U.S. patent application number 10/458162 was filed with the patent office on 2003-11-06 for kinesin-like motor protein.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Corley, Neil C., Guegler, Karl J., Patterson, Chandra, Tang, Y. Tom.
Application Number | 20030207318 10/458162 |
Document ID | / |
Family ID | 22585352 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207318 |
Kind Code |
A1 |
Tang, Y. Tom ; et
al. |
November 6, 2003 |
Kinesin-like motor protein
Abstract
The invention provides a human kinesin-like motor protein
(KLIMP) and polynucleotides which identify and encode KLIMP. The
invention also provides expression vectors, host cells, antibodies,
agonists, and antagonists. The invention also provides methods for
diagnosing, treating or preventing disorders associated with
expression of KLIMP.
Inventors: |
Tang, Y. Tom; (San Jose,
CA) ; Corley, Neil C.; (Castro Valley, CA) ;
Guegler, Karl J.; (Menlo Park, CA) ; Patterson,
Chandra; (San Jose, 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: |
22585352 |
Appl. No.: |
10/458162 |
Filed: |
June 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10458162 |
Jun 9, 2003 |
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09847874 |
May 2, 2001 |
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09847874 |
May 2, 2001 |
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09467946 |
Dec 21, 1999 |
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6248594 |
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09467946 |
Dec 21, 1999 |
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09162373 |
Sep 28, 1998 |
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6013454 |
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Current U.S.
Class: |
435/6.11 ;
435/183; 435/320.1; 435/325; 435/69.1; 514/1.2; 514/17.7; 514/19.3;
530/388.26; 536/23.2 |
Current CPC
Class: |
A61P 25/18 20180101;
A61P 21/04 20180101; A61P 25/22 20180101; A61P 31/04 20180101; A61P
25/16 20180101; A61P 37/02 20180101; A61P 21/00 20180101; A61P 9/08
20180101; A61P 1/04 20180101; A61P 25/02 20180101; A61P 35/00
20180101; C07K 14/47 20130101; A61P 3/10 20180101; A61P 37/08
20180101; A61P 25/28 20180101; A61P 13/00 20180101; A61P 27/02
20180101; A61P 17/00 20180101; A61P 5/18 20180101; A61P 25/00
20180101; A61P 3/06 20180101; A61P 25/14 20180101; A61P 31/00
20180101; A61P 31/18 20180101; A61P 5/16 20180101; A61P 29/02
20180101; A61P 19/00 20180101; A61P 25/08 20180101; A61P 19/02
20180101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/183; 435/320.1; 435/325; 530/388.26; 536/23.2; 514/12 |
International
Class: |
C12Q 001/68; C12P
021/02; C12N 005/06; C07K 014/47; C07H 021/04; C12N 009/00 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence of SEQ ID NO: 1,
b) a polypeptide comprising a naturally occurring an amino acid
sequence at least 90% identical to an amino acid sequence of SEQ ID
NO:1, c) a biologically active fragment of a polypeptide having an
amino acid sequence of SEQ ID NO:1, and d) an immunogenic fragment
of a polypeptide having an amino acid sequence of SEQ ID NO:1.
2. An isolated polypeptide of claim 1, comprising an 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 a
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 a polynucleotide sequence of SEQ
ID NO:2, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to a 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 an
amino acid sequence of SEQ ID NO:1.
19. A method for treating a disease or condition associated with
decreased expression of functional KLIMP, 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 KLIMP, 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 KLIMP, 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, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) contacting a sample comprising the target
polynucleotide with, 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 KLIMP 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 II and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of KLIMP 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 KLIMP 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 an amino acid
sequence of SEQ ID NO:1, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibodies
from said animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide comprising an 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 an amino acid sequence
of SEQ ID NO:1, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibody
producing cells from the animal, c) fusing the antibody producing
cells with immortalized cells to form monoclonal antibody-producing
hybridoma cells, d) culturing the hybridoma cells, and e) isolating
from the culture monoclonal antibody which binds specifically to a
polypeptide comprising an amino acid sequence of SEQ ID NO:1.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the monoclonal 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 an amino acid
sequence of SEQ ID NO:1 in a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide comprising an amino acid sequence of
SEQ ID NO:1 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence of SEQ ID NO:1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) separating the antibody from the sample and obtaining the
purified polypeptide comprising an amino acid sequence of SEQ ID
NO:1.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method 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 continuation application of U.S.
application Ser. No. 09/847,874, filed May 2, 2001, which is a
divisional application of U.S. application Ser. No. 09/467,946,
filed Dec. 21, 1999, issued Jun. 19, 2001 as U.S. Pat. No.
6,248,594, which is a divisional application of U.S. application
Ser. No. 09/162,373, filed Sep. 28, 1998, issued Jan. 11, 2000, as
U.S. Pat. No. 6,013,454, all entitled KINESIN-LIKE MOTOR PROTEIN,
all of which are hereby expressly incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a kinesin-like motor protein and to the use of these
sequences in the diagnosis, treatment, and prevention of cancer,
neurological disorders, and disorders of vesicular transport.
BACKGROUND OF THE INVENTION
[0003] Translocation of components within the cell is critical for
maintaining cell structure and function. Cellular components such
as proteins and membrane-bound organelles are transported along
well-defined routes to specific subcellular compartments.
Intracellular transport mechanisms utilize microtubules which are
filamentous polymers that serve as tracks for directing the
movement of molecules. Molecular transport is driven by the
microtubule-based motor proteins, kinesin and dynein. These
proteins use the energy derived from ATP hydrolysis to power their
movement unidirectionally along microtubules and to transport
molecular cargo to specific destinations.
[0004] Kinesin defines a ubiquitous, conserved family of over 50
proteins that can be classified into at least 8 subfamilies based
on primary amino acid sequence, domain structure, velocity of
movement, and cellular function. (Reviewed in Moore, J. D. and
Endow, S. A. (1996) Bioessays 18:207-219; and Hoyt, A. M. (1994)
Curr. Opin. Cell Biol. 6:63-68.) The prototypical kinesin molecule
is involved in the transport of membrane-bound vesicles and
organelles. This function is particularly important for axonal
transport in neurons. Protein-containing vesicles are constantly
transported from the neuronal cell body along microtubules that
span the length of the axon leading to the synaptic terminal.
Failure to supply the synaptic terminal with these vesicles blocks
the transmission of neural signals. In the fruit fly Drosophila
melanogaster, for example, mutations in kinesin cause severe
disruption of axonal transport in larval nerves which leads to
progressive paralysis (Hurd, D. D. and Saxton, W. M. (1996)
Genetics 144:1075-1085). This phenotype mimics the pathology of
some vertebrate motor neuron diseases, such as amyotrophic lateral
sclerosis (ALS). In addition to axonal transport, kinesin is also
important in all cell types for the transport of vesicles from the
Golgi complex to the endoplasmic reticulum. This role is critical
for maintaining the identity and functionality of these secretory
organelles.
[0005] Members of the more divergent subfamilies of kinesin are
called kinesin-related proteins (KRPs), many of which function
during mitosis in eukaryotes as divergent as yeast and human (Hoyt,
supra). Some KRPs are required for assembly of the mitotic spindle.
In vivo and in vitro analyses suggest that these KRPs exert force
on microtubules that comprise the mitotic spindle, resulting in the
separation of spindle poles. Phosphorylation of KRP is required for
this activity. Failure to assemble the mitotic spindle results in
abortive mitosis and chromosomal aneuploidy, the latter condition
being characteristic of cancer cells. In addition, a unique KRP,
centromere protein E, localizes to the kinetochore of human mitotic
chromosomes and may play a role in their segregation to opposite
spindle poles.
[0006] The prototypical kinesin molecule is a heterotetramer
comprised of two heavy polypeptide chains (KHCs) and two light
polypeptide chains (KLCs). The KHC subunits are typically referred
to as "kinesin." KHC is about 1000 amino acids in length, and KLC
is about 550 amino acids in length. Two KHCs dimerize to form a
rod-shaped molecule with three distinct regions of secondary
structure. At one end of the molecule is a globular motor domain
that functions in ATP hydrolysis and microtubule binding. Kinesin
motor domains are highly conserved and share over 70% identity.
Beyond the motor domain is an .alpha.-helical coiled-coil region
which mediates dimerization. At the other end of the molecule is a
fan-shaped tail that associates with molecular cargo. The tail is
formed by the interaction of the KHC C-termini with the two
KLCs.
[0007] The nematode Unc-104 kinesin-like protein defines a distinct
kinesin subfamily whose members may function monomerically (Moore
and Endow, supra). Members of this subfamily are important for
synaptic transport and mitochondrial translocation and are
characterized by movement at relatively high velocities of about 40
to 90 microns/minute. Nematodes with mutations in the Unc-104 gene
exhibit defects in locomotion and feeding behaviors, and at the
molecular level, in synaptic vesicle transport.
[0008] The discovery of a new kinesin-like motor protein and the
polynucleotides encoding it satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of cancer, neurological disorders, and
disorders of vesicular transport.
SUMMARY OF THE INVENTION
[0009] The invention is based on the discovery of a new human
kinesin-like motor protein (KLIMP), the polynucleotides encoding
KLIMP, and the use of these compositions for the diagnosis,
treatment, or prevention of cancer, neurological disorders, and
disorders of vesicular transport.
[0010] 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.
[0011] The invention further provides a substantially purified
variant having at least 90% amino acid sequence identity to the
amino acid 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 80%
polynucleotide sequence identity to the polynucleotide encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1.
[0012] 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 having a sequence which is
complementary to the polynucleotide encoding the polypeptide
comprising the amino acid sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1.
[0013] The invention also provides an isolated and purified
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified
polynucleotide variant having at least 80% polynucleotide sequence
identity to the polynucleotide comprising the polynucleotide
sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention
also provides an isolated and purified polynucleotide having a
sequence complementary to the polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID
NO:2.
[0014] The invention also provides a method for detecting a
polynucleotide in a sample containing nucleic acids, the method
comprising the steps of (a) hybridizing the complement of the
polynucleotide sequence to at least one of the polynucleotides of
the 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 in the sample. In one aspect, the method further
comprises amplifying the polynucleotide prior to hybridization.
[0015] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising the 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.
[0016] The invention also provides a method for producing a
polypeptide, the method comprising the steps of: (a) culturing the
host cell containing an expression vector containing at least a
fragment of a polynucleotide under conditions suitable for the
expression of the polypeptide; and (b) recovering the polypeptide
from the host cell culture.
[0017] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the sequence
of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a
suitable pharmaceutical carrier.
[0018] The invention further includes a purified antibody which
binds to a polypeptide comprising 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.
[0019] The invention also provides a method for treating or
preventing a disorder associated with decreased expression or
activity of KLIMP, the method comprising administering to a subject
in need of such treatment an effective amount of a pharmaceutical
composition comprising a substantially purified polypeptide having
the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID
NO:1, in conjunction with a suitable pharmaceutical carrier.
[0020] The invention also provides a method for treating or
preventing a disorder associated with increased expression or
activity of KLIMP, the method comprising administering to a subject
in need of such treatment an effective amount of an antagonist of
the polypeptide having the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
[0021] FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J show the
amino acid sequence (SEQ ID NO:1) and nucleic acid sequence (SEQ ID
NO:2) of KLIMP. The alignment was produced using MACDNASIS PRO
software (Hitachi Software Engineering, S. San Francisco
Calif.).
[0022] FIG. 2 shows electronic northern analysis of SEQ ID NO:2
using the LIFESEQ sequence database (Incyte Pharmaceuticals, Palo
Alto Calif.).
[0023] Table 1 shows the programs, their descriptions, references,
and threshold parameters used to analyze KLIMP.
DESCRIPTION OF THE INVENTION
[0024] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular machines, materials and methods
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.
[0025] 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.
[0026] 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 machines, materials, and methods similar or equivalent to those
described herein can be used to practice or test the present
invention, the preferred machines, materials and methods are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, protocols,
reagents and vectors 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.
[0027] Definitions
[0028] "KLIMP" refers to the amino acid sequences of substantially
purified KLIMP 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.
[0029] The term "agonist" refers to a molecule which, when bound to
KLIMP, increases or prolongs the duration of the effect of KLIMP.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules which bind to and modulate the effect of KLIMP.
[0030] An "allelic variant" is an alternative form of the gene
encoding KLIMP. Allelic variants may result from at least one
mutation in the nucleic acid sequence and may result in altered
mRNAs or in polypeptides whose structure or function may or may not
be altered. Any given natural or recombinant gene may have none,
one, or many allelic forms. Common mutational changes which give
rise to allelic variants are generally ascribed to natural
deletions, additions, or substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the
others, one or more times in a given sequence.
[0031] "Altered" nucleic acid sequences encoding KLIMP include
those sequences with deletions, insertions, or substitutions of
different nucleotides, resulting in a polypeptide the same as KLIMP
or a polypeptide with at least one functional characteristic of
KLIMP. Included within this definition are polymorphisms which may
or may not be readily detectable using a particular oligonucleotide
probe of the polynucleotide encoding KLIMP, and improper or
unexpected hybridization to allelic variants, with a locus other
than the normal chromosomal locus for the polynucleotide sequence
encoding KLIMP. 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 KLIMP. 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 KLIMP 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.
[0032] The terms "amino acid" or "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. In this context, "fragments," "immunogenic fragments,"
or "antigenic fragments" refer to fragments of KLIMP which are
preferably at least 5 to about 15 amino acids in length, most
preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of KLIMP. Where
"amino acid sequence" is recited to refer to an amino acid sequence
of a naturally occurring protein molecule, "amino acid sequence"
and like terms are not meant to limit the amino acid sequence to
the complete native amino acid sequence associated with the recited
protein molecule.
[0033] "Amplification" relates to the production of additional
copies of a nucleic acid sequence. Amplification is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art.
[0034] The term "antagonist" refers to a molecule which, when bound
to KLIMP, decreases the amount or the duration of the effect of the
biological or immunological activity of KLIMP. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of KLIMP.
[0035] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fab, F(ab').sub.2, and Fv fragments,
which are capable of binding the epitopic determinant. Antibodies
that bind KLIMP polypeptides can be prepared using intact
polypeptides or using fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can
be derived from the translation of RNA, or synthesized chemically,
and can be conjugated to a carrier protein if desired. Commonly
used carriers that are chemically coupled to peptides include
bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0036] The term "antigenic determinant" 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.
[0037] The term "antisense" refers to any composition containing a
nucleic acid sequence which is complementary to the "sense" strand
of a specific nucleic acid sequence. Antisense molecules may be
produced by any method including synthesis or transcription. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0038] The term "biologically active," refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" refers to
the capability of the natural, recombinant, or synthetic KLIMP, or
of any oligopeptide thereof, to induce a specific immune response
in appropriate animals or cells and to bind with specific
antibodies.
[0039] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence "5' A-G-T 3'" bonds to the complementary sequence "3'
T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial," such that only some of the nucleic acids bind, or
it may be "complete," such that total complementarity exists
between the single stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of the hybridization between
the nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands, and in the design and use of peptide nucleic acid
(PNA) molecules.
[0040] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence" refer
broadly to any composition containing the given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation
or an aqueous solution. Compositions comprising polynucleotide
sequences encoding KLIMP or fragments of KLIMP may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g.,
sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.). "Consensus
sequence" refers to a nucleic acid sequence which has been
resequenced to resolve uncalled bases, extended using XL-PCR kit
(Perkin-Elmer, Norwalk Conn.) in the 5' and/or the 3' direction,
and resequenced, or which has been assembled from the overlapping
sequences of more than one Incyte Clone using 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.
[0041] 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 KLIMP, by
northern analysis is indicative of the presence of nucleic acids
encoding KLIMP in a sample, and thereby correlates with expression
of the transcript from the polynucleotide encoding KLIMP.
[0042] A "deletion" refers to a change in the amino acid or
nucleotide sequence that results in the absence of one or more
amino acid residues or nucleotides.
[0043] The term "derivative" refers to the chemical modification of
a polypeptide sequence, or a polynucleotide sequence. Chemical
modifications of a polynucleotide sequence can include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A derivative polynucleotide encodes a polypeptide which retains at
least one biological or immunological function of the natural
molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the polypeptide
from which it was derived.
[0044] The term "similarity" refers to a degree of complementarity.
There may be partial similarity or complete similarity. The word
"identity" may substitute for the word "similarity." A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to as "substantially similar." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or northern blot, solution hybridization, and the like)
under conditions of reduced stringency. A substantially similar
sequence or hybridization probe will compete for and inhibit the
binding of a completely similar (identical) sequence to the target
sequence under conditions of reduced stringency. This is not to say
that conditions of reduced stringency are such that non-specific
binding is permitted, as reduced stringency conditions require that
the binding of two sequences to one another be a specific (i.e., a
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
similarity or identity). In the absence of non-specific binding,
the substantially similar sequence or probe will not hybridize to
the second non-complementary target sequence.
[0045] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MEGALIGN program
(DNASTAR, Madison Wis.) which creates alignments between two or
more sequences according to methods selected by the user, e.g., the
clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988)
Gene 73:237-244.) The clustal algorithm groups sequences into
clusters by examining the distances between all pairs. The clusters
are aligned pairwise and then in groups. The percentage similarity
between two amino acid sequences, e.g., sequence A and sequence B,
is calculated by dividing the length of sequence A, minus the
number of gap residues in sequence A, minus the number of gap
residues in sequence B, into the sum of the residue matches between
sequence A and sequence B, times one hundred. Gaps of low or of no
similarity between the two amino acid sequences are not included in
determining percentage similarity. Percent identity between nucleic
acid sequences can also be counted or calculated by other methods
known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J.
(1990) Methods Enzymol. 183:626-645.) Identity between sequences
can also be determined by other methods known in the art, e.g., by
varying hybridization conditions. "Human artificial chromosomes"
(HACs) are linear microchromosomes which may contain DNA sequences
of about 6 kb to 10 Mb in size, and which contain all of the
elements required for stable mitotic chromosome segregation and
maintenance.
[0046] The term "humanized antibody" refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
[0047] "Hybridization" refers to any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing.
[0048] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary bases. A hybridization complex
may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid sequence present in solution and another
nucleic acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or their nucleic acids have
been fixed).
[0049] The words "insertion" or "addition" refer to changes in an
amino acid or nucleotide sequence resulting in the addition of one
or more amino acid residues or nucleotides, respectively, to the
sequence found in the naturally occurring molecule.
[0050] "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.
[0051] The term "microarray" refers to an arrangement of distinct
polynucleotides on a substrate.
[0052] The terms "element" or "array element" in a microarray
context, refer to hybridizable polynucleotides arranged on the
surface of a substrate.
[0053] The term "modulate" refers to a change in the activity of
KLIMP. For example, modulation may cause an increase or a decrease
in protein activity, binding characteristics, or any other
biological, functional, or immunological properties of KLIMP.
[0054] The phrases "nucleic acid" or "nucleic acid sequence" refer
to a nucleotide, oligonucleotide, polynucleotide, or any fragment
thereof. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which, when translated, would produce polypeptides retaining some
functional characteristic, e.g., antigenicity, or structural domain
characteristic, e.g., ATP-binding site, of the full-length
polypeptide.
[0055] The terms "operably associated" or "operably linked" refer
to functionally related nucleic acid sequences. A promoter is
operably associated or operably linked with a coding sequence if
the promoter controls the translation of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in the same reading frame, certain genetic
elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0056] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to 60 nucleotides, preferably about
15 to 30 nucleotides, and most preferably about 20 to 25
nucleotides, which can be used in PCR amplification or in a
hybridization assay or microarray. "Oligonucleotide" is
substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the
art.
[0057] "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises an oligonucleotide of at least
about 5 nucleotides in length linked to a peptide backbone of amino
acid residues ending in lysine. The terminal lysine confers
solubility to the composition. PNAs preferentially bind
complementary single stranded DNA or RNA and stop transcript
elongation, and may be pegylated to extend their lifespan in the
cell.
[0058] The term "sample" is used in its broadest sense. A sample
suspected of containing nucleic acids encoding KLIMP, or fragments
thereof, or KLIMP 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
substrate; a tissue; a tissue print; etc.
[0059] The terms "specific binding" or "specifically binding" refer
to that interaction between a protein or peptide and an agonist, an
antibody, or an antagonist. The interaction is dependent upon the
presence of a particular structure of the protein, e.g., the
antigenic determinant or epitope, recognized by the binding
molecule. For example, if an antibody is specific for epitope "A,"
the presence of a polypeptide containing the epitope A, or the
presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will reduce the amount of labeled A that binds
to the antibody.
[0060] The term "stringent conditions" refers to conditions which
permit hybridization between polynucleotides and the claimed
polynucleotides. Stringent conditions can be defined by salt
concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0061] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90%
free from other components with which they are naturally
associated.
[0062] A "substitution" refers to the replacement of one or more
amino acids or nucleotides by different amino acids or nucleotides,
respectively.
[0063] "Substrate" refers to any suitable rigid or semi-rigid
support including membranes, filters, chips, slides, wafers,
fibers, magnetic or nonmagnetic beads, gels, tubing, plates,
polymers, microparticles and capillaries. The substrate can have a
variety of surface forms, such as wells, trenches, pins, channels
and pores, to which polynucleotides or polypeptides are bound.
[0064] "Transformation" describes a process by which exogenous DNA
enters and changes a recipient cell. Transformation may occur under
natural or artificial conditions according to various methods well
known in the art, and may rely on any known method for the
insertion of foreign nucleic acid sequences into a prokaryotic or
eukaryotic host cell. The method for transformation is selected
based on the type of host cell being transformed and may include,
but is not limited to, viral infection, electroporation, heat
shock, lipofection, and particle bombardment. The term
"transformed" cells includes stably transformed cells in which the
inserted DNA is capable of replication either as an autonomously
replicating plasmid or as part of the host chromosome, as well as
transiently transformed cells which express the inserted DNA or RNA
for limited periods of time.
[0065] A "variant" of KLIMP polypeptides refers to an amino acid
sequence that is altered by one or more amino acid residues. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software (DNASTAR).
[0066] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to KLIMP. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
MRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) in which the
polynucleotide sequence varies by one base. The presence of SNPs
may be indicative of, for example, a certain population, a disease
state, or a propensity for a disease state.
[0067] The Invention
[0068] The invention is based on the discovery of a new human
kinesin-like motor protein (KLIMP), the polynucleotides encoding
KLIMP, and the use of these compositions for the diagnosis,
treatment, or prevention of cancer, neurological disorders, and
disorders of vesicular transport.
[0069] Nucleic acids encoding the KLIMP of the present invention
were identified in Incyte Clone 1281811 from the colon cDNA library
(COLNNOT16) using a computer search for nucleotide and/or 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 1281827H1 and 1281811H1 (COLNNOT16),
3098665H1 (CERVNOT03), 3402610H1 (ESOGNOT03), 893899X28F1 and
964318X27 (BRSTNOT05), 3386436H1 (LUNGTUT17), 660598X15
(BRAINOT03), 2791542F6 and 2791542T6 (COLNTUT16), 3046745H1
(HEAANOT01), 1988294R6 (LUNGAST01), and 1257207F1 (MENITUT03).
[0070] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J. KLIMP is 1103
amino acids in length and has six potential N-glycosylation sites
at N33, N133, N211, N272, N455, and N851; two potential cAMP- and
cGMP-dependent protein kinase phosphorylation sites at S295 and
S33; eighteen potential casein kinase II phosphorylation sites at
T20, T60, S61, S145, S188, T234, S245, S326, S385, T457, S676,
S684, S694, T743, T806, S852, S919, and S1092; nineteen potential
protein kinase C phosphorylation sites at S5, S165, S214, T226,
S238, S252, S290, T338, S396, T447, S494, S680, S688, S694, S716,
S852, S905, S910, and S1022; and one potential tyrosine kinase
phosphorylation site at Y300. MOTIFS analysis shows that KLIMP
contains an ATP-binding motif from G97 to S104. PROFILESCAN and
PFAM indicate the presence of a kinesin motor domain from R11 to
L377. Within this region, MOTIFS analysis identifies a kinesin
motor domain signature sequence from S242 to E253; BLOCKS analysis
indicates that KLIMP contains five out of eight protein domain
blocks which are characteristic of kinesin motor domains and which
are most closely related to those blocks found in Unc-104; and
PRINTS analysis indicates that KLIMP contains four out of four
protein fingerprints which are likewise characteristic of kinesin
motor domains. A fragment of SEQ ID NO:2 from about nucleotide 1518
to about nucleotide 1547 is useful in hybridization or
amplification technologies to identify SEQ ID NO:2 and to
distinguish between SEQ ID NO:2 and a related sequence.
[0071] Northern analysis shows the expression of this sequence in
various libraries, at least 68% of which are associated with cancer
or cell proliferation. In particular, 27% of the libraries
expressing KLIMP are derived from reproductive tissue and 19% are
derived from neural tissue. FIG. 2 shows the three cDNA libraries
from the LIFESEQ database in which SEQ ID NO:2 is most abundantly
expressed. Abundance refers to the number of times SEQ ID NO:2
appears in each of the libraries listed, and percent abundance
refers to the abundance divided by the total number of sequences
examined in a given library. Of particular note is that these three
cDNA libraries are all derived from neurodegenerative tissue:
MUSCNOT11 is derived from diseased muscle tissue from a patient who
died of amyotrophic lateral sclerosis, BRAYDIT01 is derived from
hypothalamus tissue from a patient with Huntington's disease, and
PONSAZT01 is derived from diseased pons tissue removed from the
brain of a patient who died of Alzheimer's disease.
[0072] The invention also encompasses KLIMP variants. A preferred
KLIMP 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 KLIMP amino acid sequence, and which
contains at least one functional or structural characteristic of
KLIMP.
[0073] The invention also encompasses polynucleotides which encode
KLIMP. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2,
which encodes KLIMP.
[0074] The invention also encompasses a variant of a polynucleotide
sequence encoding KLIMP. 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 KLIMP. 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 KLIMP.
[0075] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding KLIMP, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
sequence that could be made by selecting combinations based on
possible codon choices. These combinations are made in accordance
with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally occurring KLIMP, and all such
variations are to be considered as being specifically
disclosed.
[0076] Although nucleotide sequences which encode KLIMP and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring KLIMP under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding KLIMP or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. Codons may be selected to
increase the rate at which expression of the peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the
frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence
encoding KLIMP and its derivatives without altering the encoded
amino acid sequences include the production of RNA transcripts
having more desirable properties, such as a greater half-life, than
transcripts produced from the naturally occurring sequence.
[0077] The invention also encompasses production of DNA sequences
which encode KLIMP and KLIMP derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding KLIMP or any fragment thereof.
[0078] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:2, or to 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; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.) For example, stringent salt concentration will
ordinarily be less than about 750 mM NaCl and 75 mM trisodium
citrate, preferably less than about 500 mM NaCl and 50 mM trisodium
citrate, and most preferably less than about 250 mM NaCl and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic solvent, e.g., formamide, while high
stringency hybridization can be obtained in the presence of at
least about 35% formamide, and most preferably at least about 50%
formamide. Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0079] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 MM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 420C, and
most preferably of at least about 68.degree. C. In a preferred
embodiment, wash steps will occur at 25.degree. C. in 30 mM NaCl, 3
mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment,
wash steps will occur at 42.degree. C. in 15 mM NaCl, 1.5 mM
trisodium citrate, and 0.1% SDS. In a most preferred embodiment,
wash steps will occur at 68.degree. C. in 15 mM NaCl, 1.5 mM
trisodium citrate, and 0.1% SDS. Additional variations on these
conditions will be readily apparent to those skilled in the
art.
[0080] Methods for DNA sequencing are well known in the art and may
be used to practice any of the embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA
polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq
polymerase (Perkin-Elmer), thermostable T7 polymerase (Amersham
Pharmacia Biotech, Piscataway N.J.), or combinations of polymerases
and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg Md.).
Preferably, sequence preparation is automated with machines such as
the Robbins Hydra microdispenser (Robbins Scientific, Sunnyvale
Calif.), Hamilton MICROLAB 2200 (Hamilton, Reno Nev.), Peltier
thermal cycler 200 (PTC200; MJ Research, Watertown Mass.) and the
ABI CATALYST 800 (Perkin-Elmer). Sequencing is then carried out
using either ABI 373 or 377 DNA sequencing systems (Perkin-Elmer)
or the MEGABACE 1000 DNA sequencing system (Molecular Dynamics,
Sunnyvale Calif.). The resulting sequences are analyzed using a
variety of algorithms which are well known in the art. (See, e.g.,
Ausubel, F. M. (1997) Short Protocols in Molecular Biology, John
Wiley & Sons, New York N.Y., unit 7.7; Meyers, R.A. (1995)
Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp.
856-853.)
[0081] The nucleic acid sequences encoding KLIMP may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based methods known in the art to detect upstream sequences,
such as promoters and regulatory elements. For example, one method
which may be employed, restriction-site PCR, uses universal and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1: 11-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This
procedure avoids the need to screen libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers
may be designed using commercially available software, such as
OLIGO 4.06 primer analysis software (National Biosciences, Plymouth
Minn.) or another appropriate program, to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the template at temperatures of about 68.degree.
C. to 72.degree. C.
[0082] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are preferable for situations
in which an oligo d(T) library does not yield a full-length cDNA.
Genomic libraries may be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0083] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g., GENOTYPER
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 sequencing small DNA fragments which may be present
in limited amounts in a particular sample.
[0084] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode KLIMP may be cloned in
recombinant DNA molecules that direct expression of KLIMP, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced and used to express
KLIMP.
[0085] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter KLIMP-encoding sequences for a variety of purposes including,
but not limited to, modification of the cloning, processing, and/or
expression of the gene product. DNA shuffling by random
fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be used to engineer the nucleotide sequences.
For example, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0086] In another embodiment, sequences encoding KLIMP 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, KLIMP itself or a fragment
thereof may be synthesized using chemical methods. For example,
peptide synthesis can be performed using various solid-phase
techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI
431A peptide synthesizer (Perkin-Elmer). Additionally, the amino
acid sequence of KLIMP, 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.
[0087] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1984) Proteins,
Structures and Molecular Properties, W H Freeman, New York N.Y.) In
order to express a biologically active KLIMP, the nucleotide
sequences encoding KLIMP or derivatives thereof may be inserted
into an appropriate expression vector, i.e., a vector which
contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding KLIMP. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding KLIMP.
Such signals include the ATG initiation codon and adjacent
sequences, e.g. the Kozak sequence. In cases where sequences
encoding KLIMP and its initiation codon and upstream regulatory
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including an in-frame ATG initiation codon should be provided by
the vector. Exogenous translational elements and initiation codons
may be of various origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
enhancers appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0088] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding KLIMP 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; Ausubel, F. M. et al. (1995) Current
Protocols in Molecular Biology, John Wiley & Sons, New York
N.Y., ch. 9, 13, and 16.)
[0089] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding KLIMP. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0090] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding KLIMP. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding KLIMP can be achieved using a multifunctional E. coli
vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding KLIMP
into the vector's multiple cloning site disrupts the lacZ gene,
allowing a colorimetric screening procedure for identification of
transformed bacteria containing recombinant molecules. In addition,
these vectors may be useful for in vitro transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.
and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large
quantities of KLIMP are needed, e.g. for the production of
antibodies, vectors which direct high level expression of KLIMP may
be used. For example, vectors containing the strong, inducible T5
or T7 bacteriophage promoter may be used.
[0091] Yeast expression systems may be used for production of
KLIMP. A number of vectors containing constitutive or inducible
promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or
Pichia pastoris. In addition, such vectors direct either the
secretion or intracellular retention of expressed proteins and
enable integration of foreign sequences into the host genome for
stable propagation. (See, e.g., Ausubel, 1995, supra; Grant et al.
(1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (1994)
Bio/Technology 12:181-184.)
[0092] Plant systems may also be used for expression of KLIMP.
Transcription of sequences encoding KLIMP may be driven by viral
promoters, e.g., the 35S and 19S promoters of CaMV used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as
the small subunit of RUBISCO or heat shock promoters may be used.
(See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie,
R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991)
Results Probl. Cell Differ. 17:85-105.) These constructs can be
introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York
N.Y., pp. 191-196.)
[0093] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding KLIMP 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 infective virus which expresses KLIMP in host cells. (See,
e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0094] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from a plasmid. HACs of about 6 kb to 10 Mb are
constructed and delivered via conventional delivery methods
(liposomes, polycationic amino polymers, or vesicles) for
therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997)
Nat. Genet. 15:345-355.)
[0095] For long term production of recombinant proteins in
mammalian systems, stable expression of KLIMP in cell lines is
preferred. For example, sequences encoding KLIMP can be transformed
into cell lines using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for about 1 to 2 days in enriched media before being switched
to selective media. The purpose of the selectable marker is to
confer resistance to a selective agent, and its presence allows
growth and recovery of cells which successfully express the
introduced sequences. Resistant clones of stably transformed cells
may be propagated using tissue culture techniques appropriate to
the cell type.
[0096] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase genes, for use in tk.sup.- or apr.sup.-
cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell
11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or herbicide resistance can be used as
the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides,
neomycin and G-418; and als or pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol.
150:1-14.) Additional selectable genes have been described, e.g.,
trpB and hisD, which alter cellular requirements for metabolites.
(See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.
Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins,
green fluorescent proteins (GFP; Clontech), .beta. glucuronidase
and its substrate .beta.-glucuronide, or luciferase and its
substrate luciferin may be used. These markers can be used not only
to identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system. (See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol.
55:121-131.)
[0097] 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 KLIMP is inserted within a marker gene
sequence, transformed cells containing sequences encoding KLIMP can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding KLIMP 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.
[0098] In general, host cells that contain the nucleic acid
sequence encoding KLIMP and that express KLIMP may be identified by
a variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR amplification, and protein bioassay or
immunoassay techniques which include membrane, solution, or chip
based technologies for the detection and/or quantification of
nucleic acid or protein sequences.
[0099] Immunological methods for detecting and measuring the
expression of KLIMP using either specific polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques
include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
KLIMP is preferred, but a competitive binding assay may be
employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory
Manual, APS Press, St Paul Minn., Sect. IV; Coligan, J. E. et al.
(1997) Current Protocols in Immunology, Greene Pub. Associates and
Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0100] 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 KLIMP include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding KLIMP, 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 Amersham Pharmacia Biotech, Promega (Madison Wis.), and
US Biochemical. 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.
[0101] Host cells transformed with nucleotide sequences encoding
KLIMP may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or retained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode KLIMP may be designed to
contain signal sequences which direct secretion of KLIMP through a
prokaryotic or eukaryotic cell membrane.
[0102] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and W138), 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.
[0103] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding KLIMP may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric KLIMP protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of KLIMP activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate fusion proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin
(HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the KLIMP encoding sequence and the heterologous protein
sequence, so that KLIMP may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra,
ch 10). A variety of commercially available kits may also be used
to facilitate expression and purification of fusion proteins.
[0104] In a further embodiment of the invention, synthesis of
radiolabeled KLIMP may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract systems (Promega). These
systems couple transcription and translation of protein-coding
sequences operably associated with the T7, T3, or SP6 promoters.
Translation takes place in the presence of a radiolabeled amino
acid precursor, preferably .sup.35S-methionine.
[0105] Fragments of KLIMP may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI
431A peptide synthesizer (Perkin-Elmer). Various fragments of KLIMP
may be synthesized separately and then combined to produce the full
length molecule.
[0106] Therapeutics
[0107] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of KLIMP and the motor
domain of kinesin. In addition, the expression of KLIMP is closely
associated with cancerous and proliferating cells and
neurodegenerative tissue. Therefore, KLIMP appears to be associated
with cancer, neurological disorders, and disorders of vesicular
transport. In the treatment of cancer, neurological disorders, and
disorders of vesicular transport associated with increased KLIMP
activity, it is desirable to decrease the expression or activity of
KLIMP. In the treatment of the above conditions associated with
decreased KLIMP activity, it is desirable to provide the protein or
to increase the expression of KLIMP.
[0108] Therefore, in one embodiment, KLIMP or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of KLIMP. Examples of such disorders include, but are not limited
to, a cancer 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; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other extrapyramidal disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias,
multiple sclerosis and other demyelinating diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and
radiculitis, viral central nervous system disease, prion diseases
including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,
nutritional and metabolic diseases of the nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis, encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and
polymyositis, inherited, metabolic, endocrine, and toxic
myopathies, myasthenia gravis, periodic paralysis, mental disorders
including mood, anxiety, and schizophrenic disorders, akathesia,
amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; and a disorder of vesicular transport such as
cystic fibrosis, glucose-galactose malabsorption syndrome,
hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-
and hypoglycemia, Grave's disease, goiter, Cushing's disease,
Addison's disease, gastrointestinal disorders including ulcerative
colitis, gastric and duodenal ulcers, other conditions associated
with abnormal vesicle trafficking including acquired
immunodeficiency syndrome (AIDS), allergic reactions, autoimmune
hemolytic anemia, proliferative glomerulonephritis, inflammatory
bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid
arthritis, osteoarthritis, scleroderma, Chediak-Higashi syndrome,
Sjogren's syndrome, systemic lupus erythematosus, toxic shock
syndrome, traumatic tissue damage, and viral, bacterial, fungal,
helminthic, and protozoal infections.
[0109] In another embodiment, a vector capable of expressing KLIMP
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a disorder associated with decreased
expression or activity of KLIMP including, but not limited to,
those described above.
[0110] In a further embodiment, a pharmaceutical composition
comprising a substantially purified KLIMP in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a disorder associated with decreased expression or
activity of KLIMP including, but not limited to, those provided
above.
[0111] In still another embodiment, an agonist which modulates the
activity of KLIMP may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of KLIMP including, but not limited to, those listed above.
[0112] In a further embodiment, an antagonist of KLIMP may be
administered to a subject to treat or prevent a disorder associated
with increased expression or activity of KLIMP. Such disorders may
include, but are not limited to, those discussed above. In one
aspect, an antibody which specifically binds KLIMP 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 KLIMP.
[0113] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding KLIMP may be administered
to a subject to treat or prevent a disorder associated with
increased expression or activity of KLIMP including, but not
limited to, those described above.
[0114] 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.
[0115] An antagonist of KLIMP may be produced using methods which
are generally known in the art. In particular, purified KLIMP may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
KLIMP. Antibodies to KLIMP 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.
[0116] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with KLIMP 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.
[0117] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to KLIMP 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 KLIMP amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0118] Monoclonal antibodies to KLIMP may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0119] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
KLIMP-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc.
Natol. Acad. Sci. 88:10134-10137.)
[0120] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al.
(1991) Nature 349:293-299.)
[0121] Antibody fragments which contain specific binding sites for
KLIMP 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.)
[0122] 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 KLIMP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering KLIMP
epitopes is preferred, but a competitive binding assay may also be
employed (Pound, supra).
[0123] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for KLIMP. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
KLIMP-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple KLIMP epitopes,
represents the average affinity, or avidity, of the antibodies for
KLIMP. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular KLIMP epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
l/mole are preferred for use in immunoassays in which the
KLIMP-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 l/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of KLIMP, preferably in active form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington D.C.; Liddell, J. E. and Cryer, A. (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York N.Y.).
[0124] The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml,
is preferred for use in procedures requiring precipitation of
KLIMP-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0125] In another embodiment of the invention, the polynucleotides
encoding KLIMP, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding KLIMP 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 KLIMP. Thus, complementary molecules or
fragments may be used to modulate KLIMP 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 KLIMP.
[0126] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding KLIMP. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
[0127] Genes encoding KLIMP can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide, or fragment thereof, encoding KLIMP. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector, and may last even longer if appropriate replication
elements are part of the vector system.
[0128] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding KLIMP. 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, Mt. Kisco N.Y., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0129] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding KLIMP.
[0130] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0131] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding KLIMP. 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.
[0132] 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.
[0133] 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.)
[0134] 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.
[0135] 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 KLIMP, antibodies to KLIMP, and
mimetics, agonists, antagonists, or inhibitors of KLIMP. 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.
[0136] 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.
[0137] 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, Easton
Pa.).
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. 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.
[0146] 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 KLIMP, such
labeling would include amount, frequency, and method of
administration.
[0147] 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.
[0148] 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.
[0149] A therapeutically effective dose refers to that amount of
active ingredient, for example KLIMP or fragments thereof,
antibodies of KLIMP, and agonists, antagonists or inhibitors of
KLIMP, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of therapeutic to toxic
effects is the therapeutic index, and it can be expressed as the
ED.sub.50/LD.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0150] 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.
[0151] 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.
[0152] Diagnostics
[0153] In another embodiment, antibodies which specifically bind
KLIMP may be used for the diagnosis of disorders characterized by
expression of KLIMP, or in assays to monitor patients being treated
with KLIMP or agonists, antagonists, or inhibitors of KLIMP.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for KLIMP include methods which utilize the antibody and a label to
detect KLIMP 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.
[0154] A variety of protocols for measuring KLIMP, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of KLIMP expression.
Normal or standard values for KLIMP expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to KLIMP under conditions
suitable for complex formation. The amount of standard complex
formation may be quantitated by various methods, preferably by
photometric means. Quantities of KLIMP expressed in subject,
control, and disease samples from biopsied tissues are compared
with the standard values. Deviation between standard and subject
values establishes the parameters for diagnosing disease.
[0155] In another embodiment of the invention, the polynucleotides
encoding KLIMP 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 KLIMP may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of KLIMP, and to
monitor regulation of KLIMP levels during therapeutic
intervention.
[0156] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding KLIMP or closely related molecules may be used
to identify nucleic acid sequences which encode KLIMP. 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 KLIMP, allelic variants, or related
sequences.
[0157] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the KLIMP 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 KLIMP gene.
[0158] Means for producing specific hybridization probes for DNAs
encoding KLIMP include the cloning of polynucleotide sequences
encoding KLIMP or KLIMP 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 35S, or by enzymatic labels, such
as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0159] Polynucleotide sequences encoding KLIMP may be used for the
diagnosis of disorders associated with expression of KLIMP.
Examples of such disorders include, but are not limited to, a
cancer 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; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other extrapyramidal disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias,
multiple sclerosis and other demyelinating diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and
radiculitis, viral central nervous system disease, prion diseases
including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,
nutritional and metabolic diseases of the nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis, encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and
polymyositis, inherited, metabolic, endocrine, and toxic
myopathies, myasthenia gravis, periodic paralysis, mental disorders
including mood, anxiety, and schizophrenic disorders, akathesia,
amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; and a disorder of vesicular transport such as
cystic fibrosis, glucose-galactose malabsorption syndrome,
hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-
and hypoglycemia, Grave's disease, goiter, Cushing's disease,
Addison's disease, gastrointestinal disorders including ulcerative
colitis, gastric and duodenal ulcers, other conditions associated
with abnormal vesicle trafficking including acquired
immunodeficiency syndrome (AIDS), allergic reactions, autoimmune
hemolytic anemia, proliferative glomerulonephritis, inflammatory
bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid
arthritis, osteoarthritis, scleroderma, Chediak-Higashi syndrome,
Sjogren's syndrome, systemic lupus erythematosus, toxic shock
syndrome, traumatic tissue damage, and viral, bacterial, fungal,
helminthic, and protozoal infections. The polynucleotide sequences
encoding KLIMP may be used in Southern or northern analysis, dot
blot, or other membrane-based technologies; in PCR technologies; in
dipstick, pin, and multiformat ELISA-like assays; and in
microarrays utilizing fluids or tissues from patients to detect
altered KLIMP expression. Such qualitative or quantitative methods
are well known in the art.
[0160] In a particular aspect, the nucleotide sequences encoding
KLIMP may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding KLIMP 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 KLIMP 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.
[0161] In order to provide a basis for the diagnosis of a disorder
associated with expression of KLIMP, 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 KLIMP, 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.
[0162] 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.
[0163] With respect to cancer, the presence of an abnormal amount
of transcript (either under- or over-expressed) 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.
[0164] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding KLIMP 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 KLIMP, or a fragment of a
polynucleotide complementary to the polynucleotide encoding KLIMP,
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.
[0165] Methods which may also be used to quantitate the expression
of KLIMP 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; Duplaa, C. et al. (1993)
Anal. Biochem. 229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in an ELISA format
where the oligomer of interest is presented in various dilutions
and a spectrophotometric or calorimetric response gives rapid
quantitation.
[0166] 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.
[0167] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0168] In another embodiment of the invention, nucleic acid
sequences encoding KLIMP 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., Harrington, J. J. et al. (1997) Nat. Genet.
15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.
J. (1991) Trends Genet. 7:149-154.)
[0169] 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, supra, 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
KLIMP 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.
[0170] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, 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.
[0171] In another embodiment of the invention, KLIMP, 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 KLIMP and the agent being tested may be
measured.
[0172] 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. The test
compounds are reacted with KLIMP, or fragments thereof, and washed.
Bound KLIMP is then detected by methods well known in the art.
Purified KLIMP 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.
[0173] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding KLIMP specifically compete with a test compound for binding
KLIMP. In this manner, antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with KLIMP.
[0174] In additional embodiments, the nucleotide sequences which
encode KLIMP 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.
[0175] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0176] I. cDNA Library Construction
[0177] The COLNNOT16 library was constructed using RNA isolated
from sigmoid colon tissue removed from a 62-year-old Caucasian male
during a sigmoidectomy and permanent colostomy. Pathology for the
associated tumor tissue indicated invasive grade 2 adenocarcinoma.
One lymph node contained metastasis with extranodal extension.
Patient history included hyperlipidemia, cataract disorder,
dermatitis, cholecystectomy, and inguinal hernia repair. Family
history included benign hypertension, atherosclerotic coronary
artery disease, hyperlipidemia, breast cancer, and prostate
cancer.
[0178] Frozen tissue was homogenized and lysed in guanidinium
isothiocyanate solution using a Brinkmann Homogenizer Polytron
PT-3000 (Brinkmann Instruments, Westbury N.Y.). The lysate was
centrifuged over a CsCl cushion to isolate RNA. The RNA was
extracted with acid phenol, precipitated with sodium acetate and
ethanol, resuspended in RNase-free water, and treated with DNase.
The RNA was re-extracted with acid phenol and reprecipitated with
sodium acetate and ethanol. Poly(A+) RNA was isolated using the
OLIGOTEX mRNA purification kit (QIAGEN, Chatsworth Calif.).
[0179] Poly(A+) RNA was used for cDNA synthesis and construction of
the cDNA library according to the recommended protocols in the
SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were
fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia
Biotech), and those cDNAs exceeding 400 bp were ligated into pINCY
(Incyte Pharmaceuticals). Recombinant plasmids were transformed
into DH5.alpha. competent cells (Life Technologies).
[0180] II. Isolation of cDNA Clones
[0181] Plasmid DNA was released from the cells and purified using
the R.E.A.L. PREP 96 plasmid kit (QIAGEN). The recommended protocol
was employed except for the following changes: 1) the bacteria were
cultured in 1 ml of sterile Terrific Broth (Life Technologies) with
carbenicillin at 25 mg/l and glycerol at 0.4%; 2) after the
cultures were incubated for 19 hours, the cells were lysed with 0.3
ml of lysis buffer; and 3) following isopropanol precipitation, the
plasmid DNA pellets were each resuspended in 0.1 ml of distilled
water. The DNA samples were stored at 4.degree. C.
[0182] III. Sequencing and Analysis
[0183] The cDNAs were prepared for sequencing using the ABI
CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Robbins
Scientific) or MICROLAB 2200 (Hamilton) systems in combination with
the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced
using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer)
and standard ABI protocols, base calling software, and kits. In one
alternative, cDNAs were sequenced using the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were amplified and sequenced using the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In
yet another alternative, cDNAs were sequenced using solutions and
dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs
were determined using standard methods (reviewed in Ausubel, 1997,
supra, unit 7.7). Some of the cDNA sequences were selected for
extension using the techniques disclosed in Example V.
[0184] The polynucleotide sequences derived from cDNA, extension,
and shotgun sequencing were assembled and analyzed using a
combination of software programs which utilize algorithms well
known to those skilled in the art. Table 1 summarizes the software
programs, descriptions, references, and threshold parameters used.
The first column of Table 1 shows the tools, programs, and
algorithms used, the second column provides a brief description
thereof, the third column presents the references which are
incorporated by reference herein, and the fourth column presents,
where applicable, the scores, probability values, and other
parameters used to evaluate the strength of a match between two
sequences (the higher the probability the greater the homology).
Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software Engineering) and LASERGENE software (DNASTAR).
[0185] The polynucleotide sequences were validated by removing
vector, linker, and polyA sequences and by masking ambiguous bases,
using algorithms and programs based on BLAST, dynamic programming,
and dinucleotide nearest neighbor analysis. The sequences were then
queried against a selection of public databases such as GenBank
primate, rodent, mammalian, vertebrate, and eukaryote databases,
and BLOCKS to acquire annotation, using programs based on BLAST,
FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened for open reading frames using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide
sequences were translated to derive the corresponding full length
amino acid sequences, and these full length sequences were
subsequently analyzed by querying against databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS,
PFAM, and Prosite.
[0186] The programs described above for the assembly and analysis
of full length polynucleotide and amino acid sequences were used to
identify polynucleotide sequence fragments from SEQ ID NO:2.
Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and amplification technologies were described in
The Invention section above.
[0187] IV. Northern Analysis
[0188] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and
16.)
[0189] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in nucleotide databases
such as the GenBank or LIFESEQ database (Incyte Pharmaceuticals,
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 similar. The basis of the search
is the product score, which is defined as: 1 % sequence identity
.times. % maximum BLAST score 100
[0190] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0191] The results of northern analyses are reported a percentage
distribution of libraries in which the transcript encoding KLIMP
occurred. Analysis involved the categorization of cDNA libraries by
organ/tissue and disease. The organ/tissue categories included
cardiovascular, dermatologic, developmental, endocrine,
gastrointestinal, hematopoietic/immune, musculoskeletal, nervous,
reproductive, and urologic. The disease categories included cancer,
inflammation/trauma, fetal, neurological, and pooled. For each
category, the number of libraries expressing the sequence of
interest was counted and divided by the total number of libraries
across all categories. Percentage values of tissue-specific and
disease expression are reported in the description of the
invention.
[0192] V. Extension of KLIMP Encoding Polynucleotides
[0193] The full length nucleic acid sequence of SEQ ID NO:2 was
produced by extension of an appropriate fragment of the full length
molecule using oligonucleotide primers designed from this fragment.
One primer was synthesized to initiate 5' extension of the known
fragment, and the other primer, to initiate 3' extension of the
known fragment. The initial primers were designed using OLIGO 4.06
software (National Biosciences), or another appropriate program, to
be about 22 to 30 nucleotides in length, to have a GC content of
about 50% or more, and to anneal to the target sequence at
temperatures of about 68.degree. C. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0194] Selected human cDNA libraries were used to extend the
sequence. If more than one extension was necessary or desired,
additional or nested sets of primers were designed.
[0195] High fidelity amplification was obtained by PCR using
methods well known in the art. PCR was performed in 96-well plates
using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction
buffer containing Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4, and
.beta.-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair PCI A and PCI B: Step 1: 94.degree. C., 3 min; Step 2:
94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68.degree. C., 5 min; Step 7: storage at 4.degree. C. In
the alternative, the parameters for primer pair T7 and SK+ were as
follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15
sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. C., 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C.,
5 min; Step 7: storage at 4.degree. C.
[0196] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1.times. TE
and 0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA
to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of
the sample and to quantify the concentration of DNA. A 5 .mu.l to
10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a 1% agarose mini-gel to determine which
reactions were successful in extending the sequence.
[0197] The extended nucleotides were desalted and concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison Wis.), and
sonicated or sheared prior to religation into pUC 18 vector
(Amersham Pharmacia Biotech). For shotgun sequencing, the digested
nucleotides were separated on low concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar
ACE (Promega). Extended clones were religated using T4 ligase (New
England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction site overhangs, and transfected into competent
E. coli cells. Transformed cells were selected on
antibiotic-containing media, individual colonies were picked and
cultured overnight at 37.degree. C. in 384-well plates in
LB/2.times. carb liquid media.
[0198] The cells were lysed, and DNA was amplified by PCR using Taq
DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated 29
times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the same conditions as described above. Samples were diluted
with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (Perkin-Elmer).
[0199] 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 such extension, and an appropriate
genomic library.
[0200] VI. Labeling and Use of Individual Hybridization Probes
[0201] 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 [.sup.32P]-adenosine triphosphate (Amersham Pharmacia
Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston Mass.).
The labeled oligonucleotides are substantially purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column
(Amersham Pharmacia Biotech). An aliquot containing 10.sup.7counts
per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of human genomic DNA digested with one of
the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba1, or
Pvu II (DuPont NEN).
[0202] 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. Hybridization patterns are visualized
using autoradiography or an alternative imaging means and
compared.
[0203] VII. Microarrays
[0204] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0205] 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 software (DNASTAR).
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; 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.
[0206] VIII. Complementary Polynucleotides
[0207] Sequences complementary to the KLIMP-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring KLIMP. 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 (National Biosciences) and the
coding sequence of KLIMP. 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 KLIMP-encoding transcript.
[0208] IX. Expression of KLIMP
[0209] Expression and purification of KLIMP are achieved using
bacterial or virus-based expression systems. For expression of
KLIMP in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express KLIMP upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of KLIMP
in eukaryotic cells is achieved by infecting insect or mammalian
cell lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding KLIMP by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0210] In most expression systems, KLIMP is synthesized as a fusion
protein with, e.g., glutathione S-transferase (GST) or a peptide
epitope tag, such as FLAG or 6-His, permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from
crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma
japonicum, enables the purification of fusion proteins on
immobilized glutathione under conditions that maintain protein
activity and antigenicity (Amersham Pharmacia Biotech). Following
purification, the GST moiety can be proteolytically cleaved from
KLIMP at specifically engineered sites. FLAG, an 8-amino acid
peptide, enables immunoaffinity purification using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues,
enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression and purification are discussed in Ausubel (1995,
supra, ch 10 and 16). Purified KLIMP obtained by these methods can
be used directly in the following activity assay.
[0211] X. Demonstration of KLIMP Activity
[0212] A microtubule motility assay for KLIMP activity measures
motor domain function. In this assay, recombinant KLIMP is
immobilized onto a glass slide or similar substrate.
Taxol-stabilized bovine brain microtubules (commercially available)
in a solution containing ATP and cytosolic extract are perfused
onto the slide. Movement of microtubules as driven by KLIMP motor
activity can be visualized and quantified using video-enhanced
light microscopy and image analysis techniques. KLIMP activity is
directly proportional to the frequency and velocity of microtubule
movement.
[0213] XI. Functional Assays
[0214] KLIMP function is assessed by expressing the sequences
encoding KLIMP at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life
Technologies) and PCR3.1 (Invitrogen, Carlsbad Calif.), both of
which contain the cytomegalovirus promoter. 5-10 .mu.g of
recombinant vector are transiently transfected into a human cell
line, preferably of endothelial or hematopoietic origin, using
either liposome formulations or electroporation. 1-2 .mu.g of an
additional plasmid containing sequences encoding a marker protein
are co-transfected. Expression of a marker protein provides a means
to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA expression from the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP, and to
evaluate cellular properties, for example, their apoptotic state.
FCM detects and quantifies the uptake of fluorescent molecules that
diagnose events preceding or coincident with cell death. These
events include changes in nuclear DNA content as measured by
staining of DNA with propidium iodide; changes in cell size and
granularity as measured by forward light scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by
decrease in bromodeoxyuridine uptake; alterations in expression of
cell surface and intracellular proteins as measured by reactivity
with specific antibodies; and alterations in plasma membrane
composition as measured by the binding of fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry
are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New
York N.Y.
[0215] The influence of KLIMP on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding KLIMP and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding KLIMP and other genes of interest can
be analyzed by northern analysis or microarray techniques.
[0216] XII. Production of KLIMP Specific Antibodies
[0217] KLIMP substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488-495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0218] Alternatively, the KLIMP amino acid sequence is analyzed
using LASERGENE software (DNASTAR) 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, 1995, supra, ch. 11.)
[0219] Typically, oligopeptides 15 residues in length are
synthesized using an ABI 431A peptide synthesizer (Perkin-Elmer)
using fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis
Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995,
supra.) Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity by, for example, binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio-iodinated goat anti-rabbit
IgG.
[0220] XIII. Purification of Naturally Occurring KLIMP Using
Specific Antibodies
[0221] Naturally occurring or recombinant KLIMP is substantially
purified by immunoaffinity chromatography using antibodies specific
for KLIMP. An immunoaffinity column is constructed by covalently
coupling anti-KLIMP antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
After the coupling, the resin is blocked and washed according to
the manufacturer's instructions.
[0222] Media containing KLIMP are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of KLIMP (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/KLIMP 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 KLIMP is collected.
[0223] XIV. Identification of Molecules Which Interact with
KLIMP
[0224] KLIMP, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
KLIMP, washed, and any wells with labeled KLIMP complex are
assayed. Data obtained using different concentrations of KLIMP are
used to calculate values for the number, affinity, and association
of KLIMP with the candidate molecules. 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
2 1 1103 PRT Homo sapiens 1281811 1 Met Ala Gly Ala Ser Val Lys Val
Ala Val Arg Val Arg Pro Phe 1 5 10 15 Asn Ala Arg Glu Thr Ser Gln
Asp Ala Lys Cys Val Val Ser Met 20 25 30 Gln Gly Asn Thr Thr Ser
Ile Ile Asn Pro Lys Gln Ser Lys Asp 35 40 45 Ala Pro Lys Ser Phe
Thr Phe Asp Tyr Ser Tyr Trp Ser His Thr 50 55 60 Ser Thr Glu Asp
Pro Gln Phe Ala Ser Gln Gln Gln Val Tyr Arg 65 70 75 Asp Ile Gly
Glu Glu Met Leu Leu His Ala Phe Glu Gly Tyr Asn 80 85 90 Val Cys
Ile Phe Ala Tyr Gly Gln Thr Gly Ala Gly Lys Ser Tyr 95 100 105 Thr
Met Met Gly Arg Gln Glu Pro Gly Gln Gln Gly Ile Val Pro 110 115 120
Gln Leu Cys Glu Asp Leu Phe Ser Arg Val Ser Glu Asn Gln Ser 125 130
135 Ala Gln Leu Ser Tyr Ser Val Glu Val Ser Tyr Met Glu Ile Tyr 140
145 150 Cys Glu Arg Val Arg Asp Leu Leu Asn Pro Lys Ser Arg Gly Ser
155 160 165 Leu Arg Val Arg Glu His Pro Ile Leu Gly Pro Tyr Val Gln
Asp 170 175 180 Leu Ser Lys Leu Ala Val Thr Ser Tyr Ala Asp Ile Ala
Asp Leu 185 190 195 Met Asp Cys Gly Asn Lys Ala Arg Thr Val Ala Ala
Thr Asn Met 200 205 210 Asn Glu Thr Ser Ser Arg Ser His Ala Val Phe
Thr Ile Val Phe 215 220 225 Thr Gln Arg Cys His Asp Gln Leu Thr Gly
Leu Asp Ser Glu Lys 230 235 240 Val Ser Lys Ile Ser Leu Val Asp Leu
Ala Gly Ser Glu Arg Ala 245 250 255 Asp Ser Ser Gly Ala Arg Gly Met
Gly Leu Lys Glu Gly Ala Asn 260 265 270 Ile Asn Lys Ser Leu Thr Thr
Leu Gly Lys Val Ile Ser Ala Leu 275 280 285 Ala Asp Met Gln Ser Lys
Lys Arg Lys Ser Asp Phe Ile Pro Tyr 290 295 300 Arg Asp Ser Val Leu
Thr Trp Leu Leu Lys Glu Asn Leu Gly Gly 305 310 315 Asn Ser Arg Thr
Ala Met Ile Ala Ala Leu Ser Pro Ala Asp Ile 320 325 330 Asn Tyr Glu
Glu Thr Leu Ser Thr Leu Arg Tyr Ala Asp Arg Thr 335 340 345 Lys Gln
Ile Arg Cys Asn Ala Ile Ile Asn Glu Asp Pro Asn Ala 350 355 360 Arg
Leu Ile Arg Glu Leu Gln Glu Glu Val Ala Arg Leu Arg Glu 365 370 375
Leu Leu Met Ala Gln Gly Leu Ser Ala Ser Ala Leu Glu Gly Leu 380 385
390 Lys Thr Glu Glu Gly Ser Val Arg Gly Ala Leu Pro Ala Val Ser 395
400 405 Ser Pro Pro Ala Pro Val Ser Pro Ser Ser Pro Thr Thr His Asn
410 415 420 Gly Glu Leu Glu Pro Ser Phe Ser Pro Asn Thr Glu Ser Gln
Ile 425 430 435 Gly Pro Glu Glu Ala Met Glu Arg Leu Gln Glu Thr Glu
Lys Ile 440 445 450 Ile Ala Glu Leu Asn Glu Thr Trp Glu Glu Lys Leu
Arg Lys Thr 455 460 465 Glu Ala Leu Arg Met Glu Arg Glu Ala Leu Leu
Ala Glu Met Gly 470 475 480 Val Ala Val Arg Glu Asp Gly Gly Thr Val
Gly Val Phe Ser Pro 485 490 495 Lys Lys Thr Pro His Leu Val Asn Leu
Asn Glu Asp Pro Leu Met 500 505 510 Ser Glu Cys Leu Leu Tyr His Ile
Lys Asp Gly Val Thr Arg Val 515 520 525 Gly Gln Val Asp Met Asp Ile
Lys Leu Thr Gly Gln Phe Ile Arg 530 535 540 Glu Gln His Cys Leu Phe
Arg Ser Ile Pro Gln Pro Asp Gly Glu 545 550 555 Val Val Val Thr Leu
Glu Pro Cys Glu Gly Ala Glu Thr Tyr Val 560 565 570 Asn Gly Lys Leu
Val Thr Glu Pro Leu Val Leu Lys Ser Gly Asn 575 580 585 Arg Ile Val
Met Gly Lys Asn His Val Phe Arg Phe Asn His Pro 590 595 600 Glu Gln
Ala Arg Leu Glu Arg Glu Arg Gly Val Pro Pro Pro Pro 605 610 615 Gly
Pro Pro Ser Glu Pro Val Asp Trp Asn Phe Ala Gln Lys Glu 620 625 630
Leu Leu Glu Gln Gln Gly Ile Asp Ile Lys Leu Glu Met Glu Lys 635 640
645 Arg Leu Gln Asp Leu Glu Asn Gln Tyr Arg Lys Glu Lys Glu Glu 650
655 660 Ala Asp Leu Leu Leu Glu Gln Gln Arg Leu Tyr Ala Asp Ser Asp
665 670 675 Ser Gly Asp Asp Ser Asp Lys Arg Ser Cys Glu Glu Ser Trp
Arg 680 685 690 Leu Ile Thr Ser Leu Arg Glu Gln Leu Pro Pro Thr Thr
Val Gln 695 700 705 Thr Ile Val Lys Arg Cys Gly Leu Pro Ser Ser Gly
Lys Arg Arg 710 715 720 Ala Pro Arg Arg Val Tyr Gln Ile Pro Gln Arg
Arg Arg Leu Gln 725 730 735 Gly Lys Asp Pro Arg Trp Ala Thr Met Ala
Asp Leu Lys Met Gln 740 745 750 Ala Val Lys Glu Ile Cys Tyr Glu Val
Ala Leu Ala Asp Phe Arg 755 760 765 His Gly Arg Ala Glu Ile Glu Ala
Leu Ala Ala Leu Lys Met Arg 770 775 780 Glu Leu Cys Arg Thr Tyr Gly
Lys Pro Asp Gly Pro Gly Asp Ala 785 790 795 Trp Arg Ala Val Ala Arg
Asp Val Trp Asp Thr Val Gly Glu Glu 800 805 810 Glu Gly Gly Gly Ala
Gly Ser Gly Gly Gly Ser Glu Glu Gly Ala 815 820 825 Arg Gly Ala Glu
Val Glu Asp Leu Arg Ala His Ile Asp Lys Leu 830 835 840 Thr Gly Ile
Leu Gln Glu Val Lys Leu Gln Asn Ser Ser Lys Asp 845 850 855 Arg Glu
Leu Gln Ala Leu Arg Asp Arg Met Leu Arg Met Glu Arg 860 865 870 Val
Ile Pro Leu Ala Gln Asp His Glu Asp Glu Asn Glu Glu Gly 875 880 885
Gly Glu Val Pro Trp Ala Pro Pro Glu Gly Ser Glu Ala Ala Glu 890 895
900 Glu Ala Ala Pro Ser Asp Arg Met Pro Ser Ala Arg Pro Pro Ser 905
910 915 Pro Pro Leu Ser Ser Trp Glu Arg Val Ser Arg Leu Met Glu Glu
920 925 930 Asp Pro Ala Phe Arg Arg Gly Arg Leu Arg Trp Leu Lys Gln
Glu 935 940 945 Gln Leu Arg Leu Gln Gly Leu Gln Gly Ser Gly Gly Arg
Gly Gly 950 955 960 Gly Leu Arg Arg Pro Pro Ala Arg Phe Val Pro Pro
His Asp Cys 965 970 975 Lys Leu Arg Phe Pro Phe Lys Ser Asn Pro Gln
His Arg Glu Ser 980 985 990 Trp Pro Gly Met Gly Ser Gly Glu Ala Pro
Thr Pro Leu Gln Pro 995 1000 1005 Pro Glu Glu Val Thr Pro His Pro
Ala Thr Pro Ala Arg Arg Pro 1010 1015 1020 Pro Ser Pro Arg Arg Ser
His His Pro Arg Arg Asn Ser Leu Asp 1025 1030 1035 Gly Gly Gly Arg
Ser Arg Gly Ala Gly Ser Ala Gln Pro Glu Pro 1040 1045 1050 Gln His
Phe Gln Pro Lys Lys His Asn Ser Tyr Pro Gln Pro Pro 1055 1060 1065
Gln Pro Tyr Pro Ala Gln Arg Pro Pro Gly Pro Arg Tyr Pro Pro 1070
1075 1080 Tyr Thr Thr Pro Pro Arg Met Arg Arg Gln Arg Ser Ala Pro
Asp 1085 1090 1095 Leu Lys Glu Ser Gly Ala Ala Val 1100 2 3930 DNA
Homo sapiens 1281811 2 gtggcagcca gaactgatac agcccccctg gtctggggcc
aggacgccag ctgaggaggg 60 caggagtgtc tggagctatg gctggtgcct
cggtgaaagt ggcagtgagg gttcggccct 120 ttaacgcccg tgagaccagc
caggatgcca agtgtgtggt cagcatgcag ggcaacacca 180 cctccatcat
caatcctaaa cagagcaagg atgcccccaa aagcttcacc tttgactact 240
cctactggtc acacacttcg acggaggacc cccagtttgc atctcagcag caagtgtatc
300 gggacattgg agaagagatg ctgctccacg cctttgaagg ctacaacgtg
tgcatctttg 360 cctatgggca gaccggggct gggaaatcct ataccatgat
ggggcgacag gagccagggc 420 agcagggcat cgtgccccag ctctgtgagg
acctcttctc tcgcgttagt gagaaccaga 480 gtgctcagct atcctactct
gtggaggtga gctatatgga gatctactgt gagcgggtac 540 gagacctctt
gaaccccaag agtcggggtt ctctgcgggt ccgggagcac cccatcctgg 600
gcccgtacgt gcaggacctg tccaaattgg ctgtgacctc ctacgcagac attgctgacc
660 tcatggactg tggaaataaa gcacggactg tggctgccac caacatgaat
gagaccagca 720 gccgttccca tgccgtcttt accatcgtct tcacacagcg
ctgccatgac cagctcacgg 780 ggctggactc ggagaaggtc agtaagatca
gtttggtgga ccttgctggg agtgagcgag 840 ccgactcctc aggggcccgg
ggcatgggcc tgaaggaagg agccaacatc aataagtccc 900 tgactacact
agggaaagtg atctcggccc ttgcagatat gcaatcaaag aagcgaaagt 960
cggattttat cccctacagg gactctgtgc tcacctggct gctcaaggaa aatttggggg
1020 ggaactcacg cacagccatg attgcagccc tgagccctgc tgacatcaat
tacgaggaga 1080 ctctcagcac cctcaggtat gctgaccgca ccaagcaaat
ccgctgcaat gccatcatca 1140 acgaggaccc taatgcccgg ctgattagag
agctgcagga ggaagtagcc cggctgcggg 1200 aactgctgat ggctcaggga
ctgtcagcct ctgctctgga aggcctgaag acggaagaag 1260 ggagtgtcag
aggcgccctg ccagctgtgt catctccccc agctccagtt tcaccctcat 1320
cacccaccac acataatggg gagctggagc cgtcattctc ccccaacacg gagtcccaga
1380 ttgggcctga ggaagccatg gagaggctgc aggagacaga gaagattata
gctgagctga 1440 acgagacatg ggaggagaag ctacgcaaga cagaagccct
gaggatggag agagaagcat 1500 tgctggctga gatgggggtg gccgtccggg
aggatggggg aactgtgggc gtcttctctc 1560 caaagaagac tccccacctg
gtgaacctga acgaagaccc tctgatgtct gagtgtctgc 1620 tctaccacat
caaagatggc gtcaccaggg tcggccaagt agatatggac atcaagctga 1680
ccggacagtt cattcgggag caacactgtc tgttccggag catcccccag ccagatggag
1740 aagtggtggt cactctggag ccttgtgaag gagctgagac atatgtgaat
gggaagcttg 1800 tgacggagcc gctggtgctg aagtcaggga ataggattgt
gatgggcaag aaccacgttt 1860 tccgcttcaa ccacccggag caggcaaggc
tggaacggga acgaggggtc cccccacccc 1920 caggaccgcc ctctgagcca
gtcgactgga actttgccca gaaggaactg ctggagcagc 1980 aaggcatcga
cataaagctg gaaatggaga agaggctgca ggatctggag aatcagtacc 2040
ggaaagaaaa ggaagaagcc gatcttctgc tggagcagca gcgactgtat gcagactcgg
2100 acagcgggga tgactctgac aagcgctctt gtgaagagag ctggaggctc
atcacctcct 2160 tgcgggagca gctgccgccc accacggtcc agaccattgt
caaacgctgt ggtctgccca 2220 gcagtggcaa gcgcagggcc cctcgcaggg
tttatcagat cccccagcgg cgcaggctgc 2280 agggcaaaga cccccgctgg
gccaccatgg ctgacctgaa gatgcaggcg gtgaaggaga 2340 tctgctacga
ggtggccctg gctgacttcc gccacgggcg ggctgagatt gaggccctgg 2400
ccgccctcaa gatgcgggag ctgtgtcgca cctatggcaa gccagacggc cccggagacg
2460 cctggagggc tgtggcccgg gatgtctggg acactgtagg cgaggaggaa
ggaggtggag 2520 ctggcagtgg tggtggcagt gaggagggag cccgaggggc
ggaggtggag gacctccggg 2580 cccacatcga caagctgacg gggattctgc
aggaggtgaa gctgcagaac agcagcaagg 2640 accgggagct gcaggccctg
cgggaccgca tgctccgcat ggagagggtc atccccctgg 2700 cccaggatca
tgaggatgag aatgaagaag gtggtgaggt cccctgggcc ccgcctgaag 2760
gatcagaggc agcagaggag gcagccccca gtgaccgcat gccgtcagcc cggcccccct
2820 cgccgccact gtcaagctgg gagcgggtgt cacggctcat ggaggaggac
cctgccttcc 2880 gtcgtggtcg tcttcgctgg ctcaagcagg agcagctacg
gctgcaggga ctgcagggct 2940 ctgggggccg gggcgggggg ctgcgcaggc
ccccagcccg ctttgtgccc cctcacgact 3000 gcaagctacg cttccccttc
aagagcaacc cccagcaccg ggagtcttgg ccagggatgg 3060 ggagcgggga
ggctccaact ccgctccaac cccctgagga ggtcactccc catccagcca 3120
cccctgcccg ccggcctccg agtccccgaa ggtcccacca tccccgcagg aactccctgg
3180 atggaggggg ccgatcccgg ggagcgggtt ctgcacagcc tgaaccccag
cacttccagc 3240 ccaaaaagca caactcttat ccccagccac cccaacccta
cccagcccag cggcccccag 3300 ggccccgcta ccccccatac actactcccc
cacgaatgag acggcagcgt tctgcccctg 3360 acctcaagga gagtggggca
gctgtgtgag tcccacatcc tgggcagagg gcctggtggg 3420 gccccttgct
aggagaaggg aagacgcccg agacgctgct tccccagaag tgctggggca 3480
gggaggccca ggagatgaga gagaaggtcc gagtaggtga tagaagacaa gggggagacc
3540 gagccggagg ctgaggaaag gaagagggca cggagttgcc aggagcaaac
caaagtgaag 3600 agagagatag gaagctgcct cggggccacc ccttgcaaag
ggggtgtgtc ccacaaacgc 3660 tgctatgggt ggggtggggg gctggggtgc
tgcgtagcca gtgtttgact ttcttttcaa 3720 gtgggggaaa gtgggagagg
actgagagtg aggcaagttc tccccagccc ctgtccgtct 3780 gtctgtctct
gtctgtggtg gtttctgttt cttgggaggc atggtaggat cataagtcat 3840
tcccctcccc ttccaggcct cctgctatat ttgggggacc tgactggttt ggctggagtc
3900 ccgatgagga tgtggccctt actataggta 3930
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