U.S. patent application number 09/969959 was filed with the patent office on 2002-07-04 for vacuolar proton atpase subunits.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Bandman, Olga, Corley, Neil C., Guegler, Karl J., Lal, Preeti, Patterson, Chandra, Tang, Y. Tom.
Application Number | 20020086394 09/969959 |
Document ID | / |
Family ID | 22270904 |
Filed Date | 2002-07-04 |
United States Patent
Application |
20020086394 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
July 4, 2002 |
Vacuolar proton ATPase subunits
Abstract
The invention provides human vacuolar proton ATPase subunits
(VATPS) and polynucleotides which identify and encode VATPS. The
invention also provides expression vectors, host cells, antibodies,
agonists, and antagonists. The invention also provides methods for
diagnosing, treating or preventing disorders associated with
expression of VATPS.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Tang, Y. Tom; (San Jose, CA) ;
Lal, Preeti; (Santa Clara, CA) ; Corley, Neil C.;
(Mountain View, CA) ; Guegler, Karl J.; (Menlo
Park, CA) ; Patterson, Chandra; (Mountain View,
CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
22270904 |
Appl. No.: |
09/969959 |
Filed: |
October 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09969959 |
Oct 1, 2001 |
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09590756 |
Jun 8, 2000 |
|
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09590756 |
Jun 8, 2000 |
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09098789 |
Jun 17, 1998 |
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Current U.S.
Class: |
435/196 ;
435/252.3; 435/320.1; 435/325; 435/69.1; 536/23.2; 800/8 |
Current CPC
Class: |
C12N 9/14 20130101; A61K
38/00 20130101 |
Class at
Publication: |
435/196 ;
435/69.1; 435/320.1; 435/325; 435/252.3; 800/8; 536/23.2 |
International
Class: |
C12N 009/16; A01K
067/00; C07H 021/04; C12P 021/02; C12N 005/06; C12N 001/21; C12N
009/14 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO: 1-2, b) a polypeptide comprising
a naturally occurring amino acid sequence at least 90% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-2, c) a biologically active fragment of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1-2, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO: 1-2.
2. An isolated polypeptide of claim 1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-2.
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 comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:3-4.
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 an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-2.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:3-4, b) a
polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:3-4, c) a
polynucleotide complementary to a polynucleotide of a), d) a
polynucleotide 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 selected from the group consisting of SEQ ID NO:
1-2.
19. A method for treating a disease or condition associated with
decreased expression of functional VATPS, 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 VATPS, 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 VATPS, 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 VATPS 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 VATPS 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 VATPS 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, the method comprising: a)
immunizing an animal with a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-2, 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 selected from the group
consisting of SEQ ID NO: 1-2.
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, the method comprising: a) immunizing
an animal with a polypeptide having an amino acid sequence selected
from the group consisting of SEQ ID NO: 1-2, 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 selected from the group consisting of SEQ ID NO:
1-2.
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 of detecting a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-2 in 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) detecting specific binding,
wherein specific binding indicates the presence of a polypeptide
having an amino acid sequence selected from the group consisting of
SEQ ID NO: 1-2 in the sample.
45. A method of purifying a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-2 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 selected from the group consisting of SEQ ID
NO: 1-2.
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. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:3.
58. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:4.
Description
[0001] This application is a continuation application of U.S.
application Ser. No. 09/590,756, filed Jun. 8, 2000, which is a
divisional application of U.S. application Ser. No. 09/098,789,
filed Jun. 17, 1998, now U.S. Pat. No. 6,180,342, issued Jan. 30,
2001, both entitled VACUOLAR PROTON ATPASE SUBUNITS, all of which
applications and patents are hereby incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of vacuolar proton ATPase subunits and to the use of
these sequences in the diagnosis, treatment, and prevention of cell
proliferative and neurological disorders.
BACKGROUND OF THE INVENTION
[0003] Proton-translocating adenosine triphosphatases (proton
ATPases) are a large class of membrane-proteins that use the energy
of ATP hydrolysis to generate an electrochemical proton gradient
across a membrane. The resultant gradient may be used to transport
other ions across the membrane (Na.sup.+, K.sup.+, or Cl.sup.-) or
to maintain an acidic environment important to the function of many
cellular vesicles (Mellman, I. et al. (1986) Ann. Rev. Biochem.
55:663-700). Proton ATPases are further subdivided into the
mitochondrial F-ATPases, the plasma membrane ATPases, and the
vacuolar ATPases.
[0004] The vacuolar proton ATPases (vp-ATPases) provide most of the
energy required for transport processes in the vacuolar system in
eukaryotic cells. vp-ATPases establish and maintain an acidic pH
within various vesicles involved in the processes of endocytosis
and exocytosis. Such vesicles include phagosomes, lysosomes,
endosomes, and secretory vesicles. Endocytosis is the process in
cells of internalizing nutrients, solutes or small particles
(pinocytosis), or large particles such as internalized receptors,
viruses, bacteria, or bacterial toxins (phagocytosis). Exocytosis
is the process of transporting molecules to the cell surface.
Exocytosis facilitates the placement or localization of
membrane-bound receptors or other membrane proteins and the
secretion of hormones, neurotransmitters, digestive enzymes, and
wastes. Endocytosis and exocytosis are fundamental to the function
of all types of cells.
[0005] Alterations in both endocytosis and exocytosis play a role
in a variety of disorders. For example, synaptic vesicles play a
major role in neural transmission at nerve terminals through
storage and controlled release of neurotransmitters.
Neurotransmitter uptake into synaptic vesicles is driven by the
electrochemical proton gradient generated by vp-ATPase.
Inactivation of vp-ATPase has been shown to inhibit glutamate
uptake by synaptic vesicles, decreasing neurotransmitter release
during episodes of oxidative stress or in response to second
messenger signaling. Additionally, inactivation of vp-ATPase has
been shown to trigger apoptosis in a variety of immortalized and
primary cell lines. Activation of vp-ATPases has been found to
delay apoptosis (Wang, Y. et al. (1998) J. Neurochem. 70:646-652;
Nishihara, T. et al. (1995) Biochem. Biophys. Res. Commun. 212:
255-262; and Niessen, H. et al. (1997) Blood 90: 4598-4601.)
[0006] The vp-ATPases and the F-ATPases, which function in ATP
synthesis and hydrolysis in mitochondria, are related in both their
subunit structure and evolutionary origin. Both contain distinct
catalytic and membrane sectors, and each sector contains multiple
subunits. The catalytic sector of vp-ATPase consists of five
subunits designated A (72 kDa), B (57 kDa), C (41 kDa), D (34 kDa),
and E (33 kDa) (Nelson, H. et al. (1995) Proc. Natl. Acad. Sci.
92:497-501). Three subunits, ACl 15, AC39, and a proteolipid
component, have been identified in the membrane sector of vp-ATPase
from various sources. The proteolipid subunit has been implicated
in the mechanism of energy transfer in the enzyme. The membrane
sector has several functions including proton conduction across the
membrane, energy coupling with the catalytic sector, communication
with the lumen, and modulation of enzyme activity. Mutational
studies in yeast have shown that, while the membrane sector may be
assembled independently of the catalytic sector, assembly of the
catalytic sector is absolutely dependent on previous assembly of
the membrane sector (Noumi, T. et al. (1991) Proc. Natl. Acad. Sci.
88: 1938-42; Ludwig, J. et al. (1998) J. Biol. Chem. 273:
10939-10947; and Supekova, L. et al. (1996) 199: 1147-1156). Thus,
expression and assembly of the membrane sector subunits control the
overall activity of the enzyme complex.
[0007] The discovery of new vacuolar proton ATPase subunits and the
polynucleotides encoding them satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of cell proliferative and neurological
disorders.
SUMMARY OF THE INVENTION
[0008] The invention features substantially purified polypeptides,
vacuolar proton ATPase subunits, referred to collectively as
"VATPS" and individually as "VATPS-1" and "VATPS-2." In one aspect,
the invention provides a substantially purified polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO:
1, and a fragment of SEQ ID NO:2.
[0009] The invention further provides a substantially purified
variant having at least 90% amino acid identity to the amino acid
sequences of SEQ ID NO: 1 or SEQ ID NO:2, or to a fragment of
either of these sequences. The invention also provides an isolated
and purified polynucleotide encoding the polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of
SEQ ID NO:2. The invention also includes an isolated and purified
polynucleotide variant having at least 70% polynucleotide sequence
identity to the polynucleotide encoding the polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO:2, a fragment of SEQ ID NO: 1, and a fragment of
SEQ ID NO:2.
[0010] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO: 1,
SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID
NO:2, as well as an isolated and purified polynucleotide having a
sequence which is complementary to the polynucleotide encoding the
polypeptide comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:l, SEQ ID NO:2, a fragment of SEQ ID
NO:1, and a fragment of SEQ ID NO:2.
[0011] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ
ID NO:3, and a fragment of SEQ ID NO:4. The invention further
provides an isolated and purified polynucleotide variant having at
least 70% polynucleotide sequence identity to the polynucleotide
sequence comprising a polynucleotide sequence selected from the
group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID
NO:3, and a fragment of SEQ ID NO:4, as well as an isolated and
purified polynucleotide having a sequence which is complementary to
the polynucleotide comprising a polynucleotide sequence selected
from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment
of SEQ ID NO:3, and a fragment of SEQ ID NO:4.
[0012] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID
NO: 1, and a fragment of SEQ ID NO:2. In another aspect, the
expression vector is contained within a host cell.
[0013] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO:2, a fragment of SEQ ID
NO:1, and a fragment of SEQ ID NO:2, the method comprising the
steps of: (a) culturing the host cell containing an expression
vector containing at least a fragment of a polynucleotide encoding
the polypeptide under conditions suitable for the expression of the
polypeptide; and (b) recovering the polypeptide from the host cell
culture.
[0014] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO: 1,
SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID
NO:2 in conjunction with a suitable pharmaceutical carrier.
[0015] The invention further includes a purified antibody which
binds to a polypeptide comprising the amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, a fragment
of SEQ ID NO:1, and a fragment of SEQ ID NO:2, as well as a
purified agonist and a purified antagonist to the polypeptide.
[0016] The invention also provides a method for treating or
preventing a neurological disorder associated with reduced
expression or activity of VATPS, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising a substantially
purified polypeptide having the amino acid sequence selected from
the group consisting of SEQ ID NO: 1 through 5, and fragments
thereof in conjunction with a suitable pharmaceutical carrier.
[0017] The invention also provides a method for treating or
preventing a neurological disorder associated with increased
expression or activity of VATPS, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO:2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID
NO:2.
[0018] The invention also provides a method for treating or
preventing a cell proliferative disorder associated with reduced
expression or activity of VATPS, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising a substantially
purified polypeptide having the amino acid sequence selected from
the group consisting of SEQ ID NO: 1 through 5, and fragments
thereof in conjunction with a suitable pharmaceutical carrier.
[0019] The invention also provides a method for treating or
preventing a cell proliferative disorder associated with increased
expression or activity of VATPS, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO:2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID
NO:2.
[0020] The invention also provides a method for detecting a
polynucleotide encoding the polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO:2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:2 in
a biological sample containing nucleic acids, the method comprising
the steps of: (a) hybridizing the complement of the polynucleotide
sequence encoding the polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO:2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:2 to
at least one of the nucleic acids of the biological sample, thereby
forming a hybridization complex; and (b) detecting the
hybridization complex, wherein the presence of the hybridization
complex correlates with the presence of a polynucleotide encoding
the polypeptide in the biological sample. In one aspect, the method
further comprises amplifying the polynucleotide prior to
hybridization.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
[0021] FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID
NO: 1) and nucleic acid sequence (SEQ ID NO:3) of VATPS-1. The
alignment was produced using MacDNASIS PRO.TM. software (Hitachi
Software Engineering Co. Ltd., San Bruno, Calif.).
[0022] FIG. 2 shows the amino acid sequence alignments between
VATPS-1 (Incyte Clone number 2246348; SEQ ID NO:1) and bovine
vacuolar ATPase subunit (GI 1699359; SEQ ID NO:14), produced using
the multisequence alignment program of LASERGENE.TM. software
(DNASTAR Inc, Madison Wis.).
[0023] FIGS. 3A, 3B, 3C and 3D show the amino acid sequence (SEQ ID
NO:2) and nucleic acid sequence (SEQ ID NO:4) of VATPS-2. The
alignment was produced using MacDNASIS PRO.TM. software.
[0024] FIG. 4 shows the amino acid sequence alignments between
VATPS-2 (Incyte Clone number 2246348; SEQ ID NO:2) and human
vacuolar ATPase subunit (GI 2584789; SEQ ID NO:15), produced using
the multisequence alignment program of LASERGENE.TM. software.
[0025] TABLE 1 describes the programs, algorithms, databases, and
scores for analyzing VATPS.
DESCRIPTION OF THE INVENTION
[0026] 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.
[0027] 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, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0028] 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 cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and 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
[0029] "VATPS" refers to the amino acid sequences of substantially
purified VATPS obtained from any species, particularly a mammalian
species, including bovine, ovine, porcine, murine, equine, and
preferably the human species, from any source, whether natural,
synthetic, semi-synthetic, or recombinant.
[0030] The term "agonist" refers to a molecule which, when bound to
VATPS, increases or prolongs the duration of the effect of VATPS.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules which bind to and modulate the effect of VATPS.
[0031] An "allelic variant" is an alternative form of the gene
encoding VATPS. Allelic variants may result from at least one
mutation in the nucleic acid sequence and may result in altered
mRNAs or in polypeptides whose structure or function may or may not
be altered. Any given natural or recombinant gene may have none,
one, or many allelic forms. Common mutational changes which give
rise to allelic variants 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.
[0032] "Altered" nucleic acid sequences encoding VATPS include
those sequences with deletions, insertions, or substitutions of
different nucleotides, resulting in a polynucleotide the same as
VATPS or a polypeptide with at least one functional characteristic
of VATPS. Included within this definition are polymorphisms which
may or may not be readily detectable using a particular
oligonucleotide probe of the polynucleotide encoding VATPS, and
improper or unexpected hybridization to allelic variants, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding VATPS. The encoded protein may
also be "altered," and may contain deletions, insertions, or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent VATPS. 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 or immunological activity of VATPS 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; and phenylalanine and
tyrosine.
[0033] The terms "amino acid" or "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. In this context, "fragments," "immunogenic fragments,"
or "antigenic fragments" refer to fragments of VATPS which are
preferably at least 5 to about 15 amino acids in length, most
preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of VATPS. Where
"amino acid sequence" is recited to refer to an amino acid sequence
of a naturally occurring protein molecule, "amino acid sequence"
and like terms are not meant to limit the amino acid sequence to
the complete native amino acid sequence associated with the recited
protein molecule. "Amplification" relates to the production of
additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art.
[0034] The term "antagonist" refers to a molecule which, when bound
to VATPS, decreases the amount or the duration of the effect of the
biological or immunological activity of VATPS. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of VATPS.
[0035] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fab, F(ab').sub.2, and Fv fragments,
which are capable of binding the epitopic determinant. Antibodies
that bind VATPS polypeptides can be prepared using intact
polypeptides or using fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can
be derived from the translation 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, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0036] The term "antigenic determninant" refers to that fragment of
a molecule (i.e., an epitope) that makes contact with a particular
antibody. When a protein or a 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 antigenic
determinants (given regions or three-dimensional structures on the
protein). An antigenic determinant may compete with the intact
antigen (i.e., the imrnunogen used to elicit the immune response)
for binding to an antibody.
[0037] The term "antisense" refers to any composition containing a
nucleic acid sequence which is complementary to the "sense" strand
of a specific nucleic acid sequence. Antisense molecules may be
produced by any method including synthesis or transcription. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0038] The term "biologically active," 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 VATPS, or
of any oligopeptide thereof, to induce a specific immune response
in appropriate animals or cells and to bind with specific
antibodies.
[0039] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence "5' A-G-T 3"' binds to the complementary sequence "3'
T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial," such that only some of the nucleic acids bind, or
it may be "complete," such that 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 the hybridization between
the nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands, and in the design and use of peptide nucleic acid
(PNA) molecules.
[0040] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence" refer
broadly to any composition containing the given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation
or an aqueous solution. Compositions comprising polynucleotide
sequences encoding VATPS or fragments of VATPS may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts, e.g., NaCl, detergents, e.g.,
sodium dodecyl sulfate (SDS), and other components, e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.
[0041] "Consensus sequence" refers to a nucleic acid sequence which
has been resequenced to resolve uncalled bases, extended using
XL-PCR.TM. (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 a
computer program for fragment assembly, such as the GELVIEW.TM.
Fragment Assembly system (GCG, Madison, Wis.). Some sequences have
been both extended and assembled to produce the consensus
sequence.
[0042] The term "correlates with expression of a polynucleotide"
indicates that the detection of the presence of nucleic acids, the
same or related to a nucleic acid sequence encoding VATPS, by
Northern analysis is indicative of the presence of nucleic acids
encoding VATPS in a sample, and thereby correlates with expression
of the transcript from the polynucleotide encoding VATPS.
[0043] A "deletion" refers to a change in the amino acid or
nucleotide sequence that results in the absence of one or more
amino acid residues or nucleotides. The term "derivative" refers to
the chemical modification of a polypeptide sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide
sequence can include, for example, replacement of hydrogen by an
alkyl, acyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains at least one biological or immunological
function of the natural molecule. A derivative polypeptide is one
modified by glycosylation, pegylation, or any similar process that
retains at least one biological or immunological function of the
polypeptide from which it was derived.
[0044] The term "similarity" refers to a degree of complementarity.
There may be partial similarity or complete similarity. The word
"identity" may substitute for the word "similarity." A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to as "substantially similar." 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 reduced stringency. A substantially similar
sequence or hybridization probe will compete for and inhibit the
binding of a completely similar (identical) sequence to the target
sequence under conditions of reduced stringency. This is not to say
that conditions of reduced stringency are such that non-specific
binding is permitted, as reduced stringency conditions require that
the binding of two sequences to one another be a specific (i.e., a
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%
similarity or identity). In the absence of non-specific binding,
the substantially similar sequence or probe will not hybridize to
the second non-complementary target sequence.
[0045] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MegAlign.TM. program
(DNASTAR, Inc., Madison Wis.). The MegAlign.TM. program can create
alignments between two or more sequences according to different
methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and
P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups
sequences into clusters by examining the distances between all
pairs. The clusters are aligned pairwise and then in groups. The
percentage similarity between two amino acid sequences, e.g.,
sequence A and sequence B, is calculated by dividing the length of
sequence A, minus the number of gap residues in sequence A, minus
the number of gap residues in sequence B, into the sum of the
residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no similarity between the two amino acid
sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted
or calculated by other methods known in the art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol.
183:626-645.) Identity between sequences can also be determined by
other methods known in the art, e.g., by varying hybridization
conditions.
[0046] "Human artificial chromosomes" (HACs) are linear
microchromosomes which may contain DNA sequences of about 6 kb to
10 Mb in size, and which contain all of the elements required for
stable mitotic chromosome segregation and maintenance. (See, e.g.,
Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.)
[0047] The term "humanized antibody" refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
[0048] "Hybridization" refers to any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing.
[0049] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary bases. A hybridization complex
may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid sequence present in solution and another
nucleic acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or their nucleic acids have
been fixed).
[0050] The words "insertion" or "addition" refer to changes in an
amino acid or nucleotide sequence resulting in the addition of one
or more amino acid residues or nucleotides, respectively, to the
sequence found in the naturally occurring molecule.
[0051] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0052] The term "microarray" refers to an arrangement of distinct
polynucleotides arrayed on a substrate, e.g., paper, nylon or any
other type of membrane, filter, chip, glass slide, or any other
suitable solid support.
[0053] The terms "element" or "array element" in a microarray
context, refer to hybridizable polynucleotides arranged on the
surface of a substrate.
[0054] The term "modulate" refers to a change in the activity of
VATPS. For example, modulation may cause an increase or a decrease
in protein activity, binding characteristics, or any other
biological, functional, or immunological properties of VATPS.
[0055] The phrases "nucleic acid" or "nucleic acid sequence" refer
to a nucleotide, oligonucleotide, polynucleotide, or any fragment
thereof. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which, when translated, would produce polypeptides retaining some
functional characteristic, e.g., antigenicity, or structural domain
characteristic, e.g., ATP-binding site, of the full-length
polypeptide.
[0056] The terms "operably associated" or "operably linked" refer
to functionally related nucleic acid sequences. A promoter is
operably associated or operably linked with a coding sequence if
the promoter controls the translation of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in the same reading frame, certain genetic
elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0057] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to 60 nucleotides, preferably about
15 to 30 nucleotides, and most preferably about 20 to 25
nucleotides, which can be used in PCR amplification or in a
hybridization assay or microarray. "Oligonucleotide" is
substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the
art.
[0058] "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises an oligonucleotide of at least
about 5 nucleotides in length linked to a peptide backbone of amino
acid residues ending in lysine. The terminal lysine confers
solubility to the composition. PNAs preferentially bind
complementary single stranded DNA or RNA and stop transcript
elongation, and may be pegylated to extend their lifespan in the
cell.
[0059] The term "sample" is used in its broadest sense. A
biological sample suspected of containing nucleic acids encoding
VATPS, or fragments thereof, or VATPS itself, may comprise a bodily
fluid; an extract from a cell, chromosome, organelle, or membrane
isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in
solution or bound to a solid support; a tissue; a tissue print;
etc.
[0060] The terms "specific binding" or "specifically binding" refer
to that interaction between a protein or peptide and an agonist, an
antibody, or an antagonist. The interaction is dependent upon the
presence of a particular structure of the protein, e.g., the
antigenic determinant or epitope, recognized by the binding
molecule. For example, if an antibody is specific for epitope "A,"
the presence of a polypeptide containing the epitope A, or the
presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will reduce the amount of labeled A that binds
to the antibody.
[0061] The term "stringent conditions" refers to conditions which
permit hybridization between polynucleotides and the claimed
polynucleotides. Stringent conditions can be defined by salt
concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0062] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90%
free from other components with which they are naturally
associated.
[0063] A "substitution" refers to the replacement of one or more
amino acids or nucleotides by different amino acids or nucleotides,
respectively.
[0064] "Transformation" describes a process by which exogenous DNA
enters and changes a recipient cell. Transformation may occur under
natural or artificial conditions according to various methods well
known in the art, and may rely on any known method for the
insertion of foreign nucleic acid sequences into a prokaryotic or
eukaryotic host cell. The method for transformation is selected
based on the type of host cell being transformed and may include,
but is not limited to, viral infection, electroporation, heat
shock, lipofection, and particle bombardment. The term
"transformed" cells includes 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, as well as
transiently transformed cells which express the inserted DNA or RNA
for limited periods of time.
[0065] A "variant" of VATPS polypeptides refers to an amino acid
sequence that is altered by one or more amino acid residues. 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 glycine with
tryptophan). Analogous 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.TM. software.
[0066] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to VATPS. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) in which the
polynucleotide sequence varies by one base. The presence of SNPs
may be indicative of, for example, a certain population, a disease
state, or a propensity for a disease state.
THE INVENTION
[0067] The invention is based on the discovery of a new human
vacuolar proton ATPase subunits (VATPS), the polynucleotides
encoding VATPS, and the use of these compositions for the
diagnosis, treatment, or prevention of cell proliferative and
neurological disorders.
[0068] Nucleic acids encoding the VATPS-1 of the present invention
were first identified in Incyte Clone 2246348 from the hippocampus
cDNA library (HIPONON02) using a computer search, e.g., BLAST, for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:3,
was derived from the following overlapping and/or extended nucleic
acid sequences: Incyte Clones 2246348CT1 (HIPONON02), and 659609H1
(BRAINOT03).
[0069] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A, lB, and 1C. VATPS-1 is 118 amino acids in length and has
a potential N-glycosylation site at residue N68 and a potential
protein kinase C phosphorylation site at residue T77. As shown in
FIG. 2, VATPS-1 has chemical and structural similarity with bovine
vacuolar ATPase subunit (GI 1699359; SEQ ID NO:14). In particular,
VATPS-1 and bovine vacuolar proton ATPase subunit share 69%
identity. Northern analysis shows the expression of this sequence
in various libraries, at least 58% of which involve neurological
disorders and at least 36% are immortalized or cancerous. Of
particular note is the expression of VATPS-1 in neural tissues.
[0070] Nucleic acids encoding the VATPS-2 of the present invention
were first identified in Incyte Clone from the testicular tumor
cDNA library (TESTTUT02) using a computer search, e.g., BLAST, for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:4,
was derived from the following overlapping and/or extended nucleic
acid sequences: Incyte Clones 2346304H11 (TESTTUT02), 1616336T6
(BRAITUT12), 1661631F6 (BRSTNOT09), 1811952F6 (PROSTUT12),
2155351T6 (BRAINOT09), 2481208H1 (SMCANOT01), and 3110132F6
(BRSTTUT15).
[0071] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, as shown in
FIGS. 3A, 3B, 3C and 3D. VAT?S-2 is 81 amino acids in length and
has a potential N-glycosylation site at residue N70. As shown in
FIG. 4, VATPS-2 has chemical and structural similarity with human
vacuolar proton ATPase subunit (GI 2584789; SEQ ID NO: 15). In
particular, VATPS-2 and human vacuolar proton ATPase subunit share
70% identity. Northern analysis shows the expression of this
sequence in various libraries, at least 58% of which are
immortalized or cancerous and at least 22% of which involve an
immune response. Of particular note is the expression of VATPS-2 in
neural tissues.
[0072] The invention also encompasses VATPS variants. A preferred
VATPS variant is one which has at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the VATPS amino acid sequence, and which
contains at least one functional or structural characteristic of
VATPS.
[0073] The invention also encompasses polynucleotides which encode
VATPS. In a particular embodiment, the invention encompasses a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3, and a fragment of
SEQ ID NO:4.
[0074] The invention also encompasses a variant of a polynucleotide
sequence encoding VATPS. In particular, such a variant
polynucleotide sequence will have at least about 70%, more
preferably at least about 80%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding VATPS. A particular aspect of the invention encompasses a
variant of a nucleic acid sequence selected from the group
consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3,
and a fragment of SEQ ID NO:4 which has at least about 70%, more
preferably at least about 80%, and most preferably at least about
95% polynucleotide sequence identity to a nucleic acid sequence
selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a
fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4. Any one of
the polynucleotide variants described above can encode an amino
acid sequence which contains at least one functional or structural
characteristic of VATPS.
[0075] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding VATPS, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
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
polynucleotide sequence of naturally occurring VATPS, and all such
variations are to be considered as being specifically
disclosed.
[0076] Although nucleotide sequences which encode VATPS and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring VATPS under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding VATPS or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. 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 VATPS 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.
[0077] The invention also encompasses production of DNA sequences
which encode VATPS and VATPS derivatives, or fragments thereof,
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 well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding VATPS or any fragment thereof.
[0078] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:3, SEQ ID NO:4, or a fragment of SEQ ID NO:3, or a fragment
of SEQ ID NO:4 under various conditions of stringency. (See e.g.,
Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407;
Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.) Methods for DNA
sequencing are well known in the art and may be used to practice
any of the embodiments of the invention. The methods may employ
such enzymes as the Klenow fragment of DNA polymerase I,
SEQUENASE.RTM. (US Biochemical Corp., Cleveland, Ohio), Taq
polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham
Pharmacia Biotech, Piscataway, N.J.), or combinations of
polymerases and proofreading exonucleases such as those found in
the ELONGASE amplification system (Life Technologies, Gaithersburg
Md.). Preferably, sequence preparation 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
CATALYST 800 (Perkin Elmer). Sequencing is then carried out using
either ABI 373 or 377 DNA Sequencing Systems (Perkin Elmer) or
capillary electrophoresis (Molecular Dynamics). The resulting
sequences are analyzed using a variety of algorithms which are well
known in the art. (See, e.g., Ausubel, supra, ch. 7.7; and Meyers,
R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, Inc.,
New York, N.Y., pp. 856-853.) The nucleic acid sequences encoding
VATPS may be extended utilizing a partial nucleotide sequence and
employing various PCR-based 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 and nested primers to amplify unknown sequence from
genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993)
PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses
primers that extend in divergent directions to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising a known genomic locus and
surrounding sequences. (See, e.g., Triglia, T. et al. (1988)
Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves
PCR amplification of DNA fragments adjacent to known sequences in
human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method,
multiple restriction enzyme digestions and ligations may be used to
insert an engineered double-stranded sequence into a region of
unknown sequence before performing PCR. Other methods which may be
used to retrieve unknown sequences are known in the art. (See,
e.g., Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and
PromoterFinder.TM. libraries to walk genomic DNA (Clontech, Palo
Alto, Calif.). This procedure avoids the need to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based
methods, primers may be designed using commercially available
software, such as OLIGO.TM. 4.06 Primer Analysis software (National
Biosciences Inc., Plymouth, Minn.) or another appropriate program,
to be about 22 to 30 nucleotides in length, to have a GC content of
about 50% or more, and to anneal to the template at temperatures of
about 68.degree. C. to 72.degree. C.
[0079] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are 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 5'
non-transcribed regulatory regions.
[0080] 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 nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g.,
Genotyper.TM. and Sequence Navigator.TM., 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 sequencing small DNA
fragments which may be present in limited amounts in a particular
sample.
[0081] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode VATPS may be cloned in
recombinant DNA molecules that direct expression of VATPS, or
fragments or functional equivalents thereof, 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 used to express
VATPS.
[0082] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter VATPS-encoding sequences for a variety of purposes including,
but not limited to, modification of 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, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0083] In another embodiment, sequences encoding VATPS may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232.) Alternatively, VATPS itself or a fragment
thereof may be synthesized using chemical methods. For example,
peptide synthesis can be performed using various solid-phase
techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI
431A Peptide Synthesizer (Perkin Elmer). Additionally, the amino
acid sequence of VATPS, or any part thereof, may be altered during
direct synthesis and/or combined with sequences from other
proteins, or any part thereof, to produce a variant
polypeptide.
[0084] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1984) Proteins.
Structures and Molecular Properties, W H Freeman and Co., New York,
N.Y.)
[0085] In order to express a biologically active VATPS, the
nucleotide sequences encoding VATPS or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding VATPS. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding VATPS.
Such signals include the ATG initiation codon and adjacent
sequences, e.g. the Kozak sequence. In cases where sequences
encoding VATPS and its initiation codon and upstream regulatory
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 fragment
thereof, is inserted, exogenous translational control signals
including an in-frame ATG initiation codon should be provided by
the vector. 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 appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.) Methods which are well known to those skilled in the
art may be used to construct expression vectors containing
sequences encoding VATPS and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,
Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.
(1995, and periodic supplements) Current Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., ch. 9, 13, and
16.)
[0086] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding VATPS. 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 viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0087] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding VATPS. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding VATPS can be achieved using a multifunctional E. coli
vector such as Bluescript.RTM. (Stratagene) or pSport1.TM. plasmid
(Life Technologies). Ligation of sequences encoding VATPS into the
vector's multiple cloning site disrupts the lacZ gene, allowing a
colorimetric screening procedure for identification of transformed
bacteria containing recombinant molecules. In addition, these
vectors may be useful for in vitro transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.
and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large
quantities of VATPS are needed, e.g. for the production of
antibodies, vectors which direct high level expression of VATPS may
be used. For example, vectors containing the strong, inducible T5
or T7 bacteriophage promoter may be used.
[0088] Yeast expression systems may be used for production of
VATPS. A number of vectors containing constitutive or inducible
promoters, such as alpha factor, alcohol oxidase, and PGH, may be
used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors direct either the secretion or intracellular
retention of expressed proteins and enable integration of foreign
sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, supra; and Grant et al. (1987) Methods Enzymol.
153:516-54; Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)
Plant systems may also be used for expression of VATPS.
Transcription of sequences encoding VATPS may be driven viral
promoters, e.g., the 35S and 19S promoters of CaMV 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.
(See, e.g., 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. (See, e.g., Hobbs, S. or Murry, L.
E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New York, N.Y.; pp. 191-196.)
[0089] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding VATPS may be ligated into an
25 adenovirus transcription/translation complex consisting of the
late promoter and tripartite leader sequence. Insertion in a
non-essential El or E3 region of the viral genome may be used to
obtain infective virus which expresses VATPS in host cells. (See,
e.g., 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. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0090] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from a plasmid. HACs of about 6 kb to 10 Mb are
constructed and delivered via conventional delivery methods
(liposomes, polycationic amino polymers, or vesicles) for
therapeutic purposes.
[0091] For long term production of recombinant proteins in
mammalian systems, stable expression of VATPS in cell lines is
preferred. For example, sequences encoding VATPS can be transformed
into cell lines 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 about 1 to 2 days in enriched media before being switched
to selective media. The purpose of the selectable marker is to
confer resistance to a selective agent, and its presence allows
growth and recovery of cells which successfully express the
introduced sequences. Resistant clones of stably transformed cells
may be propagated using tissue culture techniques appropriate to
the cell type.
[0092] 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 and adenine
phosphoribosyltransferase genes, for use in tk.sup.- or apr.sup.-
cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell
11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or herbicide resistance can be used as
the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides
neomycin and G-418; and als or pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol. Biol.
150:1-14; and Murry, supra.) Additional selectable genes have been
described, e.g., trpB and hisD, which alter cellular requirements
for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan
(1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,
anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo
Alto, Calif.), 6 glucuronidase and its substrate B-D-glucuronoside,
or luciferase and its substrate luciferin may be used. These
markers can be used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system. (See, e.g., Rhodes, C. A.
et al. (1995) Methods Mol. Biol. 55:121-131.)
[0093] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding VATPS is inserted within a marker gene
sequence, transformed cells containing sequences encoding VATPS can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding VATPS 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.
[0094] In general, host cells that contain the nucleic acid
sequence encoding VATPS and that express VATPS 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, PCR amplification, 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 sequences.
[0095] Immunological methods for detecting and measuring the
expression of VATPS using either specific polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques
include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
VATPS is preferred, but a competitive binding assay may be
employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory
Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E. et
al. (1997 and periodic supplements) Current Protocols in
Immunology, Greene Pub. Associates and Wiley-Interscience, New
York, N.Y.; and Maddox, D. E. et al. (1983) J. Exp. Med.
158:1211-1216).
[0096] 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 VATPS include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding VATPS, or any
fragments 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, such as those
provided by Pharmacia & Upjohn (Kalamazoo, MI), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0097] Host cells transformed with nucleotide sequences encoding
VATPS may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or retained 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 VATPS may be designed to
contain signal sequences which direct secretion of VATPS through a
prokaryotic or eukaryotic cell membrane.
[0098] In addition, a host cell strain may be chosen for its
ability to modulate 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 specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and WI38), are available from the American Type
Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0099] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding VATPS may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric VATPS protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of VATPS activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate fusion proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin
(HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the VATPS encoding sequence and the heterologous protein
sequence, so that VATPS may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel, F. M. et al.
(1995 and periodic supplements) Current Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., ch 10. A variety of
commercially available kits may also be used to facilitate
expression and purification of fusion proteins.
[0100] In a further embodiment of the invention, synthesis of
radiolabeled VATPS may be achieved in vitro using the TNT.TM.
rabbit reticulocyte lysate or wheat germ extract systems (Promega,
Madison, Wis.).
[0101] These systems couple transcription and translation of
protein-coding sequences operably associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid precursor, preferably
.sup.35S-methionine.
[0102] Fragments of VATPS may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the Applied
Biosystems 43 1A Peptide Synthesizer (Perkin Elmer). Various
fragments of VATPS may be synthesized separately and then combined
to produce the full length molecule.
THERAPEUTICS
[0103] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between VATPS-1 and bovine vacuolar
ATPase subunit (GI 1699359), and between VATPS-2 and human vacuolar
ATPase subunit (GI 2584789). In addition, the expression of VATPS
is closely associated with cell proliferation and neural tissues.
Therefore, in cell proliferative and neurological disorders where
VATPS is an activator, or enhancer, and is promoting cell
proliferative and neurological disorders, it is desirable to
decrease the expression of VATPS. In cell proliferative and
neurological disorders where VATPS is an inhibitor, or suppressor
of cell proliferative and neurological disorders, it is desirable
to increase the expression of VATPS.
[0104] Therefore, in one embodiment, VATPS or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a cell proliferative disorder. Such disorders can include,
but are not limited to, actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective
tissue disease, myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma, teratocarcinoma, and, in particular, cancers of the
adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus.
[0105] In another embodiment, a vector capable of expressing VATPS
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a cell proliferative disorder
including, but not limited to, those described above.
[0106] In a further embodiment, a pharmaceutical composition
comprising a substantially purified VATPS in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a cell proliferative disorder including, but not
limited to, those provided above.
[0107] In still another embodiment, an agonist which modulates the
activity of VATPS may be administered to a subject to treat or
prevent a cell proliferative disorder including, but not limited
to, those listed above.
[0108] In a further embodiment, an antagonist of VATPS may be
administered to a subject to treat or prevent a cell proliferative
disorder. Such a disorder may include, but is not limited to, those
discussed above. In one aspect, an antibody which specifically
binds VATPS 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 VATPS.
[0109] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding VATPS may be administered
to a subject to treat or prevent a cell proliferative disorder
including, but not limited to, those described above.
[0110] In an additional embodiment, VATPS or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a neurological disorder. Such a disorder may include, but
is not limited to, akathesia, Alzheimer's disease, amnesia,
amyotrophic lateral sclerosis, bipolar disorder, catatonia,
cerebral neoplasms, dementia, depression, diabetic neuropathy,
Down's syndrome, tardive dyskinesia, dystonias, epilepsy,
Huntington's disease, peripheral neuropathy, multiple sclerosis,
neurofibromatosis, Parkinson's disease, paranoid psychoses,
postherpetic neuralgia, schizophrenia, and Tourette's disorder. In
one aspect, an antibody which specifically binds VATPS 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 VATPS.
[0111] In another embodiment, a vector capable of expressing VATPS
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a neurological disorder including, but
not limited to, those described above.
[0112] In a further embodiment, a pharmaceutical composition
comprising a substantially purified VATPS in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a neurological disorder including, but not limited
to, those provided above.
[0113] In another embodiment, an agonist which modulates the
activity of VATPS may be administered to a subject to treat or
prevent a neurological disorder including, but not limited to,
those listed above.
[0114] In a further embodiment, an antagonist of VATPS may be
administered to a subject to treat or prevent a neurological
disorder. Such a disorder may include, but is not limited to, those
discussed above. In one aspect, an antibody which specifically
binds VATPS 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 VATPS.
[0115] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding VATPS may be administered
to a subject to treat or prevent a neurological disorder including,
but not limited to, those described above.
[0116] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0117] An antagonist of VATPS may be produced using methods which
are generally known in the art. In particular, purified VATPS may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
VATPS. Antibodies to VATPS may also be generated using methods that
are well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain
antibodies, 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.
[0118] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with VATPS or with 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, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0119] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to VATPS have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of VATPS amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0120] Monoclonal antibodies to VATPS 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. (See, e.g., 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; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0121] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and 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
VATPS-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
[0122] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et
al. (1991) Nature 349:293-299.)
[0123] Antibody fragments which contain specific binding sites for
VATPS may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments 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. (See, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0124] 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 VATPS and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering VATPS
epitopes is preferred, but a competitive binding assay may also be
employed. (Maddox, supra.)
[0125] In another embodiment of the invention, the polynucleotides
encoding VATPS, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding VATPS 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 VATPS. Thus, complementary molecules or
fragments may be used to modulate VATPS activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding VATPS.
[0126] Expression vectors derived from retroviruses, adenoviruses,
or 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 vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding VATPS. (See, e.g., Sambrook, sunra; and Ausubel,
supra.)
[0127] Genes encoding VATPS can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide, or fragment thereof, encoding VATPS. 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 may last even longer if appropriate replication
elements are part of the vector system.
[0128] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding VATPS. Oligonucleotides derived from
the transcription initiation site, e.g., between about 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. (See, e.g., 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., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of MRNA by preventing the
transcript from binding to ribosomes.
[0129] 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. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding VATPS.
[0130] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including 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.
[0131] Complementary ribonucleic acid 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.
[0132] Alternatively, RNA molecules may be generated by in vitro
and in vivo transcription of DNA sequences encoding VATPS. Such DNA
sequences may be incorporated into a wide variety of vectors with
suitable RNA polymerase promoters such as T7 or SP6.
[0133] Alternatively, these cDNA constructs that synthesize
complementary RNA, constitutively or inducibly, can be introduced
into cell lines, cells, or tissues.
[0134] 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 rather than phosphodiesterase 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
[0135] acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0136] 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,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
[0137] 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.
[0138] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of VATPS, antibodies to VATPS, and
mimetics, agonists, antagonists, or inhibitors of VATPS. The
compositions may be administered alone or in combination with at
least one other agent, such as a 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.
[0139] 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.
[0140] 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.).
[0141] 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.
[0142] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and 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, and alginic acid or a salt thereof, such as
sodium alginate.
[0143] 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.
[0144] 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 fillers
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.
[0145] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks's 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, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0146] 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.
[0147] 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.
[0148] 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, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms.
[0149] In other cases, the preferred preparation may be a
lyophilized powder which may contain any or all of the following: 1
mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol,
at a pH range of 4.5 to 5.5, that is combined with buffer prior to
use.
[0150] 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 VATPS, such
labeling would include amount, frequency, and method of
administration.
[0151] 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.
[0152] 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 such as mice, rats, rabbits,
dogs, or pigs. An 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.
[0153] A therapeutically effective dose refers to that amount of
active ingredient, for example VATPS or fragments thereof,
antibodies of VATPS, and agonists, antagonists or inhibitors of
VATPS, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of therapeutic to toxic
effects is the therapeutic index, and it can be expressed as the
ED.sub.50/LD.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0154] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring 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,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long- acting pharmaceutical compositions may be administered every
3 to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0155] Normal dosage amounts may vary from about 0.1 Ig to 100,000
ug, up to a total dose of about 1 gram, 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.
DIAGNOSTICS
[0156] In another embodiment, antibodies which specifically bind
VATPS may be used for the diagnosis of disorders characterized by
expression of VATPS, or in assays to monitor patients being treated
with VATPS or agonists, antagonists, or inhibitors of VATPS.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for VATPS include methods which utilize the antibody and a label to
detect VATPS in human body fluids or in extracts of cells or
tissues. The antibodies may be used with or without modification,
and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A wide variety of reporter molecules, several of
which are described above, are known in the art and may be
used.
[0157] A variety of protocols for measuring VATPS, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of VATPS expression.
Normal or standard values for VATPS expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to VATPS under conditions
suitable for complex formation The amount of standard complex
formation may be quantitated by various methods, preferably by
photometric means. Quantities of VATPS expressed in subject,
control, and disease samples from biopsied tissues are compared
with the standard values. Deviation between standard and subject
values establishes the parameters for diagnosing disease.
[0158] In another embodiment of the invention, the polynucleotides
encoding VATPS may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of VATPS may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of VATPS, and to
monitor regulation of VATPS levels during therapeutic
intervention.
[0159] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding VATPS or closely related molecules may be used
to identify nucleic acid sequences which encode VATPS. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding VATPS, allelic variants, or related
sequences.
[0160] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the VATPS encoding sequences. The hybridization
probes of the subject invention may be DNA or RNA and may be
derived from the sequence of SEQ ID NO:3, SEQ ID NO:4 or from
genomic sequences including promoters, enhancers, and introns of
the VATPS gene.
[0161] Means for producing specific hybridization probes for DNAs
encoding VATPS include the cloning of polynucleotide sequences
encoding VATPS or VATPS derivatives into vectors for the production
of MRNA probes. Such vectors are known in the art, are 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, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0162] Polynucleotide sequences encoding VATPS may be used for the
diagnosis of a cell proliferative or neurological disorder
associated with expression of VATPS. Examples of such disorders
include, but are not limited to, actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia; cancers including adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; and neurological
disorders such as akathesia, Alzheimer's disease, amnesia,
amyotrophic lateral sclerosis, bipolar disorder, catatonia,
cerebral neoplasms, dementia, depression, diabetic neuropathy,
Down's syndrome, tardive dyskinesia, dystonias, epilepsy,
Huntington's disease, peripheral neuropathy, multiple sclerosis,
neurofibromatosis, Parkinson's disease, paranoid psychoses,
postherpetic neuralgia, schizophrenia, and Tourette's disorder. The
polynucleotide sequences encoding VATPS may be used in Southern or
Northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies; in dipstick, pin, and ELISA assays; and in
microarrays utilizing fluids or tissues from patients to detect
altered VATPS expression. Such qualitative or quantitative methods
are well known in the art.
[0163] In a particular aspect, the nucleotide sequences encoding
VATPS may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding VATPS 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 patient sample is significantly altered in
comparison to a control sample then the presence of altered levels
of nucleotide sequences encoding VATPS in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0164] In order to provide a basis for the diagnosis of a disorder
associated with expression of VATPS, 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,
encoding VATPS, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained in this manner may
be compared with values obtained from samples from patients who are
symptomatic for a disorder. Deviation from standard values is used
to establish the presence of a disorder.
[0165] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0166] 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.
[0167] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding VATPS may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding VATPS, or a fragment of a
polynucleotide complementary to the polynucleotide encoding VATPS,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantitation of
closely related DNA or RNA sequences.
[0168] Methods which may also be used to quantitate the expression
of VATPS include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al.
(1993) Anal. Biochem. 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 calorimetric response gives
rapid quantitation.
[0169] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0170] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0171] In another embodiment of the invention, nucleic acid
sequences encoding VATPS may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial PI constructions, or single chromosome cDNA
libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet.
15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B.
J. (1991) Trends Genet. 7:149-154.) Fluorescent in situ
hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data. (See, e.g.,
Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular
Biology and Biotechnology, VCH Publishers New York, N.Y., pp.
965-968.) Examples of genetic map data can be found in various
scientific journals or at the Online Mendelian Inheritance in Man
(OMIM) site. Correlation between the location of the gene encoding
VATPS on a physical chromosomal map and a specific disorder, or a
predisposition to a specific disorder, may help define the region
of DNA associated with that disorder. The nucleotide sequences of
the invention may be used to detect differences in gene sequences
among normal, carrier, and affected individuals.
[0172] 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 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, e.g., ataxia-telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. (See, e.g., Gatti, R.
A. et al. (1988) Nature 336:577-580.) 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.
[0173] In another embodiment of the invention, VATPS, 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 VATPS and the agent being tested may be
measured.
[0174] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84103564.) In this method, 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 VATPS, or fragments thereof, and washed. Bound VATPS is then
detected by methods well known in the art. Purified VATPS 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.
[0175] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding VATPS specifically compete with a test compound for binding
VATPS. In this manner, antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with VATPS.
[0176] In additional embodiments, the nucleotide sequences which
encode VATPS 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.
[0177] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0178] I. cDNA Library Construction
[0179] The HIPONON02 normalized brain library was constructed from
1.13 million independent clones from a hippocampus tissue library.
Starting RNA was made from the hippocampus tissue of a 72-year-old
Caucasian female, who died from a cerebrovascular accident. The
library was oligo(dT)-primed, and cDNAs were cloned directionally
into the pSPORT1 vector (Life Technologies) using Sall (5') and
Notl (3'). The normalization and hybridization conditions were
adapted from Soares et al. (Proc. Natl. Acad. Sci. (1994)
91:9228-9232), except that a significantly longer (48-hour)
reannealing hybridization was used.
[0180] The TESTTUT02 library was constructed using RNA isolated
from a testicular tumor removed from a 31-year-old Caucasian male
during unilateral orchiectomy. Pathology indicated embryonal
carcinoma forming a largely necrotic mass involving the entire
testicle. Rare foci of residual testicle showed intralobular germ
cell neoplasia. cDNA synthesis was initiated using a NotI-oligo(dT)
primer. Double-stranded cDNA was blunted, ligated to EcoRI
adaptors, digested with NotI, size-selected, and cloned into the
NotI and EcoRI sites of the pINCY vector (Incyte Pharmaceuticals,
Palo Alto Calif.).
[0181] II. Isolation and Sequencing of cDNA Clones
[0182] Plasmids were recovered from host cells by in vivo excision
(UniZAP vector system, Stratagene) or by cell lysis. Plasmids were
purified using the MAGIC MINIPREPS DNA purification system
(Promega, Madison, Wis.); Miniprep kit (Advanced Genetic
Technologies Corporation, Gaithersburg, Md.); QIAwell-8 Plasmid,
QIAwell PLUS DNA, or QIAwell ULTRA DNA purification systems; or
REAL Prep 96 plasmid kit (QIAGEN Inc) using the recommended
protocol. Following precipitation, plasmids were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization,
at 4 .degree. C.
[0183] Alternatively, plasmid DNA was amplified from host cell
lysates using direct link PCR (Rao, V. B. (1994) Anal. Biochem.
216:1-14) in a high-throughput format. Host cell lysis and thermal
cycling steps were carried out in a single reaction mixture.
Samples were processed and stored in 384-well plates (Genetix Ltd,
Christchurch UK) and concentration of amplified plasmid DNA was
quantified fluorometrically using Pico Green Dye (Molecular Probes,
Eugene Ore.) and a Fluoroscan II fluorescence scanner (Labsystems
Oy, Helsinki, Finland).
[0184] III. Sequencing and Analysis
[0185] The cDNAs were prepared for sequencing using either an ABI
Catalyst 800 (Perkin Elmer) or a Hamilton Micro Lab 2200 (Hamilton,
Reno, Nev.) in combination with Peltier Thermal Cyclers (PTC200; M
J Research, Watertown Mass.). The cDNAs were sequenced on the ABI
373 or 377 DNA Sequencing systems (Perkin Elmer) by the method of
Sanger F and A. R. Coulson (1975; J. Mol. Biol. 94:441-448) using
standard ABI protocols, base calling software, and kits.
Alternatively, cDNAs were sequenced using solutions and dyes from
Amersham Pharmacia Biotech. Reading frame was determined using
standard methods (Ausubel, supra).
[0186] The cDNA sequences and the full length nucleotide and amino
acid sequences disclosed in the Sequence Listing were queried
against databases such as GenBank primate (pri), rodent (rod),
mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp)
databases, SwissProt, BLOCKS, and other databases which contain
previously identified and annotated motifs and sequences.
Algorithms such as Smith Waterman which deal with primary sequence
patterns and secondary structure gap penalties (Smith, T. et al.
(1992) Protein Engineering 5:35-51) and programs and algorithms
such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F.
(1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol.
Biol. 215:403-410), and HMM (Hidden Markov Models; Eddy, S. R.
(1996) Cur. Opin. Str. Biol. 6:361-365 and Sonnhammer, E.L.L. et
al. (1997) Proteins 28:405-420) were used to assemble and analyze
nucleotide and amino acid sequences. The databases, programs,
algorithms, methods and tools are available, well known in the art,
and described in Ausubel (supra, unit 7.7), in Meyers, R. A. (1995;
Molecular Biology and Biotechnology, Wiley VCH, Inc, New York N.Y.,
p 856-853), in documentation provided with software (Genetics
Computer Group (GCG), Madison Wis.), and on the world wide web
(www). Two comprehensive websites which list, describe, and/or link
many of the databases and tools are: 1) the www resource in
practical sequence analysis (http:H/genome.wustl.edu/), and 2) the
bibliography of computational gene recognition
(http://linkage.rockefeller.edu/wli/gene/ programs.html). For
example, the first website links PFAM as a database
(http:Hlgenome.wustl.edu/Pfam/) and as an HMM search tool
(http://genome.wustl.edu/eddy/cgi-bin/hmm-page.cgi).
[0187] TABLE 1 summarizes the databases and tools used herein. The
first column of TABLE 1 shows the tool, program, or algorithm; the
second column, the database; the third column, a brief description;
and the fourth column (where applicable), scores for determining
the strength of a match between two sequences (the higher the
value, the more homologous).
[0188] IV. Northern Analysis
[0189] 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.
(See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and
16.)
[0190] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ.TM. 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 similar.
[0191] The basis of the search is the product score, which is
defined as:
% sequence identity.times.% maximum BLAST score/100
[0192] 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% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0193] The results of Northern analysis are reported as a list of
libraries in which the transcript encoding VATPS 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.
[0194] V. Extension of VATPS Encoding Polynucleotides
[0195] The nucleic acid sequence of Incyte Clones 2246348 and
2346304 were used to design oligonucleotide primers for extending
partial nucleotide sequence to full length. For each nucleic acid
sequence, one primer was synthesized to initiate extension of an
antisense polynucleotide, and the other was synthesized to initiate
extension of a sense polynucleotide. Primers were 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 were designed from the CDNA using
OLIGO.TM. 4.06 (National Biosciences, Plymouth, Minn.), or another
appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of about 50% or more, and to anneal to the target
sequence at temperatures of about 68.degree. C. to about 72.degree.
C. Any stretch of nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
[0196] Selected human cDNA libraries (GIBCO BRL) were used to
extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0197] High fidelity amplification was obtained by following the
instructions for the XL-PCRT kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
Peltier Thermal Cycler (PTC200; M. J. Research, Watertown, Mass.),
beginning with 40 pmol of each primer and the recommended
concentrations of all other components of the kit, with 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 steps 4 through 6 for an
additional 15 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 steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0198] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low 35 concentration (about 0.6%
to 0.8%) agarose mini-gel to determine which reactions were
successful in extending the sequence. Bands thought to contain the
largest products were excised from the gel, purified using
QIAQUICK.TM. (QIAGEN Inc.), and trimmed of overhangs using Klenow
enzyme to facilitate religation and cloning.
[0199] After ethanol precipitation, the products were redissolved
in 13 Hl of ligation buffer, 1 .mu.l T4-40 DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 gl of SOC medium. (See, e.g., Sambrook, supra,
Appendix A, p. 2.) After incubation for one hour at 37.degree. C.,
the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin
(2x carb). The following day, several colonies were randomly picked
from each plate and cultured in 150 .mu.l of liquid LB/2x carb
medium placed in an individual well of an appropriate
commercially-available sterile 96-well microtiter plate. The
following day, 5 .mu.l of each overnight culture was transferred
into a non-sterile 96-well plate and, after dilution 1:10 with
water, 5 ltl from each sample was transferred into a PCR array.
[0200] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3x) containing 4 units of rTth DNA polymerase, a vector
primer, and one or both of the gene specific primers used for the
extension reaction were added to each well. Amplification was
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 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0201] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0202] In like manner, the nucleotide sequence of SEQ ID NO:3, SEQ
ID NO:4 are used to obtain 5' regulatory sequences using the
procedure above, oligonucleotides designed for 5' extension, and an
appropriate genomic library.
[0203] VI. Labeling and Use of Individual Hybridization Probes
[0204] Hybridization probes derived from SEQ ID NO:3, SEQ ID NO:4
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 nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO.TM. 4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham
Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN.RTM.,
Boston, Mass.). The labeled oligonucleotides are substantially
purified using a Sephadex.TM. G-25 superfine size exclusion dextran
bead column (Amersham Pharmacia Biotech). An aliquot containing 107
counts per minute of the labeled probe 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, Xbal, or Pvu II (DuPont NEN, Boston, Mass.).
[0205] The DNA from each digest is fractionated on a 0.7% 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 x saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT AR.TM. film (Kodak, Rochester,
N.Y.) is exposed to the blots for several hours, hybridization
patterns are compared visually.
[0206] VII. Microarrays
[0207] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0208] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE. Full-length cDNAs, ESTs,
or fragments thereof corresponding to one of the nucleotide
sequences of the present invention, or selected at random from a
cDNA library relevant to the present invention, are arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to
the slide using, e.g., UV cross-linking followed by thermal and
chemical treatments and 2X subsequent drying. (See, e.g., Schena,
M. et al. (1995) Science 270:467-470; and Shalon, D. et al. (1996)
Genome Res. 6:639-645.) Fluorescent probes are prepared and used
for hybridization to the elements on the substrate. The substrate
is analyzed by procedures described above.
[0209] VIII. Complementary Polynucleotides
[0210] Sequences complementary to the VATPS-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring VATPS. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO.TM. 4.06 software and the coding sequence of
VATPS. To inhibit transcription, a complementary oligonucleotide is
designed from the most unique 5' sequence and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal
binding to the VATPS-encoding transcript.
[0211] IX. Expression of VATPS
[0212] Expression and purification of VATPS is achieved using
bacterial or virus-based expression systems. For expression of
VATPS in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express VATPS upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of VATPS
in eukaryotic cells is achieved by infecting insect or mammalian
cell lines with recombinant Autographica califomica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding VATPS by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.) In most expression systems, VATPS is
synthesized as a fusion protein with, e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or
6-His, permitting rapid, single-step, affinity- based purification
of recombinant fusion protein from crude cell lysates. GST, a
26-kilodalton enzyme lia from Schistosoma japonicum, enables the
purification of fusion proteins on immobilized glutathione under
conditions that maintain protein activity and antigenicity
(Amersham Pharmacia Biotech). Following purification, the GST
moiety can be proteolytically cleaved from VATPS at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification using commercially available monoclonal
and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester,
N.Y.). 6-His, a stretch of six consecutive histidine residues,
enables purification on metal-chelate resins (QIAGEN Inc,
Chatsworth, Calif.). Methods for protein expression and
purification are discussed in Ausubel, F. M. et al. (1995 and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., ch 10, 16. Purified VATPS
obtained by these methods can be used directly in the following
activity assay.
[0213] X. Demonstration of VATPS Activity
[0214] The activity of VATPS is demonstrated by the measurement of
proton flux in phospholipid vesicles containing vp-ATPase
reconstituted from its constituent polypeptides, including VATPS
(Zhang, J. et al. (1992) J. Biol. Chem. 267: 9773-78).
Reconstituted vp-ATPase is incorporated into phospholipid vesicles.
The vesicles are incubated in 20 mM HEPES buffer, pH 7.0,
containing 2 .mu.M 9-amino-6-chloro-2 methoxyacridine (ACMA).
Proton flux is initiated by the addition of 20 nM valinomycin, and
measured by fluorescence quenching of ACMA in a fluorescence
spectrophotometer using excitation and emission wavelengths of 410
nm and 490 nm, respectively. A negative control assay is performed
using vp-ATPase reconstituted in the absence of VATPS. A positive
control assay is performed using native vp-ATPase assayed in an
identical manner. Quenching of ACMA fluorescence in the vp-ATPase
assay containing reconstituted VATPS compared to a lack of
quenching in the negative control is evidence of VATPS activity.
The level of ACMA quenching is proportional to the amount of
vp-ATPase and hence VATPS present in the assayed sample.
[0215] XI. Functional Assays
[0216] VATPS function is assessed by expressing the sequences
encoding VATPS at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT.TM. (Life
Technologies, Gaithersburg, Md.) and pCRTm 3.1 (Invitrogen,
Carlsbad, Calif., both of which contain the cytomegalovirus
promoter. 5-10 .mu.g of recombinant vector are transiently
transfected into a human cell line, preferably of endothelial or
hematopoietic origin, using either liposome formulations or
electroporation. 1-2 yg of an additional plasmid containing
sequences encoding a marker protein are co-transfected. Expression
of a marker protein provides a means to distinguish transfected
cells from nontransfected cells and is a reliable predictor of cDNA
expression from the recombinant vector. Marker proteins of choice
include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo
Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP, and to
evaluate properties, for example, their apoptotic state. FCM
detects and quantifies the uptake of fluorescent molecules that
diagnose events preceding or coincident with cell death. These
events include changes in nuclear DNA content as measured by
staining of DNA with propidium iodide; changes in cell size and
granularity as measured by forward light scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by
decrease in bromodeoxyuridine uptake; alterations in expression of
cell surface and intracellular proteins as measured by reactivity
with specific antibodies; and alterations in plasma membrane
composition as measured by the binding of fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry
are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New
York, N.Y.
[0217] The influence of VATPS on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding VATPS and either CD64 or CD64-GFP. CD64 and
CD64- GFP are expressed on the surface of transfected cells and
bind to conserved regions of human immunoglobulin G (IgG).
Transfected cells are efficiently separated from nontransfected
cells using magnetic beads coated with either human IgG or antibody
against CD64 (DYNAL, Lake Success, N.Y.). mRNA can be purified from
the cells using methods well known by those of skill in the art.
Expression of mRNA encoding VATPS and other genes of interest can
be analyzed by Northern analysis or microarray techniques.
[0218] XII. Production of VATPS Specific Antibodies
[0219] VATPS substantially purified using polyacrylamide gel
electrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488-495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0220] Alternatively, the VATPS amino acid sequence is analyzed
using LASERGENE.TM. software (DNASTAR Inc.) to determine regions of
high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Methods for selection of appropriate epitopes,
such as those near the C-terminus or in hydrophilic regions are
well described in the art. (See, e.g., Ausubel supra, ch. 11.)
[0221] Typically, oligopeptides 15 residues in length are
synthesized using an Applied Biosystems Peptide Synthesizer Model
43 1A using fmoc-chemistry and coupled to KLH (Sigma, St. Louis,
Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase immunogenicity. (See, e.g., Ausubel supra.)
Rabbits are immunized with the oligopeptide-KLH complex in complete
Freund's adjuvant. Resulting antisera are tested for antipeptide
activity by, for example, binding the peptide to plastic, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG.
[0222] XIII. Purification of Naturally Occurring VATPS Using
Specific Antibodies
[0223] Naturally occurring or recombinant VATPS is substantially
purified by immunoaffinity chromatography using antibodies specific
for VATPS. An immunoaffinity column is constructed by covalently
coupling anti-VATPS 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.
[0224] Media containing VATPS are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of VATPS (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/VATPS binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and VATPS is collected.
[0225] XIV. Identification of Molecules Which Interact with
VATPS
[0226] VATPS, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., 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
VATPS, washed, and any wells with labeled VATPS complex are
assayed. Data obtained using different concentrations of VATPS are
used to calculate values for the number, affinity, and association
of VATPS with the candidate molecules.
[0227] Various modifications and variations of the described
methods and systems 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.
3TABLE 1 Program/algorithm Databases Description Useful Parameters
ESTs Smith Waterman GenBank Local alignment algorithm for homology
searching min length = 49 nt < 12% uncalled bases FASTA GenBank
Fast nucleotide sequence database searching program for UNIX, VMS
BLAST GenBank Ultra-fast database searching program for UNIX, VMS C
source Log likelihood for exact matches is .about. 10.sup.25 and
for homologs > 10.sup.-8 Full Length Phred Reads trace data from
sequencing runs, makes base calls for assembly of cDNA sequences,
produces quality scores Phrap Quality-score based assembly program
for shotgun sequences match > 56 score>120 CONSED Graphical
tool for editing Phrap contigs GCG Assembly, GenBank Wisconsing
PackagePrograms for the assembly, editing, and Motifs, Profilescan,
PROSITE characterization of nucleotide sequences Spscan Examines
proteins for secretory, signal sequences >7 strong, 4.5-7
suggestive GENEMARK Statistical analysis of nucleotide sequences to
identify open reading frame BLAST GenBank Ultra-fast database
searching program for UNIX, VMS C source score > 100, P <
1c-5 SwissProt FASTX GenBank Fast amino acid sequence database
searching program log likelihood > 17 SwissProt for UNIX, VMS
BLIMPS BLOCKS Weighted matrix analysis for prediction of protein
family >1300 strong, 1000- PRINTS 1300 suggestive, P < 1c-3
PFAM PROSITE Analyses sequences 3-60 amino acids long which
correspond to Score > 11 strong, 8-10 highly conserved regions
of a protein family suggestive HMM Probabilistic approaches and
modeling of the primary structure Score > 11 strong, 8-10 of
protein families suggestive McDNAsis Pro Software for sequence
analysis LASERGENE Software programs (EditSeq, MegAlign,
PrimerSelect, Protean, SeqMan, etc.) for sequence analysis
[0228]
Sequence CWU 1
1
* * * * *
References