U.S. patent application number 11/023453 was filed with the patent office on 2005-05-19 for isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Beasley, Ellen M., Di Francesco, Valentina, Guegler, Karl, Ketchum, Karen A., Merkulov, Gennady V., Wei, Ming-Hui, Woodage, Trevor.
Application Number | 20050106622 11/023453 |
Document ID | / |
Family ID | 26904298 |
Filed Date | 2005-05-19 |
United States Patent
Application |
20050106622 |
Kind Code |
A1 |
Wei, Ming-Hui ; et
al. |
May 19, 2005 |
Isolated human kinase proteins, nucleic acid molecules encoding
human kinase proteins, and uses thereof
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the kinase
peptides of the present invention. The present invention
specifically provides isolated peptide and nucleic acid molecules,
methods of identifying orthologs and paralogs of the kinase
peptides, and methods of identifying modulators of the kinase
peptides.
Inventors: |
Wei, Ming-Hui; (Germantown,
MD) ; Guegler, Karl; (Menlo Park, CA) ;
Ketchum, Karen A.; (Germantown, MD) ; Merkulov,
Gennady V.; (Baltimore, MD) ; Woodage, Trevor;
(Washington, DC) ; Di Francesco, Valentina;
(Rockville, MD) ; Beasley, Ellen M.; (Darnestown,
MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Norwalk
CT
|
Family ID: |
26904298 |
Appl. No.: |
11/023453 |
Filed: |
December 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11023453 |
Dec 29, 2004 |
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10153917 |
May 24, 2002 |
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6852519 |
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10153917 |
May 24, 2002 |
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09732025 |
Dec 8, 2000 |
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6416990 |
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60209585 |
Jun 6, 2000 |
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Current U.S.
Class: |
435/6.16 ;
435/194; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61K 38/00 20130101; C12N 9/1205 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/194; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/12 |
Claims
That which is claimed is:
1. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
2. An isolated peptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence shown in
SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said allelic
variant is encoded by a nucleic acid molecule that hybridizes under
stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of
an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein
said ortholog is encoded by a nucleic acid molecule that hybridizes
under stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino
acid sequence shown in SEQ ID NO:2, wherein said fragment comprises
at least 10 contiguous amino acids.
3. An isolated antibody that selectively binds to a peptide of
claim 2.
4. An isolated nucleic acid molecule consisting of a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
6. A gene chip comprising a nucleic acid molecule of claim 5.
7. A transgenic non-human animal comprising a nucleic acid molecule
of claim 5.
8. A nucleic acid vector comprising a nucleic acid molecule of
claim 5.
9. A host cell containing the vector of claim 8.
10. A method for producing any of the peptides of claim 1
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
11. A method for producing any of the peptides of claim 2
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
12. A method for detecting the presence of any of the peptides of
claim 2 in a sample, said method comprising contacting said sample
with a detection agent that specifically allows detection of the
presence of the peptide in the sample and then detecting the
presence of the peptide.
13. A method for detecting the presence of a nucleic acid molecule
of claim 5 in a sample, said method comprising contacting the
sample with an oligonucleotide that hybridizes to said nucleic acid
molecule under stringent conditions and determining whether the
oligonucleotide binds to said nucleic acid molecule in the
sample.
14. A method for identifying a modulator of a peptide of claim 2,
said method comprising contacting said peptide with an agent and
determining if said agent has modulated the function or activity of
said peptide.
15. The method of claim 14, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
16. A method for identifying an agent that binds to any of the
peptides of claim 2, said method comprising contacting the peptide
with an agent and assaying the contacted mixture to determine
whether a complex is formed with the agent bound to the
peptide.
17. A pharmaceutical composition comprising an agent identified by
the method of claim 16 and a pharmaceutically acceptable carrier
therefor.
18. A method for treating a disease or condition mediated by a
human kinase protein, said method comprising administering to a
patient a pharmaceutically effective amount of an agent identified
by the method of claim 16.
19. A method for identifying a modulator of the expression of a
peptide of claim 2, said method comprising contacting a cell
expressing said peptide with an agent, and determining if said
agent has modulated the expression of said peptide.
20. An isolated human kinase peptide having an amino acid sequence
that shares at least 70% homology with an amino acid sequence shown
in SEQ ID NO:2.
21. A peptide according to claim 20 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO:2.
22. An isolated nucleic acid molecule encoding a human kinase
peptide, said nucleic acid molecule sharing at least 80 percent
homology with a nucleic acid molecule shown in SEQ ID NOS:1 or
3.
23. A nucleic acid molecule according to claim 22 that shares at
least 90 percent homology with a nucleic acid molecule shown in SEQ
ID NOS:1 or 3.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of kinase proteins
that are related to the MAP/extracellular signal-regulated kinase
subfamily, recombinant DNA molecules, and protein production. The
present invention specifically provides novel peptides and proteins
that effect protein phosphorylation and nucleic acid molecules
encoding such peptide and protein molecules, all of which are
useful in the development of human therapeutics and diagnostic
compositions and methods.
BACKGROUND OF THE INVENTION
[0002] Protein Kinases
[0003] Kinases regulate many different cell proliferation,
differentiation, and signaling processes by adding phosphate groups
to proteins. Uncontrolled signaling has been implicated in a
variety of disease conditions including inflammation, cancer,
arteriosclerosis, and psoriasis. Reversible protein phosphorylation
is the main strategy for controlling activities of eukaryotic
cells. It is estimated that more than 1000 of the 10,000 proteins
active in a typical mammalian cell are phosphorylated. The high
energy phosphate, which drives activation, is generally transferred
from adenosine triphosphate molecules (ATP) to a particular protein
by protein kinases and removed from that protein by protein
phosphatases. Phosphorylation occurs in response to extracellular
signals (hormones, neurotransmitters, growth and differentiation
factors, etc), cell cycle checkpoints, and environmental or
nutritional stresses and is roughly analogous to turning on a
molecular switch. When the switch goes on, the appropriate protein
kinase activates a metabolic enzyme, regulatory protein, receptor,
cytoskeletal protein, ion channel or pump, or transcription
factor.
[0004] The kinases comprise the largest known protein group, a
superfamily of enzymes with widely varied functions and
specificities. They are usually named after their substrate, their
regulatory molecules, or some aspect of a mutant phenotype. With
regard to substrates, the protein kinases may be roughly divided
into two groups; those that phosphorylate tyrosine residues
(protein tyrosine kinases, PTK) and those that phosphorylate serine
or threonine residues (serine/threonine kinases, STK). A few
protein kinases have dual specificity and phosphorylate threonine
and tyrosine residues. Almost all kinases contain a similar 250-300
amino acid catalytic domain. The N-terminal domain, which contains
subdomains I-IV, generally folds into a two-lobed structure, which
binds and orients the ATP (or GTP) donor molecule. The larger C
terminal lobe, which contains subdomains VI A-XI, binds the protein
substrate and carries out the transfer of the gamma phosphate from
ATP to the hydroxyl group of a serine, threonine, or tyrosine
residue. Subdomain V spans the two lobes.
[0005] The kinases may be categorized into families by the
different amino acid sequences (generally between 5 and 100
residues) located on either side of, or inserted into loops of, the
kinase domain. These added amino acid sequences allow the
regulation of each kinase as it recognizes and interacts with its
target protein. The primary structure of the kinase domains is
conserved and can be further subdivided into 11 subdomains. Each of
the 11 subdomains contains specific residues and motifs or patterns
of amino acids that are characteristic of that subdomain and are
highly conserved (Hardie, G. and Hanks, S. (1995) The Protein
Kinase Facts Books, Vol I:7-20 Academic Press, San Diego,
Calif.).
[0006] The second messenger dependent protein kinases primarily
mediate the effects of second messengers such as cyclic AMP (cAMP),
cyclic GMP, inositol triphosphate, phosphatidylinositol,
3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid,
diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent
protein kinases (PKA) are important members of the STK family.
Cyclic-AMP is an intracellular mediator of hormone action in all
prokaryotic and animal cells that have been studied. Such
hormone-induced cellular responses include thyroid hormone
secretion, cortisol secretion, progesterone secretion, glycogen
breakdown, bone resorption, and regulation of heart rate and force
of heart muscle contraction. PKA is found in all animal cells and
is thought to account for the effects of cyclic-AMP in most of
these cells. Altered PKA expression is implicated in a variety of
disorders and diseases including cancer, thyroid disorders,
diabetes, atherosclerosis, and cardiovascular disease (Isselbacher,
K. J. et al. (1994) Harrison's Principles of Internal Medicine,
McGraw-Hill, New York, N.Y., pp. 416-431, 1887).
[0007] Calcium-calmodulin (CaM) dependent protein kinases are also
members of STK family. Calmodulin is a calcium receptor that
mediates many calcium regulated processes by binding to target
proteins in response to the binding of calcium. The principle
target protein in these processes is CaM dependent protein kinases.
CaM-kinases are involved in regulation of smooth muscle contraction
(MLC kinase), glycogen breakdown (phosphorylase kinase), and
neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I
phosphorylates a variety of substrates including the
neurotransmitter related proteins synapsin I and II, the gene
transcription regulator, CREB, and the cystic fibrosis conductance
regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal
14:3679-86). CaM II kinase also phosphorylates synapsin at
different sites, and controls the synthesis of catecholamines in
the brain through phosphorylation and activation of tyrosine
hydroxylase. Many of the CaM kinases are activated by
phosphorylation in addition to binding to CaM. The kinase may
autophosphorylate itself, or be phosphorylated by another kinase as
part of a "kinase cascade".
[0008] Another ligand-activated protein kinase is 5'-AMP-activated
protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol. Chem.
15:8675-81). Mammalian AMPK is a regulator of fatty acid and sterol
synthesis through phosphorylation of the enzymes acetyl-CoA
carboxylase and hydroxymethylglutaryl-CoA reductase and mediates
responses of these pathways to cellular stresses such as heat shock
and depletion of glucose and ATP. AMPK is a heterotrimeric complex
comprised of a catalytic alpha subunit and two non-catalytic beta
and gamma subunits that are believed to regulate the activity of
the alpha subunit. Subunits of AMPK have a much wider distribution
in non-lipogenic tissues such as brain, heart, spleen, and lung
than expected. This distribution suggests that its role may extend
beyond regulation of lipid metabolism alone.
[0009] PRK (proliferation-related kinase) is a serum/cytokine
inducible STK that is involved in regulation of the cell cycle and
cell proliferation in human megakaroytic cells (Li, B. et al.
(1996) J. Biol. Chem. 271:19402-8). PRK is related to the polo
(derived from humans polo gene) family of STKs implicated in cell
division. PRK is downregulated in lung tumor tissue and may be a
proto-oncogene whose deregulated expression in normal tissue leads
to oncogenic transformation.
[0010] The cyclin-dependent protein kinases (CDKs) are another
group of STKs that control the progression of cells through the
cell cycle. Cyclins are small regulatory proteins that act by
binding to and activating CDKs that then trigger various phases of
the cell cycle by phosphorylating and activating selected proteins
involved in the mitotic process. CDKs are unique in that they
require multiple inputs to become activated. In addition to the
binding of cyclin, CDK activation requires the phosphorylation of a
specific threonine residue and the dephosphorylation of a specific
tyrosine residue.
[0011] Protein tyrosine kinases, PTKs, specifically phosphorylate
tyrosine residues on their target proteins and may be divided into
transmembrane, receptor PTKs and nontransmembrane, non-receptor
PTKs. Transmembrane protein-tyrosine kinases are receptors for most
growth factors. Binding of growth factor to the receptor activates
the transfer of a phosphate group from ATP to selected tyrosine
side chains of the receptor and other specific proteins. Growth
factors (GF) associated with receptor PTKs include; epidermal GF,
platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and
insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage
colony stimulating factor.
[0012] Non-receptor PTKs lack transmembrane regions and, instead,
form complexes with the intracellular regions of cell surface
receptors. Such receptors that function through non-receptor PTKs
include those for cytokines, hormones (growth hormone and
prolactin) and antigen-specific receptors on T and B
lymphocytes.
[0013] Many of these PTKs were first identified as the products of
mutant oncogenes in cancer cells where their activation was no
longer subject to normal cellular controls. In fact, about one
third of the known oncogenes encode PTKs, and it is well known that
cellular transformation (oncogenesis) is often accompanied by
increased tyrosine phosphorylation activity (Carbonneau H and Tonks
NK (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK
activity may therefore be an important strategy in controlling some
types of cancer.
[0014] Extracellular Signal-Regulated Kinases
(ERKs)/Mitogen-Activated Protein (MAP Kinases
[0015] The protein provided by the present invention is a novel
human mitogen-activated protein (MAP) kinase, also referred to as
extracellular signal-regulated kinases (ERKs). The MAP kinases are
members of the STK family. MAP kinases regulate numerous cellular
signaling pathways and mediate signal transduction from the cell
surface to the nucleus via phosphorylation cascades. Several
subgroups have been identified, and each manifests different
substrate specificities and responds to distinct extracellular
stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature
365:781-783). MAP kinase signaling pathways are present in
mammalian cells as well as in yeast. The extracellular stimuli that
activate mammalian pathways include epidermal growth factor (EGF),
ultraviolet light, hyperosmolar medium, heat shock, endotoxic
lipopolysaccharide (LPS), and pro-inflammatory cytokines such as
tumor necrosis factor (TNF) and interleukin-1 (IL-1). Altered MAP
kinase expression is implicated in a variety of disease conditions
including cancer, inflammation, immune disorders, and disorders
affecting growth and development.
[0016] MAP kinases may be the central integration point for
numerous biochemical signals because they are activated by a wide
variety of extracellular signals, are highly phosphorylated at
threonine and tyrosine residues, and are highly conserved between
species (Crews et al., Science 258: 478-480, 1992).
[0017] MEK1 and MEK2 are also ERKs/MAP kinases. Constitutive
activation of MEK1 causes cellular transformation and therefore
MEK1 is an ideal drug target for treating proliferative diseases.
Furthermore, inhibition of MEK1 results in up to 80% reduction in
colon carcinoma tumor growth, with no toxic side effects
(Sebolt-Leopold et al., Nature Med. 5: 810-816, 1999). Thus,
inhibitors of MEK and other ERKs/MAP kinases are useful as safe,
effective treatments for cancers such as colon cancer.
[0018] The ERK protein provided by the present invention shows a
high degree of structural similarity to ERK7. ERK7 is
constitutively active in serum-starved cells, and this activity is
dependent on the presence of a C-terminal tail, which regulates the
nuclear localization and growth inhibiting functions of ERK7. ERK7
therefore represents a novel type of MAP kinase characterized by
the importance of interactions via its C-terminal tail, rather than
extracellular signal-mediated activation cascades, in regulating
its activity, localization, and function (Abe et al., Mol Cell Biol
1999 February; 19(2):1301-12).
[0019] For a further review of ERKs/MAP kinases, see Crews et al.,
Science 258: 478-480, 1992; Orth et al., Science 285: 1920-1923,
1999; Rampoldi et al., Cytogenet. Cell Genet. 78: 301-303, 1997;
Ryan et al., Nature 404: 892-897, 2000; Sebolt-Leopold et al.,
Nature Med. 5: 810-816, 1999; Seger et al., FASEB J.9: 726-735,
1995; Seger et al., J. Biol. Chem. 267: 25628-25631, 1992; and
Zheng et al., J. Biol. Chem. 268: 11435-11439, 1993.
[0020] Kinase proteins, particularly members of the
MAP/extracellular signal-regulated kinase subfamily, are a major
target for drug action and development. Accordingly, it is valuable
to the field of pharmaceutical development to identify and
characterize previously unknown members of this subfamily of kinase
proteins. The present invention advances the state of the art by
providing previously unidentified human kinase proteins that have
homology to members of the MAP/extracellular signal-regulated
kinase subfamily.
SUMMARY OF THE INVENTION
[0021] The present invention is based in part on the identification
of amino acid sequences of human kinase peptides and proteins that
are related to the MAP/extracellular signal-regulated kinase
subfamily, as well as allelic variants and other mammalian
orthologs thereof. These unique peptide sequences, and nucleic acid
sequences that encode these peptides, can be used as models for the
development of human therapeutic targets, aid in the identification
of therapeutic proteins, and serve as targets for the development
of human therapeutic agents that modulate kinase activity in cells
and tissues that express the kinase. Experimental data as provided
in FIG. 1 indicates expression in humans in the larynx, kidney
(adult and fetal), pancreas, fetal heart, uterus, and prostate.
DESCRIPTION OF THE FIGURE SHEETS
[0022] FIG. 1 provides the nucleotide sequence of a cDNA molecule
that encodes the kinase protein of the present invention. (SEQ ID
NO:1) In addition, structure and functional information is
provided, such as ATG start, stop and tissue distribution, where
available, that allows one to readily determine specific uses of
inventions based on this molecular sequence. Experimental data as
provided in FIG. 1 indicates expression in humans in the larynx,
kidney (adult and fetal), pancreas, fetal heart, uterus, and
prostate.
[0023] FIG. 2 provides the predicted amino acid sequence of the
kinase of the present invention. (SEQ ID NO:2) In addition
structure and functional information such as protein family,
function, and modification sites is provided where available,
allowing one to readily determine specific uses of inventions based
on this molecular sequence.
[0024] FIG. 3 provides genomic sequences that span the gene
encoding the kinase protein of the present invention. (SEQ ID NO:3)
In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As illustrated in FIG.
3, five SNPs were identified, including one SNP 5' of the ORF that
may affect control/regulatory elements.
DETAILED DESCRIPTION OF THE INVENTION
[0025] General Description
[0026] The present invention is based on the sequencing of the
human genome. During the sequencing and assembly of the human
genome, analysis of the sequence information revealed previously
unidentified fragments of the human genome that encode peptides
that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art
as being a kinase protein or part of a kinase protein and are
related to the MAP/extracellular signal-regulated kinase subfamily.
Utilizing these sequences, additional genomic sequences were
assembled and transcript and/or cDNA sequences were isolated and
characterized. Based on this analysis, the present invention
provides amino acid sequences of human kinase peptides and proteins
that are related to the MAP/extracellular signal-regulated kinase
subfamily, nucleic acid sequences in the form of transcript
sequences, cDNA sequences and/or genomic sequences that encode
these kinase peptides and proteins, nucleic acid variation (allelic
information), tissue distribution of expression, and information
about the closest art known protein/peptide/domain that has
structural or sequence homology to the kinase of the present
invention.
[0027] In addition to being previously unknown, the peptides that
are provided in the present invention are selected based on their
ability to be used for the development of commercially important
products and services. Specifically, the present peptides are
selected based on homology and/or structural relatedness to known
kinase proteins of the MAP/extracellular signal-regulated kinase
subfamily and the expression pattern observed. Experimental data as
provided in FIG. 1 indicates expression in humans in the larynx,
kidney (adult and fetal), pancreas, fetal heart, uterus, and
prostate. The art has clearly established the commercial importance
of members of this family of proteins and proteins that have
expression patterns similar to that of the present gene. Some of
the more specific features of the peptides of the present
invention, and the uses thereof, are described herein, particularly
in the Background of the Invention and in the annotation provided
in the Figures, and/or are known within the art for each of the
known MAP/extracellular signal-regulated kinase family or subfamily
of kinase proteins.
[0028] Specific Embodiments
[0029] Peptide Molecules
[0030] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the kinase family of proteins and are related to the
MAP/extracellular signal-regulated kinase subfamily (protein
sequences are provided in FIG. 2, transcript/cDNA sequences are
provided in FIG. 1 and genomic sequences are provided in FIG. 3).
The peptide sequences provided in FIG. 2, as well as the obvious
variants described herein, particularly allelic variants as
identified herein and using the information in FIG. 3, will be
referred herein as the kinase peptides of the present invention,
kinase peptides, or peptides/proteins of the present invention.
[0031] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the kinase peptides disclosed in the FIG.
2, (encoded by the nucleic acid molecule shown in FIG. 1,
transcript/cDNA or FIG. 3, genomic sequence), as well as all
obvious variants of these peptides that are within the art to make
and use. Some of these variants are described in detail below.
[0032] As used herein, a peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
free of chemical precursors or other chemicals. The peptides of the
present invention can be purified to homogeneity or other degrees
of purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components (the features of an
isolated nucleic acid molecule is discussed below).
[0033] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0034] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the kinase peptide having less than about
30% (by dry weight) chemical precursors or other chemicals, less
than about 20% chemical precursors or other chemicals, less than
about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0035] The isolated kinase peptide can be purified from cells that
naturally express it, purified from cells that have been altered to
express it (recombinant), or synthesized using known protein
synthesis methods. Experimental data as provided in FIG. 1
indicates expression in humans in the larynx, kidney (adult and
fetal), pancreas, fetal heart, uterus, and prostate. For example, a
nucleic acid molecule encoding the kinase peptide is cloned into an
expression vector, the expression vector introduced into a host
cell and the protein expressed in the host cell. The protein can
then be isolated from the cells by an appropriate purification
scheme using standard protein purification techniques. Many of
these techniques are described in detail below.
[0036] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence
of such a protein is provided in FIG. 2. A protein consists of an
amino acid sequence when the amino acid sequence is the final amino
acid sequence of the protein.
[0037] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues,
for example from about 1 to about 100 or so additional residues,
typically from 1 to about 20 additional residues in the final
protein.
[0038] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2),
for example, proteins encoded by the transcript/cDNA nucleic acid
sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences
provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid
sequence when the amino acid sequence is at least part of the final
amino acid sequence of the protein. In such a fashion, the protein
can be only the peptide or have additional amino acid molecules,
such as amino acid residues (contiguous encoded sequence) that are
naturally associated with it or heterologous amino acid
residues/peptide sequences. Such a protein can have a few
additional amino acid residues or can comprise several hundred or
more additional amino acids. The preferred classes of proteins that
are comprised of the kinase peptides of the present invention are
the naturally occurring mature proteins. A brief description of how
various types of these proteins can be made/isolated is provided
below.
[0039] The kinase peptides of the present invention can be attached
to heterologous sequences to form chimeric or fusion proteins. Such
chimeric and fusion proteins comprise a kinase peptide operatively
linked to a heterologous protein having an amino acid sequence not
substantially homologous to the kinase peptide. "Operatively
linked" indicates that the kinase peptide and the heterologous
protein are fused in-frame. The heterologous protein can be fused
to the N-terminus or C-terminus of the kinase peptide.
[0040] In some uses, the fusion protein does not affect the
activity of the kinase peptide per se. For example, the fusion
protein can include, but is not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig
fusions. Such fusion proteins, particularly poly-His fusions, can
facilitate the purification of recombinant kinase peptide. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased by using a heterologous
signal sequence.
[0041] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A kinase peptide-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the kinase peptide.
[0042] As mentioned above, the present invention also provides and
enables obvious variants of the amino acid sequence of the proteins
of the present invention, such as naturally occurring mature forms
of the peptide, allelic/sequence variants of the peptides,
non-naturally occurring recombinantly derived variants of the
peptides, and orthologs and paralogs of the peptides. Such variants
can readily be generated using art-known techniques in the fields
of recombinant nucleic acid technology and protein biochemistry. It
is understood, however, that variants exclude any amino, acid
sequences disclosed prior to the invention.
[0043] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the kinase peptides
of the present invention. The degree of homology/identity present
will be based primarily on whether the peptide is a functional
variant or non-functional variant, the amount of divergence present
in the paralog family and the evolutionary distance between the
orthologs.
[0044] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment, at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of
a reference sequence is aligned for comparison purposes. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0045] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0046] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0047] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the kinase peptides of the present invention as
well as being encoded by the same genetic locus as the kinase
peptide provided herein. The gene provided by the present invention
is located on a genome component that has been mapped to human
chromosome 8 (as indicated in FIG. 3), which is supported by
multiple lines of evidence, such as STS and BAC map data.
[0048] Allelic variants of a kinase peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the kinase peptide as well as being encoded by the same genetic
locus as the kinase peptide provided herein. Genetic locus can
readily be determined based on the genomic information provided in
FIG. 3, such as the genomic sequence mapped to the reference human.
The gene provided by the present invention is located on a genome
component that has been mapped to human chromosome 8 (as indicated
in FIG. 3), which is supported by multiple lines of evidence, such
as STS and BAC map data. As used herein, two proteins (or a region
of the proteins) have significant homology when the amino acid
sequences are typically at least about 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous. A significantly
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence that will hybridize to a
kinase peptide encoding nucleic acid molecule under stringent
conditions as more fully described below.
[0049] FIG. 3 provides information on SNPs that have been found in
the gene encoding the kinase protein of the present invention. The
following variations were identified: T1004G, G1822T, A2023G,
A2562G, and C6624A. SNPs such as these that are located in introns
and 5' of the ORF may affect control/regulatory elements.
[0050] Paralogs of a kinase peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the kinase peptide, as being encoded by a gene
from humans, and as having similar activity or function. Two
proteins will typically be considered paralogs when the amino acid
sequences are typically at least about 60% or greater, and more
typically at least about 70% or greater homology through a given
region or domain. Such paralogs will be encoded by a nucleic acid
sequence that will hybridize to a kinase peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0051] Orthologs of a kinase peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the kinase peptide as well as being encoded by a
gene from another organism. Preferred orthologs will be isolated
from mammals, preferably primates, for the development of human
therapeutic targets and agents. Such orthologs will be encoded by a
nucleic acid sequence that will hybridize to a kinase peptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins.
[0052] Non-naturally occurring variants of the kinase peptides of
the present invention can readily be generated using recombinant
techniques. Such variants include, but are not limited to
deletions, additions and substitutions in the amino acid sequence
of the kinase peptide. For example, one class of substitutions are
conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a kinase peptide by another
amino acid of like characteristics. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr; exchange of the acidic residues Asp
and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues Lys and Arg; and replacements among
the aromatic residues Phe and Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al., Science 247:1306-1310 (1990).
[0053] Variant kinase peptides can be fully functional or can lack
function in one or more activities, e.g. ability to bind substrate,
ability to phosphorylate substrate, ability to mediate signaling,
etc. Fully functional variants typically contain only conservative
variation or variation in non-critical residues or in non-critical
regions. FIG. 2 provides the result of protein analysis and can be
used to identify critical domains/regions. Functional variants can
also contain substitution of similar amino acids that result in no
change or an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0054] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0055] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the results
provided in FIG. 2. The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as kinase
activity or in assays such as an in vitro proliferative activity.
Sites that are critical for binding partner/substrate binding can
also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312
(1992)).
[0056] The present invention further provides fragments of the
kinase peptides, in addition to proteins and peptides that comprise
and consist of such fragments, particularly those comprising the
residues identified in FIG. 2. The fragments to which the invention
pertains, however, are not to be construed as encompassing
fragments that may be disclosed publicly prior to the present
invention.
[0057] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a kinase peptide.
Such fragments can be chosen based on the ability to retain one or
more of the biological activities of the kinase-peptide or could be
chosen for the ability to perform a function, e.g. bind a substrate
or act as an immunogen. Particularly important fragments are
biologically active fragments, peptides that are, for example,
about 8 or more amino acids in length. Such fragments will
typically comprise a domain or motif of the kinase peptide, e.g.,
active site, a transmembrane domain or a substrate-binding domain.
Further, possible fragments include, but are not limited to, domain
or motif containing fragments, soluble peptide fragments, and
fragments containing immunogenic structures. Predicted domains and
functional sites are readily identifiable by computer programs well
known and readily available to those of skill in the art (e.g.,
PROSITE analysis). The results of one such analysis are provided in
FIG. 2.
[0058] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in kinase peptides are described
in basic texts, detailed monographs, and the research literature,
and they are well known to those of skill in the art (some of these
features are identified in FIG. 2).
[0059] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0060] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[0061] Accordingly, the kinase peptides of the present invention
also encompass derivatives or analogs in which a substituted amino
acid residue is not one encoded by the genetic code, in which a
substituent group is included, in which the mature kinase peptide
is fused with another compound, such as a compound to increase the
half-life of the kinase peptide (for example, polyethylene glycol),
or in which the additional amino acids are fused to the mature
kinase peptide, such as a leader or secretory sequence or a
sequence for purification of the mature kinase peptide or a
pro-protein sequence.
[0062] Protein/Peptide Uses
[0063] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in the Figures; to raise antibodies or to
elicit another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein (or its binding partner or ligand) in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a
kinase-effector protein interaction or kinase-ligand interaction),
the protein can be used to identify the binding partner/ligand so
as to develop a system to identify inhibitors of the binding
interaction. Any or all of these uses are-capable of being
developed into reagent grade or kit format for commercialization as
commercial products.
[0064] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0065] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, kinases isolated from
humans and their human/mammalian orthologs serve as targets for
identifying agents for use in mammalian therapeutic applications,
e.g. a human drug, particularly in modulating a biological or
pathological response in a cell or tissue that expresses the
kinase. Experimental data as provided in FIG. 1 indicates that
kinase proteins of the present invention are expressed in humans in
the larynx, kidney (adult and fetal), pancreas, fetal heart,
uterus, and prostate. Specifically, a virtual northern blot shows
expression in the larynx, kidney, and pancreas. In addition,
PCR-based tissue screening panels indicate expression in the fetal
heart, fetal kidney, uterus, prostate, and pancreas. A large
percentage of pharmaceutical agents are being developed that
modulate the activity of kinase proteins, particularly members of
the MAP/extracellular signal-regulated kinase subfamily (see
Background of the Invention). The structural and functional
information provided in the Background and Figures provide specific
and substantial uses for the molecules of the present invention,
particularly in combination with the expression information
provided in FIG. 1. Experimental data as provided in FIG. 1
indicates expression in humans in the larynx, kidney (adult and
fetal), pancreas, fetal heart, uterus, and prostate. Such uses can
readily be determined using the information provided herein, that
which is known in the art, and routine experimentation.
[0066] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to kinases that
are related to members of the MAP/extracellular signal-regulated
kinase subfamily. Such assays involve any of the known kinase
functions or activities or properties useful for diagnosis and
treatment of kinase-related conditions that are specific for the
subfamily of kinases that the one of the present invention belongs
to, particularly in cells and tissues that express the kinase.
Experimental data as provided in FIG. 1 indicates that kinase
proteins of the present invention are expressed in humans in the
larynx, kidney (adult and fetal), pancreas, fetal heart, uterus,
and prostate. Specifically, a virtual northern blot shows
expression in the larynx, kidney, and pancreas. In addition,
PCR-based tissue screening panels indicate expression in the fetal
heart, fetal kidney, uterus, prostate, and pancreas.
[0067] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the kinase, as a biopsy or expanded in cell culture. Experimental
data as provided in FIG. 1 indicates expression in humans in the
larynx, kidney (adult and fetal), pancreas, fetal heart, uterus,
and prostate. In an alternate embodiment, cell-based assays involve
recombinant host cells expressing the kinase protein.
[0068] The polypeptides can be used to identify compounds that
modulate kinase activity of the protein in its natural state or an
altered form that causes a specific disease or pathology associated
with the kinase. Both the kinases of the present invention and
appropriate variants and fragments can be used in high-throughput
screens to assay candidate compounds for the ability to bind to the
kinase. These compounds can be further screened against a
functional kinase to determine the effect of the compound on the
kinase activity. Further, these compounds can be tested in animal
or invertebrate systems to determine activity/effectiveness.
Compounds can be identified that activate (agonist) or inactivate
(antagonist) the kinase to a desired degree.
[0069] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the kinase protein and a molecule that normally
interacts with the kinase protein, e.g. a substrate or a component
of the signal pathway that the kinase protein normally interacts
(for example, another kinase). Such assays typically include the
steps of combining the kinase protein with a candidate compound
under conditions that allow the kinase protein, or fragment, to
interact with the target molecule, and to detect the formation of a
complex between the protein and the target or to detect the
biochemical consequence of the interaction with the kinase protein
and the target, such as any of the associated effects of signal
transduction such as protein phosphorylation, cAMP turnover, and
adenylate cyclase activation, etc.
[0070] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides. (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0071] One candidate compound is a soluble fragment of the receptor
that competes for substrate binding. Other candidate compounds
include mutant kinases or appropriate fragments containing
mutations that affect kinase function and thus compete for
substrate. Accordingly, a fragment that competes for substrate, for
example with a higher affinity, or a fragment that binds substrate
but does not allow release, is encompassed by the invention.
[0072] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) kinase
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate kinase activity. Thus,
the phosphorylation of a substrate, activation of a protein, a
change in the expression of genes that are up- or down-regulated in
response to the kinase protein dependent signal cascade can be
assayed.
[0073] Any of the biological or biochemical functions mediated by
the kinase can be used as an endpoint assay. These include all of
the biochemical or biochemical/biological events described herein,
in the references cited herein, incorporated by reference for these
endpoint assay targets, and other functions known to those of
ordinary skill in the art or that can be readily identified using
the information provided in the Figures, particularly FIG. 2.
Specifically, a biological function of a cell or tissues that
expresses the kinase can be assayed. Experimental data as provided
in FIG. 1 indicates that kinase proteins of the present invention
are expressed in humans in the larynx, kidney (adult and fetal),
pancreas, fetal heart, uterus, and prostate. Specifically, a
virtual northern blot shows expression in the larynx, kidney, and
pancreas. In addition, PCR-based tissue screening panels indicate
expression in the fetal heart, fetal kidney, uterus, prostate, and
pancreas.
[0074] Binding and/or activating compounds can also be screened by
using chimeric kinase proteins in which the amino terminal
extracellular domain, or parts thereof, the entire transmembrane
domain or subregions, such as any of the seven transmembrane
segments or any of the intracellular or extracellular loops and the
carboxy terminal intracellular domain, or parts thereof, can be
replaced by heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate then that which is recognized by the native
kinase. Accordingly, a different set of signal transduction
components is available as an end-point assay for activation. This
allows for assays to be performed in other than the specific host
cell from which the kinase is derived.
[0075] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the kinase (e.g. binding partners
and/or ligands). Thus, a compound is exposed to a kinase
polypeptide under conditions that allow the compound to bind or to
otherwise interact with the polypeptide. Soluble kinase polypeptide
is also added to the mixture. If the test compound interacts with
the soluble kinase polypeptide, it decreases the amount of complex
formed or activity from the kinase target. This type of assay is
particularly useful in cases in which compounds are sought that
interact with specific regions of the kinase. Thus, the soluble
polypeptide that competes with the target kinase region is designed
to contain peptide sequences corresponding to the region of
interest.
[0076] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the kinase protein, or fragment, or
its target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0077] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of kinase-binding protein found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques. For example, either the polypeptide or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively,
antibodies reactive with the protein but which do not interfere
with binding of the protein to its target molecule can be
derivatized to the wells of the plate, and the protein trapped in
the wells by antibody conjugation. Preparations of a kinase-binding
protein and a candidate compound are incubated in the kinase
protein-presenting wells and the amount of complex trapped in the
well can be quantitated. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the kinase protein target molecule, or which are
reactive with kinase protein and compete with the target molecule,
as well as enzyme-linked assays which rely on detecting an
enzymatic activity associated with the target molecule.
[0078] Agents that modulate one of the kinases of the present
invention can be identified using one or more of the above assays,
alone or in combination. It is generally preferable to use a
cell-based or cell free system first and then confirm activity in
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0079] Modulators of kinase protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the kinase pathway, by treating cells or
tissues that express the kinase. Experimental data as provided in
FIG. 1 indicates expression in humans in the larynx, kidney (adult
and fetal), pancreas, fetal heart, uterus, and prostate. These
methods of treatment include the steps of administering a modulator
of kinase activity in a pharmaceutical composition to a subject in
need of such treatment, the modulator being identified as described
herein.
[0080] In yet another aspect of the invention, the kinase proteins
can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with the
kinase and are involved in kinase activity. Such kinase-binding
proteins are also likely to be involved in the propagation of
signals by the kinase proteins or kinase targets as, for example,
downstream elements of a kinase-mediated signaling pathway.
Alternatively, such kinase-binding proteins are likely to be kinase
inhibitors.
[0081] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a kinase
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a kinase-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the kinase protein.
[0082] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a kinase-modulating
agent, an antisense kinase nucleic acid molecule, a kinase-specific
antibody, or a kinase-binding partner) can be used in an animal or
other model to determine the efficacy, toxicity, or side effects of
treatment with such an agent. Alternatively, an agent identified as
described herein can be used in an animal or other model to
determine the mechanism of action of such an agent. Furthermore,
this invention pertains to uses of novel agents identified by the
above-described screening assays for treatments as described
herein.
[0083] The kinase proteins of the present invention are also useful
to provide a target for diagnosing a disease or predisposition to
disease mediated by the peptide. Accordingly, the invention
provides methods for detecting the presence, or levels of, the
protein (or encoding mRNA) in a cell, tissue, or organism.
Experimental data as provided in FIG. 1 indicates expression in
humans in the larynx, kidney (adult and fetal), pancreas, fetal
heart, uterus, and prostate. The method involves contacting a
biological sample with a compound capable of interacting with the
kinase protein such that the interaction can be detected. Such an
assay can be provided in a single detection format or a
multi-detection format such-as an antibody chip array.
[0084] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject. The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered kinase activity in cell-based or
cell-free assay, alteration in substrate or antibody-binding
pattern, altered isoelectric point, direct amino acid sequencing,
and any other of the known assay techniques useful for detecting
mutations in a protein. Such an assay can be provided in a single
detection format or a multi-detection format such as an antibody
chip array.
[0085] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0086] The peptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic ortherapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
kinase protein in which one or more of the kinase functions in one
population is different from those in another population. The
peptides thus allow a target to ascertain a genetic predisposition
that can affect treatment modality. Thus, in a ligand-based
treatment, polymorphism may give rise to amino terminal
extracellular domains and/or other substrate-binding regions that
are more or less active in substrate binding, and kinase
activation. Accordingly, substrate dosage would necessarily be
modified to maximize the therapeutic effect within a given
population containing a polymorphism. As an alternative to
genotyping, specific polymorphic peptides could be identified.
[0087] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in FIG. 1
indicates expression in humans in the larynx, kidney (adult and
fetal), pancreas, fetal heart, uterus, and prostate. Accordingly,
methods for treatment include the use of the kinase protein or
fragments.
[0088] Antibodies
[0089] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof. As used herein, an antibody selectively binds a target
peptide when it binds the target peptide and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0090] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0091] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0092] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. The full-length protein, an
antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments' are those covering functional
domains, such as the domains identified in FIG. 2, and domain of
sequence homology or divergence amongst the family, such as those
that can readily be identified using protein alignment methods and
as presented in the Figures.
[0093] Antibodies are preferably prepared from regions or discrete
fragments of the kinase proteins. Antibodies can be prepared from
any region of the peptide as described herein. However, preferred
regions will include those involved in function/activity and/or
kinase/binding partner interaction. FIG. 2 can be used to identify
particularly important regions while sequence alignment can be used
to identify conserved and unique sequence fragments.
[0094] An antigenic fragment will typically comprise at least 8
contiguous amino acid residues. The antigenic peptide can comprise,
however, at least 10, 12, 14, 16 or more amino acid residues. Such
fragments can be selected on a physical property, such as fragments
correspond to regions that are located on the surface of the
protein, e.g., hydrophilic regions or can be selected based on
sequence uniqueness (see FIG. 2).
[0095] Detection on an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0096] Antibody Uses
[0097] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Experimental data as provided in FIG. 1 indicates that kinase
proteins of the present invention are expressed in humans in the
larynx, kidney (adult and fetal), pancreas, fetal heart, uterus,
and prostate. Specifically, a virtual northern blot shows
expression in the larynx, kidney, and pancreas. In addition,
PCR-based tissue screening panels indicate expression in the fetal
heart, fetal kidney, uterus, prostate, and pancreas. Further, such
antibodies can be used to detect protein in situ, in vitro, or in a
cell lysate or supernatant in order to evaluate the abundance and
pattern of expression. Also, such antibodies can be used to assess
abnormal tissue distribution or abnormal expression during
development or progression of a biological condition. Antibody
detection of circulating fragments of the full length protein can
be used to identify turnover.
[0098] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in FIG. 1 indicates expression in humans in the larynx, kidney
(adult and fetal), pancreas, fetal heart, uterus, and prostate. If
a disorder is characterized by a specific mutation in the protein,
antibodies specific for this mutant protein can be used to assay
for the presence of the specific mutant protein.
[0099] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in FIG. 1 indicates
expression in humans in the larynx, kidney (adult and fetal),
pancreas, fetal heart, uterus, and prostate. The diagnostic uses
can be applied, not only in genetic testing, but also in monitoring
a treatment modality. Accordingly, where treatment is ultimately
aimed at correcting expression; level or the presence of aberrant
sequence and aberrant tissue distribution or developmental
expression, antibodies directed against the protein or relevant
fragments can be used to monitor therapeutic efficacy.
[0100] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0101] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in the larynx, kidney (adult and fetal), pancreas, fetal
heart, uterus, and prostate. Thus, where a specific protein has
been correlated with expression in a specific tissue, antibodies
that are specific for this protein can be used to identify a tissue
type.
[0102] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the kinase peptide
to a binding partner such as a substrate. These uses can also be
applied in a therapeutic context in which treatment involves
inhibiting the protein's function. An antibody can be used, for
example, to block binding, thus modulating (agonizing or
antagonizing) the peptides activity. Antibodies can be prepared
against specific fragments containing sites required for function
or against intact protein that is associated with a cell or cell
membrane. See FIG. 2 for structural information relating to the
proteins of the present invention.
[0103] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a standard;
and instructions for use. Such a kit can be supplied to detect a
single protein or epitope or can be configured to detect one of a
multitude of epitopes, such as in an antibody detection array.
Arrays are described in detail below for nucleic acid arrays and
similar methods have been developed for antibody arrays.
[0104] Nucleic Acid Molecules
[0105] The present invention further provides isolated nucleic acid
molecules that encode a kinase peptide or protein of the present
invention (cDNA, transcript and genomic sequence). Such nucleic
acid molecules will consist of, consist essentially of, or comprise
a nucleotide sequence that encodes one of the kinase peptides of
the present invention, an allelic variant thereof, or an ortholog
or paralog thereof.
[0106] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0107] Moreover, an "isolated" nucleic acid molecule, such as a
transcript/cDNA molecule, can be substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0108] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0109] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 1
or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists
of a nucleotide sequence when the nucleotide sequence is the
complete nucleotide sequence of the nucleic acid molecule.
[0110] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3,
genomic sequence), or any nucleic acid molecule that encodes the
protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a
nucleotide sequence is present with only a few additional nucleic
acid residues in the final nucleic acid molecule.
[0111] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 1 or
3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises
a nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleic acid residues, such
as nucleic acid residues that are naturally associated with it or
heterologous nucleotide sequences. Such a nucleic acid molecule can
have a few additional nucleotides or can comprises several hundred
or more additional nucleotides. A brief description of how various
types of these nucleic acid molecules can be readily made/isolated
is provided below.
[0112] In FIGS. 1 and 3, both coding and non-coding sequences are
provided. Because of the source of the present invention, humans
genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1),
the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5' and 3' non-coding sequences, gene regulatory
regions and non-coding intergenic sequences. In general such
sequence features are either noted in FIGS. 1 and 3 or can readily
be identified using computational tools known in the art. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0113] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0114] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the kinase
peptide alone, the sequence encoding the mature peptide and
additional coding sequences, such as a leader or secretory sequence
(e.g., a pre-pro or pro-protein sequence), the sequence encoding
the mature peptide, with or without the additional coding
sequences, plus additional non-coding sequences, for example
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding and stability of mRNA. In addition, the nucleic
acid molecule may be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0115] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0116] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention as well
as nucleic acid molecules that encode obvious variants of the
kinase proteins of the present invention that are described above.
Such nucleic acid molecules may be naturally occurring, such as
allelic variants (same locus), paralogs (different locus), and
orthologs (different organism), or may be constructed by
recombinant DNA methods or by chemical synthesis. Such
non-naturally occurring variants may be made by mutagenesis
techniques, including those applied to nucleic acid molecules,
cells, or organisms. Accordingly, as discussed above, the variants
can contain nucleotide substitutions, deletions, inversions and
insertions. Variation can occur in either or both the coding and
non-coding regions. The variations can produce both conservative
and non-conservative amino acid substitutions.
[0117] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence provided in
FIG. 3.
[0118] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0119] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive nucleotides.
[0120] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. Allelic variants can readily be
determined by genetic locus of the encoding gene. The gene provided
by the present invention is located on a genome component that has
been mapped to human chromosome 8 (as indicated in FIG. 3), which
is supported by multiple lines of evidence, such as STS and BAC map
data.
[0121] FIG. 3 provides information on SNPs that have been found in
the gene encoding the kinase protein of the present invention. The
following variations were identified: T1004G, G1822T, A2023G,
A2562G, and C6624A. SNPs such as these that are located in introns
and 5' of the ORF may affect control/regulatory elements.
[0122] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45 C, followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
[0123] Nucleic Acid Molecule Uses
[0124] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2. As illustrated in
FIG. 3, five SNPs were identified, including one SNP 5' of the ORF
that may affect control/regulatory elements.
[0125] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Figures.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0126] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0127] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0128] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0129] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. The gene provided by the
present invention is located on a genome component that has been
mapped to human chromosome 8 (as indicated in FIG. 3), which is
supported by multiple lines of evidence, such as STS and BAC map
data.
[0130] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0131] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein.
[0132] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0133] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0134] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0135] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form and distribution
of nucleic acid expression. Experimental data as provided in FIG. 1
indicates that kinase proteins of the present invention are
expressed in humans in the larynx, kidney (adult and fetal),
pancreas, fetal heart, uterus, and prostate. Specifically, a
virtual northern blot shows expression in the larynx, kidney, and
pancreas. In addition, PCR-based tissue screening panels indicate
expression in the fetal heart, fetal kidney, uterus, prostate, and
pancreas. Accordingly, the probes can be used to detect the
presence of, or to determine levels of, a specific nucleic acid
molecule in cells, tissues, and in organisms. The nucleic acid
whose level is determined can be DNA or RNA. Accordingly, probes
corresponding to the peptides described herein can be used to
assess expression and/or gene copy number in a given cell, tissue,
or organism. These uses are relevant for diagnosis of disorders
involving an increase or decrease in kinase protein expression
relative to normal results.
[0136] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro-techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0137] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a kinase protein, such as
by measuring a level of a kinase-encoding nucleic acid in a sample
of cells from a subject e.g., mRNA or genomic DNA, or determining
if a kinase gene has been mutated. Experimental data as provided in
FIG. 1 indicates that kinase proteins of the present invention are
expressed in humans in the larynx, kidney (adult and fetal),
pancreas, fetal heart, uterus, and prostate. Specifically, a
virtual northern blot shows expression in the larynx, kidney, and
pancreas. In addition, PCR-based tissue screening-panels indicate
expression in the fetal heart, fetal kidney, uterus, prostate, and
pancreas.
[0138] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate kinase nucleic acid
expression.
[0139] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the kinase gene, particularly biological
and pathological processes that are mediated by the kinase in cells
and tissues that express it. Experimental data as provided in FIG.
1 indicates expression in humans in the larynx, kidney (adult and
fetal), pancreas, fetal heart, uterus, and prostate. The method
typically includes assaying the ability of the compound to modulate
the expression of the kinase nucleic acid and thus identifying a
compound that can be used to treat a disorder characterized by
undesired kinase nucleic acid expression. The assays can be
performed in cell-based and cell-free systems. Cell-based assays
include cells naturally expressing the kinase nucleic acid or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[0140] The assay for kinase nucleic acid expression can involve
direct assay of nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in the signal pathway. Further, the
expression of genes that are up- or down-regulated in response to
the kinase protein signal pathway can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0141] Thus, modulators of kinase gene expression can be identified
in a method wherein a cell is contacted with a candidate compound
and the expression of mRNA determined. The level of expression of
kinase mRNA in the presence of the candidate compound is compared
to the level of expression of kinase mRNA in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of nucleic acid expression based on this comparison
and be used, for example to treat a disorder characterized by
aberrant nucleic acid expression. When expression of mRNA is
statistically significantly greater in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of nucleic acid expression. When nucleic
acid expression is statistically significantly less in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of nucleic acid
expression.
[0142] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound identified through
drug screening as a gene modulator to modulate kinase nucleic acid
expression in cells and tissues that express the kinase.
Experimental data as provided in FIG. 1 indicates that kinase
proteins of the present invention are expressed in humans in the
larynx, kidney (adult and fetal), pancreas, fetal heart, uterus,
and prostate. Specifically, a virtual northern blot shows
expression in the larynx, kidney, and pancreas. In addition,
PCR-based tissue screening panels indicate expression in the fetal
heart, fetal kidney, uterus, prostate, and pancreas. Modulation
includes both up-regulation (i.e. activation or agonization) or
down-regulation (suppression or antagonization) or nucleic acid
expression.
[0143] Alternatively, a modulator for kinase nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the kinase nucleic acid expression in the cells
and tissues that express the protein. Experimental data as provided
in FIG. 1 indicates expression in humans in the larynx, kidney
(adult and fetal), pancreas, fetal heart, uterus, and prostate.
[0144] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the kinase gene in clinical trials or in a treatment
regimen. Thus, the gene expression pattern can serve as a barometer
for the continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance. The gene expression pattern can also serve as a marker
indicative of a physiological response of the affected cells to the
compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0145] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in kinase nucleic acid expression,
and particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in kinase
genes and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally
occurring genetic mutations in the kinase gene and thereby to
determine whether a subject with the mutation is at risk for a
disorder caused by the mutation. Mutations include deletion,
addition, or substitution of one or more nucleotides in the gene,
chromosomal rearrangement, such as inversion or transposition,
modification of genomic DNA, such as aberrant methylation patterns
or changes in gene copy number, such as amplification. Detection of
a mutated form of the kinase gene associated with a dysfunction
provides a diagnostic tool for an active disease or susceptibility
to disease when the disease results from overexpression,
underexpression, or altered expression of a kinase protein.
[0146] Individuals carrying mutations in the kinase gene can be
detected at the nucleic acid level by a variety of techniques. FIG.
3 provides information on SNPs that have been found in the gene
encoding the kinase protein of the present invention. The following
variations were identified: T1004G, G1822T, A2023G, A2562G, and
C6624A. SNPs such as these that are located in introns and 5' of
the ORF may affect control/regulatory elements. The gene provided
by the present invention is located on a genome component that has
been mapped to human chromosome 8 (as indicated in FIG. 3), which
is supported by multiple lines of evidence, such as STS and BAC map
data. Genomic DNA can be analyzed directly or can be amplified by
using PCR prior to analysis. RNA or cDNA can be used in the same
way. In some uses, detection of the mutation involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.
Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,
or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,
Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et
al., PNAS 91:360-364 (1994)), the latter of which can be
particularly useful for detecting point mutations in the gene (see
Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method
can include the steps of collecting a sample of cells from a
patient, isolating nucleic acid (e.g., genomic, mRNA or both) from
the cells of the sample, contacting the nucleic acid sample with
one or more primers which specifically hybridize to a gene under
conditions such that hybridization and amplification of the gene
(if present) occurs, and detecting the presence or absence of an
amplification product, or detecting the size of the amplification
product and comparing the length to a control sample. Deletions and
insertions can be detected by a change in size of the amplified
product compared to the normal genotype. Point mutations can be
identified by hybridizing amplified DNA to normal RNA or antisense
DNA sequences.
[0147] Alternatively, mutations in a kinase gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0148] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0149] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant kinase gene and a wild-type gene can be determined
by direct DNA sequencing. A variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
(Naeve, C. W., (1995) Biotechniques 19:448), including sequencing
by mass spectrometry (see, e.g., PCT International Publication No.
WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and
Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).
[0150] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0151] The nucleic acid molecules are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the kinase gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment. FIG. 3 provides information on SNPs that
have been found in the gene encoding the kinase protein of the
present invention. The following variations were identified:
T1004G, G1822T, A2023G, A2562G, and C6624A. SNPs such as these that
are located in introns and 5' of the ORF may affect
control/regulatory elements.
[0152] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0153] The nucleic acid molecules are thus useful as antisense
constructs to control kinase gene expression in cells, tissues, and
organisms. A DNA antisense nucleic acid molecule is designed to be
complementary to a region of the gene involved in transcription,
preventing transcription and hence production of kinase protein. An
antisense RNA or DNA nucleic acid molecule would hybridize to the
mRNA and thus block translation of mRNA into kinase protein.
[0154] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of kinase nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired kinase nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the kinase protein, such as
substrate binding.
[0155] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in kinase
gene expression. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired kinase protein to treat the individual.
[0156] The invention also encompasses kits for detecting the
presence of a kinase nucleic acid in a biological sample.
Experimental data as provided in FIG. 1 indicates that kinase
proteins of the present invention are expressed in humans in the
larynx, kidney (adult and fetal), pancreas, fetal heart, uterus,
and prostate. Specifically, a virtual northern blot shows
expression in the larynx, kidney, and pancreas. In addition,
PCR-based tissue screening panels indicate expression in the fetal
heart, fetal kidney, uterus, prostate, and pancreas. For example,
the kit can comprise reagents such as a labeled or labelable
nucleic acid or agent capable of detecting kinase nucleic acid in a
biological sample; means for determining the amount of kinase
nucleic acid in the sample; and means for comparing the amount of
kinase nucleic acid in the sample with a standard. The compound or
agent can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect kinase protein
mRNA or DNA.
[0157] Nucleic Acid Arrays
[0158] The present invention further provides nucleic acid
detection kits, such as arrays or microarrays of nucleic acid
molecules that are based on the sequence information provided in
FIGS. 1 and 3 (SEQ ID NOS:1 and 3).
[0159] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by Brown et al., U.S.
Pat. No. 5,807,522.
[0160] The microarray or detection kit is preferably composed of a
large number of unique, single-stranded nucleic acid sequences,
usually either synthetic antisense oligonucleotides or fragments of
cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60 nucleotides in length, more preferably 15-30
nucleotides in length, and most preferably about 20-25 nucleotides
in length. For a certain type of microarray or detection kit, it
may be preferable to use oligonucleotides that are only 7-20
nucleotides in length. The microarray or detection kit may contain
oligonucleotides that cover the known 5', or 3', sequence,
sequential oligonucleotides which cover the full length sequence;
or unique oligonucleotides selected from particular areas along the
length of the: sequence. Polynucleotides used in the microarray or
detection kit may be oligonucleotides that are specific to a gene
or genes of interest.
[0161] In order to produce oligonucleotides to a known sequence for
a microarray or detection kit, the gene(s) of interest (or an ORF
identified from the contigs of the present invention) is typically
examined using a computer algorithm which starts at the 5' or at
the 3' end of the nucleotide sequence. Typical algorithms will then
identify oligomers of defined length that are unique to the gene,
have a GC content within a range suitable for hybridization, and
lack predicted secondary structure that may interfere with
hybridization. In certain situations it may be appropriate to use
pairs of oligonucleotides on a microarray or detection kit. The
"pairs" will be identical, except for one nucleotide that
preferably is located in the center of the sequence. The second
oligonucleotide in the pair (mismatched by one) serves as a
control. The number of oligonucleotide pairs may range from two to
one million. The oligomers are synthesized at designated areas on a
substrate using a light-directed chemical process. The substrate
may be paper, nylon or other type of membrane, filter, chip, glass
slide or any other suitable solid support.
[0162] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0163] In order to conduct sample analysis using a microarray or
detection kit, the RNA or DNA from a biological sample is made into
hybridization probes. The mRNA is isolated, and cDNA is produced
and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the presence of fluorescent nucleotides, and labeled
probes are incubated with the microarray or detection kit so that
the probe sequences hybridize to complementary oligonucleotides of
the microarray or detection kit. Incubation conditions are adjusted
so that hybridization occurs with precise complementary matches or
with various degrees of less complementarity. After removal of
nonhybridized probes; a scanner is used to determine the levels and
patterns of fluorescence. The scanned images are examined to
determine degree of complementarity and the relative abundance of
each oligonucleotide sequence on the microarray or detection kit.
The biological samples may be obtained from any bodily fluids (such
as blood, urine, saliva, phlegm, gastric juices; etc.), cultured
cells, biopsies, or other tissue preparations. A detection system
may be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large-scale correlation studies on the
sequences, expression patterns, mutations, variants, or
polymorphisms among samples.
[0164] Using such arrays, the present invention provides methods to
identify the expression of the kinase proteins/peptides of the
present invention. In detail, such methods comprise incubating a
test sample with one or more nucleic acid molecules and assaying
for binding of the nucleic acid molecule with components within the
test sample. Such assays will typically involve arrays comprising
many genes, at least one of which is a gene of the present
invention and or alleles of the kinase gene of the present
invention. FIG. 3 provides information on SNPs that have been found
in the gene encoding the kinase protein of the present invention.
The following variations were identified: T1004G, G1822T, A2023G,
A2562G, and C6624A. SNPs such as these that are located in introns
and 5' of the ORF may affect control/regulatory elements.
[0165] Conditions for incubating a nucleic acid molecule with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid molecule used in the assay. One
skilled in the art will recognize that any one of the commonly
available hybridization, amplification or array assay formats can
readily be adapted to employ the novel fragments of the Human
genome disclosed herein. Examples of such assays can be found in
Chard, T, An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The
test samples of the present invention include cells, protein or
membrane extracts of cells. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts-used as
the sample to be assayed. Methods for preparing nucleic acid
extracts or of cells are well known in the art and can be readily
be adapted in order to obtain a sample that is compatible with the
system utilized.
[0166] In another embodiment of the present invention, kits-are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0167] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the nucleic acid
molecules that can bind to a fragment of the Human genome disclosed
herein; and (b) one or more other containers comprising one or more
of the following: wash reagents, reagents capable of detecting
presence of a bound nucleic acid.
[0168] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers, strips of
plastic, glass or paper, or arraying material such as silica. Such
containers allows one to efficiently transfer reagents from one
compartment to another compartment such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample, a container which contains the
nucleic acid probe, containers which contain wash reagents (such as
phosphate buffered saline, Tris-buffers, etc.), and containers
which contain the reagents used to detect the bound probe. One
skilled in the art will readily recognize that the previously
unidentified kinase gene of the present invention can be routinely
identified using the sequence information disclosed herein can be
readily incorporated into one of the established kit formats which
are well known in the art, particularly expression arrays.
[0169] Vectors/Host Cells
[0170] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0171] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the nucleic acid molecules. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the nucleic acid molecules when the host cell
replicates.
[0172] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[0173] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
[0174] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0175] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0176] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[0177] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors; for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989).
[0178] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0179] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0180] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0181] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enterokinase. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New
England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,
N.J.) which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, to the target recombinant
protein. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0182] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).
Alternatively, the sequence of the nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, for example E. coli. (Wada et al., Nucleic
Acids Res. 20:2111-2118 (1992)).
[0183] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kujan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0184] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0185] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0186] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the nucleic acid molecules described
herein. These are found for example in Sambrook, J., Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0187] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0188] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0189] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989).
[0190] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the nucleic acid molecule
vector.
[0191] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0192] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0193] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0194] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as kinases, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0195] Where the peptide is not secreted into the medium, which is
typically the case with kinases, the protein can be isolated from
the host cell by standard disruption procedures, including freeze
thaw, sonication, mechanical disruption, use of lysing agents and
the like. The peptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0196] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
[0197] Uses of Vectors and Host Cells
[0198] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a kinase protein or peptide that can be further purified
to produce desired amounts of kinase protein or fragments. Thus,
host cells containing expression vectors are useful for peptide
production.
[0199] Host cells are also useful for conducting cell-based assays
involving the kinase protein or kinase protein fragments, such as
those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a native kinase protein is
useful for assaying compounds that stimulate or inhibit kinase
protein function.
[0200] Host cells are also useful for identifying kinase protein
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant kinase protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native kinase protein.
[0201] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA which is integrated into
the genome of a cell from which a transgenic animal develops and
which remains in the genome of the mature animal in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a kinase protein and
identifying and evaluating modulators of kinase protein activity.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, and amphibians.
[0202] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the kinase
protein nucleotide sequences can be introduced as a transgene into
the genome of a non-human animal, such as a mouse.
[0203] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the kinase
protein to particular cells.
[0204] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0205] In another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0206] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter Go phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0207] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
effect substrate binding, kinase protein activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo kinase protein function,
including substrate interaction, the effect of specific mutant
kinase proteins on kinase protein function and substrate
interaction, and the effect of chimeric kinase proteins. It is also
possible to assess the effect of null mutations, that is, mutations
that substantially or completely eliminate one or more kinase
protein functions.
[0208] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system 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 above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
4 1 1878 DNA Homo sapien 1 ggacactgac atggactgaa ggagtagaaa
aaccgactca acagtaaggc cccgcgggcg 60 tcctggccgc catgtgcacc
gtagtggacc ctcgcattgt ccggagatac ctactcaggc 120 ggcagctcgg
gcagggggcc tatggcattg tgtggaaggc agtggaccgg aggactggtg 180
aggtcgtggc catcaagaaa atctttgatg cttttaggga taagacagat gcccagagaa
240 cattccggga aatcacgctc ctccaggagt ttggggacca tcccaacatc
atcagcctcc 300 ttgacgtgat ccgggcagag aacgacaggg acatttacct
ggtgtttgag tttatggaca 360 ctgacctgaa cgcagtcatc cggaagggcg
gcctgctgca ggacgtccac gtgcgctcca 420 tcttctacca gctcctgcgg
gccacccggt tcctccactc ggggcacgtt gtgcaccggg 480 accagaagcc
gtccaatgtg ctcctggatg ccaactgcac agtgaagctg tgtgactttg 540
gcctggcccg ctccctgggc gacctccccg aggggcctga ggaccaggcc gtgacagagt
600 acgtggccac acgctggtac cgagcaccgg aggtgctgct ctcttcgcac
cgatacaccc 660 ttggggtgga catgtggagt ctgggctgta tcctggggga
gatgctgcgg gggagacccc 720 tgttccccgg cacgtccacc ctccaccagc
tggagctgat cctggagacc atcccaccgc 780 catctgagga ggacacctcc
ccagaggcct tggacctcct taggcgactc ctggtgttcg 840 ccccggacaa
gcggttaagc gcgacccagg cactgcagca cccctacgtg cagaggttcc 900
actgccccag cgacgagtgg gcacgagagg cagatgtgcg gccccgggca cacgaagggg
960 tccagctctc tgtgcctgag taccgcagcc gcgtctatca gatgatcctg
gagtgtggag 1020 gcagcagcgg cacctcgaga gagaagggcc cggagggtgt
ctccccaagc caggcacacc 1080 tgcacaaacc cagagccgac cctcagctgc
cttctaggac acctgtgcag ggtcccagac 1140 ccaggcccca gagcagccca
ggccatgacc ctgccgagca cgagtccccc cgtgcagcca 1200 agaacgttcc
caggcagaac tccgctcccc tgctccaaac tgctctccta gggaatgggg 1260
aaaggccccc tggggcgaag gaagcgcccc ccttgacact ctcgctggtg aagccaagcg
1320 ggaggggagc tgcgccctcc ctgacctccc aggctgcggc tcaggtggcc
aaccaggccc 1380 tgatccgggg tgactggaac cggggcggtg gggtgagggt
ggccagcgta caacaggtcc 1440 ctccccggct tcctccggag gcccggcccg
gccggaggat gttcagcccc tctgccttgc 1500 agggtgccca ggggggtgcc
agggctttgc ttggaggcta ctcccaagcc tacgggactg 1560 tttgcccctc
ggcactgggc cccctgcccc tgctggaggg gccccatatg tgagccgccc 1620
tactcccttc acctggccct ctgttcctgc cccagcccct tccccagacc cctttccagt
1680 ttcctgcccc ccttagccct ccctgctttg cctggcccgt tgaagttcca
gggagcttgc 1740 ccgggtctcc tcgggggagc aaatgagggc cctgcccccg
cccccctgac ttcctccaat 1800 aaagtcatgt ttgcccccca aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860 aaaaaaaaaa aaaaaaaa 1878 2
513 PRT Homo sapien 2 Met Cys Thr Val Val Asp Pro Arg Ile Val Arg
Arg Tyr Leu Leu Arg 1 5 10 15 Arg Gln Leu Gly Gln Gly Ala Tyr Gly
Ile Val Trp Lys Ala Val Asp 20 25 30 Arg Arg Thr Gly Glu Val Val
Ala Ile Lys Lys Ile Phe Asp Ala Phe 35 40 45 Arg Asp Lys Thr Asp
Ala Gln Arg Thr Phe Arg Glu Ile Thr Leu Leu 50 55 60 Gln Glu Phe
Gly Asp His Pro Asn Ile Ile Ser Leu Leu Asp Val Ile 65 70 75 80 Arg
Ala Glu Asn Asp Arg Asp Ile Tyr Leu Val Phe Glu Phe Met Asp 85 90
95 Thr Asp Leu Asn Ala Val Ile Arg Lys Gly Gly Leu Leu Gln Asp Val
100 105 110 His Val Arg Ser Ile Phe Tyr Gln Leu Leu Arg Ala Thr Arg
Phe Leu 115 120 125 His Ser Gly His Val Val His Arg Asp Gln Lys Pro
Ser Asn Val Leu 130 135 140 Leu Asp Ala Asn Cys Thr Val Lys Leu Cys
Asp Phe Gly Leu Ala Arg 145 150 155 160 Ser Leu Gly Asp Leu Pro Glu
Gly Pro Glu Asp Gln Ala Val Thr Glu 165 170 175 Tyr Val Ala Thr Arg
Trp Tyr Arg Ala Pro Glu Val Leu Leu Ser Ser 180 185 190 His Arg Tyr
Thr Leu Gly Val Asp Met Trp Ser Leu Gly Cys Ile Leu 195 200 205 Gly
Glu Met Leu Arg Gly Arg Pro Leu Phe Pro Gly Thr Ser Thr Leu 210 215
220 His Gln Leu Glu Leu Ile Leu Glu Thr Ile Pro Pro Pro Ser Glu Glu
225 230 235 240 Asp Thr Ser Pro Glu Ala Leu Asp Leu Leu Arg Arg Leu
Leu Val Phe 245 250 255 Ala Pro Asp Lys Arg Leu Ser Ala Thr Gln Ala
Leu Gln His Pro Tyr 260 265 270 Val Gln Arg Phe His Cys Pro Ser Asp
Glu Trp Ala Arg Glu Ala Asp 275 280 285 Val Arg Pro Arg Ala His Glu
Gly Val Gln Leu Ser Val Pro Glu Tyr 290 295 300 Arg Ser Arg Val Tyr
Gln Met Ile Leu Glu Cys Gly Gly Ser Ser Gly 305 310 315 320 Thr Ser
Arg Glu Lys Gly Pro Glu Gly Val Ser Pro Ser Gln Ala His 325 330 335
Leu His Lys Pro Arg Ala Asp Pro Gln Leu Pro Ser Arg Thr Pro Val 340
345 350 Gln Gly Pro Arg Pro Arg Pro Gln Ser Ser Pro Gly His Asp Pro
Ala 355 360 365 Glu His Glu Ser Pro Arg Ala Ala Lys Asn Val Pro Arg
Gln Asn Ser 370 375 380 Ala Pro Leu Leu Gln Thr Ala Leu Leu Gly Asn
Gly Glu Arg Pro Pro 385 390 395 400 Gly Ala Lys Glu Ala Pro Pro Leu
Thr Leu Ser Leu Val Lys Pro Ser 405 410 415 Gly Arg Gly Ala Ala Pro
Ser Leu Thr Ser Gln Ala Ala Ala Gln Val 420 425 430 Ala Asn Gln Ala
Leu Ile Arg Gly Asp Trp Asn Arg Gly Gly Gly Val 435 440 445 Arg Val
Ala Ser Val Gln Gln Val Pro Pro Arg Leu Pro Pro Glu Ala 450 455 460
Arg Pro Gly Arg Arg Met Phe Ser Pro Ser Ala Leu Gln Gly Ala Gln 465
470 475 480 Gly Gly Ala Arg Ala Leu Leu Gly Gly Tyr Ser Gln Ala Tyr
Gly Thr 485 490 495 Val Cys Pro Ser Ala Leu Gly Pro Leu Pro Leu Leu
Glu Gly Pro His 500 505 510 Met 3 8285 DNA Homo sapien misc_feature
(1)...(8285) n = A,T,C or G 3 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nttgttcctt ttccttcttt
ttttgaattc 300 tttttgagca agtagtttgt gttgtggttg ttgtttgaga
cagggtctgg ctctgtcacc 360 caggctggag tgcagtggcg caatccaggc
tcactgcaac ctctgcctcc cggctcaagc 420 gatcctccta cctcagcctc
ccaagtagct gggacaacag gctcatgtca ccacacccag 480 ctaattttcc
tatttttttt ttttaataga aatgaggttt tatgttgccg aagctggtct 540
ccaattcctg agtcattagc cacgcccggc taatttttgt atttttagtg gagacggggt
600 ttcaccacgt tggccaggct ggtcttgaac ccttgacctc gggtgatcca
cccgcctcgg 660 cctcccagag tgttgggatt acaggcgtga accaccgtgt
cccgcccaaa taataatata 720 ctattaatac ttcacatgta acttaagaac
cttacaatac atattctcat gttattttgt 780 aatagtataa atgtgtattt
ccattatccc ccttcacttt ttgctattgg tgtcatgcat 840 tttacttcta
caagttatag agtccacaac agatagttct tgtttctact ttagtcagct 900
gggctgggcg tggtcctgcg aggaggtggg cggggcgcac tgtggggcgg ggccggtggg
960 gacgtgggcg gggcgccatt gaggggaggg gcctgcgggg aggttgggtg
ggcccactgt 1020 ggggcggagc cggggcctgc cgggggcggg gggtgttggg
aggggcgccc cgaggggcgg 1080 ggccgggccg ccgtcggttc ccacggcaac
cgactcaaca gtaaggcccc gcgggcgtcc 1140 tggccgccat gtgcaccgta
gtggaccctc gcattgtccg gagataccta ctcaggcggc 1200 agctcgggca
gggggtgagt gcctgggggt gcgtccgcgc gccgaggggc gcggcatatc 1260
tgcggataga ggacctgnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnncc cgggtcactg
aaagaagggc ggaccccagg ctcaggtagc acaggggcga 1560 ggcccgagaa
gggcctgagc ggttatgggg tgggcgcaga gtgaagggca gagccttgtg 1620
tatctgtgtg tgtgtgtgag catgtaagcc tgtgtgtgtg tgcgtgggtg tgtggggggg
1680 tgttcgaggg tgccatgggg gaggggagga agagccttcc aggcagtgca
gacggtaagt 1740 gcgtaggccc agtgcagggt tgtgtatgtg caactggata
ggagatggag agagacaggt 1800 gagtggtgag ggtccgatcg tgtgggagct
ttggggaact tccaagactt tggtttttac 1860 tgttgctgag gctgggagct
gtagcagctg ctggtgtcac tttacaaggc ccacccctgt 1920 gctgaggacc
taccgtgggt gtgcacggga gcggcagacg gagatgagtt aaggggttag 1980
cgtagccacg cagcgagaga tgccagaggc tgggaccagg gtaggggcag aagagaccgt
2040 ggcaggggct agattctgga ggaatctgaa ggtagggcca atgggattgg
gggtggatgg 2100 ggtgtgagag aaagggaggg agagtgcctg ggcagctgga
aggatgatag ggcatccccg 2160 agcttcattt cctgcccaga cgctcccctc
tgtggcctcc tttcctccag ggcctcgcca 2220 gctctcaccc tcccttccct
ctacctcccc tcctctggaa gatgtcggag tctagggcag 2280 cctgcagttg
cgggagccca cactcccatc ccctctcggg acccaggatg ggaaggagga 2340
gcctcatgtc tgtagggaca atctgggtgg gcaggggatg gggggaaggg gctggccctg
2400 tgtgacggca ctccttccca ggcctatggc attgtgtgga aggcagtgga
ccggaggact 2460 ggtgaggtcg tggccatcaa gaaaatcttt gatgctttta
gggataagac agatgcccag 2520 gtgagtgtgt ggggagaagc gtgggagagg
atgggggcag gaaggggcag ccccttgccc 2580 tggtgcctgg aagctcaggt
gggagctgga gcccagtcat agcagatgtt ctggcctgtc 2640 tcggaacact
gcccccttgc cacgcctggt ctggtgggta ttgggtgaca gacatcagct 2700
cctttgggtc ctctcaggac atgggcttcc ttcttgctcc acccacccac acacctgtgt
2760 ttctgtctct tcagagaaca ttccgggaaa tcacgctcct ccaggtgagt
ggcctgggcc 2820 ctccagtcca atccccttgc ccaggtacag atctctccag
acaggagaga aactggcctt 2880 cttgggcccc agagcacagc ccctcctggc
cttccagccg cctccgactc tctccccagg 2940 agtttgggga ccatcccaac
atcatcagcc tccttgacgt gatccgggca gagaacgaca 3000 gggacattta
cctggtgttt gagtttatgg gtgagtgagg ccccggccag cgccccagcc 3060
ccacctctgt tctgtcctga cgccgtctgc gggtccctct gcgtgtccct ctgcgtgtcc
3120 ctctgcagct ggcccacagt ggcttgctcc ctcaccatgt accctggact
cagggacaga 3180 cagctgacta gtgtcagcct ccagagccag cagcgacccc
tttcgtccca cctgccccag 3240 gctcctgctc tgaccacagt ttgcagttgc
gttctccttt ttcttctcat tttatgaaac 3300 aaaggcaaca tgaaataaag
tgttaaaact cctgcagacc tcaccgctgt gcccacaggc 3360 agtgcacagg
atggaggagc ggggcggcca ggccgtgggc tggttcaaag tgggacagac 3420
ctgccaggtg cccctctccc actcccccca ggttgccccc ccagcccccc acccccgact
3480 gcagtgcgca ccctctctgc agacactgac ctgaacgcag tcatccggaa
gggcggcctg 3540 ctgcaggacg tccacgtgcg ctccatcttc taccagctcc
tgcgggccac ccggttcctc 3600 cactcggggc acgttgtgca ccgggaccag
aaggtgcggt tcccccgccc ccgctatgcc 3660 acgtggcccg gctcccggcc
ccacccagcc ccggggcctc agcctgcctc ctctctgcag 3720 ccgtccaatg
tgctcctgga tgccaactgc acagtgaagc tgtgtgactt tggcctggcc 3780
cgctccctgg gcgacctccc tgaggggcct gaggaccagg ccgtgacaga gtacgtggcc
3840 acacgctggt accgagcacc ggaggtgctg ctctcttcgc accggtaata
gcgagacatc 3900 cccaaccccc ctccacctcc ctgctgccct cctgcccagc
cagggctccc aggcctcccg 3960 tactccgacc ctgccttggt ccacaagtgt
tcccccattc accccccagc aaccccaccc 4020 ccacctctgc ctctgggtct
ctccatgcct acaccgcttc ctgccccaga tacacccttg 4080 gggtggacat
gtggagtctg ggctgtatcc tgggggagat gctgcggggg agacccctgt 4140
tccccggcac gtccaccctc caccagctgg agctgatcct ggagaccatc ccaccgccat
4200 ctgaggaggg tgagccaggc tgctggggct gggcaccagg aatgctgcag
gtcagacagc 4260 acagctgtgg ggagacagca gctgacaggc taggactgtg
ctgagaggag ggacggggac 4320 agggaggatc cagaggatgg ggcaggagcc
ccaggaagac cgactggtga tgggggccca 4380 ggaggagctg ctgggggtgg
gtgtgggcaa ggcagcacct ggcacagtca ccatgagagc 4440 caagcagtga
ccgtgaaggg gccagcaggc tggacaaggt ccccaaggga ttcgggtagc 4500
aggggcaggg actgtcactg tgccgggagc tggggtgtgc agagacagct gggcaggaga
4560 gattcaggtg ctgagggaag aggtggagga aggcagtggt agaggggcca
tgggggtcac 4620 tcttgagggt gggggcaaga gggagctgca ccgccaggca
tagctgcttg tctgggtgga 4680 gcctcctggg ccgtggaggt gggcgccagc
atccacttct gtgagcacac cccagggcca 4740 ggtgcccgag tgtggagcag
gggtcatgtg cgggtgctcc cgtgcacagg ctgggtggca 4800 cgccctggtg
atggggtgtt tgagccccgc cagacagcag aaaccctgta gagaggctgt 4860
gctccctggg gctggaagag atgactggcc ccagatgccc tgagccgccc cagccgacca
4920 ggcctgcctg ggtcacacca ccttctgctg ccccagacct cctggctctc
ggctcaggct 4980 gccgtgcctc tgtgctgcac cagctggggt cccggtgagt
gggggcactt cggtgagggt 5040 gacagggtgg cctatctcaa gggagcaggg
ccaccttcct gcaagtttac tggggccagt 5100 ttgtaccagt tcagattctg
cctgttttca agatggcagt cccaaaccca acaactgttg 5160 gccacactga
aagcaggagc ccctctggtg ctcctagagg gtggcccaga ggagctgtgc 5220
cagggcgtgg agaggagggc accagggggc cgcaggggtc tctccaccct gcaggggccc
5280 agactgcctg caggtcaggc acaggggcat ctacctagac aggacagcag
ggtggacccc 5340 agtttggaag ctgagccccc agccacgaac atggatctga
ggaggggccc ttgggtcggg 5400 ccctggagac gacacacggc agcccacagg
ccacgacaga cgctggatgc cctcctaccg 5460 ccagacacct ccccagaggc
cttggacctc cttaggcgac tcctggtgtt cgccccggac 5520 aagcggttaa
gcgcgaccca ggcactgcag cacccctacg tgcagaggtg ggggtgggag 5580
agagtccccc aagtgcgggg ggacagaggt gggggcagga gagagccagc ccatgaggga
5640 cagcccccac agcagggacc ctgctgtgac ggcttgaggg gctcccttgg
ccgcagcccg 5700 ggccccacct ccctggctcc ctgcaggttc cactgcccca
gcgacgagtg ggcacgagag 5760 gcagatgtgc ggccccgggc acacgaaggg
gtccagctct ctgtgcctga gtaccgcagc 5820 cgcgtctatc aggtgctccg
gctctcgacc cctatcatcc cctgtctact gcaccctgga 5880 ggctgcctcc
tatgtcagag acccccaaac gccccatgcc caggctgtga cctctgagca 5940
cccttcccct cccgcagatg atcctggagt gtggaggcag cagcggcacc tcgagagaga
6000 agggcccgga gggtgtctcc ccaagccagg cacacctgca caaacccaga
gccgaccctc 6060 agctgccttc taggacacct gtgcagggtc ccagacccag
gccccagagc agcccaggcc 6120 atgaccctgc cgagcacggt gtgtgatctt
tgctggccgc ccacgcggag catggcccgg 6180 gccccttctg cctgtgctgc
caactatgcg cagcattcgg ttcctgaccc tggggttgac 6240 ccactgaccc
cggggttgac ccactgaccc cacagagtcc ccccgtgcag ccaagaacgt 6300
tcccaggcag aactccgctc ccctgctcca aactgctctc ctagggaatg gggaaaggcc
6360 ccctggggcg aaggaagcgc cccccttgac actctcgctg gtaagtcatg
gtggggcggg 6420 cacaggaggg acccctcctc tgcacctttc agtgaccctg
tgacatggcc cttcccaggt 6480 gaagccaagc gggaggggag ctgcgccctc
cctgacctcc caggctgcgg ctcaggtggc 6540 caaccaggcc ctgatccggg
gtgactggaa ccggggcggt ggggtgaggg tggccagcgt 6600 acaacaggta
agcccggccc agtctgcccc cgtcccctca tcctcctttc ccctttcccc 6660
ttcccccctg cttttccctc ccttccccat gcttcccatt gcccctccaa tgtccagttc
6720 aaatctctcg aggacctcaa ggcctcccct ccactgcacc ccctctgatg
gcccctttat 6780 gtgaccctca actgtacaca ggtccctccc cggcttcctc
cggaggcccg gcccggccgg 6840 aggatgttca gcacctctgc cttgcagggt
gcccaggggg gtgccagggc tttgcttgga 6900 ggctactccc aagcctacgg
gactgtctgc cactcggcac tgggccacct gcccctgctg 6960 gaggggcacc
atgtgtgagc cgccctactc ccttcacctg gccctctgtt cctgccccag 7020
ccccttcccc agacccctct ccagtctcct gcacccctta gccctccctg ctttgcctgg
7080 cccgttgaag ttccagggag cttgcccggg tctcctcggg ggagcagatg
agggccctgc 7140 ccccgcccca ctgacttcct ccaataaagt catgtctgcc
cccaacctaa gcagccatcg 7200 ttcctcccct cccctctgag gtcacagcat
ccactagctg ggggccccgg cccctttcct 7260 gaagcctcca ctcctctgag
gaccccaccc cacccccgtc ctgaaacctc caccccagag 7320 cccagtgccg
ccccctagag gccctgccca ctgcacatcc agcactgggc ttttccctcc 7380
aggtttgcct ggggcagctt cttgttcttt gtccatcatt tccttacctg ctgtggcttc
7440 agggtccagg ctgcccccca gggtggtcct gtggggtagg gacgtagggt
caccccctgg 7500 ccatgtttgt gactctgagc cagaggagag aaggggagag
agaaggggga cacccctccc 7560 cctgctgtca gggactgcag cctgcgcccc
ctagtatggc cactgcacct gatctgtctt 7620 caggtctccg taggtgaggg
tgggagacag acatctcgcg aggtcagggt tacctcctct 7680 tgtcaccccc
aggcaaggtc cctggtgtga gttcaggcca gggctgtgca gggctgcaaa 7740
gatcaaaggg gccctgtggg cacagacctg tgtcctaggg tgccaggtgt cctcagctgc
7800 acctgcccat gggttggggt tggaacacaa ggaggcagct ggaaagctca
caggctggag 7860 gagctcacag tctaaagggc gcggcctgtg ctgtcggtgg
cggagttggg ctgccaggct 7920 cacagtctgg gaagctcata ggccggagga
gctcacagtt tgaagggtgc ggcctgtgct 7980 gtggtcggtg ttgggctgcc
aggagagggg cgctgctggg ttgtggaagc cattgccacc 8040 atgggggagg
gcggggaagg acaagatgtg ggtgggggag ctgagcagaa ggtgagagct 8100
ggcgctgccc tggtgctgga ccaggcacct gcaagagact cagaaaggga ggctgggttt
8160 gggagaaggt tggaggaggc ggaggaggga tcgggagggc ccgaggaagc
ggtgagccag 8220 tcagagaccc agcccagggg ctgtttcctg agggggctgc
cgagggaggt gcttgttgag 8280 cttca 8285 4 544 PRT Rattus norvegicus 4
Met Cys Ala Ala Glu Val Asp Arg His Val Ser Gln Arg Tyr Leu Ile 1 5
10 15 Lys Arg Arg Leu Gly Lys Gly Ala Tyr Gly Ile Val Trp Lys Ala
Met 20 25 30 Asp Arg Arg Thr Gly Glu Val Val Ala Ile Lys Lys Ile
Phe Asp Ala 35 40 45 Phe Arg Asp Gln Thr Asp Ala Gln Arg Thr Phe
Arg Glu Ile Met Leu 50 55 60 Leu Arg Glu Phe Gly Gly His Pro Asn
Ile Ile Arg Leu Leu Asp Val 65 70 75 80 Ile Pro Ala Lys Asn Asp Arg
Asp Ile Tyr Leu Val Phe Glu Ser Met 85 90 95 Asp Thr Asp Leu Asn
Ala Val Ile Gln Lys Gly Arg Leu Leu Glu Asp 100 105 110 Ile His Lys
Arg Cys Ile Phe Tyr Gln Leu Leu Arg Ala Thr Lys Phe 115 120 125 Ile
His Ser Gly Arg Val Ile His Arg Asp Gln Lys Pro Ala Asn Val 130 135
140 Leu Leu Asp Ala Ala Cys Arg Val Lys Leu Cys Asp Phe Gly Leu Ala
145 150 155 160 Arg Ser Leu Ser Asp Phe Pro Glu Gly Leu Gly Gln Ala
Leu Thr Glu 165 170 175 Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu
Val Leu Leu Ser Ser 180 185 190 Arg Trp Tyr Thr Pro Gly Val Asp Met
Trp Ser Leu Gly Cys Ile Leu 195 200 205 Gly Glu Met Leu Arg Gly Gln
Pro Leu Phe Pro Gly Thr Ser Thr Phe 210 215 220 His Gln Leu Glu Leu
Ile Leu Glu Thr Ile Pro Leu Pro Ser Met Glu 225 230 235 240 Glu Leu
Gln Gly Leu Gly Ser Asp Tyr Ser Ala Leu Ile Leu
Gln Asn 245 250 255 Leu Gly Ser Arg Pro Arg Gln Thr Leu Asp Ala Leu
Leu Pro Pro Asp 260 265 270 Thr Pro Pro Glu Ala Leu Asp Leu Leu Lys
Arg Leu Leu Ala Phe Ala 275 280 285 Pro Asp Lys Arg Leu Ser Ala Glu
Gln Ala Leu Gln His Pro Tyr Val 290 295 300 Gln Arg Phe His Cys Pro
Asp Arg Glu Trp Thr Arg Gly Ser Asp Val 305 310 315 320 Arg Leu Pro
Val His Glu Gly Asp Gln Leu Ser Ala Pro Glu Tyr Arg 325 330 335 Asn
Arg Leu Tyr Gln Met Ile Leu Glu Arg Arg Arg Asn Ser Arg Ser 340 345
350 Pro Arg Glu Glu Asp Leu Gly Val Val Ala Ser Arg Ala Glu Leu Arg
355 360 365 Ala Ser Gln Arg Gln Ser Leu Lys Pro Gly Val Leu Pro Gln
Val Leu 370 375 380 Ala Glu Thr Pro Ala Arg Lys Arg Gly Pro Lys Pro
Gln Asn Gly His 385 390 395 400 Gly His Asp Pro Glu His Val Glu Val
Arg Arg Gln Ser Ser Asp Pro 405 410 415 Leu Tyr Gln Leu Pro Pro Pro
Gly Ser Gly Glu Arg Pro Pro Gly Ala 420 425 430 Thr Gly Glu Pro Pro
Ser Ala Pro Ser Gly Val Lys Thr His Val Arg 435 440 445 Ala Val Ala
Pro Ser Leu Thr Ser Gln Ala Ala Ala Gln Ala Ala Asn 450 455 460 Gln
Pro Leu Ile Arg Ser Asp Pro Ala Arg Gly Gly Gly Pro Arg Ala 465 470
475 480 Val Gly Ala Arg Arg Val Pro Ser Arg Leu Pro Arg Glu Ala Pro
Glu 485 490 495 Pro Arg Pro Gly Arg Arg Met Phe Gly Ile Ser Val Ser
Gln Gly Ala 500 505 510 Gln Gly Ala Ala Arg Ala Ala Leu Gly Gly Tyr
Ser Gln Ala Tyr Gly 515 520 525 Thr Val Cys Arg Ser Ala Leu Gly Arg
Leu Pro Leu Leu Pro Gly Pro 530 535 540
* * * * *
References