U.S. patent application number 09/969836 was filed with the patent office on 2002-07-04 for human apoptosis-related calcium-binding protein.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Goli, Surya K., Hillman, Jennifer L..
Application Number | 20020086830 09/969836 |
Document ID | / |
Family ID | 25076950 |
Filed Date | 2002-07-04 |
United States Patent
Application |
20020086830 |
Kind Code |
A1 |
Hillman, Jennifer L. ; et
al. |
July 4, 2002 |
Human apoptosis-related calcium-binding protein
Abstract
The present invention provides a human apoptosis-related
calcium-binding protein (HARC) and polynucleotides which identify
and encode HARC. The invention also provides genetically engineered
expression vectors and host cells comprising the nucleic acid
sequences encoding HARC and a method for producing HARC. The
invention also provides for agonists, antibodies, or antagonists
specifically binding HARC, and their use, in the prevention and
treatment of diseases associated with expression of HARC.
Additionally, the invention provides for the use of antisense
molecules to polynucleotides encoding HARC for the treatment of
diseases associated with the expression of HARC. The invention also
provides diagnostic assays which utilize the polynucleotide, or
fragments or the complement thereof, and antibodies specifically
binding HARC.
Inventors: |
Hillman, Jennifer L.; (San
Jose, CA) ; Goli, Surya K.; (Sunnyvale, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
25076950 |
Appl. No.: |
09/969836 |
Filed: |
October 1, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09969836 |
Oct 1, 2001 |
|
|
|
09328687 |
Jun 9, 1999 |
|
|
|
09328687 |
Jun 9, 1999 |
|
|
|
09094212 |
Jun 9, 1998 |
|
|
|
09094212 |
Jun 9, 1998 |
|
|
|
08766605 |
Dec 12, 1996 |
|
|
|
Current U.S.
Class: |
514/18.9 ;
435/226; 435/252.3; 435/320.1; 435/325; 435/69.1; 514/19.3;
536/23.2; 800/8 |
Current CPC
Class: |
A61P 37/04 20180101;
C07K 14/4702 20130101; A61P 9/10 20180101; A61P 37/06 20180101;
A61P 25/16 20180101; A61P 31/14 20180101; A61P 35/00 20180101; C07K
14/4747 20130101; A61P 1/16 20180101; A61P 29/00 20180101; A61P
1/00 20180101; A61P 13/12 20180101; A61P 31/20 20180101; A61P 25/28
20180101; A61P 19/02 20180101; A61K 38/00 20130101; A61P 17/06
20180101; A61P 25/00 20180101; A61P 27/02 20180101; A61P 43/00
20180101; A61P 31/22 20180101 |
Class at
Publication: |
514/12 ;
435/69.1; 435/226; 435/320.1; 435/325; 435/252.3; 800/8;
536/23.2 |
International
Class: |
A01K 067/00; A61K
038/17; C07H 021/04; C12N 009/64; C12P 021/02; C12N 005/06; C12N
001/21 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: a) a polypeptide comprising
an amino acid sequence of SEQ ID NO: 1, b) a naturally occurring
polypeptide comprising an amino acid sequence at least 90%
identical to an amino acid sequence of SEQ ID NO: 1, c) a
biologically active fragment of a polypeptide having an amino acid
sequence of SEQ ID NO: 1, and d) an immunogenic fragment of a
polypeptide having an amino acid sequence of SEQ ID NO: 1.
2. An isolated polypeptide of claim 1, having a sequence of SEQ ID
NO: 1.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4, having a sequence of SEQ
ID NO: 2 .
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide has the sequence
of SEQ ID NO: 1.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide comprising a sequence selected from
the group consisting of: a) a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 2, b) a naturally occurring
polynucleotide comprising a polynucleotide sequence at least 90%
identical to a polynucleotide sequence of SEQ ID NO: 2, c) a
polynucleotide having a sequence complementary to a polynucleotide
of a), d) a polynucleotide having a sequence complementary to a
polynucleotide of b) and e) an RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide has an amino
acid sequence of SEQ ID NO: 1.
19. A method for treating a disease or condition associated with
decreased expression of functional HARC, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional HARC, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional HARC, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, said method comprising the steps of: a)
combining the polypeptide of claim 1 with at least one test
compound under suitable conditions, and b) detecting binding of the
polypeptide of claim 1 to the test compound, thereby identifying a
compound that specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method for assessing toxicity of a test compound, said method
comprising: a) treating a biological sample containing nucleic
acids with the test compound; b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof; c)
quantifying the amount of hybridization complex; and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of HARC in a biological sample comprising the steps
of: a) combining the biological sample with an antibody of claim
11, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex; and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of HARC in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of HARC in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11 comprising: a) immunizing
an animal with a polypeptide having an amino acid sequence of SEQ
ID NO: 1, or an immunogenic fragment thereof, under conditions to
elicit an antibody response; b) isolating antibodies from said
animal; and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide having an amino acid sequence of SEQ
ID NO: 1.
37. An antibody produced by a method of claim 36.
38. A composition comprising the antibody of claim 37 and a
suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11 comprising: a) immunizing an animal
with a polypeptide having an amino acid sequence of SEQ ID NO: 1,
or an immunogenic fragment thereof, under conditions to elicit an
antibody response; b) isolating antibody producing cells from the
animal; c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridoma cells; d)
culturing the hybridoma cells; and e) isolating from the culture
monoclonal antibody which binds specifically to a polypeptide
having an amino acid sequence of SEQ ID NO: 1.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the antibody of claim 40 and a
suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method for detecting a polypeptide having an amino acid
sequence of SEQ ID NO: 1 in a sample, comprising the steps of: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide; and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide having an amino acid sequence of SEQ
ID NO: 1 in the sample.
45. A method of purifying a polypeptide having an amino acid
sequence of SEQ ID NO: 1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide; and
b) separating the antibody from the sample and obtaining the
purified polypeptide having an amino acid sequence of SEQ ID NO:
1.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method for generating a transcript image of a sample which
contains polynucleotides, the method comprising the steps of: a)
labeling the polynucleotides of the sample, b) contacting the
elements of the microarray of claim 46 with the labeled
polynucleotides of the sample under conditions suitable for the
formation of a hybridization complex, and c) quantifying the
expression of the polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, said target
polynucleotide having a sequence of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, which is a microarray.
52. An array of claim 48, further comprising said target
polynucleotide hybridized to said first oligonucleotide or
polynucleotide.
53. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
54. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules having the
same sequence, and each distinct physical location on the substrate
contains nucleotide molecules having a sequence which differs from
the sequence of nucleotide molecules at another physical location
on the substrate.
55. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO: 1.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO: 2.
57. An isolated antibody of claim 11 which specifically binds to a
polypeptide comprising an amino acid sequence selected from the
group consisting of a) a biologically-active fragment of at least
10 amino acids of the amino acid sequence of SEQ ID NO: 1, said
fragment having a calcium-binding domain and having calcium-binding
activity, and b) an immunologically-active fragment of at least 10
amino acids of the amino acid sequence of SEQ ID NO: 1, said
fragment having a calcium-binding domain.
Description
[0001] This application is a continuation application of U.S. Ser.
No. 09/328,687, filed Jun. 9, 1999, which is a divisional
application of U.S. Ser. No. 09/094,212, filed Jun. 9, 1998, now
U.S. Pat. No. 5,935,931, which is a divisional application of U.S.
Ser. No. 08/766,605, filed Dec. 12, 1996, now U.S. Pat. No.
5,763,220, the contents each of which is hereby incorporated by
reference, and each application entitled "Human Apoptosis-Related
Calcium-Binding Protein".
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a novel human apoptosis-related calcium-binding
protein and to the use of these sequences in the diagnosis,
prevention, and treatment of diseases associated with decreased or
increased apoptosis.
BACKGROUND OF THE INVENTION
[0003] Normal development, growth, and homeostasis in multicellular
organisms require a careful balance between the production and
destruction of cells in tissues throughout the body. Cell division
is a carefully coordinated process with numerous checkpoints and
control mechanisms. These mechanisms are designed to regulate DNA
replication and to prevent inappropriate or excessive
proliferation. In contrast, programmed cell death is the
genetically controlled process by which unneeded or damaged cells
can be eliminated without causing the tissue destruction and
inflammatory responses that are often associated with acute injury
and necrosis.
[0004] The term "apoptosis" was first used by Kerr, J. F. et al.
(1972; Br. J. Cancer 26:239-257) to describe the morphological
changes that characterize cells undergoing programmed cell death.
Apoptotic cells have a shrunken appearance with an altered membrane
lipid content and highly condensed nuclei. Apoptotic cells are
rapidly phagocytosed by neighboring cells or macrophages without
leaking their potentially damaging contents into the surrounding
tissue.
[0005] The processes and mechanisms regulating apoptosis are highly
conserved throughout the phylogenetic tree. Indeed, much of our
current knowledge about apoptosis is derived from studies of the
nematode, Caenorhabditis elegans and the fruit fly, Drosophila
melanogaster (See for example, Steller, H. (1995) Science
267:1445-1449, and references therein). Dysregulation of apoptosis
has recently been recognized as a significant factor in the
pathogenesis of human disease. For example, inappropriate cell
survival can cause or contribute to many diseases such as cancer,
autoimmune diseases, and inflammatory diseases. In contrast,
increased apoptosis can cause immunodeficiency diseases such as
AIDS, neurodegenerative disorders, and myelodysplastic syndromes
(reviewed by Thompson, C. B. (1995) Science 267:1456-1462).
[0006] A variety of ligands and their cellular receptors, enzymes,
tumor suppressors, viral gene products, pharmacological agents, and
inorganic ions have important positive or negative roles in
regulating and implementing the apoptotic destruction of a cell
(Steller, H., supra; Thompson, C. B., supra). Although many
different extra- and intracellular signals can trigger apoptosis
(cf. Raff, M. C. (1992) Nature 356:397-400, Raff, M. C. et al.
(1993) Science 262:695-700, and Steller, H. and M. E. Grether,
(1994) Neuron 13:1269-1274), these signals probably all converge on
a common mechanism that ultimately causes the cell to die.
[0007] The mouse apoptosis-linked gene 2 (ALG-2) is one of six
clones that has been identified using an in vitro "death trap"
model for apoptosis. In this model, 3DO hybridoma cells are
transformed with cloned cDNAs and then induced to undergo
programmed cell death by cross-linking their T cell receptors.
Certain cDNAs afford protection to the transformed 3DO cells either
by expressing a protein which inhibits apoptosis or by expressing
an antisense RNA which blocks the synthesis of a required protein
(Vito, P. et al. (1996) Science 271:521-525).
[0008] Analysis of the recovered ALG-2 clone indicates that it is
of the latter type, thereby identifying ALG-2 as a protein that is
essential for programmed cell death. Northern blot analyses detect
a single, .about.1.3 KB ALG-2 transcript that is constitutively
expressed in all adult mouse tissues; expression is highest in the
thymus and liver and lowest in the testes and skeletal muscles.
Constitutive expression in normal tissue implies that ALG-2 is
probably in an inactive state until the apoptosis pathway is
triggered. Apoptosis induced by T cell receptor cross-linking,
Fas-Fas ligand interactions, and glucocorticoid treatment all
depend on functional ALG-2, which is likely to be a part of the
common pathway leading to cell death (Vito, P. et al., supra).
[0009] The ALG-2 sequence predicts an acidic protein of 191 amino
acids with 2 EF-hand Ca.sup.2+-binding domains; both EF-hand
domains are required for Ca.sup.2+ binding. ALG-2 is the first
Ca.sup.2+-binding protein that has been shown to be directly
required in the apoptosis pathway (Vito, P. et al., supra). A
requirement for Ca.sup.2+ in apoptosis was previously suggested by
studies showing its involvement in DNA cleavage (Hewish, D. R. and
L. A. Burgoyne (1973) Biochem. Biophys. Res. Comm. 52:504-510).
Other studies show that: 1) intracellular Ca.sup.2+ concentrations
increase when apoptosis is triggered in thymocytes by either T cell
receptor cross-linking or by glucocorticoids, and 2) cell death can
be prevented by blocking this increase in intracellular Ca.sup.2+
(McConkey, D. J. et al. (1989) J. Immunol. 143:1801-1806; McConkey,
D. J. et al. (1989) Arch. Biochem. Biophys. 269:365-370).
Additional support for the role of Ca.sup.2+ in apoptosis comes
from work on Fas-mediated cell death (Vignaux, F. et al. (1995) J.
Exp. Med. 181:781-786; Oshimi, Y. and S. Miyazaki (1995) J.
Immunol. 154:599-609).
[0010] The discovery of polynucleotides encoding human
apoptosis-related calcium-binding protein, and the molecules
themselves, provide a means to investigate the regulation of
programmed cell death and apoptosis. Discovery of molecules related
to mouse ALG-2 satisfies a need in the art by providing new
diagnostic or therapeutic compositions useful in the detection,
prevention, and treatment of cancer, autoimmune diseases,
lymphoproliferative disorders, psoriasis, atherosclerosis,
restenosis, AIDS, immunodeficiency diseases, ischemic injuries,
neurodegenerative diseases, osteoporosis, myelodysplastic
syndromes, toxin-induced diseases, cachexia, and viral
infections.
SUMMARY OF THE INVENTION
[0011] The present invention features a novel human
apoptosis-related calcium-binding protein hereinafter designated
HARC and characterized as having similarity to mouse
apoptosis-linked gene 2, ALG-2.
[0012] Accordingly, the invention features a substantially purified
HARC having the amino acid sequence shown in SEQ ID NO: 1.
[0013] One aspect of the invention features isolated and
substantially purified polynucleotides that encode HARC. In a
particular aspect, the polynucleotide is the nucleotide sequence of
SEQ ID NO: 2.
[0014] The invention also relates to a polynucleotide sequence
comprising the complement of SEQ ID NO: 2 or variants thereof. In
addition, the invention features polynucleotide sequences which
hybridize under stringent conditions to SEQ ID NO: 2.
[0015] The invention additionally features nucleic acid sequences
encoding polypeptides, oligonucleotides, peptide nucleic acids
(PNA), fragments, portions or antisense molecules thereof, and
expression vectors and host cells comprising polynucleotides that
encode HARC. The present invention also features antibodies which
bind specifically to HARC, and pharmaceutical compositions
comprising substantially purified HARC. The invention also features
the use of agonists and antagonists of HARC.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO: 1)
and nucleic acid sequence (SEQ ID NO: 2) of HARC. The alignment was
produced using MacDNASIS PRO.TM. software (Hitachi Software
Engineering Co., Ltd., San Bruno, Calif.).
[0017] FIG. 2 shows the amino acid sequence alignments between HARC
(SEQ ID NO: 1) and mouse ALG-2 (GI1213520; SEQ ID NO: 3). The
alignment was produced using the multisequence alignment program of
LASERGENE software (DNASTAR Inc, Madison Wis.).
[0018] FIG. 3 shows the hydrophobicity plot (MacDNASIS PRO
software) for HARC, SEQ ID NO: 1; the positive X axis reflects
amino acid position, and the negative Y axis, hydrophobicity.
[0019] FIG. 4 shows the hydrophobicity plot for ALG-2, SEQ ID NO:
3.
[0020] FIGS. 5A and 5B show the northern analysis for SEQ ID NO: 2.
The northern analysis was produced electronically using the LIFESEQ
database (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.).
DESCRIPTION OF THE INVENTION
[0021] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described as these may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0022] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to the "antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0023] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the cell lines, vectors, and methodologies which are reported in
the publications which might be used in connection with the
invention. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
DEFINITIONS
[0024] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide, or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which may be single- or double-stranded, and represent the sense or
antisense strand. Similarly, "amino acid sequence" as used herein
refers to an oligopeptide, peptide, polypeptide, or protein
sequence, and fragments or portions thereof, and to naturally
occurring or synthetic molecules.
[0025] Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms, such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule.
[0026] "Peptide nucleic acid", as used herein, refers to a molecule
which comprises an oligomer to which an amino acid residue, such as
lysine, and an amino group have been added. These small molecules,
also designated anti-gene agents, stop transcript elongation by
binding to their complementary strand of nucleic acid (Nielsen, P.
E. et al. (1993) Anticancer Drug Des. 8:53-63).
[0027] HARC, as used herein, refers to the amino acid sequences of
substantially purified HARC obtained from any species, particularly
mammalian, including bovine, ovine, porcine, murine, equine, and
preferably human, from any source whether natural, synthetic,
semi-synthetic, or recombinant.
[0028] "Consensus", as used herein, refers to a nucleic acid
sequence which has been resequenced to resolve uncalled bases, or
which has been extended using XL-PCR kit (Perkin Elmer, Norwalk,
Conn.) in the 5' and/or the 3' direction and resequenced, or which
has been assembled from the overlapping sequences of more than one
Incyte clone using the GELVIEW fragment assembly system (GCG,
Madison, Wis.), or which has been both extended and assembled.
[0029] A "variant" of HARC, as used herein, refers to an amino acid
sequence that is altered by one or more amino acids. The variant
may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties, e.g., replacement of
leucine with isoleucine. More rarely, a variant may have
"nonconservative" changes, e.g., replacement of a glycine with a
tryptophan. Similar minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software.
[0030] A "deletion", as used herein, refers to a change in either
amino acid or nucleotide sequence in which one or more amino acid
or nucleotide residues, respectively, are absent.
[0031] An "insertion" or "addition", as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid or nucleotide residues,
respectively, as compared to the naturally occurring molecule.
[0032] A "substitution", as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0033] The term "biologically active", as used herein, refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
HARC, or any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0034] The term "agonist", as used herein, refers to a molecule
which, when bound to HARC, causes a change in HARC which modulates
the activity of HARC. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to HARC.
[0035] The terms "antagonist" or "inhibitor", as used herein, refer
to a molecule which, when bound to HARC, blocks or modulates the
biological or immunological activity of HARC. Antagonists and
inhibitors may include proteins, nucleic acids, carbohydrates, or
any other molecules which bind to HARC.
[0036] The term "modulate", as used herein, refers to a change or
an alteration in the biological activity of HARC. Modulation may be
an increase or a decrease in protein activity, a change in binding
characteristics, or any other change in the biological, functional
or immunological properties of HARC.
[0037] The term "mimetic", as used herein, refers to a molecule,
the structure of which is developed from knowledge of the structure
of HARC or portions thereof and, as such, is able to effect some or
all of the actions of ALG-2-like molecules.
[0038] The term "derivative", as used herein, refers to the
chemical modification of a nucleic acid encoding HARC or the
encoded HARC. Illustrative of such modifications would be
replacement of hydrogen by an alkyl, acyl, or amino group. A
nucleic acid derivative would encode a polypeptide which retains
essential biological characteristics of the natural molecule.
[0039] The term "substantially purified", as used herein, refers to
nucleic or amino acid sequences that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably 75% free, and most preferably 90% free from other
components with which they are naturally associated.
[0040] "Amplification" as used herein refers to the production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR
Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y.).
[0041] The term "hybridization", as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0042] The term "hybridization complex", as used herein, refers to
a complex formed between two nucleic acid sequences by virtue of
the formation of hydrogen bonds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., C.sub.ot or R.sub.ot analysis) or between one
nucleic acid sequence present in solution and another nucleic acid
sequence immobilized on a solid support (e.g., membranes, filters,
chips, pins or glass slides to which cells have been fixed for in
situ hybridization).
[0043] The terms "complementary" or "complementarity", as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing. For
example, for the sequence "A-G-T" binds to the complementary
sequence "T-C-A". Complementarity between two single-stranded
molecules may be "partial", in which only some of the nucleic acids
bind, or it may be complete when total complementarity exists
between the single-stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands.
[0044] The term "homology", as used herein, refers to a degree of
complementarity. There may be partial homology or complete homology
(i.e., identity). A partially complementary sequence is one that at
least partially inhibits an identical sequence from hybridizing to
a target nucleic acid; it is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous sequence or probe to the target sequence
under conditions of low stringency. This is not to say that
conditions of low stringency are such that non-specific binding is
permitted; low stringency conditions require that the binding of
two sequences to one another be a specific (i.e., selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% identity); in
the absence of non-specific binding, the probe will not hybridize
to the second non-complementary target sequence.
[0045] As known in the art, numerous equivalent conditions may be
employed to comprise either low or high stringency conditions.
Factors such as the length and nature (DNA, RNA, base composition)
of the sequence, nature of the target (DNA, RNA, base composition,
presence in solution or immobilization, etc.), and the
concentration of the salts and other components (e.g., the presence
or absence of formamide, dextran sulfate and/or polyethylene
glycol) are considered and the hybridization solution may be varied
to generate conditions of either low or high stringency different
from, but equivalent to, the above listed conditions.
[0046] The term "stringent conditions", as used herein, is the
"stringency" which occurs within a range from about Tm-5.degree. C.
(5.degree. C. below the melting temperature (Tm) of the probe) to
about 20.degree. C. to 25.degree. C. below Tm. As will be
understood by those of skill in the art, the stringency of
hybridization may be altered in order to identify or detect
identical or related polynucleotide sequences.
[0047] The term "antisense", as used herein, refers to nucleotide
sequences which are complementary to a specific DNA or RNA
sequence. The term "antisense strand" is used in reference to a
nucleic acid strand that is complementary to the "sense" strand.
Antisense molecules may be produced by any method, including
synthesis by ligating the gene(s) of interest in a reverse
orientation to a viral promoter which permits the synthesis of a
complementary strand. Once introduced into a cell, this transcribed
strand combines with natural sequences produced by the cell to form
duplexes. These duplexes then block either the further
transcription or translation. In this manner, mutant phenotypes may
be generated. The designation "negative" is sometimes used in
reference to the antisense strand, and "positive" is sometimes used
in reference to the sense strand.
[0048] The term "portion", as used herein, with regard to a protein
(as in "a portion of a given protein") refers to fragments of that
protein. The fragments may range in size from four amino acid
residues to the entire amino acid sequence minus one amino acid.
Thus, a protein "comprising at least a portion of the amino acid
sequence of SEQ ID NO: 1" encompasses the full-length human HARC
and fragments thereof.
[0049] "Transformation", as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method is selected based
on the host cell being transformed and may include, but is not
limited to, viral infection, electroporation, lipofection, and
particle bombardment. Such "transformed" cells include stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome. They also include cells which
transiently express the inserted DNA or RNA for limited periods of
time.
[0050] The term "antigenic determinant", as used herein, refers to
that portion of a molecule that makes contact with a particular
antibody (i.e., an epitope). When a protein or fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to a given region or three-dimensional structure on
the protein; these regions or structures are referred to as
antigenic determinants. An antigenic determinant may compete with
the intact antigen (i.e., the immunogen used to elicit the immune
response) for binding to an antibody.
[0051] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody and a
protein or peptide, mean that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words, the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general. For example, if an antibody is specific for
epitope "A", the presence of a protein containing epitope A (or
free, unlabeled A) in a reaction containing labeled "A" and the
antibody will reduce the amount of labeled A bound to the
antibody.
[0052] The term "sample", as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acid
encoding HARC or fragments thereof may comprise a cell, chromosomes
isolated from a cell (e.g., a spread of metaphase chromosomes),
genomic DNA (in solution or bound to a solid support such as for
Southern analysis), RNA (in solution or bound to a solid support
such as for northern analysis), cDNA (in solution or bound to a
solid support), an extract from cells or a tissue, and the
like.
[0053] The term "correlates with expression of a polynucleotide",
as used herein, indicates that the detection of the presence of
ribonucleic acid that is similar to SEQ ID NO: 2 by northern
analysis is indicative of the presence of mRNA encoding HARC in a
sample and thereby correlates with expression of the transcript
from the polynucleotide encoding the protein.
[0054] "Alterations" in the polynucleotide of SEQ ID NO: 2, as used
herein, comprise any alteration in the sequence of polynucleotides
encoding HARC including deletions, insertions, and point mutations
that may be detected using hybridization assays. Included within
this definition is the detection of alterations to the genomic DNA
sequence which encodes HARC (e.g., by alterations in the pattern of
restriction fragment length polymorphisms capable of hybridizing to
SEQ ID NO: 2), the inability of a selected fragment of SEQ ID NO: 2
to hybridize to a sample of genomic DNA (e.g., using
allele-specific oligonucleotide probes), and improper or unexpected
hybridization, such as hybridization to a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
HARC (e.g., using fluorescent in situ hybridization (FISH) to
metaphase chromosomes spreads).
[0055] As used herein, the term "antibody" refers to intact
molecules as well as fragments thereof, such as Fab, F(ab').sub.2,
and Fv, which are capable of binding the epitopic determinant.
Antibodies that bind HARC polypeptides can be prepared using intact
polypeptides or fragments containing small peptides of interest as
the immunizing antigen. The polypeptide or peptide used to immunize
an animal can be derived from the transition of RNA or synthesized
chemically, and can be conjugated to a carrier protein, if desired.
Commonly used carriers that are chemically coupled to peptides
include bovine serum albumin and thyroglobulin. The coupled peptide
is then used to immunize the animal (e.g., a mouse, a rat, or a
rabbit).
[0056] The term "humanized antibody", as used herein, refers to
antibody molecules in which amino acids have been replaced in the
non-antigen binding regions in order to more closely resemble a
human antibody, while still retaining the original binding
ability.
THE INVENTION
[0057] The invention is based on the discovery of a novel human
apoptosis-related calcium-binding protein (HARC), the
polynucleotides encoding HARC, and the use of these compositions
for the diagnosis, prevention, and treatment of cancer, autoimmune
diseases, lymphoproliferative disorders, psoriasis,
atherosclerosis, restenosis, AIDS, immunodeficiency diseases,
ischemic injuries, neurodegenerative diseases, osteoporosis,
myelodysplastic syndromes, toxin-induced diseases, cachexia, and
viral infections.
[0058] Nucleic acids encoding the human HARC of the present
invention were first identified in Incyte Clone 036596 from the
human umbilical cord vein endothelial cell line cDNA library
(HUVENOB01) through a computer-generated search for amino acid
sequence alignments. A consensus sequence, SEQ ID NO: 2, was
derived from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 036596 (HUVENOB01), 179146 (PLACNOB01),
334277 (EOSIHET02), 1304460 (PLACNOT02), and 1752846
(LIVRTUT01).
[0059] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO: 1, as shown in
FIG. 1. HARC is 214 amino acids in length and has two potential
EF-hand calcium-binding domains at residues 53-69 and 117-137. HARC
has chemical and structural homology with ALG-2 (GI1213520; SEQ ID
NO: 3). In particular, HARC and ALG-2 share 47% overall identity,
and 58% identity in each of the two calcium-binding domains. As
illustrated by FIGS. 3 and 4, HARC and ALG-2 have similar
hydrophobicity plots, and both are very acidic proteins with
isoelectric points of 5.26 and 5.01, respectively. Northern
analysis (FIG. 5) shows the expression of this sequence in various
libraries, 55% of which are from cancers, tissues associated with
inflammatory diseases, fetal tissues, and cell lines. Of particular
note is the expression of HARC in normal brain (5/44) and prostate
(3/44) cDNA libraries.
[0060] The invention also encompasses HARC variants. A preferred
HARC variant is one having at least 80%, and more preferably 90%,
amino acid sequence similarity to the HARC amino acid sequence (SEQ
ID NO: 1). A most preferred HARC variant is one having at least 95%
amino acid sequence similarity to SEQ ID NO: 1.
[0061] The invention also encompasses polynucleotides which encode
HARC. Accordingly, any nucleic acid sequence which encodes the
amino acid sequence of HARC can be used to generate recombinant
molecules which express HARC. In a particular embodiment, the
invention encompasses the polynucleotide comprising the nucleic
acid sequence of SEQ ID NO: 2 as shown in FIGS. 1A and 1B.
[0062] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
nucleotide sequences encoding HARC, some bearing minimal homology
to the nucleotide sequences of any known and naturally occurring
gene, may be produced. Thus, the invention contemplates each and
every possible variation of nucleotide sequence that could be made
by selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet
genetic code as applied to the nucleotide sequence of naturally
occurring HARC, and all such variations are to be considered as
being specifically disclosed.
[0063] Although nucleotide sequences which encode HARC and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring HARC under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding HARC or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding HARC and its derivatives without altering the
encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0064] The invention also encompasses production of DNA sequences,
or portions thereof, which encode HARC and its derivatives,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art at the time of the filing of this application. Moreover,
synthetic chemistry may be used to introduce mutations into a
sequence encoding HARC or any portion thereof.
[0065] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those shown in SEQ ID NO: 2, under
various conditions of stringency. Hybridization conditions are
based on the melting temperature (Tm) of the nucleic acid binding
complex or probe, as taught in Wahl, G. M. and S. L. Berger (1987;
Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods
Enzymol. 152:507-511), and may be used at a defined stringency.
[0066] Altered nucleic acid sequences encoding HARC which are
encompassed by the invention include deletions, insertions, or
substitutions of different nucleotides resulting in a
polynucleotide that encodes the same or a functionally equivalent
HARC. The encoded protein may also contain deletions, insertions,
or substitutions of amino acid residues which produce a silent
change and result in a functionally equivalent HARC. Deliberate
amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
biological activity of HARC is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid;
positively charged amino acids may include lysine and arginine; and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; phenylalanine and tyrosine.
[0067] Also included within the scope of the present invention are
alleles of the genes encoding HARC. As used herein, an "allele" or
"allelic sequence" is an alternative form of the gene which may
result from at least one mutation in the nucleic acid sequence.
Alleles may result in altered mRNAs or polypeptides whose structure
or function may or may not be altered. Any given gene may have
none, one, or many allelic forms. Common mutational changes which
give rise to alleles are generally ascribed to natural deletions,
additions, or substitutions of nucleotides. Each of these types of
changes may occur alone, or in combination with the others, one or
more times in a given sequence.
[0068] Methods for DNA sequencing which are well known and
generally available in the art may be used to practice any
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, SEQUENASE (US
Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of recombinant polymerases and proofreading
exonucleases such as the amplification system (GIBCOIBRL,
Gaithersburg, Md.). Preferably, the process is automated with
machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno, Nev.),
Peltier thermal cycler (PTC200; M. J. Research, Watertown, Mass.)
and the ABI 377 DNA sequencers (Perkin Elmer).
[0069] The nucleic acid sequences encoding HARC may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences such as
promoters and regulatory elements. For example, one method which
may be employed, "restriction-site" PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus (Sarkar, G.
(1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA
is first amplified in the presence of primer to linker sequence and
a primer specific to the known region. The amplified sequences are
then subjected to a second round of PCR with the same linker primer
and another specific primer internal to the first one. Products of
each round of PCR are transcribed with an appropriate RNA
polymerase and sequenced using reverse transcriptase.
[0070] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region (Triglia, T. et al.
(1988) Nucleic Acids Res. 16:8186). The primers may be designed
using OLIGO 4.06 Primer Analysis software (National Biosciences
Inc., Plymouth, Minn.), or another appropriate program, to be 22-30
nucleotides in length, to have a GC content of 50% or more, and to
anneal to the target sequence at temperatures about
68.degree.-72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0071] Another method which may be used is capture PCR which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA (Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119). In this method,
multiple restriction enzyme digestions and ligations may also be
used to place an engineered double-stranded sequence into an
unknown portion of the DNA molecule before performing PCR.
[0072] Another method which may be used to retrieve unknown
sequences is that of Parker, J. D. et al. (1991; Nuc. Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries to walk in genomic DNA (Clontech, Palo
Alto, Calif.). This process avoids the need to screen libraries and
is useful in finding intron/exon junctions.
[0073] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable, in that they will
contain more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into the 5' and 3' non-transcribed regulatory regions.
[0074] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and detection of the emitted
wavelengths by a charge coupled device camera. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g. SEQUENCE NAVIGATOR and GENOTYPER, Perkin Elmer) and
the entire process from loading of samples to computer analysis and
electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0075] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode HARC, or fusion
proteins or functional equivalents thereof, may be used in
recombinant DNA molecules to direct expression of HARC in
appropriate host cells. Due to the inherent degeneracy of the
genetic code, other DNA sequences which encode substantially the
same or a functionally equivalent amino acid sequence may be
produced and these sequences may be used to clone and express
HARC.
[0076] As will be understood by those of skill in the art, it may
be advantageous to produce HARC-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce a
recombinant RNA transcript having desirable properties, such as a
half-life which is longer than that of a transcript generated from
the naturally occurring sequence.
[0077] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter HARC encoding sequences for a variety of reasons, including
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, or introduce mutations,
and so forth.
[0078] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding HARC may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of HARC activity, it may
be useful to encode a chimeric HARC protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the HARC
encoding sequence and the heterologous protein sequence, so that
HARC may be cleaved and purified away from the heterologous
moiety.
[0079] In another embodiment, sequences encoding HARC may be
synthesized, in whole or in part, using chemical methods well known
in the art (see Caruthers, M. H. et al. (1980) Nuc. Acids Res.
Symp. Ser. 7:215-223, Horn, T. et al. (1980) Nuc. Acids Res. Symp.
Ser. 7:225-232). Alternatively, the protein itself may be produced
using chemical methods to synthesize the amino acid sequence of
HARC, or a portion thereof. For example, peptide synthesis can be
performed using various solid-phase techniques (Roberge, J. Y. et
al. (1995) Science 269:202-204) and automated synthesis may be
achieved, for example, using the ABI 431A Peptide Synthesizer
(Perkin Elmer).
[0080] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins, Structures and Molecular Principles,
W H Freeman and Co., New York, N.Y.). The composition of the
synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure; Creighton,
supra). Additionally, the amino acid sequence of HARC, or any part
thereof, may be altered during direct synthesis and/or combined
using chemical methods with sequences from other proteins, or any
part thereof, to produce a variant polypeptide.
[0081] In order to express a biologically active HARC, the
nucleotide sequences encoding HARC or functional equivalents, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0082] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding HARC and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y.
[0083] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding HARC. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems.
[0084] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector-enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1
plasmid (GIBCO/BRL) and the like may be used. The baculovirus
polyhedrin promoter may be used in insect cells. Promoters or
enhancers derived from the genomes of plant cells (e.g., heat
shock, RUBISCO; and storage protein genes) or from plant viruses
(e.g., viral promoters or leader sequences) may be cloned into the
vector. In mammalian cell systems, promoters from mammalian genes
or from mammalian viruses are preferable. If it is necessary to
generate a cell line that contains multiple copies of the sequence
encoding HARC, vectors based on SV40 or EBV may be used with an
appropriate selectable marker.
[0085] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for HARC. For example,
when large quantities of HARC are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, the multifunctional E. coli
cloning and expression vectors such as BLUESCRIPT phagemid
(Stratagene), in which the sequence encoding HARC may be ligated
into the vector in frame with sequences for the amino-terminal Met
and the subsequent 7 residues of .beta.-galactosidase so that a
hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.
Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. PGEX
vectors (Promega, Madison, Wis.) may also be used to express
foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor XA protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will.
[0086] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol.
153:516-544.
[0087] In cases where plant expression vectors are used, the
expression of sequences encoding HARC may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196.)
[0088] An insect system may also be used to express HARC. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding HARC may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of HARC will
render the polyhedrin gene inactive and produce recombinant virus
lacking coat protein. The recombinant viruses may then be used to
infect, for example, S. frugiperda cells or Trichoplusia larvae in
which HARC may be expressed (Engelhard, E. K. et al. (1994) Proc.
Nat. Acad. Sci. 91:3224-3227).
[0089] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding HARC may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing HARC in
infected host cells (Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. 81:3655-3659). In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0090] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding HARC. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding HARC, its initiation codon, and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a portion
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0091] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells such as CHO, HeLa, MDCK, HEK293, and W138, which have
specific cellular machinery and characteristic mechanisms for such
post-translational activities, may be chosen to ensure the correct
modification and processing of the foreign protein.
[0092] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express HARC may be transformed using expression
vectors which may contain viral origins of replication and/or
endogenous expression elements and a selectable marker gene on the
same or on a separate vector. Following the introduction of the
vector, cells may be allowed to grow for 1-2 days in an enriched
media before they are switched to selective media. The purpose of
the selectable marker is to confer resistance to selection, and its
presence allows growth and recovery of cells which successfully
express the introduced sequences. Resistant clones of stably
transformed cells may be proliferated using tissue culture
techniques appropriate to the cell type.
[0093] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1980) Cell 22:817-23) genes which can be employed in tk.sup.-
or aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic
or herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which
confers resistance to the aminoglycosides neomycin and (G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-51). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta. glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin,
being widely used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131).
[0094] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. For example, if the
sequence encoding HARC is inserted within a marker gene sequence,
recombinant cells containing sequences encoding HARC can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding HARC
under the control of a single promoter. Expression of the marker
gene in response to induction or selection usually indicates
expression of the tandem gene as well.
[0095] Alternatively, host cells which contain the nucleic acid
sequence encoding HARC and express HARC may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein.
[0096] The presence of polynucleotide sequences encoding HARC can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or portions or fragments of polynucleotides encoding
HARC. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding HARC
to detect transformants containing DNA or RNA encoding HARC. As
used herein "oligonucleotides" or "oligomers" refer to a nucleic
acid sequence of at least about 10 nucleotides and as many as about
60 nucleotides, preferably about 15 to 30 nucleotides, and more
preferably about 20-25 nucleotides, which can be used as a probe or
amplimer.
[0097] A variety of protocols for detecting and measuring the
expression of HARC, using either polyclonal or monoclonal
antibodies specific for the protein are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on HARC is preferred, but
a competitive binding assay may be employed. These and other assays
are described, among other places, in Hampton, R. et al. (1990;
Serological Methods, a Laboratory Manual, APS Press, St Paul,
Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
158:1211-1216).
[0098] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding HARC include oligolabeling, nick
translation, end-labeling or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding HARC, or any
portions thereof may be cloned into a vector for the production of
an mRNA probe. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a
variety of commercially available kits (Pharmacia & Upjohn,
(Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical
Corp., Cleveland, Ohio). Suitable reporter molecules or labels,
which may be used, include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0099] Host cells transformed with nucleotide sequences encoding
HARC may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a recombinant cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode HARC may be designed to
contain signal sequences which direct secretion of HARC through a
prokaryotic or eukaryotic cell membrane. Other recombinant
constructions may be used to join sequences encoding HARC to
nucleotide sequence encoding a polypeptide domain which will
facilitate purification of soluble proteins. Such purification
facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp.,
Seattle, Wash.). The inclusion of cleavable linker sequences such
as those specific for Factor XA or enterokinase (Invitrogen, San
Diego, Calif.) between the purification domain and HARC may be used
to facilitate purification. One such expression vector provides for
expression of a fusion protein containing HARC and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography as described in Porath, J. et al. (1992, Prot. Exp.
Purif. 3: 263-281) while the enterokinase cleavage site provides a
means for purifying HARC from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
[0100] In addition to recombinant production, fragments of HARC may
be produced by direct peptide synthesis using solid-phase
techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154).
Protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various
fragments of HARC may be chemically synthesized separately and
combined using chemical methods to produce the full length
molecule.
[0101] THERAPEUTICS
[0102] Based on the chemical and structural homology between HARC
(SEQ ID NO: 1) and mouse ALG-2 (SEQ ID NO: 3), HARC appears to play
a role in the development of diseases related to decreased or
increased apoptosis. Diseases arising from decreased apoptosis
include, but are not limited to, cancer of the breast, prostate,
and ovary as well as lymphomas and carcinomas, autoimmune disorders
such as systemic lupus erythematosus, glomerulonephritis, Sjogren's
syndrome, Graves disease, multiple sclerosis, rheumatoid arthritis,
and diabetes, inflammatory diseases such as osteoarthritis, Crohn's
disease, inflammatory bowel disease, and colitis, proliferative
disorders such as atherosclerosis, restenosis, psoriasis,
lymphadenopathy, and viral infections such as by herpesviruses,
poxviruses, and adenoviruses.
[0103] HARC-associated diseases that are related to increased
apoptosis include, but are not limited to, AIDS and other
infectious or genetic immunodeficiencies, neurodegenerative
diseases such as Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, retinitis pigmentosa, and cerebellar
degeneration, myelodysplastic syndromes such as aplastic anemia,
ischemic injuries such as myocardial infarction, stroke, and
reperfusion injury, toxin-induced diseases such as alcohol-induced
liver damage, cirrhosis, and lathyrism, wasting diseases such as
cachexia, viral infections such as by hepatitis B and C, and
osteoporosis.
[0104] Therefore, in one embodiment, HARC or a fragment or
derivative thereof may be administered to a subject to treat the
disorders and diseases resulting from decreased apoptosis
including, but not limited to, those listed above.
[0105] In another embodiment, a vector capable of expressing HARC,
or a fragment or a derivative thereof, may also be administered to
a subject to treat the disorders and diseases resulting from
decreased apoptosis including, but not limited to, those described
above.
[0106] In another embodiment, agonists of HARC may be administered
to a subject to treat or prevent the disorders and diseases
resulting from decreased apoptosis including, but not limited to,
those described above.
[0107] In another embodiment, antagonists or inhibitors of HARC may
be administered to a subject to treat or prevent the disorders and
diseases resulting from increased apoptosis including, but not
limited to, those described above.
[0108] In another embodiment, a vector expressing antisense of the
polynucleotide encoding HARC may be administered to a subject to
treat or prevent the disorders and diseases resulting from
increased apoptosis including, but not limited to, those described
above.
[0109] In one aspect, antibodies which are specific for HARC may be
used directly as an antagonist. In another aspect, antibodies may
be used to deliver a pharmaceutical agent to cells or tissue which
express HARC.
[0110] In other embodiments, any of the therapuetic compositions
described above may be administered in combination with other
conventional therapeutic agents. The combination of therapeutic
agents having different mechanisms of action will have synergistic
effects allowing for the use of lower effective doses of each agent
and thereby lessening side effects.
[0111] Antagonists or inhibitors of HARC may be produced using
methods which are generally known in the art. In particular,
purified HARC may be used to produce antibodies or to screen
libraries of pharmaceutical agents to identify those which
specifically bind HARC.
[0112] Antibodies which are specific for HARC may be used directly
as an antagonist, or indirectly as a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue
which express HARC. The antibodies may be generated using methods
that are well known in the art. Such antibodies may include, but
are not limited to, polyclonal, monoclonal, chimeric, single chain,
Fab fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies, (i.e., those which inhibit dimer
formation) are especially preferred for therapeutic use.
[0113] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with HARC or any fragment or oligopeptide thereof which
has immunogenic properties. Depending on the host species, various
adjuvants may be used to increase immunological response. Such
adjuvants include, but are not limited to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable.
[0114] It is preferred that the peptides, fragments, or
oligopeptides used to induce antibodies to HARC have an amino acid
sequence consisting of at least five amino acids, and more
preferably at least 10 amino acids. It is also preferable that they
are identical to a portion of the amino acid sequence of the
natural protein, and they may contain the entire amino acid
sequence of a small, naturally occurring molecule. Short stretches
of HARC amino acids may be fused with those of another protein such
as keyhole limpet hemocyanin and antibody produced against the
chimeric molecule.
[0115] Monoclonal antibodies to HARC may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.
80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120).
[0116] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison, S. L. et
al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et
al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature
314:452-454). Alternatively, techniques described for the
production of single chain antibodies may be adapted, using methods
known in the art, to produce HARC-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic
composition, may be generated by chain shuffling from random
combinatorial immunoglobin libraries (Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:11120-3).
[0117] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991)
Nature 349:293-299).
[0118] Antibody fragments which contain specific binding sites for
HARC may also be generated. For example, such fragments include,
but are not limited to, the F(ab')2 fragments which can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 254:1275-1281).
[0119] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between HARC and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering HARC epitopes
is preferred, but a competitive binding assay may also be employed
(Maddox, supra).
[0120] In another embodiment of the invention, the polynucleotides
encoding HARC, or any fragment thereof, or antisense molecules, may
be used for therapeutic purposes. In one aspect, antisense to the
polynucleotide encoding HARC may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding HARC. Thus, antisense molecules may be
used to modulate HARC activity, or to achieve regulation of gene
function. Such technology is now well known in the art, and sense
or antisense oligomers or larger fragments, can be designed from
various locations along the coding or control regions of sequences
encoding HARC.
[0121] Expression vectors derived from retroviruses, adenovirus,
herpes or vaccinia viruses, or from various bacterial plasmids may
be used for delivery of nucleotide sequences to the targeted organ,
tissue or cell population. Methods which are well known to those
skilled in the art can be used to construct recombinant vectors
which will express antisense molecules complementary to the
polynucleotides of the gene encoding HARC. These techniques are
described both in Sambrook et al. (supra) and in Ausubel et al.
(supra).
[0122] Genes encoding HARC can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide or fragment thereof which encodes HARC. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector and even longer if appropriate replication elements are part
of the vector system.
[0123] As mentioned above, modifications of gene expression can be
obtained by designing antisense molecules, DNA, RNA, or PNA, to the
control regions of the gene encoding HARC, i.e., the promoters,
enhancers, and introns. Oligonucleotides derived from the
transcription initiation site, e.g., between positions -10 and +10
from the start site, are preferred. Similarly, inhibition can be
achieved using "triple helix" base-pairing methodology. Triple
helix pairing is useful because it causes inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I.
Carr, Molecular and Immunologic Approaches, Futura Publishing Co.,
Mt. Kisco, N.Y.). The antisense molecules may also be designed to
block translation of mRNA by preventing the transcript from binding
to ribosomes.
[0124] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Examples which may be used include engineered hammerhead
motif ribozyme molecules that can specifically and efficiently
catalyze endonucleolytic cleavage of sequences encoding HARC.
[0125] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0126] Antisense molecules and ribozymes of the invention may be
prepared by any method known in the art for the synthesis of
nucleic acid molecules. These include techniques for chemically
synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis. Alternatively, RNA molecules may be generated
by in vitro and in vivo transcription of DNA sequences encoding
HARC. Such DNA sequences may be incorporated into a wide variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA constructs that synthesize antisense RNA
constitutively or inducibly can be introduced into cell lines,
cells, or tissues.
[0127] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule or the use of phosphorothioate or
2'-O-methyl phosphodiester linkages within the backbone of the
molecule. This concept is inherent in the production of PNAs and
can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0128] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection
and by liposome injections may be achieved using methods which are
well known in the art.
[0129] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0130] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of HARC, antibodies to HARC, mimetics, agonists,
antagonists, or inhibitors of HARC. The compositions may be
administered alone or in combination with at least one other agent,
such as stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0131] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0132] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0133] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0134] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0135] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0136] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0137] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0138] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0139] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0140] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents than are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder which
may contain any or all of the following: 1-50 mM histidine, 0.1%-2%
sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is
combined with buffer prior to use.
[0141] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of HARC, such
labeling would include amount, frequency, and method of
administration.
[0142] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0143] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually mice, rabbits, dogs,
or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0144] A therapeutically effective dose refers to that amount of
active ingredient, for example HARC or fragments thereof,
antibodies of HARC, agonists, antagonists or inhibitors of HARC,
which ameliorates the symptoms or condition. Therapeutic efficacy
and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., ED50
(the dose therapeutically effective in 50% of the population) and
LD50 (the dose lethal to 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index, and
it can be expressed as the ratio, LD50/ED50.
[0145] Pharmaceutical compositions which exhibit large therapeutic
indices are preferred. The data obtained from cell culture assays
and animal studies is used in formulating a range of dosage for
human use. The dosage contained in such compositions is preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage varies within this range
depending upon the dosage form employed, sensitivity of the
patient, and the route of administration.
[0146] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active moiety or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0147] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0148] DIAGNOSTICS
[0149] In another embodiment, antibodies which specifically bind
HARC may be used for the diagnosis of conditions or diseases
characterized by expression of HARC, or in assays to monitor
patients being treated with HARC, agonists, antagonists or
inhibitors. The antibodies useful for diagnostic purposes may be
prepared in the same manner as those described above for
therapeutics. Diagnostic assays for HARC include methods which
utilize the antibody and a label to detect HARC in human body
fluids or extracts of cells or tissues. The antibodies may be used
with or without modification, and may be labeled by joining them,
either covalently or non-covalently, with a reporter molecule. A
wide variety of reporter molecules which are known in the art may
be used, several of which are described above.
[0150] A variety of protocols including ELISA, RIA, and FACS for
measuring HARC are known in the art and provide a basis for
diagnosing altered or abnormal levels of HARC expression. Normal or
standard values for HARC expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to HARC under conditions suitable
for complex formation The amount of standard complex formation may
be quantified by various methods, but preferably by photometric,
means. Quantities of HARC expressed in subject samples from
biopsied tissues are compared with the standard values. Deviation
between standard and subject values establishes the parameters for
diagnosing disease.
[0151] In another embodiment of the invention, the polynucleotides
encoding HARC may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, antisense RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of HARC may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
HARC, and to monitor regulation of HARC levels during therapeutic
intervention.
[0152] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding HARC or closely related molecules, may be used
to identify nucleic acid sequences which encode HARC. The
specificity of the probe, whether it is made from a highly specific
region, e.g., 10 unique nucleotides in the 5' regulatory region, or
a less specific region, e.g., especially in the 3' coding region,
and the stringency of the hybridization or amplification (maximal,
high, intermediate, or low) will determine whether the probe
identifies only naturally occurring sequences encoding HARC,
alleles, or related sequences.
[0153] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides from any of the HARC encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
derived from the nucleotide sequence of SEQ ID NO: 2 or from
genomic sequence including promoter, enhancer elements, and introns
of the naturally occurring HARC.
[0154] Means for producing specific hybridization probes for DNAs
encoding HARC include the cloning of nucleic acid sequences
encoding HARC or HARC derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, radionuclides
such as 32P or 35S, or enzymatic labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
[0155] Polynucleotide sequences encoding HARC may be used for the
diagnosis of conditions or diseases which are associated with
expression of HARC. Examples of such conditions or diseases include
cancer, autoimmune diseases, lymphoproliferative disorders,
psoriasis, atherosclerosis, restenosis, AIDS, immunodeficiency
diseases, ischemic injuries, neurodegenerative diseases,
osteoporosis, myelodysplastic syndromes, toxin-induced diseases,
cachexia, and viral infections. The polynucleotide sequences
encoding HARC may be used in Southern or northern analysis, dot
blot, or other membrane-based technologies; in PCR technologies; or
in dip stick, pin, ELISA or chip assays utilizing fluids or tissues
from patient biopsies to detect altered HARC expression. Such
qualitative or quantitative methods are well known in the art.
[0156] In a particular aspect, the nucleotide sequences encoding
HARC may be useful in assays that detect activation or induction of
various cancers, particularly those mentioned above. The nucleotide
sequences encoding HARC may be labeled by standard methods, and
added to a fluid or tissue sample from a patient under conditions
suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is
quantitated and compared with a standard value. If the amount of
signal in the biopsied or extracted sample is significantly altered
from that of a comparable control sample, the nucleotide sequences
have hybridized with nucleotide sequences in the sample, and the
presence of altered levels of nucleotide sequences encoding HARC in
the sample indicates the presence of the associated disease. Such
assays may also be used to evaluate the efficacy of a particular
therapeutic treatment regimen in animal studies, in clinical
trials, or in monitoring the treatment of an individual
patient.
[0157] In order to provide a basis for the diagnosis of disease
associated with expression of HARC, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
which encodes HARC, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with those from
an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for disease. Deviation between
standard and subject values is used to establish the presence of
disease.
[0158] Once disease is established and a treatment protocol is
initiated, hybridization assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that which is observed in the normal patient. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0159] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0160] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding HARC may involve the use of PCR. Such
oligomers may be chemically synthesized, generated enzymatically,
or produced from a recombinant source. Oligomers will preferably
consist of two nucleotide sequences, one with sense orientation
(5'.fwdarw.3') and another with antisense (3'.rarw.5'), employed
under optimized conditions for identification of a specific gene or
condition. The same two oligomers, nested sets of oligomers, or
even a degenerate pool of oligomers may be employed under less
stringent conditions for detection and/or quantitation of closely
related DNA or RNA sequences.
[0161] Methods which may also be used to quantitate the expression
of HARC include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and standard curves onto
which the experimental results are interpolated (Melby, P. C. et
al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al.
(1993) Anal. Biochem. 212:229-236). The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
[0162] In another embodiment of the invention, the nucleic acid
sequences which encode HARC may also be used to generate
hybridization probes which are useful for mapping the naturally
occurring genomic sequence. The sequences may be mapped to a
particular chromosome or to a specific region of the chromosome
using well known techniques. Such techniques include FISH, FACS, or
artificial chromosome constructions, such as yeast artificial
chromosomes, bacterial artificial chromosomes, bacterial P1
constructions or single chromosome cDNA libraries as reviewed in
Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J. (1991)
Trends Genet. 7:149-154.
[0163] FISH (as described in Verma et al. (1988) Human Chromosomes:
A Manual of Basic Techniques, Pergamon Press, New York, N.Y.) may
be correlated with other physical chromosome mapping techniques and
genetic map data. Examples of genetic map data can be found in the
1994 Genome Issue of Science (265:1981f). Correlation between the
location of the gene encoding HARC on a physical chromosomal map
and a specific disease, or predisposition to a specific disease,
may help delimit the region of DNA associated with that genetic
disease. The nucleotide sequences of the subject invention may be
used to detect differences in gene sequences between normal,
carrier, or affected individuals.
[0164] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms, or parts
thereof, by physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, for example, AT to 11q22-23 (Gatti, R. A. et al.
(1988) Nature 336:577-580), any sequences mapping to that area may
represent associated or regulatory genes for further investigation.
The nucleotide sequence of the subject invention may also be used
to detect differences in the chromosomal location due to
translocation, inversion, etc. among normal, carrier, or affected
individuals.
[0165] In another embodiment of the invention, HARC, its catalytic
or immunogenic fragments or oligopeptides thereof, can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes, between HARC and the agent being tested, may be
measured.
[0166] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564. In this method, as applied to
HARC, large numbers of different small test compounds are
synthesized on a solid substrate, such as plastic pins or some
other surface. The test compounds are reacted with HARC, or
fragments thereof, and washed. Bound HARC is then detected by
methods well known in the art. Purified HARC can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0167] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding HARC specifically compete with a test compound for binding
HARC. In this manner, the antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with HARC.
[0168] In additional embodiments, the nucleotide sequences which
encode HARC may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0169] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0170] I HUVENOB01 cDNA Library Construction
[0171] Incyte clone 036596 was identified in the HUVENOB01 cDNA
library prepared from control (or untreated) HUVEC cells. The HUVEC
cell line is a normal, homogeneous, well-characterized early
passage endothelial cell culture from human umbilical vein (Cell
Systems Corp., Kirkland, Wash.).
[0172] The induced human endothelial cell (HUVEC) cDNA library was
custom constructed by Stratagene (Cat. #937207). cDNA synthesis was
primed using either oligo d(T) or random hexamers. Synthetic
adaptor oligonucleotides were ligated onto cDNA ends enabling
insertion into the lambda vector. The HUVEC cDNA library was
constructed using the UNIZAP vector system (Stratagene). Finally,
the two libraries were combined into a single library by mixing
equal numbers of bacteriophage.
[0173] The custom-constructed library phage particles were infected
into E. coli host strain XL1-BLUE (Stratagene) which has a high
transformation efficiency, increasing the probability of obtaining
rare, under-represented clones in the cDNA library. Alternative
unidirectional vectors include, but are not limited to, pcDNAI
(Invitrogen, San Diego, Calif.) and pSHlox-1 (Novagen, Madison,
Wis.).
[0174] II Isolation and Sequencing of cDNA Clones
[0175] The phagemid forms of individual cDNA clones were obtained
by the in vivo excision process in which the host bacterial strain
was coinfected with both the lambda library phage and an f1 helper
phage. Polypeptides derived from both the library-containing phage
and the helper phage nicked the lambda DNA, initiated new DNA
synthesis from defined sequences on the lambda target DNA and
created a smaller, single-stranded circular phagemid DNA molecule
that included all DNA sequences of the BLUESCRIPT plasmid
(Stratagene) and the cDNA insert. The phagemid DNA was secreted
from the cells, purified, and used to re-infect fresh host cells
where the double-stranded phagemid DNA was produced. Because the
phagemid carries the gene for .beta.-lactamase, the
newly-transformed bacteria were selected on media containing
ampicillin.
[0176] Phagemid DNA was purified using the MAGIC MINIPREPS DNA
Purification System (Cat. #A7100; Promega Corp., Madison, Wis.).
This small-scale process provides a simple and reliable method for
lysing the bacterial cells and rapidly isolating purified phagemid
DNA using a proprietary DNA-binding resin.
[0177] Phagemid DNA was also purified using the QIAwell-8 Plasmid,
QIAwell Plus and QIAwell Ultra DNA purification systems (QIAGEN,
Chatsworth, Calif.). This product line provides a convenient,
rapid, and reliable high-throughput method for lysing the bacterial
cells and isolating highly purified phagemid DNA. The DNA was
eluted from the purification and binding resins already prepared
for DNA sequencing and other analytical manipulations.
[0178] The cDNA inserts from random isolates of the induced and
control HUVEC libraries were sequenced in part using the method of
Sanger et al. (1975; J. Mol. Biol. 94:441f). Conventional enzymatic
methods employ DNA polymerase Klenow fragments, SEQUENASE or Taq
polymersae to extend DNA chains from an oligonucleotide primer
annealed to the DNA template of interest. Methods have been
developed for the use of both single- and double-stranded
templates. The chain termination reaction products are separated by
electrophoreses through urea-acrylamide gels and are detected by
fluorescence.
[0179] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0180] Each cDNA was compared to sequences in GenBank using two
search algorithms. The first was originally developed by D. J.
Lipman and W. R. Pearson, (1985; Science 227:1435-1441). In this
algorithm, the homologous regions are searched in a two-step
manner. In the first step, the highest homologous regions are
determined by calculating a matching score using a homology score
table. The parameter `Ktup` is used in this step to establish the
minimum window size to be shifted for comparing two sequences. Ktup
also sets the number of bases that must match to extract the
highest homologous region among the sequences. In this step, no
insertion or deletions are applied, and the homology is displayed
as an initial (INIT) value.
[0181] In the second step, the homologous regions are aligned to
obtain the highest matching score by inserting a gap in order to
add a probable deleted portion. The matching score obtained in the
first step is recalculated using the homology score table and the
insertion score table to an optimized (OPT) value in the final
output.
[0182] DNA homologies between two sequences can be examined
graphically using the Harr method of constructing dot matrix
homology plots (Needleman, S. B. and C. O. Wunsch (1970) J. Mol.
Biol. 48:443-453). This method produces a two-dimensional plot
which can be useful in determining regions of homology versus
regions of repetition.
[0183] The second algorithm was developed by Applied Biosystems and
incorporated into the INHERIT 670 sequence analysis system. In this
algorithm, Pattern Specification Language (TRW Inc., Los Angeles,
Calif.) was used to determine regions of homology. The three
parameters that determine how the sequence comparisons run were
window size, window offset, and error tolerance. Using a
combination of these three parameters, the DNA database was
searched for sequences containing regions of homology to the query
sequence, and the appropriate sequences were scored with an initial
value. Subsequently, these homologous regions were examined using
dot matrix homology plots to distinguish regions of homology from
chance matches. Smith-Waterman alignments were used to display the
results of the homology search.
[0184] Peptide and protein sequence homologies were ascertained
using the INHERIT-670 sequence analysis system using the methods
similar to those used in DNA sequence homologies. Pattern
Specification Language and parameter windows were used to search
protein databases for sequences containing regions of homology
which were scored with an initial value. Dot-matrix homology plots
were examined to distinguish regions of significant homology from
chance matches.
[0185] BLAST, which stands for Basic Local Alignment Search Tool
(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al.
(1990) J. Mol. Biol. 215:403-410), was used to search for local
sequence alignments. BLAST produces alignments of both nucleotide
and amino acid sequences to determine sequence similarity. Because
of the local nature of the alignments, BLAST is especially useful
in determining exact matches or in identifying homologs. BLAST is
useful for matches which do not contain gaps. The fundamental unit
of BLAST algorithm output is the High-scoring Segment Pair
(HSP).
[0186] An HSP consists of two sequence fragments of arbitrary but
equal lengths whose alignment is locally maximal and for which the
alignment score meets or exceeds a threshold or cutoff score set by
the user. The BLAST approach is to look for HSPs between a query
sequence and a database sequence, to evaluate the statistical
significance of any matches found, and to report only those matches
which satisfy the user-selected threshold of significance. The
parameter E establishes the statistically significant threshold for
reporting database sequence matches. E is interpreted as the upper
bound of the expected frequency of chance occurrence of an HSP (or
set of HSPs) within the context of the entire database search. Any
database sequence whose match satisfies E is reported in the
program output.
[0187] IV Northern Analysis
[0188] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound
(Sambrook et al., supra).
[0189] Analogous computer techniques using BLAST (Altschul, S. F.
1993 and 1990, supra) are used to search for identical or related
molecules in nucleotide databases such as GenBank or the LIFESEQ
database (Incyte Pharmaceuticals). This analysis is much faster
than multiple, membrane-based hybridizations. In addition, the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or
homologous.
[0190] The basis of the search is the product score which is
defined as:
% sequence identity.times.% maximum BLAST score
100
[0191] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1-2% error; and at 70, the match will be exact.
Homologous molecules are usually identified by selecting those
which show product scores between 15 and 40, although lower scores
may identify related molecules.
[0192] The results of northern analysis are reported as a list of
libraries in which the transcript encoding HARC occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0193] V Extension of HARC-Encoding Polynucleotides to Full Length
or to Recover Regulatory Sequences
[0194] Full length HARC-encoding nucleic acid sequence (SEQ ID NO:
2) is used to design oligonucleotide primers for extending a
partial nucleotide sequence to full length or for obtaining 5' or
3', intron or other control sequences from genomic libraries. One
primer is synthesized to initiate extension in the antisense
direction (XLR) and the other is synthesized to extend sequence in
the sense direction (XLF). Primers are used to facilitate the
extension of the known sequence "outward" generating amplicons
containing new, unknown nucleotide sequence for the region of
interest. The initial primers are designed from the cDNA using
OLIGO 4.06 (National Biosciences), or another appropriate program,
to be 22-30 nucleotides in length, to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures about
68.degree.-72.degree. C. Any stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations is
avoided.
[0195] The original, selected cDNA libraries, or a human genomic
library are used to extend the sequence; the latter is most useful
to obtain 5' upstream regions. If more extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0196] By following the instructions for the XL-PCR kit (Perkin
Elmer) and thoroughly mixing the enzyme and reaction mix, high
fidelity amplification is obtained. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR is performed using the Peltier thermal cycler
(PTC200; M. J. Research, Watertown, Mass.) and the following
parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat step 4-6 for 15 additional
cycles Step 8 94.degree. C. for 15 sec Step 9 65.degree. C. for 1
min Step 10 68.degree. C. for 7:15 min Step 11 Repeat step 8-10 for
12 cycles Step 12 72.degree. C. for 8 min Step 13 4.degree. C. (and
holding)
[0197] A 5-10 .mu.l aliquot of the reaction mixture is analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
mini-gel to determine which reactions were successful in extending
the sequence. Bands thought to contain the largest products are
selected and removed from the gel. Further purification involves
using a commercial gel extraction method such as QIAQUICK (QIAGEN).
After recovery of the DNA, Klenow enzyme is used to trim
single-stranded, nucleotide overhangs creating blunt ends which
facilitate religation and cloning.
[0198] After ethanol precipitation, the products are redissolved in
13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1
.mu.l T4 polynucleotide kinase are added, and the mixture is
incubated at room temperature for 2-3 hours or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) are transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook et al., supra). After
incubation for one hour at 37.degree. C., the whole transformation
mixture is plated on Luria Bertani (LB)-agar (Sambrook et al.,
supra) containing 2.times.Carb. The following day, several colonies
are randomly picked from each plate and cultured in 150 .mu.l of
liquid LB/2.times.Carb medium placed in an individual well of an
appropriate, commercially-available, sterile 96-well microtiter
plate. The following day, 5 82 l of each overnight culture is
transferred into a non-sterile 96-well plate and after dilution
1:10 with water, 5 .mu.l of each sample is transferred into a PCR
array.
[0199] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction are added to each well. Amplification is
performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0200] Aliquots of the PCR reactions are run on agarose gels
together with molecular weight markers. The sizes of the PCR
products are compared to the original partial cDNAs, and
appropriate clones are selected, ligated into plasmid, and
sequenced.
[0201] VI Labeling and Use of Hybridization Probes
[0202] Hybridization probes derived from SEQ ID NO: 2 are employed
to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base-pairs, is
specifically described, essentially the same procedure is used with
larger cDNA fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 (National
Biosciences), labeled by combining 50 pmol of each oligomer and 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham) and
T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeled
oligonucleotides are substantially purified with Sephadex G-25
superfine resin column (Pharmacia & Upjohn). A portion
containing 10.sup.7 counts per minute of each of the sense and
antisense oligonucleotides is used in a typical membrane based
hybridization analysis of human genomic DNA digested with one of
the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,
or Pvu II; DuPont NEN).
[0203] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (Nytran Plus,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times.saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film
(Kodak, Rochester, N.Y.) is exposed to the blots in a PHOSPHOIMAGER
cassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,
hybridization patterns are compared visually.
[0204] VII Antisense Molecules
[0205] Antisense molecules to the HARC-encoding sequence, or any
part thereof, is used to inhibit in vivo or in vitro expression of
naturally occurring HARC. Although use of antisense
oligonucleotides, comprising about 20 base-pairs, is specifically
described, essentially the same procedure is used with larger cDNA
fragments. An oligonucleotide based on the coding sequences of
HARC, as shown in FIGS. 1A and 1B, is used to inhibit expression of
naturally occurring HARC. The complementary oligonucleotide is
designed from the most unique 5' sequence as shown in FIGS. 1A and
1B and used either to inhibit transcription by preventing promoter
binding to the upstream nontranslated sequence or translation of an
HARC-encoding transcript by preventing the ribosome from binding.
Using an appropriate portion of the signal and 5' sequence of SEQ
ID NO: 2, an effective antisense oligonucleotide includes any 15-20
nucleotides spanning the region which translates into the signal or
5' coding sequence of the polypeptide as shown in FIGS. 1A and
1B.
[0206] VIII Expression of HARC
[0207] Expression of HARC is accomplished by subcloning the cDNAs
into appropriate vectors and transforming the vectors into host
cells. In this case, the cloning vector, pSport, previously used
for the generation of the cDNA library is used to express HARC in
E. coli. Upstream of the cloning site, this vector contains a
promoter for .beta.-galactosidase, followed by sequence containing
the amino-terminal Met, and the subsequent seven residues of
.beta.-galactosidase. Immediately following these eight residues is
a bacteriophage promoter useful for transcription and a linker
containing a number of unique restriction sites.
[0208] Induction of an isolated, transformed bacterial strain with
IPTG using standard methods produces a fusion protein which
consists of the first eight residues of .beta.-galactosidase, about
5 to 15 residues of linker, and the full length protein. The signal
residues direct the secretion of HARC into the bacterial growth
media which can be used directly in the following assay for
activity.
[0209] IX Demonstration of HARC Activity
[0210] HARC can be expressed by transforming a mammalian cell line
such as COS7, HeLa or CHO with an eukaryotic expression vector
encoding HARC. Eukaryotic expression vectors are commercially
available, and the techniques to introduce them into cells are well
known to those skilled in the art. The cells are incubated for
48-72 hours after transformation under conditions appropriate for
the cell line to allow expression and accumulation of HARC.
[0211] Extracts containing solubilized proteins are prepared from
cells expressing HARC by methods well known in the art. Portions of
the extract containing HARC are applied to the wells of a
polyacrylamide gel and electrophoresed at constant current using
techniques well known in the art until a suitable tracking dye,
such as bromophenol blue (Sigma), has migrated to the bottom of the
gel. Appropriate control samples, prepared from extracts of
untransformed cells and/or cells transformed with vector sequences
alone, are electrophoresed in parallel lanes of the gel. Protein
standards of known molecular weight (BioRad, Hercules, Calif.) are
run in adjacent lanes to calibrate the gel.
[0212] The separated proteins are blotted onto a nitrocellulose
membrane and assayed for binding to Ca.sup.2+ using the method
described by Maruyama, K. et al. (1984; J. Biochem. (Tokyo)
95:511-519). After incubation in the presence of .sup.45Ca.sup.2+,
the membrane is washed to remove nonspecifically bound radionuclide
and exposed against Kodak XOMAT film for an appropriate period of
time. A band will be visible on the film at a position that is
indicative of a protein of the size predicted for HARC. A band of
similar mobility will not be present in control samples prepared
from extracts of untransformed cells or cells transformed with
vector sequence alone.
[0213] The presence of HARC in the band is confirmed using an
antibody specific for HARC by the western blot procedure that is
well known in the art. The membrane is incubated with HARC-specific
antibodies derived from an animal such as rabbit. After washing to
remove unbound antibodies, the membrane is incubated with tagged
goat anti-rabbit immunoglobulins. The tag may consist of any of a
number of chromogenic, fluorescent or enzymatic molecules that can
be attached to immunoglobulins by techniques well known in the art.
The presence of tagged goat immunoglobulins bound to rabbit
anti-HARC antibodies may be detected using techniques appropriate
to the nature of the tag. Pre-immune sera or unrelated antisera may
be used as suitable controls for nonspecific binding to the
membrane.
[0214] X Production of HARC Specific Antibodies
[0215] HARC that is substantially purified using PAGE
electrophoresis (Sambrook, supra), or other purification
techniques, is used to immunize rabbits and to produce antibodies
using standard protocols. The amino acid sequence deduced from SEQ
ID NO: 2 is analyzed using LASERGENE software (DNASTAR Inc) to
determine regions of high immunogenicity and a corresponding
oligopolypeptide is synthesized and used to raise antibodies by
means known to those of skill in the art. Selection of appropriate
epitopes, such as those near the C-terminus or in hydrophilic
regions, is described by Ausubel et al. (supra), and others.
[0216] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems 431A peptide synthesizer
using fmoc-chemistry, and coupled to keyhole limpet hemocyanin
(KLH, Sigma, St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,
supra). Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. The resulting antisera are tested for
antipeptide activity, for example, by binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radioiodinated, goat anti-rabbit
IgG.
[0217] XI Purification of Naturally Occurring HARC Using Specific
Antibodies
[0218] Naturally occurring or recombinant HARC is substantially
purified by immunoaffinity chromatography using antibodies specific
for HARC. An immunoaffinity column is constructed by covalently
coupling HARC antibody to an activated chromatographic resin, such
as CnBr-activated SEPHAROSE (Pharmacia & Upjohn). After the
coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0219] Media containing HARC is passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of HARC (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/HARC binding (e.g., a buffer of pH
2-3 or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and HARC is collected.
[0220] XII Identification of Molecules Which Interact with HARC
[0221] HARC or biologically active fragments thereof are labeled
with .sup.125I Bolton-Hunter reagent (Bolton et al. (1973) Biochem.
J. 133: 529). Candidate molecules previously arrayed in the wells
of a multi-well plate are incubated with the labeled HARC, washed
and any wells with labeled HARC complex are assayed. Data obtained
using different concentrations of HARC are used to calculate values
for the number, affinity, and association of HARC with the
candidate molecules.
[0222] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
* * * * *