U.S. patent application number 10/618538 was filed with the patent office on 2004-01-22 for humanena/vasp-like protein splice variant.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Corley, Neil C., Guegler, Karl J., Lal, Preeti.
Application Number | 20040013670 10/618538 |
Document ID | / |
Family ID | 21832659 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040013670 |
Kind Code |
A1 |
Lal, Preeti ; et
al. |
January 22, 2004 |
Humanena/VASP-like protein splice variant
Abstract
The invention provides a human ena/VASP-like protein splice
variant (EVL1) and polynucleotides which identify and encode EVL1.
The invention also provides expression vectors, host cells,
antibodies, agonists, and antagonists. The invention also provides
methods for treating or preventing disorders associated with
expression of EVL1.
Inventors: |
Lal, Preeti; (Santa Clara,
CA) ; Guegler, Karl J.; (Menlo Park, CA) ;
Corley, Neil C.; (Castro Valley, CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Corporation
Palo Alto
CA
|
Family ID: |
21832659 |
Appl. No.: |
10/618538 |
Filed: |
July 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10618538 |
Jul 10, 2003 |
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09387811 |
Sep 1, 1999 |
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6645499 |
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09387811 |
Sep 1, 1999 |
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09227420 |
Jan 8, 1999 |
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5990087 |
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09227420 |
Jan 8, 1999 |
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09026587 |
Feb 20, 1998 |
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5912128 |
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Current U.S.
Class: |
424/146.1 ;
435/226; 435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
Y10S 530/849 20130101;
Y10T 436/143333 20150115; C07K 14/4702 20130101 |
Class at
Publication: |
424/146.1 ;
435/69.1; 435/226; 435/320.1; 435/325; 530/388.26; 536/23.2 |
International
Class: |
C07H 021/04; A61K
039/395; C12N 009/64; C12P 021/02; C12N 005/06; C07K 016/40 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1,
b) a polypeptide comprising a naturally occurring amino acid
sequence at least 95% identical to the amino acid sequence of SEQ
ID NO:1, c) a biologically active fragment of a polypeptide having
the amino acid sequence of SEQ ID NO:1, and d) an immunogenic
fragment of a polypeptide having the amino acid sequence of SEQ ID
NO:1.
2. An isolated polypeptide of claim 1 comprising the amino acid
sequence of SEQ ID NO:1.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising the
polynucleotide sequence of SEQ ID NO:2.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of 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 comprises the
amino acid sequence of SEQ ID NO:1.
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 the polynucleotide sequence of
SEQ ID NO:2, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to the
polynucleotide sequence of SEQ ID NO:2, 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 of 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 of 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 comprises
the amino acid sequence of SEQ ID NO:1.
19. A method for treating a disease or condition associated with
decreased expression of functional EVL1, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
contacting a sample comprising a polypeptide of claim 1 with 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 EVL1, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
contacting a sample comprising a polypeptide of claim 1 with 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 EVL1, 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, the method comprising: 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, the 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 of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) contacting a sample comprising the target
polynucleotide with 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 of screening for potential toxicity of a test
compound, the 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 indicates
potential toxicity of the test compound.
30. A method for a diagnostic test for a condition or disease
associated with the expression of EVL1 in a biological sample, the
method comprising: 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 EVL1 in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, further comprising a label.
35. A method of diagnosing a condition or disease associated with
the expression of EVL1 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 consisting of the amino
acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof,
under conditions to elicit an antibody response, b) isolating
antibodies from the animal, and c) screening the isolated
antibodies with the polypeptide, thereby identifying a polyclonal
antibody which specifically binds to a polypeptide comprising the
amino acid sequence of SEQ ID NO:1.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal 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 consisting of the amino acid sequence
of SEQ ID NO:1, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibody
producing cells from the animal, c) fusing the antibody producing
cells with immortalized cells to form monoclonal antibody-producing
hybridoma cells, d) culturing the hybridoma cells, and e) isolating
from the culture monoclonal antibody which specifically binds to a
polypeptide comprising the amino acid sequence of SEQ ID NO:1.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal 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 comprising the amino acid
sequence of SEQ ID NO:1 in a sample, the method comprising: a)
incubating the antibody of claim 11 with the 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 comprising the
amino acid sequence of SEQ ID NO:1 in the sample.
45. A method of purifying a polypeptide comprising the amino acid
sequence of SEQ ID NO:1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with the 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 comprising the amino acid
sequence of SEQ ID NO:1.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: 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, and wherein
said target polynucleotide is a polynucleotide 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, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have 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 distinct physical location on the substrate.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/387,811, filed Sep. 1, 1999, which is a divisional of U.S.
application Ser. No. 09/227,420, filed on Jan. 8, 1999, issued Nov.
23, 1999, as U.S. Pat. No. 5,990,087, which is a divisional of U.S.
application Ser. No. 09/026,587, filed Feb. 20, 1998, issued Jun.
15, 1999, as U.S. Pat. No. 5,912,128, 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 a human ena/VASP-like protein splice variant and to
the use of these sequences in the diagnosis, treatment, and
prevention of reproductive, immunological, vesicle trafficking,
nervous system, developmental, and neoplastic disorders.
BACKGROUND OF THE INVENTION
[0003] The control of cell morphology and motility requires the
coupling of external stimuli to processes that alter the
cytoskeletal architecture. The mechanical forces that drive
morphological change and migration arise initially from the
microfilament-based cytoskeleton. A large body of evidence links
various signal transduction pathways to the formation of cellular
outgrowths. The migration of neuronal growth cones is a
well-studied mechanism for the actin-driven formation of membrane
protrusions. In one example, the processes of axonal outgrowth are
mediated by the Drosophila homolog of the c-Abl tyrosine kinase
(Abl) and the product of the Disabled gene (Dab). Homozygous
mutants of Abl and Dab make few or no proper axonal connections.
The defects caused by loss of Abl and Dab in Drosophila are
ameliorated by mutations in the Enabled (Ena) gene. Ena protein is
tyrosine phosphorylated and has a proline-rich core which binds to
the SH3 domains of Abl protein and Src protein in vitro. The murine
homolog of Ena (Mena) and ena/VASP-like protein have recently been
described and are members of a family of related molecules that
include vasoactive-stimulated phosphoprotein (VASP). These proteins
share three distinct regions of similarity: an amino-terminal 115
amino acids (EVH1 domain); a proline-rich core; and a
carboxy-terminal 226 amino acids (EVH2 domain). Mena has
phosphotyrosine and phosphoserine moieties and binds Abl and Src
SH3 domains. (Gertler, F. B. et al. (1996) Cell 87:227-239.)
[0004] Human platelet activation is inhibited by agents such as
prostaglandins and nitric oxide donors, which elevate intracellular
cAMP or cGMP levels. Activation of platelets is associated with
increased formation of intracellular F-actin. VASP is an abundant
in vivo substrate for cyclic nucleotide-dependent protein kinases
in platelets. VASP is a ligand for profilin, an actin-monomer
binding protein that can stimulate the formation of F-actin. VASP
is organized into three distinct domains. A central proline-rich
domain contains a GPPPPP motif as a single copy and as a 3-fold
tandem repeat, as well as three conserved phosphorylation sites for
cyclic nucleotide-dependent protein kinases. A C-terminal domain
contains a repetitive mixed-charge cluster which is predicted to
form an alpha-helix. VASP expression in transiently transfected
BHK21 cells was predominantly detected at stress fibers, at focal
adhesions, and in F-actin-containing cell surface protrusions. In
contrast, truncated VASP lacking the C-terminal domain was no
longer concentrated at focal adhesions. These data indicate that
the C-terminal domain is required for anchoring VASP at focal
adhesion sites, while the central domain may mediate VASP
interaction with profilin. (Ermekova, K. S. et al. (1997) J. Biol.
Chem. 272:32869-32877.)
[0005] In comparison, Mena binds FE65, a neuronal protein which
binds to the cytoplasmic portion of the .beta.-amyloid precursor
protein (.beta.-APP). .beta.-APP is a precursor to .beta.-amyloid
peptide, the major constituent of the extracellular plaques present
in brain tissue from Alzheimer disease patients. Both VASP and Mena
bind their respective adapter proteins (profilin or FE65) via
distinct proline-rich regions and thus regulate adapter
interaction(s) with other molecules. (Ermekova et al, supra.)
Proline-rich domains and proline clusters have been identified in
many proteins, particularly those that are associated with synaptic
vesicles and other secretory organelles. These domains and clusters
act as protein-protein interaction modules. (Linial, M. (1994)
Neuroreport 5:2009-2015.) Two members of ena/VASP-like proteins
have been recently isolated from mouse and rat and share 98.5%
sequence identity. (Gertler et al., supra; and Ohta, S. et al.
(1997) Biochem. Biophys. Res. Comm. 237:307-312.)
[0006] The discovery of a new human ena/VASP-like protein splice
variant and the polynucleotides encoding it satisfies a need in the
art by providing new compositions which are useful in the
diagnosis, treatment, and prevention of reproductive,
immunological, vesicle trafficking, nervous system, developmental,
and neoplastic disorders.
SUMMARY OF THE INVENTION
[0007] The invention is based on the discovery of a new human
ena/VASP-like protein splice variant (EVL1), the polynucleotides
encoding EVL1, and the use of these compositions for the diagnosis,
treatment, or prevention of reproductive, immunological, vesicle
trafficking, nervous system, developmental, and neoplastic
disorders.
[0008] The invention features a substantially purified polypeptide,
comprising the amino acid sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1.
[0009] The invention further provides a substantially purified
variant having at least 90% amino acid identity to the sequence of
SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also
provides an isolated and purified polynucleotide encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1. The invention also includes an isolated
and purified polynucleotide variant having at least 90%
polynucleotide identity to the polynucleotide encoding the
polypeptide consisting of the sequence of SEQ ID NO:1 or a fragment
of SEQ ID NO:1.
[0010] Additionally, the invention provides a composition
comprising a polynucleotide encoding the polypeptide comprising the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified
polynucleotide which hybridizes under stringent conditions to the
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an
isolated and purified polynucleotide which is complementary to the
polynucleotide encoding the polypeptide consisting of the sequence
of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0011] The invention also provides an isolated and purified
polynucleotide comprising a sequence of SEQ ID NO:2 or a fragment
of SEQ ID NO:2, and an isolated and purified polynucleotide variant
having at least 90% polynucleotide identity to the polynucleotide
comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID
NO:2. The invention also provides an isolated and purified
polynucleotide which is complementary to the polynucleotide
comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID
NO:2.
[0012] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1. 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 of SEQ ID NO:1 or a
fragment of SEQ ID NO:1, 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
consisting of a substantially purified polypeptide comprising the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in
conjunction with a suitable pharmaceutical carrier.
[0015] The invention further includes a purified antibody which
binds to a polypeptide consisting of the sequence of SEQ ID NO:1 or
a fragment of SEQ ID NO:1, as well as a purified agonist and a
purified antagonist of the polypeptide.
[0016] The invention also provides a method for treating or
preventing a reproductive disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified polypeptide comprising the amino acid sequence of SEQ ID
NO:1 or a fragment of SEQ ID NO:1.
[0017] The invention also provides a method for treating or
preventing an immunological disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified polypeptide comprising the amino sequence of SEQ ID NO:1
or a fragment of SEQ ID NO:1.
[0018] The invention also provides a method for treating or
preventing a vesicle trafficking disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified polypeptide comprising the amino acid sequence of SEQ ID
NO:1 or a fragment of SEQ ID NO:1.
[0019] The invention also provides a method for treating or
preventing a nervous system disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified polypeptide comprising the amino acid sequence of SEQ ID
NO:1 or a fragment of SEQ ID NO:1.
[0020] The invention also provides a method for treating or
preventing a developmental disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified polypeptide comprising the amino acid sequence of SEQ ID
NO:1 or a fragment of SEQ ID NO:1.
[0021] The invention also provides a method for treating or
preventing a neoplastic disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide comprising the amino
acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0022] The invention also provides a method for detecting a
polynucleotide encoding a polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a
biological sample containing nucleic acids, the method comprising
the steps of: (a) hybridizing the complement of the polynucleotide
encoding the polypeptide comprising the amino acid sequence of SEQ
ID NO:1 or a fragment of SEQ ID NO:1 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 nucleic acids of the biological sample
are amplified by the polymerase chain reaction prior to the
hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence
(SEQ ID NO:1) and nucleic acid sequence (SEQ ID NO:2) of EVL1. The
alignment was produced using MACDNASIS PRO software (Hitachi
Software Engineering Co. Ltd., San Bruno, Calif.).
[0024] FIGS. 2A, 2B, and 2C show the amino acid sequence alignments
among EVL1 (3089412; SEQ ID NO:1), mouse ena/VASP-like protein (GI
1644453; SEQ ID NO:3), and human VASP (GI 624964; SEQ ID NO:4),
produced using the multisequence alignment program of DNASTAR
software (DNASTAR Inc., Madison, Wis.).
[0025] FIGS. 3A and 3B show the hydrophobicity plots for EVL1 (SEQ
ID NO:1) and mouse ena/VASP-like protein (SEQ ID NO:3),
respectively; the positive X axis reflects amino acid position and
the negative Y axis reflects hydrophobicity (MACDNASIS PRO
software).
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.
[0029] Definitions
[0030] "EVL1," as used herein, refers to the amino acid sequences
of substantially purified EVL1 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.
[0031] The term "agonist," as used herein, refers to a molecule
which, when bound to EVL1, increases or prolongs the duration of
the effect of EVL1. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of EVL1.
[0032] An "allele" or an "allelic sequence," as these terms are
used herein, is an alternative form of the gene encoding EVL1.
Alleles 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 alleles are generally
ascribed to natural deletions, additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in
combination with the others, one or more times in a given
sequence.
[0033] "Altered" nucleic acid sequences encoding EVL1, as described
herein, include those sequences with deletions, insertions, or
substitutions of different nucleotides, resulting in a
polynucleotide the same EVL1 or a polypeptide with at least one
functional characteristic of EVL1. Included within this definition
are polymorphisms which may or may not be readily detectable using
a particular oligonucleotide probe of the polynucleotide encoding
EVL1, and improper or unexpected hybridization to alleles, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding EVL1. 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 EVL1. 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 EVL1 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.
[0034] The terms "amino acid" or "amino acid sequence," as used
herein, 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 EVL1
which are preferably about 5 to about 15 amino acids in length and
which retain some biological activity or immunological activity of
EVL1. Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms are not meant to limit the
amino acid sequence to the complete native amino acid sequence
associated with the recited protein molecule.
[0035] "Amplification," as used herein, 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. (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer: A Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y., pp. 1-5.)
[0036] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to EVL1, decreases the amount or the
duration of the effect of the biological or immunological activity
of EVL1. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of EVL1.
[0037] As used herein, 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 EVL1 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.
[0038] The term "antigenic determinant," as used herein, 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
immunogen used to elicit the immune response) for binding to an
antibody.
[0039] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to a specific nucleic acid sequence. The term
"antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense molecules
may be produced by any method including synthesis 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.
[0040] As used herein, 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
EVL1, or of any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0041] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A." 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.
[0042] A "composition comprising a given polynucleotide sequence"
or a "composition consisting of a given amino acid sequence," as
these terms are used herein, refer broadly to any composition
containing the given polynucleotide or amino acid sequence. The
composition may comprise a dry formulation, an aqueous solution, or
a sterile composition. Compositions comprising polynucleotide
sequences encoding EVL1 or fragments of EVL1 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.,
SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
[0043] The phrase "consensus sequence," as used herein, refers to a
nucleic acid sequence which has been resequenced to resolve
uncalled bases, extended using the XL-PCR kit (Perkin Elmer,
Norwalk, Conn.) in the 5' and/or the 3' direction, and resequenced,
or which has been assembled from the overlapping sequences of more
than one Incyte Clone using a computer program for fragment
assembly, such as the GELVIEW fragment assembly system (GCG,
Madison, Wis.). Some sequences have been both extended and
assembled to produce the consensus sequence.
[0044] As used herein, 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 EVL1, by northern analysis is indicative of the presence
of nucleic acids encoding EVL1 in a sample, and thereby correlates
with expression of the transcript from the polynucleotide encoding
EVL1.
[0045] A "deletion," as the term is used herein, 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.
[0046] The term "derivative," as used herein, refers to the
chemical modification of EVL1, of a polynucleotide sequence
encoding EVL1, or of a polynucleotide sequence complementary to a
polynucleotide sequence encoding EVL1. 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.
[0047] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete
homology. The word "identity" may substitute for the word
"homology." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially homologous."
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous 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% homology or
identity). In the absence of non-specific binding, the
substantially homologous sequence or probe will not hybridize to
the second non-complementary target sequence.
[0048] 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 program
(Lasergene software package, DNASTAR, Inc., Madison, Wis.). The
MEGALIGN program can create alignments between two or more
sequences according to different methods, e.g., the clustal method.
(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 100. Gaps of low or of no homology between the two amino acid
sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be
calculated by the clustal method, or by other methods known in the
art, such as 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.
[0049] "Human artificial chromosomes" (HACs), as described herein,
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.)
[0050] The term "humanized antibody," as used herein, 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.
[0051] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0052] As used herein, 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., C.sub.0t or
R.sub.0t 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).
[0053] The words "insertion" or "addition," as used herein, 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.
[0054] "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.
[0055] The term "microarray," as used herein, refers to an array of
distinct polynucleotides or oligonucleotides arrayed on a
substrate, such as paper, nylon or any other type of membrane,
filter, chip, glass slide, or any other suitable solid support.
[0056] The term "modulate," as it appears herein, refers to a
change in the activity of EVL1. For example, modulation may cause
an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of EVL1.
[0057] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to an oligonucleotide, nucleotide,
polynucleotide, or any fragment thereof, 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 are greater than about 60 nucleotides in length, and most
preferably are at least about 100 nucleotides, at least about 1000
nucleotides, or at least about 10,000 nucleotides in length.
[0058] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related nucleic acid sequences.
A promoter is operably associated or operably linked with a coding
sequence if the promoter controls the transcription of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in reading frame, certain
genetic elements, e.g., repressor genes, are not contiguously
linked to the encoded polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0059] The term "oligonucleotide," as used herein, 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. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimers," "primers," "oligomers," and "probes," as
these terms are commonly defined in the art.
[0060] "Peptide nucleic acid" (PNA), as used herein, 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 and RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993)
Anticancer Drug Des. 8:53-63.)
[0061] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding EVL1, or fragments thereof, or EVL1 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.
[0062] As used herein, 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 recognized by the binding molecule (i.e.,
the antigenic determinant or epitope). 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.
[0063] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotide
sequences and the claimed polynucleotide sequences. Suitably
stringent conditions can be defined by, for example, the
concentrations of salt or formamide in the prehybridization and
hybridization solutions, or by the hybridization temperature, and
are 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.
[0064] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS, and 200 .mu.g/ml sheared and denatured
salmon sperm DNA. Hybridization could occur under reduced
stringency conditions as described above, but in 35% formamide at a
reduced temperature of 35.degree. C. The temperature range
corresponding to a particular level of stringency can be further
narrowed by calculating the purine to pyrimidine ratio of the
nucleic acid of interest and adjusting the temperature accordingly.
Variations on the above ranges and conditions are well known in the
art.
[0065] The term "substantially purified," as used herein, 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.
[0066] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0067] "Transformation," as defined herein, 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, and refers to cells which transiently
express the inserted DNA or RNA for limited periods of time.
[0068] A "variant" of EVL1, as used herein, refers to an amino acid
sequence that is altered by one or more amino acids. The variant
may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties (e.g., replacement of
leucine with isoleucine). More rarely, a variant may have
"nonconservative" changes (e.g., replacement of 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,
DNASTAR software.
[0069] The Invention
[0070] The invention is based on the discovery of a new human
ena/VASP-like protein splice variant (EVL1), the polynucleotides
encoding EVL1, and the use of these compositions for the diagnosis,
treatment, or prevention of reproductive, immunological, vesicle
trafficking, nervous system, developmental, and neoplastic
disorders.
[0071] Nucleic acids encoding the EVL1 of the present invention
were first identified in Incyte Clone 3089412 from the aorta cDNA
library (HEAONOT03) using a computer search for amino acid sequence
alignments. A consensus sequence, SEQ ID NO:2, was derived from the
following overlapping and/or extended nucleic acid sequences:
Incyte Clones 3089412 (HEAONOT03), 2836864 (TLYMNOT03), 1822064
(GBLATUT01), 1446806 (PLACNOT02), 1556238 (BLADTUT04), 1209813
(BRSTNOT02), and the shotgun sequence SAEA02787.
[0072] In one embodiment, the invention encompasses a polypeptide
consisting of the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A, 1B, 1C, 1D, and 1E. EVL1 is 418 amino acids in length and
has two potential N-glycosylation sites at residues N64 and N319;
one potential cAMP- and cGMP-dependent protein kinase
phosphorylation site at residue S160; eight potential casein kinase
II phosphorylation sites at residues S96, S132, S215, S216, S253,
S285, S298 and S371; six potential protein kinase C phosphorylation
sites at residues T21, S22, T48, S123, T252 and S287; and a proline
cluster from about residue P346 to about residue P368 and a
predicted turn-coil-turn three-fold repeat structure from about
residue T345 to about residue D374, indicative of a protein-protein
interacting domain. As shown in FIGS. 2A, 2B, and 2C, EVL1 has
chemical and structural homology with mouse ena/VASP-like protein
(GI 1644453; SEQ ID NO:3), and human VASP (GI 624964; SEQ ID NO:4).
In particular, EVL1 and mouse ena/VASP-like protein share 92%
identity, two potential N-glycosylation sites, one potential cAMP-
and cGMP-dependent protein kinase phosphorylation site, seven
potential casein kinase II phosphorylation sites, and six potential
protein kinase C phosphorylation sites. In addition, EVL1 and mouse
ena/VASP-like protein have similar isoelectric points, 9.2 and 8.7,
respectively. As illustrated by FIGS. 3A and 3B, EVL1 and mouse
ena/VASP-like protein have rather similar hydrophobicity plots. The
fragment of SEQ ID NO:2 from about nucleotide 1161 to about
nucleotide 1197 is useful for designing oligonucleotides or to be
used directly as a hybridization probe. Northern analysis shows the
expression of this sequence in various libraries, at least 49% of
which are immortalized or cancerous and at least 26% of which
involve immune response. Of particular note is the expression of
EVL1 in gastrointestinal, cardiovascular, neural, and developmental
tissue; and in prostate, breast, ovary, and uterus tissue.
[0073] The invention also encompasses EVL1 variants. A preferred
EVL1 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 EVL1 amino acid sequence, and which
contains at least one functional or structural characteristic of
EVL1.
[0074] The invention also encompasses polynucleotides which encode
EVL1. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2,
which encodes an EVL1.
[0075] The invention also encompasses a variant of a polynucleotide
sequence encoding EVL1. In particular, such a variant
polynucleotide sequence will have at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding EVL1. A particular aspect of the invention encompasses a
variant of SEQ ID NO:2 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:2. Any one of the
polynucleotide variants described above can encode an amino acid
sequence which contains at least one functional or structural
characteristic of EVL1.
[0076] 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 EVL1, some bearing minimal
homology 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 EVL1, and all such variations are
to be considered as being specifically disclosed.
[0077] Although nucleotide sequences which encode EVL1 and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring EVL1 under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding EVL1 or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding EVL1 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.
[0078] The invention also encompasses production of DNA sequences
which encode EVL1 and EVL1 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 that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding EVL1 or any fragment
thereof.
[0079] 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:2, or a fragment of SEQ ID NO:2, under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; and Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.)
[0080] Methods for DNA sequencing are well known and generally
available 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 (U.S.
Biochemical Corp., Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE amplification system (GIBCO BRL,
Gaithersburg, Md.). Preferably, the process is automated with
machines such as the MICRO LAB 2200 liquid transfer system
(Hamilton, Reno, Nev.), PTC200 thermal cycler (M. J. Research,
Watertown, Mass.) and the ABI CATALYST and 373 and 377 DNA
sequencers (Perkin Elmer).
[0081] The nucleic acid sequences encoding EVL1 may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences, such as
promoters and regulatory elements. For example, one method which
may be employed, restriction-site PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus. (See, e.g.,
Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) In particular,
genomic DNA is first amplified in the presence of a primer
complementary to a linker sequence within the vector and a primer
specific to the region predicted to encode the gene. The amplified
sequences are then subjected to a second round of PCR with the same
linker primer and another specific primer internal to the first
one. Products of each round of PCR are transcribed with an
appropriate RNA polymerase and sequenced using reverse
transcriptase.
[0082] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region. (See, e.g.,
Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) The primers
may be designed using commercially available software such as OLIGO
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 target sequence at temperatures of about
68.degree. C. to 72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0083] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA. (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 place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule 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. 19:3055-3060.) Additionally, one may use PCR, nested primers,
and PROMOTERFINDER libraries to walk genomic DNA (Clontech, Palo
Alto, Calif.). This process avoids the need to screen libraries and
is useful in finding intron/exon junctions.
[0084] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo-d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into 5' non-transcribed regulatory regions.
[0085] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and 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 and SEQUENCE NAVIGATOR, Perkin Elmer),
and the entire process from loading of samples to computer analysis
and electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0086] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode EVL1 may be used in
recombinant DNA molecules to direct expression of EVL1, 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 these sequences
may be used to clone and express EVL1.
[0087] As will be understood by those of skill in the art, it may
be advantageous to produce EVL1-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0088] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter EVL1-encoding sequences for a variety of reasons including,
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0089] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding EVL1 may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of EVL1 activity, it may
be useful to encode a chimeric EVL1 protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the EVL1
encoding sequence and the heterologous protein sequence, so that
EVL1 may be cleaved and purified away from the heterologous
moiety.
[0090] In another embodiment, sequences encoding EVL1 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, the protein itself may be
produced using chemical methods to synthesize the amino acid
sequence of EVL1, or a fragment thereof. 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).
[0091] The newly synthesized 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. E. (1993) Proteins: Structures and Molecular
Properties, W. H. Freeman and Co., New York, N.Y.) Additionally,
the amino acid sequence of EVL1, 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.
[0092] In order to express a biologically active EVL1, the
nucleotide sequences encoding EVL1 or derivatives thereof may be
inserted into appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0093] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding EVL1 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.)
[0094] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding EVL1. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) 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.
[0095] The "control elements" or "regulatory sequences" are those
non-translated regions, e.g., enhancers, promoters, and 5' and 3'
untranslated regions, of the vector and polynucleotide sequences
encoding EVL1 which interact with host cellular proteins to carry
out transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used. For example, when cloning in bacterial systems, inducible
promoters, e.g., hybrid lacZ promoter of the BLUESCRIPT phagemid
(Stratagene, La Jolla, Calif.) or PSPORT plasmid (GIBCO BRL), may
be used. The baculovirus polyhedrin promoter may be used in insect
cells. Promoters or enhancers derived from the genomes of plant
cells (e.g., heat shock, RUBISCO, and storage protein genes) or
from plant viruses (e.g., viral promoters or leader sequences) may
be cloned into the vector. In mammalian cell systems, promoters
from mammalian genes or from mammalian viruses are preferable. If
it is necessary to generate a cell line that contains multiple
copies of the sequence encoding EVL1, vectors based on SV40 or EBV
may be used with an appropriate selectable marker.
[0096] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for EVL1. For example,
when large quantities of EVL1 are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as BLUESCRIPT (Stratagene), in which
the sequence encoding EVL1 may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced, and pIN vectors. (See, e.g., Van Heeke, G. and S. M.
Schuster (1989) J. Biol. Chem. 264:5503-5509.) pGEX vectors
(Pharmacia Biotech, Uppsala, Sweden) may also be used to express
foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor XA protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will.
[0097] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel
et al., supra; and Bitter, G. A. et al. (1987) Methods Enzymol.
153:516-544.)
[0098] In cases where plant expression vectors are used, the
expression of sequences encoding EVL1 may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.
6:307-311.) Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used. (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. Such techniques are described in a number of
generally available reviews. (See, e.g., Hobbs, S. or Murry, L. E.
(1992) in McGraw Hill Yearbook of Science and Technology, McGraw
Hill, New York, N.Y., pp. 191-196.)
[0099] An insect system may also be used to express EVL1. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding EVL1 may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of sequences
encoding EVL1 will render the polyhedrin gene inactive and produce
recombinant virus lacking coat protein. The recombinant viruses may
then be used to infect, for example, S. frugiperda cells or
Trichoplusia larvae in which EVL1 may be expressed. (See, e.g.,
Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. USA
91:3224-3227.)
[0100] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding EVL1 may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing EVL1 in
infected host cells. (See, e.g., Logan, J. and T. Shenk (1984)
Proc. Natl. Acad. Sci. USA 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.
[0101] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in 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.
[0102] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding EVL1. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding EVL1 and its initiation codon and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers appropriate for the particular cell
system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
[0103] 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
facilitate correct insertion, folding, and/or function. 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, Manassas, Va.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0104] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing EVL1 can be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for 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
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0105] 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 genes and adenine
phosphoribosyltransferase genes, which can be employed 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; npt 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. USA 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, which allows cells to utilize
indole in place of tryptophan, or hisD, which allows cells to
utilize histinol in place of histidine. (See, e.g., Hartman, S. C.
and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.)
Recently, the use of visible markers has gained popularity with
such markers as anthocyanins, .beta.-glucuronidase and its
substrate GUS, luciferase and its substrate luciferin. Green
fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.) are also
used. (See, e.g., Chalfie, M. et al. (1994) Science 263:802-805.)
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.)
[0106] 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 EVL1 is inserted within a marker gene
sequence, transformed cells containing sequences encoding EVL1 can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding EVL1 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.
[0107] Alternatively, host cells which contain the nucleic acid
sequence encoding EVL1 and express EVL1 may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein
sequences.
[0108] The presence of polynucleotide sequences encoding EVL1 can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
EVL1. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding EVL1
to detect transformants containing DNA or RNA encoding EVL1.
[0109] A variety of protocols for detecting and measuring the
expression of EVL1, using either polyclonal or monoclonal
antibodies specific for the protein, 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
EVL1 is preferred, but a competitive binding assay may be employed.
These and other assays are well described in the art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods: A Laboratory Manual,
APS Press, St Paul, Minn., Section IV; and Maddox, D. E. et al.
(1983) J. Exp. Med. 158:1211-1216.)
[0110] 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 EVL1 include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding EVL1, 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, Mich.), 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.
[0111] Host cells transformed with nucleotide sequences encoding
EVL1 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 contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode EVL1 may be designed to
contain signal sequences which direct secretion of EVL1 through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding EVL1 to nucleotide sequences
encoding a polypeptide domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences, such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.), between the purification domain and the EVL1 encoding
sequence may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing EVL1 and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification on immobilized metal ion
affinity chromatography (IMIAC). (See, e.g., Porath, J. et al.
(1992) Prot. Exp. Purif. 3: 263-281.) The enterokinase cleavage
site provides a means for purifying EVL1 from the fusion protein.
(See, e.g., Kroll, D. J. et al. (1993) DNA Cell Biol.
12:441-453.)
[0112] Fragments of EVL1 may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, T. E. (1984) Proteins:
Structures and Molecular Principles, W. H. Freeman and Co., New
York, N.Y., pp. 55-60.) Protein synthesis may be performed by
manual techniques or by automation. Automated synthesis may be
achieved, for example, using the ABI 431A peptide synthesizer
(Perkin Elmer). Various fragments of EVL1 may be synthesized
separately and then combined to produce the full length
molecule.
[0113] Therapeutics
[0114] Chemical and structural homology exists among EVL1, mouse
ena/VASP-like protein (GI 1644453), and human VASP (GI 624964). In
addition, EVL1 is expressed in cancer and the immune response; in
gastrointestinal, cardiovascular, neural, and developmental tissue;
and in prostate, breast, ovary, and uterus tissue. Therefore, EVL1
appears to play a role in reproductive, immunological, vesicle
trafficking, nervous system, developmental, and neoplastic
disorders.
[0115] Therefore, in one embodiment, EVL1 or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a reproductive disorder. Such reproductive disorders can
include, but are not limited to, disorders of prolactin production,
infertility, including tubal disease, ovulatory defects, and
endometriosis, disruptions of the estrous cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors,
autoimmune disorders, ectopic pregnancy, and teratogenesis, cancer
of the breast, fibrocystic breast disease, and galactorrhea,
disruptions of spermatogenesis, abnormal sperm physiology, cancer
of the testis, cancer of the prostate, benign prostatic
hyperplasia, and prostatitis, carcinoma of the male breast and
gynecomastia.
[0116] In another embodiment, a vector capable of expressing EVL1
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a reproductive disorder including, but
not limited to, those described above.
[0117] In a further embodiment, a pharmaceutical composition
comprising a substantially purified EVL1 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a reproductive disorder including, but not limited
to, those provided above.
[0118] In still another embodiment, an agonist which modulates the
activity of EVL1 may be administered to a subject to treat or
prevent a reproductive disorder including, but not limited to,
those listed above.
[0119] In another embodiment, EVL1 or a fragment or derivative
thereof may be administered to a subject to treat or prevent an
immunological disorder. Such immunological disorders can include,
but are not limited to, AIDS, Addison's disease, adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune thyroiditis, bronchitis, cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,
scleroderma, Sjoren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, ulcerative colitis, Werner
syndrome, and complications of cancer, hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections, and trauma.
[0120] In another embodiment, a vector capable of expressing EVL1
or a fragment or derivative thereof may be administered to a
subject to treat or prevent an immunological disorder including,
but not limited to, those described above.
[0121] In a further embodiment, a pharmaceutical composition
comprising a substantially purified EVL1 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent an immunological disorder including, but not
limited to, those provided above.
[0122] In still another embodiment, an agonist which modulates the
activity of EVL1 may be administered to a subject to treat or
prevent an immunological disorder including, but not limited to,
those listed above.
[0123] In another embodiment, EVL 1 or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
vesicle trafficking disorder. Such vesicle trafficking disorders
can include, but are not limited to, cystic fibrosis,
glucose-galactose malabsorption syndrome, hypercholesterolemia,
diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia,
Grave's disease, goiter, Cushing's disease, and Addison's disease,
gastrointestinal disorders including ulcerative colitis, gastric
and duodenal ulcers, other conditions associated with abnormal
vesicle trafficking including AIDS, allergies including hay fever,
asthma, and urticaria (hives), autoimmune hemolytic anemia,
proliferative glomerulonephritis, inflammatory bowel disease,
multiple sclerosis, myasthenia gravis, rheumatoid and
osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's
syndromes, systemic lupus erythematosus, toxic shock syndrome,
traumatic tissue damage, and viral, bacterial, fungal, helminth,
and protozoal infections.
[0124] In another embodiment, a vector capable of expressing EVL1
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a vesicle trafficking disorder
including, but not limited to, those described above.
[0125] In a further embodiment, a pharmaceutical composition
comprising a substantially purified EVL1 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a vesicle trafficking disorder including, but not
limited to, those provided above.
[0126] In still another embodiment, an agonist which modulates the
activity of EVL1 may be administered to a subject to treat or
prevent a vesicle trafficking disorder including, but not limited
to, those listed above.
[0127] In another embodiment, EVL1 or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
nervous system disorder. Such nervous system disorders can include,
but are 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, multiple sclerosis, neurofibromatosis,
Parkinson's disease, paranoid psychoses, post-herpetic neuralgia,
schizophrenia, and Tourette's disorder, angina, anaphylactic shock,
arrhythmias, asthma, cardiovascular shock, Cushing's syndrome,
hypertension, hypoglycemia, myocardial infarction, migraine, and
pheochromocytoma.
[0128] In another embodiment, a vector capable of expressing EVL1
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a nervous system disorder including,
but not limited to, those described above.
[0129] In a further embodiment, a pharmaceutical composition
comprising a substantially purified EVL1 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a nervous system disorder including, but not
limited to, those provided above.
[0130] In still another embodiment, an agonist which modulates the
activity of EVL1 may be administered to a subject to treat or
prevent a nervous system disorder including, but not limited to,
those listed above.
[0131] In another embodiment, EVL1 or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
developmental disorder. The term "developmental disorder" refers to
any disorder associated with development or function of a tissue,
organ, or system of a subject (such as the brain, adrenal gland,
kidney, skeletal or reproductive system). Such developmental
disorders can include, but are not limited to, renal tubular
acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome, Smith-Magenis syndrome, myelodysplastic
syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and neurofibromatosis, hypothyroidism, hydrocephalus,
seizure disorders such as Syndenham's chorea and cerebral palsy,
spinal bifida, and congenital glaucoma, cataract, or sensorineural
hearing loss.
[0132] In another embodiment, a vector capable of expressing EVL1
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a developmental disorder including, but
not limited to, those described above.
[0133] In a further embodiment, a pharmaceutical composition
comprising a substantially purified EVL1 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a developmental disorder including, but not
limited to, those provided above.
[0134] In still another embodiment, an agonist which modulates the
activity of EVL1 may be administered to a subject to treat or
prevent a developmental disorder including, but not limited to,
those listed above.
[0135] In a further embodiment, an antagonist of EVL1 may be
administered to a subject to treat or prevent a neoplastic
disorder. Such a neoplastic disorder may include, but is not
limited to, 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.
[0136] In one aspect, an antibody which specifically binds EVL1 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 EVL1.
[0137] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding EVL1 may be administered
to a subject to treat or prevent a neoplastic disorder including,
but not limited to, those described above.
[0138] 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.
[0139] An antagonist of EVL1 may be produced using methods which
are generally known in the art. In particular, purified EVL1 may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind EVL1. Antibodies
to EVL1 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.
[0140] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with EVL1 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.
[0141] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to EVL1 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 EVL1 amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0142] Monoclonal antibodies to EVL1 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. USA 80:2026-2030; and Cole, S. P. et al. (1984) Mol.
Cell. Biol. 62:109-120.)
[0143] 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. USA
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
EVL1-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., Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA 88:11120-11123.)
[0144] 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. USA 86:3833-3837; and Winter, G.
et al. (1991) Nature 349:293-299.)
[0145] Antibody fragments which contain specific binding sites for
EVL1 may also be generated. For example, such fragments include,
but are not limited to, F(ab').sub.2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab').sub.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.)
[0146] 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 EVL1 and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering EVL1 epitopes
is preferred, but a competitive binding assay may also be employed.
(Maddox et al., supra.)
[0147] In another embodiment of the invention, the polynucleotides
encoding EVL1, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding EVL1 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 EVL1. Thus, complementary molecules or
fragments may be used to modulate EVL1 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 EVL1.
[0148] 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 which
will express nucleic acid sequences complementary to the
polynucleotides of the gene encoding EVL1. (See, e.g., Sambrook et
al., supra; and Ausubel et al., supra.)
[0149] Genes encoding EVL1 can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding EVL1. 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.
[0150] 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 EVL1. 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.
[0151] 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 EVL1.
[0152] 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.
[0153] 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. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding EVL1. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell lines, cells, or tissues.
[0154] 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 acetyl-, methyl-, thio-, and similarly
modified forms of adenine, cytidine, guanine, thymine, and uridine
which are not as easily recognized by endogenous endonucleases.
[0155] 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.)
[0156] 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.
[0157] 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 EVL1, antibodies to EVL1, and mimetics,
agonists, antagonists, or inhibitors of EVL1. 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.
[0158] 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.
[0159] 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.).
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, 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.
[0165] 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.
[0166] 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.
[0167] 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 acids. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 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.
[0168] 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 EVL1, such
labeling would include amount, frequency, and method of
administration.
[0169] 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.
[0170] 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.
[0171] A therapeutically effective dose refers to that amount of
active ingredient, for example EVL1 or fragments thereof,
antibodies of EVL1, and agonists, antagonists or inhibitors of
EVL1, 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 ED50 (the dose therapeutically effective in 50%
of the population) or LD50 (the dose lethal to 50% of the
population) statistics. The dose ratio of toxic to therapeutic
effects is the therapeutic index, which can be expressed as the
LD50/ED50 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 ED50 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.
[0172] 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.
[0173] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, 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.
[0174] Diagnostics
[0175] In another embodiment, antibodies which specifically bind
EVL1 may be used for the diagnosis of disorders characterized by
expression of EVL1, or in assays to monitor patients being treated
with EVL1 or agonists, antagonists, or inhibitors of EVL1.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for EVL1 include methods which utilize the antibody and a label to
detect EVL1 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.
[0176] A variety of protocols for measuring EVL1, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of EVL1 expression. Normal or
standard values for EVL1 expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to EVL1 under conditions suitable
for complex formation. The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of EVL1 expressed in subject samples from biopsied
tissues are compared with the standard values. Deviation between
standard and subject values establishes the parameters for
diagnosing disease.
[0177] In another embodiment of the invention, the polynucleotides
encoding EVL1 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 EVL1 may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of EVL1, and to
monitor regulation of EVL1 levels during therapeutic
intervention.
[0178] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding EVL1 or closely related molecules may be used
to identify nucleic acid sequences which encode EVL1. 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 EVL1, alleles, or related
sequences.
[0179] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides from any of the EVL1 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:2 or from genomic
sequences including promoters, enhancers, and introns of the EVL1
gene.
[0180] Means for producing specific hybridization probes for DNAs
encoding EVL1 include the cloning of polynucleotide sequences
encoding EVL1 or EVL1 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.
[0181] Polynucleotide sequences encoding EVL1 may be used for the
diagnosis of a disorder associated with expression of EVL1.
Examples of such a disorder include, but are not limited to: a
reproductive disorder, such as disorders of prolactin production,
infertility, including tubal disease, ovulatory defects, and
endometriosis, disruptions of the estrous cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors,
autoimmune disorders, ectopic pregnancy, and teratogenesis, cancer
of the breast, fibrocystic breast disease, and galactorrhea,
disruptions of spermatogenesis, abnormal sperm physiology, cancer
of the testis, cancer of the prostate, benign prostatic
hyperplasia, and prostatitis, carcinoma of the male breast and
gynecomastia; an immunological disorder, such as AIDS, Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis,
hypereosinophilia, irritable bowel syndrome, lupus erythematosus,
multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis, ulcerative colitis, Werner syndrome, and
complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; a vesicle trafficking disorder,
such as cystic fibrosis, glucose-galactose malabsorption syndrome,
hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-
and hypoglycemia, Grave's disease, goiter, Cushing's disease, and
Addison's disease, gastrointestinal disorders including ulcerative
colitis, gastric and duodenal ulcers, other conditions associated
with abnormal vesicle trafficking including AIDS, allergies
including hay fever, asthma, and urticaria (hives), autoimmune
hemolytic anemia, proliferative glomerulonephritis, inflammatory
bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid
and osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's
syndromes, systemic lupus erythematosus, toxic shock syndrome,
traumatic tissue damage, and viral, bacterial, fungal, helminth,
and protozoal infections; a nervous system disorder, 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, multiple
sclerosis, neurofibromatosis, Parkinson's disease, paranoid
psychoses, postherpetic neuralgia, schizophrenia, and Tourette's
disorder, angina, anaphylactic shock, arrhythmias, asthma,
cardiovascular shock, Cushing's syndrome, hypertension,
hypoglycemia, myocardial infarction, migraine, and
pheochromocytoma; a developmental disorder, such as renal tubular
acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome, Smith-Magenis syndrome, myelodysplastic
syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and neurofibromatosis, hypothyroidism, hydrocephalus,
seizure disorders such as Syndenham's chorea and cerebral palsy,
spinal bifida, and congenital glaucoma, cataract, or sensorineural
hearing loss; and a neoplastic disorder, such as 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. The polynucleotide
sequences encoding EVL1 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 EVL1 expression. Such qualitative or quantitative methods
are well known in the art.
[0182] In a particular aspect, the nucleotide sequences encoding
EVL1 may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding EVL1 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 EVL1 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.
[0183] In order to provide a basis for the diagnosis of a disorder
associated with expression of EVL1, 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 EVL1, 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.
[0184] 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.
[0185] 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.
[0186] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding EVL1 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 EVL1, or a fragment of a
polynucleotide complementary to the polynucleotide encoding EVL1,
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.
[0187] Methods which may also be used to quantitate the expression
of EVL1 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. 212:229-236.) The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
[0188] 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.
[0189] 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. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT
application WO95/251116; Shalon, D. et al. (1995) PCT application
WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA
94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No.
5,605,662.)
[0190] In another embodiment of the invention, nucleic acid
sequences encoding EVL1 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 P1 constructions, or single chromosome cDNA
libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;
and Trask, B. J. (1991) Trends Genet. 7:149-154.)
[0191] 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 EVL1 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.
[0192] 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 (AT) 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.
[0193] In another embodiment of the invention, EVL1, 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 EVL1 and the agent being tested may be
measured.
[0194] 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 WO84/03564.) 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 EVL1, or fragments thereof, and washed. Bound EVL1 is then
detected by methods well known in the art. Purified EVL1 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.
[0195] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding EVL1 specifically compete with a test compound for binding
EVL1. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
EVL1.
[0196] In additional embodiments, the nucleotide sequences which
encode EVL1 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.
[0197] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0198] I. HEAONOT03 cDNA Library Construction
[0199] The HEAONOT03 cDNA library was constructed from normal aorta
tissue obtained from a 27-year-old Caucasian female who died from
intracranial bleeding.
[0200] The frozen tissue was homogenized and lysed in TRIZOL
reagent (1 g tissue/10 ml TRIZOL), a monophasic solution of phenol
and guanidine isothiocyanate, using a POLYTRON PT-3000 homogenizer
(Brinkmann Instruments, Westbury, N.Y.). After a brief incubation
on ice, chloroform was added (1:5 v/v) and the lysate was
centrifuged. The upper aqueous layer was removed to a fresh tube
and the RNA precipitated with isopropanol, resuspended in
DEPC-treated water, and treated with DNAse for 25 min at 37.degree.
C. The RNA was extracted and precipitated as described before. The
mRNA was then isolated using the OLIGOTEX mRNA purification kit
(QIAGEN, Inc., Chatsworth, Calif.) and used to construct the cDNA
library.
[0201] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system (GIBCO BRL). cDNA synthesis was
initiated with a Not I-oligo-d(T) primer. Double-stranded cDNA was
blunted, ligated to EcoR I adaptors, digested with Not I,
fractionated on a SEPHAROSE CL4B column (Pharmacia), and those
cDNAs exceeding 400 bp were ligated into the Not I and EcoR I sites
of the pINCY 1 vector (Incyte). The plasmid pINCY 1 was
subsequently transformed into DH5 a competent cells (GIBCO
BRL).
[0202] II. Isolation and Sequencing of cDNA Clones
[0203] Plasmid DNA was released from the cells and purified using
the R.E.A.L. PREP 96 plasmid kit (QIAGEN, Inc.). The recommended
protocol was employed except for the following changes: 1) the
bacteria were cultured in 1 ml of sterile Terrific Broth (GIBCO
BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after
inoculation, the cultures were incubated for 19 hours and at the
end of incubation, the cells were lysed with 0.3 ml of lysis
buffer; and 3) following isopropanol precipitation, the plasmid DNA
pellet was resuspended in 0.1 ml of distilled water. After the last
step in the protocol, samples were transferred to a 96-well block
for storage at 4.degree. C.
[0204] The cDNAs were sequenced by the method of Sanger et al.
(1975; J. Mol. Biol. 94:441f), using a MICROLAB 2200 liquid
transfer system (Hamilton, Reno, Nev.) in combination with PTC200
thermal cyclers (M. J. Research, Watertown, Mass.) and ABI 377 DNA
sequencing systems (Perkin Elmer), and the reading frame was
determined.
[0205] III. Homology Searching of cDNA Clones and Their Deduced
Proteins
[0206] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SWISSPROT, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool). (See, e.g., Altschul, S. F. (1993) J. Mol. Evol.
36:290-300; and Altschul, S. F. et al. (1990) J. Mol. Biol.
215:403-410.)
[0207] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms could have been used when dealing with
primary sequence patterns and secondary structure gap penalties.
(See, e.g., Smith, T. et al. (1992) Protein Engineering 5:35-51.)
The sequences disclosed in this application have lengths of at
least 49 nucleotides and have no more than 12% uncalled bases
(where N is recorded rather than A, C, G, or T).
[0208] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-8 for peptides.
[0209] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam). Deduced amino acid sequences from the
same clones were then searched against GenBank functional protein
databases, mammalian (mamp), vertebrate (vrtp), and eukaryote
(eukp), for homology.
[0210] Additionally, sequences identified from cDNA libraries may
be analyzed to identify those gene sequences encoding conserved
protein motifs using an appropriate analysis program, e.g., the
Block 2 Bioanalysis Program (Incyte Corp., Palo Alto, Calif.). This
motif analysis program, based on sequence information contained in
the SWISS PROT database and PROSITE, is a method of determining the
function of uncharacterized proteins translated from genomic or
cDNA sequences. (See, e.g., Bairoch, A. et al. (1997) Nucleic Acids
Res. 25:217-221; and Attwood, T. K. et al. (1997) J. Chem. Inf.
Comput. Sci. 37:417-424.) PROSITE may be used to identify common
functional or structural domains in divergent proteins. The method
is based on weight matrices. Motifs identified by this method are
then calibrated against the SWISSPROT database in order to obtain a
measure of the chance distribution of the matches.
[0211] In another alternative, Hidden Markov models (HMMs) may be
used to find protein domains, each defined by a dataset of proteins
known to have a common biological function. (See, e.g., Pearson, W.
R. and D. J. Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448;
and Smith, T. F. and M. S. Waterman (1981) J. Mol. Biol.
147:195-197.) HMMs were initially developed to examine speech
recognition patterns, but are now being used in a biological
context to analyze protein and nucleic acid sequences as well as to
model protein structure. (See, e.g., Krogh, A. et al. (1994) J.
Mol. Biol. 235:1501-1531; and Collin, M. et al. (1993) Protein Sci.
2:305-314.) HMMs have a formal probabilistic basis and use
position-specific scores for amino acids or nucleotides. The
algorithm continues to incorporate information from newly
identified sequences to increase its motif analysis
capabilities.
[0212] IV. Northern Analysis
[0213] 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 et al., supra, ch. 7; and Ausubel et al.,
supra, ch. 4 and 16.) Analogous computer techniques applying BLAST
are used to search for identical or related molecules in nucleotide
databases such as GenBank or LIFESEQ (Incyte Corp., Palo Alto,
Calif.). This analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or homologous.
[0214] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0215] 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. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0216] The results of northern analysis are reported as a list of
libraries in which the transcript encoding EVL1 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.
[0217] V. Extension of EVL1 Encoding Polynucleotides
[0218] The nucleic acid sequence of Incyte Clone 3089412 was used
to design oligonucleotide primers for extending a partial
nucleotide sequence to full length. 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 4.06 software (National
Biosciences, Plymouth, Minn.), or another appropriate program, to
be about 22 to 30 nucleotides in length, to have a G+C 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.
[0219] 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.
[0220] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
PTC200 thermal cycler (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)
[0221] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low 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 columns
(QIAGEN Inc., Chatsworth, Calif.), and trimmed of overhangs using
Klenow enzyme to facilitate religation and cloning.
[0222] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-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 .mu.l of SOC medium. (See, e.g., Sambrook et al.,
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 et al., supra, Appendix A, p. 1)
containing 2.times. Carb. The following day, several colonies were
randomly picked from each plate and cultured in 150 .mu.l of liquid
LB/2.times. Carb medium placed in an individual well of an
appropriate commercially-available sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and, after dilution
1:10 with water, 5 .mu.l from each sample was transferred into a
PCR array.
[0223] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction 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)
[0224] 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.
[0225] In like manner, the nucleotide sequence of SEQ ID NO:2 is
used to obtain 5' regulatory sequences using the procedure above,
oligonucleotides designed for 5' extension, and an appropriate
genomic library.
[0226] VI. Labeling and Use of Individual Hybridization Probes
[0227] Hybridization probes derived from SEQ ID NO:2 are employed
to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham,
Chicago, Ill.), and T4 polynucleotide kinase (DuPont NEN, Boston,
Mass.). The labeled oligonucleotides are substantially purified
using a SEPHADEX G-25 superfine resin column (Pharmacia &
Upjohn, Kalamazoo, Mich.). An aliquot containing 10.sup.7 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, EcoR I, Pst I, Xba 1,
or Pvu II (DuPont NEN, Boston, Mass.).
[0228] 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.times.saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT AR film (Kodak, Rochester,
N.Y.) is exposed to the blots, hybridization patterns are compared
visually.
[0229] VII. Microarrays
[0230] 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 et al., 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.
[0231] 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 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.
[0232] VIII. Complementary Polynucleotides
[0233] Sequences complementary to the EVL1-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring EVL1. 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 4.06 software and the coding sequence of EVL1.
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 EVL1-encoding transcript.
[0234] IX. Expression of EVL1
[0235] Expression of EVL1 is accomplished by subcloning the cDNA
into an appropriate vector and transforming the vector into host
cells. This vector contains an appropriate promoter, e.g.,
.beta.-galactosidase upstream of the cloning site, operably
associated with the cDNA of interest. (See, e.g., Sambrook et al.,
supra, pp. 404-433; and Rosenberg, M. et al. (1983) Methods
Enzymol. 101: 123-138.)
[0236] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopyranoside (IPTG) using standard
methods produces a fusion protein which consists of the first 8
residues of .beta.-galactosidase, about 5 to 15 residues of linker,
and the full length protein. The signal residues direct the
secretion of EVL1 into bacterial growth media which can be used
directly in the following assay for activity.
[0237] X. Demonstration of EVL1 Activity
[0238] The assay for human ena/VASP-like protein splice variant
(EVL1) is based upon the binding affinity of mouse ena/VASP for the
neural protein FE65. (Ermekova et al., supra.) Monkey cell line
COS-7 (ATCC CRL 1651) cells are grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin mixture in 5% CO.sub.2 atmosphere at
37.degree. C. 3.times.10.sup.6 cells are transfected by
electroporation with 20 .mu.g of CMV-hemagglutinin-FE65 plasmid and
20 .mu.g of CMV-EVL1 plasmid as known in the art. 62 h after the
transfection, the cells are harvested in ice-cold PBS and
centrifuged at 2000 rpm at 4.degree. C., and the pellet is
dissolved in lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1
mM sodium vanadate; 50 mM NaF, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, and 10 .mu.g/ml each of aprotinin,
leupeptin, and pepstatin). The extracts are clarified by
centrifugation at 16,000.times.g at 4.degree. C., and 4 mg of
supernatant are incubated for 1 h at 4.degree. C. with an
anti-hemagglutinin monoclonal antibody or with an unrelated
monoclonal antibody. Thereafter, 30 .mu.l of protein A-SEPHAROSE
resin (Pharmacia) are added to each sample of the extract-antibody
mixture, and the immunocomplexes are eluted with 50 mM Tris-HCl pH
6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol, 0.01% bromphenol
blue. The proteins are resolved by 7.5% SDS-PAGE and transferred to
IMMOBILON-P membranes (Millipore). The filter is blocked in 5%
nonfat dry milk in Tris-buffered saline, 0.5% Tween buffer (TBS-T)
and incubated with anti-EVL1 antibodies at 1:1000 dilution for 1 h
at room temperature. After washing in TBS-T, the filter is exposed
to horseradish peroxidase-conjugated protein A (Amersham Corp.) at
a dilution of 1:5000 for 30 min at room temperature. The signals
are detected by chemiluminescence using the ECL system (Amersham
Corp.). The signal response is proportional to the activity of EVL1
in the preparation.
[0239] XI. Production of EVL1 Specific Antibodies
[0240] EVL1 substantially purified using PAGE electrophoresis (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. The EVL1 amino acid
sequence is analyzed using DNASTAR 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 et al., supra, ch. 11.)
[0241] Typically, the oligopeptides are 15 residues in length, and
are synthesized using an ABI 431A peptide synthesizer (Perkin
Elmer) 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 et al.,
supra.) Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity, for example, by binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio-iodinated goat anti-rabbit
IgG.
[0242] XII. Purification of Naturally Occurring EVL1 Using Specific
Antibodies
[0243] Naturally occurring or recombinant EVL1 is substantially
purified by immunoaffinity chromatography using antibodies specific
for EVL1. An immunoaffinity column is constructed by covalently
coupling anti-EVL1 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.
[0244] Media containing EVL1 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of EVL1 (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/EVL1 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 EVL1 is collected.
[0245] XIII. Identification of Molecules Which Interact with
EVL1
[0246] EVL1, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton, A. E. and
W. M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated
with the labeled EVL1, washed, and any wells with labeled EVL1
complex are assayed. Data obtained using different concentrations
of EVL1 are used to calculate values for the number, affinity, and
association of EVL1 with the candidate molecules.
[0247] 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.
Sequence CWU 1
1
4 1 418 PRT Homo sapiens misc_feature Incyte ID No 3089412 1 Met
Ala Thr Ser Glu Gln Ser Ile Cys Gln Ala Arg Ala Ser Val 1 5 10 15
Met Val Tyr Asp Asp Thr Ser Lys Lys Trp Val Pro Ile Lys Pro 20 25
30 Gly Gln Gln Gly Phe Ser Arg Ile Asn Ile Tyr His Asn Thr Ala 35
40 45 Ser Asn Thr Phe Arg Val Val Gly Val Lys Leu Gln Asp Gln Gln
50 55 60 Val Val Ile Asn Tyr Ser Ile Val Lys Gly Leu Lys Tyr Asn
Gln 65 70 75 Ala Thr Pro Thr Phe His Gln Trp Arg Asp Ala Arg Gln
Val Tyr 80 85 90 Gly Leu Asn Phe Ala Ser Lys Glu Glu Ala Thr Thr
Phe Ser Asn 95 100 105 Ala Met Leu Phe Ala Leu Asn Ile Met Asn Ser
Gln Glu Gly Gly 110 115 120 Pro Ser Ser Gln Arg Gln Val Gln Asn Gly
Pro Ser Pro Asp Glu 125 130 135 Met Asp Ile Gln Arg Arg Gln Val Met
Glu Gln His Gln Gln Gln 140 145 150 Arg Gln Glu Ser Leu Glu Arg Arg
Thr Ser Ala Thr Gly Pro Ile 155 160 165 Leu Pro Pro Gly His Pro Ser
Ser Ala Ala Ser Ala Pro Val Ser 170 175 180 Cys Ser Gly Pro Pro Pro
Pro Pro Pro Pro Leu Val Pro Pro Pro 185 190 195 Pro Thr Gly Ala Thr
Pro Pro Pro Pro Pro Pro Leu Pro Ala Gly 200 205 210 Gly Ala Gln Gly
Ser Ser His Asp Glu Ser Ser Met Ser Gly Leu 215 220 225 Ala Ala Ala
Ile Ala Gly Ala Lys Leu Arg Arg Val Gln Arg Pro 230 235 240 Glu Asp
Ala Ser Gly Gly Ser Ser Pro Ser Gly Thr Ser Lys Ser 245 250 255 Asp
Ala Asn Arg Ala Ser Ser Gly Gly Gly Gly Gly Gly Leu Met 260 265 270
Glu Glu Met Asn Lys Leu Leu Ala Lys Arg Arg Lys Ala Ala Ser 275 280
285 Gln Ser Asp Lys Pro Ala Glu Lys Lys Glu Asp Glu Ser Gln Met 290
295 300 Glu Asp Pro Ser Thr Ser Pro Ser Pro Gly Thr Arg Ala Ala Ser
305 310 315 Gln Pro Pro Asn Ser Ser Glu Ala Gly Arg Lys Pro Trp Glu
Arg 320 325 330 Ser Asn Ser Val Glu Lys Pro Val Ser Ser Ile Leu Ser
Arg Thr 335 340 345 Pro Ser Val Ala Lys Ser Pro Glu Ala Lys Ser Pro
Leu Gln Ser 350 355 360 Gln Pro His Ser Arg Met Lys Pro Ala Gly Ser
Val Asn Asp Met 365 370 375 Ala Leu Asp Ala Phe Asp Leu Asp Arg Met
Lys Gln Glu Ile Leu 380 385 390 Glu Glu Val Val Arg Glu Leu His Lys
Val Lys Glu Glu Ile Ile 395 400 405 Asp Ala Ile Arg Gln Glu Leu Ser
Gly Ile Ser Thr Thr 410 415 2 1889 DNA Homo sapiens misc_feature
Incyte ID No 3089412 2 tttaagtagg ctataaaaat caagttgctg tcttcagagg
gtctgtggtc ctctgatcaa 60 cataggctgg tgggagtaca ggactcgcct
cctcagggtt ccctgtgctg ccacttttca 120 gccatggcca caagtgaaca
gagtatctgc caagcccggg cttccgtgat ggtctacgat 180 gacaccagta
agaaatgggt accaatcaaa cctggccagc agggattcag ccggatcaac 240
atctaccaca acactgccag caacaccttc agagtcgttg gagtcaagtt gcaggatcag
300 caggttgtga tcaattattc aatcgtgaaa gggctgaagt acaatcaggc
cacgccaacc 360 ttccaccagt ggcgagatgc ccgccaggtc tacggcttaa
actttgcaag taaagaagag 420 gcaaccacgt tctccaatgc aatgctgttt
gccctgaaca tcatgaattc ccaagaagga 480 ggcccctcca gccagcgtca
ggtgcagaat ggcccctctc ctgatgagat ggacatccag 540 agaagacaag
tgatggagca gcaccagcag cagcgtcagg aatctctaga aagaagaacc 600
tcggccacag ggcccatcct cccaccagga catccttcat ctgcagccag cgcccccgtc
660 tcatgtagtg ggcctccacc gcccccccca cctctagtcc cacctccacc
cactggggct 720 accccacctc ccccaccccc actgccagcc ggaggagccc
aggggtccag ccacgacgag 780 agctccatgt caggactggc cgctgccata
gctggggcca agctgagaag agtccaacgg 840 ccagaagacg catctggagg
ctccagtccc agtgggacct caaagtccga tgccaaccgg 900 gcaagcagcg
ggggtggcgg aggaggcctc atggaggaaa tgaacaaact gctggccaag 960
aggagaaaag cagcctccca gtcagacaag ccagccgaga agaaggaaga tgaaagccaa
1020 atggaagatc ctagtacctc cccctctccg gggacccgag cagccagcca
gccacctaac 1080 tcctcagagg ctggccggaa gccctgggag cggagcaact
cggtggagaa gcctgtgtcc 1140 tcgattctgt ccagaacccc gtctgtggca
aagagccccg aagctaagag cccccttcag 1200 tcgcagcctc actctaggat
gaagcctgct gggagcgtga atgacatggc cctggatgcc 1260 ttcgacttgg
accggatgaa gcaggagatc ctagaggagg tggtgagaga gctccacaag 1320
gtgaaggagg agatcatcga cgccatcagg caggagctga gtgggatcag caccacgtaa
1380 ggggccggcc tcgctgcgct gattcgtcga gcccatccgg cgacagagga
cagccagaag 1440 cccagccagc cccagactcc agtgcaccag agcacgcaca
ggagcctggg cgcgctgctg 1500 tgaaacgtcc tgacctgtga tcacacatga
cagtgaggaa accaagtgca actcctgggt 1560 tttttttaga ttctgcctga
cacggaacac caggtctgct cgtctttttt gtgttttata 1620 tttgcttatt
taaggtacat ttctttgggt ttctagagac gcccctaagt cacctgcttc 1680
attagacggt ttccaggttt tctcccaggt gacgctgtta gcgcctcagc tggcggtgac
1740 agccggccca gcgtggcgcc accacacacc gcagagctgt ccaggcacag
ctccgtcccc 1800 agcgctcatg gtgttgaaac tgtctgtcat gcaccacggt
gtctgtgtcc acacagtaat 1860 aaacggttta ctgtccgcaa aaaaaaaaa 1889 3
393 PRT Homo sapiens misc_feature GenBank ID No g1644453 3 Met Ser
Glu Gln Ser Ile Cys Gln Ala Arg Ala Ser Val Met Val 1 5 10 15 Tyr
Asp Asp Thr Ser Lys Lys Trp Val Pro Ile Lys Pro Gly Gln 20 25 30
Gln Gly Phe Ser Arg Ile Asn Ile Tyr His Asn Thr Ala Ser Ser 35 40
45 Thr Phe Arg Val Val Gly Val Lys Leu Gln Asp Gln Gln Val Val 50
55 60 Ile Asn Tyr Ser Ile Val Lys Gly Leu Lys Tyr Asn Gln Ala Thr
65 70 75 Pro Thr Phe His Gln Trp Arg Asp Ala Arg Gln Val Tyr Gly
Leu 80 85 90 Asn Phe Ala Ser Lys Glu Glu Ala Thr Thr Phe Ser Asn
Ala Met 95 100 105 Leu Phe Ala Leu Asn Ile Met Asn Ser Gln Glu Gly
Gly Pro Ser 110 115 120 Thr Gln Arg Gln Val Gln Asn Gly Pro Ser Pro
Glu Glu Met Asp 125 130 135 Ile Gln Arg Arg Gln Val Met Glu Gln Gln
His Arg Gln Glu Ser 140 145 150 Leu Glu Arg Arg Ile Ser Ala Thr Gly
Pro Ile Leu Pro Pro Gly 155 160 165 His Pro Ser Ser Ala Ala Ser Thr
Thr Leu Ser Cys Ser Gly Pro 170 175 180 Pro Pro Pro Pro Pro Pro Pro
Val Pro Pro Pro Pro Thr Gly Ser 185 190 195 Thr Pro Pro Pro Pro Pro
Pro Leu Pro Ala Gly Gly Ala Gln Gly 200 205 210 Thr Asn His Asp Glu
Ser Ser Ala Ser Gly Leu Ala Ala Ala Leu 215 220 225 Ala Gly Ala Lys
Leu Arg Arg Val Gln Arg Pro Glu Asp Ala Ser 230 235 240 Gly Gly Ser
Ser Pro Ser Gly Thr Ser Lys Ser Asp Ala Asn Arg 245 250 255 Ala Ser
Ser Gly Gly Gly Gly Gly Gly Leu Met Glu Glu Met Asn 260 265 270 Lys
Leu Leu Ala Lys Arg Arg Lys Ala Ala Ser Gln Thr Asp Lys 275 280 285
Pro Ala Asp Arg Lys Glu Asp Glu Ser Gln Thr Glu Asp Pro Ser 290 295
300 Thr Ser Pro Ser Pro Gly Thr Arg Ala Thr Ser Gln Pro Pro Asn 305
310 315 Ser Ser Glu Ala Gly Arg Lys Pro Trp Glu Arg Ser Asn Ser Val
320 325 330 Glu Lys Pro Val Ser Ser Leu Leu Ser Arg Val Lys Pro Ala
Gly 335 340 345 Ser Val Asn Asp Val Gly Leu Asp Ala Leu Asp Leu Asp
Arg Met 350 355 360 Lys Gln Glu Ile Leu Glu Glu Val Val Arg Glu Leu
His Lys Val 365 370 375 Lys Glu Glu Ile Ile Asp Ala Ile Arg Gln Glu
Leu Ser Gly Ile 380 385 390 Ser Thr Thr 4 380 PRT Homo sapiens
misc_feature GenBank ID No g624964 4 Met Ser Glu Thr Val Ile Cys
Ser Ser Arg Ala Thr Val Met Leu 1 5 10 15 Tyr Asp Asp Gly Asn Lys
Arg Trp Leu Pro Ala Gly Thr Gly Pro 20 25 30 Gln Ala Phe Ser Arg
Val Gln Ile Tyr His Asn Pro Thr Ala Asn 35 40 45 Ser Phe Arg Val
Val Gly Arg Lys Met Gln Pro Asp Gln Gln Val 50 55 60 Val Ile Asn
Cys Ala Ile Val Arg Gly Val Lys Tyr Asn Gln Ala 65 70 75 Thr Pro
Asn Phe His Gln Trp Arg Asp Ala Arg Gln Val Trp Gly 80 85 90 Leu
Asn Phe Gly Ser Lys Glu Asp Ala Ala Gln Phe Ala Ala Gly 95 100 105
Met Ala Ser Ala Leu Glu Ala Leu Glu Gly Gly Gly Pro Pro Pro 110 115
120 Pro Pro Ala Leu Pro Thr Trp Ser Val Pro Asn Gly Pro Ser Pro 125
130 135 Glu Glu Val Glu Gln Gln Lys Arg Gln Gln Pro Gly Pro Ser Glu
140 145 150 His Ile Glu Arg Arg Val Ser Asn Ala Gly Gly Pro Pro Ala
Pro 155 160 165 Pro Ala Gly Gly Pro Pro Pro Pro Pro Gly Pro Pro Pro
Pro Pro 170 175 180 Gly Pro Pro Pro Pro Pro Gly Leu Pro Pro Ser Gly
Val Pro Ala 185 190 195 Ala Ala His Gly Ala Gly Gly Gly Pro Pro Pro
Ala Pro Pro Leu 200 205 210 Pro Ala Ala Gln Gly Pro Gly Gly Gly Gly
Ala Gly Ala Pro Gly 215 220 225 Leu Ala Ala Ala Ile Ala Gly Ala Lys
Leu Arg Lys Val Ser Lys 230 235 240 Gln Glu Glu Ala Ser Gly Gly Pro
Thr Ala Pro Lys Ala Glu Ser 245 250 255 Gly Arg Ser Gly Gly Gly Gly
Leu Met Glu Glu Met Asn Ala Met 260 265 270 Leu Ala Arg Arg Arg Lys
Ala Thr Gln Val Gly Glu Lys Thr Pro 275 280 285 Lys Asp Glu Ser Ala
Asn Gln Glu Glu Pro Glu Ala Arg Val Pro 290 295 300 Ala Gln Ser Glu
Ser Val Arg Arg Pro Trp Glu Lys Asn Ser Thr 305 310 315 Thr Leu Pro
Arg Met Lys Ser Ser Ser Ser Val Thr Thr Ser Glu 320 325 330 Thr Gln
Pro Cys Thr Pro Ser Ser Ser Asp Tyr Ser Asp Leu Gln 335 340 345 Arg
Val Lys Gln Glu Leu Leu Glu Glu Val Lys Lys Glu Leu Gln 350 355 360
Lys Val Lys Glu Glu Ile Ile Glu Ala Phe Val Gln Glu Leu Arg 365 370
375 Lys Arg Gly Ser Pro 380
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