Accession No.
______; and c) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, 6,
11, 17, or 22, or the amino acid sequence encoded by the cDNA
insert of the plasmid deposited with the ATCC as Accession No.
______, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21, or 23; comprising culturing
the host cell of claim 5 under conditions in which the nucleic acid
molecule is expressed.
13. A method for detecting the presence of a polypeptide of claim 8
in a sample, comprising: a) contacting the sample with a compound
which selectively binds to a polypeptide of claim 8; and b)
determining whether the compound binds to the polypeptide in the
sample.
14. The method of claim 13, wherein the compound which binds to the
polypeptide is an antibody.
15. A kit comprising a compound which selectively binds to a
polypeptide of claim 8 and instructions for use.
16. 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.
17. The method of claim 16, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 and instructions for use.
19. A method for identifying a compound which binds to a
polypeptide of claim 8 comprising the steps of: a) contacting a
polypeptide, or a cell expressing a polypeptide of claim 8 with a
test compound; and b) determining whether the polypeptide binds to
the test compound.
20. The method of claim 19, 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; and, b) detection
of binding using a competition binding assay.
21. A method for modulating the activity of a polypeptide of claim
8 comprising contacting a polypeptide or a cell expressing a
polypeptide of claim 8 with a compound which binds to the
polypeptide in a sufficient concentration to modulate the activity
of the polypeptide.
22. A method for identifying a compound which modulates the
activity of a polypeptide of claim 8, comprising: a) contacting a
polypeptide of claim 8 with a test compound; and b) determining the
effect of the test compound on the activity of the polypeptide to
thereby identify a compound which modulates the activity of the
polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of 10/165,800, filed Jun.
7, 2002, which is a continuation-in-part of 09/781,677, filed Feb.
12, 2001, which claims the benefit of U.S. Provisional Application
No. 60/181,705, filed Feb. 10, 2000, now abandoned; and a
continuation-in-part of 09/795,038, filed Feb. 26, 2001, now
abandoned, which claims the benefit of U.S. Provisional Application
No. 60/186,234, filed Feb. 29, 2000, now abandoned; and a
continuation-in-part of 09/790,180, filed Feb. 21, 2001, now
abandoned, which claims the benefit of U.S. Provisional Application
No. 60/185,947, filed Feb. 29, 2000, now abandoned; and a
continuation-in-part of 09/790,838, filed Feb. 22, 2001, now U.S.
Pat. No. 6,489,152, which claims the benefit of U.S. Provisional
Application No. 60/185,946, filed Feb. 29, 2000, now abandoned; and
a continuation-in-part of 09/790,179, filed Feb. 21, 2001, now U.S.
Pat. No. 6,479,268, which claims the benefit of U.S. Provisional
60/185,609, filed Feb. 29, 2000, now abandoned; all of which are
hereby incorporated herein in their entirety by 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 7970, Novel ATPase-Like Molecule and Uses Thereof
[0004] i) SEQ ID NOS:1-4 [0005] ii) FIGS. 1-12 [0006] iii)
Continuation-In-Part of Ser. No. 09/790,179, filed Feb. 21, 2001,
which claims the benefit of U.S. Provisional 60/185,609, filed Feb.
29, 2000 [0007] Chapter 2 32670, Novel Human Phosphatidylserine
Synthase-Like Molecules and Uses Thereof [0008] i) SEQ ID NOS: 5-9
[0009] ii) FIGS. 13-15 [0010] iii) Continuation-In-Part of Ser. No.
09/790,838, filed Feb. 22, 2001, which claims the benefit of U.S.
Provisional 60/185,946, filed Feb. 29, 2000 [0011] Chapter 3 5698,
A DNA Fragmentation Factor-Like Molecule and Uses Thereof [0012] i)
SEQ ID NOS: 10-15 [0013] ii) FIGS. 16-20B [0014] iii)
Continuation-In-Part of Ser. No. 09/790,180, filed Feb. 21, 2001,
which claims the benefit of U.S. Provisional Application No.
60/185,947, filed Feb. 29, 2000 [0015] Chapter 4 32621, Novel Human
Phospholipid Scramblase-Like Molecules and Uses Thereof [0016] i)
SEQ ID NOS: 16-20 [0017] ii) FIGS. 21-27 [0018] iii)
Continuation-In-Part of Ser. No. 09/795,038, filed Feb. 26, 2001,
which claims the benefit of U.S. Provisional Application No.
60/186,234, filed Feb. 29, 2000 [0019] Chapter 5 27802, A Novel
Adenylate Kinase [0020] i) SEQ ID NOS: 21-25 [0021] ii) FIGS.
28-35B [0022] iii) Continuation-In-Part of 09/781, 677 filed Feb.
12, 2001, which claims the benefit of U.S. Provisional Application
No. 60/181,705, filed Feb. 10, 2000
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts a hydropathy plot of a human ATPase-like
molecule. 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:2) of human ATPase-like
sequence 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.
[0024] FIG. 2 depicts an alignment of the AAA (ATPases Associated
to a variety of cellular Activities) domain of the human
ATPase-like sequence of the invention with a consensus amino acid
sequence derived from a hidden Markov model. The upper sequence is
the consensus amino acid sequence (SEQ ID NO:4), while the lower
amino acid sequence corresponds to amino acids 128 to 312 of SEQ ID
NO:2.
[0025] FIG. 3 shows an analysis of the 7970 amino acid
sequence:.alpha..beta. turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0026] FIGS. 4A-B show the expression level of the 7970 mRNA
transcript in various tissues and cell lines.
[0027] FIG. 5 shows the expression level of the 7970 mRNA
transcript in various normal and tumorous tissues and cell
lines.
[0028] FIGS. 6A-B show the expression level of the 7970 mRNA
transcript in various normal and tumorous tissues.
[0029] FIG. 7 shows the expression level of the 7970 mRNA
transcript in clinical angiogenic samples.
[0030] FIG. 8 shows the expression level of the 7970 transcript in
clinical colon and liver samples.
[0031] FIG. 9 shows the expression level of the 7970 transcript in
a non-small cell lung cancer cell line (H640) in the presence and
absence of the p16 gene.
[0032] FIG. 10 shows the expression of the 7970 transcript in
clinical breast samples.
[0033] FIG. 11 shows the expression of the 7970 transcript in
clinical ovary samples.
[0034] FIG. 12 shows the expression of the 7970 transcript in
clinical lung samples.
[0035] FIG. 13 shows the amino acid sequence alignment for the
protein (32670; SEQ ID NO:6) encoded by human 32670 (SEQ ID NO:5)
with the phosphatidylserine synthase II from Cricetulus griseus (GI
2190007; NCBI Accession No. BAA20355; SEQ ID NO:8) and the
phosphatidylserine synthase-2 from Mus musculus (GI 4063700; NCBI
Accession Number AAC98383; SEQ ID NO:9). The sequence alignment was
generated using the Clustal method. The 32670 protein shares
approximately 85% identity with the phosphatidylserine synthase II
from Cricetulus griseus and approximately 86% identity with the
murine phosphatidylserine synthase-2 as determined by pairwise
alignment.
[0036] FIGS. 14A-B provide the nucleotide and amino acid sequence
(SEQ ID NO:5 and 6, respectively) for clone 32670. The coding
sequence for 32670 is shown in SEQ ID NO:7.
[0037] FIG. 15 shows a hydropathy plot of the 32670 polypeptide
shown in SEQ ID NO:6. 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:6) of human 32670 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.
[0038] FIG. 16 depicts a hydropathy plot of human a DFF-like
molecule. 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:11) of human DFF-like
molecule 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.
[0039] FIG. 17 depicts an alignment of the CAD domain of the human
DFF-like molecule with a consensus amino acid sequence derived from
a hidden Markov model. The upper sequence is the consensus amino
acid sequence (SEQ ID NO:13), while the lower amino acid sequence
corresponds to amino acids 36 to 108 of SEQ ID NO:11.
[0040] FIG. 18 shows an analysis of the 5698 amino acid sequence:
.alpha..beta.turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index; and surface probability
plot.
[0041] FIG. 19 shows the amino acid sequence alignment for the
protein (5698; SEQ ID NO:11) encoded by human 5698 (SEQ ID NO:10 or
12) with the Mus musculus cell death activator CIDE-B (SP Accession
No. 3114594; Genbank Accession Number AAC34986; SEQ ID NO:14) and
with the Homo sapiens cell death activator CIDE-A (SP Accession No.
3114596; Genbank Accession No. AAC34987; SEQ ID NO:15). The
sequence alignment was generated using the Clustal method. The 5698
protein shares approximately 83% identity with the murine CIDE-B
and approximately 40% identity with the human CIDE-A amino acid
sequence as determined by pairwise alignment.
[0042] FIGS. 20A-B show the expression level of the 5698 mRNA
transcript in various normal and diseased human tissues.
[0043] FIG. 21 shows the amino acid sequence alignment for the
protein (32621; SEQ ID NO:17) encoded by human 32621 (SEQ ID NO:16)
with the murine phospholipid scramblase-like (SP Accession No.
2935163; Genbank Accession No. AAC40053; SEQ ID NO:19), and human
Mm-1 cell derived transplantability-associated gene 1b (hMmTRA1b;
SP Accession No. 3510297; Genbank Accession No. BAA32568; SEQ ID
NO:20). The sequence alignment was generated using the Clustal
method. The 32621 protein shares approximately 45% identity to the
Mus musculus phospholipid scramblase-like and approximately 41%
identity to the Homo sapiens hMmTRA1b protein as determined by
pairwise alignment.
[0044] FIGS. 22A-B provide the nucleotide and amino acid sequence
(SEQ ID NO:16 and 17, respectively) for clone 32621.
[0045] FIG. 23 depicts a hydropathy plot of human 32621. 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:17) of human 32621 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.
[0046] FIGS. 24A-B depict relative expression levels of 32621 in
various human tissues and cells: artery (column 1); vein (column
2); aortic SMC, smooth muscle cells, early (column 3); aortic SMC
late (column 4); static HUVEC, human umbilical vein endothelial
cells (column 5); shear HUVEC (column 6); heart (column 7); heart
CHF, congestive heart failure heart tissue (column 8); kidney
(column 9); skeletal muscle (column 10); adipose (column 11);
pancreas (column 12); primary osteoblasts (column 13); osteoclasts
(column 14); skin (column 15); spinal cord (column 16); brain
cortex (column 17); brain hypothalamus (column 18); nerve (column
19); DRG, dorsal root ganglion (column 20); glial cells (column
21); glioblastoma (column 22); breast (column 23); breast tumor
(column 24); ovary (column 25); ovarian tumor (column 26); prostate
(column 27); prostate tumor (column 28); prostate epithelial cells
(column 29); colon (column 30); colon tumor (column 31); lung
(column 32); lung tumor (column 33); lung COPD, chronic obstructive
pulmonary diseased lung (column 34); colon IBD, inflammatory bowel
diseased colon (column 35); liver (column 36); liver fibrosis
(column 37); dermal cells (column 38); spleen (column 39); tonsil
(column 40); lymph node (column 41); thymus (column 42);
skin-decubitis (column 43); synovium (column 44); bone marrow
mononuclear cells (column 45); and activated peripheral blood
mononuclear cells (column 46). 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.
[0047] FIGS. 25A-B depict relative expression levels of 32621 in
various organs: conf HMVEC, human microvascular endothelial cells
(column 1); human fetal heart (column 2); human normal atrium
(column 3); human normal atrium (column 4); human normal ventricle
(column 5); human normal ventricle (column 6); human normal
ventricle (column 7); human normal ventricle (column 8); human
normal ventricle (column 9); human heart diseased ventricle (column
10); human heart diseased ventricle (column 11); human heart
diseased ventricle (column 12); normal human kidney (column 13);
normal human kidney (column 14); normal human kidney (column 15);
normal human kidney (column 16); human kidney HT (column 17); human
kidney HT (column 18); human kidney HT (column 19); human kidney HT
(column 20); human skeletal muscle (column 21); human skeletal
muscle (column 22); human liver (column 23); human liver with
inflammation (column 24); fetal adrenal (column 25); Wilms Tumor
(column 26); Wilms Tumor (column 27); normal human spinal cord
(column 28); diseased human cartilage (column 29); normal mouse
atrium (column 30); normal mouse atrium (column 31); normal mouse
ventricle (column 32); and normal mouse ventricle (column 33).
Relative expression levels were determined as described in FIGS.
24A-B.
[0048] FIG. 26 depicts relative expression levels of 32621 in
various organ and liver samples including liver samples from
animals fed modified diets: normal human heart (column 1); normal
human kidney (column 2); normal human skeletal muscle (column 3);
normal human liver (column 4); normal human liver (column 5);
normal human liver (column 6); normal human liver (column 7);
normal human liver (column 8); normal human liver (column 9);
normal human liver (column 10); diseased human liver (column 11);
diseased human liver (column 12); diseased human liver (column 13);
diseased human liver (column 14); MK liver (chow diet) (column 15);
MK liver (poly diet without chol., cholesterol) (column 16); MK
liver (poly diet with chol.) (column 17); MK liver (chow diet)
(column 18); MK liver (Sat. Diet without chol.) (column 19); and MK
liver (Sat diet with chol.) (column 20). Relative expression levels
were determined as described in FIGS. 24A-B.
[0049] FIG. 27 depicts 32621 expression in various cell types:
aortic smooth muscle cells (ASMC)-A1PO, (column 1); ASMC-A2P3
(column 2); ASMC-A3P4 (column 3); ASMC-AL (column 4); coronary
artery smooth muscle cells (CASMC)-C1P3 (column 5); CASMC-C2P3
(column 6); CASMC-C5PO (column 7); CASMC-C1P6 (column 8);
macrophage cells (column 9); macrophage cells treated with
interferon .gamma. (column 10); CD40+ macrophage cells (column 11);
macrophage cells treated with lipopolysaccharide (column 12);
HMVEC, human umbilical vein endothelial cells (column 13); HMVEC,
human microvascular endothelial cells (column 14); HAEC1, human
aortic endothelial cells (column 15); HCAEC3, human coronary
arterial endothelial cells (column 16); HCRE (column 17); RPTE,
renal proximal tubule epithelial cells (column 18); MC (column 19);
SKM1, myelogenous leukemia cells (column 20); and HLF,
hepatocellular carcinoma cell line (column 21). Relative expression
levels were determined as described in FIGS. 24A-B.
[0050] FIG. 28 shows the 27802 nucleotide sequence (SEQ ID NO:21)
and the deduced amino acid sequence (SEQ ID NO:22).
[0051] FIG. 29 shows an analysis of the 27802 amino acid sequence:
.alpha..beta. turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index; and surface probability
plot.
[0052] FIG. 30 shows a 27802 receptor 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 27802 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 an N-glycosylation
site.
[0053] FIG. 31 shows an analysis of the 27802 open reading frame
for amino acids corresponding to specific functional sites. These
sites are relevant with regard to providing fragments of the 27802
nucleic acid or peptide as disclosed herein.
[0054] FIG. 32 shows PSORT prediction of protein localization
showing a high score in the cytoplasm and significant scores in
other cellular locations.
[0055] FIG. 33 shows a description of ProDom matches for the 27802
protein.
[0056] FIG. 34 depicts an alignment of the adenylate kinase domains
of human 27802 with two consensus amino acid sequences derived from
hidden Markov models. The upper sequence for domain 1 is the
consensus amino acid sequence (SEQ ID NO:24) and the lower amino
acid sequence corresponds to amino acids 41-120 of SEQ ID NO:22.
The upper sequence for domain 2 is the consensus amino acid
sequence (SEQ ID NO:25) and the lower amino acid sequence
corresponds to amino acids 201-251 of SEQ ID NO:22.
[0057] FIGS. 35A-B display the expression levels of 27802 in
various tissues determined by quantitative PCR. The highest levels
of expression of 27802 were observed in artery, kidney, brain
cortex and brain hypothalamus, ovary, lung (tumor), and tonsil. The
tissue types are as follows from left to right: Artery Normal,
Aorta Diseased, Vein Normal, Coronary SMC, HUVEC, Hemangioma, Heart
Normal, Heart CHF, Kidney, Skeletal Muscle, Adipose Normal,
Pancreas, Primary Osteoblasts, Osteoclasts (diff), Skin Normal,
Spinal Cord Normal, Brain Cortex Normal, Brain Hypothalamus Normal,
Nerve, DRG (Dorsal Root Ganglion), Breast Normal, Breast Tumor,
Ovary Normal, Ovary Tumor, Prostate Normal, Prostate Tumor,
Salivary Glands, Colon Normal, Colon Tumor, Lung Normal, Lung
Tumor, Lung COPD, Colon IBD, Liver Normal, Liver Fibrosis, Spleen
Normal, Tonsil Normal, Lymph Node Normal, Small Intestine Normal,
Macrophages, Synovium, BM-MNC, Activated PBMC, Neutrophils,
Megakaryocytes, Erythroid, Positive Control.
Chapter 1
7970, A Novel ATPase-Like Molecule and Uses Thereof
BACKGROUND OF THE INVENTION
[0058] Enzymes that bind to and hydrolyze ATP play a pivotal role
in translating chemically stored energy into biological activity.
ATPases can function in a variety of cellular processes including,
selective ion transport events, actin-based motility, membrane
traffic and numerous biosynthetic pathways. Multiple ATPase
families exist, including ion pumps, DEAD box-helicases, ABC
transporters, and AAA (ATPases Associated to a variety of cellular
Activities).
[0059] AAA proteins play essential roles in cellular housekeeping,
cell division and differentiation and have been identified in
prokaryotes and eukaryotes. All members of the AAA family are
Mg.sup.2+ dependent ATPases and comprise a conserved region that
binds ATP. Cytosolic, transmembrane, as well as,
membrane-associated AAA family members have been identified in
various cellular locations and multimeric states.
[0060] The biological role of the AAA family members in the cell is
diverse. Currently, members of this ATPase family are known to be
involved in organelle biogenesis, cell-cycle regulation,
vesicle-mediated transport, assembly of proteins through membranes,
peroxisome biogenesis, gene expression in yeast and in human, and
26S proteasome function. For a review, see, Confalonieri et al.
(1995) BioEssays 17:639-650.
[0061] The SEC18 gene product from S. cerevisiae is an AAA family
member that influences the transport of proteins between the
endoplasmic reticulum and the golgi complex. It has been shown that
SEC18 is an essential component of a multisubunit 20S "fusion
machine" that promotes membrane bilayer fusion coupled to ATP
hydrolysis. The 20S fusion machine has been proposed to be involved
in the assembly, fusion or division of a variety of other
membrane-bound subcellular compartments such as vacuoles, nuclei,
mitochondria, or peroxisomes (Wilson et al. (1992) J. Cell. Bio.
117:531-538). Other AAA family members are involved in
mitochondrial function. YME1 is a putative ATP and zinc-dependent
protease. Its inactivation leads to several morphological and
functional defects, such as the escape of DNA from mitochondria
(Thorsness et al. (1993) Mol Cell Biol 13: 5418-5426).
[0062] MSP1 is another AAA ATPase protein family member from yeast
that influences mitochondrial function. MSP1 is an intrinsic
mitochondrial outer membrane protein with an apparent molecular
mass of 40 KDa. MSP1 is known to influence intramitochondrial
protein sorting. Nakai et al. have demonstrated that the 61 mC1
fusion protein, normally localized to the outer mitochondrial
membrane, is mislocalized to the inner membrane of the mitochondria
upon overexpression of MSP1 in yeast cell (Nakai et al. (1993) J.
Biol. Chem. 268:24262-9).
[0063] Several members of the AAA family are involved in the
biogenesis of peroxisomes. These organelles contain enzymes
responsible for fatty acid oxidation and the elimination of
peroxides. AAA family members, such as the PAS genes of S.
cerevisiae, appear to be required for peroxisome growth, and
proliferation (Subramani et al. (1993) Annu. Rev. Cell Biol.
9:445-478). Furthemmore, mutations in the AAA proteins Pex1p or
Pex6p accumulate abnormal peroxisomal vesicles, suggesting a defect
in vesicle fusion during peroxisome assembly (Song et al. (1993) J.
Cell Biol. 123:535-548 and Heyman et al. (1994) J. Cell Biol.
127:1269-1273).
[0064] AAA family members are also known to regulate transcription.
Nelbock et al. described the TBP1 protein that binds human HIV TAT
transactivator, thus impairing its activity in cotransfection
experiments (Nelbock et al. (1990) Science 248: 1650-1653). TBP1
has since been identified as an AAA family member which acts as a
transcriptional activator for various promoters (Ohana et al.
(1993) Proc. Natl. Acad. Sci. 90:138-142).
[0065] Various ATP-dependent protease, such as the regulatory
components Lon and Clp, are also members of the AAA ATPase family.
Evidence suggests the Lon and Clp proteases are involved in DNA
replication, recombination and restriction. For instance, human Lon
binds specifically to single-stranded DNA in a region of the
mitochondrial genome involved in regulation of DNA replication and
transcription. It has been suggested that Lon may target and
remodel specific DNA binding proteins either for selective
degradation or for assembly (Fu et al. (1998) Biochemistry
37:1905-1909).
[0066] Dubiel et al. discovered that subunit 4 of the human
proteasome was in fact a member of the AAA family (Dubiel et al.
(1992) J. Biol. Chem. 267:22699-22702). Subsequently, at least 5 of
the 26S-proteasome subunits already described as transcription
factors or cell cycle proteins have now been identified as
representatives of the AAA family. Therefore, members of the family
are likely to play an essential role in ATP-dependent and
ubiquitin-dependent degradation of abnormal proteins and
short-lived regulatory proteins and in antigen processing.
[0067] Macromolecular machines (protein complexes) carry out nearly
every major process in a cell with highly coordinated moving parts
driven by energy dependent conformational changes. Examples of such
structures include the proteasomes, spliceosomes, ribosomes,
peroxisomes and chromosomal replicases. The intricacy of these
machines require additional devices to assist in their assembly.
The AAA family of ATPase is thought of as a class of molecular
chaperones that assist in the noncovalent assembly of other
proteins or protein complexes. Thus, the AAA family members play
critical regulatory roles in the assembly or regulation of various
molecular machines associated with diverse cellular activities.
Accordingly, it is valuable to the field of pharmaceutical
development to identify and characterize novel ATPases. The present
invention advances the state of the art by providing a novel human
ATPase-like nucleic acid and polypeptide.
SUMMARY OF THE INVENTION
[0068] Isolated nucleic acid molecules corresponding to ATPase-like
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 sequences shown in SEQ ID NO:2. Further provided are
ATPase-like polypeptides having an amino acid sequence encoded by a
nucleic acid molecule described herein.
[0069] 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.
[0070] The ATPase molecules of the present invention are useful for
modulating agents in a variety of cellular processes including
organelle biogenesis, cell-cycle regulation, vesicle-mediated
transport, assembly of proteins through membranes, peroxisome
biogenesis, protein sorting, gene expression, and 26S proteasome
function. The molecules are also useful for the diagnosis and
treatment of a variety of clinical conditions.
[0071] Accordingly, in one aspect, this invention provides isolated
nucleic acid molecules encoding ATPase-like proteins or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection of ATPase-like-encoding nucleic acids.
[0072] Another aspect of this invention features isolated or
recombinant ATPase-like proteins and polypeptides. Preferred
ATPase-like proteins and polypeptides possess at least one
biological activity possessed by naturally occurring ATPase
proteins.
[0073] 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.
[0074] Antibodies and antibody fragments that selectively bind the
ATPase-like polypeptides and fragments are provided. Such
antibodies are useful in detecting the ATPase-like polypeptides as
well as in regulating the cellular activities influenced by the
ATPase-like polypeptide.
[0075] In another aspect, the present invention provides a method
for detecting the presence of ATPase-like activity or expression in
a biological sample by contacting the biological sample with an
agent capable of detecting an indicator of ATPase-like activity
such that the presence of ATPase-like activity is detected in the
biological sample.
[0076] In yet another aspect, the invention provides a method for
modulating ATPase-like activity comprising contacting a cell with
an agent that modulates (inhibits or stimulates) ATPase-like
activity or expression such that ATPase-like activity or expression
in the cell is modulated. In one embodiment, the agent is an
antibody that specifically binds to ATPase-like proteins. In
another embodiment, the agent modulates expression of ATPase-like
protein by modulating transcription of an ATPase-like gene,
splicing of an ATPase-like mRNA, or translation of an ATPase-like
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 ATPase-like mRNA or the ATPase-like gene.
[0077] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
ATPase-like protein activity or nucleic acid expression by
administering an agent that is an ATPase-like modulator to the
subject. In one embodiment, the ATPase-like modulator is an
ATPase-like protein. In another embodiment, the ATPase-like
modulator is an ATPase-like nucleic acid molecule. In other
embodiments, the ATPase-like modulator is a peptide,
peptidomimetic, or other small molecule.
[0078] 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 ATPase-like protein;
(2) misregulation of a gene encoding an ATPase-like protein; and
(3) aberrant post-translational modification of an ATPase-like
protein, wherein a wild-type form of the gene encodes a protein
with an ATPase-like activity.
[0079] In another aspect, the invention provides a method for
identifying a compound that binds to or modulates the activity of
an ATPase-like protein. In general, such methods entail measuring a
biological activity of an ATPase-like protein in the presence and
absence of a test compound and identifying those compounds that
alter the activity of the ATPase-like protein.
[0080] The invention also features methods for identifying a
compound that modulates the expression of ATPase-like genes by
measuring the expression of the ATPase-like sequences in the
presence and absence of the compound.
[0081] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0082] 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.
[0083] 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.
[0084] The present invention provides ATPase-like molecules. By
"ATPase-like molecules" is intended a novel human sequence referred
to as 7970, and variants and fragments thereof. These full-length
gene sequences or fragments thereof are referred to as
"ATPase-like" sequences, indicating they share sequence similarity
with ATPase genes. Isolated nucleic acid molecules comprising
nucleotide sequences encoding the 7970 polypeptide whose amino acid
sequence is given in SEQ ID NO:2, or a variant or fragment thereof,
are provided. A nucleotide sequence encoding the 7970 polypeptide
is set forth in SEQ ID NO:1 and 3. The sequences are members of the
secretin family of ATPases.
[0085] A novel human ATPase-like gene sequence, referred to as
7970, is provided. This gene sequence and variants and fragments
thereof are encompassed by the term "ATPase-like" molecules or
sequences as used herein. The ATPase-like sequences find use in
modulating a ATPase function. By "modulating" is intended the
upregulating or downregulating of a response. The sequences of the
invention find use in modulating organelle biogenesis, cell-cycle
regulation, protein degradation, vesicle-mediated transport,
assembly of proteins through membranes, peroxisome biogenesis, gene
expression, and 26S proteasome function response. That is, the
compositions of the invention, affect the targeted activity in
either a positive or negative fashion.
[0086] Proteins and/or antibodies of the invention are also useful
in modulating the above mentioned cellular process.
[0087] The present invention provides isolated nucleic acid
molecules comprising nucleotide sequences encoding the ATPase-like
polypeptides whose amino acid sequences are given in SEQ ID NO:2,
or a variant or fragment of the polypeptides. Nucleotide sequences
encoding the ATPase-like polypeptides of the invention are set
forth in SEQ ID NO:1 and 3.
[0088] The disclosed invention relates to methods and compositions
for the modulation, diagnosis, and treatment of a variety of
disorders. 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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 leukernia/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.
[0095] 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.
[0096] 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,
Imunopathology 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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,
lynphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[0101] 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.
[0102] 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 glomeruloscierosis,
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
(hypernephroma, adenocarcinoma of kidney), which includes
urothelial carcinomas of renal pelvis.
[0103] 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.
[0104] Disorders in the male breast include, but are not limited
to, gynecomastia and carcinoma.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] The ATPase-like gene, clone 7970, was identified in a cDNA
library. Clone 7970 encodes a mRNA transcript having the
corresponding cDNA set forth in SEQ ID NO:1. This transcript has a
1086 nucleotide open reading frame (nucleotides 79-1165 of SEQ ID
NO:1), which encodes a 361 amino acid protein (SEQ ID NO:2). An
analysis of the full-length 7970 polypeptide predicts that the
N-terminal 41 amino acids represent a signal peptide. Transmembrane
segments from amino acids (aa) 16-34 and 173-191 were predicted by
MEMSAT. Transmembrane segments were also predicted from aa 133-151
of the presumed mature peptide sequence. Prosite program analysis
was used to predict various sites within the 7970 protein.
N-glycosylation sites were predicted at aa 200-203 and 316-319.
Protein kinase C phosphorylation sites were predicted at aa 33-35,
44-46, 146-148, 163-165, 170-172 and 273-239. Casein kinase II
phosphorylation sites were predicted at aa 12-15, 70-73, 89-92,
146-149, 161-164, 202-205, 218-212, 295-298, 317-320, and 322-325.
An N-myristoylation site is predicted at aa 136-141. An
ATP/GTP-binding site motif A (P-loop) was predicted at aa 133-140.
A Leucine zipper pattern was predicted at aa 109-130.
[0116] The ATPase-like protein possesses a NB-ARC domain, from aa
131-145, an AAA domain from aa 128-312, an adenylate kinase domain
from aa 131-139, a RNA helicase domain from aa 7-337, as predicted
by HMMer, Version 2. 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
http//www.psc.edu/general/software/packages/pfam/pfam.html. The
NB-ARC domain is a novel signaling motif shared by plant resistant
gene products and regulators of cell death in animals. See for
example, Van der Biezen et al. (1998) Curr Biol 8:229-227.
Adenylate kinase is a small monomeric enzyme that catalyzes the
reversible transfer of MgATP to AMP. In mammals there are three
different isozymes: AK1 (or myokinase), which is cytosolic; AK2,
which is located in the outer compartment of mitochondria; and, AK3
(or GTP:AMP phosphotransferase), which is located in the
mitochondrial matrix and which uses MgGTP instead of MgATP. The RNA
helices domain is found in a family of RNA helices thought to be
involved in duplex unwinding during viral RNA replication. Members
of this family are found in a variety of single stranded RNA
viruses. See for example, Gorbalenya et al. (1989) NAR
17:4713-4730. The AAA domain (ATPase Associated with various
cellular Activities) is found in a family of proteins that often
perform chaperone-like functions that assist in the assembly,
operation, or disassembly of protein complexes. See for example,
Confalonieri et al. (1995) Bioessays 17:639-650 and Neuwald et al.
(1999) Genome Research 9:27-43.
[0117] The 7970 protein displays 37% identity from aa 202-312, 34%
identity from aa 154-292, and 57% identity to aa 128-165 to a
Prodom consensus sequence found in members of the Lon family of
ATP-dependent proteases; 25% identity from aa 13-127 to a Prodom
consensus sequence found in the MSP1 protein homolog; 25% identity
from aa 126-336 to a Prodom consensus sequence found in a putative
cell division protein from Treponema pallidum; 22% identity from aa
130-347 to a Prodom consensus sequence found in a probable
Peroxin-6 protein which may play a role in biogenesis of
peroxisomes; 35% identity from aa 77-351 and 31% identity from aa
324-351 to a Prodom consensus sequence found in members of the
peptidase family 16S; 20% identity from aa 129-355 to a Prodom
consensus sequence found in a chromosomal replication initiator
protein; and, 32% identity from aa 117-198 to a Prodom consensus
sequence found in a TAT-binding homolog. Furthermore, the amino
acid sequence of the 7970 sequence shares approximately 47%
sequence identity with the C. elegans MSPI protein homolog (Genbank
Accession No. P54815). This sequence alignment was generated using
the clustal method.
[0118] The ATPase-like sequences of the invention are members of a
family of molecules (the "ATPase family") 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.
[0119] As used herein, the term "AAA domain" includes an amino acid
sequence of about 80-184 amino acid residues in length and having a
bit score for the alignment of the sequence to the AAA domain (HMM)
of at least 8. Preferably, an AAA domain includes at least about
50-200 amino acids, more preferably about 100-185 amino acid
residues, or about 81-185 amino acids and has a bit score for the
alignment of the sequence to the AAA domain (HMM) of at least 16 or
greater. The AAA domain (HMM) has been assigned the PFAM Accession
No. PF00004 (http://pfam.wustl.edu/). An alignment of the AAA
domain (amino acids 128 to 312 of SEQ ID NO:2) of the human
ATPase-like sequence with a consensus amino acid sequence derived
from a hidden Markov model is depicted in FIG. 3.
[0120] In a preferred embodiment ATPase-like polypeptide or protein
has a "AAA 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 "AAA domain," e.g., the AAA domain
of human ATPase-like molecule (e.g., amino acid residues 128-312 of
SEQ ID NO:2).
[0121] To identify the presence of an "AAA" domain in an
ATPase-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
(http://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.
[0122] In one embodiment, an ATPase-like protein includes at least
one transmembrane domain. As used herein, the term "transmembrane
domain" includes an amino acid sequence of about 15 amino acid
residues in length that spans a phospholipid membrane. More
preferably, a transmembrane domain includes about at least 18, 20,
22, 24, 25, 30, 30.5 or 40 amino acid residues and spans a
phospholipid membrane. Transmembrane domains are rich in
hydrophobic residues, and typically have an .alpha.-helical
structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%,
90%, 95% or more of the amino acids of a transmembrane domain are
hydrophobic, e.g., leucines, isoleucines, tyrosines, or
tryptophans. Transmembrane domains are described in, for example,
http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N.
et al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of
which are incorporated herein by reference.
[0123] In a preferred embodiment, an ATPase-like] polypeptide or
protein has at least one transmembrane domain or a region which
includes at least 18, 20, 22, 24, 25, 30, 35 or 40 amino acid
residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%
sequence identity with a "transmembrane domain," e.g., at least one
transmembrane domain of human ATPase-like (e.g., amino acid
residues 18 of SEQ ID NO:2).
[0124] In another embodiment, an ATPase-like protein includes at
least one "non-transmembrane domain." As used herein,
"non-transmembrane domains" are domains that reside outside of the
membrane. When referring to plasma membranes, non-transmembrane
domains include extracellular domains (i.e., outside of the cell)
and intracellular domains (i.e., within the cell). When referring
to membrane-bound proteins found in intracellular organelles (e.g.,
mitochondria, endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally occurring ATPase-like, or ATPase-like protein.
[0125] In a preferred embodiment, an ATPase-like polypeptide or
protein has a "non-transmembrane domain" or a region which includes
at least about 1-170, preferably about 100-170, about 50-160, and
about 20-125 amino acid residues, and has at least about 60%, 70%
80% 90% 95%, 99% or 100% sequence identity with a
"non-transmembrane domain", e.g., a non-transmembrane domain of
human ATPase-like (e.g., residues 191 and 361 of SEQ ID NO:2).
Preferably, a non-transmembrane domain is capable of catalytic
activity (e.g., ATPase-like activity).
[0126] A non-transmembrane domain located at the N-terminus of an
ATPase-like protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-350, preferably about 30-325, more
preferably about 50-320, or even more preferably about 80-310 amino
acid residues in length and is located outside the boundaries of a
membrane. For example, an N-terminal non-transmembrane domain is
located at about amino acid residues 1-15 of SEQ ID NO:2.
[0127] Similarly, a non-transmembrane domain located at the
C-terminus of an ATPase-like protein or polypeptide is referred to
herein as a "C-terminal non-transmembrane domain." As used herein,
an "C-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-300, preferably about 15-290, preferably
about 20-270, more preferably about 25-255 amino acid residues in
length and is located outside the boundaries of a membrane. For
example, an C-terminal non-transmembrane domain is located at about
amino acid residues 191-361 of SEQ ID NO:2.
[0128] An ATPase-like molecule can further include a signal
sequence. As used herein, a "signal sequence" refers to a peptide
of about 20-80 amino acid residues in length which occurs at the
N-terminus of secretory and integral membrane proteins and which
contains a majority of hydrophobic amino acid residues. For
example, a signal sequence contains at least about 12-25 amino acid
residues, preferably about 30-70 amino acid residues, more
preferably about 61 amino acid residues, and has at least about
40-70%, preferably about 50-65%, and more preferably about 55-60%
hydrophobic amino acid residues (e.g., alanine, valine, leucine,
isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such
a "signal sequence", also referred to in the art as a "signal
peptide", serves to direct a protein containing such a sequence to
a lipid bilayer. For example, in one embodiment, an ATPase-like
protein contains a signal sequence of about amino acids 1-41 of SEQ
ID NO:2. The "signal sequence" is cleaved during processing of the
mature protein. The mature ATPase-like protein corresponds to amino
acids 42-361 of SEQ ID NO:2.
[0129] Preferred ATPase-like 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.
[0130] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e., percent identity=number of identical
positions/total number 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.
[0131] The 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
http://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
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. 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.
[0132] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of Karlin and
Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, 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. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12, to obtain nucleotide sequences homologous
to ATPase-like 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 ATPase-like 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.
Alternatively, PSI-Blast can be used to perform an iterated search
that detects distant relationships between molecules. See Altschul
et al. (1997) supra. 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
http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting
example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Myers and Miller (1988) CABIOS
4:11-17. Such an algorithm is incorporated into the ALIGN program
(version 2.0), which is part of the GCG 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.
[0133] Accordingly, another embodiment of the invention features
isolated ATPase-like proteins and polypeptides having an
ATPase-like protein activity. As used interchangeably herein, a
"ATPase-like protein activity", "biological activity of an
ATPase-like protein", or "functional activity of an ATPase-like
protein" refers to an activity exerted by an ATPase-like protein,
polypeptide, or nucleic acid molecule on an ATPase-like responsive
cell as determined in vivo, or in vitro, according to standard
assay techniques. An ATPase-like 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 signaling
activity mediated by interaction of the ATPase-like protein with a
second protein. In a preferred embodiment, an ATPase-like activity
includes at least one or more of the following activities: (1)
modulating (stimulating and/or enhancing or inhibiting) cellular
division; (2) modulating organelle biogenesis; (3) modulating
protein sorting; (4) modulating gene expression; (5) modulating
protein degradation; and (6) modulating the function of the 26S
proteosome.
[0134] An "isolated" or "purified" ATPase-like 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
ATPase-like 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 ATPase-like
protein that is substantially free of cellular material includes
preparations of ATPase-like protein having less than about 30%,
20%, 10%, or 5% (by dry weight) of non-ATPase protein (also
referred to herein as a "contaminating protein"). When the
ATPase-like 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 ATPase-like 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-ATPase chemicals.
[0135] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[0136] One aspect of the invention pertains to isolated nucleic
acid molecules comprising nucleotide sequences encoding ATPase-like
proteins and polypeptides or biologically active portions thereof,
as well as nucleic acid molecules sufficient for use as
hybridization probes to identify ATPase-like-encoding nucleic acids
(e.g., ATPase-like mRNA) and fragments for use as PCR primers for
the amplification or mutation of ATPase-like 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.
[0137] Nucleotide sequences encoding the ATPase-like proteins of
the present invention include sequences set forth in SEQ ID NO:1,
3, 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 ATPase-like protein
encoded by these nucleotide sequences is set forth in SEQ ID NO:2.
The invention also encompasses nucleic acid molecules comprising
nucleotide sequences encoding partial-length ATPase-like proteins,
including the sequence set forth in SEQ ID NO:1, 3, and complements
thereof.
[0138] Nucleic acid molecules that are fragments of these
ATPase-like nucleotide sequences are also encompassed by the
present invention. By "fragment" is intended a portion of the
nucleotide sequence encoding an ATPase-like protein. A fragment of
an ATPase-like nucleotide sequence may encode a biologically active
portion of an ATPase-like 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 ATPase-like
protein can be prepared by isolating a portion of one of the
ATPase-like nucleotide sequences of the invention, expressing the
encoded portion of the ATPase-like protein (e.g., by recombinant
expression in vitro), and assessing the activity of the encoded
portion of the ATPase-like protein. Nucleic acid molecules that are
fragments of an ATPase-like 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 ATPase-like nucleotide sequence disclosed
herein (for example, 1748 nucleotides for SEQ ID NO:1 and 1086
nucleotides for SEQ ID NO:3) depending upon the intended use.
Alternatively, a nucleic acid molecules that is a fragment of an
ATPase-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,
900-1086, 1086-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,
1500-1600, 1600-1700, or 1700-1748 of SEQ ID NO:1 or 3.
[0139] 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.
[0140] A fragment of an ATPase-like nucleotide sequence that
encodes a biologically active portion of an ATPase-like protein of
the invention will encode at least 15, 25, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 contiguous amino acids, or up to the
total number of amino acids present in a full-length ATPase-like
protein of the invention (for example, 362 amino acids). Fragments
of an ATPase-like nucleotide sequence that are useful as
hybridization probes for PCR primers generally need not encode a
biologically active portion of an ATPase-like protein.
[0141] Nucleic acid molecules that are variants of the ATPase-like
nucleotide sequences disclosed herein are also encompassed by the
present invention. "Variants" of the ATPase-like nucleotide
sequences include those sequences that encode the ATPase-like
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 ATPase-like
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
ATPase-like nucleotide sequence will encode an ATPase-like 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
ATPase-like protein disclosed herein.
[0142] In addition to the ATPase-like nucleotide sequences shown in
SEQ ID NOS:1 and 3, it will be appreciated by those skilled in the
art that DNA sequence polymorphisms that lead to changes in the
amino acid sequences of ATPase-like proteins may exist within a
population (e.g., the human population). Such genetic polymorphism
in an ATPase-like 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
ATPase-like protein, preferably a mammalian ATPase-like protein. As
used herein, the phrase "allelic variant" refers to a nucleotide
sequence that occurs at an ATPase-like 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 ATPase-like gene. Any and all such nucleotide variations and
resulting amino acid polymorphisms or variations in an ATPase-like
sequence that are the result of natural allelic variation and that
do not alter the functional activity of ATPase-like proteins are
intended to be within the scope of the invention.
[0143] Moreover, nucleic acid molecules encoding ATPase-like
proteins from other species (ATPase-like homologues), which have a
nucleotide sequence differing from that of the ATPase-like
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 ATPase-like
cDNA of the invention can be isolated based on their identity to
the human ATPase-like 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.
[0144] In addition to naturally-occurring allelic variants of the
ATPase-like 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
ATPase-like proteins, without altering the biological activity of
the ATPase-like proteins. Thus, an isolated nucleic acid molecule
encoding an ATPase-like 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.
[0145] 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 ATPase-like 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 growth factor and cytokine receptor signature 2 sequence and
the U-PAR/Ly-6 domain sequence of SEQ ID NO:2, where such residues
are essential for protein activity.
[0146] Alternatively, variant ATPase-like nucleotide sequences can
be made by introducing mutations randomly along all or part of an
ATPase-like coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for ATPase-like 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.
[0147] Thus the nucleotide sequences of the invention include the
sequences disclosed herein as well as fragments and variants
thereof. The ATPase-like nucleotide sequences of the invention, and
fragments and variants thereof, can be used as probes and/or
primers to identify and/or clone ATPase-like homologues in other
cell types, e.g., from other tissues, as well as ATPase-like
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 ATPase-like
protein, such as by measuring levels of an ATPase-like-encoding
nucleic acid in a sample of cells from a subject, e.g., detecting
ATPase-like mRNA levels or determining whether a genomic
ATPase-like gene has been mutated or deleted.
[0148] 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). ATPase-like nucleotide sequences
isolated based on their sequence identity to the ATPase-like
nucleotide sequences set forth herein or to fragments and variants
thereof are encompassed by the present invention.
[0149] In a hybridization method, all or part of a known
ATPase-like 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 ATPase-like nucleotide sequence disclosed
herein. Degenerate primers designed on the basis of conserved
nucleotides or amino acid residues in a known ATPase-like
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 ATPase-like 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.
[0150] For example, in one embodiment, a previously unidentified
ATPase-like nucleic acid molecule hybridizes under stringent
conditions to a probe that is a nucleic acid molecule comprising
one of the ATPase-like nucleotide sequences of the invention or a
fragment thereof. In another embodiment, the previously unknown
ATPase-like 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 ATPase-like nucleotide sequences disclosed
herein or a fragment thereof.
[0151] Accordingly, in another embodiment, an isolated previously
unknown ATPase-like 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, 3, or a complement, fragment, or variant thereof.
[0152] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences 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, 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 that hybridizes under stringent
conditions to an ATPase-like 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).
[0153] Thus, in addition to the ATPase-like 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 ATPase-like nucleotide sequences disclosed herein
or variants and fragments thereof.
[0154] 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 ATPase-like 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 ATPase-like protein. The noncoding
regions are the 5' and 3' sequences that flank the coding region
and are not translated into amino acids.
[0155] Given the coding-strand sequence encoding an ATPase-like
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 ATPase-like
mRNA, but more preferably is an oligonucleotide that is antisense
to only a portion of the coding or noncoding region of ATPase-like
mRNA. For example, the antisense oligonucleotide can be
complementary to the region surrounding the translation start site
of ATPase-like 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.
[0156] 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).
[0157] 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 ATPase-like 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 m promoter are
preferred.
[0158] 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).
[0159] 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 ATPase-like mRNA
transcripts to thereby inhibit translation of ATPase-like mRNA. A
ribozyme having specificity for an ATPase-like-encoding nucleic
acid can be designed based upon the nucleotide sequence of an
ATPase-like 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, ATPase-like 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.
[0160] The invention also encompasses nucleic acid molecules that
form triple helical structures. For example, ATPase-like gene
expression can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the ATPase-like protein
(e.g., the ATPase-like promoter and/or enhancers) to form triple
helical structures that prevent transcription of the ATPase-like
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.
[0161] 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.
[0162] PNAs of an ATPase-like 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).
[0163] In another embodiment, PNAs of an ATPase-like 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 ATPase-like Proteins and Anti-ATPase-Like
Antibodies
[0164] ATPase-like proteins are also encompassed within the present
invention. By "ATPase-like 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.
[0165] "Fragments" or "biologically active portions" include
polypeptide fragments suitable for use as immunogens to raise
anti-ATPase-like antibodies. Fragments include peptides comprising
amino acid sequences sufficiently identical to or derived from the
amino acid sequence of an ATPase-like protein, or partial-length
protein, of the invention and exhibiting at least one activity of
an ATPase-like protein, but which include fewer amino acids than
the full-length (SEQ ID NO:2) ATPase-like protein disclosed herein.
Typically, biologically active portions comprise a domain or motif
with at least one activity of the ATPase-like protein. A
biologically active portion of an ATPase-like protein can be a
polypeptide which 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 ATPase-like 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.
[0166] 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 a nucleic acid molecule that hybridizes to the nucleic acid
molecule of SEQ ID NO:2, 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:2. 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 generally retain the
functional activity of the ATPase-like proteins of the invention.
Variants include polypeptides that differ in amino acid sequence
due to natural allelic variation or mutagenesis.
[0167] The invention also provides ATPase-like chimeric or fusion
proteins. As used herein, an ATPase-like "chimeric protein" or
"fusion protein" comprises an ATPase-like polypeptide operably
linked to a non-ATPase polypeptide. An "ATPase-like polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to an ATPase-like protein, whereas a "non-ATPase-like polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to a protein that is not substantially identical to the ATPase-like
protein, e.g., a protein that is different from the ATPase-like
protein and which is derived from the same or a different organism.
Within an ATPase-like fusion protein, the ATPase-like polypeptide
can correspond to all or a portion of an ATPase-like protein,
preferably at least one biologically active portion of an
ATPase-like protein. Within the fusion protein, the term "operably
linked" is intended to indicate that the ATPase-like polypeptide
and the non-ATPase-like polypeptide are fused in-frame to each
other. The non-ATPase-like polypeptide can be fused to the
N-terminus or C-terminus of the ATPase-like polypeptide.
[0168] One useful fusion protein is a GST-ATPase-like fusion
protein in which the ATPase-like sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant ATPase-like
proteins.
[0169] In yet another embodiment, the fusion protein is an
ATPase-like-immunoglobulin fusion protein in which all or part of
an ATPase-like protein is fused to sequences derived from a member
of the immunoglobulin protein family. The
ATPase-like-immunoglobulin fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject to inhibit an interaction between an ATPase-like ligand and
an ATPase-like protein on the surface of a cell, thereby
suppressing ATPase-like-mediated signal transduction in vivo. The
ATPase-like-immunoglobulin fusion proteins can be used to affect
the bioavailability of an ATPase-like cognate ligand. Inhibition of
the ATPase-like ligand/ATPase-like interaction may be useful
therapeutically, both for treating proliferative and
differentiative disorders and for modulating (e.g., promoting or
inhibiting) cell survival. Moreover, the ATPase-like-immunoglobulin
fusion proteins of the invention can be used as immunogens to
produce anti-ATPase-like antibodies in a subject, to purify
ATPase-like ligands, and in screening assays to identify molecules
that inhibit the interaction of an ATPase-like protein with an
ATPase-like ligand.
[0170] Preferably, an ATPase-like 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 ATPase-like-encoding nucleic acid can be cloned into a
commercially available expression vector such that it is linked
in-frame to an existing fusion moiety. Variants of the ATPase-like
proteins can function as either ATPase-like agonists (mimetics) or
as ATPase-like antagonists. Variants of the ATPase-like protein can
be generated by mutagenesis, e.g., discrete point mutation or
truncation of the ATPase-like protein. An agonist of the
ATPase-like protein can retain substantially the same, or a subset,
of the biological activities of the naturally occurring form of the
ATPase-like protein. An antagonist of the ATPase-like protein can
inhibit one or more of the activities of the naturally occurring
form of the ATPase-like protein by, for example, competitively
binding to a downstream or upstream member of a cellular signaling
cascade that includes the ATPase-like 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 ATPase-like
proteins.
[0171] Variants of an ATPase-like protein that function as either
ATPase-like agonists or as ATPase-like antagonists can be
identified by screening combinatorial libraries of mutants, e.g.,
truncation mutants, of an ATPase-like protein for ATPase-like
protein agonist or antagonist activity. In one embodiment, a
variegated library of ATPase-like variants is generated by
combinatorial mutagenesis at the nucleic acid level and is encoded
by a variegated gene library. A variegated library of ATPase-like
variants can be produced by, for example, enzymatically ligating a
mixture of synthetic oligonucleotides into gene sequences such that
a degenerate set of potential ATPase-like sequences is expressible
as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of
ATPase-like sequences therein. There are a variety of methods that
can be used to produce libraries of potential ATPase-like 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 ATPase-like 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).
[0172] In addition, libraries of fragments of an ATPase-like
protein coding sequence can be used to generate a variegated
population of ATPase-like fragments for screening and subsequent
selection of variants of an ATPase-like protein. In one embodiment,
a library of coding sequence fragments can be generated by treating
a double-stranded PCR fragment of an ATPase-like 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 ATPase-like protein.
[0173] 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 ATPase-like 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 ATPase-like variants (Arkin
and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;
Delgrave et al. (1993) Protein Engineering 6(3):327-331).
[0174] An isolated ATPase-like polypeptide of the invention can be
used as an immunogen to generate antibodies that bind ATPase-like
proteins using standard techniques for polyclonal and monoclonal
antibody preparation. The full-length ATPase-like protein can be
used or, alternatively, the invention provides antigenic peptide
fragments of ATPase-like proteins for use as immunogens. The
antigenic peptide of an ATPase-like 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
ATPase-like protein such that an antibody raised against the
peptide forms a specific immune complex with the ATPase-like
protein. Preferred epitopes encompassed by the antigenic peptide
are regions of a ATPase-like protein that are located on the
surface of the protein, e.g., hydrophilic regions.
[0175] Accordingly, another aspect of the invention pertains to
anti-ATPase-like polyclonal and monoclonal antibodies that bind an
ATPase-like protein. Polyclonal anti-ATPase-like antibodies can be
prepared by immunizing a suitable subject (e.g., rabbit, goat,
mouse, or other mammal) with an ATPase-like immunogen. The
anti-ATPase-like 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 ATPase-like
protein. At an appropriate time after immunization, e.g., when the
anti-ATPase-like 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).
[0176] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-ATPase-like antibody can be
identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with an ATPase-like protein to thereby isolate immunoglobulin
library members that bind the ATPase-like 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.TM. Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods
and reagents particularly amenable for use in generating and
screening antibody display library can be found in, for example,
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.
[0177] Additionally, recombinant anti-ATPase-like 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.
[0178] 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.
[0179] 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).
[0180] An anti-ATPase-like antibody (e.g., monoclonal antibody) can
be used to isolate ATPase-like proteins by standard techniques,
such as affinity chromatography or immunoprecipitation. An
anti-ATPase-like antibody can facilitate the purification of
natural ATPase-like protein from cells and of recombinantly
produced ATPase-like protein expressed in host cells. Moreover, an
anti-ATPase-like antibody can be used to detect ATPase-like protein
(e.g., in a cellular lysate or cell supernatant) in order to
evaluate the abundance and pattern of expression of the ATPase-like
protein. Anti-ATPase-like 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.
[0181] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). 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.
[0182] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84:Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
III. Recombinant Expression Vectors and Host Cells
[0183] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an ATPase-like 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).
[0184] 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.
[0185] 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., ATPase-like proteins, mutant forms of
ATPase-like proteins, 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 such codon usage are
well known in the art.
[0186] The recombinant expression vectors of the invention can be
designed for expression of ATPase-like 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 11d (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.
[0187] Suitable eukaryotic host cells include insect cells
(examples of Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers (1989) Virology 170:31-39));
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.
[0188] 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. 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.
[0189] 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 (Banerji et al. (1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application 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.
[0190] 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 ATPase-like 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).
[0191] 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.
[0192] 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 ATPase-like 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).
[0193] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) ATPase-like protein. Accordingly, the invention further
provides methods for producing ATPase-like 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 ATPase-like protein has been
introduced, in a suitable medium such that ATPase-like protein is
produced. In another embodiment, the method further comprises
isolating ATPase-like protein from the medium or the host cell.
[0194] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which ATPase-like-coding sequences have been introduced.
Such host cells can then be used to create nonhuman transgenic
animals in which exogenous ATPase-like sequences have been
introduced into their genome or homologous recombinant animals in
which endogenous ATPase-like sequences have been altered. Such
animals are useful for studying the function and/or activity of
ATPase-like genes and proteins and for identifying and/or
evaluating modulators of ATPase-like 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 ATPase-like 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.
[0195] A transgenic animal of the invention can be created by
introducing ATPase-like-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 ATPase-like cDNA sequence
can be introduced as a transgene into the genome of a nonhuman
animal. Alternatively, a homologue of the mouse ATPase-like 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 ATPase-like transgene to direct expression of
ATPase-like 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 ATPase-like transgene in its genome
and/or expression of ATPase-like 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 ATPase-like gene can further
be bred to other transgenic animals carrying other transgenes.
[0196] To create a homologous recombinant animal, one prepares a
vector containing at least a portion of an ATPase-like 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 ATPase-like gene. In a preferred
embodiment, the vector is designed such that, upon homologous
recombination, the endogenous ATPase-like 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 ATPase-like 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 ATPase-like protein). In the homologous recombination
vector, the altered portion of the ATPase-like gene is flanked at
its 5' and 3' ends by additional nucleic acid of the ATPase-like
gene to allow for homologous recombination to occur between the
exogenous ATPase-like gene carried by the vector and an endogenous
ATPase-like gene in an embryonic stem cell. The additional flanking
ATPase-like 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 ATPase-like
gene has homologously recombined with the endogenous ATPase-like
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.
[0197] 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.
[0198] 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
[0199] The ATPase-like nucleic acid molecules, ATPase-like
proteins, and anti-ATPase-like 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the knowledge 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.
[0204] 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.
[0205] 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.TM. (BASF; Parsippany, N.J.), or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as 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.
[0206] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an ATPase-like protein or
anti-ATPase-like 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
V. Uses and Methods of the Invention
[0213] 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
ATPase-like protein (e.g., via a recombinant expression vector in a
host cell in gene therapy applications), to detect ATPase-like mRNA
(e.g., in a biological sample) or a genetic lesion in an
ATPase-like gene, and to modulate ATPase-like activity. In
addition, the ATPase-like proteins can be used to screen drugs or
compounds that modulate the cellular activities described above as
well as to treat disorders characterized by insufficient or
excessive production of ATPase-like protein or production of
ATPase-like protein forms that have decreased or aberrant activity
compared to ATPase-like wild type protein. In addition, the
anti-ATPase-like antibodies of the invention can be used to detect
and isolate ATPase-like proteins and modulate ATPase-like
activity.
[0214] A. Screening Assays
[0215] 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 ATPase-like proteins or
have a stimulatory or inhibitory effect on, for example,
ATPase-like expression or ATPase-like activity.
[0216] 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).
[0217] 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.
[0218] 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).
[0219] Determining the ability of the test compound to bind to the
ATPase-like 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 ATPase-like 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 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.
[0220] In a similar manner, one may determine the ability of the
ATPase-like protein to bind to or interact with an ATPase-like
target molecule. By "target molecule" is intended a molecule with
which an ATPase-like protein binds or interacts in nature. In a
preferred embodiment, the ability of the ATPase-like protein to
bind to or interact with an ATPase-like 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 alterations in protein sorting, cell division, protein
degradation, organelle biogenesis, etc., detecting
catalytic/enzymatic activity of the target on an appropriate
substrate, detecting the induction of a reporter gene (e.g., an
ATPase-like-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.
[0221] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting an ATPase-like protein
or biologically active portion thereof with a test compound and
determining the ability of the test compound to bind to the
ATPase-like protein or biologically active portion thereof. Binding
of the test compound to the ATPase-like protein can be determined
either directly or indirectly as described above. In a preferred
embodiment, the assay includes contacting the ATPase-like protein
or biologically active portion thereof with a known compound that
binds ATPase-like 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 ATPase-like protein or
biologically active portion thereof as compared to the known
compound.
[0222] In another embodiment, an assay is a cell-free assay
comprising contacting ATPase-like 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 ATPase-like protein or biologically active portion
thereof. Determining the ability of the test compound to modulate
the activity of an ATPase-like protein can be accomplished, for
example, by determining the ability of the ATPase-like protein to
bind to an ATPase-like 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 ATPase-like protein can be accomplished by
determining the ability of the ATPase-like protein to further
modulate an ATPase-like target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as previously
described.
[0223] In yet another embodiment, the cell-free assay comprises
contacting the ATPase-like protein or biologically active portion
thereof with a known compound that binds an ATPase-like 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 ATPase-like
target molecule.
[0224] In the above-mentioned assays, it may be desirable to
immobilize either an ATPase-like 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/ATPase-like
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 ATPase-like 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 ATPase-like binding or activity determined
using standard techniques.
[0225] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either ATPase-like protein or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated ATPase-like 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 ATPase-like protein or
target molecules but which do not interfere with binding of the
ATPase-like protein to its target molecule can be derivatized to
the wells of the plate, and unbound target or ATPase-like 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 ATPase-like protein or target
molecule, as well as enzyme-linked assays that rely on detecting an
enzymatic activity associated with the ATPase-like protein or
target molecule.
[0226] In another embodiment, modulators of ATPase-like expression
are identified in a method in which a cell is contacted with a
candidate compound and the expression of ATPase-like mRNA or
protein in the cell is determined relative to expression of
ATPase-like 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 ATPase-like 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
ATPase-like mRNA or protein expression. The level of ATPase-like
mRNA or protein expression in the cells can be determined by
methods described herein for detecting ATPase-like mRNA or
protein.
[0227] In yet another aspect of the invention, the ATPase-like
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 ATPase-like protein ("ATPase-like-binding proteins"
or "ATPase-like-bp") and modulate ATPase-like activity. Such
ATPase-like-binding proteins are also likely to be involved in the
propagation of signals by the ATPase-like proteins as, for example,
upstream or downstream elements of the ATPase-like pathway.
[0228] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0229] B. Detection Assays
[0230] 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.
[0231] 1. Chromosome Mapping
[0232] The isolated complete or partial ATPase-like gene sequences
of the invention can be used to map their respective ATPase-like
genes on a chromosome, thereby facilitating the location of gene
regions associated with genetic disease. Computer analysis of
ATPase-like 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 ATPase-like
sequences will yield an amplified fragment.
[0233] 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.
[0234] Other mapping strategies that can similarly be used to map
an ATPase-like 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.
[0235] 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.
[0236] Another strategy to map the chromosomal location of
ATPase-like genes uses ATPase-like polypeptides and fragments and
sequences of the present invention and antibodies specific thereto.
This mapping can be carried out by specifically detecting the
presence of a ATPase-like polypeptide in members of a panel of
somatic cell hybrids between cells of a first species of animal
from which the protein originates and cells from a second species
of animal, and then determining which somatic cell hybrid(s)
expresses the polypeptide and noting the chromosomes(s) from the
first species of animal that it contains. For examples of this
technique, see Pajunen et al. (1988) Cytogenet. Cell. Genet.
47:37-41 and Van Keuren et al. (1986) Hum. Genet. 74:34-40.
Alternatively, the presence of a ATPase-like polypeptide in the
somatic cell hybrids can be determined by assaying an activity or
property of the polypeptide, for example, enzymatic activity, as
described in Bordelon-Riser et al. (1979) Somatic Cell Genetics
5:597-613 and Owerbach et al. (1978) Proc. Natl. Acad. Sci. USA
75:5640-5644.
[0237] 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.
[0238] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the ATPase-like 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.
[0239] 2. Tissue Typing
[0240] The ATPase-like 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).
[0241] 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 ATPase-like sequences of the invention can be
used to prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0242] 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 ATPase-like 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:2, is used, a more appropriate number of
primers for positive individual identification would be 500 to
2,000.
[0243] 3. Use of Partial ATPase-Like Sequences in Forensic
Biology
[0244] 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.
[0245] 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 ATPase-like 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.
[0246] The ATPase-like 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 ATPase-like probes, can be used to
identify tissue by species and/or by organ type.
[0247] In a similar fashion, these reagents, e.g., ATPase-like
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).
[0248] C. Predictive Medicine
[0249] 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.
[0250] 1. Diagnostic Assays
[0251] One aspect of the present invention relates to diagnostic
assays for detecting ATPase-like protein and/or nucleic acid
expression as well as ATPase-like activity, in the context of a
biological sample. An exemplary method for detecting the presence
or absence of ATPase-like 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 ATPase-like protein or nucleic acid (e.g., mRNA, genomic
DNA) that encodes ATPase-like protein such that the presence of
ATPase-like 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.
[0252] "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.
[0253] A preferred agent for detecting ATPase-like mRNA or genomic
DNA is a labeled nucleic acid probe capable of hybridizing to
ATPase-like mRNA or genomic DNA. The nucleic acid probe can be, for
example, a full-length ATPase-like nucleic acid, such as the
nucleic acid of SEQ ID NOS:1, 3, 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 ATPase-like mRNA or genomic DNA.
Other suitable probes for use in the diagnostic assays of the
invention are described herein.
[0254] A preferred agent for detecting ATPase-like protein is an
antibody capable of binding to ATPase-like 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.
[0255] 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 ATPase-like
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
ATPase-like mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of ATPase-like
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence. In
vitro techniques for detection of ATPase-like genomic DNA include
Southern hybridizations. Furthermore, in vivo techniques for
detection of ATPase-like protein include introducing into a subject
a labeled anti-ATPase-like 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.
[0256] 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.
[0257] The invention also encompasses kits for detecting the
presence of ATPase-like 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 ATPase-like protein. For
example, the kit can comprise a labeled compound or agent capable
of detecting ATPase-like protein or mRNA in a biological sample and
means for determining the amount of an ATPase-like protein in the
sample (e.g., an anti-ATPase-like antibody or an oligonucleotide
probe that binds to DNA encoding an ATPase-like protein, e.g., SEQ
ID NOS:1 and 3). 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
ATPase-like sequences if the amount of ATPase-like protein or mRNA
is above or below a normal level.
[0258] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) that binds
to ATPase-like protein; and, optionally, (2) a second, different
antibody that binds to ATPase-like 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
ATPase-like nucleic acid sequence or (2) a pair of primers useful
for amplifying an ATPase-like nucleic acid molecule.
[0259] 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 ATPase-like proteins.
[0260] 2. Other Diagnostic Assays
[0261] 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 an ATPase-like, 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
ATPase-like nucleic acid, polypeptide, or antibody.
[0262] 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.
[0263] The method can include contacting the ATPase-like 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.
[0264] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of ATPase-like. 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.
[0265] The method can be used to detect SNPs, as described
above.
[0266] 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
ATPase-like or from a cell or subject in which an ATPase-like
mediated response has been elicited, e.g., by contact of the cell
with ATPase-like nucleic acid or protein, or administration to the
cell or subject ATPase-like 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 ATPase-like 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
ATPase-like (or does not express as highly as in the case of the
ATPase-like positive plurality of capture probes) or from a cell or
subject which in which an ATPase-like 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 an ATPase-like 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.
[0267] In another aspect, the invention features, a method of
analyzing ATPase-like, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing an ATPase-like nucleic acid or amino
acid sequence, e.g., a nucleotide sequence from 300-1916 or a
portion thereof; comparing the ATPase-like 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 ATPase-like.
[0268] The method can include evaluating the sequence identity
between an ATPase-like sequence and a database sequence. The method
can be performed by accessing the database at a second site, e.g.,
over the internet.
[0269] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of an ATPase-like gene. 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 identical in sequence with one
another (except for differences in length). The oligonucleotides
can be provided with differential labels, such that an
oligonucleotides which hybridizes to one allele provides a signal
that is distinguishable from an oligonucleotides which hybridizes
to a second allele.
[0270] 3. Prognostic Assays
[0271] 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
ATPase-like protein, ATPase-like nucleic acid expression, or
ATPase-like 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 ATPase-like protein, ATPase-like nucleic acid
expression, or ATPase-like activity.
[0272] Thus, the present invention provides a method in which a
test sample is obtained from a subject, and ATPase-like protein or
nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the
presence of ATPase-like protein or nucleic acid is diagnostic for a
subject having or at risk of developing a disease or disorder
associated with aberrant ATPase-like 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.
[0273] 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 ATPase-like activity) to effectively treat
a disease or disorder associated with aberrant ATPase-like
expression or activity. In this manner, a test sample is obtained
and ATPase-like protein or nucleic acid is detected. The presence
of ATPase-like protein or nucleic acid is diagnostic for a subject
that can be administered the agent to treat a disorder associated
with aberrant ATPase-like expression or activity.
[0274] The methods of the invention can also be used to detect
genetic lesions or mutations in an ATPase-like gene, thereby
determining if a subject with the lesioned gene is at risk for a
disorder characterized by aberrant cellular processes including,
altered protein sorting, altered gene expression, altered cell
division, altered protein stability, etc. 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 ATPase-like-protein, or the
misexpression of the ATPase-like 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 ATPase-like gene; (2) an addition of one or more nucleotides to
an ATPase-like gene; (3) a substitution of one or more nucleotides
of an ATPase-like gene; (4) a chromosomal rearrangement of an
ATPase-like gene; (5) an alteration in the level of a messenger RNA
transcript of an ATPase-like gene; (6) an aberrant modification of
an ATPase-like 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 ATPase gene; (8) a
non-wild-type level of an ATPase-like-protein; (9) an allelic loss
of an ATPase-like gene; and (10) an inappropriate
post-translational modification of an ATPase-like-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
ATPase-like gene. Any cell type or tissue in which ATPase-like
proteins are expressed may be utilized in the prognostic assays
described herein.
[0275] 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 ATPase-like-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.
[0276] 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.
[0277] In an alternative embodiment, mutations in an ATPase-like
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.
[0278] In other embodiments, genetic mutations in an ATPase-like
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 ATPase-like gene and detect mutations by comparing the
sequence of the sample ATPase-like 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).
[0279] Other methods for detecting mutations in the ATPase-like
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.
[0280] 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 ATPase-like 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 ATPase-like sequence, e.g., a wild-type ATPase-like 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.
[0281] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in ATPase-like 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).
[0282] 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).
[0283] 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.
[0284] 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 3N 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 3N end of the SN 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.
[0285] 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 ATPase-like gene.
[0286] 4. Pharmacogenomics
[0287] Agents, or modulators that have a stimulatory or inhibitory
effect on ATPase-like activity (e.g., ATPase-like gene expression)
as identified by a screening assay described herein, can be
administered to individuals to treat (prophylactically or
therapeutically) disorders associated with aberrant ATPase-like
activity as well as to modulate a cellular phenotype associated
with aberrant ATPase-like activity. 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 ATPase-like protein, expression of ATPase-like nucleic
acid, or mutation content of ATPase-like genes in an individual can
be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual.
[0288] 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.
[0289] Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician may consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a ATPase-like molecule or ATPase-like
modulator as well as tailoring the dosage and/or therapeutic
regimen of treatment with a ATPase-like molecule or ATPase-like
modulator.
[0290] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0291] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., an ATPase-like protein of the present
invention), all common variants of that gene can be fairly easily
identified in the population and it can be determined if having one
version of the gene versus another is associated with a particular
drug response.
[0292] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., an ATPase-like molecule or ATPase-like modulator of
the present invention) can give an indication whether gene pathways
related to toxicity have been turned on.
[0293] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with an ATPase-like molecule or ATPase-like
modulator, such as a modulator identified by one of the exemplary
screening assays described herein.
[0294] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the ATPase-like genes of
the present invention, wherein these products may be associated
with resistance of the cells to a therapeutic agent. Specifically,
the activity of the proteins encoded by the ATPase-like genes of
the present invention can be used as a basis for identifying agents
for overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells will become sensitive
to treatment with an agent that the unmodified target cells were
resistant to.
[0295] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a ATPase-like protein can be applied in
clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
ATPase-like gene expression, protein levels, or upregulate
ATPase-like activity, can be monitored in clinical trials of
subjects exhibiting decreased ATPase-like gene expression, protein
levels, or downregulated ATPase-like activity. Alternatively, the
effectiveness of an agent determined by a screening assay to
decrease ATPase-like gene expression, protein levels, or
downregulate ATPase-like activity, can be monitored in clinical
trials of subjects exhibiting increased ATPase-like gene
expression, protein levels, or upregulated ATPase-like activity. In
such clinical trials, the expression or activity of a ATPase-like
gene, and preferably, other genes that have been implicated in, for
example, a ATPase-like-associated disorder can be used as a "read
out" or markers of the phenotype of a particular cell.
[0296] 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.
[0297] Thus, the activity of ATPase-like protein, expression of
ATPase-like nucleic acid, or mutation content of ATPase-like 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 ATPase-like modulator; such as a
modulator identified by one of the exemplary screening assays
described herein.
[0298] 5. Monitoring of Effects During Clinical Trials
[0299] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of ATPase-like genes 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
ATPase-like gene expression, protein levels, or protein activity,
can be monitored in clinical trials of subjects exhibiting
decreased or increased ATPase-like gene expression, protein levels,
or protein activity. In such clinical trials, ATPase-like
expression or activity and preferably that of other genes that have
been implicated in influencing ATPase-like expression or activity,
can be used as a marker of the immune responsiveness of a
particular cell.
[0300] 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 ATPase-like activity (e.g.,
as identified in a screening assay described herein) can be
identified. Thus, to study the effect of agents on cellular
disorders resulting from aberrant ATPase-like activity, for
example, in a clinical trial, cells can be isolated and RNA
prepared and analyzed for the levels of expression of ATPase-like
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 ATPase-like 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.
[0301] 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; [0302] (2)
detecting the level of expression of an ATPase-like 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 ATPase-like protein,
mRNA, or genomic DNA in the postadministration samples; (5)
comparing the level of expression or activity of the ATPase-like
protein, mRNA, or genomic DNA in the preadministration sample with
the ATPase-like 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 ATPase-like
protein.
[0303] C. Methods of Treatment
[0304] 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 ATPase-like expression or activity. Additionally, the
compositions of the invention find use in the treatment of
disorders described herein. Thus, therapies for disorders
associated with ATPase-like molecules are encompassed herein.
"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.
[0305] "Treatment" is herein 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. A "therapeutic agent" includes, but is not limited
to, small molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0306] 1. Prophylactic Methods
[0307] In one aspect, the invention provides a method for
preventing in a subject a disease or condition associated with an
aberrant ATPase-like expression or activity by administering to the
subject an agent that modulates ATPase-like expression or at least
one ATPase-like gene activity. Subjects at risk for a disease that
is caused, or contributed to, by aberrant ATPase-like 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 ATPase-like aberrancy, such that a
disease or disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of ATPase-like aberrancy, for
example, an ATPase-like agonist or ATPase-like antagonist agent can
be used for treating the subject. The appropriate agent can be
determined based on screening assays described herein.
[0308] 2. Therapeutic Methods
[0309] Another aspect of the invention pertains to methods of
modulating ATPase-like 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 ATPase-like protein activity associated with the
cell. An agent that modulates ATPase-like protein activity can be
an agent as described herein, such as a nucleic acid or a protein,
a naturally-occurring cognate ligand of an ATPase-like protein, a
peptide, an ATPase-like peptidomimetic, or other small molecule. In
one embodiment, the agent stimulates one or more of the biological
activities of ATPase-like protein. Examples of such stimulatory
agents include active ATPase-like protein and a nucleic acid
molecule encoding an ATPase-like protein that has been introduced
into the cell. In another embodiment, the agent inhibits one or
more of the biological activities of ATPase-like protein. Examples
of such inhibitory agents include antisense ATPase-like nucleic
acid molecules and anti-ATPase-like antibodies.
[0310] 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 ATPase-like 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) ATPase-like expression or activity. In another
embodiment, the method involves administering an ATPase-like
protein or nucleic acid molecule as therapy to compensate for
reduced or aberrant ATPase-like expression or activity.
[0311] Stimulation of ATPase-like activity is desirable in
situations in which an ATPase-like protein is abnormally
downregulated and/or in which increased ATPase-like activity is
likely to have a beneficial effect. Conversely, inhibition of
ATPase-like activity is desirable in situations in which
ATPase-like activity is abnormally upregulated and/or in which
decreased ATPase-like activity is likely to have a beneficial
effect.
[0312] This invention is further illustrated by the following
examples, which should not be construed as limiting.
EXPERIMENTAL
Example 1
Tissue Distribution of ATPase-Like mRNA
[0313] Northern blot hybridizations with various RNA samples can be
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 ATPase-like cDNA (SEQ ID
NO:1 or 3) can be used. The DNA was 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.) can be probed in
ExpressHyb hybridization solution (Clontech) and washed at high
stringency according to manufacturer's recommendations.
[0314] TAQMAN analysis of the 7970 sequence revealed expression in
a number of tissues as shown in FIG. 4. High levels of 7970
transcripts are seen in the fetal heart, normal heart, heart (CHF),
brain cortex, brain hypothalamus, glial cells, and epithelial
cells. Moderate levels of expression are found in kidney, fetal
liver, aortic SMC early, aortic SMC late, shear HUVEC, and static
HUVEC. Low levels of expression were found in the aorta, vein,
spinal cord, brain, gioblastoma, breast, ovary, tumorous breast,
tumorous ovary, pancreas, prostate, tumorous prostate, colon,
tumorous colon, liver, liver fibrosis, lung, tumorous lung, lung
(COPD), spleen, tonsil, lymph node, thymus, endothelial cells
(aortic), skeletal muscle, fibroblasts, skin, adipose, osteoblasts
(primary), osteoblasts (undifferentiated), osteoblasts
(differentiated), and osteoclasts.
[0315] The expression of the 7970 sequence in various tumorous and
normal tissues was also determined. FIGS. 5-8 and FIGS. 10-12 show
the relative expression levels of the 7970 transcript in various
tissues including, for example, normal and tumorous colon tissues,
normal liver and metastatic liver tissues, normal brain and
tumorous brain tissues, normal breast and tumorous breast tissues,
normal ovary and tumorous ovary tissues, normal lung and tumorous
lung tissues.
[0316] FIG. 9 further shows the expression level of the 7970
transcript in a non-small cell lung cancer cell line (H640) in the
presence and the absence of the p16 gene.
Example 2
Recombinant Expression of ATPase-Like in Bacterial Cells
[0317] In this example, the ATPase-like sequence is expressed as a
recombinant glutathione-S-transferase (GST) fusion polypeptide in
E. coli and the fusion polypeptide is isolated and characterized.
Specifically, the ATPase-like sequence is fused to GST and this
fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
Expression of the GST-ATPase-like fusion protein in PEB199 is
induced with IPTG. The recombinant fusion polypeptide is purified
from crude bacterial lysates of the induced PEB 199 strain by
affinity chromatography on glutathione beads. Using polyacrylamide
gel electrophoretic analysis of the polypeptide purified from the
bacterial lysates, the molecular weight of the resultant fusion
polypeptide is determined.
Example 3
Expression of Recombinant ATPase-Like Protein in COS Cells
[0318] To express the ATPase-like 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
ATPase-like 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.
[0319] To construct the plasmid, the ATPase-like 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 ATPase-like 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 ATPase-like 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 ATPase-like 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.
[0320] COS cells are subsequently transfected with the
ATPase-like-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 ATPase-like 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.
[0321] Alternatively, DNA containing the ATPase-like 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 ATPase-like polypeptide is
detected by radiolabelling and immunoprecipitation using a
ATPase-like specific monoclonal antibody.
[0322] 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.
[0323] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Chapter 2
32670, Novel Human Phosphatidylserine Synthase-Like Molecules and
Uses Thereof
BACKGROUND OF THE INVENTION
[0324] The membranes of eukaryotic cells contain not only large
amounts of cholesterol but a variety of phospholipids. For example,
the major phospholipids in the human erythrocyte include
phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,
and sphingomyelin. The four major phospholipids in the yeast
Saccharomyces cerevisiae are phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylinositol (PI), and
phosphatidylserine (PS) (Poole et al. (1986) J. of Bacteriology
168(2):668-672). PS accounts for 4 to 8% of the total membrane
phospholipids in S. cerevisiae and is important to overall lipid
metabolism (Atkinson et al. (1980) J. Biol. Chem. 255: 6653-6661).
PS is the normal precursor to PE and PC. The synthesis of
phospholipids in eukaryotic cells involves both cytoplasmic and
membrane-associated enzymes, a number of which are coordinately
regulated (Henry et al., (1984) Annu. Rev. Genet. 18:207-231).
[0325] Phosphatidylserine (PS) is an essential phospholipid for the
growth of mammalian cells, comprising approximately 10% of the
total membrane of various mammalian tissues and cultured cells
(Kuge et al. (1986) J. Biol. Chem. 261:5790-5794).
[0326] The enzyme responsible for the biosynthesis of PS in S.
cerevisiae is CDPdiacylglycerol:L-serine O-phoshphatidyl
transferase (Phosphatidylserine synthase; EC 2.7.8.8). PS synthase
catalyzes the formation of PS and CMP from CDP-diacylglycerol
(CDP-DG) and serine by a sequential Bi Bi reaction mechanism
(Bae-Lee et al. (1984) J. Biol. Chem. 259: 10857-10862). PS
synthase is an integral membrane protein. Detailed biochemical
studies have shown that PS synthase activity is present in both the
mitochondria and endoplasmic reticulum (Kuchler et al. (1986) J.
Bacteriol. 165:901-910).
[0327] Phosphatidylserine (PS) synthase in Chinese Hamster Ovary
cells (CHO) exists in two forms--(PSS) I and II. PSS I and PSS II
are encoded by two genes pssA and pssB, respectively (Kuge et al.
(1997) J. Biol. Chem. 272: 19133-19139). PSS I is responsible for
the conversion of phosphatidylcholine to phosphatidylserine and PSS
II is responsible for the conversion of phosphatidylethanolamine to
phosphatidylserine (Saito et al. (1998) J. Biol. Chem.
273:17199-17205). PS biosynthesis in CHO-K1 cells is inhibited upon
the addition of PS to the culture medium suggesting that feedback
control is involved in the regulation of PS biosynthesis (Nishijima
et al. (1986) J. Biol. Chem. 261:5784-5789).
[0328] Various CHO mutants have been identified which exhibit
defective synthesis in PSS I and PSS II. PSS I and PSS II are
similar in sequence to each other; there is a 38% amino acid
identity between the two PS synthases (Kuge et al. (1997) J. Biol.
Chem. 272:19133-19139). Results obtained by Kuge et al. indicate
that PSS II in CHO-K1 cells is inhibited by exogenous PS and that
the activity of over-produced PSS II in CHO-K1 cells is depressed
for maintenance of the normal PS biosynthetic rate, probably
through molecular mechanisms different from those for the exogenous
PS-mediated inhibition. Also, the work of Kuge et al. demonstrated
that the ARG-97 residue of PSS II is critical for both the
exogenous PS-mediated inhibition of PS II and the depression of
overproduced PSS II activity.
[0329] Because phospholipids such as PS are important components of
eukaryotic membranes their proper biosynthesis is critical to cell
homeostasis and function. Defects in PS synthase may yield
membranes with altered functionality which could have implications
in a wide range of disease states. Accordingly, PS synthases are a
major target for drug action and development. Accordingly, it is
valuable to the field of pharmaceutical development to identify and
characterize novel PS synthases and tissues and disorders in which
PS synthases are differentially expressed. The present invention
advances the state of the art by providing novel human PS synthase
molecules and the uses thereof.
SUMMARY OF THE INVENTION
[0330] Isolated nucleic acid molecules corresponding to human
phosphatidylserine synthase-like 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 sequences shown in SEQ
ID NO:6. Further provided are human phosphatidylserine
synthase-like polypeptides having an amino acid sequence encoded by
a nucleic acid molecule described herein.
[0331] 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.
[0332] The human phosphatidylserine synthase-like molecules of the
present invention are useful for modulating the biosynthetic
pathway involving the synthesis of the membrane phospholipid
phosphatidylserine (PS). The molecules may be useful for a wide
variety of human disorders as herein described.
[0333] Accordingly, in one aspect, this invention provides isolated
nucleic acid molecules encoding human phosphatidylserine
synthase-like proteins or biologically active portions thereof, as
well as nucleic acid fragments suitable as primers or hybridization
probes for the detection of human phosphatidylserine synthase-like
encoding nucleic acids.
[0334] Another aspect of this invention features isolated or
recombinant human phosphatidylserine synthase-like proteins and
polypeptides. Preferred human phosphatidylserine synthase-like
proteins and polypeptides possess at least one biological activity
possessed by naturally occurring human phosphatidylserine
synthase-like proteins.
[0335] 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.
[0336] Antibodies and antibody fragments that selectively bind the
human phosphatidylserine synthase-like polypeptides and fragments
are provided. Such antibodies are useful in detecting the human
phosphatidylserine synthase-like polypeptides.
[0337] In another aspect, the present invention provides a method
for detecting the presence of human phosphatidylserine
synthase-like activity or expression in a biological sample by
contacting the biological sample with an agent capable of detecting
an indicator of human phosphatidylserine synthase-like activity
such that the presence of human phosphatidylserine synthase-like
activity is detected in the biological sample.
[0338] In yet another aspect, the invention provides a method for
modulating human phosphatidylserine synthase-like activity
comprising contacting a cell with an agent that modulates (inhibits
or stimulates) human phosphatidylserine synthase-like activity or
expression such that human phosphatidylserine synthase-like
activity or expression in the cell is modulated. In one embodiment,
the agent is an antibody that specifically binds to human
phosphatidylserine synthase-like protein. In another embodiment,
the agent modulates expression of human phosphatidylserine
synthase-like protein by modulating transcription of a human
phosphatidylserine synthase-like gene, splicing of a human
phosphatidylserine synthase-like mRNA, or translation of a human
phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like mRNA or the human
phosphatidylserine synthase-like gene.
[0339] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
human phosphatidylserine synthase-like protein activity or nucleic
acid expression by administering an agent that is a human
phosphatidylserine synthase-like modulator to the subject. In one
embodiment, the human phosphatidylserine synthase-like modulator is
a human phosphatidylserine synthase-like protein. In another
embodiment, the human phosphatidylserine synthase-like modulator is
a human phosphatidylserine synthase-like nucleic acid molecule. In
other embodiments, the human phosphatidylserine synthase-like
modulator is a peptide, peptidomimetic, or other small
molecule.
[0340] 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 a human
phosphatidylserine synthase-like protein; (2) misregulation of a
gene encoding a human phosphatidylserine synthase-like protein; and
(3) aberrant post-translational modification of a human
phosphatidylserine synthase-like protein, wherein a wild-type form
of the gene encodes a protein with a human phosphatidylserine
synthase-like activity.
[0341] In another aspect, the invention provides a method for
identifying a compound that binds to or modulates the activity of a
human phosphatidylserine synthase-like protein. In general, such
methods entail measuring a biological activity of a human
phosphatidylserine synthase-like protein in the presence and
absence of a test compound and identifying those compounds that
alter the activity of the human phosphatidylserine synthase-like
protein.
[0342] The invention also features methods for identifying a
compound that modulates the expression of human phosphatidylserine
synthase-like genes by measuring the expression of the human
phosphatidylserine synthase-like sequences in the presence and
absence of the compound.
[0343] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0344] 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.
[0345] 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.
[0346] The present invention provides phosphatidylserine
synthase-like molecules. By "phosphatidylserine synthase-like
molecules" is intended a novel human sequence referred to as 32670,
and variants and fragments thereof. These full-length gene
sequences or fragments thereof are referred to as
"phosphatidylserine synthase-like" sequences indicating they share
sequence similarity with phosphatidylserine synthase genes.
Isolated nucleic acid molecules comprising nucleotide sequences
encoding the 32670 polypeptide whose amino acid sequence is given
in SEQ ID NO:6, or a variant or fragment thereof, are provided. A
nucleotide sequence encoding the 32670 polypeptide is set forth in
SEQ ID NO:5, and a coding sequence encoding the 32670 polypeptide
is set: forth in SEQ ID NO:7.
[0347] The disclosed invention relates to methods and compositions
for the modulation, diagnosis, and treatment of the disorders of
the various cells and tissues herein described.
[0348] 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.
[0349] 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.
[0350] 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.
[0351] 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.
[0352] 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.
[0353] 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.
[0354] 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.
[0355] 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.
[0356] 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,
Imunopathology 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.
[0357] 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.
[0358] 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.
[0359] 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 B.sub.12
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.
[0360] 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,
lynphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[0361] 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.
[0362] 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
(hypernephroma, adenocarcinoma of kidney), which includes
urothelial carcinomas of renal pelvis.
[0363] 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.
[0364] Disorders in the male breast include, but are not limited
to, gynecomastia and carcinoma.
[0365] 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.
[0366] 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.
[0367] 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.
[0368] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0369] 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.
[0370] 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.
[0371] 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.
[0372] 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 lymphoma4a), intestinal T-cell lymphoma, adult
T-cell leukemia/lymphoma, and anaplastic large cell lymphoma.
[0373] 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, the
coma-fibromas, and roblastomas, hill cell tumors, and
gonadoblastoma; and metastatic tumors such as Krukenberg
tumors.
[0374] 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.
[0375] A novel human phosphatidylserine synthase gene sequence,
referred to as 32670, is provided. This gene sequence and variants
and fragments thereof are encompassed by the term
"phosphatidylserine synthase-like" molecules or sequences as used
herein. The phosphatidylserine synthase-like sequences find use in
modulating a phosphatidylserine synthase function. By "modulating"
is intended the upregulating or downregulating of a response. That
is, the compositions of the invention affect the targeted activity
in either a positive or negative fashion.
[0376] The human phosphatidylserine synthase-like gene, clone 32670
was identified in a human osteoblast cDNA library. Clone 32670
encodes an mRNA transcript having the corresponding cDNA set forth
in SEQ ID NO:5. This transcript has a 1461 nucleotide open reading
frame (nucleotides 14-1474 of SEQ ID NO:5; SEQ ID NO:7), which
encodes a 487 amino acid protein (SEQ ID NO:6).
[0377] In one embodiment, a 32670 protein includes at least one
transmembrane domain. As used herein, the term "transmembrane
domain" includes an amino acid sequence of about 17-22 amino acid
residues in length that spans a phospholipid membrane.
Transmembrane domains are rich in hydrophobic residues, and
typically have an .alpha.-helical structure. In a preferred
embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the
amino acids of a transmembrane domain are hydrophobic, e.g.,
leucines, isoleucines, tyrosines, or tryptophans. Transmembrane
domains are described in, for example,
http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta et
al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of which
are incorporated herein by reference.
[0378] In a preferred embodiment, a 32670 polypeptide or protein
has at least one transmembrane domain or a region which includes at
least 17, 18, 19, 20, 21, or 22 amino acid residues and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity
with a "transmembrane domain," e.g., at least one transmembrane
domain of human 32670 (e.g., amino acid residues 66-82, 96-112,
127-144, 246-262, 314-334, 345-362, 382-399, or 410-420 of SEQ ID
NO:6).
[0379] In one embodiment, a 32670 protein includes at least one
"non-transmembrane domain." As used herein, "non-transmembrane
domains" are domains that reside outside of the membrane. When
referring to plasma membranes, non-transmembrane domains include
extracellular domains (i.e., outside of the cell) and intracellular
domains (i.e., within the cell). When referring to membrane-bound
proteins found in intracellular organelles (e.g., mitochondria,
endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally occurring 32670, or 32670-like protein.
[0380] In a preferred embodiment, a 32670 polypeptide or protein
has a "non-transmembrane domain" or a region which includes at
least about 1-200, preferably about 5-150, more preferably about
10-105 amino acids, and has at least about 60%, 70% 80% 90% 95%,
99% or 100% sequence identity with a "non-transmembrane domain",
e.g., a non-transmembrane domain of human 32670 (e.g., residues
1-65, 83-95, 113-126, 145-245, 263-313, 335-344, 363-381, 400-409,
or 430-487 of SEQ ID NO:6). Preferably, a non-transmembrane domain
is capable of catalytic activity (e.g., phosphatidylserine synthase
activity).
[0381] A non-transmembrane domain located at the N-terminus of a
32670 protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-120, 25-105, or preferably 45-85 amino acid
residues in length and is located outside the boundaries of a
membrane. For example, an N-terminal non-transmembrane domain is
located at about amino acid residues 1-65 of SEQ ID NO:6.
[0382] Similarly, a non-transmembrane domain located at the
C-terminus of a 32670 protein or polypeptide is referred to herein
as a "C-terminal non-transmembrane domain." As used herein, an
"C-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-120, preferably about 20-100, more
preferably about 40-80 amino acid residues in length and is located
outside the boundaries of a membrane. For example, an C-terminal
non-transmembrane domain is located at about amino acid residues
430-487 of SEQ ID NO:6.
[0383] A 32670 polypeptide can further include a signal sequence.
As used herein, a "signal sequence" refers to a peptide of about
5-70 amino acid residues in length which occurs at the N-terminus
of secretory and integral membrane proteins and which contains a
majority of hydrophobic amino acid residues. For example, a signal
sequence contains at least about 5-60 amino acid residues,
preferably about 6-20 amino acid residues, more preferably about 7
amino acid residues, and has at least about 40-70%, preferably
about 50-65%, and more preferably about 55-60% hydrophobic amino
acid residues (e.g., alanine, valine, leucine, isoleucine,
phenylalanine, tyrosine, tryptophan, or proline). Such a "signal
sequence", also referred to in the art as a "signal peptide",
serves to direct a protein containing such a sequence to a lipid
bilayer. For example, in one embodiment, a 32670 protein contains a
signal sequence of about amino acids 1-7 of SEQ ID NO:6. The
"signal sequence" is cleaved during processing of the mature
protein. The mature 32670 protein corresponds to amino acids 8-487
of SEQ ID NO:6.
[0384] Prosite program analysis was used to predict various
consensus sites within the 32670 protein. N-glycosylation sites
were predicted at aa 181-184 and 237-240. A cAMP- and
cGMP-dependent protein kinase phosphorylation site was predicted at
aa 44-47. Protein kinase C phosphorylation sites were predicted at
aa 93-95, 269-271, 272-274, 283-285, 310-312, and 437-439. Casein
kinase II phosphorylation sites were predicted at aa 24-27, 47-50,
80-83, 85-88, 146-149, and 389-392. A tyrosine kinase
phosphorylation site was predicted at aa 44-52. N-myristoylation
sites were predicted at aa 96-101, 108-113, 257-262, 419-424,
455-460, and 476-481. An amidation site was predicted at aa 42-45.
The human phosphatidylserine synthase-like protein possesses a type
III leader peptidase domain from aa 314-338 as predicted by Mer,
Version 2.
[0385] The 32670 protein shares approximately 85% identity with the
phosphatidylserine synthase II from Cricetulus griseus and
approximately 86% identity with the murine phosphatidylserine
synthase-2 as determined by pairwise alignment (FIG. 13).
[0386] The 32670 displays approximately 52% identity from aa 45-325
and approximately 33% identity from aa 305-464 to Prodom consensus
sequences found in phosphatidylserine synthase I from Cricetulus
longicaudatus (ProDom Accession No. PDO11831). H32670 also shares
approximately 40% identity from aa 446 to a Prodom consensus
sequence found in Hepatocyte Nuclear Factor 3 Forkhead Homolog 1
(HFH-1) from Rattus norvegicus and in the murine Fork Head
Transcription Factor (Pro Dom Accession No. PD31440).
Phosphatidylserine synthase I is a serine exchange enzyme (EC
2.7.8.8) involved in the synthesis of phosphatidylserine.
Phosphatidylserine synthase I functions in a base-exchange between
free L-serine and the polar head groups of pre-existing
phospholipids. It can utilize phosphatidylcholine as a phosphatidyl
donor. It also catalyzes the choline and ethanolamine base-exchange
reactions. See, for example, Kuge et al. (1991) J. Biol. Chem.
266:24184-24189. The HFH-1 and the Fork Head Transcription Factor
contain a conserved "fork head" domain involved in DNA-binding.
See, for example, Haecker et al. (1995) EMBO J. 14:5306-5317,
Clevidence et al. (1993) Proc. Natl. Acad. Sci. 90:3948-3952.
[0387] Preferred human phosphatidylserine synthase-like
polypeptides of the present invention have an amino acid sequence
sufficiently identical to the amino acid sequence of SEQ ID NO:6.
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 60% identity, preferably 65% identity, more preferably 70%,
75%, or 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more identity are defined herein as sufficiently identical.
[0388] 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%, 90%, 100% 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.
[0389] 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 http://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 http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. 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.
[0390] 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.
[0391] 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 32670 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 32670 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al. (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0392] Accordingly, another embodiment of the invention features
isolated human phosphatidylserine synthase-like proteins and
polypeptides having a human phosphatidylserine synthase-like
protein activity. As used interchangeably herein, a "human
phosphatidylserine synthase-like protein activity", "biological
activity of a human phosphatidylserine synthase-like protein", or
"functional activity of a human phosphatidylserine synthase-like
protein" refers to an activity exerted by a human
phosphatidylserine synthase-like protein, polypeptide, or nucleic
acid molecule on a human phosphatidylserine synthase-like
responsive cell as determined in vivo, or in vitro, according to
standard assay techniques. A human phosphatidylserine synthase-like
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 signaling activity mediated by interaction of
the human phosphatidylserine synthase-like protein with a second
protein. In a preferred embodiment, human phosphatidylserine
synthase-like activity includes at least one or more modulating
activities which may include stimulating and/or enhancing or
inhibiting phosphatidylserine synthesis.
[0393] An "isolated" or "purified" human phosphatidylserine
synthase-like 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 human phosphatidylserine
synthase-like 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. A human
phosphatidylserine synthase-like protein that is substantially free
of cellular material includes preparations of human
phosphatidylserine synthase-like protein having less than about
30%, 20%, 10%, or 5% (by dry weight) of non-human
phosphatidylserine synthase-like protein (also referred to herein
as a "contaminating protein"). When the human phosphatidylserine
synthase-like 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 human phosphatidylserine synthase-like 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-human phosphatidylserine synthase-like
chemicals.
[0394] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[0395] One aspect of the invention pertains to isolated nucleic
acid molecules comprising nucleotide sequences encoding human
phosphatidylserine synthase-like proteins and polypeptides or
biologically active portions thereof, as well as nucleic acid
molecules sufficient for use as hybridization probes to identify
human phosphatidylserine synthase-like encoding nucleic acids
(e.g., human phosphatidylserine synthase-like mRNA) and fragments
for use as PCR primers for the amplification or mutation of human
phosphatidylserine synthase-like 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.
[0396] Nucleotide sequences encoding the human phosphatidylserine
synthase-like proteins of the present invention include sequences
set forth in SEQ ID NO:5, SEQ ID NO:7, 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 human
phosphatidylserine synthase-like protein encoded by these
nucleotide sequences is set forth in SEQ ID NO:6. The invention
also encompasses nucleic acid molecules comprising nucleotide
sequences encoding partial-length human phosphatidylserine
synthase-like proteins, including the sequence set forth in SEQ ID
NO:6, and complements thereof.
[0397] Nucleic acid molecules that are fragments of these human
phosphatidylserine synthase-like nucleotide sequences are also
encompassed by the present invention. By "fragment" is intended a
portion of the nucleotide sequence encoding a human
phosphatidylserine synthase-like protein. A fragment of a human
phosphatidylserine synthase-like nucleotide sequence may encode a
biologically active portion of a human phosphatidylserine
synthase-like 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 a human phosphatidylserine
synthase-like protein can be prepared by isolating a portion of one
of the human phosphatidylserine synthase-like nucleotide sequences
of the invention, expressing the encoded portion of the human
phosphatidylserine synthase-like protein (e.g., by recombinant
expression in vitro), and assessing the activity of the encoded
portion of the human phosphatidylserine synthase-like protein.
Nucleic acid molecules that are fragments of a human
phosphatidylserine synthase-like 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, 1450, 1500, 1550, 1600, 1650, 1700, 1750,
1800 nucleotides, or up to the number of nucleotides present in a
full-length human phosphatidylserine synthase-like nucleotide
sequence disclosed herein (for example, 1852 nucleotides for SEQ ID
NO:5) depending upon the intended use.
[0398] Alternatively, a nucleic acid molecule that is a fragment of
a human phosphatidylserine synthase-like nucleotide sequence of the
present invention comprises a nucleotide sequence consisting of
nucleotides 1-100, 101-200, 201-300, 301-400, 401-500, 501-600,
601-700, 701-800, 801-900, 901-1000, 1001-1100, 1101-1200,
1201-1300, 1301-1400, 1401-1500, 1501-1600, 1601-1700, 1701-1800,
or 1801-1852 of SEQ ID NO:5.
[0399] 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.
[0400] A fragment of a human phosphatidylserine synthase-like
nucleotide sequence that encodes a biologically active portion of a
human phosphatidylserine synthase-like protein of the invention
will encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200,
250, or 300 contiguous amino acids, or up to the total number of
amino acids present in a full-length human phosphatidylserine
synthase-like protein of the invention (for example, 487 amino
acids for SEQ ID NO:6). Fragments of a human phosphatidylserine
synthase-like nucleotide sequence that are useful as hybridization
probes for PCR primers generally need not encode a biologically
active portion of a human phosphatidylserine synthase-like
protein.
[0401] Nucleic acid molecules that are variants of the human
phosphatidylserine synthase-like nucleotide sequences disclosed
herein are also encompassed by the present invention. "Variants" of
the human phosphatidylserine synthase-like nucleotide sequences
include those sequences that encode the human phosphatidylserine
synthase-like 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 human
phosphatidylserine synthase-like proteins disclosed in the present
invention as discussed below. 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%, 99% or more
identity to a particular nucleotide sequence disclosed herein. A
variant human phosphatidylserine synthase-like nucleotide sequence
will encode a human phosphatidylserine synthase-like protein that
has an amino acid sequence having at least 45%, 55%, 65%, 75%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the
amino acid sequence of a human phosphatidylserine synthase-like
protein disclosed herein.
[0402] In addition to the human phosphatidylserine synthase-like
nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7, it will
be appreciated by those skilled in the art that DNA sequence
polymorphisms that lead to changes in the amino acid sequences of
human phosphatidylserine synthase-like proteins may exist within a
population (e.g., the human population). Such genetic polymorphism
in a human phosphatidylserine synthase-like 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 a human phosphatidylserine
synthase-like protein, preferably a mammalia human
phosphatidylserine synthase-like protein. As used herein, the
phrase "allelic variant" refers to a nucleotide sequence that
occurs at a human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like
gene. Any and all such nucleotide variations and resulting amino
acid polymorphisms or variations in a human phosphatidylserine
synthase-like sequence that are the result of natural allelic
variation and that do not alter the functional activity of human
phosphatidylserine synthase-like proteins are intended to be within
the scope of the invention.
[0403] Moreover, nucleic acid molecules encoding human
phosphatidylserine synthase-like proteins from other species (human
phosphatidylserine synthase-like homologues), which have a
nucleotide sequence differing from that of the human
phosphatidylserine synthase-like 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 huma human phosphatidylserine synthase-like
cDNA of the invention can be isolated based on their identity to
the huma human phosphatidylserine synthase-like 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.
[0404] In addition to naturally-occurring allelic variants of the
human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like
proteins, without altering the biological activity of the human
phosphatidylserine synthase-like proteins. Thus, an isolated
nucleic acid molecule encoding a human phosphatidylserine
synthase-like protein having a sequence that differs from that of
SEQ ID NO:5 or SEQ ID NO:7 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.
[0405] 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 a human phosphatidylserine synthase-like protein (e.g.,
the sequence of SEQ ID NO:6) 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).
[0406] Alternatively, variant human phosphatidylserine
synthase-like nucleotide sequences can be made by introducing
mutations randomly along all or part of a human phosphatidylserine
synthase-like coding sequence, such as by saturation mutagenesis,
and the resultant mutants can be screened for human
phosphatidylserine synthase-like 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.
[0407] Thus the nucleotide sequences of the invention include the
sequences disclosed herein as well as fragments and variants
thereof. The human phosphatidylserine synthase-like nucleotide
sequences of the invention, and fragments and variants thereof, can
be used as probes and/or primers to identify and/or clone human
phosphatidylserine synthase-like homologues in other cell types,
e.g., from other tissues, as well as human phosphatidylserine
synthase-like 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 a human phosphatidylserine synthase-like protein, such
as by measuring levels of a human phosphatidylserine
synthase-like-encoding nucleic acid in a sample of cells from a
subject, e.g., detecting human phosphatidylserine synthase-like
mRNA levels or determining whether a genomic human
phosphatidylserine synthase-like gene has been mutated or
deleted.
[0408] 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). Human phosphatidylserine
synthase-like nucleotide sequences isolated based on their sequence
identity to the human phosphatidylserine synthase-like nucleotide
sequences set forth herein or to fragments and variants thereof are
encompassed by the present invention.
[0409] In a hybridization method, all or part of a known human
phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like nucleotide sequence disclosed
herein. Degenerate primers designed on the basis of conserved
nucleotides or amino acid residues in a known human
phosphatidylserine synthase-like 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 a human phosphatidylserine
synthase-like 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.
[0410] For example, in one embodiment, a previously unidentified
human phosphatidylserine synthase-like nucleic acid molecule
hybridizes under stringent conditions to a probe that is a nucleic
acid molecule comprising one of the human phosphatidylserine
synthase-like nucleotide sequences of the invention or a fragment
thereof. In another embodiment, the previously unknown human
phosphatidylserine synthase-like 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 human phosphatidylserine
synthase-like nucleotide sequences disclosed herein or a fragment
thereof.
[0411] Accordingly, in another embodiment, an isolated previously
unknown human phosphatidylserine synthase-like 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,
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:5 (shown in SEQ ID NO:7) or
a complement, fragment, or variant thereof.
[0412] 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:5 or SEQ ID NO:7 corresponds to a
naturally-occurring nucleic acid molecule.
[0413] 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).
[0414] Thus, in addition to the human phosphatidylserine
synthase-like 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 human
phosphatidylserine synthase-like nucleotide sequences disclosed
herein or variants and fragments thereof.
[0415] 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 human phosphatidylserine synthase-like
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 a human
phosphatidylserine synthase-like protein. The noncoding regions are
the 5' and 3' sequences that flank the coding region and are not
translated into amino acids.
[0416] Given the coding-strand sequence encoding a human
phosphatidylserine synthase-like protein disclosed herein (e.g.,
SEQ ID NO:6), 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 human phosphatidylserine synthase-like
mRNA, but more preferably is an oligonucleotide that is antisense
to only a portion of the coding or noncoding region of human
phosphatidylserine synthase-like mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of human phosphatidylserine synthase-like
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.
[0417] 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).
[0418] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a human phosphatidylserine synthase-like 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.
[0419] An antisense nucleic acid molecule of the invention can be
an .alpha.-anomeric nucleic acid molecule. An a-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).
[0420] 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 human
phosphatidylserine synthase-like mRNA transcripts to thereby
inhibit translation of human phosphatidylserine synthase-like mRNA.
A ribozyme having specificity for a human phosphatidylserine
synthase-like-encoding nucleic acid can be designed based upon the
nucleotide sequence of a human phosphatidylserine synthase-like
cDNA disclosed herein (e.g., SEQ ID NO:5 or SEQ ID NO:7). 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, human phosphatidylserine
synthase-like 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.
[0421] The invention also encompasses nucleic acid molecules that
form triple helical structures. For example, human
phosphatidylserine synthase-like gene expression can be inhibited
by targeting nucleotide sequences complementary to the regulatory
region of the human phosphatidylserine synthase-like protein (e.g.,
the human phosphatidylserine synthase-like promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the human phosphatidylserine synthase-like 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.
[0422] 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.
[0423] PNAs of a human phosphatidylserine synthase-like 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).
[0424] In another embodiment, PNAs of a human phosphatidylserine
synthase-like 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 Human Phosphatidylserine Synthase-Like Proteins and
Anti-Human Phosphatidylserine Synthase-Like Antibodies
[0425] Human phosphatidylserine synthase-like proteins are also
encompassed within the present invention. By "human
phosphatidylserine synthase-like 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.
[0426] "Fragments" or "biologically active portions" include
polypeptide fragments suitable for use as immunogens to raise
anti-human phosphatidylserine synthase-like antibodies. Fragments
include peptides comprising amino acid sequences sufficiently
identical to or derived from the amino acid sequence of a human
phosphatidylserine synthase-like protein, or partial-length
protein, of the invention and exhibiting at least one activity of a
human phosphatidylserine synthase-like protein, but which include
fewer amino acids than the full-length (SEQ ID NO:6) human
phosphatidylserine synthase-like protein disclosed herein.
Typically, biologically active portions comprise a domain or motif
with at least one activity of the human phosphatidylserine
synthase-like protein. A biologically active portion of a human
phosphatidylserine synthase-like protein can be a polypeptide which
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 human phosphatidylserine synthase-like
protein. As used here, a fragment comprises at least 5 contiguous
amino acids of SEQ ID NO:6. The invention encompasses other
fragments, however, such as any fragment in the protein greater
than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19 or 20 amino
acids.
[0427] 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%, 90%, 91%, 923, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to the amino acid sequence of SEQ ID NO:6.
Variants also include polypeptides encoded by a nucleic acid
molecule that hybridizes to the nucleic acid molecule of SEQ ID
NO:5, SEQ ID NO:7, 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:6. 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 generally retain the
functional activity of the 32670 proteins of the invention.
Variants include polypeptides that differ in amino acid sequence
due to natural allelic variation or mutagenesis.
[0428] The invention also provides human phosphatidylserine
synthase-like chimeric or fusion proteins. As used herein, a human
phosphatidylserine synthase-like "chimeric protein" or "fusion
protein" comprises a human phosphatidylserine synthase-like
polypeptide operably linked to a non-human phosphatidylserine
synthase-like polypeptide. A "human phosphatidylserine
synthase-like polypeptide" refers to a polypeptide having an amino
acid sequence corresponding to a human phosphatidylserine
synthase-like protein, whereas a "non-human phosphatidylserine
synthase-like polypeptide" refers to a polypeptide having an amino
acid sequence corresponding to a protein that is not substantially
identical to the human phosphatidylserine synthase-like protein,
e.g., a protein that is different from the human phosphatidylserine
synthase-like protein and which is derived from the same or a
different organism. Within a human phosphatidylserine synthase-like
fusion protein, the human phosphatidylserine synthase-like
polypeptide can correspond to all or a portion of a human
phosphatidylserine synthase-like protein, preferably at least one
biologically active portion of a human phosphatidylserine
synthase-like protein. Within the fusion protein, the term
"operably linked" is intended to indicate that the human
phosphatidylserine synthase-like polypeptide and the non-human
phosphatidylserine synthase-like polypeptide are fused in-frame to
each other. The non-human phosphatidylserine synthase-like
polypeptide can be fused to the N-terminus or C-terminus of the
human phosphatidylserine synthase-like polypeptide.
[0429] One useful fusion protein is a GST-human phosphatidylserine
synthase-like fusion protein in which the human phosphatidylserine
synthase-like sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of
recombinant human phosphatidylserine synthase-like proteins.
[0430] In yet another embodiment, the fusion protein is a human
phosphatidylserine synthase-like-immunoglobulin fusion protein in
which all or part of a human phosphatidylserine synthase-like
protein is fused to sequences derived from a member of the
immunoglobulin protein family. The human phosphatidylserine
synthase-like-immunoglobulin fusion proteins of the invention can
be incorporated into pharmaceutical compositions and administered
to a subject to inhibit an interaction between a human
phosphatidylserine synthase-like ligand and a human
phosphatidylserine synthase-like protein on the surface of a cell,
thereby suppressing human phosphatidylserine synthase-like-mediated
signal transduction in vivo. The human phosphatidylserine
synthase-like-immunoglobulin fusion proteins can be used to affect
the bioavailability of a human phosphatidylserine synthase-like
cognate ligand. Inhibition of the human phosphatidylserine
synthase-like ligand/human phosphatidylserine synthase-like
interaction may be useful therapeutically. Moreover, the human
phosphatidylserine synthase-like-immunoglobulin fusion proteins of
the invention can be used as immunogens to produce anti-human
phosphatidylserine synthase-like antibodies in a subject, to purify
human phosphatidylserine synthase-like ligands, and in screening
assays to identify molecules that inhibit the interaction of a
human phosphatidylserine synthase-like protein with a human
phosphatidylserine synthase-like ligand.
[0431] Preferably, a human phosphatidylserine synthase-like
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, a human phosphatidylserine
synthase-like-encoding nucleic acid can be cloned into a
commercially available expression vector such that it is linked
in-frame to an existing fusion moiety. Variants of the human
phosphatidylserine synthase-like proteins can function as either
human phosphatidylserine synthase-like agonists (mimetics) or as
human phosphatidylserine synthase-like antagonists. Variants of the
human phosphatidylserine synthase-like protein can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the
human phosphatidylserine synthase-like protein. An agonist of the
human phosphatidylserine synthase-like protein can retain
substantially the same, or a subset, of the biological activities
of the naturally occurring form of the human phosphatidylserine
synthase-like protein. An antagonist of the human
phosphatidylserine synthase-like protein can inhibit one or more of
the activities of the naturally occurring form of the human
phosphatidylserine synthase-like protein by, for example,
competitively binding to a downstream or upstream member of a
cellular signaling cascade that includes the human
phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like proteins.
[0432] Variants of a human phosphatidylserine synthase-like protein
that function as either human phosphatidylserine synthase-like
agonists or as human phosphatidylserine synthase-like antagonists
can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of a human phosphatidylserine
synthase-like protein for human phosphatidylserine synthase-like
protein agonist or antagonist activity. In one embodiment, a
variegated library of human phosphatidylserine synthase-like
variants is generated by combinatorial mutagenesis at the nucleic
acid level and is encoded by a variegated gene library. A
variegated library of human phosphatidylserine synthase-like
variants can be produced by, for example, enzymatically ligating a
mixture of synthetic oligonucleotides into gene sequences such that
a degenerate set of potential human phosphatidylserine
synthase-like sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of human phosphatidylserine
synthase-like sequences therein. There are a variety of methods
that can be used to produce libraries of potential human
phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like 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).
[0433] In addition, libraries of fragments of a human
phosphatidylserine synthase-like protein coding sequence can be
used to generate a variegated population of human
phosphatidylserine synthase-like fragments for screening and
subsequent selection of variants of a human phosphatidylserine
synthase-like protein. In one embodiment, a library of coding
sequence fragments can be generated by treating a double-stranded
PCR fragment of a human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like protein.
[0434] 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 human phosphatidylserine synthase-like 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 human phosphatidylserine
synthase-like variants (Arkin and Yourvan (1992) Proc. Natl. Acad.
Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering
6(3):327-331).
[0435] An isolated human phosphatidylserine synthase-like
polypeptide of the invention can be used as an immunogen to
generate antibodies that bind human phosphatidylserine
synthase-like proteins using standard techniques for polyclonal and
monoclonal antibody preparation. The full-length human
phosphatidylserine synthase-like protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of human phosphatidylserine synthase-like proteins for use as
immunogens. The antigenic peptide of a human phosphatidylserine
synthase-like protein comprises at least 8, preferably 10, 15, 20,
or 30 amino acid residues of the amino acid sequence shown in SEQ
ID NO:6 and encompasses an epitope of a human phosphatidylserine
synthase-like protein such that an antibody raised against the
peptide forms a specific immune complex with the human
phosphatidylserine synthase-like protein. Preferred epitopes
encompassed by the antigenic peptide are regions of a human
phosphatidylserine synthase-like protein that are located on the
surface of the protein, e.g., hydrophilic regions.
[0436] Accordingly, another aspect of the invention pertains to
anti-human phosphatidylserine synthase-like polyclonal and
monoclonal antibodies that bind a human phosphatidylserine
synthase-like protein. Polyclonal anti-human phosphatidylserine
synthase-like antibodies can be prepared by immunizing a suitable
subject (e.g., rabbit, goat, mouse, or other mammal) with a human
phosphatidylserine synthase-like immunogen. The anti-human
phosphatidylserine synthase-like 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
human phosphatidylserine synthase-like protein. At an appropriate
time after immunization, e.g., when the anti-human
phosphatidylserine synthase-like 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 266:550-52;
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).
[0437] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-human phosphatidylserine
synthase-like antibody can be identified and isolated by screening
a recombinant combinatorial immunoglobulin library (e.g., an
antibody phage display library) with a human phosphatidylserine
synthase-like protein to thereby isolate immunoglobulin library
members that bind the human phosphatidylserine synthase-like
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.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening antibody display library can be found in,
for example, 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/Techniques 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.
[0438] Additionally, recombinant anti-human phosphatidylserine
synthase-like 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 86/101533 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.
[0439] 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.
[0440] 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).
[0441] An anti-human phosphatidylserine synthase-like antibody
(e.g., monoclonal antibody) can be used to isolate human
phosphatidylserine synthase-like proteins by standard techniques,
such as affinity chromatography or immunoprecipitation. An
anti-human phosphatidylserine synthase-like antibody can facilitate
the purification of natural human phosphatidylserine synthase-like
protein from cells and of recombinantly produced human
phosphatidylserine synthase-like protein expressed in host cells.
Moreover, an anti-human phosphatidylserine synthase-like antibody
can be used to detect human phosphatidylserine synthase-like
protein (e.g., in a cellular lysate or cell supernatant) in order
to evaluate the abundance and pattern of expression of the human
phosphatidylserine synthase-like protein. Anti-human
phosphatidylserine synthase-like 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.
[0442] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). 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.
[0443] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84:Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
III. Recombinant Expression Vectors and Host Cells
[0444] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
human phosphatidylserine synthase-like 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.
[0445] 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., human phosphatidylserine synthase-like
proteins, mutant forms of human phosphatidylserine synthase-like
proteins, fusion proteins, etc.).
[0446] 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 such codon usage are well known in the art.
[0447] The recombinant expression vectors of the invention can be
designed for expression of human phosphatidylserine synthase-like
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 11d
(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.
[0448] Suitable eukaryotic host cells include insect cells
(examples of Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers (1989) Virology 170:31-39));
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.
[0449] 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.
[0450] 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.
[0451] 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 (Banerji et al. (1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application Patent
Publication No. 264,166). Developmentally-regulated promoters are
also encompassed, for example the murine homeobox (hox) promoters
(Kessel and Gruss (1990) Science 249:374-379), the a-fetoprotein
promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546), and the
like.
[0452] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an anti sense 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 human phosphatidylserine
synthase-like 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).
[0453] 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 (2nd ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.) and other laboratory
manuals.
[0454] 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 a human phosphatidylserine synthase-like
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).
[0455] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) human phosphatidylserine synthase-like protein.
Accordingly, the invention further provides methods for producing
human phosphatidylserine synthase-like protein using the host cells
of the invention. In one embodiment, the method comprises culturing
the host cell of the invention, into which a recombinant expression
vector encoding a human phosphatidylserine synthase-like protein
has been introduced, in a suitable medium such that human
phosphatidylserine synthase-like protein is produced. In another
embodiment, the method further comprises isolating human
phosphatidylserine synthase-like protein from the medium or the
host cell.
[0456] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which human phosphatidylserine synthase-like-coding
sequences have been introduced. Such host cells can then be used to
create nonhuman transgenic animals in which exogenous human
phosphatidylserine synthase-like sequences have been introduced
into their genome or homologous recombinant animals in which
endogenous human phosphatidylserine synthase-like sequences have
been altered. Such animals are useful for studying the function
and/or activity of human phosphatidylserine synthase-like genes and
proteins and for identifying and/or evaluating modulators of human
phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like 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.
[0457] A transgenic animal of the invention can be created by
introducing human phosphatidylserine synthase-like-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 human
phosphatidylserine synthase-like cDNA sequence can be introduced as
a transgene into the genome of a nonhuman animal. Alternatively, a
homologue of the mouse human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like transgene to
direct expression of human phosphatidylserine synthase-like 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
human phosphatidylserine synthase-like transgene in its genome
and/or expression of human phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like gene can further be bred to other
transgenic animals carrying other transgenes.
[0458] To create a homologous recombinant animal, one prepares a
vector containing at least a portion of a human phosphatidylserine
synthase-like 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 human phosphatidylserine
synthase-like gene. In a preferred embodiment, the vector is
designed such that, upon homologous recombination, the endogenous
human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like
protein). In the homologous recombination vector, the altered
portion of the human phosphatidylserine synthase-like gene is
flanked at its 5' and 3' ends by additional nucleic acid of the
human phosphatidylserine synthase-like gene to allow for homologous
recombination to occur between the exogenous human
phosphatidylserine synthase-like gene carried by the vector and an
endogenous human phosphatidylserine synthase-like gene in an
embryonic stem cell. The additional flanking human
phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like gene has
homologously recombined with the endogenous human
phosphatidylserine synthase-like 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/Techniques 2:823-829 and in PCT Publication Nos. WO 90/11354,
WO 91/01140, WO 92/0968, and WO 93/04169.
[0459] 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.
[0460] 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
[0461] The human phosphatidylserine synthase-like nucleic acid
molecules, human phosphatidylserine synthase-like proteins, and
anti-human phosphatidylserine synthase-like 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.
[0462] The compositions of the invention are useful to treat any of
the disorders discussed herein. 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.
[0463] 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.
[0464] 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.
[0465] 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.
[0466] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the skill 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.
[0467] 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.
[0468] 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.TM. (BASF; Parsippany, N.J.), or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as 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.
[0469] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a human phosphatidylserine
synthase-like protein or anti-human phosphatidylserine
synthase-like 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.
[0470] 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.
[0471] 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.
[0472] 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.
[0473] 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.
[0474] 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.
[0475] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
V. Uses and Methods of the Invention
[0476] 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
human phosphatidylserine synthase-like protein (e.g., via a
recombinant expression vector in a host cell in gene therapy
applications), to detect human phosphatidylserine synthase-like
mRNA (e.g., in a biological sample) or a genetic lesion in a human
phosphatidylserine synthase-like gene, and to modulate human
phosphatidylserine synthase-like activity. In addition, the human
phosphatidylserine synthase-like proteins can be used to screen
drugs or compounds in disorders characterized by insufficient or
excessive production of human phosphatidylserine synthase-like
protein or production of human phosphatidylserine synthase-like
protein forms that have decreased or aberrant activity compared to
human phosphatidylserine synthase-like wild type protein. In
addition, the anti-human phosphatidylserine synthase-like
antibodies of the invention can be used to detect and isolate human
phosphatidylserine synthase-like proteins and modulate human
phosphatidylserine synthase-like activity.
[0477] A. Screening Assays
[0478] 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 human phosphatidylserine
synthase-like proteins or have a stimulatory or inhibitory effect
on, for example, human phosphatidylserine synthase-like expression
or human phosphatidylserine synthase-like activity.
[0479] 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).
[0480] 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.
[0481] 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).
[0482] Determining the ability of the test compound to bind to the
human phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like 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.
[0483] In a similar manner, one may determine the ability of the
human phosphatidylserine synthase-like protein to bind to or
interact with a human phosphatidylserine synthase-like target
molecule. By "target molecule" is intended a molecule with which a
human phosphatidylserine synthase-like protein binds or interacts
in nature. In a preferred embodiment, the ability of the human
phosphatidylserine synthase-like protein to bind to or interact
with a human phosphatidylserine synthase-like 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 catalytic/enzymatic activity of the target on an
appropriate substrate, detecting the induction of a reporter gene
(e.g., a human phosphatidylserine synthase-like-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.
[0484] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting a human
phosphatidylserine synthase-like protein or biologically active
portion thereof with a test compound and determining the ability of
the test compound to bind to the human phosphatidylserine
synthase-like protein or biologically active portion thereof.
Binding of the test compound to the human phosphatidylserine
synthase-like protein can be determined either directly or
indirectly as described above. In a preferred embodiment, the assay
includes contacting the human phosphatidylserine synthase-like
protein or biologically active portion thereof with a known
compound that binds human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like
protein or biologically active portion thereof as compared to the
known compound.
[0485] In another embodiment, an assay is a cell-free assay
comprising contacting human phosphatidylserine synthase-like
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 human phosphatidylserine
synthase-like protein or biologically active portion thereof.
Determining the ability of the test compound to modulate the
activity of a human phosphatidylserine synthase-like protein can be
accomplished, for example, by determining the ability of the human
phosphatidylserine synthase-like protein to bind to a human
phosphatidylserine synthase-like 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 a human phosphatidylserine synthase-like protein can be
accomplished by determining the ability of the human
phosphatidylserine synthase-like protein to further modulate a
human phosphatidylserine synthase-like target molecule. For
example, the catalytic/enzymatic activity of the target molecule on
an appropriate substrate can be determined as previously
described.
[0486] In yet another embodiment, the cell-free assay comprises
contacting the human phosphatidylserine synthase-like protein or
biologically active portion thereof with a known compound that
binds a human phosphatidylserine synthase-like 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 a human phosphatidylserine
synthase-like target molecule.
[0487] In the above-mentioned assays, it may be desirable to
immobilize either a human phosphatidylserine synthase-like 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/human phosphatidylserine synthase-like
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 human phosphatidylserine
synthase-like 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 human phosphatidylserine
synthase-like binding or activity determined using standard
techniques.
[0488] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either human phosphatidylserine synthase-like protein or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin. Biotinylated human phosphatidylserine synthase-like
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 a human
phosphatidylserine synthase-like protein or target molecules but
which do not interfere with binding of the human phosphatidylserine
synthase-like protein to its target molecule can be derivatized to
the wells of the plate, and unbound target or human
phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like protein or
target molecule, as well as enzyme-linked assays that rely on
detecting an enzymatic activity associated with the human
phosphatidylserine synthase-like protein or target molecule.
[0489] In another embodiment, modulators of human
phosphatidylserine synthase-like expression are identified in a
method in which a cell is contacted with a candidate compound and
the expression of human phosphatidylserine synthase-like mRNA or
protein in the cell is determined relative to expression of human
phosphatidylserine synthase-like 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 human phosphatidylserine
synthase-like 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 human
phosphatidylserine synthase-like mRNA or protein expression. The
level of human phosphatidylserine synthase-like mRNA or protein
expression in the cells can be determined by methods described
herein for detecting human phosphatidylserine synthase-like mRNA or
protein.
[0490] In yet another aspect of the invention, the human
phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like protein ("human phosphatidylserine
synthase-like-binding proteins" or "human phosphatidylserine
synthase-like-bp") and modulate human phosphatidylserine
synthase-like activity. Such human phosphatidylserine
synthase-like-binding proteins are also likely to be involved in
the propagation of signals by the human phosphatidylserine
synthase-like proteins as, for example, upstream or downstream
elements of the human phosphatidylserine synthase-like pathway.
[0491] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0492] B. Detection Assays
[0493] 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.
[0494] 1. Chromosome Mapping
[0495] The isolated complete or partial human phosphatidylserine
synthase-like gene sequences of the invention can be used to map
their respective human phosphatidylserine synthase-like genes on a
chromosome, thereby facilitating the location of gene regions
associated with genetic disease. Computer analysis of human
phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like
sequences will yield an amplified fragment.
[0496] 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.
[0497] Other mapping strategies that can similarly be used to map a
human phosphatidylserine synthase-like 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 et
al. (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.
[0498] 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.
[0499] Another strategy to map the chromosomal location of human
phosphatidylserine synthase-like genes uses human
phosphatidylserine synthase-like polypeptides and fragments and
sequences of the present invention and antibodies specific thereto.
This mapping can be carried out by specifically detecting the
presence of a human phosphatidylserine synthase-like polypeptide in
members of a panel of somatic cell hybrids between cells of a first
species of animal from which the protein originates and cells from
a second species of animal, and then determining which somatic cell
hybrid(s) expresses the polypeptide and noting the chromosomes(s)
from the first species of animal that it contains. For examples of
this technique, see Pajunen et al. (1988) Cytogenet. Cell. Genet.
47:37-41 and Van Keuren et al. (1986) Hum. Genet. 74:34-40.
Alternatively, the presence of a human phosphatidylserine
synthase-like polypeptide in the somatic cell hybrids can be
determined by assaying an activity or property of the polypeptide,
for example, enzymatic activity, as described in Bordelon-Riser et
al. (1979) Somatic Cell Genetics 5:597-613 and Owerbach et al.
(1978) Proc. Natl. Acad. Sci. USA 75:5640-5644.
[0500] 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.
[0501] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the human phosphatidylserine synthase-like 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.
[0502] 2. Tissue Typing
[0503] The human phosphatidylserine synthase-like 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).
[0504] 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 human phosphatidylserine synthase-like sequences
of the invention can be used to prepare two PCR primers from the 5'
and 3' ends of the sequences. These primers can then be used to
amplify an individual's DNA and subsequently sequence it.
[0505] 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 human
phosphatidylserine synthase-like 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:5 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:5,
is used, a more appropriate number of primers for positive
individual identification would be 500 to 2,000.
[0506] 3. Use of Partial Human Phosphatidylserine Synthase-Like
Sequences in Forensic Biology
[0507] 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.
[0508] 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:5
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 human phosphatidylserine
synthase-like sequences or portions thereof, e.g., fragments
derived from the noncoding regions of SEQ ID NO:5 having a length
of at least 20 or 30 bases.
[0509] The human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like probes, can be used to
identify tissue by species and/or by organ type.
[0510] In a similar fashion, these reagents, e.g., human
phosphatidylserine synthase-like 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).
[0511] C. Predictive Medicine
[0512] 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.
[0513] 1. Diagnostic Assays
[0514] One aspect of the present invention relates to diagnostic
assays for detecting human phosphatidylserine synthase-like protein
and/or nucleic acid expression as well as human phosphatidylserine
synthase-like activity, in the context of a biological sample. An
exemplary method for detecting the presence or absence of human
phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like protein
or nucleic acid (e.g., mRNA, genomic DNA) that encodes human
phosphatidylserine synthase-like protein such that the presence of
human phosphatidylserine synthase-like 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.
[0515] A preferred agent for detecting human phosphatidylserine
synthase-like mRNA or genomic DNA is a labeled nucleic acid probe
capable of hybridizing to human phosphatidylserine synthase-like
mRNA or genomic DNA. The nucleic acid probe can be, for example, a
full-length human phosphatidylserine synthase-like nucleic acid,
such as the nucleic acid of SEQ ID NO:5, 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 human phosphatidylserine
synthase-like mRNA or genomic DNA. Other suitable probes for use in
the diagnostic assays of the invention are described herein.
[0516] A preferred agent for detecting human phosphatidylserine
synthase-like protein is an antibody capable of binding to human
phosphatidylserine synthase-like protein, preferably an antibody
with a detectable label. Antibodies can be polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof
(e.g., Fab or F(ab).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.
[0517] 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 human
phosphatidylserine synthase-like mRNA, protein, or genomic DNA in a
biological sample in vitro as well as in vivo. For example, in
vitro techniques for detection of human phosphatidylserine
synthase-like mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of human
phosphatidylserine synthase-like protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations,
and immunofluorescence. In vitro techniques for detection of human
phosphatidylserine synthase-like genomic DNA include Southern
hybridizations. Furthermore, in vivo techniques for detection of
human phosphatidylserine synthase-like protein include introducing
into a subject a labeled anti-human phosphatidylserine
synthase-like 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.
[0518] 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 sample containing erythrocytes, lymphocytes
and platelets which make for easily assayable cells.
[0519] The invention also encompasses kits for detecting the
presence of human phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like protein (e.g., an immunological
disorder). For example, the kit can comprise a labeled compound or
agent capable of detecting human phosphatidylserine synthase-like
protein or mRNA in a biological sample and means for determining
the amount of a human phosphatidylserine synthase-like protein in
the sample (e.g., an anti-human phosphatidylserine synthase-like
antibody or an oligonucleotide probe that binds to DNA encoding a
human phosphatidylserine synthase-like protein, e.g., SEQ ID NO:6).
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 human phosphatidylserine
synthase-like sequences if the amount of human phosphatidylserine
synthase-like protein or mRNA is above or below a normal level.
[0520] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) that binds
to human phosphatidylserine synthase-like protein; and, optionally,
(2) a second, different antibody that binds to human
phosphatidylserine synthase-like 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 a
human phosphatidylserine synthase-like nucleic acid sequence or (2)
a pair of primers useful for amplifying a human phosphatidylserine
synthase-like nucleic acid molecule.
[0521] 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 human phosphatidylserine synthase-like
proteins.
[0522] 2. Other Diagnostic Assays
[0523] 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 phosphatidylserine synthase-like
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
phosphatidylserine synthase-like nucleic acid, polypeptide, or
antibody. 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.
[0524] The method can include contacting the phosphatidylserine
synthase-like 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.
[0525] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of a phosphatidylserine synthase-like sequence of the
invention. 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. Thus, for example, the 32670 sequence
set forth in SEQ ID NO:5 and SEQ ID NO:7 encodes a
phosphatidylserine synthase-like polypeptide that is useful to
evaluate a disease or disorder wherein there is defective cell
membrane formation.
[0526] The method can be used to detect single nucleotide
polymorphisms (SNPs), as described below.
[0527] 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
a phosphatidylserine synthase-like polypeptide of the invention or
from a cell or subject in which a phosphatidylserine
synthase-like-mediated response has been elicited, e.g., by contact
of the cell with a phosphatidylserine synthase-like nucleic acid or
protein of the invention, or administration to the cell or subject
a phosphatidylserine synthase-like nucleic acid or protein of the
invention; contacting the array with one or more inquiry probes,
wherein an inquiry probe can be a nucleic acid, polypeptide, or
antibody (which is preferably other than a phosphatidylserine
synthase-like nucleic acid, polypeptide, or antibody of the
invention); 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 a phosphatidylserine synthase-like sequence of the
invention (or does not express as highly as in the case of the
phosphatidylserine synthase-like positive plurality of capture
probes) or from a cell or subject in which a phosphatidylserine
synthase-like-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 phosphatidylserine synthase-like nucleic acid,
polypeptide, or antibody of the invention), 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.
[0528] In another aspect, the invention features a method of
analyzing a phosphatidylserine synthase-like sequence of the
invention, e.g., analyzing structure, function, or relatedness to
other nucleic acid or amino acid sequences. The method includes:
providing a phosphatidylserine synthase-like nucleic acid or amino
acid sequence, e.g., the 32670 sequence set forth in SEQ ID NO:5,
SEQ ID NO:7, or a portion thereof; comparing the phosphatidylserine
synthase-like 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 the
phosphatidylserine synthase-like sequence of the invention.
[0529] The method can include evaluating the sequence identity
between a phosphatidylserine synthase-like sequence of the
invention, e.g., the 32670 sequence, and a database sequence. The
method can be performed by accessing the database at a second site,
e.g., over the internet.
[0530] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of a Phosphatidylserine synthase-like
sequence of the invention, e.g., the 15571 sequence. 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 identical in sequence with one
another (except for differences in length). The oligonucleotides
can be provided with differential labels, such that an
oligonucleotides which hybridizes to one allele provides a signal
that is distinguishable from an oligonucleotides which hybridizes
to a second allele.
[0531] 3. Prognostic Assays
[0532] 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 human
phosphatidylserine synthase-like protein, human phosphatidylserine
synthase-like nucleic acid expression, or human phosphatidylserine
synthase-like 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 human phosphatidylserine synthase-like protein,
human phosphatidylserine synthase-like nucleic acid expression, or
human phosphatidylserine synthase-like activity.
[0533] Thus, the present invention provides a method in which a
test sample is obtained from a subject, and human
phosphatidylserine synthase-like protein or nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of human
phosphatidylserine synthase-like protein or nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant human phosphatidylserine
synthase-like 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.
[0534] 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 human phosphatidylserine synthase-like
activity) to effectively treat a disease or disorder associated
with aberrant human phosphatidylserine synthase-like expression or
activity. In this manner, a test sample is obtained and human
phosphatidylserine synthase-like protein or nucleic acid is
detected. The presence of human phosphatidylserine synthase-like
protein or nucleic acid is diagnostic for a subject that can be
administered the agent to treat a disorder associated with aberrant
human phosphatidylserine synthase-like expression or activity.
[0535] The methods of the invention can also be used to detect
genetic lesions or mutations in a human phosphatidylserine
synthase-like gene. 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 a
human phosphatidylserine synthase-like-protein, or the
misexpression of the human phosphatidylserine synthase-like 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 a human phosphatidylserine
synthase-like gene; (2) an addition of one or more nucleotides to a
human phosphatidylserine synthase-like gene; (3) a substitution of
one or more nucleotides of a human phosphatidylserine synthase-like
gene; (4) a chromosomal rearrangement of a human phosphatidylserine
synthase-like gene; (5) an alteration in the level of a messenger
RNA transcript of a human phosphatidylserine synthase-like gene;
(6) an aberrant modification of a human phosphatidylserine
synthase-like gene, such as of the methylation pattern of the
genomic DNA; (7) the presence of a non-wild-type splicing pattern
of a messenger RNA transcript of a human phosphatidylserine
synthase-like gene; (8) a non-wild-type level of a human
phosphatidylserine synthase-like-protein; (9) an allelic loss of a
human phosphatidylserine synthase-like gene; and (10) an
inappropriate post-translational modification of a human
phosphatidylserine synthase-like-protein. As described herein,
there are a large number of assay techniques known in the art that
can be used for detecting lesions in a human phosphatidylserine
synthase-like gene. Any cell type or tissue, preferably peripheral
blood leukocytes, in which human phosphatidylserine synthase-like
proteins are expressed may be utilized in the prognostic assays
described herein.
[0536] 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 human phosphatidylserine synthase-like-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.
[0537] 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.
[0538] In an alternative embodiment, mutations in a human
phosphatidylserine synthase-like 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.
[0539] In other embodiments, genetic mutations in a human
phosphatidylserine synthase-like 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 human
phosphatidylserine synthase-like gene and detect mutations by
comparing the sequence of the sample human phosphatidylserine
synthase-like 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).
[0540] Other methods for detecting mutations in the human
phosphatidylserine synthase-like 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.
[0541] 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 human phosphatidylserine
synthase-like 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 a human phosphatidylserine
synthase-like sequence, e.g., a wild-type human phosphatidylserine
synthase-like 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.
[0542] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in human
phosphatidylserine synthase-like 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).
[0543] 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).
[0544] 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.
[0545] 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.
[0546] 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 a human phosphatidylserine synthase-like
gene.
[0547] 4. Pharmacogenomics
[0548] Agents, or modulators that have a stimulatory or inhibitory
effect on human phosphatidylserine synthase-like activity (e.g.,
human phosphatidylserine synthase-like gene expression) as
identified by a screening assay described herein, can be
administered to individuals to treat (prophylactically or
therapeutically) disorders associated with aberrant human
phosphatidylserine synthase-like activity. 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 human phosphatidylserine synthase-like protein,
expression of human phosphatidylserine synthase-like nucleic acid,
or mutation content of human phosphatidylserine synthase-like genes
in an individual can be determined to thereby select appropriate
agent(s) for therapeutic or prophylactic treatment of the
individual.
[0549] 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.
[0550] Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician may consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a phosphatidylserine synthase-like molecule
or phosphatidylserine synthase-like modulator of the invention as
well as tailoring the dosage and/or therapeutic regimen of
treatment with a phosphatidylserine synthase-like molecule or
phosphatidylserine synthase-like modulator of the invention.
[0551] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, an "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0552] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., a phosphatidylserine synthase-like protein
of the present invention), all common variants of that gene can be
fairly easily identified in the population and it can be determined
if having one version of the gene versus another is associated with
a particular drug response.
[0553] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a phosphatidylserine synthase-like molecule or
phosphatidylserine synthase-like modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0554] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a phosphatidylserine synthase-like
molecule or phosphatidylserine synthase-like modulator of the
invention, such as a modulator identified by one of the exemplary
screening assays described herein.
[0555] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the Phosphatidylserine
synthase-like genes of the present invention, wherein these
products may be associated with resistance of the cells to a
therapeutic agent. Specifically, the activity of the proteins
encoded by the phosphatidylserine synthase-like genes of the
present invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., hepatic
stellate cells, will become sensitive to treatment with an agent
that the unmodified target cells were resistant to.
[0556] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a phosphatidylserine synthase-like
protein can be applied in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as
described herein to increase phosphatidylserine synthase-like gene
expression, protein levels, or upregulate phosphatidylserine
synthase-like activity, can be monitored in clinical trials of
subjects exhibiting decreased phosphatidylserine synthase-like gene
expression, protein levels, or downregulated phosphatidylserine
synthase-like activity. Alternatively, the effectiveness of an
agent determined by a screening assay to decrease
phosphatidylserine synthase-like gene expression, protein levels,
or downregulate phosphatidylserine synthase-like activity, can be
monitored in clinical trials of subjects exhibiting increased
phosphatidylserine synthase-like gene expression, protein levels,
or upregulated phosphatidylserine synthase-like activity. In such
clinical trials, the expression or activity of a phosphatidylserine
synthase-like gene, and preferably, other genes that have been
implicated in, for example, a phosphatidylserine
synthase-like-associated disorder can be used as a "read out" or
markers of the phenotype of a particular cell.
[0557] 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.
[0558] Thus, the activity of human phosphatidylserine synthase-like
protein, expression of human phosphatidylserine synthase-like
nucleic acid, or mutation content of human phosphatidylserine
synthase-like 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 a human phosphatidylserine
synthase-like modulator, such as a modulator identified by one of
the exemplary screening assays described herein.
[0559] 5. Monitoring of Effects During Clinical Trials
[0560] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of human phosphatidylserine
synthase-like genes 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 human phosphatidylserine synthase-like gene
expression, protein levels, or protein activity, can be monitored
in clinical trials of subjects exhibiting decreased or increased
human phosphatidylserine synthase-like gene expression, protein
levels, or protein activity.
[0561] 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 human phosphatidylserine
synthase-like 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 human phosphatidylserine
synthase-like 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 human phosphatidylserine
synthase-like 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.
[0562] 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 a human phosphatidylserine synthase-like
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 human
phosphatidylserine synthase-like protein, mRNA, or genomic DNA in
the postadministration samples; (5) comparing the level of
expression or activity of the human phosphatidylserine
synthase-like protein, mRNA, or genomic DNA in the
preadministration sample with the human phosphatidylserine
synthase-like 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 a human
phosphatidylserine synthase-like protein.
[0563] C. Methods of Treatment
[0564] 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 human phosphatidylserine synthase-like expression or
activity. Additionally, the compositions of the invention find use
in the treatment of disorders described herein. Thus, therapies for
disorders associated with CCC are encompassed herein.
[0565] 1. Prophylactic Methods
[0566] In one aspect, the invention provides a method for
preventing in a subject a disease or condition associated with an
aberrant human phosphatidylserine synthase-like expression or
activity by administering to the subject an agent that modulates
human phosphatidylserine synthase-like expression or at least one
human phosphatidylserine synthase-like gene activity. Subjects at
risk for a disease that is caused, or contributed to, by aberrant
human phosphatidylserine synthase-like 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 human phosphatidylserine synthase-like
aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
human phosphatidylserine synthase-like aberrancy, for example, a
human phosphatidylserine synthase-like agonist or human
phosphatidylserine synthase-like antagonist agent can be used for
treating the subject. The appropriate agent can be determined based
on screening assays described herein.
[0567] 2. Therapeutic Methods
[0568] Another aspect of the invention pertains to methods of
modulating human phosphatidylserine synthase-like 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 human phosphatidylserine
synthase-like protein activity associated with the cell. An agent
that modulates human phosphatidylserine synthase-like protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring cognate ligand of a human
phosphatidylserine synthase-like protein, a peptide, a human
phosphatidylserine synthase-like peptidomimetic, or other small
molecule. In one embodiment, the agent stimulates one or more of
the biological activities of human phosphatidylserine synthase-like
protein. Examples of such stimulatory agents include active human
phosphatidylserine synthase-like protein and a nucleic acid
molecule encoding a human phosphatidylserine synthase-like protein
that has been introduced into the cell. In another embodiment, the
agent inhibits one or more of the biological activities of human
phosphatidylserine synthase-like protein. Examples of such
inhibitory agents include antisense human phosphatidylserine
synthase-like nucleic acid molecules and anti-human
phosphatidylserine synthase-like antibodies.
[0569] 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 a human phosphatidylserine synthase-like 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) human phosphatidylserine
synthase-like expression or activity. In another embodiment, the
method involves administering a human phosphatidylserine
synthase-like protein or nucleic acid molecule as therapy to
compensate for reduced or aberrant human phosphatidylserine
synthase-like expression or activity.
[0570] Stimulation of human phosphatidylserine synthase-like
activity is desirable in situations in which a human
phosphatidylserine synthase-like protein is abnormally
downregulated and/or in which increased human phosphatidylserine
synthase-like activity is likely to have a beneficial effect.
Conversely, inhibition of human phosphatidylserine synthase-like
activity is desirable in situations in which human
phosphatidylserine synthase-like activity is abnormally upregulated
and/or in which decreased human phosphatidylserine synthase-like
activity is likely to have a beneficial effect.
[0571] This invention is further illustrated by the following
examples, which should not be construed as limiting.
EXAMPLES
Example 1
Identification and Characterization of Human 32670 cDNAs
[0572] The human 32670 sequence (FIGS. 14A-B; SEQ ID NO:5), which
is approximately 1852 nucleotides long including untranslated
regions, contains a predicted methionine-initiated coding sequence
of about 1464 nucleotides (nucleotides 14-1477 of SEQ ID NO:5; SEQ
ID NO:7). The coding sequence encodes a 487 amino acid protein (SEQ
ID NO:6).
Example 2
Tissue Distribution of 32670 mRNA
[0573] 32670 expression was determined by the PCR in cDNA libraries
generated from various human tissues and cell types. 32670
expression was detectable in cDNA libraries generated from the
following tissues: microvascular endothelial cells, umbilical vein
endothelial cells, U937 cells, CaCo cells, HeLa cells, fetal brain,
bronchial epithelium, astrocytes, prostate epithelium, primary
osteoblasts, keratinocytes, melanocytes, coronary smooth muscle
cells, cerebellum, pituitary, aortic endothelial cells, fetal
kidney, fetal liver, mengial, bone marrow, fetal thymus, fetal
heart, mammary gland, tissue from a subject with congestive heart
failure, prostate smooth muscle cells, osteoblasts treated with LPS
for 6 hours, fetal heart, tissue from a subject with Burkin's
lymphoma, mammary epithelium, umbilical smooth muscle, bronchial
smooth muscle, fetal spleen, esophagus, fetal liver, fetal skin,
fetal adrenal gland, lung carcinoma tissue, A549 cells, fetal
testes, pulmonary artery smooth muscle, erythroleukemia cells,
embryonic keratinocytes, tongue squamous cell carcinoma, fetal
hypothalamus, CD3 treated T cells, HPKII cells, testes, H160 cells,
placenta, skeletal muscle, kidney, HPK cells, uterus, 9 week fetus,
salivary gland, testes, K563 cells, lung, thymus, skeletal muscle,
prostate, Hep-G2 insulinoma cells, normal breast epithelia, normal
ovarian epithelia, normal megakaryocytes, Th-2 induced T cell,
fetal dorsal spinal cord, colon to live metastasis, colon
carcinoma, lung squamous cell carcinoma, d8 dendritic cells, skin,
ovarian ascites, IBD colon, dorsal root ganglia, brain subcortical
white matter, prostate tumor xenograft cell cline K10, prostate
cancer to liver metastasis, umbilical vein endothelial cells grown
under hypoxic conditions, melanoma G361 cell line, lumbosacaral
spinal chord, and adult bone marrow CD34 positive cells.
[0574] 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 32670 cDNA (SEQ ID NO:5 or
SEQ ID NO:7) 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 3
Recombinant Expression of 32670 in Bacterial Cells
[0575] In this example, 32670 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
32670 is fused to GST and this fusion polypeptide is expressed in
E. coli, e.g., strain PEB199. Expression of the GST-32670 fusion
protein in PEB199 is induced with IPTG. The recombinant fusion
polypeptide is purified from crude bacterial lysates of the induced
PEB199 strain by affinity chromatography on glutathione beads.
Using polyacrylamide gel electrophoretic analysis of the
polypeptide purified from the bacterial lysates, the molecular
weight of the resultant fusion polypeptide is determined.
Example 4
Expression of Recombinant 32670 Protein in COS Cells
[0576] To express the 32670 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 32670 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.
[0577] To construct the plasmid, the 32670 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 32670 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 32670 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 32670 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.
[0578] COS cells are subsequently transfected with the
32670-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 32670 polypeptide is detected by radiolabelling
(.sup.35S-methionine or 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 35S-methionine (or 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.
[0579] Alternatively, DNA containing the 32670 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 32670 polypeptide is detected by radiolabelling
and immunoprecipitation using a 32670 specific monoclonal
antibody.
[0580] 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.
[0581] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Chapter 3
5698, A DNA Fragmentation Factor-Like Molecule and Uses Thereof
BACKGROUND OF THE INVENTION
[0582] Apoptosis is fundamentally important in a variety of
physiological and pathological processes. Apoptotic cells undergo
an orchestrated cascade of events characterized by distinct
morphological changes including membrane blebbing, cytoplasmic and
nuclear degredation, chromatin aggregation, and formation of
apoptotic bodies. A key molecular event in the process of apoptosis
is the activation of the caspase cascade. Caspases are a family of
serine proteases identified in mammalian cells. Caspase activation
leads to cleavage of target protein and execution of the apoptotic
program. Apoptotic signals, including growth factor and interleukin
deprivation, activation of Fas, ionizing radiation, and a series of
chemicals acting as upstream signals, can convert the precursors of
caspases into the protease active enzymes.
[0583] One of the downstream substrates of caspase is a subunit of
the DNA Fragmentation Factor (DFF) complex. DFF is a heterodimeric
protein complex composed of DFF45 and DFF40 subunits. DFF45 has
been found to be the substrate of caspase-3 and DFF40 has also been
cloned and found to be a DNA fragmentation nuclease. Studies have
shown that DFF45 can mediate the correct folding of DFF40 and
remains associated with DFF40 to prevent DFF40 from being activated
until a specific signal (the activation of caspase-3) is received.
DFF45 therefore acts as a specific molecular chaperon and appears
to provide a double safety control to prevent unwanted activation
of DFF40. Following cleavage, the DFF45 dissociates from DFF40. The
active component of DFF then triggers both DNA fragmentation and
chromatin condensation during apoptosis (Gu et al. (1999) The
Journal of Biological Chemistry 274:20759-20762).
[0584] DFF homologs have also been identified in the mouse. The
mouse DFF is composed of three molecules: one caspase-activated
DNAse (CAD) and two forms of CAD inhibitors (ICAD-L and ICAD-S).
Mouse CAD and ICAD-L are apparently the counterpart of huma DFF40
(CPAN) and DFF45, respectively, whereas the human counterpart of
mouse ICAD-S has not been identified.
[0585] In addition, cell death-inducing DFF45-like effector A and B
(CIDE-A and CIDE-B) encode highly related proteins with homology to
the N-terminal region of DFF45. CIDE-A and CIDE-B activate
apoptosis and appear to function as positive effectors of the
apoptotic pathway. Inohara et al. have demonstrated that CIDE-A and
CIDE-B induce DNA fragmentation as well as other morphological
features of apoptosis including nuclear condensation and membrane
blebbing (Inohara et al. (1998) EMBO J. 17:2526-2533).
[0586] The proteins of the DFF complex influence DNA fragmentation
and ultimately apoptosis and therefore play a role in various
biological and pathological processes. For example, during normal
CNS development, a significant proportion of neurons die by
apoptosis to permit the matching of cells with their targets
(Oppenheim et al. (1991) Annu. Rev. Neuroscience 14: 453-501).
Apoptotic events also play an important role in specific
pathological conditions including Alzheimer's and Huntington's
disease (Portera-Calliau et al. (1995) J. Neurosci 15:3775-3787 and
Samle et al. (1995) Exp. Neruo. 133:225-230), cerebral ischemia
(MacManus et al. (1995) J. Cereb. Blood Flow Metab. 15:728-737),
and HIV encephalitis (Petito et al. (1995) Am. J. Pathol.
146:1121-1130).
[0587] Furthermore, recent studies have demonstrated that apoptotic
cell death occurs after traumatic spinal cord injury and following
traumatic brain injury (Li et al. (1996) J. Neruopathol. Exp.
Neruol 55:280-289). The apoptotic event is characterized by the
activation of caspase-3 in the injured rat cortex and hippocampus.
Regional and temporal changes in DFF-like proteins are observed
following these trauma events. Zhang et al. observed that
DFF45-like proteins labeled with the anti-human DFF45 antibody
significantly decrease in the cortex after brain trauma. These
changes in the DFF45-like proteins are suggestive of an early
signal of DNA fragmentation and that the DFF proteins are playing a
role during apoptosis following a traumatic brain injury (Zhang et
al. (1999) Journal of Neurochemistry 73:1650-1659).
[0588] Evidence indicates DFF complex plays an important role in
cellular apoptosis. Accordingly, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown DFF-like polypeptides. The present invention advances the
state of the art by providing previously unidentified DNA
fragmentation factor-like nucleic acid and polypeptides.
SUMMARY OF THE INVENTION
[0589] Isolated nucleic acid molecules corresponding to DFF-like
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 sequences shown in SEQ ID NO:11. Further provided are DFF-like
polypeptides having an amino acid sequence encoded by a nucleic
acid molecule described herein.
[0590] 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.
[0591] The DFF-like molecules of the present invention are useful
for modulating apoptotic events, including DNA fragmentation. The
molecules are useful for the diagnosis and treatment of disorders
associated with dysregulated apoptosis. Such disorders include
cancers, autoimmune disorders, neurodegenerative diseases, ischemic
injuries, and virus induced lymphocyte depletion. Additionally, the
molecules of the invention are useful as modulating agents in a
variety of cellular processes including DNA fragmentation,
intracellular signaling, membrane blebbing, cytoplasmic and nuclear
degradation, chromatin aggregation, and formation of apoptotic
bodies. Accordingly, in one aspect, this invention provides
isolated nucleic acid molecules encoding DFF-like proteins or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection of DFF-like-encoding nucleic acids.
[0592] Another aspect of this invention features isolated or
recombinant DFF-like proteins and polypeptides. Preferred DFF-like
proteins and polypeptides possess at least one biological activity
possessed by naturally occurring DFF-like proteins.
[0593] 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.
[0594] Antibodies and antibody fragments that selectively bind the
DFF-like polypeptides and fragments are provided. Such antibodies
are useful in detecting the DFF-like polypeptides as well as in
regulating apoptotic processes.
[0595] In another aspect, the present invention provides a method
for detecting the presence of DFF-like activity or expression in a
biological sample by contacting the biological sample with an agent
capable of detecting an indicator of DFF-like activity such that
the presence of DFF-like activity is detected in the biological
sample.
[0596] In yet another aspect, the invention provides a method for
modulating DFF-like activity comprising contacting a cell with an
agent that modulates (inhibits or stimulates) DFF-like activity or
expression such that DFF-like activity or expression in the cell is
modulated. In one embodiment, the agent is an antibody that
specifically binds to DFF-like protein. In another embodiment, the
agent modulates expression of DFF-like protein by modulating
transcription of a DFF-like gene, splicing of a DFF-like mRNA, or
translation of a DFF-like 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 DFF-like mRNA or the
DFF-like gene.
[0597] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
DFF-like protein activity or nucleic acid expression by
administering an agent that is a DFF-like modulator to the subject.
In one embodiment, the DFF-like modulator is a DFF-like protein. In
another embodiment, the DFF-like modulator is a DFF-like nucleic
acid molecule. In other embodiments, the DFF-like modulator is a
peptide, peptidomimetic, or other small molecule.
[0598] 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 a DFF-like protein; (2)
misregulation of a gene encoding a DFF-like protein; and (3)
aberrant post-translational modification of a DFF-like protein,
wherein a wild-type form of the gene encodes a protein with a
DFF-like activity.
[0599] In another aspect, the invention provides a method for
identifying a compound that binds to or modulates the activity of a
DFF-like protein. In general, such methods entail measuring a
biological activity of a DFF-like protein in the presence and
absence of a test compound and identifying those compounds that
alter the activity of the DFF-like protein.
[0600] The invention also features methods for identifying a
compound that modulates the expression of DFF-like genes by
measuring the expression of the DFF-like sequences in the presence
and absence of the compound.
[0601] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0602] 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.
[0603] 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.
[0604] The present invention provides DFF-like molecules. By
"DFF-like molecules" is intended a novel human sequence referred to
as 5698, and variants and fragments thereof. These full-length gene
sequences or fragments thereof are referred to as "DFF-like"
sequences, indicating they share sequence similarity with DFF
genes. Isolated nucleic acid molecules comprising nucleotide
sequences encoding the 5698 polypeptide whose amino acid sequence
is given in SEQ ID NO:11, or a variant or fragment thereof, are
provided. A nucleotide sequence encoding the 5698 polypeptide is
set forth in SEQ ID NO:10 or 12. The sequences are members of the
DNA Fragmentation Factor family.
[0605] The disclosed invention relates to methods and compositions
for the modulation, diagnosis, and treatment of disorders
associated with dysregulated apoptosis. By "disregulted apoptosis"
is intended an alteration in the apoptotic process that results in
either an inappropirately low or high rate of apoptosis. Disorders
associated with an inappropriately low rate of apoptosis may
prolong survival of abnormal cells. These accumulated cells can
give rise to cancers, especially those carcinomas with p53
mutations, or homo-dependent tumors, such as breast, prostate, or
ovarian cancers. Autoimmune disorders also can arise if, for
example, autoreactive lymphocytes are not removed following an
immune response. The molecules are also useful for the diagnosis
and treatment of disorders associated with increased apoptosis and
excessive cell death. These disorders are characterized by a marked
loss of normal or protective cells and include: neurodegenerative
diseases, manifested by loss of specific sets of neurons, such as
in the spinal muscular atrophies; ischemic injuries such as in
myocardial infarction and stroke; and, virus induced lymphocyte
depletion, such as in acquired immune deficiency syndrome.
[0606] The molecules are also useful for the diagnosis and
treatment of disorders in tissues in which the transcript is
expressed (see Example 1 and FIGS. 20A-B). 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.
[0607] 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
(hypernephroma, adenocarcinoma of kidney), which includes
urothelial carcinomas of renal pelvis.
[0608] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0609] 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.
[0610] The DFF-like sequences of the present invention find use in
modulating a apoptosis. By "modulating" is intended the
upregulating or downregulating of a response. That is, the
compositions of the invention affect the targeted activity in
either a positive or negative fashion. The activation of apoptosis
is manifested by changes including membrane blebbing, DNA
fragmentation, cytoplasmic and nuclear degredation, chromatin
aggregation, formation of apoptotic bodies, and cell death.
[0611] Proteins and/or antibodies of the invention are also useful
in modulating the apoptotic process.
[0612] The DFF-like gene, clone 5698 was identified in a human
primary osteoblast cDNA library. Clone 5698 encodes an mRNA
transcript having the corresponding cDNA set forth in SEQ ID NO:10.
This transcript has a nucleotide open reading frame (nucleotides
169-828 of SEQ ID NO:10), which encodes a 219 amino acid protein
(SEQ ID NO:11). Prosite program analysis was used to predict
various sites within the 5698 protein. An N-glycosylation site was
predicted at aa 18-21. Protein kinase C phosphorylation sites were
predicted at aa 46-48 and 199-201. Casein kinase II phosphorylation
sites were predicted at aa 55-58 and 82-85. N-myristoylation sites
were predicted at aa 50-55 and 195-200. An amidation site was
predicted at aa 23-26. A leucine zipper pattern was predicted at aa
179-200. The DFF-like protein possesses a CAD domain, from aa
36-108 as predicted by HMMer, Version 2. 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
http://www.psc.edu/general/software/packages/pfam/pfam.html.
[0613] As used herein, the term "CAD domain" includes an amino acid
sequence of about 50-72 amino acid residues in length and having a
bit score for the alignment of the sequence to the CAD domain (HMM)
of at least 8. Preferably, an CAD domain includes at least about
1-72 amino acids, about 20-72 amino acid residues, or about 40-72
amino acids and has a bit score for the alignment of the sequence
to the CAD domain (HMM) of at least 16 or greater
(http://smart.embl-heidelberg.de/smart/selective.cgi?domains=cad&taxon_se-
lect=ALL&taxon_text=). An alignment of the CAD domain (amino
acids 36 to 108 of SEQ ID NO:11) of the human DFF-like sequence of
the invention with a consensus amino acid sequence derived from a
hidden Markov model is depicted in FIG. 17.
[0614] In a preferred embodiment a DFF-like polypeptide or protein
has a "CAD 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 "CAD domain," e.g., the CAD domain
of a human DFF-like (e.g., amino acid residues 36-108 of SEQ ID
NO:11).
[0615] To identify the presence of an "CAD" domain in a DFF-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
(http://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.
[0616] The 5698 protein displays similarity to the Mus musculus
cell death activator CIDE-B (SP Accession No. 3114594; Genbank
Accession No. AAC34986; SEQ ID NO:14) and with the Homo sapiens
cell death activator CIDE-A (SP Accession No. 3114596; Genbank
Accession No. AAC34987; SEQ ID NO:15). The sequence alignment was
generated using the Clustal method. The 5698 protein shares
approximately 83% identity with the murine CIDE-B and approximately
40% identity with the human CIDE-A amino acid sequence as
determined by pairwise alignment.
[0617] The 5698 protein displays 37% identity from aa 36-209 to a
ProDom consensus sequence found in murine FSP27 (Genbank Accession
No. P56198) and human CIDE-A (Genbank Accession No. 060543). FSP27
is an adipocyte specific protein that belongs to the DFF-45/ICAD
family. FSP27 is associated with terminal differentiation of fat
cells and its expression is regulated by the tumor necrosis
pathway. See, for example, Danesch et al. (1992) J. Biol. Chem
267:7185-7193 and Williams et al. (1992) Mol Endocrinol
6:1135-1141. CIDE-A has homology to the 45 kDa subunit of the DNA
fragmentation factor. See, for example, Inohara et al. (1998) Embo
J. 17:2526-2533. The 5698 protein also displays 46% identity to a
ProDom consensus sequence found in the hypothetical protein F-121
of human adenovirus type 2.
[0618] The DFF-like sequences of the invention are members of a
family of molecules (the "DNA fragmentation factor-like") 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.
[0619] Preferred DFF-like polypeptides of the present invention
have an amino acid sequence sufficiently identical to the amino
acid sequence of SEQ ID NO:11. 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.
[0620] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e., percent identity=number of identical
positions/total number 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.
[0621] The 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
http://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
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. 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.
[0622] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of Karlin and
Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, 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. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12, to obtain nucleotide sequences homologous
to DFF-like 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 DFF-like
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. Alternatively,
PSI-Blast can be used to perform an iterated search that detects
distant relationships between molecules. See Altschul et al. (1997)
supra. 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 http://www.ncbi.nlm.nih.gov. Another
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated
into the ALIGN program (version 2.0), which is part of the GCG
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.
[0623] Accordingly, another embodiment of the invention features
isolated DFF-like proteins and polypeptides having a DFF-like
protein activity. As used interchangeably herein, a "DFF-like
protein activity", "biological activity of a DFF-like protein", or
"functional activity of a DFF-like protein" refers to an activity
exerted by a DFF-like protein, polypeptide, or nucleic acid
molecule on a DFF-like responsive cell as determined in vivo, or in
vitro, according to standard assay techniques. A DFF-like 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 signaling activity mediated by interaction of
the DFF-like protein with a second protein. In a preferred
embodiment, a DFF-like activity includes at least one or more of
the following activities: (1) modulating (stimulating and/or
enhancing or inhibiting) apoptotic events, including DNA
fragmentation, membrane blebbing, cytoplasmic and nuclear
degredation, chromatin aggregation, and formtion of apoptotic
bodies (2) modulating the programmed destruction of cells during
embryogenesis including implantation, organogenesis, developmental
involution and metamorphosis (3) modulating hormone-dependent
involution in the adult, such as endometrial cell breakdown during
the menstrual cycle, ovarian follicular atresia in the menopause,
the regression of the lactating breast after weaning, and prostate
atrophy after castration (4) modulating cell deletion in
proliferating cell populations, such as intestinal crypt epithelia
(5) modulating cell death in tumors (6) modulating the death of
neutrophils during an acute inflammatory response (7) modulating
the death of immune cells, both B and T lymphocytes after cytokine
depletion (8) modulating the cell death induced by cytotoxic
T-cells such as in cellular immune rejection and graft-verses-host
diseases and (9) modulating atrophy in parenchymal organs after
duct obstruction, such as occurs in the pancreas, parotid gland,
and kidney.
[0624] An "isolated" or "purified" DFF-like 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
DFF-like 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. A DFF-like protein
that is substantially free of cellular material includes
preparations of DFF-like protein having less than about 30%, 20%,
10%, or 5% (by dry weight) of non-DFF-like protein (also referred
to herein as a "contaminating protein"). When the DFF-like 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 DFF-like
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-DFF-like chemicals.
[0625] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[0626] One aspect of the invention pertains to isolated nucleic
acid molecules comprising nucleotide sequences encoding DFF-like
proteins and polypeptides or biologically active portions thereof,
as well as nucleic acid molecules sufficient for use as
hybridization probes to identify DFF-like-encoding nucleic acids
(e.g., DFF-like mRNA) and fragments for use as PCR primers for the
amplification or mutation of DFF-like 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.
[0627] Nucleotide sequences encoding the DFF-like proteins of the
present invention include sequences set forth in SEQ ID NO:10, 12
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 DFF-like protein encoded by these nucleotide
sequences is set forth in SEQ ID NO:11. The invention also
encompasses nucleic acid molecules comprising nucleotide sequences
encoding partial-length DFF-like proteins, including the sequence
set forth in SEQ ID NO:10 or 12, and complements thereof.
[0628] Nucleic acid molecules that are fragments of these DFF-like
nucleotide sequences are also encompassed by the present invention.
By "fragment" is intended a portion of the nucleotide sequence
encoding a DFF-like protein. A fragment of a DFF-like nucleotide
sequence may encode a biologically active portion of a DFF-like
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 a DFF-like protein can be prepared
by isolating a portion of one of the DFF-like nucleotide sequences
of the invention, expressing the encoded portion of the DFF-like
protein (e.g., by recombinant expression in vitro), and assessing
the activity of the encoded portion of the DFF-like protein.
Nucleic acid molecules that are fragments of a DFF-like 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 DFF-like nucleotide
sequence disclosed herein (for example, 1284 nucleotides for SEQ ID
NO:10) depending upon the intended use. Alternatively, a nucleic
acid molecules that is a fragment of a DFF-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, 1000-1100, 1100-1200, 1200-1284 of SEQ
ID NO:10 or 12.
[0629] 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.
[0630] A fragment of a DFF-like nucleotide sequence that encodes a
biologically active portion of a DFF-like protein of the invention
will encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, or 200
contiguous amino acids, or up to the total number of amino acids
present in a full-length DFF-like protein of the invention (for
example, 219 amino acids for SEQ ID NO:11. Fragments of a DFF-like
nucleotide sequence that are useful as hybridization probes for PCR
primers generally need not encode a biologically active portion of
a DFF-like protein.
[0631] Nucleic acid molecules that are variants of the DFF-like
nucleotide sequences disclosed herein are also encompassed by the
present invention. "Variants" of the DFF-like nucleotide sequences
include those sequences that encode the DFF-like 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 DFF-like 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 DFF-like nucleotide sequence will
encode a DFF-like 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 a DFF-like protein disclosed herein.
[0632] In addition to the DFF-like nucleotide sequences shown in
SEQ ID NOS:1 and 3, it will be appreciated by those skilled in the
art that DNA sequence polymorphisms that lead to changes in the
amino acid sequences of DFF-like proteins may exist within a
population (e.g., the human population). Such genetic polymorphism
in a DFF-like 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 a DFF-like
protein, preferably a mammalian DFF-like protein. As used herein,
the phrase "allelic variant" refers to a nucleotide sequence that
occurs at a DFF-like 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 DFF-like
gene. Any and all such nucleotide variations and resulting amino
acid polymorphisms or variations in a DFF-like sequence that are
the result of natural allelic variation and that do not alter the
functional activity of DFF-like proteins are intended to be within
the scope of the invention.
[0633] Moreover, nucleic acid molecules encoding DFF-like proteins
from other species (DFF-like homologues), which have a nucleotide
sequence differing from that of the DFF-like 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 DFF-like cDNA of the invention
can be isolated based on their identity to the human DFF-like
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.
[0634] In addition to naturally-occurring allelic variants of the
DFF-like 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
DFF-like proteins, without altering the biological activity of the
DFF-like proteins. Thus, an isolated nucleic acid molecule encoding
a DFF-like protein having a sequence that differs from that of SEQ
ID NO:11 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.
[0635] 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 a DFF-like protein (e.g., the sequence of SEQ ID NO:11)
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 growth factor and cytokine receptor signature 2 sequence and
the U-PAR/Ly-6 domain sequence of SEQ ID NO:11, where such residues
are essential for protein activity.
[0636] Alternatively, variant DFF-like nucleotide sequences can be
made by introducing mutations randomly along all or part of a
DFF-like coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for DFF-like 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.
[0637] Thus the nucleotide sequences of the invention include the
sequences disclosed herein as well as fragments and variants
thereof. The DFF-like nucleotide sequences of the invention, and
fragments and variants thereof, can be used as probes and/or
primers to identify and/or clone DFF-like homologues in other cell
types, e.g., from other tissues, as well as DFF-like 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 a DFF-like protein, such as by
measuring levels of a DFF-like-encoding nucleic acid in a sample of
cells from a subject, e.g., detecting DFF-like mRNA levels or
determining whether a genomic DFF-like gene has been mutated or
deleted.
[0638] 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). DFF-like nucleotide sequences
isolated based on their sequence identity to the DFF-like
nucleotide sequences set forth herein or to fragments and variants
thereof are encompassed by the present invention.
[0639] In a hybridization method, all or part of a known DFF-like
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 DFF-like nucleotide sequence disclosed herein.
Degenerate primers designed on the basis of conserved nucleotides
or amino acid residues in a known DFF-like 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 a DFF-like 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.
[0640] For example, in one embodiment, a previously unidentified
DFF-like nucleic acid molecule hybridizes under stringent
conditions to a probe that is a nucleic acid molecule comprising
one of the DFF-like nucleotide sequences of the invention or a
fragment thereof. In another embodiment, the previously unknown
DFF-like 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 DFF-like nucleotide sequences disclosed herein or a
fragment thereof.
[0641] Accordingly, in another embodiment, an isolated previously
unknown DFF-like 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:10, 12, or a complement, fragment, or variant thereof.
[0642] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences 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, 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 that hybridizes under stringent
conditions to a DFF-like 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).
[0643] Thus, in addition to the DFF-like 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 DFF-like nucleotide sequences disclosed herein or
variants and fragments thereof.
[0644] 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 DFF-like 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 a DFF-like protein. The noncoding
regions are the 5' and 3' sequences that flank the coding region
and are not translated into amino acids.
[0645] Given the coding-strand sequence encoding a DFF-like protein
disclosed herein (e.g., SEQ ID NO:10 or 12), antisense nucleic
acids of the invention can be designed according to the rules of
Watson and Crick base pairing. The antisense nucleic acid molecule
can be complementary to the entire coding region of DFF-like mRNA,
but more preferably is an oligonucleotide that is antisense to only
a portion of the coding or noncoding region of DFF-like mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of DFF-like mRNA. An
anti sense 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.
[0646] 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).
[0647] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a DFF-like 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.
[0648] An antisense nucleic acid molecule of the invention can be
an a-anomeric nucleic acid molecule. An a-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).
[0649] 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 DFF-like mRNA
transcripts to thereby inhibit translation of DFF-like mRNA. A
ribozyme having specificity for a DFF-like-encoding nucleic acid
can be designed based upon the nucleotide sequence of a DFF-like
cDNA disclosed herein (e.g., SEQ ID NO:10 or 12). 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, DFF-like 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.
[0650] The invention also encompasses nucleic acid molecules that
form triple helical structures. For example, DFF-like gene
expression can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the DFF-like protein
(e.g., the DFF-like promoter and/or enhancers) to form triple
helical structures that prevent transcription of the DFF-like 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.
[0651] 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.
[0652] PNAs of a DFF-like 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).
[0653] In another embodiment, PNAs of a DFF-like 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 DFF-Like Proteins and Anti-DFF Antibodies
[0654] DFF-like proteins are also encompassed within the present
invention. By "DFF-like protein" is intended a protein having the
amino acid sequence set forth in SEQ ID NO:11, as well as
fragments, biologically active portions, and variants thereof.
[0655] "Fragments" or "biologically active portions" include
polypeptide fragments suitable for use as immunogens to raise
anti-DFF-like antibodies. Fragments include peptides comprising
amino acid sequences sufficiently identical to or derived from the
amino acid sequence of a DFF-like protein, or partial-length
protein, of the invention and exhibiting at least one activity of a
DFF-like protein, but which include fewer amino acids than the
full-length (SEQ ID NO:11) DFF-like protein disclosed herein.
Typically, biologically active portions comprise a domain or motif
with at least one activity of the DFF-like protein. A biologically
active portion of a DFF-like protein can be a polypeptide which is,
for example, 10, 25, 50, 100 or more amino acids in length.
Alternatively, a fragment of a polypeptide of the present invention
comprises an amino acid sequence consisting of amino acid residues
1-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160,
160-180, 180-200, 200-219 of SEQ ID NO:11. Such biologically active
portions can be prepared by recombinant techniques and evaluated
for one or more of the functional activities of a native DFF-like
protein. As used here, a fragment comprises at least 5 contiguous
amino acids of SEQ ID NO:11. The invention encompasses other
fragments, however, such as any fragment in the protein greater
than 6, 7, 8, or 9 amino acids.
[0656] 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:11. Variants also include polypeptides
encoded by a nucleic acid molecule that hybridizes to the nucleic
acid molecule of SEQ ID NOS:1 or 3, 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:11. 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 generally retain the
functional activity of the DFF-like proteins of the invention.
Variants include polypeptides that differ in amino acid sequence
due to natural allelic variation or mutagenesis.
[0657] The invention also provides DFF-like chimeric or fusion
proteins. As used herein, a DFF-like "chimeric protein" or "fusion
protein" comprises a DFF-like polypeptide operably linked to a
non-DFF-like polypeptide. A "DFF-like polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a DFF
protein, whereas a "non-DFF-like polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein that is not substantially identical to the DFF-like
protein, e.g., a protein that is different from the DFF-like
protein and which is derived from the same or a different organism.
Within a DFF-like fusion protein, the DFF-like polypeptide can
correspond to all or a portion of a DFF-like protein, preferably at
least one biologically active portion of a DFF-like protein. Within
the fusion protein, the term "operably linked" is intended to
indicate that the DFF-like polypeptide and the non-DFF-like
polypeptide are fused in-frame to each other. The non-DFF-like
polypeptide can be fused to the N-terminus or C-terminus of the
DFF-like polypeptide.
[0658] One useful fusion protein is a GST-DFF-like fusion protein
in which the DFF-like sequences are fused to the C-terminus of the
GST sequences. Such fusion proteins can facilitate the purification
of recombinant DFF-like proteins.
[0659] In yet another embodiment, the fusion protein is a
DFF-like-immunoglobulin fusion protein in which all or part of a
DFF-like protein is fused to sequences derived from a member of the
immunoglobulin protein family. The DFF-like-immunoglobulin fusion
proteins of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a DFF-like ligand and a DFF-like protein on the
surface of a cell, thereby suppressing DFF-like-mediated signal
transduction in vivo. The DFF-like-immunoglobulin fusion proteins
can be used to affect the bioavailability of a DFF-like cognate
ligand. Inhibition of the DFF-like ligand/DFF-like interaction may
be useful therapeutically, both for treating proliferative and
differentiative disorders and for modulating (e.g., promoting or
inhibiting) cell survival. Moreover, the DFF-like-immunoglobulin
fusion proteins of the invention can be used as immunogens to
produce anti-DFF-like antibodies in a subject, to purify DFF-like
ligands, and in screening assays to identify molecules that inhibit
the interaction of a DFF-like protein with a DFF-like ligand.
[0660] Preferably, a DFF-like 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,
a DFF-like-encoding nucleic acid can be cloned into a commercially
available expression vector such that it is linked in-frame to an
existing fusion moiety.
[0661] Variants of the DFF-like proteins can function as either
DFF-like agonists (mimetics) or as DFF-like antagonists. Variants
of the DFF-like protein can be generated by mutagenesis, e.g.,
discrete point mutation or truncation of the DFF-like protein. An
agonist of the DFF-like protein can retain substantially the same,
or a subset, of the biological activities of the naturally
occurring form of the DFF-like protein. An antagonist of the
DFF-like protein can inhibit one or more of the activities of the
naturally occurring form of the DFF-like protein by, for example,
competitively binding to a downstream or upstream member of a
cellular signaling cascade that includes the DFF-like 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 DFF-like proteins.
[0662] Variants of a DFF-like protein that function as either
DFF-like agonists or as DFF-like antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of a DFF-like protein for DFF-like protein agonist or
antagonist activity. In one embodiment, a variegated library of
DFF-like variants is generated by combinatorial mutagenesis at the
nucleic acid level and is encoded by a variegated gene library. A
variegated library of DFF-like variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential DFF-like sequences is expressible as individual
polypeptides, or alternatively, as a set of larger fusion proteins
(e.g., for phage display) containing the set of DFF-like sequences
therein. There are a variety of methods that can be used to produce
libraries of potential DFF-like 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 DFF-like 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).
[0663] In addition, libraries of fragments of a DFF-like protein
coding sequence can be used to generate a variegated population of
DFF-like fragments for screening and subsequent selection of
variants of a DFF-like protein. In one embodiment, a library of
coding sequence fragments can be generated by treating a
double-stranded PCR fragment of a DFF-like 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 DFF-like
protein.
[0664] 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 DFF-like 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 DFF-like variants (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[0665] An isolated DFF-like polypeptide of the invention can be
used as an immunogen to generate antibodies that bind DFF-like
proteins using standard techniques for polyclonal and monoclonal
antibody preparation. The full-length DFF-like protein can be used
or, alternatively, the invention provides antigenic peptide
fragments of DFF-like proteins for use as immunogens. The antigenic
peptide of a DFF-like protein comprises at least 8, preferably 10,
15, 20, or 30 amino acid residues of the amino acid sequence shown
in SEQ ID NO:11 and encompasses an epitope of a DFF-like protein
such that an antibody raised against the peptide forms a specific
immune complex with the DFF-like protein. Preferred epitopes
encompassed by the antigenic peptide are regions of a DFF-like
protein that are located on the surface of the protein, e.g.,
hydrophilic regions.
[0666] Accordingly, another aspect of the invention pertains to
anti-DFF-like polyclonal and monoclonal antibodies that bind a
DFF-like protein. Polyclonal anti-DFF-like antibodies can be
prepared by immunizing a suitable subject (e.g., rabbit, goat,
mouse, or other mammal) with a DFF-like immunogen. The
anti-DFF-like 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 DFF-like
protein. At an appropriate time after immunization, e.g., when the
anti-DFF-like 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 266:550-52; 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).
[0667] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-DFF-like antibody can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with a DFF-like protein to thereby isolate immunoglobulin library
members that bind the DFF-like 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 SufZAP.TM. Phage Display Kit,
Catalog No. 240612). Additionally, examples of methods and reagents
particularly amenable for use in generating and screening antibody
display library can be found in, for example, 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.
[0668] Additionally, recombinant anti-DFF-like 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.
[0669] 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.
[0670] 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).
[0671] An anti-DFF-like antibody (e.g., monoclonal antibody) can be
used to isolate DFF-like proteins by standard techniques, such as
affinity chromatography or immunoprecipitation. An anti-DFF-like
antibody can facilitate the purification of natural DFF-like
protein from cells and of recombinantly produced DFF-like protein
expressed in host cells. Moreover, an anti-DFF-like antibody can be
used to detect DFF-like protein (e.g., in a cellular lysate or cell
supernatant) in order to evaluate the abundance and pattern of
expression of the DFF-like protein. Anti-DFF-like 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.
[0672] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). 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.
[0673] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
III. Recombinant Expression Vectors and Host Cells
[0674] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
DFF-like 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.
[0675] 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., DFF-like proteins, mutant forms of DFF-like
proteins, fusion proteins, etc.).
[0676] 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 such codon usage are well known in the art.
[0677] The recombinant expression vectors of the invention can be
designed for expression of DFF-like 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 11d (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.
[0678] Suitable eukaryotic host cells include insect cells
(examples of Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers (1989) Virology 170:31-39));
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.
[0679] 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. 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.
[0680] 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 (Banerji et al. (1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application 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.
[0681] 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 DFF-like 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).
[0682] 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.
[0683] 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 a DFF-like 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).
[0684] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) DFF-like protein. Accordingly, the invention further
provides methods for producing DFF-like protein using the host
cells of the invention. In one embodiment, the method comprises
culturing the host cell of the invention, into which a recombinant
expression vector encoding a DFF-like protein has been introduced,
in a suitable medium such that DFF-like protein is produced. In
another embodiment, the method further comprises isolating DFF-like
protein from the medium or the host cell.
[0685] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which DFF-like-coding sequences have been introduced.
Such host cells can then be used to create nonhuman transgenic
animals in which exogenous DFF-like sequences have been introduced
into their genome or homologous recombinant animals in which
endogenous DFF-like sequences have been altered. Such animals are
useful for studying the function and/or activity of DFF-like genes
and proteins and for identifying and/or evaluating modulators of
DFF-like 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 DFF-like 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.
[0686] A transgenic animal of the invention can be created by
introducing DFF-like-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 DFF-like cDNA sequence can be introduced
as a transgene into the genome of a nonhuman animal. Alternatively,
a homologue of the mouse DFF-like 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 DFF-like transgene to direct expression of DFF-like 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
DFF-like transgene in its genome and/or expression of DFF-like 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 DFF-like
gene can further be bred to other transgenic animals carrying other
transgenes.
[0687] To create a homologous recombinant animal, one prepares a
vector containing at least a portion of a DFF-like 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 DFF-like gene. In a preferred embodiment,
the vector is designed such that, upon homologous recombination,
the endogenous DFF-like 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 DFF-like 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 DFF-like protein). In the
homologous recombination vector, the altered portion of the
DFF-like gene is flanked at its 5' and 3' ends by additional
nucleic acid of the DFF-like gene to allow for homologous
recombination to occur between the exogenous DFF-like gene carried
by the vector and an endogenous DFF-like gene in an embryonic stem
cell. The additional flanking DFF-like 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 DFF-like gene has homologously recombined with
the endogenous DFF-like 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.
[0688] 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/oxP 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.
[0689] 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
[0690] The DFF-like nucleic acid molecules, DFF-like proteins, and
anti-DFF-like 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.
[0691] 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.
[0692] 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.
[0693] 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.
[0694] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the knowledge 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.
[0695] 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.
[0696] 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.TM. (BASF; Parsippany, N.J.), or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as 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.
[0697] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a DFF-like protein or
anti-DFF-like 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.
[0698] 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.
[0699] 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.
[0700] 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.
[0701] 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.
[0702] 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.
[0703] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
V. Uses and Methods of the Invention
[0704] 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
DFF-like protein (e.g., via a recombinant expression vector in a
host cell in gene therapy applications), to detect DFF-like mRNA
(e.g., in a biological sample) or a genetic lesion in a DFF-like
gene, and to modulate DFF-like activity. In addition, the DFF-like
proteins can be used to screen drugs or compounds that modulate
apoptotic events as well as to treat disorders characterized by
insufficient or excessive production of DFF-like protein or
production of DFF-like protein forms that have decreased or
aberrant activity compared to DFF-like wild type protein. In
addition, the anti-DFF-like antibodies of the invention can be used
to detect and isolate DFF-like proteins and modulate DFF-like
activity.
[0705] A. Screening Assays
[0706] 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 DFF-like proteins or have a
stimulatory or inhibitory effect on, for example, DFF-like
expression or DFF-like activity.
[0707] 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).
[0708] 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.
[0709] 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).
[0710] Determining the ability of the test compound to bind to the
DFF-like 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 DFF-like 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, 14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemission 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.
[0711] In a similar manner, one may determine the ability of the
DFF-like protein to bind to or interact with a DFF-like target
molecule. By "target molecule" is intended a molecule with which a
DFF-like protein binds or interacts in nature. In a preferred
embodiment, the ability of the DFF-like protein to bind to or
interact with a DFF-like 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 a
morphological change induced by apoptosis (e.g., DNA fragmentation,
membrane blebbing, cytoplasmic and nuclear degradation, etc.),
detecting catalytic/enzymatic activity of the target on an
appropriate substrate, or detecting the induction of a reporter
gene (e.g., DFF-like-responsive regulatory element operably linked
to a nucleic acid encoding a detectable marker, e.g.
luciferase).
[0712] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting a DFF-like protein or
biologically active portion thereof with a test compound and
determining the ability of the test compound to bind to the
DFF-like protein or biologically active portion thereof. Binding of
the test compound to the DFF-like protein can be determined either
directly or indirectly as described above. In a preferred
embodiment, the assay includes contacting the DFF-like protein or
biologically active portion thereof with a known compound that
binds DFF-like 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 DFF-like protein or
biologically active portion thereof as compared to the known
compound.
[0713] In another embodiment, an assay is a cell-free assay
comprising contacting DFF-like 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 DFF-like protein or biologically active portion
thereof. Determining the ability of the test compound to modulate
the activity of a DFF-like protein can be accomplished, for
example, by determining the ability of the DFF-like protein to bind
to a DFF-like 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 a DFF-like
protein can be accomplished by determining the ability of the
DFF-like protein to further modulate a DFF-like target molecule.
For example, the catalytic/enzymatic activity of the target
molecule on an appropriate substrate can be determined as
previously described.
[0714] In yet another embodiment, the cell-free assay comprises
contacting the DFF-like protein or biologically active portion
thereof with a known compound that binds a DFF-like 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 a DFF-like
target molecule.
[0715] In the above-mentioned assays, it may be desirable to
immobilize either a DFF-like 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/DFF-like 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 DFF-like 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 DFF-like binding or activity determined using
standard techniques.
[0716] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either DFF-like protein or its target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated
DFF-like 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
a DFF-like protein or target molecules but which do not interfere
with binding of the DFF-like protein to its target molecule can be
derivatized to the wells of the plate, and unbound target or
DFF-like 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
DFF-like protein or target molecule, as well as enzyme-linked
assays that rely on detecting an enzymatic activity associated with
the DFF-like protein or target molecule.
[0717] In another embodiment, modulators of DFF-like expression are
identified in a method in which a cell is contacted with a
candidate compound and the expression of DFF-like mRNA or protein
in the cell is determined relative to expression of DFF-like 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 DFF-like 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 DFF-like mRNA or protein expression. The level
of DFF-like mRNA or protein expression in the cells can be
determined by methods described herein for detecting DFF-like mRNA
or protein.
[0718] In yet another aspect of the invention, the DFF-like
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 DFF-like protein ("DFF-like-binding proteins" or
"DFF-like-bp") and modulate DFF-like activity. Such
DFF-like-binding proteins are also likely to be involved in the
propagation of signals by the DFF-like proteins as, for example,
upstream or downstream elements of the DFF-like pathway.
[0719] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0720] B. Detection Assays
[0721] 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.
[0722] 1. Chromosome Mapping
[0723] The isolated complete or partial DFF-like gene sequences of
the invention can be used to map their respective DFF-like genes on
a chromosome, thereby facilitating the location of gene regions
associated with genetic disease. Computer analysis of DFF-like
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 DFF-like sequences
will yield an amplified fragment.
[0724] 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.
[0725] Other mapping strategies that can similarly be used to map a
DFF-like 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 et al. (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.
[0726] 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.
[0727] Another strategy to map the chromosomal location of DFF-like
genes uses DFF-like polypeptides and fragments and sequences of the
present invention and antibodies specific thereto. This mapping can
be carried out by specifically detecting the presence of a DFF-like
polypeptide in members of a panel of somatic cell hybrids between
cells of a first species of animal from which the protein
originates and cells from a second species of animal, and then
determining which somatic cell hybrid(s) expresses the polypeptide
and noting the chromosomes(s) from the first species of animal that
it contains. For examples of this technique, see Pajunen et al.
(1988) Cytogenet. Cell. Genet. 47:37-41 and Van Keuren et al.
(1986) Hum. Genet. 74:34-40. Alternatively, the presence of a
DFF-like polypeptide in the somatic cell hybrids can be determined
by assaying an activity or property of the polypeptide, for
example, enzymatic activity, as described in Bordelon-Riser et al.
(1979) Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978)
Proc. Natl. Acad. Sci. USA 75:5640-5644.
[0728] 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.
[0729] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the DFF-like 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.
[0730] 2. Tissue Typing
[0731] The DFF-like 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).
[0732] 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 DFF-like sequences of the invention can be used
to prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0733] 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 DFF-like 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:10
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:11, is used, a more appropriate number of
primers for positive individual identification would be 500 to
2,000.
[0734] 3. Use of Partial DFF-Like Sequences in Forensic Biology
[0735] 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.
[0736] 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:10
are particularly appropriate for this use as greater numbers of
polymorphisms occur in the noncoding regions, making it easier to
differentiate individuals using this technique. Examples of
polynucleotide reagents include the DFF-like sequences or portions
thereof, e.g., fragments derived from the noncoding regions of SEQ
ID NO:10 having a length of at least 20 or 30 bases.
[0737] The DFF-like 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 DFF-like probes, can be used to
identify tissue by species and/or by organ type.
[0738] In a similar fashion, these reagents, e.g., DFF-like 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).
[0739] C. Predictive Medicine
[0740] 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.
[0741] 1. Diagnostic Assays
[0742] One aspect of the present invention relates to diagnostic
assays for detecting DFF-like protein and/or nucleic acid
expression as well as DFF-like activity, in the context of a
biological sample. An exemplary method for detecting the presence
or absence of DFF-like 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 DFF-like protein or nucleic acid (e.g., mRNA, genomic
DNA) that encodes DFF-like protein such that the presence of
DFF-like 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.
[0743] "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.
[0744] A preferred agent for detecting DFF-like mRNA or genomic DNA
is a labeled nucleic acid probe capable of hybridizing to DFF-like
mRNA or genomic DNA. The nucleic acid probe can be, for example, a
full-length DFF-like nucleic acid, such as the nucleic acid of SEQ
ID NO:10, 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
DFF-like mRNA or genomic DNA. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
[0745] A preferred agent for detecting DFF-like protein is an
antibody capable of binding to DFF-like protein, preferably an
antibody with a detectable label. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., Fab or F(ab')2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin.
[0746] 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 DFF-like
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
DFF-like mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of DFF-like
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence. In
vitro techniques for detection of DFF-like genomic DNA include
Southern hybridizations. Furthermore, in vivo techniques for
detection of DFF-like protein include introducing into a subject a
labeled anti-DFF-like 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.
[0747] 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.
[0748] The invention also encompasses kits for detecting the
presence of DFF-like 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 DFF-like protein (e.g., a
disorder resulting in dysregulated apoptosis). For example, the kit
can comprise a labeled compound or agent capable of detecting
DFF-like protein or mRNA in a biological sample and means for
determining the amount of a DFF-like protein in the sample (e.g.,
an anti-DFF-like antibody or an oligonucleotide probe that binds to
DNA encoding a DFF-like protein, e.g., SEQ ID NO:10 or 11). 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 DFF-like sequences if the amount of
DFF-like protein or mRNA is above or below a normal level.
[0749] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) that binds
to DFF-like protein; and, optionally, (2) a second, different
antibody that binds to DFF-like 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 a
DFF-like nucleic acid sequence or (2) a pair of primers useful for
amplifying a DFF-like nucleic acid molecule.
[0750] 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 DFF-like proteins.
[0751] 2. Other Diagnostic Assays
[0752] 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 DFF-like, 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 DFF-like nucleic
acid, polypeptide, or antibody.
[0753] 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.
[0754] The method can include contacting the DFF-like 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.
[0755] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of DFF-like. 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.
[0756] The method can be used to detect SNPs, as described
above.
[0757] 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
DFF-like or from a cell or subject in which a DFF-like mediated
response has been elicited, e.g., by contact of the cell with
DFF-like nucleic acid or protein, or administration to the cell or
subject DFF-like 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 DFF-like 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 DFF-like (or does not
express as highly as in the case of the DFF-like positive plurality
of capture probes) or from a cell or subject which in which a
DFF-like 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 DFF-like 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.
[0758] In another aspect, the invention features, a method of
analyzing DFF-like, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a DFF-like nucleic acid or amino acid
sequence, comparing the DFF-like 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 DFF-like.
[0759] The method can include evaluating the sequence identity
between a DFF-like sequence and a database sequence. The method can
be performed by accessing the database at a second site, e.g., over
the internet.
[0760] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of DFF-like. 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 identical in sequence with one another (except for
differences in length). The oligonucleotides can be provided with
differential labels, such that an oligonucleotides which hybridizes
to one allele provides a signal that is distinguishable from an
oligonucleotides which hybridizes to a second allele.
[0761] 3. Prognostic Assays
[0762] 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 DFF-like
protein, DFF-like nucleic acid expression, or DFF-like 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 DFF-like protein,
DFF-like nucleic acid expression, or DFF-like activity.
[0763] Thus, the present invention provides a method in which a
test sample is obtained from a subject, and DFF-like protein or
nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the
presence of DFF-like protein or nucleic acid is diagnostic for a
subject having or at risk of developing a disease or disorder
associated with aberrant DFF-like 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.
[0764] 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 DFF-like activity) to effectively treat a
disease or disorder associated with aberrant DFF-like expression or
activity. In this manner, a test sample is obtained and DFF-like
protein or nucleic acid is detected. The presence of DFF-like
protein or nucleic acid is diagnostic for a subject that can be
administered the agent to treat a disorder associated with aberrant
DFF-like expression or activity.
[0765] The methods of the invention can also be used to detect
genetic lesions or mutations in a DFF-like gene, thereby
determining if a subject with the lesioned gene is at risk for a
disorder characterized by dysregulated apoptosis. 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 a DFF-like-protein, or the
misexpression of the DFF-like 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
a DFF-like gene; (2) an addition of one or more nucleotides to a
DFF-like gene; (3) a substitution of one or more nucleotides of a
DFF-like gene; (4) a chromosomal rearrangement of a DFF-like gene;
(5) an alteration in the level of a messenger RNA transcript of a
DFF-like gene; (6) an aberrant modification of a DFF-like gene,
such as of the methylation pattern of the genomic DNA; (7) the
presence of a non-wild-type splicing pattern of a messenger RNA
transcript of a DFF-like gene; (8) a non-wild-type level of a
DFF-like-protein; (9) an allelic loss of a DFF-like gene; and (10)
an inappropriate post-translational modification of a
DFF-like-protein. As described herein, there are a large number of
assay techniques known in the art that can be used for detecting
lesions in a DFF-like gene. Any cell type or tissue in which
DFF-like proteins are expressed may be utilized in the prognostic
assays described herein.
[0766] 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 DFF-like-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.
[0767] 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.
[0768] In an alternative embodiment, mutations in a DFF-like 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.
[0769] In other embodiments, genetic mutations in a DFF-like
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 DFF-like gene and detect mutations by comparing the
sequence of the sample DF-like 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).
[0770] Other methods for detecting mutations in the DFF-like 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.
[0771] 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 DFF-like 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 a DFF-like sequence, e.g., a wild-type DFF-like 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.
[0772] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in DFF-like 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).
[0773] 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).
[0774] 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.
[0775] 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.
[0776] 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 a DFF-like gene.
[0777] 4. Pharmacogenomics
[0778] Agents, or modulators that have a stimulatory or inhibitory
effect on DFF-like activity (e.g., DFF-like gene expression) as
identified by a screening assay described herein, can be
administered to individuals to treat (prophylactically or
therapeutically) disorders associated with aberrant DFF-like
activity as well as to modulate the phenotype of dysregulated
apoptosis. 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
DFF-like protein, expression of DFF-like nucleic acid, or mutation
content of DFF-like genes in an individual can be determined to
thereby select appropriate agent(s) for therapeutic or prophylactic
treatment of the individual.
[0779] 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.
[0780] Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician may consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a DFF-like molecule or DFF-like modulator as
well as tailoring the dosage and/or therapeutic regimen of
treatment with a DFF-like molecule or DFF-like modulator.
[0781] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0782] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., a DFF-like protein of the present
invention), all common variants of that gene can be fairly easily
identified in the population and it can be determined if having one
version of the gene versus another is associated with a particular
drug response.
[0783] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a DFF-like molecule or DFF-like modulator of the
present invention) can give an indication whether gene pathways
related to toxicity have been turned on.
[0784] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a DFF-like molecule or DFF-like
modulator, such as a modulator identified by one of the exemplary
screening assays described herein.
[0785] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the DFF-like genes of the
present invention, wherein these products may be associated with
resistance of the cells to a therapeutic agent. Specifically, the
activity of the proteins encoded by the DFF-like genes of the
present invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells will become sensitive
to treatment with an agent that the unmodified target cells were
resistant to.
[0786] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a DFF-like protein can be applied in
clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
DFF-like gene expression, protein levels, or upregulate DFF-like
activity, can be monitored in clinical trials of subjects
exhibiting decreased DFF-like gene expression, protein levels, or
downregulated DFF-like activity. Alternatively, the effectiveness
of an agent determined by a screening assay to decrease DFF-like
gene expression, protein levels, or downregulate DFF-like activity,
can be monitored in clinical trials of subjects exhibiting
increased DFF-like gene expression, protein levels, or upregulated
DFF-like activity. In such clinical trials, the expression or
activity of a DFF-like gene, and preferably, other genes that have
been implicated in, for example, a DFF-like-associated disorder can
be used as a "read out" or markers of the phenotype of a particular
cell.
[0787] 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.
[0788] Thus, the activity of DFF-like protein, expression of
DFF-like nucleic acid, or mutation content of DFF-like 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
a DFF-like modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0789] 5. Monitoring of Effects During Clinical Trials
[0790] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of DFF-like genes (e.g., the ability
to modulate apoptotic events) 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 DFF-like gene expression,
protein levels, or protein activity, can be monitored in clinical
trials of subjects exhibiting decreased or increased DFF-like gene
expression, protein levels, or protein activity. In such clinical
trials, DFF-like expression or activity and preferably that of
other genes that have been implicated in for example, a disorder
resulting in dysregulated apoptosis, can be used as a marker of an
apoptotic events of a particular cell.
[0791] 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 DFF-like activity (e.g., as
identified in a screening assay described herein) can be
identified. Thus, to study the effect of agents on cellular
disorders resulting from dysregulated apoptosis, for example, in a
clinical trial, cells can be isolated and RNA prepared and analyzed
for the levels of expression of DFF-like 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
DFF-like 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.
[0792] 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 a DFF-like 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 DFF-like protein, mRNA, or
genomic DNA in the postadministration samples; (5) comparing the
level of expression or activity of the DFF-like protein, mRNA, or
genomic DNA in the preadministration sample with the DFF-like
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
a DFF-like protein.
[0793] C. Methods of Treatment
[0794] 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 DFF-like expression or activity. "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.
Additionally, the compositions of the invention find use in the
treatment of disorders described herein. Thus, therapies for
disorders associated with a DFF-like molecule are encompassed
herein. "Treatment" is herein 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. A "therapeutic agent" includes, but is not limited
to, small molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0795] 1. Prophylactic Methods
[0796] In one aspect, the invention provides a method for
preventing in a subject a disease or condition associated with an
aberrant DFF-like expression or activity by administering to the
subject an agent that modulates DFF-like expression or at least one
DFF-like gene activity. Subjects at risk for a disease that is
caused, or contributed to, by aberrant DFF-like 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 DFF-like aberrancy, such that a
disease or disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of DFF-like aberrancy, for
example, a DFF-like agonist or DFF-like antagonist agent can be
used for treating the subject. The appropriate agent can be
determined based on screening assays described herein.
[0797] 2. Therapeutic Methods
[0798] Another aspect of the invention pertains to methods of
modulating DFF-like 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 DFF-like protein activity associated with the cell.
An agent that modulates DFF-like protein activity can be an agent
as described herein, such as a nucleic acid or a protein, a
naturally-occurring cognate ligand of a DFF-like protein, a
peptide, a DFF-like peptidomimetic, or other small molecule. In one
embodiment, the agent stimulates one or more of the biological
activities of DFF-like protein. Examples of such stimulatory agents
include active DFF-like protein and a nucleic acid molecule
encoding a DFF-like protein that has been introduced into the cell.
In another embodiment, the agent inhibits one or more of the
biological activities of DFF-like protein. Examples of such
inhibitory agents include antisense DFF-like nucleic acid molecules
and anti-DFF-like antibodies.
[0799] 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 a DFF-like 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) DFF-like expression or activity. In another
embodiment, the method involves administering a DFF-like protein or
nucleic acid molecule as therapy to compensate for reduced or
aberrant DFF-like expression or activity.
[0800] Stimulation of DFF-like activity is desirable in situations
in which a DFF-like protein is abnormally downregulated and/or in
which increased DFF-like activity is likely to have a beneficial
effect. Conversely, inhibition of DFF-like activity is desirable in
situations in which DFF-like activity is abnormally upregulated
and/or in which decreased DFF-like activity is likely to have a
beneficial effect.
[0801] This invention is further illustrated by the following
examples, which should not be construed as limiting.
Example 1
Identification and Characterization of Human DFF-Like cDNAs
[0802] The human DFF-like sequence (SEQ ID NO:10), which is
approximately 1284 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
169 nucleotides (nucleotides 169-828 of SEQ ID NO:10). The coding
sequence encodes a 219 amino acid protein (SEQ ID NO:11).
[0803] A search of the nucleotide and protein databases revealed
that 5698 encodes a precursor polypeptide that shares similarity
with several CIDE proteins. An alignment of the protein sequences
having highest similarity to the 5698 precursor polypeptide is
shown in FIG. 19. The alignment was generated using the Clustal
method with PAM250 residue weight table and sequence identities
were determined by FASTA (Pearson and Lipman (1988) Proc. Natl.
Acad. Sci. USA 85:2444-2448).
Example 2
Tissue Distribution of a DFF-Like mRNA
[0804] Northern blot hybridizations with various RNA samples can be
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 DFF-like cDNA (SEQ ID
NO:10 or 3) can be used. The DNA was 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.) can be probed in
ExpressHyb hybridization solution (Clontech) and washed at high
stringency according to manufacturer's recommendations.
[0805] TaqMan analysis of 5698 revealed expression in a number of
tissues. See, for example, FIGS. 20A-B. A high level of expression
was seen in liver, diseased and normal heart ventricle, normal
kidney, kidney HT, atrium of normal heart, diseased heart
ventricle, fetal heart, and skeletal muscle. The source number
associated with each of these tissue types is provided below in
Table 1. TABLE-US-00001 TABLE 1 Source # CV Organ MPI 1097 H Heart
Normal Atrium pit 277 H Heart Normal Atrium pit 272 H Heart Normal
Ventricle tlo.sup.-1 H Heart Normal Ventricle pit 278 H Heart
Normal Ventricle pit 204 H Heart Normal Ventricle pit 205 H Heart
Normal Ventricle eli 5 H Heart Diseased Ventricle pit 16 H Heart
Diseased Ventricle mpi 613 H Heart Diseased Ventricle bwh 4 H Fetal
Heart ndr 171 H Kidney normal ndr 179 H Kidney normal pit 289 H
Kidney normal pit 351 H Kidney normal pit 353 H Kidney normal ndr
233 H Kidney HT ndr 224 H Kidney HT ndr 248 H Kidney HT ndr 252 H
Kidney HT CHT 1176 H Kidney HT mpi 570 H Skeletal Muscle mpi 602 H
Skeletal Muscle pit 284 H Skeletal Muscle mpi 145 H Liver mpi 155 H
Liver mpi 146 H Liver MPI 90 M Heart Normal Atrium MPI 92 M Heart
Normal Atrium MPI 96 M Heart Normal Ventricle MPI 538 M Heart
Normal Ventricle
Example 3
Recombinant Expression of DFF-Like Sequences in Bacterial Cells
[0806] In this example, the DFF-like sequence is expressed as a
recombinant glutathione-S-transferase (GST) fusion polypeptide in
E. coli and the fusion polypeptide is isolated and characterized.
Specifically, the DFF-like sequence is fused to GST and this fusion
polypeptide is expressed in E. coli, e.g., strain PEB199.
Expression of the GST-DFF-like fusion protein in PEB199 is induced
with IPTG. The recombinant fusion polypeptide is purified from
crude bacterial lysates of the induced PEB199 strain by affinity
chromatography on glutathione beads. Using polyacrylamide gel
electrophoretic analysis of the polypeptide purified from the
bacterial lysates, the molecular weight of the resultant fusion
polypeptide is determined.
Example 4
Expression of Recombinant DFF-Like Protein in COS Cells
[0807] To express the DFF-like 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 DFF-like
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.
[0808] To construct the plasmid, the DFF-like 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 DFF-like 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 DFF-like 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 DFF-like 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.
[0809] COS cells are subsequently transfected with the
DFF-like-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 DFF-like 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.
[0810] Alternatively, DNA containing the DFF-like 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 DFF-like polypeptide is detected by
radiolabelling and immunoprecipitation using a DFF-like specific
monoclonal antibody.
[0811] 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.
[0812] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Chapter 4
32621, Novel Human Phospholipid Scramblase-Like Molecules and Uses
Thereof
BACKGROUND OF THE INVENTION
[0813] Phospholipid asymmetry is a well-known characteristic of
mammalian plasma membranes. The outer leaflet of the lipid bilayer
is rich in choline-phospholipids, whereas aminophospholipids are
preferentially in the inner leaflet (Bevers, E. M. et al., (1998)
Lupus Suppl. 2: S126-S131). The plasma membrane phospholipids of
erythrocytes (RBC), platelets, and vascular endothelium are
normally asymmetrically distributed. Phosphatidylserine (PS) and
phosphatidylethanolamine (PE) reside almost exclusively in the
inner leaflet, and the phosphatidylcholine (PC) and sphyingomyelin
are enriched in the outer leaflet. This asymmetric distribution of
PL is maintained by an aminophospholipid translocase (APLT) which
is a Mg.sup.+2-dependent ATPase that transports PS and PE, but not
PC, from the outer to the inner plasma membrane leaflet (Stout, J.
G., et al. (1997) J. Clin. Invest. 99(9): 2232-2238).
[0814] The APLT activity has now been identified in numerous cell
types, including platelets, lymphocytes, fibroblasts, and
synaptosomes, suggesting that the asymmetry might be a general
property of all cells (Woon, L. A., et al., (1999) Cell Calcium
25(4):313-320).
[0815] When PS and PE become exposed on the outer membrane leaflet
by various mechanisms of cell activation, the Mg.sup.+2-ATPase
activity of APLT restores phospholipid asymmetry by transporting
these lipids to the inner bilayer leaflet. A number of
physiological and pathophysiological conditions may result in the
disruption of the normal phospholipid asymmetry of the plasma
membrane leading to the exposure of PS on the surface of cells.
Exposure of PS creates a procoagulant surface on platelets,
erythrocytes, and vascular endothelial cells. Also, there is
evidence which indicates that clotting, cellular adhesion, fusion
and phagocytosis of senescent or apoptotic cells are dependent on
PS exposure (Woon et al. (1999) Cell Calcium 25(4):313-320).
[0816] A second mechanism which causes phospholipid redistribution
in the plasma membrane has been linked to a phospholipid (PL)
scramblase. The scramblase is an integral membrane protein that can
mimic the action of Ca.sup.+2 at the endothelial surface of the
erythrocyte membrane (Zhao et al. (1998) J. Biol. Chem.
273(12):6603-6606). Zhao et al. demonstrated that the propensity
for PS to become exposed at the cell surface can be manipulated by
altering the level of expression of PL scramblase through plasmid
transfection. Zhao et al. posit that the transfection of cells with
PL scramblase cDNA promotes movement of PS to the cell surface and
suggests that this protein is involved in the normal redistribution
of plasma membrane phospholipids in activated, injured, and
apoptotic cells.
[0817] Phospholipid (PL) scramblase is a plasma membrane protein
that mediates accelerated transbilayer migration of PLs, upon
binding of Ca.sup.-2, facilitating rapid mobilization of
phosphatidylserine to the cell surface upon elevation of internal
calcium. (Stout, J. G. et al., (1998) Biochemistry 37:14860-14866).
An increase in intracellular calcium due to cell activation,
injury, or apoptosis causes rapid bidirectional movement of plasma
membrane PL between leaflets. PL scramblase is responsible for this
two-way movement of PL between the membrane leaflets, resulting in
exposure of PS and PE at the cell surface (Kasukabe, T. et al.,
(1998) Biochem. and Biophys. Res. Commun. 249: 449-455). The PL
scramblase can be assayed using methods as described by Zhou (Zhou,
Q. et al., (1998) Biochemistry 37: 2356-2360).
[0818] One important clinical disorder which may be linked to
defective PL scramblase is Scott syndrome. Scott syndrome is a
congenital bleeding disorder related to defective expression of
membrane coagulant activity. Circulating blood cells show decreased
cell surface exposure of phosphatidylserine (PS) at elevated
cystolic Ca.sup.+2 indicating a defect or deficiency in PL
scramblase (Stout, J. G. et al., (1997) J. Clin. Invest.
99(9):2232-2238). Scott syndrome is an extremely rare bleeding
disorder associated with a defect of the outward transmembrane
migration of pro-coagulant phospholipids at the surface of
stimulated platelets or derived-microparticles. Scott syndrome is
transmitted as an autosomal recessive trait as demonstrated in a
familial study (Toti, F. et al., (1996) Blood 87:1409-1415).
[0819] Recently, the molecular cloning of murine and human PL
scramblases has been reported. Zhou et al. reported the cDNA
cloning of a 37-kDa human plasma membrane phospholipid scramblase
from human erythrocytes (Zhou, Q. et al., (1997) J. Biol. Chem.
272(29): 18240-18244). Antibody to the scramblase indicated an
approximately 10-fold higher abundance of the PL scramblase in
platelets as compared to erythrocytes. The work of Zhou et al.
indicated that PL scramblase mRNA is found in a variety of
hematalogic and nonhematologic cells and tissues. The resulting
exposure of PS at the cell surface is thought to play a key role in
the reticuloendothelial system, in addition to activation of both
the plasma complement and coagulation systems.
[0820] More recently, the cDNA cloning of a human plasma membrane
PL scramblase (MmTRA1b) from human monocytic U937 cells and the
chromosome mapping of the gene was reported (Kaskube, T. et al.,
(1998) Biochem. and Biophys. Res. Comm. 249: 449-455). The MmTRA1b
gene is the human homologue of the previously cloned mouse
leukemogenesis-associated gene (MmTRA1a). The mouse MmTRA1a is the
truncated form of mouse MmTRA1b. The human MmTRA1b cDNA predicted a
318 amino acid protein with a molecular weight of 35,047 Da.
[0821] The human MmTRA1b protein sequence shared a 78% amino acid
identity with the mouse counterpart (328 amino acids). The human
MmTRA1b gene was mapped to chromosome 3q23. Expression of the human
homologue was increased during differentiation of U937 cells by
most typical differentiation inducers. Also, the predicted amino
acid sequence analysis of the human MmTRA1b cDNA revealed perfect
identity with human plasma membrane phospholipid scramblase that is
required for transbilayer movement of membrane phospholipids
(Kaskukabe, T. et al., (1998) Biochem. and Biophys. Res. Comm. 249:
449-455).
[0822] According to homology searches against EMBL/Genbank/DDBJ
data bases there are at least three homologous C. elegans genes
which are more closely related with the mouse and human MmTRA1b
than previously reported, as detailed by Kasukabe et al. Therefore,
there appear to be at least two mouse genes (MmTRA1b and PL
scramblase as reported by Zhou et al. and five C. elegans genes
which constitute a whole new family of PL flip/flop genes.
[0823] The human phospholipid scramblase gene herein described may
play an important role in erythrocyte, platelet, lymphocyte and
endothelium physiology and function. It may play a particularly
important role in the treatment and diagnosis of bleeding disorders
such as Scott syndrome and other hematologic disease conditions,
including but not limited to lymphocytic disorders, plasma cell
dyscrasias, hemolytic anemias, autoimmune neutropenias, immune
thrombocytopenias, lymphocytic leukemias, leukopenia, lymphomas,
red cell disorders, platelet disorders, and coagulation
disorders.
SUMMARY OF THE INVENTION
[0824] Isolated nucleic acid molecules corresponding to human
phospholipid scramblase-like 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 sequences shown in SEQ
ID NO:17. Further provided are human phospholipid scramblase-like
polypeptides having an amino acid sequence encoded by a nucleic
acid molecule described herein.
[0825] 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.
[0826] The human phospholipid scramblase-like molecules of the
present invention are useful for modulating the immune,
hematopoietic, and blood clotting systems. The molecules are useful
for the diagnosis and treatment of disorders relevant but not
limited to erythrocytes, platelets, endothelial and other cells and
tissues known to expose plasma membrane phospholipid in response to
elevated cystolic Ca.sup.+2. Additionally, the molecules of the
invention are useful as modulating agents in a variety of cellular
processes where the transbilayer movement of phospholipids in the
plasma membrane is important for proper cellular function and
homeostasis.
[0827] Accordingly, in one aspect, this invention provides isolated
nucleic acid molecules encoding human phospholipid scramblase-like
proteins or biologically active portions thereof, as well as
nucleic acid fragments suitable as primers or hybridization probes
for the detection of human phospholipid scramblase-like encoding
nucleic acids.
[0828] Another aspect of this invention features isolated or
recombinant human phospholipid scramblase-like proteins and
polypeptides. Preferred human phospholipid scramblase-like proteins
and polypeptides possess at least one biological activity possessed
by naturally occurring human phospholipid scramblase-like
proteins.
[0829] 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.
[0830] Antibodies and antibody fragments that selectively bind the
human phospholipid scramblase-like polypeptides and fragments are
provided. Such antibodies are useful in detecting the human
phospholipid scramblase-like polypeptides.
[0831] In another aspect, the present invention provides a method
for detecting the presence of human phospholipid scramblase-like
activity or expression in a biological sample by contacting the
biological sample with an agent capable of detecting an indicator
of human phospholipid scramblase-like activity such that the
presence of human phospholipid scramblase-like activity is detected
in the biological sample.
[0832] In yet another aspect, the invention provides a method for
modulating human phospholipid scramblase-like activity comprising
contacting a cell with an agent that modulates (inhibits or
stimulates) human phospholipid scramblase-like activity or
expression such that human phospholipid scramblase-like activity or
expression in the cell is modulated. In one embodiment, the agent
is an antibody that specifically binds to human phospholipid
scramblase-like protein. In another embodiment, the agent modulates
expression of human phospholipid scramblase-like protein by
modulating transcription of a human phospholipid scramblase-like
gene, splicing of a human phospholipid scramblase-like mRNA, or
translation of a human phospholipid scramblase-like 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
human phospholipid scramblase-like mRNA or the human phospholipid
scramblase-like gene.
[0833] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
human phospholipid scramblase-like protein activity or nucleic acid
expression by administering an agent that is a human phospholipid
scramblase-like modulator to the subject. In one embodiment, the
human phospholipid scramblase-like modulator is a human
phospholipid scramblase-like protein. In another embodiment, the
human phospholipid scramblase-like modulator is a human
phospholipid scramblase-like nucleic acid molecule. In other
embodiments, the human phospholipid scramblase-like modulator is a
peptide, peptidomimetic, or other small molecule.
[0834] 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 a human phospholipid
scramblase-like protein; (2) misregulation of a gene encoding a
human phospholipid scramblase-like protein; and (3) aberrant
post-translational modification of a human phospholipid
scramblase-like protein, wherein a wild-type form of the gene
encodes a protein with a human phospholipid scramblase-like
activity.
[0835] In another aspect, the invention provides a method for
identifying a compound that binds to or modulates the activity of a
human phospholipid scramblase-like protein. In general, such
methods entail measuring a biological activity of a human
phospholipid scramblase-like protein in the presence and absence of
a test compound and identifying those compounds that alter the
activity of the human phospholipid scramblase-like protein.
[0836] The invention also features methods for identifying a
compound that modulates the expression of human phospholipid
scramblase-like genes by measuring the expression of the human
phospholipid scramblase-like sequences in the presence and absence
of the compound.
[0837] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0838] 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.
[0839] 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.
[0840] The present invention provides phospholipid scramblase-like
molecules. By "phospholipid scramblase-like" is intended a novel
human sequence referred to as 32621, and variants and fragments
thereof. These full-length gene sequences or fragments thereof are
referred to as "phospholipid scramblase-like" sequences, indicating
they share sequence similarity with phospholipid scramblase genes.
Isolated nucleic acid molecules comprising nucleotide sequences
encoding the 32621 polypeptide whose amino acid sequence is given
in SEQ ID NO:17, or a variant or fragment thereof, are provided. A
nucleotide sequence encoding the 32621 polypeptide is set forth in
SEQ ID NO:16 and SEQ ID NO:18.
[0841] The disclosed invention relates to methods and compositions
for the modulation, diagnosis, and treatment of immune,
hematopoietic, platelet, and blood coagulation disorders. Such
immune disorders include, but not limited to, lymphocytic
disorders, plasma cell dyscrasias, hemolytic anemias, autoimmune
neutropenias, immune thrombocytopenias, lymphocytic leukemias,
leukopenia, and lymphomas. The hematopoietic disorders include, but
are not limited to, all bone marrow and red blood cell disorders.
The blood coagulation disorders include, but are not limited to,
hemophilia and Von Willebrand's disease. Platelet disorders
include, but are not limited to, thrombocytopenia and Scott
syndrome.
[0842] 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.
[0843] In normal bone marrow, the myelocytic series
(polymorphonuclear 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, Imunopathology 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 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; 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.
[0844] 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.
[0845] 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.
[0846] 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 B.sub.12
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.
[0847] 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.
[0848] 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.
[0849] 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
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.
[0850] 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.
[0851] 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.
[0852] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0853] 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 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.
[0854] 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.
[0855] 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.
[0856] 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.
[0857] Disorders in the male breast include, but are not limited
to, gynecomastia and carcinoma.
[0858] 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.
[0859] 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.
[0860] 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.
[0861] 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.
[0862] 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,
lynphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[0863] 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.
[0864] A novel human phospholipid scramblase-like gene sequence,
referred to as 32621, is provided. This gene sequence and variants
and fragments thereof are encompassed by the term "phospholipid
scramblase-like" molecules or sequences as used herein. The
phospholipid scramblase-like sequences find use in modulating a
phospholipid scramblase function. By "modulating" is intended the
upregulating or downregulating of a response. That is, the
compositions of the invention affect the target activity in either
a positive or negative fashion. The sequences of the invention find
use in modulating the immune, hematopoiesis, blood coagulation, and
plasma clotting systems.
[0865] The human phospholipid scramblase-like gene, clone 32621 was
identified in a human primary osteoblast cDNA library. Clone 32621
encodes an mRNA transcript having the corresponding cDNA set forth
in SEQ ID NO:16. This transcript has a 990 nucleotide open reading
frame (nucleotides 156-1142 of SEQ ID NO:16; SEQ ID NO:18), which
encodes a 329 amino acid protein (SEQ ID NO:17). A transmembrane
segment from amino acids (aa) 304-320 was predicted by MEMSAT.
Prosite program analysis was used to predict various sites within
the h32621 protein. N-glycosylation sites were predicted at aa
18-21, 92-95, and 147-150. Protein kinase C phosphorylation sites
were predicted at aa 170-172 and 204-206. Casein kinase II
phosphorylation sites were predicted at aa 7-10, 135-138, and
259-262. A tyrosine kinase phosphorylation site was predicted at aa
146-154. N-myristoylation sites were predicted at aa 3-8, 55-60,
216-221, and 281-286.
[0866] The 32621 protein shares approximately 45% identity to the
Mus musculus phospholipid scramblase-like and approximately 41%
identity to the Homo sapiens hMmTRA1b protein as determined by
pairwise alignment (FIG. 21).
[0867] The 32621 protein displays approximately 47% identity from
aa 206-321 to a ProDom consensus sequence found in murine
phospholipid scramblase-like 1; approximately 38% identity from aa
131-190 to a ProDom consensus sequence found in human phospholipid
scramblase-like (MmTRA1b); approximately 59% identity from aa
108-129 to a ProDom consensus sequence found in murine phospholipid
scramblase-like 1, human MmTRA1b, and murine transplantability
associated protein I (TRA1); and, approximately 38% identity from
aa 59-111 to a ProDom consensus sequence found in murine SRG3 and
human BAF155. Phospholipid scramblase-like 1 is a plasma membrane
protein that mediates accelerated transbilayer migration of
phospholipids upon binding calcium ions. See for example, Zhou et
al. (1998) Biochemistry 37:2356-2360. The plasma membrane protein,
human phospholipid scramblase-like, also mediates transbilayer
migration of phospholipids upon Ca.sup.2+ binding. The human
scramblase may play a central role in the initiation of fibrin clot
formation and in the recognition of apoptotic and injured cells by
the reticuloendothelial system. Defects or deficiency of this
scramblase causes Scott syndrome and possibly other bleeding
disorders. See, for example, Zho et al. (1997) J. Biol. Chem.
272:18240-18244, Kasukabe et al. (1998) Biochem. Biophys. Res.
Commun. 249:449-455, Basse et al. (1996) J. Biol. Chem.
271:17205-17210, and Zhou et al. (1998) Biochemistry 37:2356-2360.
Murine SRG3 belongs to a family of SWI/SNF related, matrix
associated, actin dependent regulator of chromatin assembly. Human
BAF155 is the 155 kDa subunit of the SWI/SNF complex (Wang et al.
(1996) Genes and Dev. 10:2117-2130). The sequences were identified
by the ProDom program, which is available from INRA, GREG (107/94),
MESR (ACC-SV13), the CNRS "Genome Initiative" and the European
Union. The ProDom Program (http://www.toulouse.inra.fr/prodom.html)
allows analysis of domain arrangements in proteins and protein
families. A detailed description of ProDom analysis can be found in
Corpet et al. (1999) Nuc. Acids Res. 27:263-267.
[0868] The human phospholipid scramblase-like sequences of the
invention are members of a family of molecules (PL flip/flop
genes). 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.
[0869] Preferred human phospholipid scramblase-like polypeptides of
the present invention have an amino acid sequence sufficiently
identical to the amino acid sequence of SEQ ID NO:17. 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 60%, 65%, 70%, 75% 85%, 90%, 95%, 96%, 97%, 98% or 99%
identity are defined herein as sufficiently identical.
[0870] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e., percent identity=number of identical
positions/total number 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.
[0871] The 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
http://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
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. 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.
[0872] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of Karlin and
Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, 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. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12, to obtain nucleotide sequences homologous
to 32621-like 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 32621-like 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.
Alternatively, PSI-Blast can be used to perform an iterated search
that detects distant relationships between molecules. See Altschul
et al. (1997) supra. 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
http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting
example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Myers and Miller (1988) CABIOS
4:11-17. Such an algorithm is incorporated into the ALIGN program
(version 2.0), which is part of the GCG 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.
[0873] Accordingly, another embodiment of the invention features
isolated human phospholipid scramblase-like proteins and
polypeptides having a human phospholipid scramblase-like protein
activity. As used interchangeably herein, a "human phospholipid
scramblase-like protein activity", "biological activity of a human
phospholipid scramblase-like protein", or "functional activity of a
human phospholipid scramblase-like protein" refers to an activity
exerted by a human phospholipid scramblase-like protein,
polypeptide, or nucleic acid molecule on a human phospholipid
scramblase-like responsive cell as determined in vivo, or in vitro,
according to standard assay techniques. A human phospholipid
scramblase-like 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 signaling activity
mediated by interaction of the human phospholipid scramblase-like
protein with a second protein. In a preferred embodiment, a human
phospholipid scramblase-like activity includes at least one or more
of the following activities: modulating (stimulating and/or
enhancing or inhibiting) phospholipid redistribution in the plasma
membrane.
[0874] An "isolated" or "purified" human phospholipid
scramblase-like 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 human phospholipid
scramblase-like 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. A human
phospholipid scramblase-like protein that is substantially free of
cellular material includes preparations of human phospholipid
scramblase-like protein having less than about 30%, 20%, 10%, or 5%
(by dry weight) of non-human phospholipid scramblase-like protein
(also referred to herein as a "contaminating protein"). When the
human phospholipid scramblase-like 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 human phospholipid
scramblase-like 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-human
phospholipid scramblase-like chemicals.
[0875] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[0876] One aspect of the invention pertains to isolated nucleic
acid molecules comprising nucleotide sequences encoding human
phospholipid scramblase-like proteins and polypeptides or
biologically active portions thereof, as well as nucleic acid
molecules sufficient for use as hybridization probes to identify
human phospholipid scramblase-like-encoding nucleic acids (e.g.,
human phospholipid scramblase-like mRNA) and fragments for use as
PCR primers for the amplification or mutation of human phospholipid
scramblase-like 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.
[0877] Nucleotide sequences encoding the human phospholipid
scramblase-like proteins of the present invention include sequences
set forth in SEQ ID NO:17 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 human
phospholipid scramblase-like protein encoded by these nucleotide
sequences is set forth in SEQ ID NO:16. The invention also
encompasses nucleic acid molecules comprising nucleotide sequences
encoding partial-length human phospholipid scramblase-like
proteins, including the sequence set forth in SEQ ID NO:17, and
complements thereof.
[0878] Nucleic acid molecules that are fragments of these human
phospholipid scramblase-like nucleotide sequences are also
encompassed by the present invention. By "fragment" is intended a
portion of the nucleotide sequence encoding a human phospholipid
scramblase-like protein. A fragment of a human phospholipid
scramblase-like nucleotide sequence may encode a biologically
active portion of a human phospholipid scramblase-like 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 a human phospholipid scramblase-like protein can be
prepared by isolating a portion of one of the human phospholipid
scramblase-like nucleotide sequences of the invention, expressing
the encoded portion of the human phospholipid scramblase-like
protein (e.g., by recombinant expression in vitro), and assessing
the activity of the encoded portion of the human phospholipid
scramblase-like protein. Nucleic acid molecules that are fragments
of a human phospholipid scramblase-like 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, 1500 nucleotides, or up to the
number of nucleotides present in a full-length human phospholipid
scramblase-like nucleotide sequence disclosed herein (for example,
1542 nucleotides for SEQ ID NO:16) depending upon the intended use.
Alternatively, a nucleic acid molecules that is a fragment of an
32621-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,
1500-1542 of SEQ ID NO:16 or 18.
[0879] 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.
[0880] A fragment of a human phospholipid scramblase-like
nucleotide sequence that encodes a biologically active portion of a
human phospholipid scramblase-like protein of the invention will
encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,
or 300 contiguous amino acids, or up to the total number of amino
acids present in a full-length human phospholipid scramblase-like
protein of the invention (for example, 329 amino acids for SEQ ID
NO:17). Alternatively, a fragment of a polypeptide of the present
invention comprises an amino acid sequence consisting of amino acid
residues 1-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140,
140-160, 160-180, 180-200, 200-220, 220-240, 240-260, 260-280,
280-300, 300-320, 320-329 of SEQ ID NO:17. Fragments of a human
phospholipid scramblase-like nucleotide sequence that are useful as
hybridization probes for PCR primers generally need not encode a
biologically active portion of a human phospholipid scramblase-like
protein.
[0881] Nucleic acid molecules that are variants of the human
phospholipid scramblase-like nucleotide sequences disclosed herein
are also encompassed by the present invention. "Variants" of the
human phospholipid scramblase-like nucleotide sequences include
those sequences that encode the human phospholipid scramblase-like
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 human
phospholipid scramblase-like 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 human phospholipid scramblase-like
nucleotide sequence will encode a human phospholipid
scramblase-like 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 a human phospholipid scramblase-like protein
disclosed herein.
[0882] In addition to the human phospholipid scramblase-like
nucleotide sequences shown in SEQ ID NO:16 it will be appreciated
by those skilled in the art that DNA sequence polymorphisms that
lead to changes in the amino acid sequences of human phospholipid
scramblase-like proteins may exist within a population (e.g., the
human population). Such genetic polymorphism in a human
phospholipid scramblase-like 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 a human phospholipid scramblase-like protein, preferably a
mammalian human phospholipid scramblase-like protein. As used
herein, the phrase "allelic variant" refers to a nucleotide
sequence that occurs at a human phospholipid scramblase-like 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 human phospholipid scramblase-like
gene. Any and all such nucleotide variations and resulting amino
acid polymorphisms or variations in a human phospholipid
scramblase-like sequence that are the result of natural allelic
variation and that do not alter the functional activity of human
phospholipid scramblase-like proteins are intended to be within the
scope of the invention.
[0883] Moreover, nucleic acid molecules encoding human phospholipid
scramblase-like proteins from other species (human phospholipid
scramblase-like homologues), which have a nucleotide sequence
differing from that of the human phospholipid scramblase-like
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 phospholipid
scramblase-like cDNA of the invention can be isolated based on
their identity to the human phospholipid scramblase-like 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.
[0884] In addition to naturally-occurring allelic variants of the
human phospholipid scramblase-like 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 human phospholipid scramblase-like
proteins, without altering the biological activity of the human
phospholipid scramblase-like proteins. Thus, an isolated nucleic
acid molecule encoding a human phospholipid scramblase-like protein
having a sequence that differs from that of SEQ ID NO:16 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.
[0885] 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 a human phospholipid scramblase-like protein (e.g., the
sequence of SEQ ID NO:17) 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 growth factor and cytokine receptor signature 2 sequence and
the U-PAR/Ly-6 domain sequence of SEQ ID NO:17, where such residues
are essential for protein activity.
[0886] Alternatively, variant human phospholipid scramblase-like
nucleotide sequences can be made by introducing mutations randomly
along all or part of a human phospholipid scramblase-like coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for human phospholipid scramblase-like
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.
[0887] Thus the nucleotide sequences of the invention include the
sequences disclosed herein as well as fragments and variants
thereof. The human phospholipid scramblase-like nucleotide
sequences of the invention, and fragments and variants thereof, can
be used as probes and/or primers to identify and/or clone human
phospholipid scramblase-like homologues in other cell types, e.g.,
from other tissues, as well as human phospholipid scramblase-like
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 a human
phospholipid scramblase-like protein, such as by measuring levels
of a human phospholipid scramblase-like-encoding nucleic acid in a
sample of cells from a subject, e.g., detecting human phospholipid
scramblase-like mRNA levels or determining whether a genomic human
phospholipid scramblase-like gene has been mutated or deleted.
[0888] 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). Human phospholipid
scramblase-like nucleotide sequences isolated based on their
sequence identity to the human phospholipid scramblase-like
nucleotide sequences set forth herein or to fragments and variants
thereof are encompassed by the present invention.
[0889] In a hybridization method, all or part of a known human
phospholipid scramblase-like 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 human phospholipid
scramblase-like nucleotide sequence disclosed herein. Degenerate
primers designed on the basis of conserved nucleotides or amino
acid residues in a known human phospholipid scramblase-like
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 a human phospholipid scramblase-like 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.
[0890] For example, in one embodiment, a previously unidentified
human phospholipid scramblase-like nucleic acid molecule hybridizes
under stringent conditions to a probe that is a nucleic acid
molecule comprising one of the human phospholipid scramblase-like
nucleotide sequences of the invention or a fragment thereof. In
another embodiment, the previously unknown human phospholipid
scramblase-like 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 human phospholipid scramblase-like nucleotide
sequences disclosed herein or a fragment thereof.
[0891] Accordingly, in another embodiment, an isolated previously
unknown human phospholipid scramblase-like 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:17 or a complement, fragment, or
variant thereof.
[0892] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences 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, 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 that hybridizes under stringent
conditions to a 32621-like 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).
[0893] Thus, in addition to the human phospholipid scramblase-like
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 human phospholipid
scramblase-like nucleotide sequences disclosed herein or variants
and fragments thereof.
[0894] 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 human phospholipid scramblase-like
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 a human
phospholipid scramblase-like protein. The noncoding regions are the
5' and 3' sequences that flank the coding region and are not
translated into amino acids.
[0895] Given the coding-strand sequence encoding a human
phospholipid scramblase-like protein disclosed herein (e.g., SEQ ID
NO:17), 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 human phospholipid scramblase-like mRNA, but more
preferably is an oligonucleotide that is antisense to only a
portion of the coding or noncoding region of human phospholipid
scramblase-like mRNA. For example, the antisense oligonucleotide
can be complementary to the region surrounding the translation
start site of human phospholipid scramblase-like 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.
[0896] 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).
[0897] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a human phospholipid scramblase-like 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.
[0898] 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).
[0899] 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 human
phospholipid scramblase-like mRNA transcripts to thereby inhibit
translation of human phospholipid scramblase-like mRNA. A ribozyme
having specificity for a human phospholipid
scramblase-like-encoding nucleic acid can be designed based upon
the nucleotide sequence of a human phospholipid scramblase-like
cDNA disclosed herein (e.g., SEQ ID NO:17). 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, human phospholipid scramblase-like 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.
[0900] The invention also encompasses nucleic acid molecules that
form triple helical structures. For example, human phospholipid
scramblase-like gene expression can be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
human phospholipid scramblase-like protein (e.g., the human
phospholipid scramblase-like promoter and/or enhancers) to form
triple helical structures that prevent transcription of the human
phospholipid scramblase-like 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.
[0901] 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 mirnics, 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.
[0902] PNAs of a human phospholipid scramblase-like 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).
[0903] In another embodiment, PNAs of a human phospholipid
scramblase-like 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 Human Phospholipid Scramblase-Like Proteins and
Anti-Human Phospholipid Scramblase-Like Antibodies
[0904] Human phospholipid scramblase-like proteins are also
encompassed within the present invention. By "human phospholipid
scramblase-like protein" is intended a protein having the amino
acid sequence set forth in SEQ ID NO:17, as well as fragments,
biologically active portions, and variants thereof.
[0905] "Fragments" or "biologically active portions" include
polypeptide fragments suitable for use as immunogens to raise
anti-human phospholipid scramblase-like antibodies. Fragments
include peptides comprising amino acid sequences sufficiently
identical to or derived from the amino acid sequence of a human
phospholipid scramblase-like protein, or partial-length protein, of
the invention and exhibiting at least one activity of a human
phospholipid scramblase-like protein, but which include fewer amino
acids than the full-length (SEQ ID NO:17) human phospholipid
scramblase-like protein disclosed herein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the human phospholipid scramblase-like protein. A
biologically active portion of a human phospholipid scramblase-like
protein can be a polypeptide which 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 human phospholipid
scramblase-like protein. As used here, a fragment comprises at
least 5 contiguous amino acids of SEQ ID NO:17. The invention
encompasses other fragments, however, such as any fragment in the
protein greater than 6, 7, 8, or 9 amino acids.
[0906] 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%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identical to the amino acid sequence of SEQ ID NO:17.
Variants also include polypeptides encoded by a nucleic acid
molecule that hybridizes to the nucleic acid molecules of SEQ ID
NO:16, SEQ ID NO:18, 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:17. 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 generally retain the
functional activity of the 32621-like proteins of the invention.
Variants include polypeptides that differ in amino acid sequence
due to natural allelic variation or mutagenesis.
[0907] In one embodiment, a 32621-like protein includes at least
one transmembrane domain. As used herein, the term "transmembrane
domain" includes an amino acid sequence of about 15 amino acid
residues in length that spans a phospholipid membrane. More
preferably, a transmembrane domain includes about at least 16, 18,
or 20 amino acid residues and spans a phospholipid membrane.
Transmembrane domains are rich in hydrophobic residues, and
typically have an .alpha.-helical structure. In a preferred
embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the
amino acids of a transmembrane domain are hydrophobic, e.g.,
leucines, isoleucines, tyrosines, or tryptophans. Transmembrane
domains are described in, for example,
http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N.
et al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of
which are incorporated herein by reference.
[0908] In a preferred embodiment, a 32621-like polypeptide or
protein has at least one transmembrane domain or a region which
includes at least 15, 16, 18, or 20 amino acid residues and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity
with a "transmembrane domain," e.g., at least one transmembrane
domain of human 32621-like (e.g., amino acid residues 304-320 of
SEQ ID NO:17).
[0909] In another embodiment, a 32621-like protein includes at
least one "non-transmembrane domain." As used herein,
"non-transmembrane domains" are domains that reside outside of the
membrane. When referring to plasma membranes, non-transmembrane
domains include extracellular domains (i.e., outside of the cell)
and intracellular domains (i.e., within the cell). When referring
to membrane-bound proteins found in intracellular organelles (e.g.,
mitochondria, endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally occurring 32621-like, or 32621 like protein.
[0910] In a preferred embodiment, a 32621-like polypeptide or
protein has a "non-transmembrane domain" or a region which includes
at least about 1-312, preferably about 200-312, more preferably
about 230-300, and even more preferably about 240-280 amino acid
residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100%
sequence identity with a "non-transmembrane domain", e.g., a
non-transmembrane domain of human 32621-like (e.g., residues 1-303
or 321-329 of SEQ ID NO:17). Preferably, a non-transmembrane domain
is capable of catalytic activity (e.g., phospholipid scramblase
activity).
[0911] A non-transmembrane domain located at the N-terminus of a
32621-like protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-303, preferably about 30-303, more
preferably about 50-303, or even more preferably about 80-290 amino
acid residues in length and is located outside the boundaries of a
membrane. For example, an N-terminal non-transmembrane domain is
located at about amino acid residues 1-303 of SEQ ID NO:17.
[0912] Similarly, a non-transmembrane domain located at the
C-terminus of a 32621-like protein or polypeptide is referred to
herein as a "C-terminal non-transmembrane domain." As used herein,
an "C-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-300, preferably about 15-290, preferably
about 20-270, more preferably about 25-255 amino acid residues in
length and is located outside the boundaries of a membrane. For
example, an C-terminal non-transmembrane domain is located at about
amino acid residues 321-329 of SEQ ID NO:17.
[0913] The invention also provides human phospholipid
scramblase-like chimeric or fusion proteins. As used herein, a
human phospholipid scramblase-like "chimeric protein" or "fusion
protein" comprises a human phospholipid scramblase-like polypeptide
operably linked to a non-human phospholipid scramblase-like
polypeptide. A "human phospholipid scramblase-like polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to a human phospholipid scramblase-like protein, whereas a
"non-human phospholipid scramblase-like polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein that is not substantially identical to the human
phospholipid scramblase-like protein, e.g., a protein that is
different from the human phospholipid scramblase-like protein and
which is derived from the same or a different organism. Within a
human phospholipid scramblase-like fusion protein, the human
phospholipid scramblase-like polypeptide can correspond to all or a
portion of a human phospholipid scramblase-like protein, preferably
at least one biologically active portion of a human phospholipid
scramblase-like protein. Within the fusion protein, the term
"operably linked" is intended to indicate that the human
phospholipid scramblase-like polypeptide and the non-human
phospholipid scramblase-like polypeptide are fused in-frame to each
other. The non-human phospholipid scramblase-like polypeptide can
be fused to the N-terminus or C-terminus of the human phospholipid
scramblase-like polypeptide.
[0914] One useful fusion protein is a GST-human phospholipid
scramblase-like fusion protein in which the human phospholipid
scramblase-like sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of
recombinant human phospholipid scramblase-like proteins.
[0915] In yet another embodiment, the fusion protein is a human
phospholipid scramblase-like-immunoglobulin fusion protein in which
all or part of a human phospholipid scramblase-like protein is
fused to sequences derived from a member of the immunoglobulin
protein family. The human phospholipid
scramblase-like-immunoglobulin fusion proteins of the invention can
be incorporated into pharmaceutical compositions and administered
to a subject to inhibit an interaction between a human phospholipid
scramblase-like ligand and a human phospholipid scramblase-like
protein on the surface of a cell, thereby suppressing human
phospholipid scramblase-like-mediated signal transduction in vivo.
The human phospholipid scramblase-like-immunoglobulin fusion
proteins can be used to affect the bioavailability of a human
phospholipid scramblase-like cognate ligand. Inhibition of the
human phospholipid scramblase-like ligand/human phospholipid
scramblase-like interaction may be useful therapeutically.
Moreover, the human phospholipid scramblase-like-immunoglobulin
fusion proteins of the invention can be used as immunogens to
produce anti-human phospholipid scramblase-like antibodies in a
subject, to purify human phospholipid scramblase-like ligands, and
in screening assays to identify molecules that inhibit the
interaction of a human phospholipid scramblase-like protein with a
human phospholipid scramblase-like ligand.
[0916] Preferably, a human phospholipid scramblase-like 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, a human phospholipid
scramblase-like-encoding nucleic acid can be cloned into a
commercially available expression vector such that it is linked
in-frame to an existing fusion moiety.
[0917] Variants of the human phospholipid scramblase-like proteins
can function as either human phospholipid scramblase-like agonists
(mimetics) or as human phospholipid scramblase-like antagonists.
Variants of the human phospholipid scramblase-like protein can be
generated by mutagenesis, e.g., discrete point mutation or
truncation of the human phospholipid scramblase-like protein. An
agonist of the human phospholipid scramblase-like protein can
retain substantially the same, or a subset, of the biological
activities of the naturally occurring form of the human
phospholipid scramblase-like protein. An antagonist of the human
phospholipid scramblase-like protein can inhibit one or more of the
activities of the naturally occurring form of the human
phospholipid scramblase-like protein by, for example, competitively
binding to a downstream or upstream member of a cellular signaling
cascade that includes the human phospholipid scramblase-like
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 human phospholipid scramblase-like
proteins.
[0918] Variants of a human phospholipid scramblase-like protein
that function as either human phospholipid scramblase-like agonists
or as human phospholipid scramblase-like antagonists can be
identified by screening combinatorial libraries of mutants, e.g.,
truncation mutants, of a human phospholipid scramblase-like protein
for human phospholipid scramblase-like protein agonist or
antagonist activity. In one embodiment, a variegated library of
human phospholipid scramblase-like variants is generated by
combinatorial mutagenesis at the nucleic acid level and is encoded
by a variegated gene library. A variegated library of human
phospholipid scramblase-like variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential human phospholipid scramblase-like sequences is
expressible as individual polypeptides, or alternatively, as a set
of larger fusion proteins (e.g., for phage display) containing the
set of human phospholipid scramblase-like sequences therein. There
are a variety of methods that can be used to produce libraries of
potential human phospholipid scramblase-like 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 human phospholipid
scramblase-like 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).
[0919] In addition, libraries of fragments of a human phospholipid
scramblase-like protein coding sequence can be used to generate a
variegated population of human phospholipid scramblase-like
fragments for screening and subsequent selection of variants of a
human phospholipid scramblase-like protein. In one embodiment, a
library of coding sequence fragments can be generated by treating a
double-stranded PCR fragment of a human phospholipid
scramblase-like 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 human phospholipid
scramblase-like protein.
[0920] 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 human phospholipid scramblase-like 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 human phospholipid
scramblase-like variants (Arkin and Yourvan (1992) Proc. Natl.
Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
[0921] An isolated human phospholipid scramblase-like polypeptide
of the invention can be used as an immunogen to generate antibodies
that bind human phospholipid scramblase-like proteins using
standard techniques for polyclonal and monoclonal antibody
preparation. The full-length human phospholipid scramblase-like
protein can be used or, alternatively, the invention provides
antigenic peptide fragments of human phospholipid scramblase-like
proteins for use as immunogens. The antigenic peptide of a human
phospholipid scramblase-like protein comprises at least 8,
preferably 10, 15, 20, or 30 amino acid residues of the amino acid
sequence shown in SEQ ID NO:17 and encompasses an epitope of a
human phospholipid scramblase-like protein such that an antibody
raised against the peptide forms a specific immune complex with the
human phospholipid scramblase-like protein. Preferred epitopes
encompassed by the antigenic peptide are regions of a human
phospholipid scramblase-like protein that are located on the
surface of the protein, e.g., hydrophilic regions.
[0922] Accordingly, another aspect of the invention pertains to
anti-human phospholipid scramblase-like polyclonal and monoclonal
antibodies that bind a human phospholipid scramblase-like protein.
Polyclonal anti-human phospholipid scramblase-like antibodies can
be prepared by immunizing a suitable subject (e.g., rabbit, goat,
mouse, or other mammal) with a human phospholipid scramblase-like
immunogen. The anti-human phospholipid scramblase-like 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 human phospholipid scramblase-like
protein. At an appropriate time after immunization, e.g., when the
anti-human phospholipid scramblase-like 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 266:550-52; 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).
[0923] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-human phospholipid scramblase-like
antibody can be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage
display library) with a human phospholipid scramblase-like protein
to thereby isolate immunoglobulin library members that bind the
human phospholipid scramblase-like 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.TM. Phage Display Kit,
Catalog No. 240612). Additionally, examples of methods and reagents
particularly amenable for use in generating and screening antibody
display library can be found in, for example, 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.
[0924] Additionally, recombinant anti-human phospholipid
scramblase-like 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 86/01533 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.
[0925] 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.
[0926] 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).
[0927] An anti-human phospholipid scramblase-like antibody (e.g.,
monoclonal antibody) can be used to isolate human phospholipid
scramblase-like proteins by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-human phospholipid
scramblase-like antibody can facilitate the purification of natural
human phospholipid scramblase-like protein from cells and of
recombinantly produced human phospholipid scramblase-like protein
expressed in host cells. Moreover, an anti-human phospholipid
scramblase-like antibody can be used to detect human phospholipid
scramblase-like protein (e.g., in a cellular lysate or cell
supernatant) in order to evaluate the abundance and pattern of
expression of the human phospholipid scramblase-like protein.
Anti-human phospholipid scramblase-like 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.
[0928] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). 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.
[0929] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84:Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
III. Recombinant Expression Vectors and Host Cells
[0930] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
human phospholipid scramblase-like 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.
[0931] 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., human phospholipid scramblase-like
proteins, mutant forms of human phospholipid scramblase-like
proteins, 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 such codon usage are well known in
the art.
[0932] The recombinant expression vectors of the invention can be
designed for expression of human phospholipid scramblase-like
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 11d
(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.
[0933] Suitable eukaryotic host cells include insect cells
(examples of Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers (1989) Virology 170:31-39));
yeast cells (examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943),
pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and 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.
[0934] 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 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.
[0935] 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 (Banerji et al. (1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application 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 a-fetoprotein promoter
(Campes and Tilghman (1989) Genes Dev. 3:537-546), and the
like.
[0936] 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 human phospholipid
scramblase-like 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).
[0937] 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,
DEAF-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.
[0938] 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 that confer resistance to drugs, such as
G418, hygromycin, and methotrexate. Nucleic acid encoding a
selectable marker can be introduced into a host cell on the same
vector as that encoding a human phospholipid scramblase-like
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).
[0939] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) human phospholipid scramblase-like protein. Accordingly,
the invention further provides methods for producing human
phospholipid scramblase-like protein using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of the invention, into which a recombinant expression
vector encoding a human phospholipid scramblase-like protein has
been introduced, in a suitable medium such that human phospholipid
scramblase-like protein is produced. In another embodiment, the
method further comprises isolating human phospholipid
scramblase-like protein from the medium or the host cell.
[0940] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which human phospholipid scramblase-like-coding sequences
have been introduced. Such host cells can then be used to create
nonhuman transgenic animals in which exogenous human phospholipid
scramblase-like sequences have been introduced into their genome or
homologous recombinant animals in which endogenous human
phospholipid scramblase-like sequences have been altered. Such
animals are useful for studying the function and/or activity of
human phospholipid scramblase-like genes and proteins and for
identifying and/or evaluating modulators of human phospholipid
scramblase-like 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 human
phospholipid scramblase-like 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.
[0941] A transgenic animal of the invention can be created by
introducing human phospholipid scramblase-like-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 human
phospholipid scramblase-like cDNA sequence can be introduced as a
transgene into the genome of a nonhuman animal. Alternatively, a
homologue of the mouse human phospholipid scramblase-like 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 human phospholipid scramblase-like transgene to
direct expression of human phospholipid scramblase-like 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
human phospholipid scramblase-like transgene in its genome and/or
expression of human phospholipid scramblase-like 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 human phospholipid
scramblase-like gene can further be bred to other transgenic
animals carrying other transgenes.
[0942] To create a homologous recombinant animal, one prepares a
vector containing at least a portion of a human phospholipid
scramblase-like 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 human phospholipid
scramblase-like gene. In a preferred embodiment, the vector is
designed such that, upon homologous recombination, the endogenous
human phospholipid scramblase-like 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 human
phospholipid scramblase-like 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 human phospholipid scramblase-like protein). In the
homologous recombination vector, the altered portion of the human
phospholipid scramblase-like gene is flanked at its 5' and 3' ends
by additional nucleic acid of the human phospholipid
scramblase-like gene to allow for homologous recombination to occur
between the exogenous human phospholipid scramblase-like gene
carried by the vector and an endogenous human phospholipid
scramblase-like gene in an embryonic stem cell. The additional
flanking human phospholipid scramblase-like 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 human phospholipid scramblase-like gene has
homologously recombined with the endogenous human phospholipid
scramblase-like 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/Techniques 2:823-829 and in
PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169.
[0943] 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.
[0944] 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
[0945] The human phospholipid scramblase-like nucleic acid
molecules, human phospholipid scramblase-like proteins, and
anti-human phospholipid scramblase-like 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.
[0946] 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.
[0947] 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.
[0948] 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.
[0949] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the skill 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.
[0950] 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, glycerin, 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.
[0951] 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.TM. (BASF; Parsippany, N.J.), or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, and 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.
[0952] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a human phospholipid
scramblase-like protein or anti-human phospholipid scramblase-like
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.
[0953] 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.
[0954] 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.
[0955] 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.
[0956] 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.
[0957] 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.
[0958] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
V. Uses and Methods of the Invention
[0959] 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
human phospholipid scramblase-like protein (e.g., via a recombinant
expression vector in a host cell in gene therapy applications), to
detect human phospholipid scramblase-like mRNA (e.g., in a
biological sample) or a genetic lesion in a human phospholipid
scramblase-like gene, and to modulate human phospholipid
scramblase-like activity. In addition, the human phospholipid
scramblase-like proteins can be used to screen drugs or compounds
that modulate the immune, hemopoetic, and blood clotting responses
as well as to treat disorders characterized by insufficient or
excessive production of human phospholipid scramblase-like protein
or production of human phospholipid scramblase-like protein forms
that have decreased or aberrant activity compared to human
phospholipid scramblase-like wild type protein. In addition, the
anti-human phospholipid scramblase-like antibodies of the invention
can be used to detect and isolate human phospholipid
scramblase-like proteins and modulate human phospholipid
scramblase-like activity.
[0960] A. Screening Assays
[0961] 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 human phospholipid
scramblase-like proteins or have a stimulatory or inhibitory effect
on, for example, human phospholipid scramblase-like expression or
human phospholipid scramblase-like activity.
[0962] 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).
[0963] 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.
[0964] 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).
[0965] Determining the ability of the test compound to bind to the
human phospholipid scramblase-like 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 human
phospholipid scramblase-like 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.
[0966] In a similar manner, one may determine the ability of the
human phospholipid scramblase-like protein to bind to or interact
with a human phospholipid scramblase-like target molecule. By
"target molecule" is intended a molecule with which a human
phospholipid scramblase-like protein binds or interacts in nature.
In a preferred embodiment, the ability of the human phospholipid
scramblase-like protein to bind to or interact with a human
phospholipid scramblase-like target molecule(s) can be determined
by monitoring the activity of the target molecule. For example, the
activity of the PS scramblase can be monitored by detecting the
translocation of phospholipids in the plasma membrane in response
to elevated Ca.sup.+2 (Zhao, J. et al. (1998) J. Biol. Chem.
273(12): 6603-6606.
[0967] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting a human phospholipid
scramblase-like protein or biologically active portion thereof with
a test compound and determining the ability of the test compound to
bind to the human phospholipid scramblase-like protein or
biologically active portion thereof. Binding of the test compound
to the human phospholipid scramblase-like protein can be determined
either directly or indirectly as described above. In a preferred
embodiment, the assay includes contacting the human phospholipid
scramblase-like protein or biologically active portion thereof with
a known compound that binds human phospholipid scramblase-like
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 human phospholipid scramblase-like
protein or biologically active portion thereof as compared to the
known compound.
[0968] In another embodiment, an assay is a cell-free assay
comprising contacting human phospholipid scramblase-like 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 human phospholipid
scramblase-like protein or biologically active portion thereof.
Determining the ability of the test compound to modulate the
activity of a human phospholipid scramblase-like protein can be
accomplished, for example, by determining the ability of the human
phospholipid scramblase-like protein to bind to a human
phospholipid scramblase-like 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 a human phospholipid scramblase-like protein can be
accomplished by determining the ability of the human phospholipid
scramblase-like protein to further modulate a human phospholipid
scramblase-like target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as previously
described.
[0969] In yet another embodiment, the cell-free assay comprises
contacting the human phospholipid scramblase-like protein or
biologically active portion thereof with a known compound that
binds a human phospholipid scramblase-like 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 a human phospholipid scramblase-like
target molecule.
[0970] In the above-mentioned assays, it may be desirable to
immobilize either a human phospholipid scramblase-like 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/human phospholipid scramblase-like 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 human phospholipid
scramblase-like 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 human phospholipid
scramblase-like binding or activity determined using standard
techniques.
[0971] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either human phospholipid scramblase-like protein or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin. Biotinylated human phospholipid scramblase-like
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
a human phospholipid scramblase-like protein or target molecules
but which do not interfere with binding of the human phospholipid
scramblase-like protein to its target molecule can be derivatized
to the wells of the plate, and unbound target or human phospholipid
scramblase-like 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
human phospholipid scramblase-like protein or target molecule, as
well as enzyme-linked assays that rely on detecting an enzymatic
activity associated with the human phospholipid scramblase-like
protein or target molecule.
[0972] In another embodiment, modulators of human phospholipid
scramblase-like expression are identified in a method in which a
cell is contacted with a candidate compound and the expression of
human phospholipid scramblase-like mRNA or protein in the cell is
determined relative to expression of human phospholipid
scramblase-like 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 human phospholipid scramblase-like 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 human phospholipid scramblase-like mRNA or protein
expression. The level of human phospholipid scramblase-like mRNA or
protein expression in the cells can be determined by methods
described herein for detecting human phospholipid scramblase-like
mRNA or protein.
[0973] In yet another aspect of the invention, the human
phospholipid scramblase-like 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 human phospholipid
scramblase-like protein ("human phospholipid
scramblase-like-binding proteins" or "human phospholipid
scramblase-like-bp") and modulate human phospholipid
scramblase-like activity. Such human phospholipid
scramblase-like-binding proteins are also likely to be involved in
the propagation of signals by the human phospholipid
scramblase-like proteins as, for example, upstream or downstream
elements of the human phospholipid scramblase-like pathway.
[0974] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0975] B. Detection Assays
[0976] 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.
[0977] 1. Chromosome Mapping
[0978] The isolated complete or partial human phospholipid
scramblase-like gene sequences of the invention can be used to map
their respective human phospholipid scramblase-like genes on a
chromosome, thereby facilitating the location of gene regions
associated with genetic disease. Computer analysis of human
phospholipid scramblase-like 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 human phospholipid scramblase-like sequences
will yield an amplified fragment.
[0979] 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.
[0980] Other mapping strategies that can similarly be used to map a
human phospholipid scramblase-like 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 et
al. (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.
[0981] 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.
[0982] Another strategy to map the chromosomal location of human
phospholipid scramblase-like genes uses human phospholipid
scramblase-like polypeptides and fragments and sequences of the
present invention and antibodies specific thereto. This mapping can
be carried out by specifically detecting the presence of a human
phospholipid scramblase-like polypeptide in members of a panel of
somatic cell hybrids between cells of a first species of animal
from which the protein originates and cells from a second species
of animal, and then determining which somatic cell hybrid(s)
expresses the polypeptide and noting the chromosomes(s) from the
first species of animal that it contains. For examples of this
technique, see Pajunen et al. (1988) Cytogenet. Cell. Genet.
47:37-41 and Van Keuren et al. (1986) Hum. Genet. 74:34-40.
Alternatively, the presence of a human phospholipid scramblase-like
polypeptide in the somatic cell hybrids can be determined by
assaying an activity or property of the polypeptide, for example,
enzymatic activity, as described in Bordelon-Riser et al. (1979)
Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978) Proc.
Natl. Acad. Sci. USA 75:5640-5644.
[0983] 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.
[0984] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the human phospholipid scramblase-like 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.
[0985] 2. Tissue Typing
[0986] The human phospholipid scramblase-like 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).
[0987] 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 human phospholipid scramblase-like sequences of
the invention can be used to prepare two PCR primers from the 5'
and 3' ends of the sequences. These primers can then be used to
amplify an individual's DNA and subsequently sequence it.
[0988] 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 human phospholipid
scramblase-like 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:16 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:16
or SEQ ID NO:18, is used, a more appropriate number of primers for
positive individual identification would be 500 to 2,000.
[0989] 3. Use of Partial Human Phospholipid Scramblase-Like
Sequences in Forensic Biology
[0990] 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.
[0991] 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:16
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 human phospholipid
scramblase-like sequences or portions thereof, e.g., fragments
derived from the noncoding regions of SEQ ID NO:16 having a length
of at least 20 or 30 bases.
[0992] The human phospholipid scramblase-like 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 human
phospholipid scramblase-like probes, can be used to identify tissue
by species and/or by organ type.
[0993] In a similar fashion, these reagents, e.g., human
phospholipid scramblase-like 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).
[0994] C. Predictive Medicine
[0995] 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.
[0996] 1. Diagnostic Assays
[0997] One aspect of the present invention relates to diagnostic
assays for detecting human phospholipid scramblase-like protein
and/or nucleic acid expression as well as human phospholipid
scramblase-like activity, in the context of a biological sample. An
exemplary method for detecting the presence or absence of human
phospholipid scramblase-like 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 human phospholipid scramblase-like protein or
nucleic acid (e.g., mRNA, genomic DNA) that encodes human
phospholipid scramblase-like protein such that the presence of
human phospholipid scramblase-like 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.
[0998] "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.
[0999] A preferred agent for detecting human phospholipid
scramblase-like mRNA or genomic DNA is a labeled nucleic acid probe
capable of hybridizing to human phospholipid scramblase-like mRNA
or genomic DNA. The nucleic acid probe can be, for example, a
full-length human phospholipid scramblase-like nucleic acid, such
as the nucleic acid of SEQ ID NO:16, 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 human phospholipid scramblase-like
mRNA or genomic DNA. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
[1000] A preferred agent for detecting human phospholipid
scramblase-like protein is an antibody capable of binding to human
phospholipid scramblase-like protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').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.
[1001] 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 human
phospholipid scramblase-like mRNA, protein, or genomic DNA in a
biological sample in vitro as well as in vivo. For example, in
vitro techniques for detection of human phospholipid
scramblase-like mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of human
phospholipid scramblase-like protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations,
and immunofluorescence. In vitro techniques for detection of human
phospholipid scramblase-like genomic DNA include Southern
hybridizations. Furthermore, in vivo techniques for detection of
human phospholipid scramblase-like protein include introducing into
a subject a labeled anti-human phospholipid scramblase-like
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.
[1002] 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.
[1003] The invention also encompasses kits for detecting the
presence of human phospholipid scramblase-like 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 human
phospholipid scramblase-like protein such as for Scott syndrome, a
disorder in platelet clotting, or liver fibrosis. For example, the
kit can comprise a labeled compound or agent capable of detecting
human phospholipid scramblase-like protein or mRNA in a biological
sample and means for determining the amount of a human phospholipid
scramblase-like protein in the sample (e.g., an anti-human
phospholipid scramblase-like antibody or an oligonucleotide probe
that binds to DNA encoding a human phospholipid scramblase-like
protein, e.g., SEQ ID NO:17). 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 human phospholipid scramblase-like sequences if the amount of
human phospholipid scramblase-like protein or mRNA is above or
below a normal level.
[1004] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) that binds
to human phospholipid scramblase-like protein; and, optionally, (2)
a second, different antibody that binds to human phospholipid
scramblase-like 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 a human phospholipid
scramblase-like nucleic acid sequence or (2) a pair of primers
useful for amplifying a human phospholipid scramblase-like nucleic
acid molecule.
[1005] 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 human phospholipid scramblase-like
proteins.
[1006] 2. Other Diagnostic Assays
[1007] 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 phospholipid scramblase-like 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 phospholipid
scramblase-like nucleic acid, polypeptide, or antibody. 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.
[1008] The method can include contacting the phospholipid
scramblase-like 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 afirst 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.
[1009] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of a phospholipid scramblase-like sequence of the invention.
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. Thus, for example, the 32621 sequence set
forth in SEQ ID NO:16 encodes a phospholipid scramblase-like
polypeptide that is associated with blood coagulation, thus it is
useful for evaluating bleeding disorders.
[1010] The method can be used to detect single nucleotide
polymorphisms (SNPs), as described below.
[1011] 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
a phospholipid scramblase-like polypeptide of the invention or from
a cell or subject in which a phospholipid scramblase-like mediated
response has been elicited, e.g., by contact of the cell with a
phospholipid scramblase-like nucleic acid or protein of the
invention, or administration to the cell or subject a phospholipid
scramblase-like nucleic acid or protein of the invention;
contacting the array with one or more inquiry probes, wherein an
inquiry probe can be a nucleic acid, polypeptide, or antibody
(which is preferably other than a phospholipid scramblase-like
nucleic acid, polypeptide, or antibody of the invention); 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 a
phospholipid scramblase-like sequence of the invention (or does not
express as highly as in the case of the phospholipid
scramblase-like positive plurality of capture probes) or from a
cell or subject in which a phospholipid scramblase-like-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 phospholipid
scramblase-like nucleic acid, polypeptide, or antibody of the
invention), 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.
[1012] In another aspect, the invention features a method of
analyzing a phospholipid scramblase-like sequence of the invention,
e.g., analyzing structure, function, or relatedness to other
nucleic acid or amino acid sequences. The method includes:
providing a phospholipid scramblase-like nucleic acid or amino acid
sequence, e.g., the 32621 sequence set forth in SEQ ID NO:17 or a
portion thereof; comparing the phospholipid scramblase-like
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 the phospholipid scramblase-like
sequence of the invention.
[1013] The method can include evaluating the sequence identity
between a phospholipid scramblase-like sequence of the invention,
e.g., the 32621 sequence, and a database sequence. The method can
be performed by accessing the database at a second site, e.g., over
the internet.
[1014] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of a phospholipid scramblase-like
sequence of the invention, e.g., the 32621 sequence. 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 identical in sequence with one
another (except for differences in length). The oligonucleotides
can be provided with differential labels, such that an
oligonucleotides which hybridizes to one allele provides a signal
that is distinguishable from an oligonucleotides which hybridizes
to a second allele.
[1015] 3. Prognostic Assays
[1016] 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 human
phospholipid scramblase-like protein, human phospholipid
scramblase-like nucleic acid expression, or human phospholipid
scramblase-like 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 human phospholipid scramblase-like protein, human
phospholipid scramblase-like nucleic acid expression, or human
phospholipid scramblase-like activity.
[1017] Thus, the present invention provides a method in which a
test sample is obtained from a subject, and human phospholipid
scramblase-like protein or nucleic acid (e.g., mRNA, genomic DNA)
is detected, wherein the presence of human phospholipid
scramblase-like protein or nucleic acid is diagnostic for a subject
having or at risk of developing a disease or disorder associated
with aberrant human phospholipid scramblase-like 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.
[1018] 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 human phospholipid scramblase-like
activity) to effectively treat a disease or disorder associated
with aberrant human phospholipid scramblase-like expression or
activity. In this manner, a test sample is obtained and human
phospholipid scramblase-like protein or nucleic acid is detected.
The presence of human phospholipid scramblase-like protein or
nucleic acid is diagnostic for a subject that can be administered
the agent to treat a disorder associated with aberrant human
phospholipid scramblase-like expression or activity.
[1019] The methods of the invention can also be used to detect
genetic lesions or mutations in a human phospholipid
scramblase-like gene, thereby determining if a subject with the
lesioned gene is at risk for a disorder characterized by abnormal
platelet disfunction or clotting or some other immune or hemopoetic
disorder. 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 a human
phospholipid scramblase-like-protein, or the misexpression of the
human phospholipid scramblase-like 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
a human phospholipid scramblase-like gene; (2) an addition of one
or more nucleotides to a human phospholipid scramblase-like gene;
(3) a substitution of one or more nucleotides of a human
phospholipid scramblase-like gene; (4) a chromosomal rearrangement
of a human phospholipid scramblase-like gene; (5) an alteration in
the level of a messenger RNA transcript of a human phospholipid
scramblase-like gene; (6) an aberrant modification of a human
phospholipid scramblase-like gene, such as of the methylation
pattern of the genomic DNA; (7) the presence of a non-wild-type
splicing pattern of a messenger RNA transcript of a human
phospholipid scramblase-like gene; (8) a non-wild-type level of a
human phospholipid scramblase-like-protein; (9) an allelic loss of
a human phospholipid scramblase-like gene; and (10) an
inappropriate post-translational modification of a human
phospholipid scramblase-like-protein. As described herein, there
are a large number of assay techniques known in the art that can be
used for detecting lesions in a human phospholipid scramblase-like
gene. Any cell type or tissue, preferably peripheral blood cells,
in which human phospholipid scramblase-like proteins are expressed
may be utilized in the prognostic assays described herein.
[1020] 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 human phospholipid scramblase-like-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.
[1021] 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.
[1022] In an alternative embodiment, mutations in a human
phospholipid scramblase-like 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.
[1023] In other embodiments, genetic mutations in a human
phospholipid scramblase-like 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 human phospholipid
scramblase-like gene and detect mutations by comparing the sequence
of the sample human phospholipid scramblase-like 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).
[1024] Other methods for detecting mutations in the human
phospholipid scramblase-like 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.
[1025] 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 human phospholipid
scramblase-like 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 a human phospholipid
scramblase-like sequence, e.g., a wild-type human phospholipid
scramblase-like 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.
[1026] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in human phospholipid
scramblase-like 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).
[1027] 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).
[1028] 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.
[1029] 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.
[1030] 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 a human phospholipid scramblase-like gene.
[1031] 4. Pharmacogenomics
[1032] Agents, or modulators that have a stimulatory or inhibitory
effect on human phospholipid scramblase-like activity (e.g., human
phospholipid scramblase-like gene expression) as identified by a
screening assay described herein, can be administered to
individuals to treat (prophylactically or therapeutically)
disorders associated with aberrant human phospholipid
scramblase-like activity as well as to modulate the phenotype of an
immune, hemopeotic, or blood clotting 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 human phospholipid scramblase-like protein, expression
of human phospholipid scramblase-like nucleic acid, or mutation
content of human phospholipid scramblase-like genes in an
individual can be determined to thereby select appropriate agent(s)
for therapeutic or prophylactic treatment of the individual.
[1033] 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.
[1034] Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician may consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a phospholipid scramblase-like molecule or
phospholipid scramblase-like modulator of the invention as well as
tailoring the dosage and/or therapeutic regimen of treatment with a
phospholipid scramblase-like molecule or phospholipid
scramblase-like modulator of the invention.
[1035] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, an "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[1036] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., a phospholipid scramblase-like protein of
the present invention), all common variants of that gene can be
fairly easily identified in the population and it can be determined
if having one version of the gene versus another is associated with
a particular drug response.
[1037] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a phospholipid scramblase-like molecule or
phospholipid scramblase-like modulator of the present invention)
can give an indication whether gene pathways related to toxicity
have been turned on.
[1038] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a phospholipid scramblase-like
molecule or phospholipid scramblase-like modulator of the
invention, such as a modulator identified by one of the exemplary
screening assays described herein.
[1039] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the phospholipid
scramblase-like genes of the present invention, wherein these
products may be associated with resistance of the cells to a
therapeutic agent. Specifically, the activity of the proteins
encoded by the phospholipid scramblase-like genes of the present
invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., hepatic
stellate cells, will become sensitive to treatment with an agent
that the unmodified target cells were resistant to.
[1040] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a phospholipid scramblase-like protein
can be applied in clinical trials. For example, the effectiveness
of an agent determined by a screening assay as described herein to
increase phospholipid scramblase-like gene expression, protein
levels, or upregulate Phospholipid scramblase-like activity, can be
monitored in clinical trials of subjects exhibiting decreased
phospholipid scramblase-like gene expression, protein levels, or
downregulated phospholipid scramblase-like activity. Alternatively,
the effectiveness of an agent determined by a screening assay to
decrease phospholipid scramblase-like gene expression, protein
levels, or downregulate phospholipid scramblase-like activity, can
be monitored in clinical trials of subjects exhibiting increased
phospholipid scramblase-like gene expression, protein levels, or
upregulated phospholipid scramblase-like activity. In such clinical
trials, the expression or activity of a phospholipid
scramblase-like gene, and preferably, other genes that have been
implicated in, for example, a phospholipid
scramblase-like-associated disorder can be used as a "read out" or
markers of the phenotype of a particular cell.
[1041] 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.
[1042] Thus, the activity of human phospholipid scramblase-like
protein, expression of human phospholipid scramblase-like nucleic
acid, or mutation content of human phospholipid scramblase-like
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 a human phospholipid scramblase-like
modulator, such as a modulator identified by one of the exemplary
screening assays described herein.
[1043] 5. Monitoring of Effects During Clinical Trials
[1044] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of human phospholipid scramblase-like
genes 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 human phospholipid scramblase-like gene expression,
protein levels, or protein activity, can be monitored in clinical
trials of subjects exhibiting decreased or increased human
phospholipid scramblase-like gene expression, protein levels, or
protein activity. In such clinical trials, human phospholipid
scramblase-like 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.
[1045] 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 human phospholipid
scramblase-like activity (e.g., as identified in a screening assay
described herein) can be identified. Thus, to study the effect of
agents disorders, for example, in a clinical trial, cells can be
isolated and RNA prepared and analyzed for the levels of expression
of human phospholipid scramblase-like 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 human
phospholipid scramblase-like 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.
[1046] 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 a human phospholipid scramblase-like
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 human
phospholipid scramblase-like protein, mRNA, or genomic DNA in the
postadministration samples; (5) comparing the level of expression
or activity of the human phospholipid scramblase-like protein,
mRNA, or genomic DNA in the preadministration sample with the human
phospholipid scramblase-like 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 a human phospholipid
scramblase-like protein.
[1047] 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 human phospholipid scramblase-like expression or activity.
"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. Additionally, the compositions of the invention find use in
the treatment of disorders described herein. Thus, therapies for
disorders associated with aberrant human phospholipid scramblase
activity are encompassed herein. "Treatment" is herein 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. A "therapeutic agent"
includes, but is not limited to, small molecules, peptides,
antibodies, ribozymes and antisense oligonucleotides.
[1048] 1. Prophylactic Methods
[1049] In one aspect, the invention provides a method for
preventing in a subject a disease or condition associated with an
aberrant human phospholipid scramblase-like expression or activity
by administering to the subject an agent that modulates human
phospholipid scramblase-like expression or at least one human
phospholipid scramblase-like gene activity. Subjects at risk for a
disease that is caused, or contributed to, by aberrant human
phospholipid scramblase-like 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 human phospholipid scramblase-like aberrancy,
such that a disease or disorder is prevented or, alternatively,
delayed in its progression. Depending on the type of human
phospholipid scramblase-like aberrancy, for example, a human
phospholipid scramblase-like agonist or human phospholipid
scramblase-like antagonist agent can be used for treating the
subject. The appropriate agent can be determined based on screening
assays described herein.
[1050] 2. Therapeutic Methods
[1051] Another aspect of the invention pertains to methods of
modulating human phospholipid scramblase-like 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 human phospholipid scramblase-like
protein activity associated with the cell. An agent that modulates
human phospholipid scramblase-like protein activity can be an agent
as described herein, such as a nucleic acid or a protein, a
naturally-occurring cognate ligand of a human phospholipid
scramblase-like protein, a peptide, a human phospholipid
scramblase-like peptidomimetic, or other small molecule. In one
embodiment, the agent stimulates one or more of the biological
activities of human phospholipid scramblase-like protein. Examples
of such stimulatory agents include active human phospholipid
scramblase-like protein and a nucleic acid molecule encoding a
human phospholipid scramblase-like protein that has been introduced
into the cell. In another embodiment, the agent inhibits one or
more of the biological activities of human phospholipid
scramblase-like protein. Examples of such inhibitory agents include
antisense human phospholipid scramblase-like nucleic acid molecules
and anti-human phospholipid scramblase-like antibodies.
[1052] 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 a human phospholipid scramblase-like
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) human phospholipid
scramblase-like expression or activity. In another embodiment, the
method involves administering a human phospholipid scramblase-like
protein or nucleic acid molecule as therapy to compensate for
reduced or aberrant human phospholipid scramblase-like expression
or activity.
[1053] Stimulation of human phospholipid scramblase-like activity
is desirable in situations in which a human phospholipid
scramblase-like protein is abnormally downregulated and/or in which
increased human phospholipid scramblase-like activity is likely to
have a beneficial effect. Conversely, inhibition of human
phospholipid scramblase-like activity is desirable in situations in
which human phospholipid scramblase-like activity is abnormally
upregulated and/or in which decreased human phospholipid
scramblase-like activity is likely to have a beneficial effect.
[1054] This invention is further illustrated by the following
examples, which should not be construed as limiting.
EXPERIMENTAL
Example 1
Identification and Characterization of 32621 Human Scramblase
[1055] The human 32621-like sequence (SEQ ID NO:16), which is
approximately 1542 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
990 nucleotides (nucleotides 156-1145 of SEQ ID NO:16; SEQ ID
NO:18). The coding sequence encodes a 329 amino acid protein (SEQ
ID NO:17).
[1056] A search of the nucleotide and protein databases revealed
that 32621 encodes a precursor polypeptide that shares similarity
with several phospholipid scramblase proteins. An alignment of the
protein sequences having highest similarity to the 32621 precursor
polypeptide is shown in FIG. 21. The alignment was generated using
the Clustal method with PAM250 residue weight table and sequence
identities were determined by FASTA (Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85:2444-2448).
[1057] The 32621 protein displays similarity (approximately 45%
identity over the full amino acid sequence) to the murine
phospholipid scramblase-like polypeptide (SEQ ID NO:19; SP
Accession No. 2935163; Zhou et al (1998) Biochem. 37:2356-2360 (see
FIG. 21). It also displays similarity to the human Mm-1 cell
derived transplantability-associated gene 1b (approximately 41%
identity over the full amino acid sequence; SEQ ID NO:20; SP
Accession No. 3510297; Kasukabe et al. (1998) Biochem. Biophys.
Res. Comm. 249:449-455 (see FIG. 21).
Example 2
Tissue Distribution of 32621 mRNA
[1058] Expression levels of 32621 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. 24A-27.
[1059] 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 32621 cDNA (SEQ ID NO:16)
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.
[1060] TaqMan analysis of 32621 revealed expression in a number of
tissues, including the following: artery; vein; aortic SMC, smooth
muscle cells, early); aortic SMC late; static HUVEC, human
umbilical vein endothelial cells; shear HUVEC; heart; heart CHF,
congestive heart failure heart tissue; kidney; skeletal muscle;
adipose; pancreas; primary osteoblasts; osteoclasts; skin; spinal
cord; brain cortex; brain hypothalamus; nerve; DRG, dorsal root
ganglion; glial cells, astrocytes; glioblastoma; breast; breast
tumor; ovary; ovarian tumor; prostate; prostate tumor; prostate
epithelial cells; colon; colon tumor; lung; lung tumor; lung COPD,
chronic obstructive pulmonary diseased lung; colon IBD,
inflammatory bowel diseased colon; liver; liver fibrosis; dermal
cells; spleen; tonsil; lymph node; thymus; skin-decubitis;
synovium; bone marrow mononuclear cells; and activated peripheral
blood mononuclear cells. High expression of 32621 occurred in
aortic smooth muscle cells, HUVEC, brain cortex, brain
hypothalamus, normal ovary, and fibrotic liver cells. See FIGS.
24A-B.
[1061] Expression of 32621 was further observed in various cell
lines and tissues, including the following: conf HMVEC,
microvascular endothelial cells; fetal heart; normal atrium; normal
ventricle; heart diseased ventricle; normal kidney; kidney HT;
skeletal muscle; skeletal muscle; liver; liver with inflammation;
fetal adrenal; Wilms Tumor; spinal cord; and diseased cartilage.
Relative expression levels of 32621 were also determined in various
liver samples from animals fed modified diets. See FIGS. 25A-B and
26.
[1062] Expression of 32621 was observed in: aortic smooth muscle
cells (ASMC)-A1PO; ASMC-A2P3; ASMC-A3P4; ASMC-AL; coronary artery
smooth muscle cells (CASMC)-C1P3; CASMC-C2P3; CASMC-C5P0;
CASMC-C1P6; macrophage cells; macrophage cells treated with
interferon .gamma.; CD40+ macrophage cells; macrophage cells
treated with lipopolysaccharide; HUVEC, human umbilical vein
endothelial cells; HMVEC, human microvascular endothelial cells;
HAEC1, human aortic endothelial cells; HCAEC3, human coronary
arterial endothelial cells; HCRE; RPTE, renal proximal tubule
epithelial cells; MC; SKM1, myelogenous leukemia cells; and HLF,
hepatocellular carcinoma cell line. Of these cell types, 32621
expression was high in microvascular endothelial cells, with
microvascular endothelial cells exhibiting an expression level
about 1990 times higher than in macrophage cells. See FIG. 27.
Example 3
Recombinant Expression of 32621 in Bacterial Cells
[1063] In this example, the 32621-like sequence is expressed as a
recombinant glutathione-S-transferase (GST) fusion polypeptide in
E. coli and the fusion polypeptide is isolated and characterized.
Specifically, the 32621-like sequence is fused to GST and this
fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
Expression of the GST-32621-like fusion protein in PEB199 is
induced with IPTG. The recombinant fusion polypeptide is purified
from crude bacterial lysates of the induced PEB199 strain by
affinity chromatography on glutathione beads. Using polyacrylamide
gel electrophoretic analysis of the polypeptide purified from the
bacterial lysates, the molecular weight of the resultant fusion
polypeptide is determined.
Example 4
Expression of Recombinant 32621-Like Protein in COS Cells
[1064] To express the 32621-like 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 32621-like
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.
[1065] To construct the plasmid, the 32621-like 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 32621-like 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 32621-like 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 32621-like 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.
[1066] COS cells are subsequently transfected with the
32621-like-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 32621-like 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.
[1067] Alternatively, DNA containing the 32621-like 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 32621-like polypeptide is detected by
radiolabelling and immunoprecipitation using a 32621-like specific
monoclonal antibody.
[1068] 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.
[1069] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Chapter 5
27802, A Novel Adenylate Kinase
BACKGROUND OF THE INVENTION
[1070] 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 adenylate 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.
[1071] 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.
[1072] 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.
[1073] 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.
[1074] 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).
[1075] 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.
[1076] 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.
[1077] 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
[1078] 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:22. Further
provided are adenylate kinase polypeptides having an amino acid
sequence encoded by a nucleic acid molecule described herein.
[1079] 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.
[1080] 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.
[1081] 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.
[1082] 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.
[1083] 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.
[1084] 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.
[1085] 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.
[1086] 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.
[1087] 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.
[1088] 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.
[1089] 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.
[1090] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[1091] 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.
[1092] 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.
[1093] 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.
[1094] 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:22, 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:22, is
set forth in SEQ ID NO:21.
[1095] A novel human gene, termed clone h27802 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.
[1096] The novel h27802 adenylate kinase gene encodes an
approximately 1.45 Kb mRNA transcript having the corresponding cDNA
set forth in SEQ ID NO:21. This transcript encodes a 258 amino acid
protein (SEQ ID NO:22). An analysis of the full-length h27802
polypeptide predicts that the N-terminal 56 amino acids may
represent a region comprising a signal peptide. Prosite program
analysis was used to predict various sites within the h27802
protein. See FIG. 31.
[1097] The h27802 adenylate kinase protein possesses adenylate
kinase domain sequences, as shown in FIG. 34. There are 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).
[1098] Human 27802 was aligned with two consensus amino acid
sequences for adenylate kinase domains derived from hidden Markov
models. 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 the information
available at URL
www.psc.edu/general/software/packages/pfam/pfam.html. The first
adenylate kinase domain (SEQ ID NO:24) aligns with amino acids
41-120 of SEQ ID NO:22 and the second adenylate kinase domain (SEQ
ID NO:25) aligns with amino acids 201-251 of SEQ ID NO:22 (see FIG.
34).
[1099] As used herein, the term "adenylate kinase domain" includes
an amino acid sequence of about 30-200 amino acid residues in
length and having a bit score for the alignment of the sequence to
the adenylate kinase domain (HMM) of at least 8. Preferably, an
adenylate kinase domain includes at least about 40-150 amino acids,
more preferably about 50-100 amino acid residues, or about 50-80
amino acids and has a bit score for the alignment of the sequence
to the adenylate kinase domain (HMM) of at least 16 or greater. The
adenylate kinase domain (HMM) has been assigned the PFAM Accession
PF00406; see also the information available at URL
pfam.wustl.edu/).
[1100] In a preferred embodiment a 27802-like polypeptide or
protein has "adenylate kinase domains" or regions which include at
least about 30-200, more preferably about 40-100, or 50-80 amino
acid residues and has at least about 60%, 70%, 80%, 85%, 90%, 95%,
99%, or 100% sequence identity with an "adenylate kinase domain,"
e.g., the adenylate kinase domain of human 27802-like (e.g., amino
acid residues 41-120 and 201-251 of SEQ ID NO:22).
[1101] To identify the presence of an adenylate kinase domain in a
27802-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 described at the URL
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.
[1102] A BLASTN search using the 27802 cDNA clone of the invention
as the subject was performed. The 27802 clone shares 97% to 100%
identity to the top five search hits. These hits are 1) a partial
cDNA clone isolated from a brain glioblastoma (Accession No:
AI359456) with homology to maize adenylate kinase; 2) a partial 3'
cDNA clone isolated from Jia bone marrow stroma (Accession No: A
WO69362); 3) a partial cDNA clone isolated from a brain
glioblastoma (Accession No: AI362274) with homology to maize
adenylate kinase; 4) a partial cDNA clone isolated from a brain
anaplastic oligodendroglioma (Accession No: AI826091) with homology
to maize adenylate kinase; and 5) a partial cDNA clone isolated
from adult heart (Accession No: CO3497).
[1103] The expression levels of 27802 were determined in various
tissues by quantitative PCR (FIG. 35). The highest levels of
expression of 27802 were observed in artery, kidney, brain cortex
and brain hypothalamus, ovary, lung (tumor), and tonsil. The
expression of 27802 in a tissue indicates that modulation of the
expression or activity of 27802 in that tissue may be used in the
treatment of disorders involving such a tissue.
[1104] 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.
[1105] In addition, the adenylate kinase 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,
but not limited to, human brain. In addition, expression of the
gene is also observed in lymphoma. 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.
[1106] 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.
[1107] 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., lymphoma, melanoma,
prostate cancer, cervical cancer, breast cancer, colon cancer, or
sarcoma.
[1108] 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 cerebrospinal fluid in some brain tumors (Ronquist et
al. (1977) Lancet i: 1284-1286). Further, adenylate 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.
[1109] The disclosed invention relates to methods and compositions
for the modulation, diagnosis, and treatment of cellular
proliferative and/or differentiative, neurological, immune,
inflammatory, lymphatic, cardiovascular, respiratory, and
hematological disorders.
[1110] 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.
[1111] 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.
[1112] 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.
[1113] 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 VIII 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.
[1114] 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.
[1115] Preferred disorders include, but are not limited to
disorders of brain and lymph node, especially lymphoma.
[1116] The disclosed invention also relates to methods and
compositions for the modulation, diagnosis, and treatment of
disorders involving the brain and lymph nodes.
[1117] 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 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.
[1118] 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.
[1119] 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,
Imunopathology 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. Accordingly, 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.
[1120] 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 (Waldenstr{overscore
(o)}m macroglobulinemia), mantle cell lymphoma, marginal zone
lymphoma (MALToma), and hairy cell leukemia.
[1121] 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.
[1122] 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.
[1123] Preferred adenylate kinase polypeptides of the present
invention have an amino acid sequence sufficiently identical to the
amino acid sequence of SEQ ID NO:22. 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 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity are
defined herein as sufficiently identical.
[1124] 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%, 90%, 100% 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.
[1125] 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 the URL 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 http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. 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.
[1126] 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.
[1127] 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 27802 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 27802 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 information at the URL
www.ncbi.nlm.nih.gov.
[1128] Accordingly, another embodiment of the invention features
isolated adenylate kinase proteins and polypeptides having an
adenylate kinase protein activity. As used interchangeably herein,
an "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,
including Th1, Th2, T cells, natural killer T cells, lymphocytes,
leukocytes, etc., and brain, such as glial cells and neurons; (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.
[1129] 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.
[1130] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[1131] 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.
[1132] Nucleotide sequences encoding the adenylate kinase proteins
of the present invention include sequences set forth in SEQ ID
NO:21 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:22.
[1133] 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, 1452 nucleotides for SEQ ID NO:21) depending upon the
intended use.
[1134] Alternatively, a nucleic acid molecule that is a fragment of
an 27802-like nucleotide sequence of the present invention
comprises a nucleotide sequence consisting of at least 5, 10, 15,
20, 25, 30, 35, or 40 contiguous nucleotides of nucleotides
215-370, or nucleotides 843-941 of SEQ ID NO:21. A fragment of a
nucleotide sequence of the present invention comprises a nucleotide
sequence consisting of nucleotides 215-300, 300-370, 843-900,
900-941 of SEQ ID NO:21.
[1135] 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 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40,
40-45, 45-50, 50-75, 75-100 or more contiguous nucleotides. Other
regions of the nucleotide sequence may comprise fragments of
various sizes, depending upon potential homology with previously
disclosed sequences.
[1136] 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, 258 amino acids for
SEQ ID NO:22). A nucleic acid molecule that is a fragment of an
27802-like nucleotide sequence of the present invention comprises a
nucleotide sequence encoding at least 15, 20, 25, 30, 35, or 40
contiguous amino acids of amino acids 1-51, or 209-241 of SEQ ID
NO:22. A fragment of a nucleotide sequence of the present invention
comprises a nucleotide sequence encoding amino acids 1-25, 25-51,
209-241 of SEQ ID NO:22.
[1137] 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.
[1138] 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 with have at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the
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%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity to the amino acid sequence of an adenylate kinase protein
disclosed herein. Such variants retain the functional activity
(e.g., the adenylate kinase activity) of the polypeptide set forth
in SEQ ID NO:22.
[1139] In addition to the adenylate kinase nucleotide sequence
shown in SEQ ID NO:21, 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.
[1140] Moreover, nucleic acid molecules encoding adenylate kinase
proteins from other species (adenylate kinase homologs), which have
a nucleotide sequence differing from that of the adenylate kinase
sequence disclosed herein, are intended to be within the scope of
the invention. For example, nucleic acid molecules corresponding to
natural allelic variants and homologs 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.
[1141] 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:22 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.
[1142] 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:22) 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:22 (see FIG.
30), where such residues are essential for protein activity.
[1143] 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.
[1144] Thus the nucleotide sequence of the invention includes the
sequence disclosed herein as well as fragments and variants
thereof. The adenylate kinase nucleotide sequence of the invention,
and fragments and variants thereof, can be used as probes and/or
primers to identify and/or clone adenylate kinase homologs in other
cell types, e.g., from other tissues, as well as adenylate kinase
homologs 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.
[1145] 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 sequence set forth herein or to
fragments and variants thereof are encompassed by the present
invention.
[1146] 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.
[1147] 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
the adenylate kinase nucleotide sequence 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 the adenylate kinase nucleotide sequence disclosed
herein or a fragment thereof.
[1148] 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 the nucleotide sequence of the
invention, preferably the coding sequence set forth in SEQ ID NO:21
or a complement, fragment, or variant thereof.
[1149] 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, or SEQ ID NO:23, corresponds to a
naturally-occurring nucleic acid molecule.
[1150] 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).
[1151] 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.
[1152] 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.
[1153] Given the coding-strand sequence encoding an adenylate
kinase protein disclosed herein (SEQ ID NO:21), 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.
[1154] 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).
[1155] 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.
[1156] 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).
[1157] 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:21). 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.
[1158] 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.
[1159] 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) Biooganic &
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.
[1160] 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).
[1161] 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
[1162] 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:22,
as well as fragments, biologically active portions, and variants
thereof.
[1163] "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:22) 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, 10-15, 15-20,
20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 70, 80, 90,
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:22. The invention encompasses
other fragments, however, such as any fragment in the protein
greater than 5 amino acids, depending upon the intended use.
[1164] By "variants" is intended proteins or polypeptides having an
amino acid sequence that is at least about 45%, 55%, 65%,
preferably about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ
ID NO:22. Variants also include polypeptides encoded by a nucleic
acid molecule that hybridizes to the nucleic acid molecule of SEQ
ID NO:21, 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. 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 generally retain the functional activity of the
27802-like proteins of the invention. Variants include polypeptides
that differ in amino acid sequence due to natural allelic variation
or mutagenesis.
[1165] The invention also provides adenylate kinase chimeric or
fusion proteins. 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, an adenylate kinase "heterologous
protein" or "chimeric protein" or "fusion protein" comprises an
adenylate kinase polypeptide operably linked to a non-adenylate
kinase polypeptide. An "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.
[1166] 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.
[1167] 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.
[1168] 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.
[1169] 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.
[1170] 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.
[1171] 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).
[1172] 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.
[1173] 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 throughput 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).
[1174] 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, 15-20, 20-25, or 30
or more amino acid residues of the amino acid sequence shown in SEQ
ID NO:22 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.
[1175] 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).
[1176] 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.TM. Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods
and reagents particularly amenable for use in generating and
screening antibody display library can be found in, for example,
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.
[1177] 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.
[1178] 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.
[1179] 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).
[1180] 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.
[1181] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). 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.
[1182] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon 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
[1183] 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.
[1184] 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.).
[1185] 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 such codon usage are well known in the art.
[1186] 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 11d (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.
[1187] Suitable eukaryotic host cells include insect cells
(examples of Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers (1989) Virology 170:31-39));
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.
[1188] 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. 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.
[1189] 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 (Banerji et al. (1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application 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.
[1190] 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).
[1191] 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.
[1192] 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).
[1193] 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.
[1194] 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.
[1195] 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.
[1196] 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 homolog 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.
[1197] 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 5' and 3' 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.
[1198] 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.
[1199] 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
[1200] 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.
[1201] 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.
[1202] 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.
[1203] 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.
[1204] 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.
[1205] 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.
[1206] 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.TM. (BASF; Parsippany, N.J.), or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as 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.
[1207] 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.
[1208] 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.
[1209] 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.
[1210] 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.
[1211] 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.
[1212] 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.
[1213] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[1214] 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.
[1215] 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.
[1216] 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.
[1217] 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
[1218] 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.
[1219] 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.
[1220] 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.
[1221] 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.
[1222] 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.
[1223] 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.
[1224] 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).
[1225] 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).
[1226] 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
[1227] The nucleic acid molecules, proteins, protein homologs, 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 uses and
methods of the invention are particularly relevant in tissues and
cells in which the adenylate kinase is expressed and especially
where expression differs from that in a normal tissue or cell. The
uses and methods are also particularly relevant in disorders
involving such tissues and cells. Accordingly, the uses and methods
are particularly relevant for disorders involving expression of the
adenylate kinase of the invention. 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.
[1228] A. Screening Assays
[1229] 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.
[1230] 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).
[1231] 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.
[1232] 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).
[1233] 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.
[1234] 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.
[1235] 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.
[1236] 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.
[1237] 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.
[1238] 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.
[1239] 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 bibtin-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.
[1240] 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.
[1241] 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.
[1242] 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.
[1243] B. Detection Assays
[1244] 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.
[1245] 1. Chromosome Mapping
[1246] 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.
[1247] 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.
[1248] 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.
[1249] 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.
[1250] 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.
[1251] 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.
[1252] 2. Tissue Typing
[1253] 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).
[1254] 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 5' and 3' ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[1255] 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:21 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:21, is used, a more appropriate number of primers for positive
individual identification would be 500 to 2,000.
[1256] 3. Use of Partial Adenylate Kinase Sequences in Forensic
Biology
[1257] 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.
[1258] 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:21
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:21 having a length of at least 20 or 30
bases.
[1259] 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.
[1260] 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).
[1261] C. Predictive Medicine
[1262] 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.
[1263] 1. Diagnostic Assays
[1264] 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.
[1265] "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.
[1266] 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:21, or a portion thereof, such as
a nucleic acid molecule of at least about 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.
[1267] 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.
[1268] 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.
[1269] 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.
[1270] 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:21). 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.
[1271] 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.
[1272] 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.
[1273] 2. Prognostic Assays
[1274] 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.
[1275] 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.
[1276] 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.
[1277] 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.
[1278] 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.
[1279] 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.
[1280] 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.
[1281] 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).
[1282] 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.
[1283] 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.
[1284] 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).
[1285] 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).
[1286] 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.
[1287] 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.
[1288] 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.
[1289] 3. Pharmacogenomics
[1290] 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. 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.
[1291] 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.
[1292] 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.
[1293] 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.
[1294] 4. Monitoring of Effects During Clinical Trials
[1295] 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
responsiveness of a particular cell.
[1296] 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.
[1297] 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.
[1298] C. Methods of Treatment
[1299] 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. 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. Thus,
therapies for disorders associated with adenylate kinase expression
are encompassed herein.
[1300] 1. Prophylactic Methods
[1301] 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.
[1302] 2. Therapeutic Methods
[1303] 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.
[1304] 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.
[1305] 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.
[1306] 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.
Other Embodiments
[1307] 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 27802, 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 27802 nucleic acid,
polypeptide, or antibody.
[1308] 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.
[1309] The method can include contacting the 27802 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.
[1310] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of 27802. 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. 27802 is associated
with adenylate kinase activity, thus it is useful for disorders
associated with abnormal cellular growth and/or metabolism.
[1311] The method can be used to detect SNPs, as described
above.
[1312] 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 misexpress 27802 or from a cell or subject in which a 27802
mediated response has been elicited, e.g., by contact of the cell
with 27802 nucleic acid or protein, or administration to the cell
or subject 27802 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 27802 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 27802 (or does not express
as highly as in the case of the 27802 positive plurality of capture
probes) or from a cell or subject which in which a 27802 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 27802 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.
[1313] In another aspect, the invention features, a method of
analyzing 27802, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a 27802 nucleic acid or amino acid
sequence; comparing the 27802 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
27802.
[1314] Preferred databases include GenBank.TM.. The method can
include evaluating the sequence identity between a 27802 sequence
and a database sequence. The method can be performed by accessing
the database at a second site, e.g., over the internet.
[1315] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of 27802. 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.
[1316] 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.
[1317] 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.
[1318] 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.
EXPERIMENTAL
Example 1
Identification and Characterization of Human 27802 cDNAs
[1319] The human 27802 sequence (FIG. 28; SEQ ID NO:21), which is
approximately 1452 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
774 nucleotides (nucleotides 219-992 of SEQ ID NO:21; SEQ ID
NO:23). The coding sequence encodes a 258 amino acid protein (SEQ
ID NO:22).
Example 2
Tissue Distribution of 27802 mRNA
[1320] 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 27802 cDNA (SEQ ID NO:21)
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.
[1321] 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. The highest levels of expression of
27802 were observed in artery, kidney, brain cortex and brain
hypothalamus, ovary, lung (tumor), and tonsil (see FIG. 35).
Example 3
Recombinant Expression of 27802 in Bacterial Cells
[1322] In this example, 27802 polypeptide is expressed as a
recombinant glutathione-S-transferase (GST) fusion polypeptide in
E. coli and the fusion polypeptide is isolated and characterized.
Specifically, 27802 is fused to GST and this fusion polypeptide is
expressed in E. coli, e.g., strain PEB199. Expression of the
GST-27802 fusion protein in PEB199 is induced with IPTG. The
recombinant fusion polypeptide is purified from crude bacterial
lysates of the induced PEB199 strain by affinity chromatography on
glutathione beads. Using polyacrylamide gel electrophoretic
analysis of the polypeptide purified from the bacterial lysates,
the molecular weight of the resultant fusion polypeptide is
determined.
Example 4
Expression of Recombinant 27802 Protein in COS Cells
[1323] To express the 27802 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 27802 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.
[1324] To construct the plasmid, the 27802 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 27802 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 27802 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 27802 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.
[1325] COS cells are subsequently transfected with the
27802-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 27802 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.
[1326] Alternatively, DNA containing the 27802 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 27802 polypeptide is detected by radiolabelling
and immunoprecipitation using a 27802 specific monoclonal antibody.
Sequence CWU 1
1
25 1 1748 DNA Homo sapiens CDS (79)...(1164) 1 ctatagggag
tcgccccgcg tccgggccgg ctgagggcac ttgctcttgc tgtttctgcc 60
cctgggttaa cattcaag atg gta cat gct gaa gcc ttt tct cgt cct ttg 111
Met Val His Ala Glu Ala Phe Ser Arg Pro Leu 1 5 10 agt cgg aat gaa
gtt gtt ggt tta att ttc cgt ttg aca ata ttt ggt 159 Ser Arg Asn Glu
Val Val Gly Leu Ile Phe Arg Leu Thr Ile Phe Gly 15 20 25 gca gtg
aca tac ttt act atc aaa tgg atg gta gat gca att gat cca 207 Ala Val
Thr Tyr Phe Thr Ile Lys Trp Met Val Asp Ala Ile Asp Pro 30 35 40
acc aga aag caa aaa gta gaa gct cag aaa cag gca gaa aaa cta atg 255
Thr Arg Lys Gln Lys Val Glu Ala Gln Lys Gln Ala Glu Lys Leu Met 45
50 55 aag caa att gga gtg aaa aat gtg aag ctc tca gaa tat gaa atg
agt 303 Lys Gln Ile Gly Val Lys Asn Val Lys Leu Ser Glu Tyr Glu Met
Ser 60 65 70 75 att gct gct cat ctt gta gac cct ctt aat atg cat gtt
act tgg agt 351 Ile Ala Ala His Leu Val Asp Pro Leu Asn Met His Val
Thr Trp Ser 80 85 90 gat ata gca ggt tta gat gat gtc att acg gat
ctg aaa gac aca gtc 399 Asp Ile Ala Gly Leu Asp Asp Val Ile Thr Asp
Leu Lys Asp Thr Val 95 100 105 atc tta cct atc aaa aag aaa cat ttg
ttt gag aat tcc agg ctt ctg 447 Ile Leu Pro Ile Lys Lys Lys His Leu
Phe Glu Asn Ser Arg Leu Leu 110 115 120 cag cct cca aaa ggt gtt ctt
ctc tat ggg cct cca ggc tgt ggt aaa 495 Gln Pro Pro Lys Gly Val Leu
Leu Tyr Gly Pro Pro Gly Cys Gly Lys 125 130 135 acg ttg att gcc aag
gcc aca gcc aaa gaa gca ggc tgt cga ttt att 543 Thr Leu Ile Ala Lys
Ala Thr Ala Lys Glu Ala Gly Cys Arg Phe Ile 140 145 150 155 aac ctt
cag cct tcg aca ctg acc gat aag tgg tat gga gaa tct cag 591 Asn Leu
Gln Pro Ser Thr Leu Thr Asp Lys Trp Tyr Gly Glu Ser Gln 160 165 170
aaa ttg gct gct gct gtc ttc tcc ctt gcc ata aag cta caa cca tcc 639
Lys Leu Ala Ala Ala Val Phe Ser Leu Ala Ile Lys Leu Gln Pro Ser 175
180 185 atc atc ttt ata gat gaa ata gac tcc ttt cta cga aac cgt tca
agt 687 Ile Ile Phe Ile Asp Glu Ile Asp Ser Phe Leu Arg Asn Arg Ser
Ser 190 195 200 tct gac cat gaa gct aca gcc atg atg aaa gct cag ttt
atg agt ctc 735 Ser Asp His Glu Ala Thr Ala Met Met Lys Ala Gln Phe
Met Ser Leu 205 210 215 tgg gat gga ttg gat act gat cac agc tgc cag
gtc ata gta atg gga 783 Trp Asp Gly Leu Asp Thr Asp His Ser Cys Gln
Val Ile Val Met Gly 220 225 230 235 gct acc aat cgt cct cag gac ctt
gac tcg gct ata atg aga aga atg 831 Ala Thr Asn Arg Pro Gln Asp Leu
Asp Ser Ala Ile Met Arg Arg Met 240 245 250 cct aca aga ttt cat atc
aac cag cct gct tta aaa cag aga gaa gca 879 Pro Thr Arg Phe His Ile
Asn Gln Pro Ala Leu Lys Gln Arg Glu Ala 255 260 265 atc ctg aaa ctc
atc ttg aaa aat gaa aat gtg gat agg cat gta gac 927 Ile Leu Lys Leu
Ile Leu Lys Asn Glu Asn Val Asp Arg His Val Asp 270 275 280 ctg cta
gaa gtt gcc cag gaa act gat ggg ttt tca gga agt gac cta 975 Leu Leu
Glu Val Ala Gln Glu Thr Asp Gly Phe Ser Gly Ser Asp Leu 285 290 295
aaa gag atg tgt cga gat gct gcc ctc ctc tgt gtt aga gaa tat gtt
1023 Lys Glu Met Cys Arg Asp Ala Ala Leu Leu Cys Val Arg Glu Tyr
Val 300 305 310 315 aat tct aca tca gaa gaa agc cat gac gaa gat gaa
att cgg cct gtt 1071 Asn Ser Thr Ser Glu Glu Ser His Asp Glu Asp
Glu Ile Arg Pro Val 320 325 330 caa cag cag gac ctg cat cgg gca att
gaa aag atg aag aaa tca aag 1119 Gln Gln Gln Asp Leu His Arg Ala
Ile Glu Lys Met Lys Lys Ser Lys 335 340 345 gat gca gca ttt cag aat
gtt tta aca cat gtt tgt tta gat taa 1164 Asp Ala Ala Phe Gln Asn
Val Leu Thr His Val Cys Leu Asp * 350 355 360 gagtaaagat catttgtaca
gttcagtgat ctagtttggt gtgtcctctt atcagttagt 1224 ggaaatagaa
cggaaagagt gctctttaaa caatgaggga gctcagtgtt tatggtttta 1284
tactctgaat tctaagttat tgagatatag ttgttacata ggtggtatta ctgttggtca
1344 aaaatcatga ggaggaacag ttgaatccag cctgaacgtg ggtgcttgtg
tttgaccttt 1404 tcagccatat attgtacagc cttatagaat ctaagctggt
cttaaagtca taaatgattc 1464 attgggtcat tagtgagaaa cggggatgtg
gttaggtgct ggttcctaga catgtgagta 1524 tgcgtttgtg tgtgtgcgtg
tatgtatgtg tatattaaat gtatatatcc acacatttta 1584 tattgacatt
ctgtagatat gtttgaatat agaaactttt tttaccccaa ctactgaatc 1644
caggagtacc aaataatata tagtaaaact aagatttaag gttgtgtcaa aaaggtacag
1704 tggattcagc catttccatt tgtcatttgt ttcaaccttt ttta 1748 2 361
PRT Homo sapiens 2 Met Val His Ala Glu Ala Phe Ser Arg Pro Leu Ser
Arg Asn Glu Val 1 5 10 15 Val Gly Leu Ile Phe Arg Leu Thr Ile Phe
Gly Ala Val Thr Tyr Phe 20 25 30 Thr Ile Lys Trp Met Val Asp Ala
Ile Asp Pro Thr Arg Lys Gln Lys 35 40 45 Val Glu Ala Gln Lys Gln
Ala Glu Lys Leu Met Lys Gln Ile Gly Val 50 55 60 Lys Asn Val Lys
Leu Ser Glu Tyr Glu Met Ser Ile Ala Ala His Leu 65 70 75 80 Val Asp
Pro Leu Asn Met His Val Thr Trp Ser Asp Ile Ala Gly Leu 85 90 95
Asp Asp Val Ile Thr Asp Leu Lys Asp Thr Val Ile Leu Pro Ile Lys 100
105 110 Lys Lys His Leu Phe Glu Asn Ser Arg Leu Leu Gln Pro Pro Lys
Gly 115 120 125 Val Leu Leu Tyr Gly Pro Pro Gly Cys Gly Lys Thr Leu
Ile Ala Lys 130 135 140 Ala Thr Ala Lys Glu Ala Gly Cys Arg Phe Ile
Asn Leu Gln Pro Ser 145 150 155 160 Thr Leu Thr Asp Lys Trp Tyr Gly
Glu Ser Gln Lys Leu Ala Ala Ala 165 170 175 Val Phe Ser Leu Ala Ile
Lys Leu Gln Pro Ser Ile Ile Phe Ile Asp 180 185 190 Glu Ile Asp Ser
Phe Leu Arg Asn Arg Ser Ser Ser Asp His Glu Ala 195 200 205 Thr Ala
Met Met Lys Ala Gln Phe Met Ser Leu Trp Asp Gly Leu Asp 210 215 220
Thr Asp His Ser Cys Gln Val Ile Val Met Gly Ala Thr Asn Arg Pro 225
230 235 240 Gln Asp Leu Asp Ser Ala Ile Met Arg Arg Met Pro Thr Arg
Phe His 245 250 255 Ile Asn Gln Pro Ala Leu Lys Gln Arg Glu Ala Ile
Leu Lys Leu Ile 260 265 270 Leu Lys Asn Glu Asn Val Asp Arg His Val
Asp Leu Leu Glu Val Ala 275 280 285 Gln Glu Thr Asp Gly Phe Ser Gly
Ser Asp Leu Lys Glu Met Cys Arg 290 295 300 Asp Ala Ala Leu Leu Cys
Val Arg Glu Tyr Val Asn Ser Thr Ser Glu 305 310 315 320 Glu Ser His
Asp Glu Asp Glu Ile Arg Pro Val Gln Gln Gln Asp Leu 325 330 335 His
Arg Ala Ile Glu Lys Met Lys Lys Ser Lys Asp Ala Ala Phe Gln 340 345
350 Asn Val Leu Thr His Val Cys Leu Asp 355 360 3 1086 DNA Homo
sapiens 3 atggtacatg ctgaagcctt ttctcgtcct ttgagtcgga atgaagttgt
tggtttaatt 60 ttccgtttga caatatttgg tgcagtgaca tactttacta
tcaaatggat ggtagatgca 120 attgatccaa ccagaaagca aaaagtagaa
gctcagaaac aggcagaaaa actaatgaag 180 caaattggag tgaaaaatgt
gaagctctca gaatatgaaa tgagtattgc tgctcatctt 240 gtagaccctc
ttaatatgca tgttacttgg agtgatatag caggtttaga tgatgtcatt 300
acggatctga aagacacagt catcttacct atcaaaaaga aacatttgtt tgagaattcc
360 aggcttctgc agcctccaaa aggtgttctt ctctatgggc ctccaggctg
tggtaaaacg 420 ttgattgcca aggccacagc caaagaagca ggctgtcgat
ttattaacct tcagccttcg 480 acactgaccg ataagtggta tggagaatct
cagaaattgg ctgctgctgt cttctccctt 540 gccataaagc tacaaccatc
catcatcttt atagatgaaa tagactcctt tctacgaaac 600 cgttcaagtt
ctgaccatga agctacagcc atgatgaaag ctcagtttat gagtctctgg 660
gatggattgg atactgatca cagctgccag gtcatagtaa tgggagctac caatcgtcct
720 caggaccttg actcggctat aatgagaaga atgcctacaa gatttcatat
caaccagcct 780 gctttaaaac agagagaagc aatcctgaaa ctcatcttga
aaaatgaaaa tgtggatagg 840 catgtagacc tgctagaagt tgcccaggaa
actgatgggt tttcaggaag tgacctaaaa 900 gagatgtgtc gagatgctgc
cctcctctgt gttagagaat atgttaattc tacatcagaa 960 gaaagccatg
acgaagatga aattcggcct gttcaacagc aggacctgca tcgggcaatt 1020
gaaaagatga agaaatcaaa ggatgcagca tttcagaatg ttttaacaca tgtttgttta
1080 gattaa 1086 4 220 PRT Artificial Sequence Consensus sequence
for the AAA domain 4 Gly Val Leu Leu Tyr Gly Pro Pro Gly Thr Gly
Lys Thr Leu Leu Ala 1 5 10 15 Lys Ala Val Ala Asn Glu Leu Gly Ser
Leu Arg Lys Ala Pro Phe Ile 20 25 30 Ser Ile Ser Gly Tyr Ser Glu
Leu Val Ser Lys Tyr Val Gly Glu Ser 35 40 45 Glu Lys Arg Val Arg
Ala Leu Phe Glu Leu Ala Arg Glu Leu Arg Lys 50 55 60 Arg Ala Ala
Pro Cys Pro Ile Ile Phe Ile Asp Glu Ile Asp Ala Ile 65 70 75 80 Ala
Pro Lys Arg Gly Gly Glu Val Ser Arg Arg Val Val Asn Gln Leu 85 90
95 Leu Thr Glu Met Asp Leu Glu Arg Ala Gly Phe Ser Lys Asn Ser Ser
100 105 110 Arg Gly Glu Asp Thr Ile Asp Leu Ser Asn Val Leu Val Ile
Ala Ala 115 120 125 Thr Asn Arg Pro Asp Thr Leu Asp Pro Ala Leu Leu
Arg Pro Gly Arg 130 135 140 Phe Asp Arg Glu Ile Glu Ile Pro Leu Pro
Pro Asp Glu Glu Gly Arg 145 150 155 160 Leu Asp Ile Leu Lys Ile His
Leu Lys Lys Met Pro Leu Ser Ser Ser 165 170 175 Leu Lys Gln Ser Glu
Leu Ala Glu Asp Val Asp Leu Asp Glu Leu Ala 180 185 190 Glu Glu Ile
Ala Thr Arg Thr Glu Gly Phe Ser Gly Ala Asp Leu Lys 195 200 205 Ala
Leu Cys Arg Glu Ala Ala Leu Arg Ala Ile Arg 210 215 220 5 1852 DNA
Homo sapiens CDS (14)...(1477) 5 cacgcgtccg gcc atg cgg agg ggc gag
cgc agg gac gcc gga ggt ccg 49 Met Arg Arg Gly Glu Arg Arg Asp Ala
Gly Gly Pro 1 5 10 cgg ccc gag tcc ccg gtg ccc gcg ggc agg gcc tcg
ctg gag gag ccg 97 Arg Pro Glu Ser Pro Val Pro Ala Gly Arg Ala Ser
Leu Glu Glu Pro 15 20 25 cct gac ggg ccg tct gcc ggc caa gcc acc
ggg ccg ggc gag ggc cgc 145 Pro Asp Gly Pro Ser Ala Gly Gln Ala Thr
Gly Pro Gly Glu Gly Arg 30 35 40 cgc agc acc gag tcc gag gtc tac
gac gac ggc acc aac acc ttc ttc 193 Arg Ser Thr Glu Ser Glu Val Tyr
Asp Asp Gly Thr Asn Thr Phe Phe 45 50 55 60 tgg cga gcc cac acc tta
acc gtg ctc ttc atc ctc acc tgt acg ctt 241 Trp Arg Ala His Thr Leu
Thr Val Leu Phe Ile Leu Thr Cys Thr Leu 65 70 75 ggc tat gtg acg
ctg ctg gag gaa aca cct cag gac acg gcc tac aac 289 Gly Tyr Val Thr
Leu Leu Glu Glu Thr Pro Gln Asp Thr Ala Tyr Asn 80 85 90 acc aag
aga ggt att gtg gcc agt att ttg gtt ttc tta tgt ttt gga 337 Thr Lys
Arg Gly Ile Val Ala Ser Ile Leu Val Phe Leu Cys Phe Gly 95 100 105
gtc aca caa gct aaa gac ggg cca ttt tcc aga cct cat cca gct tac 385
Val Thr Gln Ala Lys Asp Gly Pro Phe Ser Arg Pro His Pro Ala Tyr 110
115 120 tgg agg ttt tgg ctc tgc gtg agt gtg gtc tac gag ctg ttt ctc
atc 433 Trp Arg Phe Trp Leu Cys Val Ser Val Val Tyr Glu Leu Phe Leu
Ile 125 130 135 140 ttt ata ctc ttc cag act gtc cag gac ggc cgg cag
ttt cta aag tat 481 Phe Ile Leu Phe Gln Thr Val Gln Asp Gly Arg Gln
Phe Leu Lys Tyr 145 150 155 gtt gac ccc aag ctg gga gtc cca ctg cca
gag aga gac tac ggg gga 529 Val Asp Pro Lys Leu Gly Val Pro Leu Pro
Glu Arg Asp Tyr Gly Gly 160 165 170 aac tgc ctc atc tac gac cca gac
aat gag act gac ccc ttt cac aac 577 Asn Cys Leu Ile Tyr Asp Pro Asp
Asn Glu Thr Asp Pro Phe His Asn 175 180 185 atc tgg gac aag ttg gat
ggc ttt gtt ccc gcg cac ttt ctt ggc tgg 625 Ile Trp Asp Lys Leu Asp
Gly Phe Val Pro Ala His Phe Leu Gly Trp 190 195 200 tac ctg aag acc
ctg atg atc cga gac tgg tgg atg tgc atg atc atc 673 Tyr Leu Lys Thr
Leu Met Ile Arg Asp Trp Trp Met Cys Met Ile Ile 205 210 215 220 agc
gtg atg ttc gag ttc ctg gag tac agc ctg gag cac cag ctg ccc 721 Ser
Val Met Phe Glu Phe Leu Glu Tyr Ser Leu Glu His Gln Leu Pro 225 230
235 aac ttc agc gag tgc tgg tgg gat cac tgg atc atg gac gtg ctc gtc
769 Asn Phe Ser Glu Cys Trp Trp Asp His Trp Ile Met Asp Val Leu Val
240 245 250 tgc aac ggg ctg ggc atc tac tgc ggc atg aag acc ctt gag
tgg ctg 817 Cys Asn Gly Leu Gly Ile Tyr Cys Gly Met Lys Thr Leu Glu
Trp Leu 255 260 265 tcc ctg aag acg tac aag tgg cag ggc ctc tgg aac
att ccg acc tac 865 Ser Leu Lys Thr Tyr Lys Trp Gln Gly Leu Trp Asn
Ile Pro Thr Tyr 270 275 280 aag ggc aag atg aag agg atc gcc ttc cag
ttc acg ccg tac agc tgg 913 Lys Gly Lys Met Lys Arg Ile Ala Phe Gln
Phe Thr Pro Tyr Ser Trp 285 290 295 300 gtt cgc ttc gag tgg aag ccg
gcc tcc agc ctg cgt cgc tgg ctg gcc 961 Val Arg Phe Glu Trp Lys Pro
Ala Ser Ser Leu Arg Arg Trp Leu Ala 305 310 315 gtg tgc ggc atc atc
ctg gtg ttc ctg ttg gca gaa ctg aac acg ttc 1009 Val Cys Gly Ile
Ile Leu Val Phe Leu Leu Ala Glu Leu Asn Thr Phe 320 325 330 tac ctg
aag ttt gtg ctg tgg atg ccc ccg gag cac tac ctg gtc ctc 1057 Tyr
Leu Lys Phe Val Leu Trp Met Pro Pro Glu His Tyr Leu Val Leu 335 340
345 ctg cgg ctc gtc ttc ttc gtg aac gtg ggt ggc gtg gcc atg cgt gag
1105 Leu Arg Leu Val Phe Phe Val Asn Val Gly Gly Val Ala Met Arg
Glu 350 355 360 atc tac gac ttc atg gat gac ccg aag ccc cac aag aag
ctg ggc ccg 1153 Ile Tyr Asp Phe Met Asp Asp Pro Lys Pro His Lys
Lys Leu Gly Pro 365 370 375 380 cag gcc tgg ctg gtg gcg gcc atc acg
gcc acg gag ctg ctc atc gtg 1201 Gln Ala Trp Leu Val Ala Ala Ile
Thr Ala Thr Glu Leu Leu Ile Val 385 390 395 gtg aag tac gac ccc cac
acg ctc acc ctg tcc ctg ccc ttc tac atc 1249 Val Lys Tyr Asp Pro
His Thr Leu Thr Leu Ser Leu Pro Phe Tyr Ile 400 405 410 tcc cag tgc
tgg acc ctc ggc tcc gtc ctg gcg ctc acc tgg acc gtc 1297 Ser Gln
Cys Trp Thr Leu Gly Ser Val Leu Ala Leu Thr Trp Thr Val 415 420 425
tgg cgc ttc ttc ctg cgg gac atc aca ttg agg tac aag gag acc cgg
1345 Trp Arg Phe Phe Leu Arg Asp Ile Thr Leu Arg Tyr Lys Glu Thr
Arg 430 435 440 tgg cag aag tgg cag aac aag gat gac cag ggc agc acc
gtc ggc aac 1393 Trp Gln Lys Trp Gln Asn Lys Asp Asp Gln Gly Ser
Thr Val Gly Asn 445 450 455 460 ggg gac cag cac cca ctg ggg ctg gac
gaa gac ctg ctg ggg cct ggg 1441 Gly Asp Gln His Pro Leu Gly Leu
Asp Glu Asp Leu Leu Gly Pro Gly 465 470 475 gtg gcc gag ggc gag gga
gca cca act cca aac tga cctgggccgt 1487 Val Ala Glu Gly Glu Gly Ala
Pro Thr Pro Asn * 480 485 ggctgcctcg tgagcctccc agagcccagg
cctccgtggc ctcctcctgt gtgagtccca 1547 ccaggagcca cgtgcccggc
cttgccctca aggttttttg cttttctcct gtgcacctgg 1607 cgaggctgaa
ggcgaggggt ggaggaggcc ccagcacagc ctcatctcca tgtgtacacg 1667
tgtgtacgtg tgtatgcgtg tgtgtacgcc gtgtgtacgc gcgtgtgtac acatgcgtgg
1727 ccgctgtggt gtgcacstgt gctctgggct ccgaggctts ttcmagarct
gggarctggc 1787 tggcgtggca agggcatgct ctggggcagt gtgttcctya
agaaccargg gtccttccty 1847 ccttt 1852 6 487 PRT Homo sapiens 6 Met
Arg Arg Gly Glu Arg Arg Asp Ala Gly Gly Pro Arg Pro Glu Ser 1 5 10
15 Pro Val Pro Ala Gly Arg Ala Ser Leu Glu Glu Pro Pro Asp Gly Pro
20 25 30 Ser Ala Gly Gln Ala Thr Gly Pro Gly Glu Gly Arg Arg Ser
Thr Glu 35 40 45 Ser Glu Val Tyr Asp Asp Gly Thr Asn Thr Phe Phe
Trp Arg Ala His 50 55 60 Thr Leu Thr Val Leu Phe Ile Leu Thr Cys
Thr Leu Gly Tyr Val Thr 65 70 75 80 Leu Leu Glu Glu Thr Pro Gln Asp
Thr Ala
Tyr Asn Thr Lys Arg Gly 85 90 95 Ile Val Ala Ser Ile Leu Val Phe
Leu Cys Phe Gly Val Thr Gln Ala 100 105 110 Lys Asp Gly Pro Phe Ser
Arg Pro His Pro Ala Tyr Trp Arg Phe Trp 115 120 125 Leu Cys Val Ser
Val Val Tyr Glu Leu Phe Leu Ile Phe Ile Leu Phe 130 135 140 Gln Thr
Val Gln Asp Gly Arg Gln Phe Leu Lys Tyr Val Asp Pro Lys 145 150 155
160 Leu Gly Val Pro Leu Pro Glu Arg Asp Tyr Gly Gly Asn Cys Leu Ile
165 170 175 Tyr Asp Pro Asp Asn Glu Thr Asp Pro Phe His Asn Ile Trp
Asp Lys 180 185 190 Leu Asp Gly Phe Val Pro Ala His Phe Leu Gly Trp
Tyr Leu Lys Thr 195 200 205 Leu Met Ile Arg Asp Trp Trp Met Cys Met
Ile Ile Ser Val Met Phe 210 215 220 Glu Phe Leu Glu Tyr Ser Leu Glu
His Gln Leu Pro Asn Phe Ser Glu 225 230 235 240 Cys Trp Trp Asp His
Trp Ile Met Asp Val Leu Val Cys Asn Gly Leu 245 250 255 Gly Ile Tyr
Cys Gly Met Lys Thr Leu Glu Trp Leu Ser Leu Lys Thr 260 265 270 Tyr
Lys Trp Gln Gly Leu Trp Asn Ile Pro Thr Tyr Lys Gly Lys Met 275 280
285 Lys Arg Ile Ala Phe Gln Phe Thr Pro Tyr Ser Trp Val Arg Phe Glu
290 295 300 Trp Lys Pro Ala Ser Ser Leu Arg Arg Trp Leu Ala Val Cys
Gly Ile 305 310 315 320 Ile Leu Val Phe Leu Leu Ala Glu Leu Asn Thr
Phe Tyr Leu Lys Phe 325 330 335 Val Leu Trp Met Pro Pro Glu His Tyr
Leu Val Leu Leu Arg Leu Val 340 345 350 Phe Phe Val Asn Val Gly Gly
Val Ala Met Arg Glu Ile Tyr Asp Phe 355 360 365 Met Asp Asp Pro Lys
Pro His Lys Lys Leu Gly Pro Gln Ala Trp Leu 370 375 380 Val Ala Ala
Ile Thr Ala Thr Glu Leu Leu Ile Val Val Lys Tyr Asp 385 390 395 400
Pro His Thr Leu Thr Leu Ser Leu Pro Phe Tyr Ile Ser Gln Cys Trp 405
410 415 Thr Leu Gly Ser Val Leu Ala Leu Thr Trp Thr Val Trp Arg Phe
Phe 420 425 430 Leu Arg Asp Ile Thr Leu Arg Tyr Lys Glu Thr Arg Trp
Gln Lys Trp 435 440 445 Gln Asn Lys Asp Asp Gln Gly Ser Thr Val Gly
Asn Gly Asp Gln His 450 455 460 Pro Leu Gly Leu Asp Glu Asp Leu Leu
Gly Pro Gly Val Ala Glu Gly 465 470 475 480 Glu Gly Ala Pro Thr Pro
Asn 485 7 1464 DNA Homo sapiens 7 atgcggaggg gcgagcgcag ggacgccgga
ggtccgcggc ccgagtcccc ggtgcccgcg 60 ggcagggcct cgctggagga
gccgcctgac gggccgtctg ccggccaagc caccgggccg 120 ggcgagggcc
gccgcagcac cgagtccgag gtctacgacg acggcaccaa caccttcttc 180
tggcgagccc acaccttaac cgtgctcttc atcctcacct gtacgcttgg ctatgtgacg
240 ctgctggagg aaacacctca ggacacggcc tacaacacca agagaggtat
tgtggccagt 300 attttggttt tcttatgttt tggagtcaca caagctaaag
acgggccatt ttccagacct 360 catccagctt actggaggtt ttggctctgc
gtgagtgtgg tctacgagct gtttctcatc 420 tttatactct tccagactgt
ccaggacggc cggcagtttc taaagtatgt tgaccccaag 480 ctgggagtcc
cactgccaga gagagactac gggggaaact gcctcatcta cgacccagac 540
aatgagactg acccctttca caacatctgg gacaagttgg atggctttgt tcccgcgcac
600 tttcttggct ggtacctgaa gaccctgatg atccgagact ggtggatgtg
catgatcatc 660 agcgtgatgt tcgagttcct ggagtacagc ctggagcacc
agctgcccaa cttcagcgag 720 tgctggtggg atcactggat catggacgtg
ctcgtctgca acgggctggg catctactgc 780 ggcatgaaga cccttgagtg
gctgtccctg aagacgtaca agtggcaggg cctctggaac 840 attccgacct
acaagggcaa gatgaagagg atcgccttcc agttcacgcc gtacagctgg 900
gttcgcttcg agtggaagcc ggcctccagc ctgcgtcgct ggctggccgt gtgcggcatc
960 atcctggtgt tcctgttggc agaactgaac acgttctacc tgaagtttgt
gctgtggatg 1020 cccccggagc actacctggt cctcctgcgg ctcgtcttct
tcgtgaacgt gggtggcgtg 1080 gccatgcgtg agatctacga cttcatggat
gacccgaagc cccacaagaa gctgggcccg 1140 caggcctggc tggtggcggc
catcacggcc acggagctgc tcatcgtggt gaagtacgac 1200 ccccacacgc
tcaccctgtc cctgcccttc tacatctccc agtgctggac cctcggctcc 1260
gtcctggcgc tcacctggac cgtctggcgc ttcttcctgc gggacatcac attgaggtac
1320 aaggagaccc ggtggcagaa gtggcagaac aaggatgacc agggcagcac
cgtcggcaac 1380 ggggaccagc acccactggg gctggacgaa gacctgctgg
ggcctggggt ggccgagggc 1440 gagggagcac caactccaaa ctga 1464 8 474
PRT Cricetulus griseus 8 Met Arg Arg Ala Glu Arg Arg Val Ala Gly
Gly Ser Gly Ser Gly Ser 1 5 10 15 Pro Leu Leu Glu Gly Arg Arg Ser
Thr Glu Ser Glu Val Tyr Asp Asp 20 25 30 Gly Thr Asn Thr Phe Phe
Trp Arg Ala His Thr Leu Thr Val Leu Phe 35 40 45 Ile Leu Thr Cys
Ser Leu Gly Tyr Val Thr Leu Leu Glu Glu Thr Pro 50 55 60 Gln Asp
Thr Ala Tyr Asn Thr Lys Arg Gly Ile Val Ala Ser Ile Leu 65 70 75 80
Val Phe Leu Cys Phe Gly Val Thr Gln Ala Lys Asp Gly Pro Phe Ser 85
90 95 Arg Pro His Pro Ala Tyr Trp Arg Phe Trp Leu Cys Val Ser Val
Val 100 105 110 Tyr Glu Leu Phe Leu Ile Phe Ile Leu Phe Gln Thr Val
Gln Asp Gly 115 120 125 Arg Gln Phe Leu Lys Tyr Val Asp Pro Arg Leu
Gly Val Pro Leu Pro 130 135 140 Glu Arg Asp Tyr Gly Gly Asn Cys Leu
Ile Tyr Asp Ala Asp Asn Lys 145 150 155 160 Thr Asp Pro Phe His Asn
Ile Trp Asp Lys Leu Asp Gly Phe Val Pro 165 170 175 Ala His Phe Ile
Gly Trp Tyr Leu Lys Thr Leu Met Ile Arg Asp Trp 180 185 190 Trp Met
Cys Met Ile Ile Ser Val Met Phe Glu Phe Leu Glu Tyr Ser 195 200 205
Leu Glu His Gln Leu Pro Asn Phe Ser Glu Cys Trp Trp Asp His Trp 210
215 220 Ile Met Asp Val Leu Ile Cys Asn Gly Leu Gly Ile Tyr Cys Gly
Met 225 230 235 240 Lys Thr Leu Glu Trp Leu Ser Leu Lys Thr Tyr Lys
Trp Gln Gly Leu 245 250 255 Trp Asn Ile Pro Thr Tyr Lys Gly Lys Met
Lys Arg Ile Ala Phe Gln 260 265 270 Phe Thr Pro Tyr Ser Trp Val Arg
Phe Glu Trp Lys Pro Ala Ser Ser 275 280 285 Leu His Arg Trp Leu Ala
Val Cys Gly Ile Ile Leu Val Phe Leu Leu 290 295 300 Ala Glu Leu Asn
Thr Phe Tyr Leu Lys Phe Val Leu Trp Met Pro Pro 305 310 315 320 Glu
His Tyr Leu Val Leu Leu Arg Leu Val Phe Phe Val Asn Val Gly 325 330
335 Gly Val Ala Met Arg Glu Ile Tyr Asp Phe Met Asp Glu Leu Lys Pro
340 345 350 His Arg Lys Leu Gly Gln Gln Ala Trp Leu Val Ala Ala Ile
Thr Val 355 360 365 Thr Glu Leu Leu Ile Val Val Lys Tyr Asp Pro His
Thr Leu Thr Leu 370 375 380 Ser Leu Pro Phe Tyr Ile Ser Gln Cys Trp
Thr Leu Gly Ser Ile Leu 385 390 395 400 Val Leu Thr Trp Thr Val Trp
Arg Phe Phe Leu Arg Asp Ile Thr Met 405 410 415 Arg Tyr Lys Glu Thr
Arg Arg Gln Lys Gln Gln Ser His Gln Gly Arg 420 425 430 Ala Ile Asn
Asn Gly Asp Gly His Pro Gly Pro Asp Asp Asp Leu Leu 435 440 445 Gly
Thr Gly Thr Ala Glu Glu Glu Gly Ser Thr Asn Asp Ser Val Pro 450 455
460 Ala Glu Lys Glu Gly Ala Ser Ala Ala Ser 465 470 9 473 PRT Mus
musculus 9 Met Arg Arg Ala Glu Arg Arg Val Ala Gly Gly Ser Gly Ser
Glu Ser 1 5 10 15 Pro Leu Leu Lys Gly Arg Arg Ser Thr Glu Ser Glu
Val Tyr Asp Asp 20 25 30 Gly Thr Asn Thr Phe Phe Trp Arg Ala His
Thr Leu Thr Val Leu Phe 35 40 45 Ile Leu Thr Cys Ala Leu Gly Tyr
Val Thr Leu Leu Glu Glu Thr Pro 50 55 60 Gln Asp Thr Ala Tyr Asn
Thr Lys Arg Gly Ile Val Ala Ser Ile Leu 65 70 75 80 Val Phe Leu Cys
Phe Gly Val Thr Gln Ala Lys Asp Gly Pro Phe Ser 85 90 95 Arg Pro
His Pro Ala Tyr Trp Arg Phe Trp Leu Cys Val Ser Val Val 100 105 110
Tyr Glu Leu Phe Leu Ile Phe Ile Leu Phe Gln Thr Val Gln Asp Gly 115
120 125 Arg Gln Phe Leu Lys Tyr Val Asp Pro Arg Leu Gly Val Pro Leu
Pro 130 135 140 Glu Arg Asp Tyr Gly Gly Asn Cys Leu Ile Tyr Asp Ala
Asp Asn Lys 145 150 155 160 Thr Asp Pro Phe His Asn Ile Trp Asp Lys
Leu Asp Gly Phe Val Pro 165 170 175 Ala His Phe Ile Gly Trp Tyr Leu
Lys Thr Leu Met Ile Arg Asp Trp 180 185 190 Trp Met Cys Met Ile Ile
Ser Val Met Phe Glu Phe Leu Glu Tyr Ser 195 200 205 Leu Glu His Gln
Leu Pro Asn Phe Ser Glu Cys Trp Trp Asp His Trp 210 215 220 Ile Met
Asp Val Leu Val Cys Asn Gly Leu Gly Ile Tyr Cys Gly Met 225 230 235
240 Lys Thr Leu Glu Trp Leu Ser Leu Lys Thr Tyr Lys Trp Gln Gly Leu
245 250 255 Trp Asn Ile Pro Thr Tyr Lys Gly Lys Met Lys Arg Ile Ala
Phe Gln 260 265 270 Phe Thr Pro Tyr Ser Trp Val Arg Phe Glu Trp Lys
Pro Ala Ser Ser 275 280 285 Leu His Arg Trp Leu Ala Val Cys Gly Ile
Ile Leu Val Phe Leu Leu 290 295 300 Ala Glu Leu Asn Thr Phe Tyr Leu
Lys Phe Val Leu Trp Met Pro Pro 305 310 315 320 Glu His Tyr Leu Val
Leu Leu Arg Leu Val Phe Phe Val Asn Val Gly 325 330 335 Gly Val Ala
Met Arg Glu Ile Tyr Asp Phe Met Asp Glu Leu Lys Pro 340 345 350 His
Arg Lys Leu Gly Gln Gln Ala Trp Leu Val Ala Ala Ile Thr Val 355 360
365 Thr Glu Leu Leu Ile Val Val Lys Tyr Asp Pro His Thr Leu Thr Leu
370 375 380 Ser Leu Pro Phe Tyr Ile Ser Gln Cys Trp Thr Leu Gly Ser
Ile Leu 385 390 395 400 Val Leu Thr Trp Thr Val Trp Arg Phe Phe Leu
Arg Asp Ile Thr Met 405 410 415 Arg Tyr Lys Glu Thr Arg Arg Gln Lys
Gln Gln Ser His Gln Ala Arg 420 425 430 Ala Val Asn Asn Arg Asp Gly
His Pro Gly Pro Asp Asp Asp Leu Leu 435 440 445 Gly Thr Gly Thr Ala
Glu Glu Glu Gly Thr Thr Asn Asp Gly Val Thr 450 455 460 Ala Glu Glu
Gly Ala Ser Ala Ala Ser 465 470 10 1284 DNA Homo sapiens CDS
(169)...(828) 10 cacgcgtccg agatgaccta agtgtgactg cagcaggcag
ctctggaaaa tgaagccaga 60 gcagtgagcc agcccctcct ccgaccaagg
aggaaggaaa gagcagcccc agcacaggag 120 agaaccaccc agcccagaag
ttccagggaa ggaactctcc ggtccacc atg gag tac 177 Met Glu Tyr 1 ctc
tca gct ctg aac ccc agt gac tta ctc agg tca gta tct aat ata 225 Leu
Ser Ala Leu Asn Pro Ser Asp Leu Leu Arg Ser Val Ser Asn Ile 5 10 15
agc tcg gag ttt gga cgg agg gtc tgg acc tca gct cca cca ccc cag 273
Ser Ser Glu Phe Gly Arg Arg Val Trp Thr Ser Ala Pro Pro Pro Gln 20
25 30 35 cga cct ttc cgt gtc tgt gat cac aag cgg acc atc cgg aaa
ggc ctg 321 Arg Pro Phe Arg Val Cys Asp His Lys Arg Thr Ile Arg Lys
Gly Leu 40 45 50 aca gct gcc acc cgc cag gag ctg cta gcc aaa gca
ttg gag acc cta 369 Thr Ala Ala Thr Arg Gln Glu Leu Leu Ala Lys Ala
Leu Glu Thr Leu 55 60 65 ctg ctg aat gga gtg cta acc ctg gtg cta
gag gag gat gga act gca 417 Leu Leu Asn Gly Val Leu Thr Leu Val Leu
Glu Glu Asp Gly Thr Ala 70 75 80 gtg gac agt gag gac ttc ttc cag
ctg ctg gag gat gac acg tgc ctg 465 Val Asp Ser Glu Asp Phe Phe Gln
Leu Leu Glu Asp Asp Thr Cys Leu 85 90 95 atg gtg ttg cag tct ggt
cag agc tgg agc cct aca agg agt gga gtg 513 Met Val Leu Gln Ser Gly
Gln Ser Trp Ser Pro Thr Arg Ser Gly Val 100 105 110 115 ctg tca tat
ggc ctg gga cgg gag agg ccc aag cac agc aag gac atc 561 Leu Ser Tyr
Gly Leu Gly Arg Glu Arg Pro Lys His Ser Lys Asp Ile 120 125 130 gcc
cga ttc acc ttt gac gtg tac aag caa aac cct cga gac ctc ttt 609 Ala
Arg Phe Thr Phe Asp Val Tyr Lys Gln Asn Pro Arg Asp Leu Phe 135 140
145 ggc agc ctg aat gtc aaa gcc aca ttc tac ggg ctc tac tct atg agt
657 Gly Ser Leu Asn Val Lys Ala Thr Phe Tyr Gly Leu Tyr Ser Met Ser
150 155 160 tgt gac ttt caa gga ctt ggc cca aag aaa gta ctc agg gag
ctc ctt 705 Cys Asp Phe Gln Gly Leu Gly Pro Lys Lys Val Leu Arg Glu
Leu Leu 165 170 175 cgt tgg acc tcc aca ctg ctg caa ggc ctg ggc cat
atg ttg ctg gga 753 Arg Trp Thr Ser Thr Leu Leu Gln Gly Leu Gly His
Met Leu Leu Gly 180 185 190 195 att tcc tcc acc ctt cgt cat gca gtg
gag ggg gct gag cag tgg cag 801 Ile Ser Ser Thr Leu Arg His Ala Val
Glu Gly Ala Glu Gln Trp Gln 200 205 210 cag aag ggc cgc ctc cat tcc
tac taa ggggctctga gcttctgccc 848 Gln Lys Gly Arg Leu His Ser Tyr *
215 ccagaatcat tccaaccgac ccactgcaaa gactatgaca gcatcaaatt
tcaggacctg 908 cagacagtac aggctagata acccacccaa tttccccact
gtcctctgat cccctcgtga 968 cagaaccttt cagcataacg cctcacatcc
caagtctata cccttacctg aagaatgctg 1028 ttctttccta gccacctttc
tagcctccca cttgccctga aaggccaaga tcaagatgtc 1088 ccccaggcat
cttgatccca gcctgactgc tgctacatct aatcccctac caatgcctcc 1148
tgtccctaaa ctccccagca tactgatgac agccctctct gactttacct tgagatctgt
1208 cttcataccc ttcccctcaa actaacaaaa acatttccaa taaaaatatc
aaatatttaa 1268 aaaaaaaaaa aaaagg 1284 11 219 PRT Homo sapiens 11
Met Glu Tyr Leu Ser Ala Leu Asn Pro Ser Asp Leu Leu Arg Ser Val 1 5
10 15 Ser Asn Ile Ser Ser Glu Phe Gly Arg Arg Val Trp Thr Ser Ala
Pro 20 25 30 Pro Pro Gln Arg Pro Phe Arg Val Cys Asp His Lys Arg
Thr Ile Arg 35 40 45 Lys Gly Leu Thr Ala Ala Thr Arg Gln Glu Leu
Leu Ala Lys Ala Leu 50 55 60 Glu Thr Leu Leu Leu Asn Gly Val Leu
Thr Leu Val Leu Glu Glu Asp 65 70 75 80 Gly Thr Ala Val Asp Ser Glu
Asp Phe Phe Gln Leu Leu Glu Asp Asp 85 90 95 Thr Cys Leu Met Val
Leu Gln Ser Gly Gln Ser Trp Ser Pro Thr Arg 100 105 110 Ser Gly Val
Leu Ser Tyr Gly Leu Gly Arg Glu Arg Pro Lys His Ser 115 120 125 Lys
Asp Ile Ala Arg Phe Thr Phe Asp Val Tyr Lys Gln Asn Pro Arg 130 135
140 Asp Leu Phe Gly Ser Leu Asn Val Lys Ala Thr Phe Tyr Gly Leu Tyr
145 150 155 160 Ser Met Ser Cys Asp Phe Gln Gly Leu Gly Pro Lys Lys
Val Leu Arg 165 170 175 Glu Leu Leu Arg Trp Thr Ser Thr Leu Leu Gln
Gly Leu Gly His Met 180 185 190 Leu Leu Gly Ile Ser Ser Thr Leu Arg
His Ala Val Glu Gly Ala Glu 195 200 205 Gln Trp Gln Gln Lys Gly Arg
Leu His Ser Tyr 210 215 12 657 DNA Homo sapiens 12 atggagtacc
tctcagctct gaaccccagt gacttactca ggtcagtatc taatataagc 60
tcggagtttg gacggagggt ctggacctca gctccaccac cccagcgacc tttccgtgtc
120 tgtgatcaca agcggaccat ccggaaaggc ctgacagctg ccacccgcca
ggagctgcta 180 gccaaagcat tggagaccct actgctgaat ggagtgctaa
ccctggtgct agaggaggat 240 ggaactgcag tggacagtga ggacttcttc
cagctgctgg aggatgacac gtgcctgatg 300 gtgttgcagt ctggtcagag
ctggagccct acaaggagtg gagtgctgtc atatggcctg 360 ggacgggaga
ggcccaagca cagcaaggac atcgcccgat tcacctttga cgtgtacaag 420
caaaaccctc gagacctctt tggcagcctg aatgtcaaag ccacattcta cgggctctac
480 tctatgagtt gtgactttca aggacttggc ccaaagaaag tactcaggga
gctccttcgt 540 tggacctcca cactgctgca aggcctgggc catatgttgc
tgggaatttc ctccaccctt 600 cgtcatgcag tggagggggc tgagcagtgg
cagcagaagg gccgcctcca ttcctac 657 13 76 PRT Artificial Sequence
Consensus sequence for the CAD domain 13 Arg Pro Phe Lys Val Arg
Asp His Asp Arg Asn Val Arg Lys Gly Val 1 5 10 15 Ala Ala Ser Ser
Leu Glu Glu Leu Leu Ser Lys Val Leu Asp Lys Leu
20 25 30 Lys Leu Pro Asp Ser Leu Glu Pro Val Thr Leu Val Leu Glu
Glu Asp 35 40 45 Gly Thr Glu Val Glu Asp Glu Glu Tyr Phe Arg Thr
Leu Pro Asn Asn 50 55 60 Thr Glu Leu Val Ala Leu Glu Gln Gly Glu
Lys Trp 65 70 75 14 219 PRT Mus musculus 14 Met Glu Tyr Leu Ser Ala
Phe Asn Pro Asn Gly Leu Leu Arg Ser Val 1 5 10 15 Ser Thr Val Ser
Ser Glu Leu Ser Arg Arg Val Trp Asn Ser Ala Pro 20 25 30 Pro Pro
Gln Arg Pro Phe Arg Val Cys Asp His Lys Arg Thr Val Arg 35 40 45
Lys Gly Leu Thr Ala Ala Ser Leu Gln Glu Leu Leu Asp Lys Val Leu 50
55 60 Glu Thr Leu Leu Leu Arg Gly Val Leu Thr Leu Val Leu Glu Glu
Asp 65 70 75 80 Gly Thr Ala Val Asp Ser Glu Asp Phe Phe Gln Leu Leu
Glu Asp Asp 85 90 95 Thr Cys Leu Met Val Leu Glu Gln Gly Gln Ser
Trp Ser Pro Lys Ser 100 105 110 Gly Met Leu Ser Tyr Gly Leu Gly Arg
Glu Lys Pro Lys His Ser Lys 115 120 125 Asp Ile Ala Arg Ile Thr Phe
Asp Val Tyr Lys Gln Asn Pro Arg Asp 130 135 140 Leu Phe Gly Ser Leu
Asn Val Lys Ala Thr Phe Tyr Gly Leu Tyr Ser 145 150 155 160 Met Ser
Cys Asp Phe Gln Gly Val Gly Pro Lys Arg Val Leu Arg Glu 165 170 175
Leu Leu Arg Gly Thr Ser Ser Gln Leu Gln Gly Leu Gly His Met Leu 180
185 190 Leu Gly Ile Ser Ser Thr Leu Arg His Val Val Glu Gly Ala Asp
Arg 195 200 205 Trp Gln Trp His Gly Gln Arg His Leu His Ser 210 215
15 219 PRT Homo sapiens 15 Met Glu Ala Ala Arg Asp Tyr Ala Gly Ala
Leu Ile Arg Pro Leu Thr 1 5 10 15 Phe Met Gly Ser Gln Thr Lys Arg
Val Leu Phe Thr Pro Leu Met His 20 25 30 Pro Ala Arg Pro Phe Arg
Val Ser Asn His Asp Arg Ser Ser Arg Arg 35 40 45 Gly Val Met Ala
Ser Ser Leu Gln Glu Leu Ile Ser Lys Thr Leu Asp 50 55 60 Ala Leu
Val Ile Ala Thr Gly Leu Val Thr Leu Val Leu Glu Glu Asp 65 70 75 80
Gly Thr Val Val Asp Thr Glu Glu Phe Phe Gln Thr Leu Gly Asp Asn 85
90 95 Thr His Phe Met Ile Leu Glu Lys Gly Gln Lys Trp Met Pro Gly
Ser 100 105 110 Gln His Val Pro Thr Cys Ser Pro Pro Lys Arg Ser Gly
Ile Ala Arg 115 120 125 Val Thr Phe Asp Leu Tyr Arg Leu Asn Pro Lys
Asp Phe Ile Gly Cys 130 135 140 Leu Asn Val Lys Ala Thr Met Tyr Glu
Met Tyr Ser Val Ser Tyr Asp 145 150 155 160 Ile Arg Cys Thr Gly Leu
Lys Gly Leu Leu Arg Ser Leu Leu Arg Phe 165 170 175 Leu Ser Tyr Ser
Ala Gln Val Thr Gly Gln Phe Leu Ile Tyr Leu Gly 180 185 190 Thr Tyr
Met Leu Arg Val Leu Asp Asp Lys Glu Glu Arg Pro Ser Leu 195 200 205
Arg Ser Gln Ala Lys Gly Arg Phe Thr Cys Gly 210 215 16 1542 DNA
Homo sapiens CDS (156)...(1145) misc_feature 1502 n = A,T,C or G 16
cacgcgtccg ggcgtctggc gtccccggtg cccagcattc tgcggggcag gcggtctcgc
60 ttgattgggt ttctcatggg tctctggcgt ttctacggcg cggctctcac
ggactcaggc 120 caggccactc gcaggattaa ttggaattct tcaaa atg tca ggt
gtg gta ccc 173 Met Ser Gly Val Val Pro 1 5 aca gcc cct gaa cag cct
gca ggt gaa atg gaa aat caa aca aaa cca 221 Thr Ala Pro Glu Gln Pro
Ala Gly Glu Met Glu Asn Gln Thr Lys Pro 10 15 20 cca gat cca agg
cct gat gct cct cct gaa tac aat tct cat ttt tta 269 Pro Asp Pro Arg
Pro Asp Ala Pro Pro Glu Tyr Asn Ser His Phe Leu 25 30 35 cca gga
ccc cct gga aca gct gtc cct cca cct act ggc tac cca gga 317 Pro Gly
Pro Pro Gly Thr Ala Val Pro Pro Pro Thr Gly Tyr Pro Gly 40 45 50
ggc ttg cct atg gga tac tac agt cca cag caa ccc agt acc ttc cct 365
Gly Leu Pro Met Gly Tyr Tyr Ser Pro Gln Gln Pro Ser Thr Phe Pro 55
60 65 70 ttg tac cag cca gtt ggt ggt atc cat cct gtc cgg tat cag
cct ggc 413 Leu Tyr Gln Pro Val Gly Gly Ile His Pro Val Arg Tyr Gln
Pro Gly 75 80 85 aaa tat cct atg cca aat cag tct gtt cca ata aca
tgg atg cca ggg 461 Lys Tyr Pro Met Pro Asn Gln Ser Val Pro Ile Thr
Trp Met Pro Gly 90 95 100 cca act cct atg gca aac tgc cct cct ggt
ctg gaa tac tta gtt cag 509 Pro Thr Pro Met Ala Asn Cys Pro Pro Gly
Leu Glu Tyr Leu Val Gln 105 110 115 ttg gac aac ata cat gtt ctt cag
cat ttt gag cct ctg gaa atg atg 557 Leu Asp Asn Ile His Val Leu Gln
His Phe Glu Pro Leu Glu Met Met 120 125 130 aca tgt ttt gaa act aat
aat aga tat gat att aaa aac aac tca gac 605 Thr Cys Phe Glu Thr Asn
Asn Arg Tyr Asp Ile Lys Asn Asn Ser Asp 135 140 145 150 cag atg gtt
tac att gta acc gaa gac aca gat gac ttt acc agg aat 653 Gln Met Val
Tyr Ile Val Thr Glu Asp Thr Asp Asp Phe Thr Arg Asn 155 160 165 gcc
tat cgg aca cta agg ccc ttc gtc ctc cgg gtc act gat tgt atg 701 Ala
Tyr Arg Thr Leu Arg Pro Phe Val Leu Arg Val Thr Asp Cys Met 170 175
180 ggc cga gaa atc atg aca atg cag aga ccc ttc aga tgc acc tgc tgt
749 Gly Arg Glu Ile Met Thr Met Gln Arg Pro Phe Arg Cys Thr Cys Cys
185 190 195 tgc ttc tgt tgc ccc tct gcc aga caa gag ctg gag gtg cag
tgt cct 797 Cys Phe Cys Cys Pro Ser Ala Arg Gln Glu Leu Glu Val Gln
Cys Pro 200 205 210 cct ggt gtc acc att ggc ttt gtt gcg gaa cat tgg
aac ctg tgc agg 845 Pro Gly Val Thr Ile Gly Phe Val Ala Glu His Trp
Asn Leu Cys Arg 215 220 225 230 gcg gtg tac agc atc caa aat gag aag
aaa gaa aat gtg atg aga gtt 893 Ala Val Tyr Ser Ile Gln Asn Glu Lys
Lys Glu Asn Val Met Arg Val 235 240 245 cgt ggg cca tgc tca acc tat
ggc tgt ggt tca gat tct gtt ttt gag 941 Arg Gly Pro Cys Ser Thr Tyr
Gly Cys Gly Ser Asp Ser Val Phe Glu 250 255 260 gtc aaa tcc ctt gat
ggc ata tcc aac atc ggc agt att atc cgg aag 989 Val Lys Ser Leu Asp
Gly Ile Ser Asn Ile Gly Ser Ile Ile Arg Lys 265 270 275 tgg aat ggt
ttg tta tca gca atg gca gat gct gac cat ttt gac att 1037 Trp Asn
Gly Leu Leu Ser Ala Met Ala Asp Ala Asp His Phe Asp Ile 280 285 290
cac ttc cca cta gac ctg gat gtg aag atg aaa gcc atg att ttt gga
1085 His Phe Pro Leu Asp Leu Asp Val Lys Met Lys Ala Met Ile Phe
Gly 295 300 305 310 gct tgc ttc ctc att gac ttc atg tat ttt gaa aga
tct cca cca caa 1133 Ala Cys Phe Leu Ile Asp Phe Met Tyr Phe Glu
Arg Ser Pro Pro Gln 315 320 325 cgt tca aga tag agagacacag
caagccatca actatggtta attttgaaaa 1185 Arg Ser Arg * atggaaaagt
tggattgggc ttacagtcag cactcagtta tttgcaagtg tatttctttg 1245
ctttgtagag tatttttatt gggtgttaac tttgacagct gaaagtgggc ttgcaagaac
1305 acaatctaaa agtgtgtttc aattgagtat ctctctagta gaataggagt
tcatcctgaa 1365 aagctgtgac tcattaaccc agtaaacata tacaaagtaa
gcttaaaaca ctataaacat 1425 gagataaggg aaaatgaatc cagagttctc
atattaatag gtagtgaaac aataaggctt 1485 tttagagcag actttgntgg
cataaaataa cctggcttct atccctaacc ctttcct 1542 17 329 PRT Homo
sapiens VARIANT 434 Xaa = Any Amino Acid 17 Met Ser Gly Val Val Pro
Thr Ala Pro Glu Gln Pro Ala Gly Glu Met 1 5 10 15 Glu Asn Gln Thr
Lys Pro Pro Asp Pro Arg Pro Asp Ala Pro Pro Glu 20 25 30 Tyr Asn
Ser His Phe Leu Pro Gly Pro Pro Gly Thr Ala Val Pro Pro 35 40 45
Pro Thr Gly Tyr Pro Gly Gly Leu Pro Met Gly Tyr Tyr Ser Pro Gln 50
55 60 Gln Pro Ser Thr Phe Pro Leu Tyr Gln Pro Val Gly Gly Ile His
Pro 65 70 75 80 Val Arg Tyr Gln Pro Gly Lys Tyr Pro Met Pro Asn Gln
Ser Val Pro 85 90 95 Ile Thr Trp Met Pro Gly Pro Thr Pro Met Ala
Asn Cys Pro Pro Gly 100 105 110 Leu Glu Tyr Leu Val Gln Leu Asp Asn
Ile His Val Leu Gln His Phe 115 120 125 Glu Pro Leu Glu Met Met Thr
Cys Phe Glu Thr Asn Asn Arg Tyr Asp 130 135 140 Ile Lys Asn Asn Ser
Asp Gln Met Val Tyr Ile Val Thr Glu Asp Thr 145 150 155 160 Asp Asp
Phe Thr Arg Asn Ala Tyr Arg Thr Leu Arg Pro Phe Val Leu 165 170 175
Arg Val Thr Asp Cys Met Gly Arg Glu Ile Met Thr Met Gln Arg Pro 180
185 190 Phe Arg Cys Thr Cys Cys Cys Phe Cys Cys Pro Ser Ala Arg Gln
Glu 195 200 205 Leu Glu Val Gln Cys Pro Pro Gly Val Thr Ile Gly Phe
Val Ala Glu 210 215 220 His Trp Asn Leu Cys Arg Ala Val Tyr Ser Ile
Gln Asn Glu Lys Lys 225 230 235 240 Glu Asn Val Met Arg Val Arg Gly
Pro Cys Ser Thr Tyr Gly Cys Gly 245 250 255 Ser Asp Ser Val Phe Glu
Val Lys Ser Leu Asp Gly Ile Ser Asn Ile 260 265 270 Gly Ser Ile Ile
Arg Lys Trp Asn Gly Leu Leu Ser Ala Met Ala Asp 275 280 285 Ala Asp
His Phe Asp Ile His Phe Pro Leu Asp Leu Asp Val Lys Met 290 295 300
Lys Ala Met Ile Phe Gly Ala Cys Phe Leu Ile Asp Phe Met Tyr Phe 305
310 315 320 Glu Arg Ser Pro Pro Gln Arg Ser Arg 325 18 990 DNA Homo
sapiens CDS (1)...(990) 18 atg tca ggt gtg gta ccc aca gcc cct gaa
cag cct gca ggt gaa atg 48 Met Ser Gly Val Val Pro Thr Ala Pro Glu
Gln Pro Ala Gly Glu Met 1 5 10 15 gaa aat caa aca aaa cca cca gat
cca agg cct gat gct cct cct gaa 96 Glu Asn Gln Thr Lys Pro Pro Asp
Pro Arg Pro Asp Ala Pro Pro Glu 20 25 30 tac aat tct cat ttt tta
cca gga ccc cct gga aca gct gtc cct cca 144 Tyr Asn Ser His Phe Leu
Pro Gly Pro Pro Gly Thr Ala Val Pro Pro 35 40 45 cct act ggc tac
cca gga ggc ttg cct atg gga tac tac agt cca cag 192 Pro Thr Gly Tyr
Pro Gly Gly Leu Pro Met Gly Tyr Tyr Ser Pro Gln 50 55 60 caa ccc
agt acc ttc cct ttg tac cag cca gtt ggt ggt atc cat cct 240 Gln Pro
Ser Thr Phe Pro Leu Tyr Gln Pro Val Gly Gly Ile His Pro 65 70 75 80
gtc cgg tat cag cct ggc aaa tat cct atg cca aat cag tct gtt cca 288
Val Arg Tyr Gln Pro Gly Lys Tyr Pro Met Pro Asn Gln Ser Val Pro 85
90 95 ata aca tgg atg cca ggg cca act cct atg gca aac tgc cct cct
ggt 336 Ile Thr Trp Met Pro Gly Pro Thr Pro Met Ala Asn Cys Pro Pro
Gly 100 105 110 ctg gaa tac tta gtt cag ttg gac aac ata cat gtt ctt
cag cat ttt 384 Leu Glu Tyr Leu Val Gln Leu Asp Asn Ile His Val Leu
Gln His Phe 115 120 125 gag cct ctg gaa atg atg aca tgt ttt gaa act
aat aat aga tat gat 432 Glu Pro Leu Glu Met Met Thr Cys Phe Glu Thr
Asn Asn Arg Tyr Asp 130 135 140 att aaa aac aac tca gac cag atg gtt
tac att gta acc gaa gac aca 480 Ile Lys Asn Asn Ser Asp Gln Met Val
Tyr Ile Val Thr Glu Asp Thr 145 150 155 160 gat gac ttt acc agg aat
gcc tat cgg aca cta agg ccc ttc gtc ctc 528 Asp Asp Phe Thr Arg Asn
Ala Tyr Arg Thr Leu Arg Pro Phe Val Leu 165 170 175 cgg gtc act gat
tgt atg ggc cga gaa atc atg aca atg cag aga ccc 576 Arg Val Thr Asp
Cys Met Gly Arg Glu Ile Met Thr Met Gln Arg Pro 180 185 190 ttc aga
tgc acc tgc tgt tgc ttc tgt tgc ccc tct gcc aga caa gag 624 Phe Arg
Cys Thr Cys Cys Cys Phe Cys Cys Pro Ser Ala Arg Gln Glu 195 200 205
ctg gag gtg cag tgt cct cct ggt gtc acc att ggc ttt gtt gcg gaa 672
Leu Glu Val Gln Cys Pro Pro Gly Val Thr Ile Gly Phe Val Ala Glu 210
215 220 cat tgg aac ctg tgc agg gcg gtg tac agc atc caa aat gag aag
aaa 720 His Trp Asn Leu Cys Arg Ala Val Tyr Ser Ile Gln Asn Glu Lys
Lys 225 230 235 240 gaa aat gtg atg aga gtt cgt ggg cca tgc tca acc
tat ggc tgt ggt 768 Glu Asn Val Met Arg Val Arg Gly Pro Cys Ser Thr
Tyr Gly Cys Gly 245 250 255 tca gat tct gtt ttt gag gtc aaa tcc ctt
gat ggc ata tcc aac atc 816 Ser Asp Ser Val Phe Glu Val Lys Ser Leu
Asp Gly Ile Ser Asn Ile 260 265 270 ggc agt att atc cgg aag tgg aat
ggt ttg tta tca gca atg gca gat 864 Gly Ser Ile Ile Arg Lys Trp Asn
Gly Leu Leu Ser Ala Met Ala Asp 275 280 285 gct gac cat ttt gac att
cac ttc cca cta gac ctg gat gtg aag atg 912 Ala Asp His Phe Asp Ile
His Phe Pro Leu Asp Leu Asp Val Lys Met 290 295 300 aaa gcc atg att
ttt gga gct tgc ttc ctc att gac ttc atg tat ttt 960 Lys Ala Met Ile
Phe Gly Ala Cys Phe Leu Ile Asp Phe Met Tyr Phe 305 310 315 320 gaa
aga tct cca cca caa cgt tca aga tag 990 Glu Arg Ser Pro Pro Gln Arg
Ser Arg * 325 19 307 PRT Mus musculus 19 Met Glu Ala Pro Arg Ser
Gly Thr Tyr Leu Pro Ala Gly Tyr Ala Pro 1 5 10 15 Gln Tyr Pro Pro
Ala Ala Val Gln Gly Pro Pro Glu His Thr Gly Arg 20 25 30 Pro Thr
Phe Gln Thr Asn Tyr Gln Val Pro Gln Ser Gly Tyr Pro Gly 35 40 45
Pro Gln Ala Ser Tyr Thr Val Ser Thr Ser Gly His Glu Gly Tyr Ala 50
55 60 Ala Thr Arg Leu Pro Ile Gln Asn Asn Gln Thr Ile Val Leu Ala
Asn 65 70 75 80 Thr Gln Trp Met Pro Ala Pro Pro Pro Ile Leu Asn Cys
Pro Pro Gly 85 90 95 Leu Glu Tyr Leu Asn Gln Ile Asp Gln Leu Leu
Ile His Gln Gln Val 100 105 110 Glu Leu Leu Glu Val Leu Thr Gly Phe
Glu Thr Asn Asn Lys Phe Glu 115 120 125 Ile Lys Asn Ser Leu Gly Gln
Met Val Tyr Val Ala Val Glu Asp Thr 130 135 140 Asp Cys Cys Thr Arg
Asn Cys Cys Glu Ala Ser Arg Pro Phe Thr Leu 145 150 155 160 Arg Ile
Leu Asp His Leu Gly Gln Glu Val Met Thr Leu Glu Arg Pro 165 170 175
Leu Arg Cys Ser Ser Cys Cys Phe Pro Cys Cys Leu Gln Glu Ile Glu 180
185 190 Ile Gln Ala Pro Pro Gly Val Pro Ile Gly Tyr Val Thr Gln Thr
Trp 195 200 205 His Pro Cys Leu Pro Lys Leu Thr Leu Gln Asn Asp Lys
Arg Glu Asn 210 215 220 Val Leu Lys Val Val Gly Pro Cys Val Ala Cys
Thr Cys Cys Ser Asp 225 230 235 240 Ile Asp Phe Glu Ile Lys Ser Leu
Asp Glu Val Thr Arg Ile Gly Lys 245 250 255 Ile Thr Lys Gln Trp Ser
Gly Cys Val Lys Glu Ala Phe Thr Asp Ser 260 265 270 Asp Asn Phe Gly
Ile Gln Phe Pro Leu Asp Leu Glu Val Lys Met Lys 275 280 285 Ala Val
Thr Leu Gly Ala Cys Phe Leu Ile Asp Tyr Met Phe Phe Glu 290 295 300
Gly Cys Glu 305 20 318 PRT Homo sapiens 20 Met Asp Lys Gln Asn Ser
Gln Met Asn Ala Ser His Pro Glu Thr Asn 1 5 10 15 Leu Pro Val Gly
Tyr Pro Pro Gln Tyr Pro Pro Thr Ala Phe Gln Gly 20 25 30 Pro Pro
Gly Tyr Ser Gly Tyr Pro Gly Pro Gln Val Ser Tyr Pro Pro 35 40 45
Pro Pro Ala Gly His Ser Gly Pro Gly Pro Ala Gly Phe Pro Val Pro 50
55 60 Asn Gln Pro Val Tyr Asn Gln Pro Val Tyr Asn Gln Pro Val Gly
Ala 65 70 75 80 Ala Gly Val Pro Trp Met Pro Ala Pro Gln Pro Pro Leu
Asn Cys Pro 85 90 95 Pro Gly Leu Glu Tyr Leu Ser Gln Ile Asp Gln
Ile Leu Ile His Gln 100 105 110 Gln Ile Glu Leu Leu Glu Val Leu Thr
Gly Phe Glu Thr Asn Asn Lys 115 120 125 Tyr Glu Ile Lys Asn Ser Phe
Gly Gln Arg Val
Tyr Phe Ala Ala Glu 130 135 140 Asp Thr Asp Cys Cys Thr Arg Asn Cys
Cys Gly Pro Ser Arg Pro Phe 145 150 155 160 Thr Leu Arg Ile Ile Asp
Asn Met Gly Gln Glu Val Ile Thr Leu Glu 165 170 175 Arg Pro Leu Arg
Cys Ser Ser Cys Cys Cys Pro Cys Cys Leu Gln Glu 180 185 190 Ile Glu
Ile Gln Ala Pro Pro Gly Val Pro Ile Gly Tyr Val Ile Gln 195 200 205
Thr Trp His Pro Cys Leu Pro Lys Phe Thr Ile Gln Asn Glu Lys Arg 210
215 220 Glu Asp Val Leu Lys Ile Ser Gly Pro Cys Val Val Cys Ser Cys
Cys 225 230 235 240 Gly Asp Val Asp Phe Glu Ile Lys Ser Leu Asp Glu
Gln Cys Val Val 245 250 255 Gly Lys Ile Ser Lys His Trp Thr Gly Ile
Leu Arg Glu Ala Phe Thr 260 265 270 Asp Ala Asp Asn Phe Gly Ile Gln
Phe Pro Leu Asp Leu Asp Val Lys 275 280 285 Met Lys Ala Val Met Ile
Gly Ala Cys Phe Leu Ile Asp Phe Met Phe 290 295 300 Phe Glu Ser Thr
Gly Ser Gln Glu Gln Lys Ser Gly Val Trp 305 310 315 21 1452 DNA
Homo sapiens CDS (219)...(995) 21 cggaattccc gggtcgaccc acgcgtccgg
agcggcgagc cgagacggct acatggacgc 60 cactatcgcc ccgcaccgta
tcccccccga gatgccccag tacggggagg agaaccacgt 120 cttcgagttg
atgcagaaca tgctggagca actcctgatc caccagcccg aagatcccat 180
ccccttcatg atccagcact tgcatagaga caacgaca atg gca atg tgg ctc tgc
236 Met Ala Met Trp Leu Cys 1 5 aaa cat ctg aac agc agt ctc ctc acc
ctg gag aac ctg atc tta aat 284 Lys His Leu Asn Ser Ser Leu Leu Thr
Leu Glu Asn Leu Ile Leu Asn 10 15 20 gag ttt tcc tat acg gcc acc
gaa gcc aga agg ctt tat ctg caa agg 332 Glu Phe Ser Tyr Thr Ala Thr
Glu Ala Arg Arg Leu Tyr Leu Gln Arg 25 30 35 aag aca gtt ccc agt
gcg ctg ctc gtc cag ctg att cag gaa cgc ctg 380 Lys Thr Val Pro Ser
Ala Leu Leu Val Gln Leu Ile Gln Glu Arg Leu 40 45 50 gct gaa gag
gat tgc atc aag cag ggc tgg att ctg gat ggc atc cct 428 Ala Glu Glu
Asp Cys Ile Lys Gln Gly Trp Ile Leu Asp Gly Ile Pro 55 60 65 70 gag
acg cgt gag cag gct ctg agg atc cag acc ctg ggg atc aca ccc 476 Glu
Thr Arg Glu Gln Ala Leu Arg Ile Gln Thr Leu Gly Ile Thr Pro 75 80
85 aga cac gtc att gtg ctg agt gct cca gac acg gtc ctg atc gag aga
524 Arg His Val Ile Val Leu Ser Ala Pro Asp Thr Val Leu Ile Glu Arg
90 95 100 aac ttg ggg aag aga atc gac cct caa act gga gag att tat
cac acc 572 Asn Leu Gly Lys Arg Ile Asp Pro Gln Thr Gly Glu Ile Tyr
His Thr 105 110 115 acc ttt gac tgg cca ccc gaa tct gaa atc cag aac
cgt ctc atg gtg 620 Thr Phe Asp Trp Pro Pro Glu Ser Glu Ile Gln Asn
Arg Leu Met Val 120 125 130 cca gag gac atc tca gag ctg gag acg gct
cag aaa ctg ctg gag tat 668 Pro Glu Asp Ile Ser Glu Leu Glu Thr Ala
Gln Lys Leu Leu Glu Tyr 135 140 145 150 cat agg aac atc gtc agg gtc
att ccc tcc tac ccc aaa atc ctc aaa 716 His Arg Asn Ile Val Arg Val
Ile Pro Ser Tyr Pro Lys Ile Leu Lys 155 160 165 gtc atc agt gct gac
cag cca tgt gtg gac gtc ttc tac cag gct ctg 764 Val Ile Ser Ala Asp
Gln Pro Cys Val Asp Val Phe Tyr Gln Ala Leu 170 175 180 acc tat gtc
caa agc aac cat cgt act aat gcc ccg ttc acc ccg agg 812 Thr Tyr Val
Gln Ser Asn His Arg Thr Asn Ala Pro Phe Thr Pro Arg 185 190 195 gtg
ctg ctg ctc ggg cct gtg ggc agt ggg aaa agt ctg cag gcc gcc 860 Val
Leu Leu Leu Gly Pro Val Gly Ser Gly Lys Ser Leu Gln Ala Ala 200 205
210 ctc ctg gcc cag aaa tac agg ctt gtc aat gtc tgc tgt ggg caa ctg
908 Leu Leu Ala Gln Lys Tyr Arg Leu Val Asn Val Cys Cys Gly Gln Leu
215 220 225 230 ctg aaa gag gct gtg gca gat agg acc acg ttt ggc gag
ctc atc cag 956 Leu Lys Glu Ala Val Ala Asp Arg Thr Thr Phe Gly Glu
Leu Ile Gln 235 240 245 ccc ttc ttt gaa aag gag atg gca ggg tgt ttt
tcc tga atgtgccatt 1005 Pro Phe Phe Glu Lys Glu Met Ala Gly Cys Phe
Ser * 250 255 tgattccatc atggagcggc tgactctgag aagaattgat
ccagtcactg gggaaaggta 1065 ccacctcatg tacaagccac ctcccaccat
ggagatccag gctcgcctcc tgcagaaccc 1125 aaaggatgct gaagagcagg
tcaagctgaa aatggacctg ttctacagga actcagctga 1185 cttggagcag
ttgtatgggt cggccatcac cctcaatggg gaccaggacc catacacagt 1245
cttcgaatac atcgagagtg ggatcattaa tcccctgccc aagaaaatcc cctgatgggt
1305 tcagagccag gagcgctgcc ccagggaaag agttaatccc ctgcccccag
ccccccagcc 1365 tcggcacagc tcccctaaaa agccaataaa gcctgctgga
tacagaaaaa aaaaaaaaaa 1425 aaaaaaaaaa aaaaaaaaaa aaaaaaa 1452 22
258 PRT Homo sapiens 22 Met Ala Met Trp Leu Cys Lys His Leu Asn Ser
Ser Leu Leu Thr Leu 1 5 10 15 Glu Asn Leu Ile Leu Asn Glu Phe Ser
Tyr Thr Ala Thr Glu Ala Arg 20 25 30 Arg Leu Tyr Leu Gln Arg Lys
Thr Val Pro Ser Ala Leu Leu Val Gln 35 40 45 Leu Ile Gln Glu Arg
Leu Ala Glu Glu Asp Cys Ile Lys Gln Gly Trp 50 55 60 Ile Leu Asp
Gly Ile Pro Glu Thr Arg Glu Gln Ala Leu Arg Ile Gln 65 70 75 80 Thr
Leu Gly Ile Thr Pro Arg His Val Ile Val Leu Ser Ala Pro Asp 85 90
95 Thr Val Leu Ile Glu Arg Asn Leu Gly Lys Arg Ile Asp Pro Gln Thr
100 105 110 Gly Glu Ile Tyr His Thr Thr Phe Asp Trp Pro Pro Glu Ser
Glu Ile 115 120 125 Gln Asn Arg Leu Met Val Pro Glu Asp Ile Ser Glu
Leu Glu Thr Ala 130 135 140 Gln Lys Leu Leu Glu Tyr His Arg Asn Ile
Val Arg Val Ile Pro Ser 145 150 155 160 Tyr Pro Lys Ile Leu Lys Val
Ile Ser Ala Asp Gln Pro Cys Val Asp 165 170 175 Val Phe Tyr Gln Ala
Leu Thr Tyr Val Gln Ser Asn His Arg Thr Asn 180 185 190 Ala Pro Phe
Thr Pro Arg Val Leu Leu Leu Gly Pro Val Gly Ser Gly 195 200 205 Lys
Ser Leu Gln Ala Ala Leu Leu Ala Gln Lys Tyr Arg Leu Val Asn 210 215
220 Val Cys Cys Gly Gln Leu Leu Lys Glu Ala Val Ala Asp Arg Thr Thr
225 230 235 240 Phe Gly Glu Leu Ile Gln Pro Phe Phe Glu Lys Glu Met
Ala Gly Cys 245 250 255 Phe Ser 23 774 DNA Homo sapiens 23
atggcaatgt ggctctgcaa acatctgaac agcagtctcc tcaccctgga gaacctgatc
60 ttaaatgagt tttcctatac ggccaccgaa gccagaaggc tttatctgca
aaggaagaca 120 gttcccagtg cgctgctcgt ccagctgatt caggaacgcc
tggctgaaga ggattgcatc 180 aagcagggct ggattctgga tggcatccct
gagacgcgtg agcaggctct gaggatccag 240 accctgggga tcacacccag
acacgtcatt gtgctgagtg ctccagacac ggtcctgatc 300 gagagaaact
tggggaagag aatcgaccct caaactggag agatttatca caccaccttt 360
gactggccac ccgaatctga aatccagaac cgtctcatgg tgccagagga catctcagag
420 ctggagacgg ctcagaaact gctggagtat cataggaaca tcgtcagggt
cattccctcc 480 taccccaaaa tcctcaaagt catcagtgct gaccagccat
gtgtggacgt cttctaccag 540 gctctgacct atgtccaaag caaccatcgt
actaatgccc cgttcacccc gagggtgctg 600 ctgctcgggc ctgtgggcag
tgggaaaagt ctgcaggccg ccctcctggc ccagaaatac 660 aggcttgtca
atgtctgctg tgggcaactg ctgaaagagg ctgtggcaga taggaccacg 720
tttggcgagc tcatccagcc cttctttgaa aaggagatgg cagggtgttt ttcc 774 24
84 PRT Artificial Sequence Adenylate kinase consensus domain 1 24
Val Pro Asp Glu Val Val Ile Gly Leu Val Lys Glu Arg Leu Glu Gln 1 5
10 15 Asn Asp Asp Cys Lys Asn Gly Phe Leu Leu Asp Gly Phe Pro Arg
Thr 20 25 30 Val Pro Gln Ala Glu Ala Leu Glu Glu Met Leu Glu Glu
Ala Gly Ile 35 40 45 Lys Leu Asp Ala Val Ile Glu Leu Asp Val Pro
Asp Glu Val Leu Val 50 55 60 Glu Arg Leu Thr Gly Arg Arg Ile His
Pro Thr Ser Gly Arg Ser Tyr 65 70 75 80 His Leu Glu Phe 25 51 PRT
Artificial Sequence Adenylate kinase consensus domain 2 25 Leu Leu
Gly Pro Pro Gly Ala Gly Lys Gly Thr Gln Ala Glu Arg Ile 1 5 10 15
Val Lys Lys Tyr Gly Ile Pro His Leu Ser Thr Gly Asp Leu Leu Arg 20
25 30 Ala Glu Val Lys Ser Gly Thr Glu Leu Gly Lys Glu Ala Lys Glu
Tyr 35 40 45 Met Asp Lys 50
* * * * *
References