U.S. patent application number 09/870962 was filed with the patent office on 2002-06-27 for protein kinase homologs.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Azimzai, Yalda, Bandman, Olga, Corley, Neil C., Gorgone, Gina A., Guegler, Karl J., Hillman, Jennifer L., Lu, Dyung Aina M., Tang, Y. Tom, Yue, Henry.
Application Number | 20020081290 09/870962 |
Document ID | / |
Family ID | 22632671 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081290 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
June 27, 2002 |
Protein kinase homologs
Abstract
The invention provides human protein kinase homologs (PKH) and
polynucleotides which identify and encode PKH. 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
PKH.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Tang, Y. Tom; (San Jose, CA) ;
Hillman, Jennifer L.; (Mountain View, CA) ; Yue,
Henry; (Sunnyvale, CA) ; Guegler, Karl J.;
(Menlo Park, CA) ; Corley, Neil C.; (Mountain
View, CA) ; Gorgone, Gina A.; (Boulder Creek, CA)
; Azimzai, Yalda; (Union City, CA) ; Lu, Dyung
Aina M.; (San Jose, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
22632671 |
Appl. No.: |
09/870962 |
Filed: |
May 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09870962 |
May 30, 2001 |
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09420915 |
Oct 20, 1999 |
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09420915 |
Oct 20, 1999 |
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09173581 |
Oct 15, 1998 |
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Current U.S.
Class: |
424/94.5 ;
435/194; 435/252.3; 435/325; 435/69.1; 536/23.2; 800/8 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
31/12 20180101; A61P 1/04 20180101; A61P 15/14 20180101; A61P 17/06
20180101; C12Q 1/6883 20130101; A61P 15/12 20180101; A61P 35/00
20180101; A61P 15/04 20180101; A61P 15/06 20180101; A61P 3/10
20180101; A61P 37/02 20180101; A61P 37/08 20180101; A61P 29/00
20180101; A61P 7/06 20180101; A61P 27/02 20180101; A61P 21/04
20180101; A61P 1/00 20180101; A61P 19/02 20180101; A61P 1/16
20180101; A61P 15/08 20180101; A61P 19/10 20180101; A61P 33/10
20180101; A61P 17/00 20180101; A61P 7/04 20180101; A61P 33/00
20180101; A61P 33/02 20180101; C12N 9/1205 20130101; A61P 33/14
20180101; A61P 15/10 20180101; A61P 11/06 20180101; A61P 31/04
20180101; C12Q 2600/158 20130101 |
Class at
Publication: |
424/94.5 ;
435/194; 435/325; 435/252.3; 800/8; 435/69.1; 536/23.2 |
International
Class: |
A01K 067/00; A61K
038/53; C07H 021/04; C12P 021/02; C12N 005/06; C12N 009/12; C12N
001/21 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:1-9, b) a naturally occurring
polypeptide comprising an amino acid sequence at least 90%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NO: 1-9, c) a biologically active fragment of
a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-9.
2. An isolated polypeptide of claim 1 selected from the group
consisting of SEQ ID NO:1-9.
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 selected from the group
consisting of SEQ ID NO:10-18.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. An isolated antibody which specifically binds to a polypeptide
of claim 1.
11. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:10-18, b) a
naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:10-18, 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).
12. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 11.
13. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 1, 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.
14. A method of claim 13, wherein the probe comprises at least 60
contiguous nucleotides.
15. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, 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.
16. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
17. A composition of claim 16, wherein the polypeptide has an amino
acid sequence selected from the group consisting of SEQ ID
NO:1-9.
18. A method for treating a disease or condition associated with
decreased expression of functional PKH, comprising administering to
a patient in need of such treatment the composition of claim
16.
19. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
20. A composition comprising an agonist compound identified by a
method of claim 19 and a pharmaceutically acceptable excipient.
21. A method for treating a disease or condition associated with
decreased expression of functional PKH, comprising administering to
a patient in need of such treatment a composition of claim 20.
22. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
23. A composition comprising an antagonist compound identified by a
method of claim 22 and a pharmaceutically acceptable excipient.
24. A method for treating a disease or condition associated with
overexpression of functional PKH, comprising administering to a
patient in need of such treatment a composition of claim 23.
25. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, said method comprising the steps of: a)
combining the polypeptide of claim 1 with at least one test
compound under suitable conditions, and b) detecting binding of the
polypeptide of claim 1 to the test compound, thereby identifying a
compound that specifically binds to the polypeptide of claim 1.
26. 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.
27. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
28. A method for assessing toxicity of a test compound, said method
comprising: a) treating a biological sample containing nucleic
acids with the test compound; b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 11 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 11 or fragment thereof; c)
quantifying the amount of hybridization complex; and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
29. A diagnostic test for a condition or disease associated with
the expression of PKH in a biological sample comprising the steps
of: a) combining the biological sample with an antibody of claim
10, 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.
30. The antibody of claim 10, 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.
31. A composition comprising an antibody of claim 10 and an
acceptable excipient.
32. A method of diagnosing a condition or disease associated with
the expression of PKH in a subject, comprising administering to
said subject an effective amount of the composition of claim
31.
33. A composition of claim 31, wherein the antibody is labeled.
34. A method of diagnosing a condition or disease associated with
the expression of PKH in a subject, comprising administering to
said subject an effective amount of the composition of claim
33.
35. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 10 comprising: a) immunizing
an animal with a polypeptide having an amino acid sequence selected
from the group consisting of SEQ ID NO:1-9, or an immunogenic
fragment thereof, under conditions to elicit an antibody response;
b) isolating antibodies from said animal; and c) screening the
isolated antibodies with the polypeptide, thereby identifying a
polyclonal antibody which binds specifically to a polypeptide
having an amino acid sequence selected from the group consisting of
SEQ ID NO:1-9.
36. An antibody produced by a method of claim 35.
37. A composition comprising the antibody of claim 36 and a
suitable carrier.
38. A method of making a monoclonal antibody with the specificity
of the antibody of claim 10 comprising: a) immunizing an animal
with a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID NO:1-9, or an immunogenic fragment
thereof, under conditions to elicit an antibody response; b)
isolating antibody producing cells from the animal; c) fusing the
antibody producing cells with immortalized cells to form monoclonal
antibody-producing hybridoma cells; d) culturing the hybridoma
cells; and e) isolating from the culture monoclonal antibody which
binds specifically to a polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO:1-9.
39. A monoclonal antibody produced by a method of claim 38.
40. A composition comprising the antibody of claim 39 and a
suitable carrier.
41. The antibody of claim 10, wherein the antibody is produced by
screening a Fab expression library.
42. The antibody of claim 10, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
43. A method for detecting a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-9 in a
sample, comprising the steps of: a) incubating the antibody of
claim 10 with a sample under conditions to allow specific binding
of the antibody and the polypeptide; and b) detecting specific
binding, wherein specific binding indicates the presence of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-9 in the sample.
44. A method of purifying a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-9 from a
sample, the method comprising: a) incubating the antibody of claim
10 with a sample under conditions to allow specific binding of the
antibody and the polypeptide; and b) separating the antibody from
the sample and obtaining the purified polypeptide having an amino
acid sequence selected from the group consisting of SEQ ID
NO:1-9.
45. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:1.
46. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:2.
47. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:3.
48. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:4.
49. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:5.
50. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:6.
51. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:7.
52. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:8.
53. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:9.
54. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:10.
55. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:11.
56. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:12.
57. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:13
58. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:14.
59. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:15.
60. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:16.
61. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:17.
62. A polynucleotide of claim 11, comprising the polynucleotide
sequence of SEQ ID NO:18.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/420,915 filed on Oct. 20, 1999, which is a
divisional application of U.S. application Ser. No. 09/173,581
filed on Oct. 15, 1998, issued on Jan. 11, 2000, as U.S. Pat. No.
6,013,455, entitled PROTEIN KINASE HOMOLOGS, the contents all of
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of protein kinase homologs and to the use of these
sequences in the diagnosis, treatment, and prevention of cancer,
autoimmune/inflammatory disorders, and reproductive disorders.
BACKGROUND OF THE INVENTION
[0003] Kinases and phosphatases are critical components of
intracellular signal transduction mechanisms. Kinases catalyze the
transfer of high energy phosphate groups from adenosine
triphosphate (ATP) to hydroxyamino acids of various target
proteins. Phosphatases, in contrast, remove phosphate groups from
proteins. Reversible protein phosphorylation is the main strategy
for regulating protein activity in eukaryotic cells. In general,
proteins are activated by phosphorylation in response to
extracellular signals such as hormones, neurotransmitters, and
growth and differentiation factors. Protein dephosphorylation
occurs when down-regulation of a signaling pathway is required. The
combined activities of kinases and phosphatases regulate key
cellular processes such as proliferation, differentiation, and cell
cycle progression.
[0004] Kinases comprise the largest known enzyme superfamily and
vary widely in their target proteins. Kinases may be categorized as
protein tyrosine kinases (PTKs), which phosphorylate tyrosine
residues, and protein serine/threonine kinases (STKs), which
phosphorylate serine and/or threonine residues. Some kinases have
dual specificity for both serine/threonine and tyrosine residues.
Almost all kinases contain a conserved 250-300 amino acid catalytic
domain. This domain can be further divided into 11 subdomains.
N-terminal subdomains I-IV fold into a two-lobed structure which
binds and orients the ATP donor molecule, and subdomain V spans the
two lobes. C-terminal subdomains VI-XI bind the protein substrate
and transfer the gamma phosphate from ATP to the hydroxyl group of
a serine, threonine, or tyrosine residue. Each of the 11 subdomains
contains specific catalytic residues or amino acid motifs
characteristic of that subdomain. For example, subdomain I contains
an 8-amino acid glycine-rich ATP binding consensus motif, subdomain
II contains a critical lysine residue required for maximal
catalytic activity, and subdomains VI through IX comprise the
highly conserved catalytic core. STKs and PTKs also contain
distinct sequence motifs in subdomains VI and VIII which may confer
hydroxyamino acid specificity. Some STKs and PTKs possess
structural characteristics of both families. In addition, kinases
may also be classified by additional amino acid sequences,
generally between 5 and 100 residues, which either flank or occur
within the kinase domain. These additional amino acid sequences
regulate kinase activity and determine substrate specificity.
(Reviewed in Hardie, G. and Hanks, S. (1995) The Protein Kinase
Facts Book, Vol 1:7-20 Academic Press, San Diego, Calif.)
[0005] PTKs may be classified as either transmembrane or
non-transmembrane proteins. Transmembrane tyrosine kinases function
as receptors for most growth factors. Binding of growth factor to
the receptor activates the transfer of a phosphate group from ATP
to selected tyrosine residues in the receptor itself and in
specific second messenger proteins. Growth factors (GF) that
associate 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.
[0006] Non-transmembrane PTKs form signaling complexes with the
cytosolic domains of plasma membrane receptors. Receptors that
signal through non-transmembrane PTKs include cytokine, hormone,
and antigen-specific lymphocytic receptors. Many PTKs were first
identified as oncogene products in cancer cells in which PTK
activation was no longer subject to normal cellular controls. In
fact, about one third of the known oncogenes encode PTKs.
Furthermore, cellular transformation (oncogenesis) is often
accompanied by increased tyrosine phosphorylation activity.
(Carbonneau, 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.
[0007] The discovery of new protein kinase homologs and the
polynucleotides encoding them satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of cancer, autoimmune/inflammatory
disorders, and reproductive disorders.
SUMMARY OF THE INVENTION
[0008] The invention features substantially purified polypeptides,
protein kinase homologs, referred to collectively as "PKH" and
individually as "PKH-1", "PKH-2", "PKH-3", "PKH-4", "PKH-5",
"PKH-6", "PKH-7", "PKH-8", and "PKH-9". In one aspect, the
invention provides a substantially purified polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, (SEQ ID NO:1-9), and
fragments thereof.
[0009] The invention further provides a substantially purified
variant having at least 90% amino acid identity to at least one of
the amino acid sequences selected from the group consisting of SEQ
ID NO:1-9, and fragments thereof. The invention also provides an
isolated and purified polynucleotide encoding the polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-9, and fragments thereof. The invention
also includes an isolated and purified polynucleotide variant
having at least 70% polynucleotide sequence identity to the
polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-9, and
fragments thereof.
[0010] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-9,
and fragments thereof. The invention also provides an isolated and
purified polynucleotide having a sequence which is complementary to
the polynucleotide encoding the polypeptide comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:1-9,
and fragments thereof.
[0011] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, (SEQ ID NO: 10-18) and fragments thereof. The
invention further provides an isolated and purified polynucleotide
variant having at least 70% polynucleotide sequence identity to the
polynucleotide sequence selected from the group consisting of SEQ
ID NO: 10-18, and fragments thereof. The invention also provides an
isolated and purified polynucleotide having a sequence which is
complementary to the polynucleotide comprising a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 10-18,
and fragments thereof.
[0012] The invention also provides a method for detecting a
polynucleotide in a sample containing nucleic acids, the method
comprising the steps of (a) hybridizing the complement of the
polynucleotide sequence to at least one of the polynucleotides of
the sample, thereby forming a hybridization complex; and (b)
detecting the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of a
polynucleotide in the sample. In one aspect, the method further
comprises amplifying the polynucleotide prior to hybridization.
[0013] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-9, and fragments thereof. In
another aspect, the expression vector is contained within a host
cell.
[0014] The invention also provides a method for producing a
polypeptide, the method comprising the steps of: (a) culturing the
host cell containing an expression vector containing at least a
fragment of a polynucleotide under conditions suitable for the
expression of the polypeptide; and (b) recovering the polypeptide
from the host cell culture.
[0015] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO:1-9,
and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
[0016] The invention further includes a purified antibody which
binds to a polypeptide selected from the group consisting of SEQ ID
NO:1-9, and fragments thereof. The invention also provides a
purified agonist and a purified antagonist to the polypeptide.
[0017] The invention also provides a method for treating or
preventing a disorder of cell proliferation associated with
decreased expression or activity of PKH, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising a substantially
purified polypeptide having the amino acid sequence selected from
the group consisting of SEQ ID NO:1-9, and fragments thereof, in
conjunction with a suitable pharmaceutical carrier.
[0018] The invention also provides a method for treating or
preventing a disorder of cell proliferation associated with
increased expression or activity of PKH, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-9, and
fragments thereof.
BRIEF DESCRIPTION OF THE TABLES
[0019] Table 1 shows nucleotide and polypeptide sequence
identification numbers (SEQ ID NO), clone identification numbers
(clone ID), cDNA libraries, and cDNA fragments used to assemble
full-length sequences encoding PKH.
[0020] Table 2 shows features of each polypeptide sequence
including potential motifs, homologous sequences, and methods and
algorithms used for characterization of PKH.
[0021] Table 3 shows the tissue-specific expression patterns of
each nucleic acid sequence as determined by northern analysis,
diseases, disorders, or conditions associated with these tissues,
and the vector into which each cDNA was cloned.
[0022] Table 4 describes the tissues used to construct the cDNA
libraries from which Incyte cDNA clones encoding PKH were
isolated.
[0023] Table 5 shows the programs, their descriptions, references,
and threshold parameters used to analyze PKH.
DESCRIPTION OF THE INVENTION
[0024] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular machines, materials and methods
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
[0025] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0026] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any machines, materials, and methods similar or equivalent to those
described herein can be used to practice or test the present
invention, the preferred machines, materials and methods are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, protocols,
reagents and vectors which are reported in the publications and
which might be used in connection with the invention. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0027] Definitions
[0028] "PKH" refers to the amino acid sequences of substantially
purified PKH obtained from any species, particularly a mammalian
species, including bovine, ovine, porcine, murine, equine, and
preferably the human species, from any source, whether natural,
synthetic, semi-synthetic, or recombinant.
[0029] The term "agonist" refers to a molecule which, when bound to
PKH, increases or prolongs the duration of the effect of PKH.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules which bind to and modulate the effect of PKH.
[0030] An "allelic variant" is an alternative form of the gene
encoding PKH. Allelic variants may result from at least one
mutation in the nucleic acid sequence and may result in altered
mRNAs or in polypeptides whose structure or function may or may not
be altered. Any given natural or recombinant gene may have none,
one, or many allelic forms. Common mutational changes which give
rise to allelic variants are generally ascribed to natural
deletions, additions, or substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the
others, one or more times in a given sequence.
[0031] "Altered" nucleic acid sequences encoding PKH include those
sequences with deletions, insertions, or substitutions of different
nucleotides, resulting in a polynucleotide the same as PKH or a
polypeptide with at least one functional characteristic of PKH.
Included within this definition are polymorphisms which may or may
not be readily detectable using a particular oligonucleotide probe
of the polynucleotide encoding PKH, and improper or unexpected
hybridization to allelic variants, with a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
PKH. 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 PKH. 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 PKH is retained. For example, negatively charged amino acids may
include aspartic acid and glutanic acid, positively charged amino
acids may include lysine and arginine, and amino acids with
uncharged polar head groups having similar hydrophilicity values
may include leucine, isoleucine, and valine; glycine and alanine;
asparagine and glutamine; serine and threonine; and phenylalanine
and tyrosine.
[0032] The terms "amino acid" or "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. In this context, "fragments," "immunogenic fragments,"
or "antigenic fragments" refer to fragments of PKH which are
preferably at least 5 to about 15 amino acids in length, most
preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of PKH. Where "amino
acid sequence" is recited to refer to an amino acid sequence of a
naturally occurring protein molecule, "amino acid sequence" and
like terms are not meant to limit the amino acid sequence to the
complete native amino acid sequence associated with the recited
protein molecule.
[0033] "Amplification" relates to the production of additional
copies of a nucleic acid sequence. Amplification is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art.
[0034] The term "antagonist" refers to a molecule which, when bound
to PKH, decreases the amount or the duration of the effect of the
biological or immunological activity of PKH. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of PKH.
[0035] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fab, F(ab').sub.2, and Fv fragments,
which are capable of binding the epitopic determinant. Antibodies
that bind PKH polypeptides can be prepared using intact
polypeptides or using fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can
be derived from the translation of RNA, or synthesized chemically,
and can be conjugated to a carrier protein if desired. Commonly
used carriers that are chemically coupled to peptides include
bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0036] The term "antigenic determinant" refers to that fragment of
a molecule (i.e., an epitope) that makes contact with a particular
antibody. When a protein or a fragment of a protein is used to
immunize a host animal, numerous regions of the protein may induce
the production of antibodies which bind specifically to antigenic
determinants (given regions or three-dimensional structures on the
protein). An antigenic determinant may compete with the intact
antigen (i.e., the immunogen used to elicit the immune response)
for binding to an antibody.
[0037] The term "antisense" refers to any composition containing a
nucleic acid sequence which is complementary to the "sense" strand
of a specific nucleic acid sequence. Antisense molecules may be
produced by any method including synthesis or transcription. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0038] The term "biologically active," refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" refers to
the capability of the natural, recombinant, or synthetic PKH, or of
any oligopeptide thereof, to induce a specific immune response in
appropriate animals or cells and to bind with specific
antibodies.
[0039] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence "5' A-G-T 3'" bonds to the complementary sequence "3'
T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial," such that only some of the nucleic acids bind, or
it may be "complete," such that total complementarity exists
between the single stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of the hybridization between
the nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands, and in the design and use of peptide nucleic acid
(PNA) molecules.
[0040] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence" refer
broadly to any composition containing the given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation
or an aqueous solution. Compositions comprising polynucleotide
sequences encoding PKH or fragments of PKH may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g.,
sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution, dry nilk, salmon sperm DNA, etc.). "Consensus
sequence" refers to a nucleic acid sequence which has been
resequenced to resolve uncalled bases, extended using XL-PCR kit
(Perkin-Elmer, Norwalk Conn.) in the 5' and/or the 3' direction,
and resequenced, or which has been assembled from the overlapping
sequences of more than one Incyte Clone using a computer program
for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison Wis.). Some sequences have been both extended and
assembled to produce the consensus sequence.
[0041] The term "correlates with expression of a polynucleotide"
indicates that the detection of the presence of nucleic acids, the
same or related to a nucleic acid sequence encoding PKH, by
northern analysis is indicative of the presence of nucleic acids
encoding PKH in a sample, and thereby correlates with expression of
the transcript from the polynucleotide encoding PKH.
[0042] A "deletion" refers to a change in the amino acid or
nucleotide sequence that results in the absence of one or more
amino acid residues or nucleotides.
[0043] The term "derivative" refers to the chemical modification of
a polypeptide sequence, or a polynucleotide sequence. Chemical
modifications of a polynucleotide sequence can include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A derivative polynucleotide encodes a polypeptide which retains at
least one biological or immunological function of the natural
molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the polypeptide
from which it was derived.
[0044] The term "similarity" refers to a degree of complementarity.
There may be partial similarity or complete similarity. The word
"identity" may substitute for the word "similarity." A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to as "substantially similar." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or northern blot, solution hybridization, and the like)
under conditions of reduced stringency. A substantially similar
sequence or hybridization probe will compete for and inhibit the
binding of a completely similar (identical) sequence to the target
sequence under conditions of reduced stringency. This is not to say
that conditions of reduced stringency are such that non-specific
binding is permitted, as reduced stringency conditions require that
the binding of two sequences to one another be a specific (i.e., a
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
similarity or identity). In the absence of non-specific binding,
the substantially similar sequence or probe will not hybridize to
the second non-complementary target sequence.
[0045] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MEGALIGN program
(DNASTAR, Madison Wis.) which creates alignments between two or
more sequences according to methods selected by the user, e.g., the
clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988)
Gene 73:237-244.) The clustal algorithm groups sequences into
clusters by examining the distances between all pairs. The clusters
are aligned pairwise and then in groups. The percentage similarity
between two amino acid sequences, e.g., sequence A and sequence B,
is calculated by dividing the length of sequence A, minus the
number of gap residues in sequence A, minus the number of gap
residues in sequence B, into the sum of the residue matches between
sequence A and sequence B, times one hundred. Gaps of low or of no
similarity between the two amino acid sequences are not included in
determining percentage similarity. Percent identity between nucleic
acid sequences can also be counted or calculated by other methods
known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J.
(1990) Methods Enzymol. 183:626-645.) Identity between sequences
can also be determined by other methods known in the art, e.g., by
varying hybridization conditions.
[0046] "Human artificial chromosomes" (HACs) are linear
microchromosomes which may contain DNA sequences of about 6 kb to
10 Mb in size, and which contain all of the elements required for
stable mitotic chromosome segregation and maintenance.
[0047] The term "humanized antibody" refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
[0048] "Hybridization" refers to any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing.
[0049] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary bases. A hybridization complex
may be formed in solution (e.g., 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" refer to changes in an
amino acid or nucleotide sequence resulting in the addition of one
or more amino acid residues or nucleotides, respectively, to the
sequence found in the naturally occurring molecule.
[0051] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0052] The term "microarray" refers to an arrangement of distinct
polynucleotides on a substrate.
[0053] The terms "element" or "array element" in a microarray
context, refer to hybridizable polynucleotides arranged on the
surface of a substrate.
[0054] The term "modulate" refers to a change in the activity of
PKH. For example, modulation may cause an increase or a decrease in
protein activity, binding characteristics, or any other biological,
functional, or immunological properties of PKH.
[0055] The phrases "nucleic acid" or "nucleic acid sequence" refer
to a nucleotide, oligonucleotide, polynucleotide, or any fragment
thereof. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which, when translated, would produce polypeptides retaining some
functional characteristic, e.g., antigenicity, or structural domain
characteristic, e.g., ATP-binding site, of the full-length
polypeptide.
[0056] The terms "operably associated" or "operably linked" refer
to functionally related nucleic acid sequences. A promoter is
operably associated or operably linked with a coding sequence if
the promoter controls the translation of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in the same reading frame, certain genetic
elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0057] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to 60 nucleotides, preferably about
15 to 30 nucleotides, and most preferably about 20 to 25
nucleotides, which can be used in PCR amplification or in a
hybridization assay or microarray. "Oligonucleotide" is
substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the
art. "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises an oligonucleotide of at least
about 5 nucleotides in length linked to a peptide backbone of amino
acid residues ending in lysine. The terminal lysine confers
solubility to the composition. PNAs preferentially bind
complementary single stranded DNA or RNA and stop transcript
elongation, and may be pegylated to extend their lifespan in the
cell.
[0058] The term "sample" is used in its broadest sense. A sample
suspected of containing nucleic acids encoding PKH, or fragments
thereof, or PKH itself, may comprise a bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a
cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a
substrate; a tissue; a tissue print; etc.
[0059] The terms "specific binding" or "specifically binding" refer
to that interaction between a protein or peptide and an agonist, an
antibody, or an antagonist. The interaction is dependent upon the
presence of a particular structure of the protein, e.g., the
antigenic determinant or epitope, recognized by the binding
molecule. For example, if an antibody is specific for epitope "A,"
the presence of a polypeptide containing the epitope A, or the
presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will reduce the amount of labeled A that binds
to the antibody.
[0060] The term "stringent conditions" refers to conditions which
permit hybridization between polynucleotides and the claimed
polynucleotides. Stringent conditions can be defined by salt
concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0061] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90%
free from other components with which they are naturally
associated.
[0062] A "substitution" refers to the replacement of one or more
amino acids or nucleotides by different amino acids or nucleotides,
respectively.
[0063] "Substrate" refers to any suitable rigid or semi-rigid
support including membranes, filters, chips, slides, wafers,
fibers, magnetic or nonmagnetic beads, gels, tubing, plates,
polymers, microparticles and capillaries. The substrate can have a
variety of surface forms, such as wells, trenches, pins, channels
and pores, to which polynucleotides or polypeptides are bound.
[0064] "Transformation" describes a process by which exogenous DNA
enters and changes a recipient cell. Transformation may occur under
natural or artificial conditions according to various methods well
known in the art, and may rely on any known method for the
insertion of foreign nucleic acid sequences into a prokaryotic or
eukaryotic host cell. The method for transformation is selected
based on the type of host cell being transformed and may include,
but is not limited to, viral infection, electroporation, heat
shock, lipofection, and particle bombardment. The term
"transformed" cells includes stably transformed cells in which the
inserted DNA is capable of replication either as an autonomously
replicating plasmid or as part of the host chromosome, as well as
transiently transformed cells which express the inserted DNA or RNA
for limited periods of time.
[0065] A "variant" of PKH polypeptides refers to an amino acid
sequence that is altered by one or more amino acid residues. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software (DNASTAR).
[0066] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to PKH. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) in which the
polynucleotide sequence varies by one base. The presence of SNPs
may be indicative of, for example, a certain population, a disease
state, or a propensity for a disease state.
[0067] The Invention
[0068] The invention is based on the discovery of new human protein
kinase homologs (PKH), the polynucleotides encoding PKH, and the
use of these compositions for the diagnosis, treatment, or
prevention of cancer, autoimmune/inflammatory disorders, and
reproductive disorders.
[0069] Table 1 lists the Incyte Clones used to derive full length
nucleotide sequences encoding PKH. Columns 1 and 2 show the
sequence identification numbers (SEQ ID NO) of the amino acid and
nucleic acid sequences, respectively. Column 3 shows the Clone ID
of the Incyte Clone in which nucleic acids encoding each PKH were
identified, and column 4, the cDNA libraries from which these
clones were isolated. Column 5 shows Incyte clones, their
corresponding cDNA libraries, and shotgun sequences. The clones and
shotgun sequences are part of the consensus nucleotide sequence of
each PKH and are useful as fragments in hybridization
technologies.
[0070] The columns of Table 2 show various properties of the
polypeptides of the invention: column 1 references the amino acid
SEQ ID NO; column 2 shows the number of amino acid residues in each
polypeptide; column 3, potential phosphorylation sites; column 4,
the amino acid residues comprising signature sequences and motifs;
column 5, the identity of each protein; and column 6, analytical
methods used to characterize and identify each protein through
sequence homology and protein motifs.
[0071] The columns of Table 3 show the tissue specificity and
diseases, disorders, or conditions associated with nucleotide
sequences encoding PKH. The first column of Table 3 lists the
nucleotide SEQ ID NO; the second column lists tissue categories
which express PKH as a fraction of total tissue categories
expressing PKH. The third column lists the diseases, disorders, or
conditions associated with those tissues expressing PKH. The fourth
column lists the vectors used to subclone the cDNA library.
[0072] The following fragments of the nucleotide sequences encoding
PKH are useful in hybridization or amplification technologies to
identify SEQ ID NO: 10-18 and to distinguish between SEQ ID NO:
10-18 and related polynucleotide sequences. The useful fragments
are the fragment of SEQ ID NO:10 from about nucleotide 473 to about
nucleotide 532; the fragment of SEQ ID NO:11 from about nucleotide
65 to about nucleotide 125; the fragment of SEQ ID NO:12 from about
nucleotide 96 to about nucleotide 155; the fragment of SEQ ID NO:13
from about nucleotide 805 to about nucleotide 864; the fragment of
SEQ ID NO:14 from about nucleotide 230 to about nucleotide 289; the
fragment of SEQ ID NO:15 from about nucleotide 154 to about
nucleotide 213; the fragment of SEQ ID NO:16 from about nucleotide
110 to about nucleotide 169; the fragment of SEQ ID NO:17 from
about nucleotide 482 to about nucleotide 541; and the fragment of
SEQ ID NO:18 from about nucleotide 115 to about nucleotide 174.
[0073] The invention also encompasses PKH variants. A preferred PKH
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 PKH amino acid sequence, and which
contains at least one functional or structural characteristic of
PKH.
[0074] The invention also encompasses polynucleotides which encode
PKH. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising a sequence selected from the
group consisting of SEQ ID NO:10-18, which encodes PKH.
[0075] The invention also encompasses a variant of a polynucleotide
sequence encoding PKH. In particular, such a variant polynucleotide
sequence will have at least about 70%, more preferably at least
about 85%, and most preferably at least about 95% polynucleotide
sequence identity to the polynucleotide sequence encoding PKH. A
particular aspect of the invention encompasses a variant of a
polynucleotide sequence comprising a sequence selected from the
group consisting of SEQ ID NO: 10-18 which has at least about 70%,
more preferably at least about 85%, and most preferably at least
about 95% polynucleotide sequence identity to a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 10-18.
Any one of the polynucleotide variants described above can encode
an amino acid sequence which contains at least one functional or
structural characteristic of PKH.
[0076] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding PKH, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
sequence that could be made by selecting combinations based on
possible codon choices. These combinations are made in accordance
with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally occurring PKH, and all such
variations are to be considered as being specifically
disclosed.
[0077] Although nucleotide sequences which encode PKH and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring PKH under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding PKH or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. Codons may be selected to
increase the rate at which expression of the peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the
frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence
encoding PKH and its derivatives without altering the encoded amino
acid sequences include the production of RNA transcripts having
more desirable properties, such as a greater half-life, than
transcripts produced from the naturally occurring sequence.
[0078] The invention also encompasses production of DNA sequences
which encode PKH and PKH derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding PKH or any fragment thereof.
[0079] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO: 10-18 and fragments thereof under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.) For example, stringent salt concentration will
ordinarily be less than about 750 mM NaCl and 75 mM trisodium
citrate, preferably less than about 500 mM NaCl and 50 mM trisodium
citrate, and most preferably less than about 250 mM NaCl and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic solvent, e.g., formamide, while high
stringency hybridization can be obtained in the presence of at
least about 35% formamide, and most preferably at least about 50%
formamide. Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0080] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 42.degree.
C., and most preferably of at least about 68.degree. C. In a
preferred embodiment, wash steps will occur at 25.degree. C. in 30
mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred
embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred
embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on
these conditions will be readily apparent to those skilled in the
art.
[0081] Methods for DNA sequencing are well known in the art and may
be used to practice any of the embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA
polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq
polymerase (Perkin-Elmer), themostable T7 polymerase (Amersham
Pharmacia Biotech, Piscataway N.J.), or combinations of polymerases
and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg Md.).
Preferably, sequence preparation is automated with machines such as
the Hamilton MICROLAB 2200 (Reno Nev.), Peltier thermal cycler 200
(PTC200; MJ Research, Watertown Mass.) and the ABI CATALYST 800
(Perkin-Elmer). Sequencing is then carried out using either ABI 373
or 377 DNA sequencing systems (Perkin-Elmer) or the MEGABACE 1000
DNA sequencing system (Molecular Dynamics, Sunnyvale Calif.). The
resulting sequences are analyzed using a variety of algorithms
which are well known in the art. (See, e.g., Ausubel, F. M. (1997)
Short Protocols in Molecular Biology, John Wiley & Sons, New
York N.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biology and
Biotechnology, Wiley VCH, New York N.Y., pp. 856-853.)
[0082] The nucleic acid sequences encoding PKH may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based methods known in the art to detect upstream sequences,
such as promoters and regulatory elements. For example, one method
which may be employed, restriction-site PCR, uses universal and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This
procedure avoids the need to screen libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers
may be designed using commercially available software, such as
OLIGO 4.06 primer analysis software (National Biosciences, Plymouth
Minn.) or another appropriate program, to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the template at temperatures of about 68.degree.
C. to 72.degree. C.
[0083] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are preferable for situations
in which an oligo d(T) library does not yield a full-length cDNA.
Genomic libraries may be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0084] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g., GENOTYPER
and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from
loading of samples to computer analysis and electronic data display
may be computer controlled. Capillary electrophoresis is especially
preferable for sequencing small DNA fragments which may be present
in limited amounts in a particular sample.
[0085] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode PKH may be cloned in
recombinant DNA molecules that direct expression of PKH, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced and used to express
PKH.
[0086] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter PKH-encoding sequences for a variety of purposes including,
but not limited to, modification of the cloning, processing, and/or
expression of the gene product. DNA shuffling by random
fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be used to engineer the nucleotide sequences.
For example, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0087] In another embodiment, sequences encoding PKH may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232.) Alternatively, PKH itself or a fragment
thereof may be synthesized using chemical methods. For example,
peptide synthesis can be performed using various solid-phase
techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI
431A peptide synthesizer (Perkin-Elmer). Additionally, the amino
acid sequence of PKH, 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.
[0088] 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, WH Freeman, New York N.Y.)
[0089] In order to express a biologically active PKH, the
nucleotide sequences encoding PKH or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding PKH. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding PKH. Such
signals include the ATG initiation codon and adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding PKH and
its initiation codon and upstream regulatory sequences are inserted
into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including an in-frame ATG initiation codon should be provided by
the vector. Exogenous translational elements and initiation codons
may be of various origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
enhancers appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0090] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding PKH and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current
Protocols in Molecular Biology, John Wiley & Sons, New York
N.Y., ch. 9, 13, and 16.)
[0091] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding PKH. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0092] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding PKH. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding PKH can be achieved using a multifunctional E. coli vector
such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding PKH
into the vector's multiple cloning site disrupts the lacZ gene,
allowing a colorimetric screening procedure for identification of
transformed bacteria containing recombinant molecules. In addition,
these vectors may be useful for in vitro transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.
and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large
quantities of PKH are needed, e.g. for the production of
antibodies, vectors which direct high level expression of PKH may
be used. For example, vectors containing the strong, inducible T5
or T7 bacteriophage promoter may be used.
[0093] Yeast expression systems may be used for production of PKH.
A number of vectors containing constitutive or inducible promoters,
such as alpha factor, alcohol oxidase, and PGH, may be used in the
yeast Saccharomyces cerevisiae or Pichia pastoris. In addition,
such vectors direct either the secretion or intracellular retention
of expressed proteins and enable integration of foreign sequences
into the host genome for stable propagation. (See, e.g., Ausubel,
1995, supra; Grant et al. (1987) Methods Enzymol. 153:516-54; and
Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.) Plant
systems may also be used for expression of PKH. Transcription of
sequences encoding PKH may be driven viral promoters, e.g., the 35S
and 19S promoters of CaMV used alone or in combination with the
omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used. (See, e.g.,
Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results
Probl. Cell Differ. 17:85-105.) These constructs can be introduced
into plant cells by direct DNA transformation or pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and
Technology (1992) McGraw Hill, New York N.Y., pp. 191-196.)
[0094] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding PKH may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain infective virus which expresses PKH in host cells. (See,
e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0095] Human artificial chromosomes (HACS) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from a plasmid. HACs of about 6 kb to 10 Mb are
constructed and delivered via conventional delivery methods
(liposornes, polycationic amino polymers, or vesicles) for
therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997)
Nat Genet. 15:345-355.)
[0096] For long term production of recombinant proteins in
mammalian systems, stable expression of PKH in cell lines is
preferred. For example, sequences encoding PKH can be transformed
into cell lines using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for about 1 to 2 days in enriched media before being switched
to selective media. The purpose of the selectable marker is to
confer resistance to a selective agent, and its presence allows
growth and recovery of cells which successfully express the
introduced sequences. Resistant clones of stably transformed cells
may be propagated using tissue culture techniques appropriate to
the cell type.
[0097] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes sinplex virus thymidine kinase and adenine
phosphoribosyltransferase genes, for use in tk.sup.- or apr.sup.-
cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell
11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or herbicide resistance can be used as
the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides,
neomycin and G-418; and als and pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol.
150:1-14.) Additional selectable genes have been described, e.g.,
trpB and hisD, which alter cellular requirements for metabolites.
(See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.
Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins,
green fluorescent proteins (GFP; Clontech), .beta. glucuronidase
and its substrate .beta.-glucuronide, or luciferase and its
substrate luciferin may be used. These markers can be used not only
to identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system (See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol.
55:121-131.)
[0098] 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 PKH is inserted within a marker gene
sequence, transformed cells containing sequences encoding PKH can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding PKH 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.
[0099] In general, host cells that contain the nucleic acid
sequence encoding PKH and that express PKH may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR amplification, and protein bioassay or
immunoassay techniques which include membrane, solution, or chip
based technologies for the detection and/or quantification of
nucleic acid or protein sequences.
[0100] Immunological methods for detecting and measuring the
expression of PKH using either specific polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques
include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
PKH is preferred, but a competitive binding assay may be employed.
These and other assays are well known in the art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual,
APS Press, St Paul Minn., Sect. IV; Coligan, J. E. et al. (1997)
Current Protocols in Immunology, Greene Pub. Associates and
Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0101] 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 anino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding PKH include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding PKH, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Amersham Pharmacia Biotech, Promega (Madison Wis.), and
US Biochemical. Suitable reporter molecules or labels which may be
used for ease of detection include radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents, as well as
substrates, cofactors, inhibitors, magnetic particles, and the
like.
[0102] Host cells transformed with nucleotide sequences encoding
PKH may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or retained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode PKH may be designed to
contain signal sequences which direct secretion of PKH through a
prokaryotic or eukaryotic cell membrane.
[0103] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and WI38), are available from the American Type
Culture Collection (ATCC, Manassas Va.) and may be chosen to ensure
the correct modification and processing of the foreign protein.
[0104] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding PKH may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric PKH protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of PKH activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate fusion proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin
(HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the PKH encoding sequence and the heterologous protein
sequence, so that PKH may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra,
ch 10). A variety of commercially available kits may also be used
to facilitate expression and purification of fusion proteins.
[0105] In a further embodiment of the invention, synthesis of
radiolabeled PKH may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract systems (Promega). These
systems couple transcription and translation of protein-coding
sequences operably associated with the T7, T3, or SP6 promoters.
Translation takes place in the presence of a radiolabeled amino
acid precursor, preferably .sup.35S-methionine.
[0106] Fragments of PKH may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI
431A peptide synthesizer (Perkin-Elmer). Various fragments of PKH
may be synthesized separately and then combined to produce the full
length molecule.
[0107] Therapeutics
[0108] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of PKH and various
protein kinase homologs. In addition, the expression of PKH is
closely associated with cancer, reproductive tissues and
hematopoietic/immune tissues. Therefore, PKH appears to play a role
in cancer, autoimmune/inflammatory disorders, and reproductive
disorders. In the treatment of cancer, autoimmune/inflammatory
disorders, and reproductive disorders associated with increased PKH
expression or activity, it is desirable to decrease the expression
or activity of PKH. In the treatment of the above conditions
associated with decreased PKH expression or activity, it is
desirable to increase the expression or activity of PKH.
[0109] Therefore, in one embodiment, PKH or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of PKH. Examples of such disorders include, but are not limited to,
a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast,
cervix, gall bladder, ganglia, gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid,
and uterus; an autoimmune/inflammatory disorder such as acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic
anemia, autoimmune thyroiditis, autoimmune
polyenodocrinopathy-candidiasi- s-ectodermal dystrophy (APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis
fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections,
and trauma.; and a reproductive disorder, such as disorders of
prolactin production; infertility, including tubal disease,
ovulatory defects, and endometriosis; disruptions of the estrous
cycle, disruptions of the menstrual cycle, polycystic ovary
syndrome, ovarian hyperstimulation syndrome, endometrial and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic
pregnancies, and teratogenesis; cancer of the breast, fibrocystic
breast disease, and galactorrhea; disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of the male breast, and
gynecomastia.
[0110] In another embodiment, a vector capable of expressing PKH or
a fragment or derivative thereof may be administered to a subject
to treat or prevent a disorder associated with decreased expression
or activity of PKH including, but not limited to, those described
above.
[0111] In a further embodiment, a pharmaceutical composition
comprising a substantially purified PKH in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a disorder associated with decreased expression or
activity of PKH including, but not limited to, those provided
above.
[0112] In still another embodiment, an agonist which modulates the
activity of PKH may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of PKH including, but not limited to, those listed above.
[0113] In a further embodiment, an antagonist of PKH may be
administered to a subject to treat or prevent a disorder associated
with increased expression or activity of PKH. Examples of such
disorders include, but are not limited to, those described above.
In one aspect, an antibody which specifically binds PKH 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 PKH.
[0114] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding PKH may be administered
to a subject to treat or prevent a disorder associated with
increased expression or activity of PKH including, but not limited
to, those described above.
[0115] 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.
[0116] An antagonist of PKH may be produced using methods which are
generally known in the art. In particular, purified PKH may be used
to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind PKH. Antibodies to
PKH 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.
[0117] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with PKH 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.
[0118] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to PKH 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 PKH amino acids may be fused
with those of another protein, such as KLH, and antibodies to the
chimeric molecule may be produced.
[0119] Monoclonal antibodies to PKH may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0120] 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, canbeused. (See, e.g.,
Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855;
Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S.
et al. (1985) Nature 314:452-454.) Alternatively, techniques
described for the production of single chain antibodies may be
adapted, using methods known in the art, to produce PKH-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.)
[0121] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al.
(1991) Nature 349:293-299.)
[0122] Antibody fragments which contain specific binding sites for
PKH 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.) 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 PKH
and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two non-interfering PKH
epitopes is preferred, but a competitive binding assay may also be
employed (Pound, supra).
[0123] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for PKH. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
PKH-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple PKH epitopes,
represents the average affinity, or avidity, of the antibodies for
PKH. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular PKH epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
L/mole are preferred for use in immunoassays in which the
PKH-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 L/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of PKH, preferably in active form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, D.C.; Liddell, J. E. and Cryer, A. (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York N.Y.).
[0124] The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/nil,
is preferred for use in procedures requiring precipitation of
PKH-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0125] In another embodiment of the invention, the polynucleotides
encoding PKH, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding PKH may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding PKH. Thus, complementary molecules or
fragments may be used to modulate PKH activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding PKH.
[0126] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding PKH. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
[0127] Genes encoding PKH can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding PKH. Such constructs
may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the absence of integration into the
DNA, such vectors may continue to transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may
last for a month or more with a non-replicating vector, and may
last even longer if appropriate replication elements are part of
the vector system.
[0128] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding PKH. Oligonucleotides derived from the
transcription initiation site, e.g., between about positions -10
and +10 from the start site, are preferred. Similarly, inhibition
can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful because it causes inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E.
and B. I. Carr, Molecular and Immunologic Approaches, Futura
Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementary sequence
or antisense molecule may also be designed to block translation of
mRNA by preventing the transcript from binding to ribosomes.
[0129] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding PKH.
[0130] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0131] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding PKH. Such DNA sequences may be incorporated into
a wide variety of vectors with suitable RNA polymerase promoters
such as T7 or SP6. Alternatively, these cDNA constructs that
synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines, cells, or tissues.
[0132] RNA molecules nay be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0133] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
[0134] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0135] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of PKH, antibodies to PKH, and mimetics,
agonists, antagonists, or inhibitors of PKH. The compositions may
be administered alone or in combination with at least one other
agent, such as a stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs, or hormones.
[0136] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0137] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing, Easton
Pa.).
[0138] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0139] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0140] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0141] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0142] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acids esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0143] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0144] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0145] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0146] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of PKH, such labeling
would include amount, frequency, and method of administration.
[0147] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0148] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0149] A therapeutically effective dose refers to that amount of
active ingredient, for example PKH or fragments thereof, antibodies
of PKH, and agonists, antagonists or inhibitors of PKH, 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 toxic to therapeutic effects is the
therapeutic index, and it can be expressed as the
LD.sub.50/ED.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0150] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0151] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0152] Diagnostics
[0153] In another embodiment, antibodies which specifically bind
PKH may be used for the diagnosis of disorders characterized by
expression of PKH, or in assays to monitor patients being treated
with PKH or agonists, antagonists, or inhibitors of PKH. Antibodies
useful for diagnostic purposes may be prepared in the same manner
as described above for therapeutics. Diagnostic assays for PKH
include methods which utilize the antibody and a label to detect
PKH in human body fluids or in extracts of cells or tissues. The
antibodies may be used with or without modification, and may be
labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of reporter molecules, several of which
are described above, are known in the art and may be used.
[0154] A variety of protocols for measuring PKH, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of PKH expression. Normal or
standard values for PKH expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to PKH under conditions suitable
for complex formation. The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of PKH expressed in subject, samples from biopsied
tissues are compared with the standard values. Deviation between
standard and subject values establishes the parameters for
diagnosing disease.
[0155] In another embodiment of the invention, the polynucleotides
encoding PKH 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 PKH may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of PKH, and to
monitor regulation of PKH levels during therapeutic
intervention.
[0156] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding PKH or closely related molecules may be used to
identify nucleic acid sequences which encode PKH. 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 PKH, allelic variants, or related sequences.
[0157] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the PKH 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: 10-18 or from genomic
sequences including promoters, enhancers, and introns of the PKH
gene.
[0158] Means for producing specific hybridization probes for DNAs
encoding PKH include the cloning of polynucleotide sequences
encoding PKH or PKH derivatives into vectors for the production of
mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0159] Polynucleotide sequences encoding PKH may be used for the
diagnosis of disorders associated with expression of PKH. Examples
of such disorders include, but are not limited to, cancers
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma, teratocarcinoma, and, in particular, cancers of the
adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
autoimmune/inflammatory disorders such as acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoinmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohi's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; and reproductive disorders
including 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, uterine fibroids, autoimmune disorders, ectopic
pregnancies, and teratogenesis; cancer of the breast, fibrocystic
breast disease, and galactorrhea; disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of the male breast, and gynecomastia.
The polynucleotide sequences encoding PKH may be used in Southern
or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; in dipstick, pin, and
multiformat ELISA-like assays; and in microarrays utilizing fluids
or tissues from patients to detect altered PKH expression. Such
qualitative or quantitative methods are well known in the art.
[0160] In a particular aspect, the nucleotide sequences encoding
PKH may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding PKH 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 PKH in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0161] In order to provide a basis for the diagnosis of a disorder
associated with expression of PKH, 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
PKH, under conditions suitable for hybridization or amplification.
Standard hybridization may be quantified by comparing the values
obtained from normal subjects with values from an experiment in
which a known amount of a substantially purified polynucleotide is
used. Standard values obtained in this manner may be compared with
values obtained from samples from patients who are symptomatic for
a disorder. Deviation from standard values is used to establish the
presence of a disorder.
[0162] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0163] With respect to cancer, the presence of an abnormal amount
of transcript (either under- or overexpressed) in biopsied tissue
from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0164] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding PKH 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 PKH, or a fragment of a polynucleotide
complementary to the polynucleotide encoding PKH, and will be
employed under optimized conditions for identification of a
specific gene or condition. Oligomers may also be employed under
less stringent conditions for detection or quantitation of closely
related DNA or RNA sequences.
[0165] Methods which may also be used to quantitate the expression
of PKH include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993)
Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in an ELISA format
where the oligomer of interest is presented in various dilutions
and a spectrophotometric or calorimetric response gives rapid
quantitation.
[0166] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0167] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0168] In another embodiment of the invention, nucleic acid
sequences encoding PKH may be used to generate hybridization probes
useful in mapping the naturally occurring genomic sequence. The
sequences may be mapped to a particular chromosome, to a specific
region of a chromosome, or to artificial chromosome constructions,
e.g., human artificial chromosomes (HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1 constructions, or single chromosome cDNA libraries.
(See, e.g., Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355;
Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991)
Trends Genet. 7:149-154.)
[0169] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp.
965-968.) Examples of genetic map data can be found in various
scientific journals or at the Online Mendelian Inheritance in Man
(OMIM) site. Correlation between the location of the gene encoding
PKH on a physical chromosomal map and a specific disorder, or a
predisposition to a specific disorder, may help define the region
of DNA associated with that disorder. The nucleotide sequences of
the invention may be used to detect differences in gene sequences
among normal, carrier, and affected individuals.
[0170] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., ataxia-telangiectasia to 11 q22-23, any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. (See, e.g., Gatti, R.
A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of
the subject invention may also be used to detect differences in the
chromosomal location due to translocation, inversion, etc., among
normal, carrier, or affected individuals.
[0171] In another embodiment of the invention, PKH, 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 PKH and the agent being tested may be
measured.
[0172] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate. The test
compounds are reacted with PKH, or fragments thereof, and washed.
Bound PKH is then detected by methods well known in the art.
Purified PKH can also be coated directly onto plates for use in the
aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support.
[0173] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding PKH specifically compete with a test compound for binding
PKH. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
PKH.
[0174] In additional embodiments, the nucleotide sequences which
encode PKH may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0175] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0176] I. Construction of cDNA Libraries
[0177] RNA was purchased from Clontech or isolated from tissues
described in Table 4. Some tissues were homogenized and lysed in
guanidinium isothiocyanate, while others were homogenized and lysed
in phenol or in a suitable mixture of denaturants, such as TRIZOL
reagent (Life Technologies), a monophasic solution of phenol and
guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCl cushions or extracted with chloroform RNA was
precipitated from the lysates with either isopropanol or sodium
acetate and ethanol, or by other routine methods.
[0178] Phenol extraction and precipitation of RNA were repeated as
necessary to increase RNA purity. In some cases, RNA was treated
with DNase. For most libraries, poly(A+) RNA was isolated using
oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN, Valencia Calif.), or an OLIGOTEX mRNA
purification kit (QIAGEN). Alternatively, RNA was isolated directly
from tissue lysates using other RNA isolation kits, e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).
[0179] In some cases, Stratagene was provided with RNA and
constructed the corresponding cDNA libraries. Otherwise, cDNA was
synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using the recommended procedures or similar methods
known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6).
Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double
stranded cDNA, and the cDNA was digested with the appropriate
restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B,
or SEPHAROSE CL4B column chromatography (Amersham Pharmacia
Biotech) or preparative agarose gel electrophoresis. cDNAs were
ligated into compatible restriction enzyme sites of the polylinker
of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene),
PSPORT1 plasmid (Life Technologies), or pINCY (Incyte
Pharmaceuticals, Palo Alto Calif.). Recombinant plasmids were
transformed into competent E. coli cells including XL1-BLUE,
XL1-BLUEMRF, or SOLR from Stratagene or DH5.alpha., DH10B, or
ELECTROMAX DH10B from Life Technologies.
[0180] II. Isolation of cDNA Clones
[0181] Plasmids were recovered from host cells by in vivo excision,
using the UNIZAP vector system (Stratagene) or cell lysis. Plasmids
were purified using at least one of the following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg Md.); and
QIAWELL 8 plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid
purification systems or the R.E.A.L. PREP 96 plasmid kit from
QIAGEN. Following precipitation, plasmids were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization,
at 4.degree. C.
[0182] Alternatively, plasmid DNA was amplified from host cell
lysates using direct link PCR in a high-throughput format (Rao, V.
B. (1994) Anal. Biochem 216:1-14). Host cell lysis and thermal
cycling steps were carried out in a single reaction mixture.
Samples were processed and stored in 384-well plates, and the
concentration of amplified plasmid DNA was quantified
fluorometrically using PICOGREEN dye (Molecular Probes, Eugene
Oreg.) and a Pluoroskan II fluorescence scanner (Labsystems Oy,
Helsinki, Finland).
[0183] III. Sequencing and Analysis
[0184] The cDNAs were prepared for sequencing using the ABI
CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Robbins
Scientific) or MICROLAB 2200 (Hamilton) systems in combination with
the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced
using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer)
and standard ABI protocols, base calling software, and kits. In one
alternative, cDNAs were sequenced using the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were amplified and sequenced using the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In
yet another alternative, cDNAs were sequenced using solutions and
dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs
were determined using standard methods (reviewed in Ausubel, 1997,
supra, unit 7.7). Some of the cDNA sequences were selected for
extension using the techniques disclosed in Example V.
[0185] The polynucleotide sequences derived from cDNA, extension,
and shotgun sequencing were assembled and analyzed using a
combination of software programs which utilize algorithms well
known to those skilled in the art. Table 5 summarizes the software
programs, descriptions, references, and threshold parameters used.
The first column of Table 5 shows the tools, programs, and
algorithms used, the second column provides a brief description
thereof, the third column presents the references which are
incorporated by reference herein, and the fourth column presents,
where applicable, the scores, probability values, and other
parameters used to evaluate the strength of a match between two
sequences (the higher the probability the greater the homology).
Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software Engineering, South San Francisco Calif.) and LASERGENE
software (DNASTAR).
[0186] The polynucleotide sequences were validated by removing
vector, linker, and polyA sequences and by masking ambiguous bases,
using algorithms and programs based on BLAST, dynamic programing,
and dinucleotide nearest neighbor analysis. The sequences were then
queried against a selection of public databases such as GenBank
primate, rodent, manmalian, vertebrate, and eukaryote databases,
and BLOCKS to acquire annotation, using programs based on BLAST,
FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened for open reading frames using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide
sequences were translated to derive the corresponding full length
amino acid sequences, and these full length sequences were
subsequently analyzed by querying against databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS,
PFAM, and Prosite.
[0187] The programs described above for the assembly and analysis
of full length polynucleotide and amino acid sequences were also
used to identify polynucleotide sequence fragments from SEQ ID NO:
10-18. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization and amplification technologies were
described in The Invention section above.
[0188] IV. Northern Analysis
[0189] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and
16.)
[0190] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). This
analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or similar. The basis of the search is the
product score, which is defined as: 1 % sequence identity .times. %
maximum BLAST score 100
[0191] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0192] The results of northern analyses are reported as a
percentage distribution of libraries in which the transcript
encoding PKH occurred. Analysis involved the categorization of cDNA
libraries by organ/tissue and disease. The organ/tissue categories
included cardiovascular, dermatologic, developmental, endocrine,
gastrointestinal, hematopoietic/immune, musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories
included cancer, inflammation/trauma, cell proliferation,
neurological, and pooled. For each category, the number of
libraries expressing the sequence of interest was counted and
divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or
condition-specific expression are reported in Table 3.
[0193] V. Extension of PKH Encoding Polynucleotides
[0194] The full length nucleic acid sequence of SEQ ID NO: 10-18
was produced by extension of an appropriate fragment of the full
length molecule using oligonucleotide primers designed from this
fragment. One primer was synthesized to initiate 5' extension of
the known fragment, and the other primer, to initiate 3' extension
of the known fragment. The initial primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate
program, to be about 22 to 30 nucleotides in length, to have a GC
content of about 50% or more, and to anneal to the target sequence
at temperatures of about 68.degree. C. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0195] Selected human cDNA libraries were used to extend the
sequence. If more than one extension was necessary or desired,
additional or nested sets of primers were designed.
[0196] High fidelity amplification was obtained by PCR using
methods well known in the art. PCR was performed in 96-well plates
using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction
buffer containing Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4, and
.beta.-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair PCI A and PCI B: Step 1: 94.degree. C., 3 min; Step 2:
94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 tines;
Step 6: 68.degree. C., 5 min; Step 7: storage at 4.degree. C. Inthe
alternative, the parameters for primer pair T7 and SK+ were as
follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15
sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. C., 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C.,
5 min; Step 7: storage at 4.degree. C.
[0197] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1.times. TE
and 0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA
to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of
the sample and to quantify the concentration of DNA. A 5 .mu.l to
10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a 1% agarose mini-gel to determine which
reactions were successful in extending the sequence.
[0198] The extended nucleotides were desalted and concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison Wis.), and
sonicated or sheared prior to religation into pUC 18 vector
(Amersham Pharmacia Biotech). For shotgun sequencing, the digested
nucleotides were separated on low concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar
ACE (Promega). Extended clones were religated using T4 ligase (New
England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction site overhangs, and transfected into competent
E. coli cells. Transformed cells were selected on
antibiotic-containing media, individual colonies were picked and
cultured overnight at 37.degree. C. in 384-well plates in
LB/2.times. carb liquid media.
[0199] The cells were lysed, and DNA was amplified by PCR using Taq
DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated 29
times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the same conditions as described above. Samples were diluted
with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (Perkin-Elmer).
[0200] In like manner, the nucleotide sequence of SEQ ID NO: 10-18
is used to obtain 5' regulatory sequences using the procedure
above, oligonucleotides designed for such extension, and an
appropriate genomic library.
[0201] VI. Labeling and Use of Individual Hybridization Probes
[0202] Hybridization probes derived from SEQ ID NO: 10-18 are
employed to screen cDNAs, genomic DNAs, or mRNAs. Although the
labeling of oligonucleotides, consisting of about 20 base pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham
Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN,
Boston Mass.). The labeled oligonucleotides are substantially
purified using a SEPHADEX G-25 superfine size exclusion dextran
bead column (Amersham Pharmacia Biotech). An aliquot containing
10.sup.7 counts per minute of the labeled probe is used in a
typical membrane-based hybridization analysis of human genomic DNA
digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
[0203] The DNA from each digest is fractionated on a 0.7% agarose
gel and transferred to nylon membranes (NYTRAN PLUS, Schleicher
& Schuell, Durham N.H.). Hybridization is carried out for 16
hours at 40.degree. C. To remove nonspecific signals, blots are
sequentially washed at room temperature under increasingly
stringent conditions up to 0.1.times. saline sodium citrate and
0.5% sodium dodecyl sulfate. After XOMAT-AR film (Eastman Kodak,
Rochester N.Y.) is exposed to the blots, hybridization patterns are
compared visually.
[0204] VII. Microarrays
[0205] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0206] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE software (DNASTAR).
Full-length cDNAs, ESTs, or fragments thereof corresponding to one
of the nucleotide sequences of the present invention, or selected
at random from a cDNA library relevant to the present invention,
are arranged on an appropriate substrate, e.g., a glass slide. The
cDNA is fixed to the slide using, e.g., UV cross-linking, followed
by thermal and chemical treatments and subsequent drying. (See,
e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et
al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared
and used for hybridization to the elements on the substrate. The
substrate is analyzed by procedures described above.
[0207] VIII. Complementary Polynucleotides
[0208] Sequences complementary to the PKH-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring PKH. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the
coding sequence of PKH. 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 PKH-encoding transcript.
[0209] IX. Expression of PKH
[0210] Expression and purification of PKH is achieved using
bacterial or virus-based expression systems. For expression of PKH
in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express PKH upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of PKH in
eukaryotic cells is achieved by infecting insect or mammalian cell
lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding PKH by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodotera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0211] In most expression systems, PKH is synthesized as a fusion
protein with, e.g., glutathione S-transferase (GST) or a peptide
epitope tag, such as FLAG or 6-His, permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from
crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma
japonicum, enables the purification of fusion proteins on
immobilized glutathione under conditions that maintain protein
activity and antigenicity (Amersham Pharmacia Biotech). Following
purification, the GST moiety can be proteolytically cleaved from
PKH at specifically engineered sites. FLAG, an 8-amino acid
peptide, enables immunoaffinity purification using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues,
enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression and purification are discussed in Ausubel (1995,
supra, ch 10 and 16). Purified PKH obtained by these methods can be
used directly in the following activity assay.
[0212] X. Demonstration of PKH Activity
[0213] An assay for PKH activity measures the phosphorylation of a
substrate in the presence of gamma-labeled .sup.32P-ATP. PKH is
incubated with an appropriate substrate and .sup.32P-ATP in a
buffered solution. .sup.32P-labeled product is separated from free
.sup.32P-ATP by gel electrophoresis or chromatographic procedures,
and the incorporated .sup.32P is quantified by phosphoimage
analysis or scintillation counter. The amount of .sup.32P detected
is proportional to the activity of PKH in this assay. The specific
amino acid residue phosphorylated by PKH may be determined by
phosphoamino acid analysis of the labeled, hydrolyzed protein.
[0214] XI. Functional Assays
[0215] PKH function is assessed by expressing the sequences
encoding PKH at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life
Technologies) and pCR3.1 (Invitrogen, Carlsbad Calif.), both of
which contain the cytomegalovirus promoter. 5-10 .mu.g of
recombinant vector are transiently transfected into a human cell
line, preferably of endothelial or hematopoietic origin, using
either liposome formulations or electroporation. 1-2 .mu.g of an
additional plasmid containing sequences encoding a marker protein
are co-transfected. Expression of a marker protein provides a means
to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA expression from the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP, and to
evaluate properties, for example, their apoptotic state. FCM
detects and quantifies the uptake of fluorescent molecules that
diagnose events preceding or coincident with cell death. These
events include changes in nuclear DNA content as measured by
staining of DNA with propidium iodide; changes in cell size and
granularity as measured by forward light scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by
decrease in bromodeoxyuridine uptake; alterations in expression of
cell surface and intracellular proteins as measured by reactivity
with specific antibodies; and alterations in plasma membrane
composition as measured by the binding of fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry
are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New
York N.Y.
[0216] The influence of PKH on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding PKH and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding PKH and other genes of interest can be
analyzed by northern analysis or microarray techniques.
[0217] XII. Production of PKH Specific Antibodies
[0218] PKH substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488-495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0219] Alternatively, the PKH amino acid sequence is analyzed using
LASERGENE software (DNASTAR) to determine regions of high
immunogenicity, and a corresponding oligopeptide is synthesized and
used to raise antibodies by means known to those of skill in the
art. Methods for selection of appropriate epitopes, such as those
near the C-terminus or in hydrophilic regions are well described in
the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
[0220] Typically, oligopeptides 15 residues in length are
synthesized using an ABI 431A peptide synthesizer (Perkin-Elmer)
using fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis
Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995,
supra.) Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity by, for example, binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio-iodinated goat anti-rabbit
IgG.
[0221] XIII. Purification of Naturally Occurring PKH Using Specific
Antibodies
[0222] Naturally occurring or recombinant PKH is substantially
purified by immunoaffinity chromatography using antibodies specific
for PKH. An immunoaffinity column is constructed by covalently
coupling anti-PKH antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
After the coupling, the resin is blocked and washed according to
the manufacturer's instructions.
[0223] Media containing PKH are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of PKH (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/PKH 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 PKH is collected.
[0224] XIV. Identification of Molecules Which Interact with PKH
[0225] PKH, 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
PKH, washed, and any wells with labeled PKH complex are assayed.
Data obtained using different concentrations of PKH are used to
calculate values for the number, affinity, and association of PKH
with the candidate molecules.
[0226] 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.
1TABLE 1 Amino Acid Nucleotide SEQ ID NO: SEQ ID NO: Clone ID
Library Fragments 1 10 119819 MUSCNOT01 119819H1 (MUSCNOT0l),
1476434H1 (CORPNOT02), 1719355H1 (BLADNOT06), 3016364F6
(MUSCNOT07), 3373249F6 (CONNTUT05) 2 11 132750 BMARNOT02 132750H1
and 132750X335D1 (BMARNOT02), 287300R1 (EOSIHET02), 1271292F1
(TESTTUT02), 3343321H1 (SPLNNOT09) 3 12 507669 TMLR3DT01 101010R6
(ADRENOT01), 5076691H1 and 507669R6 TMLR3DT01), 697674H1
(SYNORAT03), 851624T1 (NGANNOT01), 1270195F7 (BRATNOT09) 4 13
1439938 THYRNOT03 814363X11 (OVARTUT01), 822246X13 (KERANOT02),
1439938H1 (THYRNOT03), 3365318F6 (PROSBPT02) 5 14 1447427 PLACNOT02
712366R6 (SYNORAT04), 1447427H1 (PLACNOT02), 2179842F6 (SININOT01),
2697460F6 (UTRSNOT12) 6 15 1567782 UTRSNOT05 1567782H1 and
1567786F6 (UTRSNOT05), 2289257X42F1 (BRAINON01), 2839231T6
(DRGLNOT01), SAFB00259F1 7 16 2295842 BRSTNOT05 606173H1
(BRSTTUTO1), 865560R1 (BRAITUT03), 1441018F6 (THYRNOT03), 1706132T6
(DUODNOT02), 1811824F6 (PROSTUT12), 2223155H1 (LUNGNOT18),
2295842H1 (BRSTNOT05) 8 17 2605059 LUNGTUT07 381188R6 (HYPONOB01),
381188T6 (HYPONOB01), 691185T6 (LUNGTUT02), 1824201F6 (GBLATUT01),
2605059F6, 2605059H1 and 2605059X304F1 (LUNGTUT07) 9 18 3000825
TLYMNOT06 2015630F6 (ADRENOT07) , 3000825F6 and 3000825H1
(TLYMNOT06)
[0227]
2TABLE 2 Amino Acid Amino Acid Potential Signature Seq ID NO:
Residues Phosphorylation Sites Sequence Identification Analytical
Methods 1 297 S285 T29 S197 S202 S225 A117-L295 SR protein- PFAM,
BLAST, BLOCKS, S285 specific kinase MOTIFS 2 287 S283 T106 T179
S194 S31-T284 protein kinase PFAM, BLOCKS, PRINTS, S274 T69 T184
S233 S270 homolog BLAST, MOTIFS 3 346 S7 T105 S160 T232 T244
E125-D333 tyrosine kinase PFAM, BLAST, T282 S329 T336 S214 BLOCKS,
PRINTS, T255 Y231 PROFILESCAN, MOTIFS 4 90 S60 S9 S62 F28-R78
protein kinase PFAM, BLOCKS, BLAST, homolog MOTIFS 5 327 S96 T210
S277 T40 S137 Y11-N235 protein kinase PFAM, BLOCKS, PRINTS, S179
T273 Y178 Y310 homolog BLAST, MOTIFS 6 345 S327 S23 S41 S48 S123
Y4-I226 serine/threonine- PFAM, BLAST, BLOCKS, S219 T319 T166 S175
Y30 and tyrosine- PRINTS, PROFILESCAN, specific protein MOTIFS
kinase, Nek1 7 424 T97 S218 S298 T389 S413 D202-V412 protein kinase
PFAM, BLOCKS, PRINTS, T54 T62 T89 T109 S112 homolog BLAST, MOTIFS
S151 S223 S229 S286 S318 8 99 cdc2+/CDC28- BLAST related protein
kinase 9 138 T91 T24 T57 T91 T14 R72-V101 serine/threonine PFAM,
BLAST, MOTIFS protein kinase
[0228]
3TABLE 3 Nucleotide Tissue Expression Disease or Condition Seq ID
NO: (Fraction of Total) (Fraction of Total) Vector 10 Nervous
(0.400) Musculoskeletal (0.200) Cancer (0.500) Neurological (0.300)
pBluescript Cardiovascular (0.100) 11 Reproductive (0.316)
Hematopoietic/Immnune Inflammation (0.421) Cancer (0.368)
pBluescript (0.211) Gastrointestinal (0.158) 12
Hematopoietic/Immune (0.514) Gastrointestinal Inflammation (0.595)
Cancer (0.243) pBluescript (0.189) Reproductive (0.081) 13
Reproductive (0.375) Developmental (0.125) Cancer (0.375)
Inflammation (0.250) pINCY Endocrine (0.125) 14 Reproductive
(0.346) Nervous (0.269) Cancer (0.462) Inflammation (0.385) pINCY
Hematopoietic/Immune (0.231) 15 Nervous (0.500) Developmental
(0.167) Cancer (0.833) Inflammation (0.333) pINCY Musculoskeletal
(0. 167) 16 Reproductive (0.290) Gastrointestinal (0.145) Cancer
(0.420) Inflammation (0.362) pSPORT1 Nervous (0.130) 17 Nervous
(0.250) Gastrointestinal (0.167) Cancer (0.500) Inflammation
(0.250) pINCY Hematopoietic/Immune (0. 167) 18 Endocrine (0.333)
Hematopoietic/Immune (0.333) Inflammation (0.667) Cancer (0.333)
pINCY Reproductive (0.333)
[0229]
4TABLE 4 Nucleotide SEQ ID NO: Library Library Comment 10 MUSCNOT01
Library was constructed at Stratagene (STR937209), using RNA
isolated from the skeletal muscle tissue of a patient with
malignant hyperthermia. 11 BMARNOT02 Library was constructed using
RNA isolated from the bone marrow of 24 male and female Caucasian
donors, 16 to 70 years old. (RNA came from Clontech.) 12 TMLR3DT02
Library was constructed using RNA isolated from non-adherent
peripheral blood mononuclear cells collected from a pool of male
and female donors. Cells from each donor were purified on Ficoll
Hypaque, then co-cultured for 72 hours. The cells were pooled,
washed once in PBS, lysed in a buffer containing GuSCN, and spun
through CsCl to obtain RNA. PolyA RNA was isolated using a Qiagen
Oligotex kit. 13 THYRNOT03 Library was constructed using RNA
isolated from thyroid tissue removed from the left thyroid of a
28-year-old Caucasian female during a complete thyroidectomy.
Pathology Indicated a small nodule of adenomatous hyperplasia
present in the left thyroid. Pathology for the associated tumor
tissue indicated dominant follicular adenoma, forming a
well-encapsulated mass in the left thyroid. 14 PLACNOT02 Library
was constructed using RNA isolated from the placental tissue of a
Hispanic female fetus, who was prematurely delivered at 21 weeks'
gestation. Serologies of the mother's blood were positive for
cytomegalovirus. 15 UTRSNOT05 Library was constructed using RNA
isolated from the uterine tissue of a 45-year- old Caucasian female
during a total abdominal hysterectomy and total colectomy.
Pathology for the associated tumor tissue indicated multiple
lelomyomas of the myometrium. 16 BRSTNOT05 Library was constructed
using RNA isolated from breast tissue removed from a 58- year-old
Caucasian female during a unilateral extended simple mastectomy.
Pathology for the associated tumor tissue indicated multicentric
invasive grade 4 lobular carcinoma. Family history include.d breast
and prostate cancer. 17 LUNGTUT07 Library was constructed using RNA
isolated from lung tumor tissue removed from the upper lobe of a
50-year-old Caucasian male during segmental lung resection.
Pathology indicated an invasive grade 4 squamous cell
adenocarcinoma. Patient history included tobacco use. Family
history included skin cancer. 18 TLYMNOT06 Library was constructed
using RNA isolated from activated Th2 cells. These cells were
differentiated from umbilical cord CD4 T cells with IL-4 in the
presence of anti-IL-12 antibodies and B7-transfected COS cells, and
then activated for six hours with anti-CD3 and anti-CD28
antibodies.
[0230]
5TABLE 5 Program Description Reference Parameter Threshold ABI
FACTURA A program that removes vector sequences and masks
Perkin-Elmer Applied Biosystems ambigous bases in nucleic acid
sequences Foster City, CA. ABI/PARACEL FDF A Fast Data Finder
useful in comparing and annotating Perkin-Elmer Applied Biosystems
Mismatch <50% amino acid or nulceic acid sequences. Foster City,
CA; Paracel Inc., Pasadena CA. ABI AutoAssembler A program that
assembles nucleic acid sequences. Perkin-Elmer Applied Biosystems,
Foster City, CA. BLAST A Basic Local Alignment Search Tool useful
in sequence Altschul, S.F. et al. (1990) J. Mol. Biol. ESTs:
Probability value = 1.0E-8 similarity search for amino acid and
nucleic acid sequences. 215:403-410; Altscul, S.F. et al. (1997) or
less BLAST includes five functions:blastp,blastn,blastx- , Nucleic
Acids Res. 25:3389-3402. Full Lengrh sequences: tblastn, and
tblastx. Probability value = 1.0E-10 or less FASTA A Pearson and
Lipman algorithm that searches for Pearson, W.R. and D.J. Lipman
(1988) Proc. ESTs: fasta E value = 1.06E-6 similarity between a
query sequence and a group of Natl. Acad Sci. 85:2444-2448;
Pearson, W.R. Assembled ESTs: fasta Identity = sequences of the
same type. FASTA comprises as least (1990) Methods Enzymol.
183:63-98; and 95% or greater and Match five functions: fasta,
tfasta, fastx, tfastx, and search. Smith, T.F. and M.S. Waterman
(1981) Adv. length = 200 bases or greater; fastx Appl. Math.
2:482-489. E value = 1.0E-8 or less Full Length sequences: fastx
score = 100 or greater BLIMPS A BLocks IMProved Searcher that
matches a sequence Henikoff, S and J.G. Henikoff, Nucl. Acid Res.,
Score = 1000 or greater; Ratio of against those in BLOCKS and
PRINTS databases to search 19:6565-72, 1991. J.G. Henikoff and S.
Score/Strength = 0.75 or larger; for gene families, sequence
homology, and structural Henikoff (1996) Methods Enzymol.
266:88-105; and Probability value = 1.0E-3 or fingerprint regions.
and Attwood, T.K. et al. (1997) J. Chem. Inf. less Comput. Sci.
37:417-424. PFAM A Hidden Markov Models-based application useful
for Krogh, A. et al. (1994) J. Mol. Biol., 235:1501- Score = 10-50
bits, depending on protein family search. 1531; Sonnhammer, E.L.L.
et al. (1988) individual protein families Nucleic Acids Res.
26:320-322. ProfileScan An algorithm that searches for structural
and sequence Gribskov, M. et al. (1988) CABIOS 4:61-66; Score = 4.0
or greater motifs in protein sequences that match sequence patterns
Gribskov, et al. (1989) Methods Enzymol. defined in Prosite.
183:146-159; Bairoch, A. et al. (1997) Nucleic Acids Res.
25:217-221. Phred A base-calling algorithm that examines automated
Ewing, B. et al. (1998) Genome sequencer traces with high
sensitivity and probability. Res. 8:175-185; Ewing, B. and P. Green
(1998) Genome Res. 8:186-194. Phrap A Phils Revised Assembly
Program including SWAT and Smith, T.F. and M. S. Waterman (1981)
Adv. Score = 120 or greater; Match CrossMatch, programs based on
efficient implementation of Appl. Math. 2:482-489; Smith, T.F. and
M. S. length = 56 or greater the Smith-Waterman algorithm, useful
in searching Waterman (1981) J. Mol. Biol. 147:195-197; sequence
homology and assembling DNA sequences. and Green, P., University of
Washington, Seattle, WA. Consed A graphical tool for viewing and
editing Phrap assemblies Gordon, D. et al. (1998) Genome Res.
8:195-202. SPScan A weight matrix analysis program that scans
protein Nielson, H. et al. (1997) Protein Engineering Score = 5 or
greater sequences for the presence of secretory signal peptides.
10:1-6; Claverie, J.M. and S. Audic (1997) CABIOS 12:431-439.
Motifs A program that searches amino acid sequences for patterns
Bairoch et al. supra; Wisconsin that matched those defined in
Prosite. Package Program Manual, version 9, page M51-59, Genetics
Computer Group, Madison, WI.
[0231]
Sequence CWU 1
1
18 1 297 PRT Homo sapiens 119819 1 Met Arg Arg Lys Arg Lys Gln Gln
Lys Arg Leu Leu Glu Glu Arg 1 5 10 15 Leu Arg Asp Leu Gln Arg Leu
Glu Ala Met Glu Ala Ala Thr Gln 20 25 30 Ala Glu Asp Ser Gly Leu
Arg Leu Asp Gly Gly Ser Gly Ser Thr 35 40 45 Ser Ser Ser Gly Cys
His Pro Gly Gly Ala Arg Ala Gly Pro Ser 50 55 60 Pro Ala Ser Ser
Ser Pro Ala Pro Gly Gly Gly Arg Ser Leu Ser 65 70 75 Ala Gly Ser
Gln Thr Ser Gly Phe Ser Gly Ser Leu Phe Ser Pro 80 85 90 Ala Ser
Cys Ser Ile Leu Ser Gly Ser Ser Asn Gln Arg Glu Thr 95 100 105 Gly
Gly Leu Leu Ser Pro Ser Thr Pro Phe Gly Ala Ser Asn Leu 110 115 120
Leu Val Asn Pro Leu Glu Pro Gln Asn Ala Asp Lys Ile Lys Ile 125 130
135 Lys Ile Ala Asp Leu Gly Asn Ala Cys Trp Val His Lys His Phe 140
145 150 Thr Glu Asp Ile Gln Thr Arg Gln Tyr Arg Ala Val Glu Val Leu
155 160 165 Ile Gly Ala Glu Tyr Gly Pro Pro Ala Asp Ile Trp Ser Thr
Ala 170 175 180 Cys Met Ala Phe Glu Leu Ala Thr Gly Asp Tyr Leu Phe
Glu Pro 185 190 195 His Ser Gly Glu Asp Tyr Ser Arg Asp Glu Asp His
Ile Ala His 200 205 210 Ile Val Glu Leu Leu Gly Asp Ile Pro Pro Ala
Phe Ala Leu Ser 215 220 225 Gly Arg Tyr Ser Arg Glu Phe Phe Asn Arg
Arg Gly Glu Leu Arg 230 235 240 His Ile His Asn Leu Lys His Trp Gly
Leu Tyr Glu Val Leu Met 245 250 255 Glu Lys Tyr Glu Trp Pro Leu Glu
Gln Ala Thr Gln Phe Ser Ala 260 265 270 Phe Leu Leu Pro Met Asn Glu
Tyr Ile Pro Glu Lys Arg Ala Ser 275 280 285 Ala Arg Asp Cys Leu Gln
His Pro Trp Leu Gln Pro 290 295 2 287 PRT Homo sapiens 132750 2 Met
Gln Glu Ile Pro Gln Glu Gln Ile Lys Glu Ile Lys Lys Glu 1 5 10 15
Gln Leu Ser Gly Ser Pro Trp Ile Leu Leu Arg Glu Asn Glu Val 20 25
30 Ser Thr Leu Tyr Lys Gly Glu Tyr His Arg Ala Pro Val Ala Ile 35
40 45 Lys Val Phe Lys Lys Leu Gln Ala Gly Ser Ile Ala Ile Val Arg
50 55 60 Gln Thr Phe Asn Lys Glu Ile Lys Thr Met Lys Lys Phe Glu
Ser 65 70 75 Pro Asn Ile Leu Arg Ile Phe Gly Ile Cys Ile Asp Glu
Thr Val 80 85 90 Thr Pro Pro Gln Phe Ser Ile Val Met Glu Tyr Cys
Glu Leu Gly 95 100 105 Thr Leu Arg Glu Leu Leu Asp Arg Glu Lys Asp
Leu Thr Leu Gly 110 115 120 Lys Arg Met Val Leu Val Leu Gly Ala Ala
Arg Gly Leu Tyr Arg 125 130 135 Leu His His Ser Glu Ala Pro Glu Leu
His Gly Lys Ile Arg Ser 140 145 150 Ser Asn Phe Leu Val Thr Gln Gly
Tyr Gln Val Lys Leu Ala Gly 155 160 165 Phe Glu Leu Arg Lys Thr Gln
Thr Ser Met Ser Leu Gly Thr Thr 170 175 180 Arg Glu Lys Thr Asp Arg
Val Lys Ser Thr Ala Tyr Leu Ser Pro 185 190 195 Gln Glu Leu Glu Asp
Val Phe Tyr Gln Tyr Asp Val Lys Ser Glu 200 205 210 Ile Tyr Ser Phe
Gly Ile Val Leu Trp Glu Ile Ala Thr Gly Asp 215 220 225 Ile Pro Phe
Gln Gly Cys Asn Ser Glu Lys Ile Arg Lys Leu Val 230 235 240 Ala Val
Lys Arg Gln Gln Glu Pro Leu Gly Glu Asp Cys Pro Ser 245 250 255 Glu
Leu Arg Glu Ile Ile Asp Glu Cys Arg Ala His Asp Pro Ser 260 265 270
Val Arg Pro Ser Val Asp Glu Ile Leu Lys Lys Leu Ser Thr Phe 275 280
285 Ser Lys 3 346 PRT Homo sapiens 507669 3 Met Gly Cys Gly Cys Ser
Ser His Pro Glu Asp Asp Trp Met Glu 1 5 10 15 Asn Ile Asp Val Cys
Glu Asn Cys His Tyr Pro Ile Val Pro Leu 20 25 30 Asp Gly Lys Gly
Thr Leu Leu Ile Arg Asn Gly Ser Glu Val Arg 35 40 45 Asp Pro Leu
Val Thr Tyr Glu Gly Ser Asn Pro Pro Ala Ser Pro 50 55 60 Leu Gln
Asp Asn Leu Val Ile Ala Leu His Ser Tyr Glu Pro Ser 65 70 75 His
Asp Gly Asp Leu Gly Phe Glu Lys Gly Glu Gln Leu Arg Ile 80 85 90
Leu Glu Gln Ser Gly Glu Trp Trp Lys Ala Gln Ser Leu Thr Thr 95 100
105 Gly Gln Glu Gly Phe Ile Pro Phe Asn Phe Val Ala Lys Ala Asn 110
115 120 Ser Leu Glu Pro Glu Ala Asn Leu Met Lys Gln Leu Gln His Gln
125 130 135 Arg Leu Val Arg Leu Tyr Ala Val Val Thr Gln Glu Pro Ile
Tyr 140 145 150 Ile Ile Thr Glu Tyr Met Glu Asn Gly Ser Leu Val Asp
Phe Leu 155 160 165 Lys Thr Pro Ser Gly Ile Lys Leu Thr Ile Asn Lys
Leu Leu Asp 170 175 180 Met Ala Ala Gln Ile Ala Glu Gly Met Ala Phe
Ile Glu Glu Arg 185 190 195 Asn Tyr Ile His Arg Asp Leu Arg Ala Ala
Asn Ile Leu Val Ser 200 205 210 Asp Thr Leu Ser Cys Lys Ile Ala Asp
Phe Gly Leu Ala Arg Leu 215 220 225 Ile Glu Asp Asn Glu Tyr Thr Ala
Arg Glu Gly Ala Lys Phe Pro 230 235 240 Ile Lys Trp Thr Ala Pro Glu
Ala Ile Asn Tyr Gly Thr Phe Thr 245 250 255 Ile Lys Ser Asp Val Trp
Ser Phe Gly Ile Leu Leu Thr Glu Ile 260 265 270 Val Thr His Gly Arg
Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu 275 280 285 Val Ile Gln Asn
Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp 290 295 300 Asn Cys Pro
Glu Glu Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys 305 310 315 Glu Arg
Pro Glu Asp Arg Pro Thr Phe Asp Tyr Leu Arg Ser Val 320 325 330 Leu
Glu Asp Phe Phe Thr Ala Thr Glu Gly Gln Tyr Gln Pro Gln 335 340 345
Pro 4 90 PRT Homo sapiens 1439938 4 Met Pro Ala Gly Gly Arg Ala Gly
Ser Leu Lys Asp Pro Asp Val 1 5 10 15 Ala Glu Leu Phe Phe Lys Asp
Asp Pro Glu Lys Leu Phe Ser Asp 20 25 30 Leu Arg Glu Ile Gly His
Gly Ser Phe Gly Ala Val Tyr Phe Ala 35 40 45 Arg Asp Val Arg Asn
Ser Glu Val Val Ala Ile Lys Lys Met Ser 50 55 60 Tyr Ser Gly Lys
Gln Ser Asn Glu Lys Trp Gln Asp Ile Ile Lys 65 70 75 Glu Val Arg
Arg Arg Arg Arg Val Gly Arg Glu Asp Glu Glu Arg 80 85 90 5 327 PRT
Homo sapiens 1447427 5 Met Ser Ser Phe Leu Pro Glu Gly Gly Cys Tyr
Glu Leu Leu Thr 1 5 10 15 Val Ile Gly Lys Gly Phe Glu Asp Leu Met
Thr Val Asn Leu Ala 20 25 30 Arg Tyr Lys Pro Thr Gly Glu Tyr Val
Thr Val Arg Arg Ile Asn 35 40 45 Leu Glu Ala Cys Ser Asn Glu Met
Val Thr Phe Leu Gln Gly Glu 50 55 60 Leu His Val Ser Lys Leu Phe
Asn His Pro Asn Ile Val Pro Tyr 65 70 75 Arg Ala Thr Phe Ile Ala
Asp Asn Glu Leu Trp Val Val Thr Ser 80 85 90 Phe Met Ala Tyr Gly
Ser Ala Lys Asp Leu Ile Cys Thr His Phe 95 100 105 Met Asp Gly Met
Asn Glu Leu Ala Ile Ala Tyr Ile Leu Gln Gly 110 115 120 Val Leu Lys
Ala Leu Asp Tyr Ile His His Met Gly Tyr Val His 125 130 135 Arg Ser
Val Lys Ala Ser His Ile Leu Ile Ser Val Asp Gly Lys 140 145 150 Val
Tyr Leu Ser Gly Leu Arg Thr Thr Leu Ser Met Ile Ser His 155 160 165
Gly Gln Arg Gln Arg Val Val His Asp Phe Pro Lys Tyr Ser Val 170 175
180 Lys Val Leu Pro Trp Leu Ser Pro Glu Val Leu Gln Gln Asn Leu 185
190 195 Gln Gly Tyr Asp Ala Lys Ser Asp Ile Tyr Ser Val Gly Ile Thr
200 205 210 Ala Cys Glu Leu Ala Asn Gly His Val Pro Phe Lys Asp Met
Pro 215 220 225 Ala Thr Gln Met Leu Leu Glu Lys Leu Asn Gly Thr Val
Pro Cys 230 235 240 Leu Leu Asp Thr Ser Thr Ile Pro Ala Glu Glu Leu
Thr Met Ser 245 250 255 Pro Ser Arg Ser Val Ala Asn Ser Gly Leu Ser
Asp Ser Leu Thr 260 265 270 Thr Ser Thr Pro Arg Pro Ser Asn Gly Asp
Ser Pro Ser His Pro 275 280 285 Tyr His Arg Thr Phe Ser Pro His Phe
His His Phe Val Glu Gln 290 295 300 Cys Leu Gln Arg Asn Pro Asp Ala
Arg Tyr Pro Cys Trp Pro Gly 305 310 315 Pro Gly Leu Arg Glu Ser Arg
Gly Cys Ser Gly Gly 320 325 6 345 PRT Homo sapiens 1567782 6 Met
Glu Lys Tyr Val Arg Leu Gln Lys Ile Gly Glu Gly Ser Phe 1 5 10 15
Gly Lys Ala Ile Leu Val Lys Ser Thr Glu Asp Gly Arg Gln Tyr 20 25
30 Val Ile Lys Glu Ile Asn Ile Ser Arg Met Ser Ser Lys Glu Arg 35
40 45 Glu Glu Ser Arg Arg Glu Val Ala Val Leu Ala Asn Met Lys His
50 55 60 Pro Asn Ile Val Gln Tyr Arg Glu Ser Phe Glu Gly Ile Leu
Asp 65 70 75 Trp Phe Val Gln Ile Cys Leu Ala Leu Lys His Val His
Asp Arg 80 85 90 Lys Ile Leu His Arg Asp Ile Lys Ser Gln Asn Ile
Phe Leu Thr 95 100 105 Lys Asp Gly Thr Val Gln Leu Gly Asp Phe Gly
Ile Ala Arg Val 110 115 120 Leu Asn Ser Thr Val Glu Leu Ala Arg Thr
Cys Ile Gly Thr Pro 125 130 135 Tyr Tyr Leu Ser Pro Glu Ile Cys Glu
Asn Lys Pro Tyr Asn Asn 140 145 150 Lys Ser Asp Ile Trp Ala Leu Gly
Cys Val Leu Tyr Glu Leu Cys 155 160 165 Thr Leu Lys His Ala Phe Glu
Ala Gly Ser Met Lys Asn Leu Val 170 175 180 Leu Lys Ile Ile Ser Gly
Ser Phe Pro Pro Val Ser Leu His Tyr 185 190 195 Ser Tyr Asp Leu Arg
Ser Leu Val Ser Gln Leu Phe Lys Arg Asn 200 205 210 Pro Arg Asp Arg
Pro Ser Val Asn Ser Ile Leu Glu Lys Gly Phe 215 220 225 Ile Ala Lys
Arg Ile Glu Lys Phe Leu Ser Pro Gln Leu Ile Ala 230 235 240 Glu Glu
Phe Cys Leu Lys Thr Phe Ser Lys Phe Gly Ser Gln Pro 245 250 255 Ile
Pro Ala Lys Arg Pro Ala Ser Gly Gln Asn Ser Ile Ser Val 260 265 270
Met Pro Ala Gln Lys Ile Thr Lys Pro Ala Ala Lys Tyr Gly Ile 275 280
285 Pro Leu Ala Tyr Lys Lys Tyr Gly Asp Lys Lys Leu His Glu Lys 290
295 300 Lys Pro Leu Gln Lys His Lys Gln Ala His Gln Thr Pro Glu Lys
305 310 315 Arg Val Asn Thr Gly Glu Glu Arg Arg Lys Ile Ser Glu Glu
Ala 320 325 330 Ala Arg Lys Arg Arg Leu Glu Phe Ile Glu Lys Asp Lys
Glu Arg 335 340 345 7 424 PRT Homo sapiens 2295842 7 Met Ile Ser
Phe Cys Pro Asp Cys Gly Lys Ser Ile Gln Ala Ala 1 5 10 15 Phe Lys
Phe Cys Pro Tyr Cys Gly Asn Ser Leu Pro Val Glu Glu 20 25 30 His
Val Gly Ser Gln Thr Phe Val Asn Pro His Val Ser Ser Phe 35 40 45
Gln Gly Ser Gly Ser Arg Pro Pro Thr Pro Lys Ser Ser Pro Gln 50 55
60 Lys Thr Arg Lys Ser Pro Gln Val Thr Arg Gly Ser Pro Gln Lys 65
70 75 Thr Ser Cys Ser Pro Gln Lys Thr Arg Gln Ser Pro Gln Thr Leu
80 85 90 Lys Arg Ser Arg Val Thr Thr Ser Leu Glu Ala Leu Pro Thr
Gly 95 100 105 Thr Val Leu Thr Asp Lys Ser Gly Arg Gln Trp Lys Leu
Lys Ser 110 115 120 Phe Gln Thr Arg Asp Asn Gln Gly Ile Leu Tyr Glu
Ala Ala Pro 125 130 135 Thr Ser Thr Leu Thr Cys Asp Ser Gly Pro Gln
Lys Gln Lys Phe 140 145 150 Ser Leu Lys Leu Asp Ala Lys Asp Gly Arg
Leu Phe Asn Glu Gln 155 160 165 Asn Phe Phe Gln Arg Ala Ala Lys Pro
Leu Gln Val Asn Lys Trp 170 175 180 Lys Lys Leu Tyr Ser Thr Pro Leu
Leu Ala Ile Pro Thr Cys Met 185 190 195 Gly Phe Gly Val His Gln Asp
Lys Tyr Arg Phe Leu Val Leu Pro 200 205 210 Ser Leu Gly Arg Ser Leu
Gln Ser Ala Leu Asp Val Ser Pro Lys 215 220 225 His Val Leu Ser Glu
Arg Ser Val Leu Gln Val Ala Cys Arg Leu 230 235 240 Leu Asp Ala Leu
Glu Phe Leu His Glu Asn Glu Tyr Val His Gly 245 250 255 Asn Val Thr
Ala Glu Asn Ile Phe Val Asp Pro Glu Asp Gln Ser 260 265 270 Gln Val
Thr Leu Ala Gly Tyr Gly Phe Ala Phe Arg Tyr Cys Pro 275 280 285 Ser
Gly Lys His Val Ala Tyr Val Glu Gly Ser Arg Ser Pro His 290 295 300
Glu Gly Asp Leu Glu Phe Ile Ser Met Asp Leu His Lys Gly Cys 305 310
315 Gly Pro Ser Arg Arg Ser Asp Leu Gln Ser Leu Gly Tyr Cys Met 320
325 330 Leu Lys Trp Leu Tyr Gly Phe Leu Pro Trp Thr Asn Cys Leu Pro
335 340 345 Asn Thr Glu Asp Ile Met Lys Gln Lys Gln Lys Phe Val Asp
Lys 350 355 360 Pro Gly Pro Phe Val Gly Pro Cys Gly His Trp Ile Arg
Pro Ser 365 370 375 Glu Thr Leu Gln Lys Tyr Leu Lys Val Val Met Ala
Leu Thr Tyr 380 385 390 Glu Glu Lys Pro Pro Tyr Ala Met Leu Arg Asn
Asn Leu Glu Ala 395 400 405 Leu Leu Gln Asp Leu Arg Val Ser Pro Tyr
Asp Pro Ile Gly Leu 410 415 420 Pro Met Val Pro 8 99 PRT Homo
sapiens 2605059 8 Met Pro Leu Glu Glu Val Leu Pro Asp Val Ser Pro
Gln Ala Leu 1 5 10 15 Asp Leu Leu Gly Gln Phe Leu Leu Tyr Pro Pro
His Gln Arg Ile 20 25 30 Ala Ala Ser Lys Ala Leu Leu His Gln Tyr
Phe Phe Thr Ala Pro 35 40 45 Leu Pro Ala His Pro Ser Glu Leu Pro
Ile Pro Gln Arg Leu Gly 50 55 60 Gly Pro Ala Pro Lys Ala His Pro
Gly Pro Pro His Ile His Asp 65 70 75 Phe His Val Asp Arg Pro Leu
Glu Glu Ser Leu Leu Asn Ser Glu 80 85 90 Leu Ile Arg Pro Phe Ile
Leu Glu Gly 95 9 138 PRT Homo sapiens 3000825 9 Met Trp Val Val Pro
Pro Ile Gly Ala Glu Phe Leu Gly Thr Glu 1 5 10 15 Lys Gly Gly Leu
Arg Asp Gln Lys Thr Pro Asp Asp His Glu Ala 20 25 30 Glu Thr Gly
Ile Lys Ser Lys Glu Ala Arg Lys Tyr Ile Phe Asn 35 40 45 Cys Leu
Asp Ala Cys Val Gln Val Asn Met Thr Thr Asp Leu Glu 50
55 60 Gly Ser Asp Met Leu Val Glu Lys Ala Asp Arg Arg Glu Phe Ile
65 70 75 Asp Leu Leu Lys Lys Met Leu Thr Ile Asp Ala Asp Lys Arg
Ile 80 85 90 Thr Pro Ile Glu Thr Leu Asn His Pro Phe Val Thr Met
Thr His 95 100 105 Leu Leu Asp Phe Pro His Ser Thr His Val Lys Ser
Cys Phe Gln 110 115 120 Asn Met Glu Ile Cys Lys Arg Arg Val Asn Met
Tyr Asp Thr Val 125 130 135 Asn Gln Ser 10 1427 DNA Homo sapiens
119819 10 cggagccaca gtggctccac cccccacctt cacgcactcc cacggtggta
atcccgaaag 60 gctgggtggc tgggctgacg gtaattcccg gggggggtca
agtgccccaa actgctcttg 120 gtgaaaggat gctgtcttcc ccgaatggcc
acttccgcct gccttagctt gggctgagag 180 gggacagaga gcaccctgag
gcgggccggc caggtcttcc cactcctaat ggagctgtgg 240 ggagtggggc
cacaggcggg gaggcaggga gagtagtgag tagctggtgc caaggggcgc 300
tggcgccaca ttctggtgtc catgggagcc ctggggcccg gagaggcctc ttccctggcg
360 gctgtgcagg gaaacctcca cttcatgctg actggggcgg gcgacaggaa
ccctggggtg 420 accctggctc tgacagcaga ccggtaagct gtccaaaaac
aagaggaaga agatgaggcg 480 caaacggaaa cagcagaagc ggctgctgga
ggagcggctg cgggacctgc agaggctgga 540 ggccatggag gctgccaccc
aggctgagga ctctggcttg agactagacg ggggcagcgg 600 ctccacatcc
tcttcaggct gtcaccccgg gggcgccaga gcaggtccct ccccagcctc 660
ttcctccccc gccccagggg gcggccgtag cctcagcgcg ggctcacaga cctcaggctt
720 ctccggctcc ctcttctctc ctgcctcctg ctccatcctc tccggctcgt
ccaatcagcg 780 agagaccggg ggcctcctgt cgcctagcac accattcggt
gcctcgaacc tcctggtgaa 840 ccccctggag ccccaaaatg cagataagat
caagatcaag atcgcagacc tgggcaacgc 900 ctgctgggtg cacaagcact
tcacggaaga catccagact cggcagtacc gggccgtcga 960 ggtgctgatc
ggcgccgaat acggcccccc ggcagacatc tggagcacag cctgcatggc 1020
cttcgagctg gccactggtg actacctgtt cgagccgcat tctggagaag actacagtcg
1080 tgatgaggac cacatcgctc acatagtgga gcttctgggg gacatccccc
cagccttcgc 1140 cctctcaggc cgctattccc gggagttctt caaccggaga
ggagagctgc ggcacatcca 1200 caatctcaag cactggggcc tgtacgaggt
actcatggaa aagtacgagt ggcccctaga 1260 gcaggccaca cagttcagcg
cctttctgct gcccatgaat gagtacatcc ccgaaaagcg 1320 ggccagtgcc
cgtgactgcc tccagcaccc ctggctccaa ccctagggcc cggctgtggc 1380
tccacctcca gctctccgtg cctttaaggg aaaagcggga cagctcc 1427 11 1586
DNA Homo sapiens 132750 11 gctcattgac tcttttgtct tctttcctct
cgggggtgag gtcagattta ccaccaaaat 60 gcatgcagga gatcccgcaa
gagcaaatca aggagatcaa gaaggagcag ctttcaggat 120 ccccgtggat
tctgctaagg gaaaatgaag tcagcacact ttataaagga gaataccaca 180
gagctccagt ggccataaaa gtattcaaaa aactccaggc tggcagcatt gcaatagtga
240 ggcagacttt caataaggag atcaaaacca tgaagaaatt cgaatctccc
aacatcctgc 300 gtatatttgg gatttgcatt gatgaaacag tgactccgcc
tcaattctcc attgtcatgg 360 agtactgtga actcgggacc ctgagggagc
tgttggatag ggaaaaagac ctcacacttg 420 gcaagcgcat ggtcctagtc
ctgggggcag cccgaggcct ataccggcta caccattcag 480 aagcacctga
actccacgga aaaatcagaa gctcaaactt cctggtaact caaggctacc 540
aagtgaagct tgcaggattt gagttgagga aaacacagac ttccatgagt ttgggaacta
600 cgagagaaaa gacagacaga gtcaaatcta cagcatatct ctcacctcag
gaactggaag 660 atgtatttta tcaatatgat gtaaagtctg aaatatacag
ctttggaatc gtcctctggg 720 aaatcgccac tggagatatc ccgtttcaag
gctgtaattc tgagaagatc cgcaagctgg 780 tggctgtgaa gcggcagcag
gagccactgg gtgaagactg cccttcagag ctgcgggaga 840 tcattgatga
gtgccgggcc catgatccct ctgtgcggcc ctctgtggat gaaatcttaa 900
agaaactctc caccttttct aagtagtgta tcaaaatcta aaccaaggag tctctggaca
960 agaagctggg agaggcacaa actggacatc tctctctctc atatccttcg
gcattgggtt 1020 atctatggga gcaaggagtg ggcacgcttc tctgttacaa
atagaaaacg attccagtca 1080 tacaggacac atcccactcc aaatgatatt
tccaaaaaca tacctctgac agtaactttg 1140 atagatggtt tgtcaaatgt
atctttctgg gtatccacac ctcttggcaa tgaaatttgc 1200 agctcctccc
ttccataaat gaagtctctt tccccaccat ttgaatctgg gctggcactg 1260
tgacttgatt tgatcaatag aatgtggaag aagtgactgt atgccagttc caagcctagg
1320 tttcaagagg ccttataaat gtctgttgga accttaccca gccatgaaca
tgttgagtga 1380 gcatgctgga gaatgagaga ccacatgaag cagaaacatg
ctttcctagc tgaagtcata 1440 ctagcccaac caacatggca gctaacacat
gaatgaggcc aatcaagacc agaagaacca 1500 ctcaagcaga tcccagccca
aattgcccat tcacacaatc aggagctaaa taaattactg 1560 ttgtcttaac
actaaaaaaa aaaaaa 1586 12 1574 DNA Homo sapiens 507669 12
cgacggcgaa gggagctgag actgtccagg cagccaggtt aggccaggag gaccatgtga
60 atggggccag aaggctcccg ggctgggcag ggaccatggg ctgtggctgc
agctcacacc 120 cggaagatga ctggatggaa aacatcgatg tgtgtgagaa
ctgccattat cccatagtcc 180 cactggatgg caagggcacg ctgctcatcc
gaaatggctc tgaggtgcgg gacccactgg 240 ttacctacga aggctccaat
ccgccggctt ccccactgca agacaacctg gttatcgctc 300 tgcacagcta
tgagccctct cacgacggag atctgggctt tgagaagggg gaacagctcc 360
gcatcctgga gcagagcggc gagtggtgga aggcgcagtc cctgaccacg ggccaggaag
420 gcttcatccc cttcaatttt gtggccaaag cgaacagcct ggagcccgag
gccaacctca 480 tgaagcagct gcaacaccag cggctggttc ggctctacgc
tgtggtcacc caggagccca 540 tctacatcat cactgaatac atggagaatg
ggagtctagt ggattttctc aagacccctt 600 caggcatcaa gttgaccatc
aacaaactcc tggacatggc agcccaaatt gcagaaggca 660 tggcattcat
tgaagagcgg aattatattc atcgtgacct tcgggctgcc aacattctgg 720
tgtctgacac cctgagctgc aagattgcag actttggcct agcacgcctc attgaggaca
780 acgagtacac agccagggag ggggccaagt ttcccattaa gtggacagcg
ccagaagcca 840 ttaactacgg gacattcacc atcaagtcag atgtgtggtc
ttttgggatc ctgctgacgg 900 aaattgtcac ccacggccgc atcccttacc
cagggatgac caacccggag gtgattcaga 960 acctggagcg aggctaccgc
atggtgcgcc ctgacaactg tccagaggag ctgtaccaac 1020 tcatgaggct
gtgctggaag gagcgcccag aggaccggcc cacctttgac tacctgcgca 1080
gtgtgctgga ggacttcttc acggccacag agggccagta ccagcctcag ccttgagagg
1140 ccttgagagg ccctggggtt ctcccccttt ctctccagcc tgacttgggg
agatggagtt 1200 cttgtgccat agtcacatgg cctatgcaca tatggactct
gcacatgaat cccacccaca 1260 tgtgacacat atgcaccttg tgtctgtaca
cgtgtcctgt agttgcgtgg actctgcaca 1320 tgtcttgtac atgtgtagcc
tgtgcatgta tgtcttggac actgtacaag gtaccccttt 1380 ctggctctcc
catttcctga gaccacagag agaggggaga agcctgggat tgacagaagc 1440
ttctgcccac ctacttttct ttcctcagat catccagaag ttcctcaagg gccaggactt
1500 tatctaatac ctctgtgtgc tcctccttgg tgcctggcct ggcacacatc
aggagttcaa 1560 taaatgtctg ttga 1574 13 1866 DNA Homo sapiens
1439938 13 cgggaggaag agggagaggg agaccgggac gagaccgggg ctgtggtgcg
gagagaggct 60 gagacggaga agaggagagg cagagagggc gcggggaccg
tcagcagcac cttagctaca 120 atcgttcagc tattctcgga agagagaagg
gagagggagg aggccggggc gggagtgggg 180 gctgtcaccc tcggaccccg
gcgtgagagg ggccgtgcgg ccggacgtcc tcggggtggg 240 cccccagtcg
gtggccgaag acctacagct caggcccctg ggtcccaaat ttccaggctt 300
tgcccctcct cctttctcag atacccgggt aacagtcctc atagtccaga tatccgggac
360 tcgggtccca acctctctaa acctgggtct ctgtttcata gaatttcaaa
tatcaggttc 420 aggcccctgc gtgcaccagt atccggggtt cattccccgg
gcgttcagat atcggattca 480 gtctccatcc cgttcagata ttcggggttc
agaccccaca atcagaaatc cggaattcgg 540 cagctgtcgc cctcgacgag
ggggaggact ggaccgcgag gtcagattag gttgtcaccc 600 cctcccctcc
aggggaggct tcccgggccc gcccctcagg aagggcgaaa gccgaggaag 660
aggtggcaag gggaaaggtc tccttgcccc tctccctgct tggcagagcc gctggaggac
720 cccaggcgga agcggaggcg ctggggcacc atagtgaccc ctaccaggcc
aggccccact 780 ctcagggccc ccaggggcca ccatgccagc tgggggccgg
gccgggagcc tgaaggaccc 840 agatgtggct gagctcttct tcaaggatga
cccagaaaag ctcttctctg acctccggga 900 aattggccat ggcagctttg
gagccgtata ctttgcccgg gatgtccgga atagtgaggt 960 ggtggccatc
aagaagatgt cctacagtgg gaagcagtcc aatgagaaat ggcaagacat 1020
catcaaggag gtgcggagac gaaggagagt agggagggag gatgaagaga gataaggggg
1080 agaaaagaga ggggcatgag agtggagcgg agctaagaag gggtagaaga
gagagtgggt 1140 gaaggggaag agacgtagag aaagtgtgga gagaggaaag
gcatagcgag agaacgaggg 1200 agagagaagt ggaaggggga agtaagagag
gataagagga acgagaggag gggaagggtg 1260 gggacgagaa cgaagagcat
gatggagagg aaagatagag aagagaggaa gtggaggcag 1320 ttagggggca
tggaggagag agagagatga gggagagtgg gagcacgggg cggatggacg 1380
gggtggagaa gaagagaggg aggagatgag aggaggaaga ggtgggagaa ccgagcgagg
1440 gaaaagatgg aggaggcagt agagagggtg tgcaaggggt gaaaagaaag
aagaaggaaa 1500 aggatggagg gagtgaaggt aggagacgag gaggagggat
gggagagaat ggagggtagc 1560 gtgtggatgg tgagtggtag agaatagtga
gatggtgaga agcggagaaa ggcagcagag 1620 gatgggggtg aagcgggaag
caaagacaat aggggatgga ggaggagagg agcaggagga 1680 agacgaagag
cgaagggctt gaaagaggga gaagagagta gtaaggggta ggtatgtaga 1740
tgcgagtagg agaggaagag aaggaatgaa tgagagagag tagagagtag agagagaacg
1800 aaggaacggg gcagagggag aggaaggaca gaaggagaag agaacaatcg
aagaatgaga 1860 gtgttt 1866 14 1498 DNA Homo sapiens unsure 1350,
1355, 1372, 1444 a or g or c or t, unknown, or other 14 ctcccctccc
agcaaccggt ctggcggcgg cgcggcagta aaactgagga ggcggagcaa 60
gacggtcggg gctgcttgct aactccagga acaggtttaa gtttttgaaa ctgaagtagg
120 tctacacagt aggaactcat gtcatttctt gtaagtaaac ccagagcgaa
tccaggacca 180 atgatgcgag ctcagagtca atagcatcct tctctaaaca
ggaggtcatg agtagctttc 240 tgccagaggg agggtgttac gagctgctca
ctgtgatagg caaaggattt gaggacctga 300 tgactgtgaa tctagcaagg
tacaaaccaa caggagagta cgtgactgta cggaggatta 360 acctagaagc
ttgttccaat gagatggtaa cattcttgca gggcgagctg catgtctcca 420
aactcttcaa ccatcccaat atcgtgccat atcgagccac ttttattgca gacaatgagc
480 tgtgggttgt cacatcattc atggcatacg gttctgcaaa agatctcatc
tgtacacact 540 tcatggatgg catgaatgag ctggcgattg cttacatcct
gcagggggtg ctgaaggccc 600 tcgactacat ccaccacatg ggatatgtac
acaggagtgt caaagccagc cacatcctga 660 tctctgtgga tgggaaggtc
tacctgtctg gtttgcgcac aacgctcagc atgataagcc 720 atgggcagcg
gcagcgagtg gtccacgatt ttcccaagta cagtgtcaag gttctgccgt 780
ggctcagccc cgaggtcctc cagcagaatc tccagggtta tgatgccaag tctgacatct
840 acagtgtggg aatcacagcc tgtgaactgg ccaacggcca tgtccccttt
aaggatatgc 900 ctgccaccca gatgctgcta gagaaactga acggcacagt
gccctgcctg ttggatacca 960 gcaccatccc cgctgaggag ctgaccatga
gcccttcgcg ctcagtggcc aactctggcc 1020 tgagtgacag cctgaccacc
agcacccccc ggccctccaa cggtgactcg ccctcccacc 1080 cctaccaccg
aaccttctcc ccccacttcc accactttgt ggagcagtgc cttcagcgca 1140
acccggatgc caggtatccc tgctggcctg ggcctgggct tcgggagagc agagggtgct
1200 caggagggta aggccagggt gtgaagggac ttacctccca aaggttctgc
aggggaatct 1260 ggagctacac acaggaggga tcagctcctg ggtgtgtcag
aggccagcct ggggagctct 1320 ggccactgct tcccatgagc tgagggagan
ggagnaggga cccgaggctg angcataagt 1380 ggcaggattt tcggaagctg
gggacacggc agtgatgctg cggtctctcc ctcccttacc 1440 tcangctcag
tgcagcaccc tctgaacact ctttctcagc agtatcgtag ccttcgtt 1498 15 1846
DNA Homo sapiens 1567782 15 taggaattcg tcgacccacg cgatccgccg
tcagaagact gccacaccta gactgatgct 60 tattagtcat caccgttatt
cctactaacg tcctgtgtca ctgagttttt taaatgtcta 120 gcatatctgt
aaagatgcct tagaaaaaga atcatggaga agtatgttag actacagaag 180
attggagaag gttcatttgg aaaagccatt cttgttaaat ctacagaaga tggcagacag
240 tatgttatca aggaaattaa catctcaaga atgtccagta aagaaagaga
agaatcaagg 300 agagaagttg cagtattggc aaacatgaag catccaaata
ttgtccagta tagagaatca 360 tttgaaggaa ttttggactg gtttgtacag
atatgtttgg ccctgaaaca tgtacatgat 420 agaaaaattc ttcatcgaga
cattaaatct cagaacatat ttttaactaa agatggaaca 480 gtacaacttg
gagattttgg aattgctaga gttcttaata gtactgtaga gctggctcga 540
acttgcatag ggaccccata ctacttgtca cctgaaatct gtgaaaacaa accttacaat
600 aataaaagtg acatttgggc tctggggtgt gtcctttatg agctgtgtac
acttaaacat 660 gcttttgaag ctggcagtat gaaaaacctg gtactgaaga
taatatctgg atcttttcca 720 cctgtgtctt tgcattattc ctatgatctc
cgcagtttgg tgtctcagtt atttaaaaga 780 aatcctaggg atagaccatc
agtcaactcc atattggaga aaggttttat agccaaacgc 840 attgaaaagt
ttctctctcc tcagcttatt gcagaagaat tttgtctaaa aacattttcg 900
aagtttggat cacagcctat accagctaaa agaccagctt caggacaaaa ctcgatttct
960 gttatgcctg ctcagaaaat tacaaagcct gccgctaaat atggaatacc
tttagcatat 1020 aagaaatatg gagataaaaa attacacgaa aagaaaccac
tgcaaaaaca taaacaggcc 1080 catcaaactc cagagaagag agtgaatact
ggagaagaaa ggaggaaaat atctgaggaa 1140 gcagcaagaa agagaaggct
ggaatttatt gaaaaagata aggaacggta ggatcagatt 1200 attagtttaa
tgaaggctga acaaatgaaa aggcaagaca aggaaaggtt ggaaagaata 1260
aatagggcca gggaacaagg atggagaaat gtgctaagtg ctggtggaag tggtgaagta
1320 aaggtaggca ttttatacca atatggttat actaccattt tcccctccag
ttccaccttg 1380 ttctataaaa tgcatgtact tgggattttc tttctttctt
tagtgtacaa ttaattttta 1440 cctagaattc tttaacattt attatgaata
cttagctttc ctgcatgtat ctgatatgta 1500 acttgtgttg ctgttatgtg
actatactca aaattgcttt aaaagttttt tgtgaagact 1560 atgataacat
tattcctgtc aggaattttt aaaaattatg tacaattcat gacactgcag 1620
cctaaaatcg ttctgtaatt tcatgtagcc ttgaagatta agttctcaga agatgcttct
1680 taaatccgat ccctgttgtc tctccaattt catcaccatt cattccccta
ccacatactg 1740 ggaagggcct attccatggc ggaaatgaag ggccataatt
tgtaggtttt ccattaccaa 1800 taatgggggg ttggcccaaa tcctactttt
gggcctttgg aacctt 1846 16 1721 DNA Homo sapiens 2295842 16
agttggacga ggctcagtga aagttttcgc tgggcaactg agaaggtcgc tgtcaagatg
60 gagtttccaa cccagtaaat ccaagggcca gaccgtgacc tcataaagca
tgatctcctt 120 ctgtccagac tgtggcaaaa gtatccaagc ggcattcaaa
ttctgcccct actgtggaaa 180 ttctttgcct gtagaggagc atgtagggtc
ccagaccttt gtcaatccac atgtgtcatc 240 cttccaaggc tccgggagca
gacccccaac ccccaaaagc agccctcaga agaccaggaa 300 gagccctcag
gtgaccaggg gtagccctca gaagaccagc tgtagccctc agaagaccag 360
gcagagccct cagacgctga agcggagccg agtgaccacc tcacttgaag ctttgcccac
420 agggacagtg ctgacagaca agagtgggcg acagtggaag ctgaagtcct
tccagaccag 480 ggacaaccag ggcattctct atgaagctgc acccacctcc
accctcacct gtgactcagg 540 accacagaag caaaagttct cactcaaact
ggatgccaag gatgggcgct tgttcaatga 600 gcagaacttc ttccagcggg
ccgccaagcc tctgcaagtc aacaagtgga agaagctgta 660 ctcgacccca
ctgctggcca tccctacctg catgggtttc ggtgttcacc aggacaaata 720
caggttcttg gtgttaccca gcctggggag gagccttcag tcggccctgg atgtcagccc
780 aaagcatgtg ctgtcagaga ggtctgtgct gcaggtggcc tgccggctgc
tggatgccct 840 ggagttcctc catgagaatg agtatgttca tggaaatgtg
acagctgaaa atatctttgt 900 ggatccagag gaccagagtc aggtgacttt
ggcaggctat ggcttcgcct tccgctattg 960 cccaagtggc aaacacgtgg
cctacgtgga aggcagcagg agccctcacg agggggacct 1020 tgagttcatt
agcatggacc tgcacaaggg atgcgggccc tcccgccgca gcgacctcca 1080
gagcctgggc tactgcatgc tgaagtggct ctacgggttt ctgccatgga caaattgcct
1140 tcccaacact gaggacatca tgaagcaaaa acagaagttt gttgataagc
cggggccctt 1200 cgtgggaccc tgcggtcact ggatcaggcc ctcagagacc
ctgcagaagt acctgaaggt 1260 ggtgatggcc ctcacgtatg aggagaagcc
gccctacgcc atgctgagga acaacctaga 1320 agctttgctg caggatctgc
gtgtgtctcc atatgacccc attggcctcc cgatggtgcc 1380 ctaggtggaa
tccagaactt tccatttgca gtgtgcaaca gaaaaaaaaa aatgaagtaa 1440
tgtgactcaa ggcctgctgt ttaatcacag ataagcttct agaacaagcc ctggaatgtg
1500 cattcctgcc actggtttca ggatactcat cagtcctgat tagcctcccg
gagggcccca 1560 gtttccctcc cgtgaatgtg aagttcccca tcttggtggc
ctgcccttca gccagtgtcc 1620 tagcaaagct ggatggggtt gggccggccc
acagggggga cccctcctac ccttgacacc 1680 tctgtgcttt ggtaataaat
tgttttacca gaaaaaaaaa a 1721 17 1985 DNA Homo sapiens 2605059 17
ttcgcatctt gggcacccca aaccctcaag tctggccggt ttgtaggggc ccttggtgag
60 gtgggtgtgg ggcaggttta ctccactccc aacagcaagt aaccactccc
tcccctgaac 120 cttctctctc ctggccccaa ccccccttga tggacaggga
ccactgtcct ggcccaactc 180 agggcttcct ccttcctgct gtcatttggg
ttggggtaga tcctgtcctt tgtccctttt 240 caccctagta cacacatgtg
cagtgtctca gcaagctgtg cacagagtcg tcatctgaga 300 gggcaagggg
atggatgaag gaatacaggg gtgggtgagt gaatgaatga tgggtcaggg 360
agacacatgg atgggagagc accccccatg tgagtgtgtg ttaggggctg agagttgaca
420 gcagagagca tggcaagggt cgggaactac tctcattgta ccctgttcct
tctccctggc 480 ccaggagctc actgagctgc cggactacaa caagatctcc
tttaaggagc aggtgcccat 540 gcccctggag gaggtgctgc ctgacgtctc
tccccaggca ttggatctgc tgggtcaatt 600 ccttctctac cctcctcacc
agcgcatcgc agcttccaag gctctcctcc atcagtactt 660 cttcacagct
cccctgcctg cccatccatc tgagctgccg attcctcagc gtctaggggg 720
acctgccccc aaggcccatc cagggccccc ccacatccat gacttccacg tggaccggcc
780 tcttgaggag tcgctgttga actcagagct gattcggccc ttcatcctgg
aggggtgaga 840 agttggccct ggtcccgtct gcctgctcct caggaccact
cagtccacct gttcctctgc 900 cacctgcctg gcttcaccct ccaaggcctc
cccatggcca cagtgggccc acaccacacc 960 ctgcccctta gcccttgcga
gggttggtct cgaggcagag gtcatgttcc cagccaagag 1020 tatgagaaca
tccagtcgag cagaggagat tcatggcctg tgctcggtga gccttacctt 1080
ctgtgtgcta ctgacgtacc catcaggaca gtgagctctg ctgccagtca aggcctgcat
1140 atgcagaatg acgatgcctg ccttggtgct gcttccccga gtgctgcctc
ctggtcaagg 1200 agaagtgcag agagtaaggt gtccttatgt tggaaactca
agtggaagga agatttggtt 1260 tggttttatt ctcagagcca ttaaacacta
gttcagtatg tgagatatag attctaaaaa 1320 cctcaggtgg ctctgcctta
tgtctgttcc tccttcattt ctctcaaggg aaatggctaa 1380 ggtggcattg
tctcatggct ctcgtttttg gggtcatggg gagggtagca ccaggcatag 1440
ccacttttgc cctgagggac tcctgtgtgc ttcacatcac tgagcactca tttagaagtg
1500 agggagacag aagtctaggc ccagggatgg ctccagttgg ggatccagca
ggagaccctc 1560 tgcacatgag gctggtttac caacatctac tccctcagga
tgagcgtgag ccagaagcag 1620 ctgtgtattt aaggaaacaa gcgttcctgg
aattaattta taaatttaat aaatcccaat 1680 ataatcccag ctagtgcttt
ttccttatta taatttgata aggtgattat aaaagataca 1740 tggaaggaag
tggaaccaga tgcagaagag gaaatgatgg aaggacttat ggtatcagat 1800
accaatattt aaaagtttgt ataataataa agagtatgat tgtggttcaa ggataaaaac
1860 agactagaga aacttattct tagccatcct ttatttttat tttatttatt
ttttgatgga 1920 gtcttgctct gttgcccact gcaattcaag ccttggtgac
agactctggt ctcaaaaaaa 1980 aaaaa 1985 18 661 DNA Homo sapiens
3000825 18 tgaggagtga tgaaagctgc atttcaactt aactgatgaa agcaggagca
gtttacatcc 60 tgtcattcag atatatttgc aggtcccagc agcagccctc
tccccttcct ggggcacagc 120 ccctctctgc ctttcctgca gagagaaaag
ccacatcctg tgggcaatga caacatgtgg 180 gtggtgcctc ccataggggc
agagttcctg ggaactgaga aagggggctt gagagatcag 240 aagacaccag
atgaccatga agcagagaca gggattaagt caaaagaagc aagaaagtac 300
attttcaact gtttagatgc ttgcgtccag gtgaacatga cgacagattt ggaagggagc
360 gacatgttgg tagaaaaggc tgaccggcgg gagttcattg acctgttgaa
gaagatgctg 420 accattgatg ctgacaagag aatcactcca
atcgaaaccc tgaaccatcc ctttgtcacc 480 atgacacact tactcgattt
tccccacagc acacacgtca aatcatgttt ccagaacatg 540 gagatctgca
agcgtcgggt gaatatgtat gacacggtga accagagcta aacctagccc 600
caaacccctc tgccgaatat cctcgctcga gggccaaatt ccctatagtg gtcgtattac
660 g 661
* * * * *