U.S. patent application number 10/741173 was filed with the patent office on 2004-07-01 for nek-1-related protein kinase.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Bandman, Olga, Baughn, Mariah R., Corley, Neil C., Guegler, Karl J..
Application Number | 20040126805 10/741173 |
Document ID | / |
Family ID | 31996435 |
Filed Date | 2004-07-01 |
United States Patent
Application |
20040126805 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
July 1, 2004 |
NEK-1-related protein kinase
Abstract
The invention provides a human Nek1-related protein kinase
(NRPK) and polynucleotides which identify and encode NRPK. 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 NRPK.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Corley, Neil C.; (Castro Valley, CA)
; Guegler, Karl J.; (Menlo Park, CA) ; Baughn,
Mariah R.; (Los Angeles, CA) |
Correspondence
Address: |
INCYTE CORPORATION
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Corporation
Palo Alto
CA
|
Family ID: |
31996435 |
Appl. No.: |
10/741173 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10741173 |
Dec 19, 2003 |
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09481275 |
Jan 11, 2000 |
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6713060 |
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09481275 |
Jan 11, 2000 |
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09295068 |
Apr 20, 1999 |
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6030801 |
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09295068 |
Apr 20, 1999 |
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09049671 |
Mar 27, 1998 |
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5928874 |
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Current U.S.
Class: |
435/6.16 ;
435/194; 435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 9/1205 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/194; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/12; C07K 016/40 |
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 the 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 the amino acid
sequence of SEQ ID NO:1.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising the
polynucleotide sequence of SEQ ID NO:2.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises the
amino acid sequence of SEQ ID NO:1.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence of SEQ
ID NO:2, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to the
polynucleotide sequence of SEQ ID NO:2, c) a polynucleotide
complementary to a polynucleotide of a), d) a polynucleotide
complementary to a polynucleotide of b) and e) an RNA equivalent of
a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO:1.
19. A method for treating a disease or condition associated with
decreased expression of functional HSLP, 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 HSLP, 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 HSLP, 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 diagnostic test for a condition or disease associated with
the expression of HSLP in a biological sample, the method
comprising: a) combining the biological sample with an antibody of
claim 11, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex, and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of HSLP 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 HSLP in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of the amino
acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof,
under conditions to elicit an antibody response, b) isolating
antibodies from said animal, and c) screening the isolated
antibodies with the polypeptide, thereby identifying a polyclonal
antibody which binds specifically to a polypeptide comprising the
amino acid sequence of SEQ ID NO:1.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of the amino acid sequence
of SEQ ID NO:1, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibody
producing cells from the animal, c) fusing the antibody producing
cells with immortalized cells to form monoclonal antibody-producing
hybridoma cells, d) culturing the hybridoma cells, and e) isolating
from the culture monoclonal antibody which binds specifically to a
polypeptide comprising the amino acid sequence of SEQ ID NO:1.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the 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 the 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 the 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 the amino acid sequence of SEQ ID
NO:1.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:1.
57. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:2.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/481,275, filed Jan. 11, 2000, and allowed
Sep. 29, 2003, which is a divisional application of U.S.
application Ser. No. 09/295,068 filed Apr. 20, 1999, now U.S. Pat.
No. 6,030,801, issued Feb. 29, 2000, which is a divisional
application of U.S. application Ser. No. 09/049,671 filed Mar. 27,
1998, now U.S. Pat. No. 5,928,874, issued Jul. 27, 1999, the
contents all of which are hereby incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of an Nek1-related protein kinase related molecule and to
the use of these sequences in the diagnosis, treatment, and
prevention of cancer and immune and reproductive disorders.
BACKGROUND OF THE INVENTION
[0003] Kinases regulate many different processes such as cell
proliferation, differentiation, and cell signaling by adding
phosphate groups to proteins. Uncontrolled signaling has been
implicated in a variety of disease conditions such as inflammation,
cancer, arteriosclerosis, and psoriasis. Reversible protein
phosphorylation is the main strategy for controlling activities of
eukaryotic cells. It is estimated that more than 1000 of the 10,000
proteins active in a typical mammalian cell are phosphorylated. The
high energy phosphate which drives phosphorylation is generally
transferred from adenosine triphosphate molecules (ATP) to a
particular protein by protein kinases and removed from that protein
by protein phosphatases. Phosphorylation occurs in response to
extracellular signals such as hormones, neurotransmitters, growth
and differentiation factors, etc. cell cycle checkpoints, and
environmental or nutritional stresses. An appropriate protein
kinase can activate a metabolic enzyme, regulatory protein,
receptor, cytoskeletal protein, ion channel or pump, or
transcription factor.
[0004] Kinases comprise the largest known protein group, a
superfamily of enzymes with widely varied functions and
specificities. They are usually named after their substrate their
regulatory molecules, or some aspect of a mutant phenotype. With
regard to substrates, the protein kinases may be roughly divided
into two groups; those that phosphorylate tyrosine residues
(protein tyrosine kinases, PTK) and those that phosphorylate serine
or threonine residues (serine/threonine kinases, STK). A few
protein kinases have dual specificity and phosphorylate serine,
threonine and tyrosine residues. Almost all kinases contain a
conserved 250-300 amino acid catalytic domain. The N-terminal
domain, which contains subdomains I-IV, generally folds into a
two-lobed structure which binds and orients the ATP (or GTP) donor
molecule. The larger C terminal lobe, which contains subdomains
VI-XI, binds the protein substrate and carries out the transfer of
the gamma phosphate from ATP to the hydroxyl group of a serine,
threonine, or tyrosine residue. Subdomain V spans the two lobes.
The kinases may be categorized into families by the different amino
acid sequences (generally between 5 and 100 residues) located on
either side of, or inserted into loops of, the kinase domain. These
added amino acid sequences allow the regulation of each kinase as
it recognizes and interacts with its target protein. The primary
structure of the kinase domain is conserved and can be further
subdivided into 11 subdomains. Each of the 11 subdomains contain
specific residues and motifs or patterns of amino acids that are
characteristic of that subdomain and are highly conserved. (Hardie,
G. and Hanks, S. (1995) The Protein Kinase Facts Books. Vol I:7-20
Academic Press, San Diego, Calif.) In particular, two protein
kinase signature sequences have been identified in the kinase
domain, the first containing an active site lysine residue involved
in ATP binding, and the second containing an aspartate residue
important for catalytic activity.
[0005] The second messenger dependent protein kinases primarily
mediate the effects of second messengers such as cyclic AMP (cAMP),
cyclic GMP, inositol triphosphate, phosphatidylinositol,
3,4,5-triphosphate, cyclic ADPribose, arachidonic acid,
diacylglycerol, and calcium-calmodulin. The cyclic-AMP dependent
protein kinases (PKA) are important members of the STK family.
Cyclic-AMP is an intracellular mediator of hormone action in all
procaryotic and animal cells that have been studied. Such
hormone-induced cellular responses include thyroid hormone
secretion, cortisol secretion, progesterone secretion, glycogen
breakdown, bone resorption, and regulation of heart rate and force
of heart muscle contraction. PKA is found in all animal cells and
is thought to account for the effects of cyclic-AMP in most of
these cells. Altered PKA expression is implicated in a variety of
disorders and diseases including cancer, thyroid disorders,
diabetes, atherosclerosis, and cardiovascular disease.
(Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal
Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887.)
[0006] PTKs specifically phosphorylate tyrosine residues on their
target proteins and may be divided into transmembrane, receptor
PTKs and nontransmembrane, non-receptor PTKs. Transmembrane
protein-tyrosine kinases are receptors for most growth factors.
Binding of growth factor to the receptor activates the transfer of
a phosphate group from ATP to selected tyrosine side chains of the
receptor and other specific proteins. Growth factors (GF)
associated with receptor PTKs include epidermal GF,
platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and
insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage
colony stimulating factor.
[0007] Non-receptor PTKs lack transmembrane regions and form
complexes with the intracellular regions of cell surface receptors.
Such receptors that function through non-receptor PTKs include
those for cytokines, hormones, such as growth hormone and
prolactin, and antigen-specific receptors on T and B
lymphocytes.
[0008] Many of these PTKs were first identified as the products of
mutant oncogenes in cancer cells where their activation was no
longer subject to normal cellular controls. About one third of the
known oncogenes encode PTKs, and it is known that cellular
transformation (oncogenesis) is often accompanied by increased
tyrosine phosphorylation activity. (Charbonneau H. and Tonks N. K.
(1992) Annu. Rev. Cell Biol. 8:463-93.) Regulation of PTK activity
may therefore be an important strategy in controlling some types of
cancer.
[0009] Nek1 is an example of a dual specificity protein kinase from
mouse capable of phosphorylating serine, threonine, and tyrosine
residues. (Letwin, K. et al. (1992) EMBO J 11:3521-3531.) Nek1
contains an N-terminal kinase domain similar to the catalytic
domain of NIMA, a serine/threonine protein kinase which regulates
the cell cycle in the fungus Aspergillus nidulans. Nek1, however,
is able to phosphorylate exogenous substrates on tyrosine as well
as serine and threonine when expressed in bacteria. Nek1 is
expressed at high levels in both male and female germ cells,
consistent with a role in meiosis.
[0010] The discovery of a new Nek1-related protein kinase and the
polynucleotides encoding it satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
treatment, and prevention of cancer and immune and reproductive
disorders.
SUMMARY OF THE INVENTION
[0011] The invention is based on the discovery of a new human
Nek1-related protein kinase (NRPK), the polynucleotides encoding
NRPK, and the use of these compositions for the diagnosis,
treatment, or prevention of cancer and immune and reproductive
disorders.
[0012] 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.
[0013] 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 sequence of SEQ ID NO:1 or
a fragment of SEQ ID NO:1. The invention also includes an isolated
and purified polynucleotide variant having at least 90%
polynucleotide 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.
[0014] The invention further provides an isolated and purified
polynucleotide which hybridizes under stringent conditions to the
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an
isolated and purified polynucleotide which is complementary to the
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0015] 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 90% 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.
[0016] 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.
[0017] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1, the method comprising the steps of: (a)
culturing the host cell containing an expression vector containing
at least a fragment of a polynucleotide encoding the polypeptide
comprising the amino acid sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1 under conditions suitable for the expression of the
polypeptide; and (b) recovering the polypeptide from the host cell
culture.
[0018] 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.
[0019] 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.
[0020] The invention also provides a method for treating or
preventing a cancer, 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.
[0021] The invention also provides a method for treating or
preventing an immune disorder, 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.
[0022] The invention also provides a method for treating or
preventing a reproductive disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0023] The invention also provides a method for detecting a
polynucleotide encoding a polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a
biological sample containing nucleic acids, the method comprising
the steps of: (a) hybridizing the complement of the polynucleotide
encoding the polypeptide comprising the amino acid sequence of SEQ
ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic
acids of the biological sample, thereby forming a hybridization
complex; and (b) detecting the hybridization complex, wherein the
presence of the hybridization complex correlates with the presence
of a polynucleotide encoding the polypeptide comprising the amino
acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in the
biological sample. In one aspect, the nucleic acids of the
biological sample are amplified by the polymerase chain reaction
prior to the hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
[0024] 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 NRPK. The alignment was produced using MAC DNASIS PRO
software (Hitachi Software Engineering Co. Ltd., San Bruno,
Calif.).
[0025] FIGS. 2A and 2B show the amino acid sequence alignments
between NRPK (3069734; SEQ ID NO:1), and a dual-specificity,
Nek1-related protein kinase from Caenorhabditis elegans (GI
1082115; SEQ ID NO:3), produced using the multisequence alignment
program of LASERGENE software (DNASTAR Inc, Madison Wis.).
DESCRIPTION OF THE INVENTION
[0026] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0027] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and which
might be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
DEFINITIONS
[0029] "NRPK," as used herein, refers to the amino acid sequences
of substantially purified NRPK obtained from any species,
particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and preferably the human species, from any source,
whether natural, synthetic, semi-synthetic, or recombinant.
[0030] The term "agonist," as used herein, refers to a molecule
which, when bound to NRPK, increases or prolongs the duration of
the effect of NRPK. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of NRPK.
[0031] An "allele" or an "allelic sequence," as these terms are
used herein, is an alternative form of the gene encoding NRPK.
Alleles may result from at least one mutation in the nucleic acid
sequence and may result in altered mRNAs or in polypeptides whose
structure or function may or may not be altered. Any given natural
or recombinant gene may have none, one, or many allelic forms.
Common mutational changes which give rise to alleles are generally
ascribed to natural deletions, additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in
combination with the others, one or more times in a given
sequence.
[0032] "Altered" nucleic acid sequences encoding NRPK, as described
herein, include those sequences with deletions, insertions, or
substitutions of different nucleotides, resulting in a
polynucleotide the same NRPK or a polypeptide with at least one
functional characteristic of NRPK. Included within this definition
are polymorphisms which may or may not be readily detectable using
a particular oligonucleotide probe of the polynucleotide encoding
NRPK, and improper or unexpected hybridization to alleles, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding NRPK. 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 NRPK. 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 NRPK is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid,
positively charged amino acids may include lysine and arginine, and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; and phenylalanine and tyrosine.
[0033] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments", "immunogenic
fragments", or "antigenic fragments" refer to fragments of NRPK
which are preferably about 5 to about 15 amino acids in length and
which retain some biological activity or immunological activity of
NRPK. Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms are not meant to limit the
amino acid sequence to the complete native amino acid sequence
associated with the recited protein molecule.
[0034] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer. a Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y., pp. 1-5.)
[0035] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to NRPK, decreases the amount or the
duration of the effect of the biological or immunological activity
of NRPK. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of NRPK.
[0036] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fa,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant. Antibodies that bind NRPK 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.
[0037] The term "antigenic determinant," as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0038] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to a specific nucleic acid sequence. The term
"antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense molecules
may be produced by any method including synthesis or transcription.
Once introduced into a cell, the complementary nucleotides combine
with natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0039] As used herein, the term "biologically active," refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
NRPK, or of any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0040] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A." Complementarity between two single-stranded molecules may
be "partial," such that only some of the nucleic acids bind, or it
may be "complete," such that total complementarity exists between
the single stranded molecules. The degree of complementarity
between nucleic acid strands has significant effects on the
efficiency and strength of the hybridization between the nucleic
acid strands. This is of particular importance in amplification
reactions, which depend upon binding between nucleic acids strands,
and in the design and use of peptide nucleic acid (PNA)
molecules.
[0041] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, refer broadly to any composition containing
the given polynucleotide or amino acid sequence. The composition
may comprise a dry formulation, an aqueous solution, or a sterile
composition. Compositions comprising polynucleotide sequences
encoding NRPK or fragments of NRPK may be employed as hybridization
probes. The probes may be stored in freeze-dried form and may be
associated with a stabilizing agent such as a carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution
containing salts (e.g., NaCl), detergents (e.g., SDS), and other
components (e.g., Denhardt's solution, dry milk, salmon sperm DNA,
etc.).
[0042] "Consensus sequence," as used herein, refers to a nucleic
acid sequence which has been resequenced to resolve uncalled bases,
extended using XL-PCR (PE Biosystems) 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.
[0043] As used herein, the term "correlates with expression of a
polynucleotide" indicates that the detection of the presence of
nucleic acids, the same or related to a nucleic acid sequence
encoding NRPK, by northern analysis is indicative of the presence
of nucleic acids encoding NRPK in a sample, and thereby correlates
with expression of the transcript from the polynucleotide encoding
NRPK.
[0044] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0045] The term "derivative," as used herein, refers to the
chemical modification of NRPK, of a polynucleotide sequence
encoding NRPK, or of a polynucleotide sequence complementary to a
polynucleotide sequence encoding NRPK. 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.
[0046] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete
homology. The word "identity" may substitute for the word
"homology." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially homologous."
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous sequence to the target sequence under conditions of
reduced stringency. This is not to say that conditions of reduced
stringency are such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% homology or
identity). In the absence of non-specific binding, the
substantially homologous sequence or probe will not hybridize to
the second non-complementary target sequence.
[0047] 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, Inc., Madison Wis.). The MEGALIGN program can create
alignments between two or more sequences according to different
methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and
P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups
sequences into clusters by examining the distances between all
pairs. The clusters are aligned pairwise and then in groups. The
percentage similarity between two amino acid sequences, e.g.,
sequence A and sequence B, is calculated by dividing the length of
sequence A, minus the number of gap residues in sequence A, minus
the number of gap residues in sequence B, into the sum of the
residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no homology 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), as described
herein, are linear microchromosomes which may contain DNA sequences
of about 6 kb to 10 Mb in size, and which contain all of the
elements required for stable mitotic chromosome segregation and
maintenance. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet.
15:345-355.)
[0048] The term "humanized antibody," as used herein, refers to
antibody molecules in which the amino acid sequence in the
non-antigen binding regions has been altered so that the antibody
more closely resembles a human antibody, and still retains its
original binding ability. "Hybridization," as the term is used
herein, refers to any process by which a strand of nucleic acid
binds with a complementary strand through base pairing.
[0049] As used herein, the term "hybridization complex" as used
herein, refers to a complex formed between two nucleic acid
sequences by virtue of the formation of hydrogen bonds between
complementary bases. A hybridization complex may be formed in
solution (e.g., C.sub.0t or R.sub.0t analysis) or formed between
one nucleic acid sequence present in solution and another nucleic
acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or their nucleic acids have
been fixed).
[0050] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0051] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0052] The term "microarray," as used herein, refers to an
arrangement of distinct polynucleotides arrayed on a substrate,
e.g., paper, nylon or any other type of membrane, filter, chip,
glass slide, or any other suitable solid support.
[0053] The terms "element" or "array element" as used herein in a
microarray context, refer to hybridizable polynucleotides arranged
on the surface of a substrate.
[0054] The term "modulate," as it appears herein, refers to a
change in the activity of NRPK. For example, modulation may cause
an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of NRPK.
[0055] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to an oligonucleotide, nucleotide,
polynucleotide, or any fragment thereof, to DNA or RNA of genomic
or synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which are greater than about 60 nucleotides in length, and most
preferably are at least about 100 nucleotides, at least about 1000
nucleotides, or at least about 10,000 nucleotides in length.
[0056] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related nucleic acid sequences.
A promoter is operably associated or operably linked with a coding
sequence if the promoter controls the transcription of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in the same reading frame,
certain genetic elements, e.g., repressor genes, are not
contiguously linked to the sequence encoding the polypeptide but
still bind to operator sequences that control expression of the
polypeptide.
[0057] The term "oligonucleotide," as used herein, refers to a
nucleic acid sequence of at least about 6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20 to 25 nucleotides, which can be used in PCR
amplification or in a hybridization assay or microarray. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimer," "primer," "oligomer," and "probe," as these
terms are commonly defined in the art.
[0058] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least about 5 nucleotides in length linked to
a peptide backbone of amino acid residues ending in lysine. The
terminal lysine confers solubility to the composition. PNAs
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993)
Anticancer Drug Des. 8:53-63.)
[0059] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding NRPK, or fragments thereof, or NRPK itself, may comprise a
bodily fluid; an extract from a cell, chromosome, organelle, or
membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA,
in solution or bound to a solid support; a tissue; a tissue print;
etc.
[0060] As used herein, the terms "specific binding" or
"specifically binding" refer to that interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the presence of a particular
structure of the protein, e.g., the antigenic determinant or
epitope, recognized by the binding molecule. For example, if an
antibody is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0061] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotide
sequences and the claimed polynucleotide sequences. Suitably
stringent conditions can be defined by, for example, the
concentrations of salt or formamide in the prehybridization and
hybridization solutions, or by the hybridization temperature, and
are well known in the art. In particular, stringency can be
increased by reducing the concentration of salt, increasing the
concentration of formamide, or raising the hybridization
temperature.
[0062] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS, and 200 .mu.g/ml sheared and denatured
salmon sperm DNA. Hybridization could occur under reduced
stringency conditions as described above, but in 35% formamide at a
reduced temperature of 35.degree. C. The temperature range
corresponding to a particular level of stringency can be further
narrowed by calculating the purine to pyrimidine ratio of the
nucleic acid of interest and adjusting the temperature accordingly.
Variations on the above ranges and conditions are well known in the
art.
[0063] The term "substantially purified," as used herein, refers to
nucleic acid or amino acid sequences that are removed from their
natural environment and are isolated or separated, and are at least
about 60% free, preferably about 75% free, and most preferably
about 90% free from other components with which they are naturally
associated.
[0064] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively. "Transformation," as defined herein,
describes a process by which exogenous DNA enters and changes a
recipient cell. Transformation may occur under natural or
artificial conditions according to various methods well known in
the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic
host cell. The method for transformation is selected based on the
type of host cell being transformed and may include, but is not
limited to, viral infection, electroporation, heat shock,
lipofection, and particle bombardment. The term "transformed" cells
includes stably transformed cells in which the inserted DNA is
capable of replication either as an autonomously replicating
plasmid or as part of the host chromosome, as well as transiently
transformed cells which express the inserted DNA or RNA for limited
periods of time.
[0065] A "variant" of NRPK, as used herein, refers to an amino acid
sequence that is altered by one or more amino acids. The variant
may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties (e.g., replacement of
leucine with isoleucine). More rarely, a variant may have
"nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software.
THE INVENTION
[0066] The invention is based on the discovery of a new human
Nek1-related protein kinase (NRPK), the polynucleotides encoding
NRPK, and the use of these compositions for the diagnosis,
treatment, or prevention of cancer and immune and reproductive
disorders.
[0067] Nucleic acids encoding the NRPK of the present invention
were first identified in Incyte Clone 3069734 from the uterine
tissue cDNA library (UTRSNOR01) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:2, was
derived from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 3069734 (UTRSNOR01), 3824812 (BRAXNOT01),
064663, 182538, and 71904 (PLACNOB01), 2371739 (ADRENOT07), 3137279
and 3037088 (SMCCNOT01), 2482869 (SMCANOT01), 1867885 (SKINBIT01),
3321079 (PTHYNOT03), 3446145 (EPIPNOT01), 2838141 (DRGLNOT01),
1243173 (LUNGNOT03), 2059154 (OVARNOT03), and 1994372
(BRSTTUT03).
[0068] 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. NRPK is 293 amino
acids in length and has potential phosphorylation sites for casein
kinase II at residues S100 and S147, for protein kinase C at S154
and S178, and for tyrosine kinase at Y204, Y246 and Y278. NRPK also
contains a consensus sequence for ATP binding shared by many
eukaryotic protein kinases between residues 140 and K64, in which
K64 is involved in the ATP binding. Four additional sequences
associated with tyrosine kinase catalytic activity are found
between residues L111 and K124, G189 and I199, S208 and D230, and
Y254 and V276. As shown in FIGS. 2A and 2B, NRPK has chemical and
structural homology with an Nek1-related protein kinase from C.
elegans (GI 1082115; SEQ ID NO:3). In particular, NRPK and the
Nek1-related protein share 64% identity. Three of the tyrosine
kinase associated sequences found in NRPK at L111 to K124, G189 to
1199, and S208 to D230 are highly conserved in the Nek1-related
protein, as are the potential phosphorylation sites at S100, S154,
S178, Y204, and Y246. A fragment of SEQ ID NO:2 from about
nucleotide 496 to about nucleotide 572 is useful, for example, as a
hybridization probe. Northern analysis shows the expression of this
sequence in various libraries, at least 54% of which are
immortalized or cancerous, at least 35% of which involve immune
response, and at least 33% of which involve reproductive tissues.
Of particular note is expression of NRPK associated with cancers of
the kidney, uterus, blood, small intestine, prostate, paraganglion,
ovaries, lung, liver, skull, breast, and adrenals, and with
inflammatory conditions including parathyroid hyperplasia,
hypereosinophilia, erythma nodosum, prostate hyperplasia,
cholecystitis, and ulcerative colitis.
[0069] The invention also encompasses NRPK variants. A preferred
NRPK 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 NRPK amino acid sequence, and which
contains at least one functional or structural characteristic of
NRPK.
[0070] The invention also encompasses polynucleotides which encode
NRPK. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2,
which encodes an NRPK.
[0071] The invention also encompasses a variant of a polynucleotide
sequence encoding NRPK. 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 NRPK. 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 NRPK.
[0072] 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 NRPK, some bearing minimal
homology to the polynucleotide sequences of any known and naturally
occurring gene, may be produced. Thus, the invention contemplates
each and every possible variation of polynucleotide sequence that
could be made by selecting combinations based on possible codon
choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring NRPK, and all such variations are
to be considered as being specifically disclosed.
[0073] Although nucleotide sequences which encode NRPK and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring NRPK under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding NRPK or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding NRPK 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.
[0074] The invention also encompasses production of DNA sequences
which encode NRPK and NRPK derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding NRPK or any fragment
thereof.
[0075] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:2, or a fragment of SEQ ID NO:2, under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.)
[0076] Methods for DNA sequencing are well known and generally
available in the art and may be used to practice any of the
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, T7 SEQUENASE DNA
polymerase, (US Biochemical Corp., Cleveland, Ohio), Taq DNA
polymerase (PE Biosystems), and THERMOSEQUENASE (Amersham, Chicago,
Ill.), or combinations of polymerases and proofreading exonucleases
such as those found in the ELONGASE amplification system
(GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is
automated with machines such as the MICROLAB 2200 system (Hamilton,
Reno, Nev.), DNA ENGINE thermal cycler (PTC200; MJ Research,
Watertown, Mass.), ABI PRISM and 373 and 377 DNA sequencing systems
(PE Biosystems).
[0077] The nucleic acid sequences encoding NRPK may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences, such as
promoters and regulatory elements. For example, one method which
may be employed, restriction-site PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus. (See, e.g.,
Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) In particular,
genomic DNA is first amplified in the presence of a primer which is
complementary to a linker sequence within the vector and a primer
specific to a region of the nucleotide sequenc. The amplified
sequences are then subjected to a second round of PCR with the same
linker primer and another specific primer internal to the first
one. Products of each round of PCR are transcribed with an
appropriate RNA polymerase and sequenced using reverse
transcriptase.
[0078] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region. (See, e.g.,
Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) The primers
may be designed using commercially available software such as OLIGO
4.06 primer analysis software (National Biosciences Inc., Plymouth,
Minn.) or another appropriate program to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the target sequence at temperatures of about
68.degree. C. to 72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0079] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In
this method, multiple restriction enzyme digestions and ligations
may be used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR. Other
methods which may be used to retrieve unknown sequences are known
in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids
Res. 19:3055-3060.) Additionally, one may use PCR, nested primers,
and PROMOTERFINDER (Clontech, Palo Alto, Calif.) libraries to walk
genomic DNA. This process avoids the need to screen libraries and
is useful in finding intron/exon junctions.
[0080] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into 5' non-transcribed regulatory regions.
[0081] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and a charge coupled device
camera for detection of the emitted wavelengths. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems),
and the entire process from loading of samples to computer analysis
and electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0082] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode NRPK may be used in
recombinant DNA molecules to direct expression of NRPK, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced, and these sequences
may be used to clone and express NRPK.
[0083] As will be understood by those of skill in the art, it may
be advantageous to produce NRPK-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0084] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter NRPK-encoding sequences for a variety of reasons including,
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0085] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding NRPK may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of NRPK activity, it may
be useful to encode a chimeric NRPK protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the NRPK
encoding sequence and the heterologous protein sequence, so that
NRPK may be cleaved and purified away from the heterologous
moiety.
[0086] In another embodiment, sequences encoding NRPK 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. 7:215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 7:225-232.) Alternatively, the protein itself may
be produced using chemical methods to synthesize the amino acid
sequence of NRPK, or a fragment thereof. For example, peptide
synthesis can be performed using various solid-phase techniques.
(See, e.g., Roberge, J. Y. et al. (1995) Science 269:202-204.)
Automated synthesis may be achieved using an ABI 431A peptide
synthesizer (PE Biosystems). Additionally, the amino acid sequence
of NRPK, 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 and Co., New York,
N.Y.)
[0088] In order to express a biologically active NRPK, the
nucleotide sequences encoding NRPK or derivatives thereof may be
inserted into appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0089] Methods which are well known to those skilled in the art
maybe used to construct expression vectors containing sequences
encoding NRPK and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al. (1995, and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)
[0090] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding NRPK. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems.
[0091] The invention is not limited by the host cell employed.
[0092] The "control elements" or "regulatory sequences" are those
non-translated regions, e.g., enhancers, promoters, and 5' and 3'
untranslated regions, of the vector and polynucleotide sequences
encoding NRPK which interact with host cellular proteins to carry
out transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used. For example, when cloning in bacterial systems, inducible
promoters, e.g., hybrid lacZ promoter of the BLUESCRIPT phagemid
(Stratagene, La Jolla, Calif.) or PSport 1 plasmid (GIBCO/BRL), may
be used. The baculovirus polyhedrin promoter may be used in insect
cells. Promoters or enhancers derived from the genomes of plant
cells (e.g., heat shock, RUBISCO, and storage protein genes) or
from plant viruses (e.g., viral promoters or leader sequences) may
be cloned into the vector. In mammalian cell systems, promoters
from mammalian genes or from mammalian viruses are preferable. If
it is necessary to generate a cell line that contains multiple
copies of the sequence encoding NRPK, vectors based on SV40 or EBV
may be used with an appropriate selectable marker.
[0093] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for NRPK. For example,
when large quantities of NRPK are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as Bluescript (Stratagene), in which
the sequence encoding NRPK may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced, and pIN vectors. (See, e.g., Van Heeke, G. and S. M.
Schuster (1989) J. Biol. Chem. 264:5503-5509.) PGEX vectors
(Amersham Pharmacia Biotech, Uppsala, Sweden) may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor XA protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will.
[0094] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel,
supra; and Grant et al. (1987) Methods Enzymol. 153:516-544.)
[0095] In cases where plant expression vectors are used, the
expression of sequences encoding NRPK may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.
6:307-311.) Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used. (See, e.g.,
Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results
Probl. Cell Differ. 17:85-105.) These constructs can be introduced
into plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews. (See, e.g., Hobbs, S. or Murry, L. E.
in McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New York, N.Y.; pp. 191-196.)
[0096] An insect system may also be used to express NRPK. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding NRPK may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of sequences
encoding NRPK will render the polyhedrin gene inactive and produce
recombinant virus lacking coat protein. The recombinant viruses may
then be used to infect, for example, S. frugiperda cells or
Trichoplusia larvae in which NRPK may be expressed. (See, e.g.,
Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.
91:3224-3227.)
[0097] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding NRPK maybe ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential El or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing NRPK in
infected 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.
[0098] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of about 6 kb to 10 Mb are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0099] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding NRPK. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding NRPK and its initiation codon and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers appropriate for the particular cell
system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
[0100] In addition, a host cell strain may be-chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and W138), are available from the American
Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0101] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing NRPK can be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for about 1 to 2 days in
enriched media before being switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0102] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase genes and adenine
phosphoribosyltransferase genes, which can be employed in tk.sup.-
or apr.sup.- cells, respectively. (See, e.g., Wigler, M. et al.
(1977) Cell 11:223-232; and Lowy, I. et al. (1980) Cell
22:817-823.) Also, antimetabolite, antibiotic, or herbicide
resistance can be used as the basis for selection. For example,
dhfr confers resistance to methotrexate; npt confers resistance to
the aminoglycosides neomycin and G-418; and als or pat confer
resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl.
Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol.
Biol. 150:1-14; and Murry, supra.) Additional selectable genes have
been described, e.g., trpB, which allows cells to utilize indole in
place of tryptophan, or hisD, which allows cells to utilize
histinol in place of histidine. (See, e.g., Hartman, S. C. and R.
C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible
markers, e.g., anthocyanins, 1 glucuronidase and its substrate GUS,
luciferase and its substrate luciferin may be used. Green
fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.) can also
be used. These markers can be used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C. A. et al. (1995) Methods Mol. Biol.
55:121-131.)
[0103] 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 NRPK is inserted within a marker gene
sequence, transformed cells containing sequences encoding NRPK can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding NRPK 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.
[0104] Alternatively, host cells which contain the nucleic acid
sequence encoding NRPK and express NRPK may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein
sequences.
[0105] The presence of polynucleotide sequences encoding NRPK can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
NRPK. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding NRPK
to detect transformants containing DNA or RNA encoding NRPK.
[0106] A variety of protocols for detecting and measuring the
expression of NRPK, using either polyclonal or monoclonal
antibodies specific for the protein, are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELUSAs), radioimmunoassays (RIAs), and fluorescence activated cell
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
NRPK is preferred, but a competitive binding assay may be employed.
These and other assays are well described in the art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual,
APS Press, St Paul, Minn., Section IV; and Maddox, D. E. et al.
(1983) J. Exp. Med. 158:1211-1216).
[0107] 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 NRPK include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding NRPK, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0108] Host cells transformed with nucleotide sequences encoding
NRPK may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode NRPK may be designed to
contain signal sequences which direct secretion of NRPK through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding NRPK to nucleotide sequences
encoding a polypeptide domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAG
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences, such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.), between the purification domain and the NRPK encoding
sequence may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing NRPK and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification on immobilized metal ion
affinity chromatography (IMIAC). (See, e.g., Porath, J. et al.
(1992) Prot. Exp. Purif. 3: 263-281.) The enterokinase cleavage
site provides a means for purifying NRPK from the fusion protein.
(See, e.g., Kroll, D. J. et al. (1993) DNA Cell Biol.
12:441-453.)
[0109] Fragments of NRPK may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the Applied
Biosystems 431 A peptide synthesizer (PE Biosystems). Various
fragments of NRPK may be synthesized separately and then combined
to produce the full length molecule.
[0110] Therapeutics
[0111] Chemical and structural homology exists between NRPK and an
Nek1-related protein kinase from C. elegans (GI 1082115). In
addition, NRPK is expressed in cancer and immortalized cell lines,
inflammation and the immune response, and in reproductive tissues.
Therefore, NRPK appears to play a role in cancer and immune and
reproductive disorders.
[0112] In particular, increased expression or activity of NRPK
appears to be associated with these disorders.
[0113] Therefore, in one embodiment, an antagonist of NRPK may be
administered to a subject to treat or prevent a cancer. Such a
cancer may include, but is not limited to, adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma,
and, in particular, cancers of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect, an
antibody which specifically binds NRPK 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
NRPK.
[0114] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding NRPK may be administered
to a subject to treat or prevent a cancer including, but not
limited to, those described above.
[0115] In another embodiment, an antagonist of NRPK may be
administered to a subject to treat or prevent an immune disorder.
Such a disorder may include, but is not limited to, AIDS, Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema,
erythemanodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic sclerosis, ulcerative colitis, Werner
syndrome, and complications of cancer, hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections, and trauma.
[0116] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding NRPK may be administered
to a subject to treat or prevent an immune disorder including, but
not limited to, those described above.
[0117] In another embodiment, an antagonist of NRPK may be
administered to a subject to treat or prevent a reproductive
disorder. Such a disorder may include, but is not limited to,
disorders of prolactin production; infertility, including tubal
disease, ovulatory defects, and endometriosis; disruptions of the
estrous cycle, disruptions of the menstrual cycle, polycystic ovary
syndrome, ovarian hyperstimulation syndrome, endometrial and
ovarian tumors, autoimmune disorders, ectopic pregnancy, and
teratogenesis; cancer of the breast, uterine fibroids, fibrocystic
breast disease, galactorrhea; disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, carcinoma of the male breast and gynecomastia.
[0118] In still another embodiment, a vector expressing the
complement of the polynucleotide encoding NRPK may be administered
to a subject to treat or prevent a reproductive disorder including,
but not limited to, those described above.
[0119] 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.
[0120] An antagonist of NRPK may be produced using methods which
are generally known in the art. In particular, purified NRPK may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind NRPK. Antibodies
to NRPK 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.
[0121] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with NRPK 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.
[0122] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to NRPK 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 NRPK amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0123] Monoclonal antibodies to NRPK 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 81:3142; 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.)
[0124] 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 314:452454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
NRPK-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
[0125] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; and Winter, G. et
al. (1991) Nature 349:293-299.)
[0126] Antibody fragments which contain specific binding sites for
NRPK may also be generated For example, such fragments include, but
are not limited to, F(ab')2 fragments produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing
the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity. (See, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0127] 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 NRPK and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering NRPK epitopes
is preferred, but a competitive binding assay may also be employed.
(Maddox, supra.)
[0128] In another embodiment of the invention, the polynucleotides
encoding NRPK, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding NRPK maybe 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 NRPK. Thus, complementary molecules or
fragments may be used to modulate NRPK activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or anti sense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding NRPK.
[0129] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors which
will express nucleic acid sequences complementary to the
polynucleotides of the gene encoding NRPK. (See, e.g., Sambrook,
supra; and Ausubel, supra.)
[0130] Genes encoding NRPK can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding NRPK. 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.
[0131] 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 NRPK. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (See, e.g., Gee, J. E. et
al. (1994) in Huber, B. E. and B. I. Carr, Molecular and
Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0132] 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 NRPK.
[0133] 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.
[0134] 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 NRPK. 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.
[0135] 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
[0136] acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0137] 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.)
[0138] 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.
[0139] 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 NRPK, antibodies to NRPK, and mimetics,
agonists, antagonists, or inhibitors of NRPK. 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.
[0140] 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.
[0141] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0142] 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.
[0143] 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.
[0144] Dragee cores maybe 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of NRPK, such
labeling would include amount, frequency, and method of
administration.
[0151] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0152] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0153] A therapeutically effective dose refers to that amount of
active ingredient, for example NRPK or fragments thereof,
antibodies of NRPK, and agonists, antagonists or inhibitors of
NRPK, 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/LD50 ratio. Pharmaceutical compositions which exhibit
large therapeutic indices are preferred. The data obtained from
cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0154] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0155] Normal dosage amounts may vary from about 0.1 .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.
[0156] Diagnostics
[0157] In another embodiment, antibodies which specifically bind
NRPK may be used for the diagnosis of disorders characterized by
expression of NRPK, or in assays to monitor patients being treated
with NRPK or agonists, antagonists, or inhibitors of NRPK.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for NRPK include methods which utilize the antibody and a label to
detect NRPK 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.
[0158] A variety of protocols for measuring NRPK, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of NRPK expression. Normal or
standard values for NRPK expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to NRPK under conditions suitable
for complex formation The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of NRPK 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.
[0159] In another embodiment of the invention, the polynucleotides
encoding NRPK 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 NRPK may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of NRPK, and to
monitor regulation of NRPK levels during therapeutic
intervention.
[0160] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding NRPK or closely related molecules may be used
to identify nucleic acid sequences which encode NRPK. 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 NRPK, alleles, or related
sequences.
[0161] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the NRPK 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 NRPK gene.
[0162] Means for producing specific hybridization probes for DNAs
encoding NRPK include the cloning of polynucleotide sequences
encoding NRPK or NRPK derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32p or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidinibiotin
coupling systems, and the like.
[0163] Polynucleotide sequences encoding NRPK may be used for the
diagnosis of a disorder associated with expression of NRPK.
Examples of such a disorder include, but are not limited to,
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; immune disorders, such as AIDS, Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis,
hypereosinophilia, irritable bowel syndrome, lupus erythematosus,
multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, rheumatoid arthritis, scleroderma, Sjoigren's
syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis, ulcerative colitis, Werner syndrome, and
complications of cancer, hemodialysis, and extracorporaal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; and reproductive disorders, such
as disorders of prolactin production; infertility, including tubal
disease, ovulatory defects, and endometriosis; disruptions of the
estrous cycle, disruptions of the menstrual cycle, polycystic ovary
syndrome, ovarian hyperstimulation syndrome, endometrial and
ovarian tumors, autoimmune disorders, ectopic pregnancy, and
teratogenesis; cancer of the breast, uterine fibroids, fibrocystic
breast disease, galactorrhea; disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, carcinoma of the male breast and gynecomastia. The
polynucleotide sequences encoding NRPK may be used in Southern or
northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies; in dipstick, pin, and ELISA assays; and in
microarrays utilizing fluids or tissues from patients to detect
altered NRPK expression. Such qualitative or quantitative methods
are well known in the art.
[0164] In a particular aspect, the nucleotide sequences encoding
NRPK may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding NRPK 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 NRPK 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.
[0165] In order to provide a basis for the diagnosis of a disorder
associated with expression of NRPK, 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 NRPK, 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.
[0166] 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.
[0167] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0168] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding NRPK 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 NRPK, or a fragment of a
polynucleotide complementary to the polynucleotide encoding NRPK,
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.
[0169] Methods which may also be used to quantitate the expression
of NRPK include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al.
(1993) Anal. Biochem. 229-236.) The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or calorimetric response gives
rapid quantitation.
[0170] 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.
[0171] Microarrays maybe 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.)
[0172] In another embodiment of the invention, nucleic acid
sequences encoding NRPK may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;
and Trask, B. J. (1991) Trends Genet. 7:149-154.)
[0173] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulhich, et al. (1995) in Meyers, R. A.
(ed.) Molecular Biology and Biotechnology, VCH Publishers New York,
N.Y., pp. 965-968.) Examples of genetic map data can be found in
various scientific journals or at the Online Mendelian Inheritance
in Man (OMIM) site. Correlation between the location of the gene
encoding NRPK 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.
[0174] 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., AT to 11q22-23, any sequences mapping to that
area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature
336:577-580.) The nucleotide sequence of the subject invention may
also be used to detect differences in the chroriosomal location due
to translocation, inversion, etc., among normal, carrier, or
affected individuals.
[0175] In another embodiment of the invention, NRPK, 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 NRPK and the agent being tested may be
measured.
[0176] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate, such as
plastic pins or some other surface. The test compounds are reacted
with NRPK, or fragments thereof, and washed. Bound NRPK is then
detected by methods well known in the art. Purified NRPK 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.
[0177] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding NRPK specifically compete with a test compound for binding
NRPK. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
NRPK.
[0178] In additional embodiments, the nucleotide sequences which
encode NRPK 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.
[0179] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0180] I. UTRSNOR01 cDNA Library Construction
[0181] The UTRSNOR01 cDNA library was constructed from
microscopically normal uterine endometrium tissue obtained from a
29-year-old Caucasian female (specimen #0909A) during a vaginal
hysterectomy and cystocele repair. Pathology of the uterus
indicated a single intramural uterine leiomyoma. The endometrium
was in secretory phase and the cervix showed mild chronic
cervicitis with focal squamous metaplasia. Patient history included
hypothyroidism, pelvic floor relaxation, an incomplete T-12 injury
from a motor vehicle accident causing paraplegia, and
self-catheterization. Previous surgeries included a cystocele
repair, a pelvic floor relaxation, a normal delivery, a
laminectomy, and a rhinoplasty. Family history included benign
hypertension in the father; and diabetes type II and hyperlipidemia
in the mother.
[0182] The frozen tissue was homogenized and lysed in TRIZOL
reagent (1 gm tissue/10 ml ITRZOL; Gibco-BRL, Gaithersburg, Md.), a
monophasic solution of phenol and guanidine isothiocyanate, using a
Brinkmann POLYTRON homogenizer PT-3000 (Brinkmann Instruments,
Westbury, N.Y.). After a brief incubation on ice, chloroform was
added (1:5 v/v) and the lysate was centrifuged. The upper
chloroform layer was removed to a fresh tube and the RNA
precipitated with isopropanol, resuspended in DEPC-treated water,
and treated with DNase for 25 min at 37.degree. C. The mRNA was
re-extracted once with acid phenol-chloroform pH 4.7 and
precipitated using 0.3M sodium acetate and 2.5 volumes ethanol. The
mRNA was isolated and purified using OLIGOTEX mRNA purification kit
(QIAGEN, Inc., Chatsworth, Calif.), and used to construct the cDNA
library.
[0183] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system for cDNA synthesis and plasmid
Cloning (Gibco/BRL). The cDNAs were fractionated on a SEPHAROSECL4B
column (Pharmacia), and those cDNAs exceeding 400 bp were ligated
into pINCY 1. The plasmid pINCY 1 was subsequently transformed into
DH5.alpha. competent cells (Gibco/BRL).
[0184] II. Isolation and Sequencing of cDNA Clones
[0185] Plasmid DNA was released from the cells and purified using
the R.E.A.L. PREP 96 plasmid kit (QIAGEN, Inc.). This kit enabled
the simultaneous purification of 96 samples in a 96-well block
using multi-channel reagent dispensers. The recommended protocol
was employed except for the following changes: 1) the bacteria were
cultured in 1 ml of sterile Terrific Broth (Gibco/BRL) with
carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after
inoculation, the cultures were incubated for 19 hours and at the
end of incubation, the cells were lysed with 0.3 ml of lysis
buffer; and 3) following isopropanol precipitation, the plasmid DNA
pellet was resuspended in 0.1 ml of distilled water. After the last
step in the protocol, samples were transferred to a 96-well block
for storage at 4.degree. C.
[0186] The cDNAs were sequenced by the method of Sangeret al.
(1975,J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.) in combination with Peltier thermal cyclers
(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems
377 DNA sequencing systems; and the reading frame was
determined.
[0187] III. Homology Searching of cDNA Clones and Their Deduced
Proteins
[0188] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, and Pima II databases. These databases, which contain
previously identified and annotated sequences, were searched for
regions of homology using BLAST (Basic Local Alignment Search
Tool). (See, e.g., Altschul, S. F. (1993) J. Mol. Evol 36:290-300;
and Altschul et al. (1990) J. Mol. Biol. 215:403-410.)
[0189] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST is especially useful in determining
exact matches or in identifying homologs which may be of
prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms could have been used when dealing with
primary sequence patterns and secondary structure gap penalties.
(See, e.g., Smith, T. et al. (1992) Protein Engineering 5:35-51.)
The sequences disclosed in this application have lengths of at
least 49 nucleotides and have no more than 12% uncalled bases
(where N is recorded rather than A, C, G, or T).
[0190] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-8 for peptides.
[0191] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for homology.
[0192] Additionally, sequences identified from cDNA libraries may
be analyzed to identify those gene sequences encoding conserved
protein motifs using an appropriate analysis program, e.g., the
BLOCK 2 bioanalysis program (Incyte, Palo Alto, Calif.). This motif
analysis program, based on sequence information contained in the
Swiss-Prot Database and PROSITE, is a method of determining the
function of uncharacterized proteins translated from genomic or
cDNA sequences. (See, e.g., Bairoch, A. et al. (1997) Nucleic Acids
Res. 25:217-221; and Attwood, T. K. et al. (1997) J. Chem. Inf.
Comput. Sci. 37:417-424.) PROSITE may be used to identify common
functional or structural domains in divergent proteins. The method
is based on weight matrices. Motifs identified by this method are
then calibrated against the SWISS-PROT database in order to obtain
a measure of the chance distribution of the matches.
[0193] In another alternative, Hidden Markov models (HMMs) may be
used to find protein domains, each defined by a dataset of proteins
known to have a common biological function. (See, e.g., Pearson, W.
R. and D. J. Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and
Smith, T. F. and M. S. Waterman (1981) J. Mol. Biol. 147:195-197.)
HNMs were initially developed to examine speech recognition
patterns, but are now being used in a biological context to analyze
protein and nucleic acid sequences as well as to model protein
structure. (See, e.g., Krogh, A. et al. (1994) J. Mol. Biol.
235:1501-1531; and Collin, M. et al. (1993) Protein Sci.
2:305-314.) HMMs have a formal probabilistic basis and use
position-specific scores for amino acids or nucleotides. The
algorithm continues to incorporate information from newly
identified sequences to increase its motif analysis
capabilities.
[0194] IV. Northern Analysis
[0195] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and
16.)
[0196] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). This
analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or homologous.
[0197] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0198] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0199] The results of northern analysis are reported as a list of
libraries in which the transcript encoding NRPK occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0200] V. Extension of NRPK Encoding Polynucleotides
[0201] The nucleic acid sequence of Incyte Clone 3069734 was used
to design oligonucleotide primers for extending a partial
nucleotide sequence to full length. One primer was synthesized to
initiate extension of an antisense polynucleotide, and the other
was synthesized to initiate extension of a sense polynucleotide.
Primers were used to facilitate the extension of the known sequence
"outward" generating amplicons containing new unknown nucleotide
sequence for the region of interest. The initial primers were
designed from the cDNA using OLIGO 4.06 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 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.
[0202] Selected human cDNA libraries (GIBCO/BRL) were used to
extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0203] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (PE Biosystems) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, Mass.),
beginning with 40 pmol of each primer and the recommended
concentrations of all other components of the kit, with the
following parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat steps 4 through 6 for an
additional 15 cycles Step 8 94.degree. C. for 15 sec Step 9
65.degree. C. for 1 min Step 10 68.degree. C. for 7:15 min Step 11
Repeat steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0204] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQuick (QIAGEN
Inc.), and trimmed of overhangs using Klenow enzyme to facilitate
religation and cloning.
[0205] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium. (See, e.g., Sambrook, supra,
Appendix A, p. 2.) After incubation for one hour at 37.degree. C.,
the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin
(2.times. carb). The following day, several colonies were randomly
picked from each plate and cultured in 150 .mu.l of liquid
LB/2.times. Carb medium placed in an individual well of an
appropriate commercially-available sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and, after dilution
1:10 with water, 5 .mu.l from each sample was transferred into a
PCR array.
[0206] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0207] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0208] In like manner, the nucleotide sequence of SEQ ID NO:2 is
used to obtain 5'regulatory sequences using the procedure above,
oligonucleotides designed for 5'extension, and an appropriate
genomic library.
[0209] VI. Labeling and Use of Individual Hybridization Probes
[0210] 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 primer analysis
software (National Biosciences) and labeled by combining 50 pmol of
each oligomer, 250 .mu.Ci of [.gamma.-.sup.32P] adenosine
triphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide
kinase (DuPont NEN, Boston, Mass.). The labeled oligonucleotides
are substantially purified using a SEPHADEX G-25 superfine resin
column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquot
containing 10.sup.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, Boston, Mass.).
[0211] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to NYTRAN PLUS nylon membranes
(Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times. saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR
autoradiography film (Kodak, Rochester, N.Y.) is exposed to the
blots in a PHOSPHORIMAGER cassette (Molecular Dynamics, Sunnyvale,
Calif.) for several hours, hybridization patterns are compared
visually.
[0212] VII. Microarrays
[0213] A chemical coupling procedure and an inkjet 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.
[0214] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE. Full-length cDNAs, ESTs,
or fragments thereof corresponding to one of the nucleotide
sequences of the present invention, or selected at random from a
cDNA library relevant to the present invention, are arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to
the slide using, e.g., UV cross-linking followed by thermal and
chemical treatments and subsequent drying. (See, e.g., Schena, M.
et al. (1995) Science 270:467-470; and Shalon, D. et al. (1996)
Genome Res. 6:639-645.) Fluorescent probes are prepared and used
for hybridization to the elements on the substrate. The substrate
is analyzed by procedures described above.
[0215] VIII. Complementary Polynucleotides
[0216] Sequences complementary to the NRPK-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring NRPK. 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 primer analysis software and the coding
sequence of NRPK. 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 NRPK-encoding transcript.
[0217] IX. Expression of NRPK
[0218] Expression of NRPK is accomplished by subcloning the cDNA
into an appropriate vector and transforming the vector into host
cells. This vector contains an appropriate promoter, e.g.,
.beta.-galactosidase, upstream of the cloning site, operably
associated with the cDNA of interest. (See, e.g., Sambrook, supra,
pp.404-433; and Rosenberg, M. et al. (1983) Methods Enzymol.
101:123-138.)
[0219] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopyranoside (IPTG) using standard
methods produces a fusion protein which consists of the first 8
residues of .beta.-galactosidase, about 5 to 15 residues of linker,
and the full length protein. The signal residues direct the
secretion of NRPK into bacterial growth media which can be used
directly in the following assay for activity.
[0220] X. Demonstration of NRPK Activity
[0221] NRPK activity may be measured by phosphorylation of a
protein substrate using gamma-labeled .sup.32P-ATP and quantitation
of the incorporated radioactivity using a gamma radioisotope
counter. NRPK is incubated with the protein substrate,
.sup.32P-ATP, and an appropriate kinase buffer. The .sup.32P
incorporated into the product is separated from free .sup.32P-ATP
by electrophoresis and the incorporated .sup.32p is counted. The
amount of .sup.32P recovered is proportional to the activity of
HPKM in the assay. A determination of the specific amino acid
residue phosphorylated is made by phosphoamino acid analysis of the
hydrolyzed protein.
[0222] XI. Production of NRPK Specific Antibodies
[0223] NRPK substantially purified using PAGE electrophoresis (see,
e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or
other purification techniques, is used to immunize rabbits and to
produce antibodies using standard protocols.
[0224] Alternatively, the NRPK amino acid sequence is analyzed
using LASERGENE software (DNASTAR Inc.) to determine regions of
high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Methods for selection of appropriate epitopes,
such as those near the C-terminus or in hydrophilic regions are
well described in the art. (See, e.g., Ausubel supra, ch. 11.)
[0225] Typically, oligopeptides 15 residues in length are
synthesized using an ABI431A peptide synthesizer (PE Biosystems)
using fmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by
reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase immunogenicity. (See, e.g., Ausubel supra.) Rabbits are
immunized with the oligopeptide-KLH complex in complete Freund's
adjuvant. Resulting antisera are tested for antipeptide activity,
for example, by binding the peptide to plastic, blocking with 1%
BSA, reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
[0226] XII. Purification of Naturally Occurring NRPK Using Specific
Antibodies
[0227] Naturally occurring or recombinant NRPK is substantially
purified by immunoaffinity chromatography using antibodies specific
for NRPK. An immunoaffinity column is constructed by covalently
coupling anti-NRPK antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (Pharmacia & Upjohn). After
the coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0228] Media containing NRPK are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of NRPK (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/NRPK 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 NRPK is collected.
[0229] XIII. Identification of Molecules Which Interact with
NRPK
[0230] NRPK, 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
NRPK, washed, and any wells with labeled NRPK complex are assayed.
Data obtained using different concentrations of NRPK are used to,
calculate values for the number, affinity, and association of NRPK
with the candidate molecules.
[0231] 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
3 1 293 PRT Homo sapiens misc_feature Incyte ID No 3069734CD1 1 Met
Asp Glu Gln Ser Gln Gly Met Gln Gly Pro Pro Val Pro Gln 1 5 10 15
Phe Gln Pro Gln Lys Ala Leu Arg Pro Asp Met Gly Tyr Asn Thr 20 25
30 Leu Ala Asn Phe Arg Ile Glu Lys Lys Ile Gly Arg Gly Gln Phe 35
40 45 Ser Glu Val Tyr Arg Ala Ala Cys Leu Leu Asp Gly Val Pro Val
50 55 60 Ala Leu Lys Lys Val Gln Ile Phe Asp Leu Met Asp Ala Lys
Ala 65 70 75 Arg Ala Asp Cys Ile Lys Glu Ile Asp Leu Leu Lys Gln
Leu Asn 80 85 90 His Pro Asn Val Ile Lys Tyr Tyr Ala Ser Phe Ile
Glu Asp Asn 95 100 105 Glu Leu Asn Ile Val Leu Glu Leu Ala Asp Ala
Gly Asp Leu Ser 110 115 120 Arg Met Ile Lys His Phe Lys Lys Gln Lys
Arg Leu Ile Pro Glu 125 130 135 Arg Thr Val Trp Lys Tyr Phe Val Gln
Leu Cys Ser Ala Leu Glu 140 145 150 His Met His Ser Arg Arg Val Met
Phe Ile Thr Ala Thr Gly Val 155 160 165 Val Lys Leu Gly Asp Leu Gly
Leu Gly Arg Phe Phe Ser Ser Lys 170 175 180 Thr Thr Ala Ala His Ser
Leu Val Gly Thr Pro Tyr Tyr Met Ser 185 190 195 Pro Glu Arg Ile His
Glu Asn Gly Tyr Asn Phe Lys Ser Asp Ile 200 205 210 Trp Ser Leu Gly
Cys Leu Leu Tyr Glu Met Ala Ala Leu Gln Ser 215 220 225 Pro Phe Tyr
Gly Asp Lys Met Asn Leu Tyr Ser Leu Cys Lys Lys 230 235 240 Ile Glu
Gln Cys Asp Tyr Pro Pro Leu Pro Ser Asp His Tyr Ser 245 250 255 Glu
Glu Leu Arg Gln Leu Val Asn Met Cys Ile Asn Pro Asp Pro 260 265 270
Glu Lys Arg Pro Asp Val Thr Tyr Val Tyr Asp Val Ala Lys Arg 275 280
285 Met His Ala Cys Thr Ala Ser Ser 290 2 4170 DNA Homo sapiens
misc_feature Incyte ID No 3069734CB1 2 gcccttgccg ccagggggga
aaagtgggga accttcccct tggcagactt cattgagtaa 60 tttccaggcc
gccccctttt acctccatgg cggaagttgg ccgcctggca ttatcccaag 120
aacatgccct tatgggcctt cccactttgc aagtacatcg acgtattagt cctcgctatt
180 cccatgttat ggggatttgc cagtacatcc atgggcttga taagggtttg
actcgcgggg 240 atttccaagt ctccacccaa ttgacgtcaa gggaagttgt
tttggcaaca aaatcacggg 300 gacttcccaa aatgtcgtaa ctactccgcg
ccattaaccc aaatggncgg aagggttcct 360 gttgcttcag acaatggatg
agcaatcaca aggaatgcaa gggccacctg ttcctcagtt 420 ccaaccacag
aaggccttac gaccggatat gggctataat acattagcca actttcgaat 480
agaaaagaaa attggtcgcg gacaatttag tgaagtttat agagcagcct gtctcttgga
540 tggagtacca gtagctttaa aaaaagtgca gatatttgat ttaatggatg
ccaaagcacg 600 tgctgattgc atcaaagaaa tagatcttct taagcaactc
aaccatccaa atgtaataaa 660 atattatgca tcattcattg aagataatga
actaaacata gttttggaac tagcagatgc 720 tggcgaccta tccagaatga
tcaagcattt taagaagcaa aagaggctaa ttcctgaaag 780 aactgtttgg
aagtattttg ttcagctttg cagtgcattg gaacacatgc attctcgaag 840
agtcatgttc attacagcca ctggggtggt aaaacttgga gatcttgggc ttggccggtt
900 tttcagctca aaaaccacag ctgcacattc tttagttggt acgccttatt
acatgtctcc 960 agagagaata catgaaaatg gatacaactt caaatctgac
atctggtctc ttggctgtct 1020 actatatgag atggctgcat tacaaagtcc
tttctatggt gacaaaatga atttatactc 1080 actgtgtaag aagatagaac
agtgtgacta cccacctctt ccttcagatc actattcaga 1140 agaactccga
cagttagtta atatgtgcat caacccagat ccagagaagc gaccagacgt 1200
cacctatgtt tatgacgtag caaagaggat gcatgcatgc actgcaagca gctaaacatg
1260 caagatcatg aagagtgtaa ccaaagtaat tgaaagtatt ttgtgcaagt
catacctccc 1320 catttatgtc tggtgttaag attaatattt cagagctagt
gtgctttgaa tccttaacca 1380 gttttcatat aagcttcatt ttgtaccagt
cacctaaatc acctccttgc aacccccaaa 1440 tgactttgga ataactgaat
tgcatgttag gagagaaaat gaaacatgat ggttttgaat 1500 ggctaaaggt
ttatagaatt tcttacagtt ttctgctgat aaattgtgtt tagatagact 1560
gtcagtgcca aatattgaag gtgcagcttg gcacacatca gaatagactc atacctgaga
1620 aaaagtatct gaacatgtga cttgtttctt ttttagtaat ttatggacat
tgagatgaac 1680 acaattgtga acttttgtga agattttatt tttaaacgtt
tgaagtacta gttttagttc 1740 ttagcagagt agttttcaaa tatgattctt
atgataaatg tagacacaaa ctatttgaga 1800 aacatttaga actcttagct
tatacattca aaatgtaact attaaatgtg aagatttggg 1860 gacaaaatgt
gagtcagaca ctgaagagtt ttttgttttg ttttaatatt tttgatattc 1920
tctttgcatt gaaatggtat aaatgaatcc atttaaaaag tggttaagga tttgtttagc
1980 tggtgtgata ataattttta aagttgcaca ttgcccaagg ctttttttgt
gtgtttttat 2040 tgttgtttgt acatttgaaa aatattcttt gaataacctt
gcagtactat atttcaattt 2100 ctttataaat ttaagtgcat tttaactcat
aattgtacac tataatataa gcctaagttt 2160 ttattcataa gttttattga
agttctgatc ggtccccttc agaaattttt ttatattatt 2220 cttcaagtta
ctttcttatt tatattgtat gtgcatttta tccattaatg tttcatactt 2280
tctgagagta taataccctt ttaaaagata tttggtatac caatactttt cctggattga
2340 aaactttttt taaacttttt aaaatttggg ccactctgta tgcatatgtt
tggtcttgtt 2400 aaagaggaag aaaggatgtg tgttatactg tacctgtgaa
tgttgataca gttacaattt 2460 atttgacaag gttgtaattc tagaatatgc
ttaataaaat gaaaactggc catgactaca 2520 gccagaactg ttatgagatt
aacatttcta ttgagaagct tttgagtaaa gtactgtatt 2580 tgttcatgaa
gatgactgag atggtaacac ttcgtgtagc ttaaggaaat gggcagaatt 2640
tcgtaaatgc tgttgtgcag atgtgttttc cctgaatgct ttcgtattag tggcgaccag
2700 tttctcacag aattgtgaag cctgaaggcc aagaggaagt cactgttaaa
ggactctgtg 2760 ccatcttaca accttggatg aattatcctg ccaacgtgaa
aacctcatgt tcaaagaaca 2820 cttcccttta gccgatgtaa ctgctggttt
tgtttttcat atgtgttttt cttacactca 2880 tttgaatgct ttcaagcatt
tgtaaactta aaaaatgtat aaagggcaaa aagtctgaac 2940 ccttgttttc
tgaaatctaa tcagttatgt atggtttctg aagggtaatt ttattttgga 3000
ataggtaaag gaaacctgtt ttgtttgttt ttcctgaggg ctagatgcat tttttttctc
3060 acactcttaa tgacttttaa catttatact gagcatccat agatatattc
ctagaagtat 3120 gagaagaatt attcttattg accattaatg tcatgttcat
tttaatgtaa tataattgag 3180 atgaaatgtt ctctggttgg aacagatact
ctcttttttt tcttgcaatc tttaagaata 3240 catagatcta aaattcatta
gcttgacccc tcaaagtaac ttttaagtaa agattaaagc 3300 ttttcttctc
agtgaatata tctgctagaa ggaaatagct gggaagaatt taatgatcag 3360
ggaaattcat tatttctata tgtggaaact ttttgcttcg aatattgtat ctttttaaat
3420 ctaaatgttc atatttttcc tgaagaaacc actgtgtaaa aatcaaattt
taattttgaa 3480 tggaataatt tcaaagaact atgaagatga tttgaagctc
taatttatat agtcacctat 3540 aaaatgttct ttatatgtgt tcataagtaa
attttatatt gattaagtta aacttttgaa 3600 ttgatttgag gagcagtaaa
atgaaagcta tatctattct aaaccttatt tagacattgg 3660 taccagttac
ccaggtgaaa atatggagta actttgtttt gtatggtaag gtttaggaat 3720
ggtggatgaa gggtatctct atataaataa agtgctcaac aatgtgcaat gattgtaaat
3780 ttagtaagat attacagcca tttcatgaat gctttaccat tcaacatagt
atctattaca 3840 aaacaccttt cttgtatcca tatacttcag gtgttgctgt
taacatttac tatgatattt 3900 attttaacca aaatgttact cacattaaat
gtttattctt taaaatgaat gtattatgtt 3960 tttaacccac aaatgcatac
ttaccctgtg cctcatattt caatagtact gtaatatgga 4020 catcttttgt
gaaatacttt tattttgtta tgctttaaat atacatacaa aaagatttct 4080
gttattagct ttgaaaattg tataatatcc taatataaac aaaaatataa aaataaaaat
4140 gaatacagta aaatgtcaaa aaaaaaaaaa 4170 3 239 PRT Homo sapiens
misc_feature GenBank ID No g1082115 3 Val Phe Glu Met Val Asp Gln
Lys Ala Arg Gln Asp Cys Leu Lys 1 5 10 15 Glu Ile Asp Leu Leu Lys
Gln Leu Asn His Val Asn Val Ile Arg 20 25 30 Tyr Tyr Ala Ser Phe
Ile Asp Asn Asn Gln Leu Asn Ile Val Leu 35 40 45 Glu Leu Ala Glu
Ala Gly Asp Met Ser Arg Met Ile Lys His Phe 50 55 60 Lys Lys Gly
Gly Arg Leu Ile Pro Glu Lys Thr Ile Trp Lys Tyr 65 70 75 Phe Val
Gln Leu Ala Arg Ala Leu Ala His Met His Ser Lys Arg 80 85 90 Ile
Met His Arg Asp Ile Lys Pro Ala Asn Val Phe Ile Thr Gly 95 100 105
Asn Gly Ile Val Lys Leu Gly Asp Leu Gly Leu Gly Arg Phe Phe 110 115
120 Ser Ser Lys Thr Thr Ala Ala His Ser Leu Val Gly Thr Pro Tyr 125
130 135 Tyr Met Ser Pro Glu Arg Ile Gln Glu Ser Gly Tyr Asn Phe Lys
140 145 150 Ser Asp Leu Trp Ser Thr Gly Cys Leu Leu Tyr Glu Met Ala
Ala 155 160 165 Leu Gln Ser Pro Phe Tyr Gly Asp Lys Met Asn Leu Tyr
Ser Leu 170 175 180 Cys Lys Lys Ile Glu Asn Cys Glu Tyr Pro Pro Leu
Pro Ala Asp 185 190 195 Ile Tyr Ser Thr Gln Val Ser Ala Asn Leu Cys
Phe Val Gln Leu 200 205 210 Ser Ser Ala Thr Trp Tyr Pro Val Val Tyr
Phe Gln Lys Leu Gln 215 220 225 Asn Asp Gln Arg Pro Val Lys Phe Tyr
Arg Phe Val Pro Arg 230 235
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